Research ArticleEvaluation of the Ribosomal Protein S1 Gene (rpsA) as a NovelBiomarker for Mycobacterium Species Identification

Hongfei Duan Guan Liu Xiaobo Wang Yuhong Fu Qian Liang Yuanyuan ShangNaihui Chu and Hairong Huang

National Clinical Laboratory on Tuberculosis Beijing Key Laboratory on Drug-Resistant Tuberculosis Beijing Chest HospitalCapital Medical University Beijing Tuberculosis ampThoracic Tumor Research Institute 97 Beimachang Tongzhou QuBeijing 101149 China

Correspondence should be addressed to Naihui Chu dongchu1994sinacom and Hairong Huang haironghuangcngmailcom

Received 26 July 2014 Accepted 7 November 2014

Academic Editor Om Parkash

Copyright copy 2015 Hongfei Duan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objectives To evaluate the resolution and reliability of the rpsA gene encoding ribosomal protein S1 as a novel biomarker formycobacteria species identification Methods A segment of the rpsA gene (565 bp) was amplified by PCR from 42 mycobacterialreference strains 172 nontuberculosis mycobacteria clinical isolates and 16 M tuberculosis complex clinical isolates The PCRproducts were sequenced and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and theMEGA program A phylogenetic tree was constructed by the neighbor-joining method Results Comparative sequence analysisof the rpsA gene provided the basis for species differentiation within the genus Mycobacterium Slow- and rapid-growing groupsof mycobacteria were clearly separated and each mycobacterial species was differentiated as a distinct entity in the phylogenetictree The sequences discrepancy was obvious between M kansasii and M gastri M chelonae and M abscessus M avium andM intracellulare and M szulgai and M malmoense which cannot be achieved by 16S ribosomal DNA (rDNA) homologue genescomparison 183 of the 188 (973) clinical isolates consisting of 8 mycobacterial species were identified correctly by rpsA geneblast Conclusions Our study indicates that rpsA sequencing can be used effectively for mycobacteria species identification as asupplement to 16S rDNA sequence analysis

1 Introduction

Members of the genus Mycobacterium are widespread innature and range from harmless saprophytic species tostrict pathogens that cause serious human and animal dis-eases Both slow-growing mycobacteria and rapid-growingmycobacteria can cause human infections Traditionallytaxonomybased onbiochemical characteristics has been usedfor species determination of mycobacteria but this approachis limited due to the overlapping biochemical and phenotypicpatterns among the different mycobacterial species Anotherapproach using analysis of cell-wall fatty acid and mycolicacid composition is also limited by profile similarity amongsome emerging nontuberculosismycobacteria (NTM) [1] 16SrDNA homologue gene sequence comparison has been usedas an important method for the mycobacterial species iden-tification however ambiguous results have been obtainedeither due to the presence of more than one copy of the 16S

rDNA gene within the genome for example in M celatumand M terrae complex [2 3] or due to sequence homol-ogy between species [4] Therefore alternative phylogeneticmarkers which are capable of complementing 16S rRNAgene would be useful for the phylogenetic study and speciesidentification of the genusMycobacterium

Ribosomal protein S1 (rpsA) which is in the 30S ribosomesubunit contains the S1 domain that has been found in a largenumber of RNA-associated proteins RpsA is a vital proteininvolved in protein translation and the ribosome-sparingprocess of translation In addition it has been reported thatRpsA is the target of pyrazinoic acid the active form ofthe antituberculosis drug pyrazinamide [5] Mycobacteriumtuberculosis has a single copy of the rpsA gene in the genome[6] while the sequence homology among different mycobac-teria species in which the gene has already been sequencedis between 867 and 100 This suggests that rpsA may be

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 271728 8 pageshttpdxdoiorg1011552015271728

2 BioMed Research International

suitable for phylogenetic study of the genus MycobacteriumIn this paper we report an evaluation of the rpsA gene as anovel biomarker for mycobacteria species identification

2 Materials and Methods

21 Mycobacterial Reference Strains and Clinical Isolates42 type and reference strains of the genus Mycobacterium(Table 1) 172 clinical NTM isolates and 16 M tuberculosiscomplex (MTC) isolates were investigated All the type andreference strains were purchased from the American TypeCulture Collection (ATCC) and all the clinical isolates usedin this study were obtained from the Clinical Database andSample Bank of tuberculosis of Beijing National ClinicalLab on Tuberculosis Beijing Chest Hospital All the clinicalNTM isolates were identified to the species level by sequencealignment of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoBand hsp65 genes as described before [2 7ndash9] Followingsequencing the 172 NTM clinical strains were found toinclude 10 strains ofM avium 75 strains ofM intracellulare23 strains ofM kansasii 39 strains ofM abscessus 17 strainsofM fortuitum complex 7 strains ofM gordonae and 1 strainofM neoaurum

22 rpsA Gene Amplification and Sequencing DNA wasreleased from cultured mycobacteria by boiling the culturedmycobacterial suspension in TE buffer for 10min After cen-trifugation the supernatant was used for PCR amplification[10] The primers used were forward primer 51015840-CCCTAC-ATCGGCAAGGAG-31015840 position 487ndash504 in the rpsA geneof Mycobacterium tuberculosis GenBank accession num-ber NC 0009622 and reverse primer 51015840-TGTCGATGA-CCTTGACCATC-31015840 position 1032ndash1051 in the rpsA geneof Mycobacterium tuberculosis GenBank accession numberNC 0009622 The amplified product was 565 bp PCR prod-ucts were purified and sequenced by a commercial company(YINGJUN Biotech Company Beijing China) using ABI3730 DNA Analyzer (Applied Biosystems California USA)

23 Sequence Analysis and the Phylogenetic Tree Construc-tions In addition to the 42 reference strains the rpsAsequences of 5 other mycobacterial species were obtaineddirectly from GenBank including M avium subspeciesparatuberculosis (GenBank accession number NC 0029442)M ulcerans (GenBank accession number NC 0086111) Mvanbaalenii (GenBank accession number NC 0087261) Mabscessus subspeciesmassiliense (GenBank accession numberNC 0181501) and M canettii (GenBank accession numberNC 0158481) All sequences were aligned and homologywas calculated by using the multiple alignment algorithm inthe MegAlign package (Windows version 710 DNASTARMadison Wis) 527 bp of sequence (excluding 38 nucleotidesat each end of the amplicon corresponding to the primerbinding sites) was analyzed for the phylogenetic tree con-struction by the neighbor-joining method using the MEGA505 package (httpwwwmegasoftwarenetmegaphp) Abootstrap analysis (1000 repeats) using Rhodococcus equi

(GenBank accession number NC 0063611) as the outgroupwas performed to evaluate the topology of the phylogenetictree

24 Species Identification of the Clinical Isolates The 188clinical isolates were analyzed blindly Sequences minus theknown PCR primer sequences were assembled and edited byusing SeqMan software (version 710 DNASTAR MadisonWI) Isolates were identified by comparing sequences byusing a FASTA BLASTn search with MegAlign (version710 DNASTAR Madison WI) to an in-house databaseof sequences consisting of type and reference strains fromexternal culture collections

3 Results

31 rpsA Sequence Alignment of the Reference StrainsBetween 854 and 100 sequence homology (interspeciesdivergence 0 to 146) was observed among the 42tested reference strains and the 5 additional mycobacterialspecies whose sequences were obtained from the Gen-Bank database (Table 2) All the rpsA gene sequences ofthe analyzed mycobacteria strains were distinct from theoutgroup strain R equi Among the 19 reference strainsof the slow-growing Mycobacterium genus 11 strains weregreater than 97 homology including 5 M tuberculosiscomplex strains Among the 28 reference strains of the rapid-growing Mycobacterium genus 14 strains were greater than97 homology (Table 1) The pathogenic M kansasii waseasily differentiated from the nonpathogenicM gastri (958homology) while those two species were not distinguish-able by the 16S rDNA sequence alignment The sequencehomologies between various species were 918 between Mchelonae and M abscessus 956 between M avium andM intracellulare and 939 between M szulgai and Mmalmoense However the sequence homology between otherspecies was higher for example it was 996 between Mulcerans and M marinum and 987 between M abscessussubspecies massiliense and M abscessus All members ofthe M tuberculosis complex had identical sequences as didM senegalense and M thermoresistibile M parafortuitumand M trivial M diernhoferi and M duvalii and M aus-troafricanum andM terrae

32 Phylogenetic Tree Construction A phylogenetic treewhich provided the basis for species differentiation in thegenus Mycobacterium was constructed (Figure 1) The abso-lute majority tested species showed good separation Therapid-growing species were well defined from the slow-growing species in the tree M chelonae M abscessus sub-speciesmassiliense andMabscessus which are categorized inthe pathogenic taxonomic group of rapid-growing mycobac-teria formed a distinctive cluster which was much closer tothe slow-growing species compared with the nonpathogenicgroup of rapid-growing mycobacteria The reliability of thephylogenetic tree was verified by the bootstrapmethod usingR equi as the outgroup

BioMed Research International 3

Table 1 Percentage rpsA sequence match among type strain

Species Type strain First match ()a Second match ()a

M thermoresistibile ATCC19527 M senegalense (100) M porcinum (987)M senegalense ATCC35796 M thermoresistibile (100) M porcinum (987)M porcinum ATCC33776 M senegalenseM thermoresistibile (987) M fortuitum (983)

M fortuitum ATCC6481 M porcinum (983) M senegalenseM thermoresistibile(981)

M phlei ATCC11758 M porcinum (965) M fortuitum (961)M gadium ATCC27726 M porcinumM fortuitum (957) M smegmatis (953)M flavescens ATCC14474 M smegmatis (966) M vaccae (960)

M smegmatis ATCC19420 M vaccaeM parafortuitumM triviale(966)

M senegalenseM thermoresistibile(963)

M vaccae ATCC15483 M diernhoferiM duvalii (968) M parafortuitumM triviale (966)M parafortuitum ATCC19686 M triviale (100) M terraeM austroafricanum (966)M triviale ATCC23292 M parafortuitum (100) M terraeM austroafricanum (966)M neoaurum ATCC25795 M diernhoferiM duvalii (964) M porcinum (955)M diernhoferi ATCC19340 M duvalii (100) M vaccae (968)M duvalii ATCC43910 M diernhoferi (100) M vaccae (968)M aurum ATCC23366 M gilvum (939) M chubuenseM vanbaalenii (930)M chubuense ATCC27278 M gilvum (966) M vanbaalenii (960)M gilvum ATCC43909 M vanbaalenii (972) M terraeM austroafricanum (970)M vanbaalenii PYR-1 M terraeM austroafricanum (998) M gilvum (972)M terrae ATCC15755 M austroafricanum (100) M vanbaalenii (998)M austroafricanum ATCC33464 M terrae (100) M vanbaalenii (998)M rhodesiae ATCC27024 M terraeM austroafricanum (939) M obuense (937)

M obuense ATCC27023 M porcinum M fortuitum (961) M senegalenseM thermoresistibile(965)

M aichiense ATCC27280 M sphagni (958) M vaccae (947)M sphagni ATCC33027 M aichiense (958) M porcinum (955)M tokaiense ATCC27282 M porcinum (953) M smegmatis (937)M chelonae ATCC35752 M obuense (939) M phlei (935)M abscessus subspeciesmassiliense str GO 06 M abscessus (987) M sphagni (914)

M abscessus ATCC19977 M abscessus subspeciesmassiliense (987) M chubuense (920)

M tuberculosis H37Rv ATCC27294 M tuberculosisH37RaM africanumMbovis BCGM bovis (100) M canettii (998)

M tuberculosis H37Ra ATCC25177 M tuberculosisH37RvM africanumMbovis BCGM bovis (100) M canettii (998)

M africanum ATCC25420 M tuberculosisH37RaM tuberculosisRvM bovis BCGM bovis (100) M canettii (998)

M bovis BCG ATCC35735 M tuberculosisH37RaM tuberculosisH37RvM africanumM bovis (100) M canettii (998)

M bovis ATCC19210 M tuberculosisH37RaM tuberculosisH37RvM africanumM bovis BCG (100) M canettii (998)

M canettii CIPT140010059 M tuberculosisM africanumM bovisBCGM bovis (998) M asiaticum (932)

M xenopi ATCC19250 M asiaticum (930) M avium subspecies paratuberculosis(924)

M avium ATCC25291 M avium subspecies paratuberculosis(996) M asiaticum (958)

4 BioMed Research International

Table 1 Continued

Species Type strain First match ()a Second match ()a

M avium subspeciesparatuberculosis K-10 M avium (994) M asiaticum (96)

M malmoense ATCC29571 M intracellulare (943) M szulgai (939)

M intracellulare ATCC13950 M avium (956) M avium subspecies paratuberculosis(954)

M scrofulaceum ATCC19981 M asiaticum (954) M szulgai (951)M marinum ATCC927 M ulcerans (996) M szulgai (960)M ulcerans Agy99 M marinum (996) M szulgai (960)M szulgai ATCC25799 M marinumM ulcerans (960) M asiaticum (956)M gordonae ATCC14470 M asiaticum (953) M kansasii (951)M asiaticum ATCC25276 M simiae (977) M kansasii (964)M kansasii ATCC12478 M asiaticum (963) M gastri (958)M gastri ATCC15754 M kansasii (958) M asiaticum (945)Rhodococcus equi 103S M smegmatis (903) M intracellulare (901)aBeside the queried type strain itself the first matched or the second matched type strain

33 Species Identification Outcomes of the Clinical IsolatesrpsA sequence and alignment efficiently identified clinicalisolates representing 8 mycobacterial species We foundthat a criterion of first distinct rpsA sequence match gt97confirmed the identification of 183 of 188 (973) clinicalisolates 20 M abscessus strains 2 M fortuitum strainsand 10 M avium strains were identified to the subspecieslevel Identification discrepancies between rpsA and othersequences were only encountered with 4M gordonae strainsand 1 M kansasii strain (see Table 3) No discrepancies werefound in the species submitted asM intracellulareM aviumM abscessus andM tuberculosis complex

The sequence divergence among MTC members was04 The intraspecies divergence among the 188 clinicalstrains ranged from 06 to 53 (Table 3) The sequencediversity among theM abscessuswas 06 while that amongthe M gordonae clinical isolates was 53 No M gordonaeclinical strain had an identical sequence with that of theM gordonae reference strain (ATCC14470) Interestingly therpsA sequence of one strain among the 23M kansasii isolatesexhibited a relatively low level of sequence similarity (956)to that of the reference strain (ATCC 12478) while those ofall the other M kansasii isolates were identical Since thestrain was confirmed as M kansasii by rpoB and hsp65 genealignment itmight suggest a distant variant or a new subtype

4 Discussion

16S rDNA gene sequence alignment has been used as thereference method for mycobacterial species identificationHowever it has been reported that by using the 16S rDNAgene alone for species identification of clinical NTM 37of such isolates remained unclassified which illustrates theneed for additional molecular tools for proper phylogeneticassignment and accurate NTM identification [11] The com-mon assumption that bacterial isolates belong to the samespecies if they have fewer than 5ndash15 bp differences within

the 16S rDNA gene sequence [12] or if they have more than97 16S rDNA gene sequence identities [13] may not beapplicable to genus of Mycobacterium whose members aremuch more closely related to each other Furthermore somemycobacterial strains shared uniform sequence of 16S rDNAgene such asM intracellulare andM aviumM kansasii andM gastri M abscessus and M chelonae and M szulgai andM malmoense

In this study we found that the rpsA gene is useful as acomplementary method to the 16S rDNA for mycobacterialspecies identification Among the 47 reference strains andtotally 1081 paired comparisons 1081 = [119899119886

1+ 119899(119899 minus 1)1198892]

1198861= 1 119899 = 46 119889 = 1 the rpsA gene alone can

differentiate 972 (1051 out of 1081) of them if using 97sequence homology as cutoff value When referring to thephylogenetic tree the rpsA resolution might increase Forthe most commonly seen pathogenic NTM species suchas M intracellulare M avium M abscessus M chelonaeand M xenopi rpsA gene sequence could differentiate thesespecies easily Furthermore 183 of the 188 (973) clinicalisolates representing 8mycobacterial species were identifiedcorrectly by rpsA gene blast Discrepancies of identificationwere only encountered with 4 M gordonae strains and 1M kansasii strain Both these species have reportedly moreintraspecies sequence divergence [9 14ndash16] Three out of the4 M gordonae strains with discrepant identification still hadthe first distinction asM gordonae by rpsA but the sequenceidentity was lower than 97 which means the identificationmight be correct when a more complete in-house database isbeing developed

rpsA alone cannot differentiate the following betweenmembers of the M tuberculosis complex between M sene-galense and M thermoresistibile between M parafortuitumand M trivial between M diernhoferi and M duvalii andbetween M austroafricanum and M terrae however thesespecies are also difficult to be separated by other markersalone such as 16s rDNA ITS rpoB and hsp65 Several

BioMed Research International 5Ta

ble2Sequ

ence

pairdistanceso

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strainso

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min

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AR

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nWI)

Percentidentity

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

2930

3132

3334

3536

3738

3940

4142

4344

4546

4748

1mdash

1000

987

981

960

953

954

964

962

945

945

951

945

945

920

939

941

943

945

945

928

956

937

947

932

924

888

892

911

911

911

911

911

913

915

939

941

913

930

928

926

926

924

934

934

924

928

892

1Mtherm

oresistibile

200

mdash987

981

960

953

954

964

962

945

945

951

945

945

920

939

941

943

945

945

928

956

937

947

932

924

888

892

911

911

911

911

911

913

915

939

941

913

930

928

926

926

924

934

934

924

928

892

2Msenegalense

313

13mdash

983

966

958

958

968

968

947

947

956

954

954

920

941

954

949

951

951

930

962

947

956

945

930

894

898

911

911

911

911

911

913

920

945

947

920

934

935

932

932

928

937

937

930

928

894

3Mp

orcin

um

419

1917

mdash962

958

958

962

962

941

941

954

945

945

920

943

945

943

945

945

939

962

949

949

937

937

901

905

909

909

909

909

909

911

920

947

949

913

932

932

930

934

928

935

937

928

930

892

4Mfortuitu

m

541

41

35

39

mdash943

954

964

960

939

939

934

943

943

915

943

943

945

947

947

915

943

941

949

928

911

898

903

901

901

901

901

901

903

911

935

937

913

934

930

935

935

930

937

934

930

920

896

5Mp

hlei

649

49

43

43

59

mdash951

953

941

937

937

947

941

941

913

937

935

934

935

935

932

937

937

937

930

924

886

890

907

907

907

907

907

909

915

943

941

907

932

920

918

922

915

930

932

918

915

880

6Mg

adium

747

47

43

43

47

51

mdash966

960

954

954

947

947

947

930

951

953

956

958

958

932

941

949

947

924

920

901

905

924

924

924

924

924

926

928

949

947

930

941

949

937

937

935

939

943

926

934

903

7Mfl

avescens

837

37

33

39

37

49

35

mdash966

966

966

953

962

962

920

962

962

964

962

962

935

947

945

945

939

915

905

909

911

911

911

911

911

913

922

937

935

920

943

935

932

932

930

934

935

924

922

909

8Msmegmatis

939

39

33

39

41

61

41

35

mdash966

966

951

968

968

924

958

956

962

964

964

930

956

949

947

924

920

915

918

909

909

909

909

909

911

922

943

945

924

939

928

934

934

932

939

935

928

928

892

9Mv

accae

1057

57

55

61

63

65

47

35

35

mdash1000

949

962

962

930

949

958

964

966

966

930

943

928

930

924

917

898

901

899

899

899

899

899

901

915

926

924

913

932

920

911

911

913

918

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903

917

890

10Mp

arafortuitu

m

1157

57

55

61

63

65

47

35

35

00

mdash949

962

962

930

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966

966

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943

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898

901

899

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899

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926

924

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903

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890

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1251

51

45

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7055

55

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53

53

mdash964

964

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930

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899

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932

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917

917

911

918

924

909

899

882

12Mn

eoaurum

1357

57

47

57

59

61

55

39

33

39

39

37

mdash1000

926

953

958

956

958

958

941

949

941

937

928

922

913

917

894

894

894

894

894

896

907

920

918

911

930

918

915

915

913

917

918

913

905

898

13Md

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oferi

1457

57

47

57

59

61

55

39

33

39

39

37

00

mdash926

953

958

956

958

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949

941

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922

913

917

894

894

894

894

894

896

907

920

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911

930

918

915

915

913

917

918

913

905

898

14Md

uvalii

1584

84

84

84

9193

7485

80

7474

86

7878

mdash930

939

930

932

932

917

918

911

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901

892

892

892

892

892

894

888

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905

915

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905

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892

901

896

890

877

15Ma

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1663

63

61

59

59

65

51

39

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53

53

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49

49

74mdash

966

960

958

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947

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905

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907

917

932

930

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926

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926

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935

915

909

884

16Mc

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1761

61

47

57

59

68

49

39

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43

43

49

43

43

63

35

mdash972

970

970

941

956

945

941

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934

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903

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905

913

930

932

920

928

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928

928

915

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901

17Mg

ilvum

1859

59

53

59

57

7045

37

39

37

37

51

45

45

7441

29

mdash998

998

941

949

928

934

926

930

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911

901

901

901

901

901

903

913

928

926

913

924

928

920

920

913

920

924

913

920

892

18Mv

anbaalenii

1957

57

51

57

55

68

43

39

37

35

35

49

43

43

7243

31

02

mdash1000

939

951

930

935

928

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915

930

928

915

926

930

922

922

915

922

926

915

922

890

19Mterrae

2057

57

51

57

55

68

43

39

37

35

35

49

43

43

7243

31

02

00

mdash939

951

930

935

928

932

909

913

903

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903

903

903

905

915

930

928

915

926

930

922

922

915

922

926

915

922

890

20Ma

ustro

afric

anum

2176

7674

63

9172

7267

7474

7461

61

61

89

63

61

61

63

63

mdash937

918

917

911

926

888

892

894

894

894

894

894

896

903

915

917

901

909

909

899

901

896

907

913

909

899

896

21Mrhodesia

e

2245

45

39

39

59

66

61

55

45

59

59

51

53

53

87

55

45

53

51

51

65

mdash937

945

934

941

903

907

903

903

903

903

903

905

905

937

939

903

926

928

922

922

918

926

930

917

909

892

22Mo

buense

2365

65

55

53

61

65

53

57

53

7676

7661

61

9555

57

7674

7486

65

mdash958

939

930

917

920

909

909

909

909

909

911

922

937

939

917

932

926

934

937

922

937

939

924

917

890

23Ma

ichien

se

2455

55

45

53

53

65

55

57

55

7474

82

65

65

9567

61

7067

67

89

57

43

mdash937

934

913

918

915

915

915

915

915

917

915

937

941

922

930

935

928

930

930

934

941

932

924

892

24Msphagni

2572

7257

65

7674

80

63

80

80

80

7876

76115

7667

7876

7695

7063

65

mdash920

888

894

896

896

896

896

896

898

905

945

945

907

930

928

918

922

920

924

934

922

915

882

25Mtokaiense

2680

80

7465

9580

84

9184

89

89

7482

82

9580

7074

7272

7861

7470

84

mdash915

918

896

896

896

896

896

898

898

922

926

894

905

911

907

911

898

920

917

907

903

871

26Mc

helona

e

27121

121

115

106

110

124

106

101

91

110

110

104

9393

110

86

9399

9797

121

104

89

93121

91mdash

987

867

867

867

867

867

869

884

884

888

884

886

894

896

896

884

901

898

882

871

871

27Ma

bscessu

s

28117

117

110

101

104

119

101

9786

106

106

9989

89

106

80

89

9593

93117

9984

86

115

87

13mdash

875

875

875

875

875

877

888

888

892

888

890

898

899

899

890

905

901

886

875

877

28Ma

bscessu

s

2995

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

mdash1000

1000

1000

1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

29Mtub

erculosis

3095

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

mdash1000

1000

1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

30Mtub

erculosis

3195

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

00

mdash1000

1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

31Mb

ovis

3295

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

00

00

mdash1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

32Ma

fricanu

m

3395

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

00

00

00

mdash998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

33Mb

ovis

3493

9393

95104

9778

9395

106

106

106

112

112

115

99102

104

102

102

112

102

9589

111

110

144

135

02

02

02

02

02

mdash911

918

922

907

918

924

918

917

918

926

932

920

917

880

34Mc

anettii

3591

9184

84

9591

7682

82

9191

9399

99122

89

9393

9191

104

102

82

91102

110

126

121

9898

9898

9895

mdash922

924

907

917

920

911

911

905

918

930

915

909

877

35Mx

enopi

3663

63

57

55

67

59

53

65

59

7878

7284

84

106

7274

7674

7491

65

65

65

57

82

126

121

89

89

89

89

89

87

82

mdash994

930

956

947

939

943

941

951

958

947

937

886

36Ma

vium

3761

61

55

53

65

61

55

67

57

80

80

7686

86

101

7472

7876

7689

63

63

61

57

78121

117

84

84

84

84

84

82

80

06

mdash935

954

951

945

949

945

954

960

953

939

888

37Ma

vium

3893

9384

9393

9974

84

80

9393

108

9595

9195

84

9391

91106

104

89

82

99115

126

121

9797

9797

97100

9974

67

mdash943

937

935

934

924

939

930

937

930

890

38Mm

almoense

3974

7470

7270

7261

59

63

7272

84

7474

102

7876

80

7878

9778

7274

74102

123

119

89

89

89

89

89

87

89

45

47

59

mdash945

941

941

934

951

947

943

943

907

39Mintracellu

lare

4076

7667

7274

84

53

67

7684

84

84

86

86

101

80

7276

7474

9776

7867

7695

115

110

82

82

82

82

82

80

84

55

51

65

57

mdash943

943

949

951

954

939

934

896

40Mscrofulaceum

4178

7872

7468

86

65

7270

9595

89

9191

112

7878

84

82

82

108

82

7076

87

99112

108

84

84

84

84

84

87

9563

57

68

61

59

mdash996

945

960

956

956

937

896

41Mm

arinum

4278

7872

7068

82

65

7270

9595

89

9191

110

7474

84

82

82

106

82

65

7482

95112

108

87

87

87

87

87

89

9559

53

7061

59

04

mdash943

960

956

954

939

894

42Mu

lcerans

4380

80

7676

7491

67

7472

9393

9593

93110

9389

9391

91112

86

82

7484

110

126

119

84

84

84

84

84

87

101

61

57

80

7053

57

59

mdash943

953

951

934

890

43Mg

ordona

e

4470

7065

67

65

7463

7063

86

86

86

89

89

117

80

7684

82

82

9978

65

7080

84

106

101

7676

7676

7678

86

51

47

63

51

51

41

41

59

mdash956

954

945

901

44Mszulga

i

4570

7065

65

7072

59

67

67

9191

80

86

86

106

67

7680

7878

9374

63

61

7089

110

106

7070

7070

7072

7443

41

7455

47

45

45

49

45

mdash964

947

888

45Ma

siaticum

4680

80

7476

7486

7880

76104

104

9793

93112

9191

9391

9197

89

80

7282

99128

123

82

82

82

82

82

85

9155

49

65

59

63

45

47

51

47

37

mdash958

888

46Mk

ansasii

4776

7676

7484

9170

82

7689

89

108

101

101

119

9795

84

82

82

108

9789

80

91104

142

137

87

87

87

87

87

89

9765

63

7459

7066

63

7057

55

43

mdash879

47Mg

astri

48117

117

115

117

112

130

104

97117

119

119

128

110

110

135

126

106

117

119

119

112

117

119

117

128

142

142

135

130

130

130

130

130

130

135

124

121

119

99112

112

115

119

106

121

121

132

mdash48

Rhodococcusequi

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

2930

3132

3334

3536

3738

3940

4142

4344

4546

4748

6 BioMed Research International

001

99

78

93

37

11

2

10

0

1

1

1

19

99

9 99

99

99

99

99

76

52

5

17

35

67

23

23

60

57

10

32

38

32

23

99

9

66

13

16

13

24

55

M thermoresistibileM senegalense

M porcinumM fortuitum

M phleiM gadium

M flavescensM smegmatisM vaccae

M parafortuitumM triviale

M neoaurumM diernhoferiM duvalii

M aurumM chubuense

M gilvumM vanbaalenii

M terraeM austroafricanum

M rhodesiaeM obuenseM aichienseM sphagni

M tokaienseM chelonae

M abscessusM abscessus subspecies massiliense

M tuberculosis H37RvM tuberculosis H37Ra

M bovisM bovis BCG

M canettiiM xenopi

M aviumM avium subspecies paratuberculosis

M malmoenseM intracellulareM scrofulaceum

M marinumM ulcerans

M szulgaiM gordonae

M asiaticumM kansassii

M gastri

M africanum

Rhodococcus equi

Figure 1 Phylogenetic tree based on rpsA gene sequences shows the relationship of the 47 type strains of mycobacteria and 1 outgroup strainThis tree was constructed by the neighbor-joiningmethod Topology was also evaluated by bootstrap analysis (MEGA program 1000 repeatswith R equi as the outgroup)The numerical values in the tree represent bootstrap results The distance between two strains is the sum of thebranch lengths between them

BioMed Research International 7

Table 3 Comparison of mycobacteria identified by 16S rDNAITShsp65rpoB gene sequencing and by rpsA sequencing among the 188clinical isolatesa

Primary identificationb (119899)Primary identificationb (119899)

First choice Second choiceIdentification (119899) match Identification (119899) match

M intracellulare (75)

M intracellulare (4) 100 M avium (4) 956M intracellulare (1) 998 M avium (1) 953M intracellulare (53) 989 M avium (53) 951M intracellulare (10) 987 M avium (10) 949M intracellulare (1) 985 M avium (1) 953M intracellulare (3) 981 M avium (3) 947M intracellulare (1) 975 M avium (1) 932M intracellulare (2) 971 M avium (2) 961

M abscessus (39)M abscessus (9) 100 M abscessus subspecies massiliense (9) 987M abscessus (10) 998 M abscessus subspecies massiliense (10) 985M abscessus subspecies massiliense (20) 994 M abscessus (20) 991

M kansasii (23) M kansasii (22) 100 M asiaticum (22) 963M szulgai (1) 966 M marinumM ulcerans (1) 960

M avium (10)M avium subspecies paratuberculosis (5) 996 M avium (5) 994M avium subspecies paratuberculosis (4) 998 M avium (4) 992M avium subspecies paratuberculosis (1) 975 M avium (1) 970

M fortuitum complex (17)

M fortuitum (2) 100 M senegalenseM thermoresistibile (2) 981M fortuitum (11) 998 M porcinum (11) 985M fortuitum (1) 996 M porcinum (1) 983M fortuitumM porcinum (1) 989 M senegalenseM thermoresistibile (1) 983M thermoresistibileM senegalense (2) 996 M fortuitum (2) 981

M gordonae (7)

M gordonae (1) 992 M asiaticum (1) 955M gordonae (2) 985 M asiaticum (2) 963M gordonae (3) 959 M asiaticum (3) 955M szulgai (1) 959 M gordonae (1) 947

M tuberculosis complex (16)M tuberculosis (2) 100 M canetti (2) 998M tuberculosis (12) 998 M canetti (12) 996M tuberculosis (2) 996 M canetti (2) 994

M neoaurum (1) M neoaurum (1) 987 M smegmatis (1) 954aNumbers in parentheses (119899) represent the numbers of isolates identified as a particular speciesbIdentification based on sequencing of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoB and hsp65 genes

reference strains had similar homology with two or morespecies which suggests inadequate taxonomy within thecurrently described species Based on nucleotide sequencesof rpoB hsp65 and sodA in clinical isolates of the Mycobac-terium abscessus group one-fourth of isolates had discordantidentification [16] Multilocus sequence typing and sequenceanalysis of several genes facilitate the identification of closelyrelated species or subspecies [17]

In our study we have demonstrated that the rpsA homol-ogy gene is a promising marker for mycobacterial speciesidentification for both fast- and slow-growing mycobacteriaTo be a good marker for species differentiation the targetgene should be stable and sequence variations should occurrandomly additionally an extremely conserved or highlyvariable gene may not be adequate As a single-copy house-keeping gene rpsA gene could work well as a target gene for

Mycobacterium species discrimination without ambiguousidentification The 16S rDNA gene has higher homologywithin the mycobacteria compared to the rpsA gene 943to 100 compared to 854 to 100 According to our workwhen used as sole marker rpsA had better resolution than 16srDNA but had similar resolution as ITS rpoB and hsp65

Due to the high resolution power of rpsA for speciesidentification we presume it has potential of clinical useFrom our own experience we recommend that when per-forming species identification by homologue DNA sequencecomparison one should start with 16s RNA gene plus atleast one of the other markers such as ITS rpoB hsp65and rpsA When conflicting outcome or dubious outcomesyield more markers should be added further Even though16s RNA gene is inferior to the other markers considering thecapacity it should be chosen firstly since 16s RNA gene has

8 BioMed Research International

the most robust sequence database which can help to avoidbig identification errors due to unreliable database of othermarkers [18]

Themain deterrents for the primary use of rpsA sequenc-ing as a routine means of identifying mycobacteria residein the need for a comprehensive database Therefore weconstructed our own rpsA sequence database by including asmany type strains as possible and integrating rpsA sequencesdeposited in GenBank such as M avium subspecies paratu-berculosis M ulcerans M vanbaalenii M abscessus sub-speciesmassiliense andM canettii Besides type strains rpsAsequences of confirmed clinical strains were also includedWe will constantly upgrade our database and the capabilityto identify the most recent described species

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HongfeiDuan andGuanLiu contributed equally to this study

Acknowledgments

The work was supported by the research funding fromInfectious Diseases Special Project Ministry of Health ofChina (2012ZX10003002-09) and Beijing Municipal Admin-istration of Hospitals Clinical Medicine Development ofSpecial Funding (ZYLX201304) All the type strains andclinical isolates used in this study were obtained from theldquoBeijing Bio-Bank of Clinical Resources on Tuberculosis(D09050704640000)rdquo Beijing Chest Hospital

References

[1] T Adekambi P Berger D Raoult and M DrancourtldquorpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacteriumbolletii sp nov Mycobacterium phocaicum sp nov andMycobacterium aubagnense sp novrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 1 ArticleID 63969 pp 133ndash143 2006

[2] B Ninet M Monod S Emler et al ldquoTwo different 16SrRNA genes in a mycobacterial strainrdquo Journal of ClinicalMicrobiology vol 34 no 10 pp 2531ndash2536 1996

[3] U Reischl K Feldmann L Naumann et al ldquo16S rRNAsequence diversity inMycobacterium celatum strains caused bypresence of two different copies of 16S rRNA generdquo Journal ofClinical Microbiology vol 36 no 6 pp 1761ndash1764 1998

[4] D E Griffith T Aksamit B A Brown-Elliott et al ldquoAn officialATSIDSA statement diagnosis treatment and prevention ofnontuberculous mycobacterial diseasesrdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 4 pp 367ndash416 2007

[5] W Shi X Zhang X Jiang et al ldquoPyrazinamide inhibits trans-translation inMycobacterium tuberculosisrdquo Science vol 333 no6049 pp 1630ndash1632 2011

[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998

[7] A Roth M Fischer M E Hamid S Michalke W Ludwig andH Mauch ldquoDifferentiation of phylogenetically related slowlygrowing mycobacteria based on 16S-23S rRNA gene internaltranscribed spacer sequencesrdquo Journal of Clinical Microbiologyvol 36 no 1 pp 139ndash147 1998

[8] H Ringuet A Honore A Varnerot et al ldquohsp65 sequencingfor identification of rapidly growing mycobacteriardquo Journal ofClinical Microbiology vol 37 no 3 pp 852ndash857 1999

[9] B-J Kim S-H Lee M-A Lyu et al ldquoIdentification ofmycobacterial species by comparative sequence analysis of theRNA polymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 37 no 6 pp 1714ndash1720 1999

[10] J Amita T Vandana R S Guleria and R K Verma ldquoQualita-tive evaluation of mycobacterial DNA extraction protocols forpolymerase chain reactionrdquoMolecular Biology Today vol 3 no2 pp 43ndash49 2002

[11] R J Pauls C Y Turenne J N Wolfe and A Kabani ldquoAhigh proportion of novel mycobacteria species identified by16S rDNA analysis among slowly growing AccuProbe-negativestrains in a clinical settingrdquo American Journal of ClinicalPathology vol 120 no 4 pp 560ndash566 2003

[12] G E Fox J D Wisotzkey and P Jurtshuk Jr ldquoHow close isclose 16S rRNA sequence identity may not be sufficient toguarantee species identityrdquo International Journal of SystematicBacteriology vol 42 no 1 pp 166ndash170 1992

[13] M Drancourt P Berger and D Raoult ldquoSystematic 16S rRNAgene sequencing of atypical clinical isolates identified 27 newbacterial species associated with humansrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2197ndash2202 2004

[14] P Kirschner E C Bottger D T Walton and M ValescoldquoMicroheterogeneity within rRNA of Mycobacterium gor-donaerdquo Journal of Clinical Microbiology vol 30 no 4 pp 1049ndash1050 1992

[15] C Taillard G Greub R Weber et al ldquoClinical implicationsofMycobacterium kansasii species heterogeneity swiss nationalsurveyrdquo Journal of Clinical Microbiology vol 41 no 3 pp 1240ndash1244 2003

[16] E Macheras A-L Roux F Ripoll et al ldquoInaccuracy of single-target sequencing for discriminating species of the Mycobac-terium abscessus grouprdquo Journal of Clinical Microbiology vol 47no 8 pp 2596ndash2600 2009

[17] L A Almeida and R Araujo ldquoHighlights on molecular iden-tification of closely related speciesrdquo Infection Genetics andEvolution vol 13 no 1 pp 67ndash75 2013

[18] J Dai Y Chen and M Lauzardo ldquoWeb-accessible databaseof hsp65 sequences from Mycobacterium reference strainsrdquoJournal of Clinical Microbiology vol 49 no 6 pp 2296ndash23032011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

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Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 BioMed Research International

suitable for phylogenetic study of the genus MycobacteriumIn this paper we report an evaluation of the rpsA gene as anovel biomarker for mycobacteria species identification

2 Materials and Methods

21 Mycobacterial Reference Strains and Clinical Isolates42 type and reference strains of the genus Mycobacterium(Table 1) 172 clinical NTM isolates and 16 M tuberculosiscomplex (MTC) isolates were investigated All the type andreference strains were purchased from the American TypeCulture Collection (ATCC) and all the clinical isolates usedin this study were obtained from the Clinical Database andSample Bank of tuberculosis of Beijing National ClinicalLab on Tuberculosis Beijing Chest Hospital All the clinicalNTM isolates were identified to the species level by sequencealignment of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoBand hsp65 genes as described before [2 7ndash9] Followingsequencing the 172 NTM clinical strains were found toinclude 10 strains ofM avium 75 strains ofM intracellulare23 strains ofM kansasii 39 strains ofM abscessus 17 strainsofM fortuitum complex 7 strains ofM gordonae and 1 strainofM neoaurum

22 rpsA Gene Amplification and Sequencing DNA wasreleased from cultured mycobacteria by boiling the culturedmycobacterial suspension in TE buffer for 10min After cen-trifugation the supernatant was used for PCR amplification[10] The primers used were forward primer 51015840-CCCTAC-ATCGGCAAGGAG-31015840 position 487ndash504 in the rpsA geneof Mycobacterium tuberculosis GenBank accession num-ber NC 0009622 and reverse primer 51015840-TGTCGATGA-CCTTGACCATC-31015840 position 1032ndash1051 in the rpsA geneof Mycobacterium tuberculosis GenBank accession numberNC 0009622 The amplified product was 565 bp PCR prod-ucts were purified and sequenced by a commercial company(YINGJUN Biotech Company Beijing China) using ABI3730 DNA Analyzer (Applied Biosystems California USA)

23 Sequence Analysis and the Phylogenetic Tree Construc-tions In addition to the 42 reference strains the rpsAsequences of 5 other mycobacterial species were obtaineddirectly from GenBank including M avium subspeciesparatuberculosis (GenBank accession number NC 0029442)M ulcerans (GenBank accession number NC 0086111) Mvanbaalenii (GenBank accession number NC 0087261) Mabscessus subspeciesmassiliense (GenBank accession numberNC 0181501) and M canettii (GenBank accession numberNC 0158481) All sequences were aligned and homologywas calculated by using the multiple alignment algorithm inthe MegAlign package (Windows version 710 DNASTARMadison Wis) 527 bp of sequence (excluding 38 nucleotidesat each end of the amplicon corresponding to the primerbinding sites) was analyzed for the phylogenetic tree con-struction by the neighbor-joining method using the MEGA505 package (httpwwwmegasoftwarenetmegaphp) Abootstrap analysis (1000 repeats) using Rhodococcus equi

(GenBank accession number NC 0063611) as the outgroupwas performed to evaluate the topology of the phylogenetictree

24 Species Identification of the Clinical Isolates The 188clinical isolates were analyzed blindly Sequences minus theknown PCR primer sequences were assembled and edited byusing SeqMan software (version 710 DNASTAR MadisonWI) Isolates were identified by comparing sequences byusing a FASTA BLASTn search with MegAlign (version710 DNASTAR Madison WI) to an in-house databaseof sequences consisting of type and reference strains fromexternal culture collections

3 Results

31 rpsA Sequence Alignment of the Reference StrainsBetween 854 and 100 sequence homology (interspeciesdivergence 0 to 146) was observed among the 42tested reference strains and the 5 additional mycobacterialspecies whose sequences were obtained from the Gen-Bank database (Table 2) All the rpsA gene sequences ofthe analyzed mycobacteria strains were distinct from theoutgroup strain R equi Among the 19 reference strainsof the slow-growing Mycobacterium genus 11 strains weregreater than 97 homology including 5 M tuberculosiscomplex strains Among the 28 reference strains of the rapid-growing Mycobacterium genus 14 strains were greater than97 homology (Table 1) The pathogenic M kansasii waseasily differentiated from the nonpathogenicM gastri (958homology) while those two species were not distinguish-able by the 16S rDNA sequence alignment The sequencehomologies between various species were 918 between Mchelonae and M abscessus 956 between M avium andM intracellulare and 939 between M szulgai and Mmalmoense However the sequence homology between otherspecies was higher for example it was 996 between Mulcerans and M marinum and 987 between M abscessussubspecies massiliense and M abscessus All members ofthe M tuberculosis complex had identical sequences as didM senegalense and M thermoresistibile M parafortuitumand M trivial M diernhoferi and M duvalii and M aus-troafricanum andM terrae

32 Phylogenetic Tree Construction A phylogenetic treewhich provided the basis for species differentiation in thegenus Mycobacterium was constructed (Figure 1) The abso-lute majority tested species showed good separation Therapid-growing species were well defined from the slow-growing species in the tree M chelonae M abscessus sub-speciesmassiliense andMabscessus which are categorized inthe pathogenic taxonomic group of rapid-growing mycobac-teria formed a distinctive cluster which was much closer tothe slow-growing species compared with the nonpathogenicgroup of rapid-growing mycobacteria The reliability of thephylogenetic tree was verified by the bootstrapmethod usingR equi as the outgroup

BioMed Research International 3

Table 1 Percentage rpsA sequence match among type strain

Species Type strain First match ()a Second match ()a

M thermoresistibile ATCC19527 M senegalense (100) M porcinum (987)M senegalense ATCC35796 M thermoresistibile (100) M porcinum (987)M porcinum ATCC33776 M senegalenseM thermoresistibile (987) M fortuitum (983)

M fortuitum ATCC6481 M porcinum (983) M senegalenseM thermoresistibile(981)

M phlei ATCC11758 M porcinum (965) M fortuitum (961)M gadium ATCC27726 M porcinumM fortuitum (957) M smegmatis (953)M flavescens ATCC14474 M smegmatis (966) M vaccae (960)

M smegmatis ATCC19420 M vaccaeM parafortuitumM triviale(966)

M senegalenseM thermoresistibile(963)

M vaccae ATCC15483 M diernhoferiM duvalii (968) M parafortuitumM triviale (966)M parafortuitum ATCC19686 M triviale (100) M terraeM austroafricanum (966)M triviale ATCC23292 M parafortuitum (100) M terraeM austroafricanum (966)M neoaurum ATCC25795 M diernhoferiM duvalii (964) M porcinum (955)M diernhoferi ATCC19340 M duvalii (100) M vaccae (968)M duvalii ATCC43910 M diernhoferi (100) M vaccae (968)M aurum ATCC23366 M gilvum (939) M chubuenseM vanbaalenii (930)M chubuense ATCC27278 M gilvum (966) M vanbaalenii (960)M gilvum ATCC43909 M vanbaalenii (972) M terraeM austroafricanum (970)M vanbaalenii PYR-1 M terraeM austroafricanum (998) M gilvum (972)M terrae ATCC15755 M austroafricanum (100) M vanbaalenii (998)M austroafricanum ATCC33464 M terrae (100) M vanbaalenii (998)M rhodesiae ATCC27024 M terraeM austroafricanum (939) M obuense (937)

M obuense ATCC27023 M porcinum M fortuitum (961) M senegalenseM thermoresistibile(965)

M aichiense ATCC27280 M sphagni (958) M vaccae (947)M sphagni ATCC33027 M aichiense (958) M porcinum (955)M tokaiense ATCC27282 M porcinum (953) M smegmatis (937)M chelonae ATCC35752 M obuense (939) M phlei (935)M abscessus subspeciesmassiliense str GO 06 M abscessus (987) M sphagni (914)

M abscessus ATCC19977 M abscessus subspeciesmassiliense (987) M chubuense (920)

M tuberculosis H37Rv ATCC27294 M tuberculosisH37RaM africanumMbovis BCGM bovis (100) M canettii (998)

M tuberculosis H37Ra ATCC25177 M tuberculosisH37RvM africanumMbovis BCGM bovis (100) M canettii (998)

M africanum ATCC25420 M tuberculosisH37RaM tuberculosisRvM bovis BCGM bovis (100) M canettii (998)

M bovis BCG ATCC35735 M tuberculosisH37RaM tuberculosisH37RvM africanumM bovis (100) M canettii (998)

M bovis ATCC19210 M tuberculosisH37RaM tuberculosisH37RvM africanumM bovis BCG (100) M canettii (998)

M canettii CIPT140010059 M tuberculosisM africanumM bovisBCGM bovis (998) M asiaticum (932)

M xenopi ATCC19250 M asiaticum (930) M avium subspecies paratuberculosis(924)

M avium ATCC25291 M avium subspecies paratuberculosis(996) M asiaticum (958)

4 BioMed Research International

Table 1 Continued

Species Type strain First match ()a Second match ()a

M avium subspeciesparatuberculosis K-10 M avium (994) M asiaticum (96)

M malmoense ATCC29571 M intracellulare (943) M szulgai (939)

M intracellulare ATCC13950 M avium (956) M avium subspecies paratuberculosis(954)

M scrofulaceum ATCC19981 M asiaticum (954) M szulgai (951)M marinum ATCC927 M ulcerans (996) M szulgai (960)M ulcerans Agy99 M marinum (996) M szulgai (960)M szulgai ATCC25799 M marinumM ulcerans (960) M asiaticum (956)M gordonae ATCC14470 M asiaticum (953) M kansasii (951)M asiaticum ATCC25276 M simiae (977) M kansasii (964)M kansasii ATCC12478 M asiaticum (963) M gastri (958)M gastri ATCC15754 M kansasii (958) M asiaticum (945)Rhodococcus equi 103S M smegmatis (903) M intracellulare (901)aBeside the queried type strain itself the first matched or the second matched type strain

33 Species Identification Outcomes of the Clinical IsolatesrpsA sequence and alignment efficiently identified clinicalisolates representing 8 mycobacterial species We foundthat a criterion of first distinct rpsA sequence match gt97confirmed the identification of 183 of 188 (973) clinicalisolates 20 M abscessus strains 2 M fortuitum strainsand 10 M avium strains were identified to the subspecieslevel Identification discrepancies between rpsA and othersequences were only encountered with 4M gordonae strainsand 1 M kansasii strain (see Table 3) No discrepancies werefound in the species submitted asM intracellulareM aviumM abscessus andM tuberculosis complex

The sequence divergence among MTC members was04 The intraspecies divergence among the 188 clinicalstrains ranged from 06 to 53 (Table 3) The sequencediversity among theM abscessuswas 06 while that amongthe M gordonae clinical isolates was 53 No M gordonaeclinical strain had an identical sequence with that of theM gordonae reference strain (ATCC14470) Interestingly therpsA sequence of one strain among the 23M kansasii isolatesexhibited a relatively low level of sequence similarity (956)to that of the reference strain (ATCC 12478) while those ofall the other M kansasii isolates were identical Since thestrain was confirmed as M kansasii by rpoB and hsp65 genealignment itmight suggest a distant variant or a new subtype

4 Discussion

16S rDNA gene sequence alignment has been used as thereference method for mycobacterial species identificationHowever it has been reported that by using the 16S rDNAgene alone for species identification of clinical NTM 37of such isolates remained unclassified which illustrates theneed for additional molecular tools for proper phylogeneticassignment and accurate NTM identification [11] The com-mon assumption that bacterial isolates belong to the samespecies if they have fewer than 5ndash15 bp differences within

the 16S rDNA gene sequence [12] or if they have more than97 16S rDNA gene sequence identities [13] may not beapplicable to genus of Mycobacterium whose members aremuch more closely related to each other Furthermore somemycobacterial strains shared uniform sequence of 16S rDNAgene such asM intracellulare andM aviumM kansasii andM gastri M abscessus and M chelonae and M szulgai andM malmoense

In this study we found that the rpsA gene is useful as acomplementary method to the 16S rDNA for mycobacterialspecies identification Among the 47 reference strains andtotally 1081 paired comparisons 1081 = [119899119886

1+ 119899(119899 minus 1)1198892]

1198861= 1 119899 = 46 119889 = 1 the rpsA gene alone can

differentiate 972 (1051 out of 1081) of them if using 97sequence homology as cutoff value When referring to thephylogenetic tree the rpsA resolution might increase Forthe most commonly seen pathogenic NTM species suchas M intracellulare M avium M abscessus M chelonaeand M xenopi rpsA gene sequence could differentiate thesespecies easily Furthermore 183 of the 188 (973) clinicalisolates representing 8mycobacterial species were identifiedcorrectly by rpsA gene blast Discrepancies of identificationwere only encountered with 4 M gordonae strains and 1M kansasii strain Both these species have reportedly moreintraspecies sequence divergence [9 14ndash16] Three out of the4 M gordonae strains with discrepant identification still hadthe first distinction asM gordonae by rpsA but the sequenceidentity was lower than 97 which means the identificationmight be correct when a more complete in-house database isbeing developed

rpsA alone cannot differentiate the following betweenmembers of the M tuberculosis complex between M sene-galense and M thermoresistibile between M parafortuitumand M trivial between M diernhoferi and M duvalii andbetween M austroafricanum and M terrae however thesespecies are also difficult to be separated by other markersalone such as 16s rDNA ITS rpoB and hsp65 Several

BioMed Research International 5Ta

ble2Sequ

ence

pairdistanceso

f47type

strainso

fmycob

acteria

and1o

ut-group

strain

determ

ined

byusingthemultip

lealignm

enta

lgorith

min

theMegAlignpackage(versio

n710

DNAST

AR

Madiso

nWI)

Percentidentity

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

2930

3132

3334

3536

3738

3940

4142

4344

4546

4748

1mdash

1000

987

981

960

953

954

964

962

945

945

951

945

945

920

939

941

943

945

945

928

956

937

947

932

924

888

892

911

911

911

911

911

913

915

939

941

913

930

928

926

926

924

934

934

924

928

892

1Mtherm

oresistibile

200

mdash987

981

960

953

954

964

962

945

945

951

945

945

920

939

941

943

945

945

928

956

937

947

932

924

888

892

911

911

911

911

911

913

915

939

941

913

930

928

926

926

924

934

934

924

928

892

2Msenegalense

313

13mdash

983

966

958

958

968

968

947

947

956

954

954

920

941

954

949

951

951

930

962

947

956

945

930

894

898

911

911

911

911

911

913

920

945

947

920

934

935

932

932

928

937

937

930

928

894

3Mp

orcin

um

419

1917

mdash962

958

958

962

962

941

941

954

945

945

920

943

945

943

945

945

939

962

949

949

937

937

901

905

909

909

909

909

909

911

920

947

949

913

932

932

930

934

928

935

937

928

930

892

4Mfortuitu

m

541

41

35

39

mdash943

954

964

960

939

939

934

943

943

915

943

943

945

947

947

915

943

941

949

928

911

898

903

901

901

901

901

901

903

911

935

937

913

934

930

935

935

930

937

934

930

920

896

5Mp

hlei

649

49

43

43

59

mdash951

953

941

937

937

947

941

941

913

937

935

934

935

935

932

937

937

937

930

924

886

890

907

907

907

907

907

909

915

943

941

907

932

920

918

922

915

930

932

918

915

880

6Mg

adium

747

47

43

43

47

51

mdash966

960

954

954

947

947

947

930

951

953

956

958

958

932

941

949

947

924

920

901

905

924

924

924

924

924

926

928

949

947

930

941

949

937

937

935

939

943

926

934

903

7Mfl

avescens

837

37

33

39

37

49

35

mdash966

966

966

953

962

962

920

962

962

964

962

962

935

947

945

945

939

915

905

909

911

911

911

911

911

913

922

937

935

920

943

935

932

932

930

934

935

924

922

909

8Msmegmatis

939

39

33

39

41

61

41

35

mdash966

966

951

968

968

924

958

956

962

964

964

930

956

949

947

924

920

915

918

909

909

909

909

909

911

922

943

945

924

939

928

934

934

932

939

935

928

928

892

9Mv

accae

1057

57

55

61

63

65

47

35

35

mdash1000

949

962

962

930

949

958

964

966

966

930

943

928

930

924

917

898

901

899

899

899

899

899

901

915

926

924

913

932

920

911

911

913

918

915

903

917

890

10Mp

arafortuitu

m

1157

57

55

61

63

65

47

35

35

00

mdash949

962

962

930

949

958

964

966

966

930

943

928

930

924

917

898

901

899

899

899

899

899

901

915

926

924

913

932

920

911

911

913

918

915

903

917

890

11Mtriv

iale

1251

51

45

47

7055

55

49

51

53

53

mdash964

964

918

945

953

951

953

953

941

951

928

922

926

930

903

907

899

899

899

899

899

901

913

932

928

899

920

920

917

917

911

918

924

909

899

882

12Mn

eoaurum

1357

57

47

57

59

61

55

39

33

39

39

37

mdash1000

926

953

958

956

958

958

941

949

941

937

928

922

913

917

894

894

894

894

894

896

907

920

918

911

930

918

915

915

913

917

918

913

905

898

13Md

iernh

oferi

1457

57

47

57

59

61

55

39

33

39

39

37

00

mdash926

953

958

956

958

958

941

949

941

937

928

922

913

917

894

894

894

894

894

896

907

920

918

911

930

918

915

915

913

917

918

913

905

898

14Md

uvalii

1584

84

84

84

9193

7485

80

7474

86

7878

mdash930

939

930

932

932

917

918

911

911

894

911

898

901

892

892

892

892

892

894

888

901

905

915

905

905

896

898

898

892

901

896

890

877

15Ma

urum

1663

63

61

59

59

65

51

39

43

53

53

57

49

49

74mdash

966

960

958

958

939

947

947

935

928

924

918

924

905

905

905

905

905

907

917

932

930

911

926

924

926

930

913

924

935

915

909

884

16Mc

hubu

ense

1761

61

47

57

59

68

49

39

45

43

43

49

43

43

63

35

mdash972

970

970

941

956

945

941

935

934

913

917

903

903

903

903

903

905

913

930

932

920

928

932

926

930

917

928

928

915

911

901

17Mg

ilvum

1859

59

53

59

57

7045

37

39

37

37

51

45

45

7441

29

mdash998

998

941

949

928

934

926

930

907

911

901

901

901

901

901

903

913

928

926

913

924

928

920

920

913

920

924

913

920

892

18Mv

anbaalenii

1957

57

51

57

55

68

43

39

37

35

35

49

43

43

7243

31

02

mdash1000

939

951

930

935

928

932

909

913

903

903

903

903

903

905

915

930

928

915

926

930

922

922

915

922

926

915

922

890

19Mterrae

2057

57

51

57

55

68

43

39

37

35

35

49

43

43

7243

31

02

00

mdash939

951

930

935

928

932

909

913

903

903

903

903

903

905

915

930

928

915

926

930

922

922

915

922

926

915

922

890

20Ma

ustro

afric

anum

2176

7674

63

9172

7267

7474

7461

61

61

89

63

61

61

63

63

mdash937

918

917

911

926

888

892

894

894

894

894

894

896

903

915

917

901

909

909

899

901

896

907

913

909

899

896

21Mrhodesia

e

2245

45

39

39

59

66

61

55

45

59

59

51

53

53

87

55

45

53

51

51

65

mdash937

945

934

941

903

907

903

903

903

903

903

905

905

937

939

903

926

928

922

922

918

926

930

917

909

892

22Mo

buense

2365

65

55

53

61

65

53

57

53

7676

7661

61

9555

57

7674

7486

65

mdash958

939

930

917

920

909

909

909

909

909

911

922

937

939

917

932

926

934

937

922

937

939

924

917

890

23Ma

ichien

se

2455

55

45

53

53

65

55

57

55

7474

82

65

65

9567

61

7067

67

89

57

43

mdash937

934

913

918

915

915

915

915

915

917

915

937

941

922

930

935

928

930

930

934

941

932

924

892

24Msphagni

2572

7257

65

7674

80

63

80

80

80

7876

76115

7667

7876

7695

7063

65

mdash920

888

894

896

896

896

896

896

898

905

945

945

907

930

928

918

922

920

924

934

922

915

882

25Mtokaiense

2680

80

7465

9580

84

9184

89

89

7482

82

9580

7074

7272

7861

7470

84

mdash915

918

896

896

896

896

896

898

898

922

926

894

905

911

907

911

898

920

917

907

903

871

26Mc

helona

e

27121

121

115

106

110

124

106

101

91

110

110

104

9393

110

86

9399

9797

121

104

89

93121

91mdash

987

867

867

867

867

867

869

884

884

888

884

886

894

896

896

884

901

898

882

871

871

27Ma

bscessu

s

28117

117

110

101

104

119

101

9786

106

106

9989

89

106

80

89

9593

93117

9984

86

115

87

13mdash

875

875

875

875

875

877

888

888

892

888

890

898

899

899

890

905

901

886

875

877

28Ma

bscessu

s

2995

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

mdash1000

1000

1000

1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

29Mtub

erculosis

3095

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

mdash1000

1000

1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

30Mtub

erculosis

3195

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

00

mdash1000

1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

31Mb

ovis

3295

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

00

00

mdash1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

32Ma

fricanu

m

3395

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

00

00

00

mdash998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

33Mb

ovis

3493

9393

95104

9778

9395

106

106

106

112

112

115

99102

104

102

102

112

102

9589

111

110

144

135

02

02

02

02

02

mdash911

918

922

907

918

924

918

917

918

926

932

920

917

880

34Mc

anettii

3591

9184

84

9591

7682

82

9191

9399

99122

89

9393

9191

104

102

82

91102

110

126

121

9898

9898

9895

mdash922

924

907

917

920

911

911

905

918

930

915

909

877

35Mx

enopi

3663

63

57

55

67

59

53

65

59

7878

7284

84

106

7274

7674

7491

65

65

65

57

82

126

121

89

89

89

89

89

87

82

mdash994

930

956

947

939

943

941

951

958

947

937

886

36Ma

vium

3761

61

55

53

65

61

55

67

57

80

80

7686

86

101

7472

7876

7689

63

63

61

57

78121

117

84

84

84

84

84

82

80

06

mdash935

954

951

945

949

945

954

960

953

939

888

37Ma

vium

3893

9384

9393

9974

84

80

9393

108

9595

9195

84

9391

91106

104

89

82

99115

126

121

9797

9797

97100

9974

67

mdash943

937

935

934

924

939

930

937

930

890

38Mm

almoense

3974

7470

7270

7261

59

63

7272

84

7474

102

7876

80

7878

9778

7274

74102

123

119

89

89

89

89

89

87

89

45

47

59

mdash945

941

941

934

951

947

943

943

907

39Mintracellu

lare

4076

7667

7274

84

53

67

7684

84

84

86

86

101

80

7276

7474

9776

7867

7695

115

110

82

82

82

82

82

80

84

55

51

65

57

mdash943

943

949

951

954

939

934

896

40Mscrofulaceum

4178

7872

7468

86

65

7270

9595

89

9191

112

7878

84

82

82

108

82

7076

87

99112

108

84

84

84

84

84

87

9563

57

68

61

59

mdash996

945

960

956

956

937

896

41Mm

arinum

4278

7872

7068

82

65

7270

9595

89

9191

110

7474

84

82

82

106

82

65

7482

95112

108

87

87

87

87

87

89

9559

53

7061

59

04

mdash943

960

956

954

939

894

42Mu

lcerans

4380

80

7676

7491

67

7472

9393

9593

93110

9389

9391

91112

86

82

7484

110

126

119

84

84

84

84

84

87

101

61

57

80

7053

57

59

mdash943

953

951

934

890

43Mg

ordona

e

4470

7065

67

65

7463

7063

86

86

86

89

89

117

80

7684

82

82

9978

65

7080

84

106

101

7676

7676

7678

86

51

47

63

51

51

41

41

59

mdash956

954

945

901

44Mszulga

i

4570

7065

65

7072

59

67

67

9191

80

86

86

106

67

7680

7878

9374

63

61

7089

110

106

7070

7070

7072

7443

41

7455

47

45

45

49

45

mdash964

947

888

45Ma

siaticum

4680

80

7476

7486

7880

76104

104

9793

93112

9191

9391

9197

89

80

7282

99128

123

82

82

82

82

82

85

9155

49

65

59

63

45

47

51

47

37

mdash958

888

46Mk

ansasii

4776

7676

7484

9170

82

7689

89

108

101

101

119

9795

84

82

82

108

9789

80

91104

142

137

87

87

87

87

87

89

9765

63

7459

7066

63

7057

55

43

mdash879

47Mg

astri

48117

117

115

117

112

130

104

97117

119

119

128

110

110

135

126

106

117

119

119

112

117

119

117

128

142

142

135

130

130

130

130

130

130

135

124

121

119

99112

112

115

119

106

121

121

132

mdash48

Rhodococcusequi

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

2930

3132

3334

3536

3738

3940

4142

4344

4546

4748

6 BioMed Research International

001

99

78

93

37

11

2

10

0

1

1

1

19

99

9 99

99

99

99

99

76

52

5

17

35

67

23

23

60

57

10

32

38

32

23

99

9

66

13

16

13

24

55

M thermoresistibileM senegalense

M porcinumM fortuitum

M phleiM gadium

M flavescensM smegmatisM vaccae

M parafortuitumM triviale

M neoaurumM diernhoferiM duvalii

M aurumM chubuense

M gilvumM vanbaalenii

M terraeM austroafricanum

M rhodesiaeM obuenseM aichienseM sphagni

M tokaienseM chelonae

M abscessusM abscessus subspecies massiliense

M tuberculosis H37RvM tuberculosis H37Ra

M bovisM bovis BCG

M canettiiM xenopi

M aviumM avium subspecies paratuberculosis

M malmoenseM intracellulareM scrofulaceum

M marinumM ulcerans

M szulgaiM gordonae

M asiaticumM kansassii

M gastri

M africanum

Rhodococcus equi

Figure 1 Phylogenetic tree based on rpsA gene sequences shows the relationship of the 47 type strains of mycobacteria and 1 outgroup strainThis tree was constructed by the neighbor-joiningmethod Topology was also evaluated by bootstrap analysis (MEGA program 1000 repeatswith R equi as the outgroup)The numerical values in the tree represent bootstrap results The distance between two strains is the sum of thebranch lengths between them

BioMed Research International 7

Table 3 Comparison of mycobacteria identified by 16S rDNAITShsp65rpoB gene sequencing and by rpsA sequencing among the 188clinical isolatesa

Primary identificationb (119899)Primary identificationb (119899)

First choice Second choiceIdentification (119899) match Identification (119899) match

M intracellulare (75)

M intracellulare (4) 100 M avium (4) 956M intracellulare (1) 998 M avium (1) 953M intracellulare (53) 989 M avium (53) 951M intracellulare (10) 987 M avium (10) 949M intracellulare (1) 985 M avium (1) 953M intracellulare (3) 981 M avium (3) 947M intracellulare (1) 975 M avium (1) 932M intracellulare (2) 971 M avium (2) 961

M abscessus (39)M abscessus (9) 100 M abscessus subspecies massiliense (9) 987M abscessus (10) 998 M abscessus subspecies massiliense (10) 985M abscessus subspecies massiliense (20) 994 M abscessus (20) 991

M kansasii (23) M kansasii (22) 100 M asiaticum (22) 963M szulgai (1) 966 M marinumM ulcerans (1) 960

M avium (10)M avium subspecies paratuberculosis (5) 996 M avium (5) 994M avium subspecies paratuberculosis (4) 998 M avium (4) 992M avium subspecies paratuberculosis (1) 975 M avium (1) 970

M fortuitum complex (17)

M fortuitum (2) 100 M senegalenseM thermoresistibile (2) 981M fortuitum (11) 998 M porcinum (11) 985M fortuitum (1) 996 M porcinum (1) 983M fortuitumM porcinum (1) 989 M senegalenseM thermoresistibile (1) 983M thermoresistibileM senegalense (2) 996 M fortuitum (2) 981

M gordonae (7)

M gordonae (1) 992 M asiaticum (1) 955M gordonae (2) 985 M asiaticum (2) 963M gordonae (3) 959 M asiaticum (3) 955M szulgai (1) 959 M gordonae (1) 947

M tuberculosis complex (16)M tuberculosis (2) 100 M canetti (2) 998M tuberculosis (12) 998 M canetti (12) 996M tuberculosis (2) 996 M canetti (2) 994

M neoaurum (1) M neoaurum (1) 987 M smegmatis (1) 954aNumbers in parentheses (119899) represent the numbers of isolates identified as a particular speciesbIdentification based on sequencing of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoB and hsp65 genes

reference strains had similar homology with two or morespecies which suggests inadequate taxonomy within thecurrently described species Based on nucleotide sequencesof rpoB hsp65 and sodA in clinical isolates of the Mycobac-terium abscessus group one-fourth of isolates had discordantidentification [16] Multilocus sequence typing and sequenceanalysis of several genes facilitate the identification of closelyrelated species or subspecies [17]

In our study we have demonstrated that the rpsA homol-ogy gene is a promising marker for mycobacterial speciesidentification for both fast- and slow-growing mycobacteriaTo be a good marker for species differentiation the targetgene should be stable and sequence variations should occurrandomly additionally an extremely conserved or highlyvariable gene may not be adequate As a single-copy house-keeping gene rpsA gene could work well as a target gene for

Mycobacterium species discrimination without ambiguousidentification The 16S rDNA gene has higher homologywithin the mycobacteria compared to the rpsA gene 943to 100 compared to 854 to 100 According to our workwhen used as sole marker rpsA had better resolution than 16srDNA but had similar resolution as ITS rpoB and hsp65

Due to the high resolution power of rpsA for speciesidentification we presume it has potential of clinical useFrom our own experience we recommend that when per-forming species identification by homologue DNA sequencecomparison one should start with 16s RNA gene plus atleast one of the other markers such as ITS rpoB hsp65and rpsA When conflicting outcome or dubious outcomesyield more markers should be added further Even though16s RNA gene is inferior to the other markers considering thecapacity it should be chosen firstly since 16s RNA gene has

8 BioMed Research International

the most robust sequence database which can help to avoidbig identification errors due to unreliable database of othermarkers [18]

Themain deterrents for the primary use of rpsA sequenc-ing as a routine means of identifying mycobacteria residein the need for a comprehensive database Therefore weconstructed our own rpsA sequence database by including asmany type strains as possible and integrating rpsA sequencesdeposited in GenBank such as M avium subspecies paratu-berculosis M ulcerans M vanbaalenii M abscessus sub-speciesmassiliense andM canettii Besides type strains rpsAsequences of confirmed clinical strains were also includedWe will constantly upgrade our database and the capabilityto identify the most recent described species

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HongfeiDuan andGuanLiu contributed equally to this study

Acknowledgments

The work was supported by the research funding fromInfectious Diseases Special Project Ministry of Health ofChina (2012ZX10003002-09) and Beijing Municipal Admin-istration of Hospitals Clinical Medicine Development ofSpecial Funding (ZYLX201304) All the type strains andclinical isolates used in this study were obtained from theldquoBeijing Bio-Bank of Clinical Resources on Tuberculosis(D09050704640000)rdquo Beijing Chest Hospital

References

[1] T Adekambi P Berger D Raoult and M DrancourtldquorpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacteriumbolletii sp nov Mycobacterium phocaicum sp nov andMycobacterium aubagnense sp novrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 1 ArticleID 63969 pp 133ndash143 2006

[2] B Ninet M Monod S Emler et al ldquoTwo different 16SrRNA genes in a mycobacterial strainrdquo Journal of ClinicalMicrobiology vol 34 no 10 pp 2531ndash2536 1996

[3] U Reischl K Feldmann L Naumann et al ldquo16S rRNAsequence diversity inMycobacterium celatum strains caused bypresence of two different copies of 16S rRNA generdquo Journal ofClinical Microbiology vol 36 no 6 pp 1761ndash1764 1998

[4] D E Griffith T Aksamit B A Brown-Elliott et al ldquoAn officialATSIDSA statement diagnosis treatment and prevention ofnontuberculous mycobacterial diseasesrdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 4 pp 367ndash416 2007

[5] W Shi X Zhang X Jiang et al ldquoPyrazinamide inhibits trans-translation inMycobacterium tuberculosisrdquo Science vol 333 no6049 pp 1630ndash1632 2011

[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998

[7] A Roth M Fischer M E Hamid S Michalke W Ludwig andH Mauch ldquoDifferentiation of phylogenetically related slowlygrowing mycobacteria based on 16S-23S rRNA gene internaltranscribed spacer sequencesrdquo Journal of Clinical Microbiologyvol 36 no 1 pp 139ndash147 1998

[8] H Ringuet A Honore A Varnerot et al ldquohsp65 sequencingfor identification of rapidly growing mycobacteriardquo Journal ofClinical Microbiology vol 37 no 3 pp 852ndash857 1999

[9] B-J Kim S-H Lee M-A Lyu et al ldquoIdentification ofmycobacterial species by comparative sequence analysis of theRNA polymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 37 no 6 pp 1714ndash1720 1999

[10] J Amita T Vandana R S Guleria and R K Verma ldquoQualita-tive evaluation of mycobacterial DNA extraction protocols forpolymerase chain reactionrdquoMolecular Biology Today vol 3 no2 pp 43ndash49 2002

[11] R J Pauls C Y Turenne J N Wolfe and A Kabani ldquoAhigh proportion of novel mycobacteria species identified by16S rDNA analysis among slowly growing AccuProbe-negativestrains in a clinical settingrdquo American Journal of ClinicalPathology vol 120 no 4 pp 560ndash566 2003

[12] G E Fox J D Wisotzkey and P Jurtshuk Jr ldquoHow close isclose 16S rRNA sequence identity may not be sufficient toguarantee species identityrdquo International Journal of SystematicBacteriology vol 42 no 1 pp 166ndash170 1992

[13] M Drancourt P Berger and D Raoult ldquoSystematic 16S rRNAgene sequencing of atypical clinical isolates identified 27 newbacterial species associated with humansrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2197ndash2202 2004

[14] P Kirschner E C Bottger D T Walton and M ValescoldquoMicroheterogeneity within rRNA of Mycobacterium gor-donaerdquo Journal of Clinical Microbiology vol 30 no 4 pp 1049ndash1050 1992

[15] C Taillard G Greub R Weber et al ldquoClinical implicationsofMycobacterium kansasii species heterogeneity swiss nationalsurveyrdquo Journal of Clinical Microbiology vol 41 no 3 pp 1240ndash1244 2003

[16] E Macheras A-L Roux F Ripoll et al ldquoInaccuracy of single-target sequencing for discriminating species of the Mycobac-terium abscessus grouprdquo Journal of Clinical Microbiology vol 47no 8 pp 2596ndash2600 2009

[17] L A Almeida and R Araujo ldquoHighlights on molecular iden-tification of closely related speciesrdquo Infection Genetics andEvolution vol 13 no 1 pp 67ndash75 2013

[18] J Dai Y Chen and M Lauzardo ldquoWeb-accessible databaseof hsp65 sequences from Mycobacterium reference strainsrdquoJournal of Clinical Microbiology vol 49 no 6 pp 2296ndash23032011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3

Table 1 Percentage rpsA sequence match among type strain

Species Type strain First match ()a Second match ()a

M thermoresistibile ATCC19527 M senegalense (100) M porcinum (987)M senegalense ATCC35796 M thermoresistibile (100) M porcinum (987)M porcinum ATCC33776 M senegalenseM thermoresistibile (987) M fortuitum (983)

M fortuitum ATCC6481 M porcinum (983) M senegalenseM thermoresistibile(981)

M phlei ATCC11758 M porcinum (965) M fortuitum (961)M gadium ATCC27726 M porcinumM fortuitum (957) M smegmatis (953)M flavescens ATCC14474 M smegmatis (966) M vaccae (960)

M smegmatis ATCC19420 M vaccaeM parafortuitumM triviale(966)

M senegalenseM thermoresistibile(963)

M vaccae ATCC15483 M diernhoferiM duvalii (968) M parafortuitumM triviale (966)M parafortuitum ATCC19686 M triviale (100) M terraeM austroafricanum (966)M triviale ATCC23292 M parafortuitum (100) M terraeM austroafricanum (966)M neoaurum ATCC25795 M diernhoferiM duvalii (964) M porcinum (955)M diernhoferi ATCC19340 M duvalii (100) M vaccae (968)M duvalii ATCC43910 M diernhoferi (100) M vaccae (968)M aurum ATCC23366 M gilvum (939) M chubuenseM vanbaalenii (930)M chubuense ATCC27278 M gilvum (966) M vanbaalenii (960)M gilvum ATCC43909 M vanbaalenii (972) M terraeM austroafricanum (970)M vanbaalenii PYR-1 M terraeM austroafricanum (998) M gilvum (972)M terrae ATCC15755 M austroafricanum (100) M vanbaalenii (998)M austroafricanum ATCC33464 M terrae (100) M vanbaalenii (998)M rhodesiae ATCC27024 M terraeM austroafricanum (939) M obuense (937)

M obuense ATCC27023 M porcinum M fortuitum (961) M senegalenseM thermoresistibile(965)

M aichiense ATCC27280 M sphagni (958) M vaccae (947)M sphagni ATCC33027 M aichiense (958) M porcinum (955)M tokaiense ATCC27282 M porcinum (953) M smegmatis (937)M chelonae ATCC35752 M obuense (939) M phlei (935)M abscessus subspeciesmassiliense str GO 06 M abscessus (987) M sphagni (914)

M abscessus ATCC19977 M abscessus subspeciesmassiliense (987) M chubuense (920)

M tuberculosis H37Rv ATCC27294 M tuberculosisH37RaM africanumMbovis BCGM bovis (100) M canettii (998)

M tuberculosis H37Ra ATCC25177 M tuberculosisH37RvM africanumMbovis BCGM bovis (100) M canettii (998)

M africanum ATCC25420 M tuberculosisH37RaM tuberculosisRvM bovis BCGM bovis (100) M canettii (998)

M bovis BCG ATCC35735 M tuberculosisH37RaM tuberculosisH37RvM africanumM bovis (100) M canettii (998)

M bovis ATCC19210 M tuberculosisH37RaM tuberculosisH37RvM africanumM bovis BCG (100) M canettii (998)

M canettii CIPT140010059 M tuberculosisM africanumM bovisBCGM bovis (998) M asiaticum (932)

M xenopi ATCC19250 M asiaticum (930) M avium subspecies paratuberculosis(924)

M avium ATCC25291 M avium subspecies paratuberculosis(996) M asiaticum (958)

4 BioMed Research International

Table 1 Continued

Species Type strain First match ()a Second match ()a

M avium subspeciesparatuberculosis K-10 M avium (994) M asiaticum (96)

M malmoense ATCC29571 M intracellulare (943) M szulgai (939)

M intracellulare ATCC13950 M avium (956) M avium subspecies paratuberculosis(954)

M scrofulaceum ATCC19981 M asiaticum (954) M szulgai (951)M marinum ATCC927 M ulcerans (996) M szulgai (960)M ulcerans Agy99 M marinum (996) M szulgai (960)M szulgai ATCC25799 M marinumM ulcerans (960) M asiaticum (956)M gordonae ATCC14470 M asiaticum (953) M kansasii (951)M asiaticum ATCC25276 M simiae (977) M kansasii (964)M kansasii ATCC12478 M asiaticum (963) M gastri (958)M gastri ATCC15754 M kansasii (958) M asiaticum (945)Rhodococcus equi 103S M smegmatis (903) M intracellulare (901)aBeside the queried type strain itself the first matched or the second matched type strain

33 Species Identification Outcomes of the Clinical IsolatesrpsA sequence and alignment efficiently identified clinicalisolates representing 8 mycobacterial species We foundthat a criterion of first distinct rpsA sequence match gt97confirmed the identification of 183 of 188 (973) clinicalisolates 20 M abscessus strains 2 M fortuitum strainsand 10 M avium strains were identified to the subspecieslevel Identification discrepancies between rpsA and othersequences were only encountered with 4M gordonae strainsand 1 M kansasii strain (see Table 3) No discrepancies werefound in the species submitted asM intracellulareM aviumM abscessus andM tuberculosis complex

The sequence divergence among MTC members was04 The intraspecies divergence among the 188 clinicalstrains ranged from 06 to 53 (Table 3) The sequencediversity among theM abscessuswas 06 while that amongthe M gordonae clinical isolates was 53 No M gordonaeclinical strain had an identical sequence with that of theM gordonae reference strain (ATCC14470) Interestingly therpsA sequence of one strain among the 23M kansasii isolatesexhibited a relatively low level of sequence similarity (956)to that of the reference strain (ATCC 12478) while those ofall the other M kansasii isolates were identical Since thestrain was confirmed as M kansasii by rpoB and hsp65 genealignment itmight suggest a distant variant or a new subtype

4 Discussion

16S rDNA gene sequence alignment has been used as thereference method for mycobacterial species identificationHowever it has been reported that by using the 16S rDNAgene alone for species identification of clinical NTM 37of such isolates remained unclassified which illustrates theneed for additional molecular tools for proper phylogeneticassignment and accurate NTM identification [11] The com-mon assumption that bacterial isolates belong to the samespecies if they have fewer than 5ndash15 bp differences within

the 16S rDNA gene sequence [12] or if they have more than97 16S rDNA gene sequence identities [13] may not beapplicable to genus of Mycobacterium whose members aremuch more closely related to each other Furthermore somemycobacterial strains shared uniform sequence of 16S rDNAgene such asM intracellulare andM aviumM kansasii andM gastri M abscessus and M chelonae and M szulgai andM malmoense

In this study we found that the rpsA gene is useful as acomplementary method to the 16S rDNA for mycobacterialspecies identification Among the 47 reference strains andtotally 1081 paired comparisons 1081 = [119899119886

1+ 119899(119899 minus 1)1198892]

1198861= 1 119899 = 46 119889 = 1 the rpsA gene alone can

differentiate 972 (1051 out of 1081) of them if using 97sequence homology as cutoff value When referring to thephylogenetic tree the rpsA resolution might increase Forthe most commonly seen pathogenic NTM species suchas M intracellulare M avium M abscessus M chelonaeand M xenopi rpsA gene sequence could differentiate thesespecies easily Furthermore 183 of the 188 (973) clinicalisolates representing 8mycobacterial species were identifiedcorrectly by rpsA gene blast Discrepancies of identificationwere only encountered with 4 M gordonae strains and 1M kansasii strain Both these species have reportedly moreintraspecies sequence divergence [9 14ndash16] Three out of the4 M gordonae strains with discrepant identification still hadthe first distinction asM gordonae by rpsA but the sequenceidentity was lower than 97 which means the identificationmight be correct when a more complete in-house database isbeing developed

rpsA alone cannot differentiate the following betweenmembers of the M tuberculosis complex between M sene-galense and M thermoresistibile between M parafortuitumand M trivial between M diernhoferi and M duvalii andbetween M austroafricanum and M terrae however thesespecies are also difficult to be separated by other markersalone such as 16s rDNA ITS rpoB and hsp65 Several

BioMed Research International 5Ta

ble2Sequ

ence

pairdistanceso

f47type

strainso

fmycob

acteria

and1o

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determ

ined

byusingthemultip

lealignm

enta

lgorith

min

theMegAlignpackage(versio

n710

DNAST

AR

Madiso

nWI)

Percentidentity

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

2930

3132

3334

3536

3738

3940

4142

4344

4546

4748

1mdash

1000

987

981

960

953

954

964

962

945

945

951

945

945

920

939

941

943

945

945

928

956

937

947

932

924

888

892

911

911

911

911

911

913

915

939

941

913

930

928

926

926

924

934

934

924

928

892

1Mtherm

oresistibile

200

mdash987

981

960

953

954

964

962

945

945

951

945

945

920

939

941

943

945

945

928

956

937

947

932

924

888

892

911

911

911

911

911

913

915

939

941

913

930

928

926

926

924

934

934

924

928

892

2Msenegalense

313

13mdash

983

966

958

958

968

968

947

947

956

954

954

920

941

954

949

951

951

930

962

947

956

945

930

894

898

911

911

911

911

911

913

920

945

947

920

934

935

932

932

928

937

937

930

928

894

3Mp

orcin

um

419

1917

mdash962

958

958

962

962

941

941

954

945

945

920

943

945

943

945

945

939

962

949

949

937

937

901

905

909

909

909

909

909

911

920

947

949

913

932

932

930

934

928

935

937

928

930

892

4Mfortuitu

m

541

41

35

39

mdash943

954

964

960

939

939

934

943

943

915

943

943

945

947

947

915

943

941

949

928

911

898

903

901

901

901

901

901

903

911

935

937

913

934

930

935

935

930

937

934

930

920

896

5Mp

hlei

649

49

43

43

59

mdash951

953

941

937

937

947

941

941

913

937

935

934

935

935

932

937

937

937

930

924

886

890

907

907

907

907

907

909

915

943

941

907

932

920

918

922

915

930

932

918

915

880

6Mg

adium

747

47

43

43

47

51

mdash966

960

954

954

947

947

947

930

951

953

956

958

958

932

941

949

947

924

920

901

905

924

924

924

924

924

926

928

949

947

930

941

949

937

937

935

939

943

926

934

903

7Mfl

avescens

837

37

33

39

37

49

35

mdash966

966

966

953

962

962

920

962

962

964

962

962

935

947

945

945

939

915

905

909

911

911

911

911

911

913

922

937

935

920

943

935

932

932

930

934

935

924

922

909

8Msmegmatis

939

39

33

39

41

61

41

35

mdash966

966

951

968

968

924

958

956

962

964

964

930

956

949

947

924

920

915

918

909

909

909

909

909

911

922

943

945

924

939

928

934

934

932

939

935

928

928

892

9Mv

accae

1057

57

55

61

63

65

47

35

35

mdash1000

949

962

962

930

949

958

964

966

966

930

943

928

930

924

917

898

901

899

899

899

899

899

901

915

926

924

913

932

920

911

911

913

918

915

903

917

890

10Mp

arafortuitu

m

1157

57

55

61

63

65

47

35

35

00

mdash949

962

962

930

949

958

964

966

966

930

943

928

930

924

917

898

901

899

899

899

899

899

901

915

926

924

913

932

920

911

911

913

918

915

903

917

890

11Mtriv

iale

1251

51

45

47

7055

55

49

51

53

53

mdash964

964

918

945

953

951

953

953

941

951

928

922

926

930

903

907

899

899

899

899

899

901

913

932

928

899

920

920

917

917

911

918

924

909

899

882

12Mn

eoaurum

1357

57

47

57

59

61

55

39

33

39

39

37

mdash1000

926

953

958

956

958

958

941

949

941

937

928

922

913

917

894

894

894

894

894

896

907

920

918

911

930

918

915

915

913

917

918

913

905

898

13Md

iernh

oferi

1457

57

47

57

59

61

55

39

33

39

39

37

00

mdash926

953

958

956

958

958

941

949

941

937

928

922

913

917

894

894

894

894

894

896

907

920

918

911

930

918

915

915

913

917

918

913

905

898

14Md

uvalii

1584

84

84

84

9193

7485

80

7474

86

7878

mdash930

939

930

932

932

917

918

911

911

894

911

898

901

892

892

892

892

892

894

888

901

905

915

905

905

896

898

898

892

901

896

890

877

15Ma

urum

1663

63

61

59

59

65

51

39

43

53

53

57

49

49

74mdash

966

960

958

958

939

947

947

935

928

924

918

924

905

905

905

905

905

907

917

932

930

911

926

924

926

930

913

924

935

915

909

884

16Mc

hubu

ense

1761

61

47

57

59

68

49

39

45

43

43

49

43

43

63

35

mdash972

970

970

941

956

945

941

935

934

913

917

903

903

903

903

903

905

913

930

932

920

928

932

926

930

917

928

928

915

911

901

17Mg

ilvum

1859

59

53

59

57

7045

37

39

37

37

51

45

45

7441

29

mdash998

998

941

949

928

934

926

930

907

911

901

901

901

901

901

903

913

928

926

913

924

928

920

920

913

920

924

913

920

892

18Mv

anbaalenii

1957

57

51

57

55

68

43

39

37

35

35

49

43

43

7243

31

02

mdash1000

939

951

930

935

928

932

909

913

903

903

903

903

903

905

915

930

928

915

926

930

922

922

915

922

926

915

922

890

19Mterrae

2057

57

51

57

55

68

43

39

37

35

35

49

43

43

7243

31

02

00

mdash939

951

930

935

928

932

909

913

903

903

903

903

903

905

915

930

928

915

926

930

922

922

915

922

926

915

922

890

20Ma

ustro

afric

anum

2176

7674

63

9172

7267

7474

7461

61

61

89

63

61

61

63

63

mdash937

918

917

911

926

888

892

894

894

894

894

894

896

903

915

917

901

909

909

899

901

896

907

913

909

899

896

21Mrhodesia

e

2245

45

39

39

59

66

61

55

45

59

59

51

53

53

87

55

45

53

51

51

65

mdash937

945

934

941

903

907

903

903

903

903

903

905

905

937

939

903

926

928

922

922

918

926

930

917

909

892

22Mo

buense

2365

65

55

53

61

65

53

57

53

7676

7661

61

9555

57

7674

7486

65

mdash958

939

930

917

920

909

909

909

909

909

911

922

937

939

917

932

926

934

937

922

937

939

924

917

890

23Ma

ichien

se

2455

55

45

53

53

65

55

57

55

7474

82

65

65

9567

61

7067

67

89

57

43

mdash937

934

913

918

915

915

915

915

915

917

915

937

941

922

930

935

928

930

930

934

941

932

924

892

24Msphagni

2572

7257

65

7674

80

63

80

80

80

7876

76115

7667

7876

7695

7063

65

mdash920

888

894

896

896

896

896

896

898

905

945

945

907

930

928

918

922

920

924

934

922

915

882

25Mtokaiense

2680

80

7465

9580

84

9184

89

89

7482

82

9580

7074

7272

7861

7470

84

mdash915

918

896

896

896

896

896

898

898

922

926

894

905

911

907

911

898

920

917

907

903

871

26Mc

helona

e

27121

121

115

106

110

124

106

101

91

110

110

104

9393

110

86

9399

9797

121

104

89

93121

91mdash

987

867

867

867

867

867

869

884

884

888

884

886

894

896

896

884

901

898

882

871

871

27Ma

bscessu

s

28117

117

110

101

104

119

101

9786

106

106

9989

89

106

80

89

9593

93117

9984

86

115

87

13mdash

875

875

875

875

875

877

888

888

892

888

890

898

899

899

890

905

901

886

875

877

28Ma

bscessu

s

2995

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

mdash1000

1000

1000

1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

29Mtub

erculosis

3095

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

mdash1000

1000

1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

30Mtub

erculosis

3195

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

00

mdash1000

1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

31Mb

ovis

3295

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

00

00

mdash1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

32Ma

fricanu

m

3395

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

00

00

00

mdash998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

33Mb

ovis

3493

9393

95104

9778

9395

106

106

106

112

112

115

99102

104

102

102

112

102

9589

111

110

144

135

02

02

02

02

02

mdash911

918

922

907

918

924

918

917

918

926

932

920

917

880

34Mc

anettii

3591

9184

84

9591

7682

82

9191

9399

99122

89

9393

9191

104

102

82

91102

110

126

121

9898

9898

9895

mdash922

924

907

917

920

911

911

905

918

930

915

909

877

35Mx

enopi

3663

63

57

55

67

59

53

65

59

7878

7284

84

106

7274

7674

7491

65

65

65

57

82

126

121

89

89

89

89

89

87

82

mdash994

930

956

947

939

943

941

951

958

947

937

886

36Ma

vium

3761

61

55

53

65

61

55

67

57

80

80

7686

86

101

7472

7876

7689

63

63

61

57

78121

117

84

84

84

84

84

82

80

06

mdash935

954

951

945

949

945

954

960

953

939

888

37Ma

vium

3893

9384

9393

9974

84

80

9393

108

9595

9195

84

9391

91106

104

89

82

99115

126

121

9797

9797

97100

9974

67

mdash943

937

935

934

924

939

930

937

930

890

38Mm

almoense

3974

7470

7270

7261

59

63

7272

84

7474

102

7876

80

7878

9778

7274

74102

123

119

89

89

89

89

89

87

89

45

47

59

mdash945

941

941

934

951

947

943

943

907

39Mintracellu

lare

4076

7667

7274

84

53

67

7684

84

84

86

86

101

80

7276

7474

9776

7867

7695

115

110

82

82

82

82

82

80

84

55

51

65

57

mdash943

943

949

951

954

939

934

896

40Mscrofulaceum

4178

7872

7468

86

65

7270

9595

89

9191

112

7878

84

82

82

108

82

7076

87

99112

108

84

84

84

84

84

87

9563

57

68

61

59

mdash996

945

960

956

956

937

896

41Mm

arinum

4278

7872

7068

82

65

7270

9595

89

9191

110

7474

84

82

82

106

82

65

7482

95112

108

87

87

87

87

87

89

9559

53

7061

59

04

mdash943

960

956

954

939

894

42Mu

lcerans

4380

80

7676

7491

67

7472

9393

9593

93110

9389

9391

91112

86

82

7484

110

126

119

84

84

84

84

84

87

101

61

57

80

7053

57

59

mdash943

953

951

934

890

43Mg

ordona

e

4470

7065

67

65

7463

7063

86

86

86

89

89

117

80

7684

82

82

9978

65

7080

84

106

101

7676

7676

7678

86

51

47

63

51

51

41

41

59

mdash956

954

945

901

44Mszulga

i

4570

7065

65

7072

59

67

67

9191

80

86

86

106

67

7680

7878

9374

63

61

7089

110

106

7070

7070

7072

7443

41

7455

47

45

45

49

45

mdash964

947

888

45Ma

siaticum

4680

80

7476

7486

7880

76104

104

9793

93112

9191

9391

9197

89

80

7282

99128

123

82

82

82

82

82

85

9155

49

65

59

63

45

47

51

47

37

mdash958

888

46Mk

ansasii

4776

7676

7484

9170

82

7689

89

108

101

101

119

9795

84

82

82

108

9789

80

91104

142

137

87

87

87

87

87

89

9765

63

7459

7066

63

7057

55

43

mdash879

47Mg

astri

48117

117

115

117

112

130

104

97117

119

119

128

110

110

135

126

106

117

119

119

112

117

119

117

128

142

142

135

130

130

130

130

130

130

135

124

121

119

99112

112

115

119

106

121

121

132

mdash48

Rhodococcusequi

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

2930

3132

3334

3536

3738

3940

4142

4344

4546

4748

6 BioMed Research International

001

99

78

93

37

11

2

10

0

1

1

1

19

99

9 99

99

99

99

99

76

52

5

17

35

67

23

23

60

57

10

32

38

32

23

99

9

66

13

16

13

24

55

M thermoresistibileM senegalense

M porcinumM fortuitum

M phleiM gadium

M flavescensM smegmatisM vaccae

M parafortuitumM triviale

M neoaurumM diernhoferiM duvalii

M aurumM chubuense

M gilvumM vanbaalenii

M terraeM austroafricanum

M rhodesiaeM obuenseM aichienseM sphagni

M tokaienseM chelonae

M abscessusM abscessus subspecies massiliense

M tuberculosis H37RvM tuberculosis H37Ra

M bovisM bovis BCG

M canettiiM xenopi

M aviumM avium subspecies paratuberculosis

M malmoenseM intracellulareM scrofulaceum

M marinumM ulcerans

M szulgaiM gordonae

M asiaticumM kansassii

M gastri

M africanum

Rhodococcus equi

Figure 1 Phylogenetic tree based on rpsA gene sequences shows the relationship of the 47 type strains of mycobacteria and 1 outgroup strainThis tree was constructed by the neighbor-joiningmethod Topology was also evaluated by bootstrap analysis (MEGA program 1000 repeatswith R equi as the outgroup)The numerical values in the tree represent bootstrap results The distance between two strains is the sum of thebranch lengths between them

BioMed Research International 7

Table 3 Comparison of mycobacteria identified by 16S rDNAITShsp65rpoB gene sequencing and by rpsA sequencing among the 188clinical isolatesa

Primary identificationb (119899)Primary identificationb (119899)

First choice Second choiceIdentification (119899) match Identification (119899) match

M intracellulare (75)

M intracellulare (4) 100 M avium (4) 956M intracellulare (1) 998 M avium (1) 953M intracellulare (53) 989 M avium (53) 951M intracellulare (10) 987 M avium (10) 949M intracellulare (1) 985 M avium (1) 953M intracellulare (3) 981 M avium (3) 947M intracellulare (1) 975 M avium (1) 932M intracellulare (2) 971 M avium (2) 961

M abscessus (39)M abscessus (9) 100 M abscessus subspecies massiliense (9) 987M abscessus (10) 998 M abscessus subspecies massiliense (10) 985M abscessus subspecies massiliense (20) 994 M abscessus (20) 991

M kansasii (23) M kansasii (22) 100 M asiaticum (22) 963M szulgai (1) 966 M marinumM ulcerans (1) 960

M avium (10)M avium subspecies paratuberculosis (5) 996 M avium (5) 994M avium subspecies paratuberculosis (4) 998 M avium (4) 992M avium subspecies paratuberculosis (1) 975 M avium (1) 970

M fortuitum complex (17)

M fortuitum (2) 100 M senegalenseM thermoresistibile (2) 981M fortuitum (11) 998 M porcinum (11) 985M fortuitum (1) 996 M porcinum (1) 983M fortuitumM porcinum (1) 989 M senegalenseM thermoresistibile (1) 983M thermoresistibileM senegalense (2) 996 M fortuitum (2) 981

M gordonae (7)

M gordonae (1) 992 M asiaticum (1) 955M gordonae (2) 985 M asiaticum (2) 963M gordonae (3) 959 M asiaticum (3) 955M szulgai (1) 959 M gordonae (1) 947

M tuberculosis complex (16)M tuberculosis (2) 100 M canetti (2) 998M tuberculosis (12) 998 M canetti (12) 996M tuberculosis (2) 996 M canetti (2) 994

M neoaurum (1) M neoaurum (1) 987 M smegmatis (1) 954aNumbers in parentheses (119899) represent the numbers of isolates identified as a particular speciesbIdentification based on sequencing of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoB and hsp65 genes

reference strains had similar homology with two or morespecies which suggests inadequate taxonomy within thecurrently described species Based on nucleotide sequencesof rpoB hsp65 and sodA in clinical isolates of the Mycobac-terium abscessus group one-fourth of isolates had discordantidentification [16] Multilocus sequence typing and sequenceanalysis of several genes facilitate the identification of closelyrelated species or subspecies [17]

In our study we have demonstrated that the rpsA homol-ogy gene is a promising marker for mycobacterial speciesidentification for both fast- and slow-growing mycobacteriaTo be a good marker for species differentiation the targetgene should be stable and sequence variations should occurrandomly additionally an extremely conserved or highlyvariable gene may not be adequate As a single-copy house-keeping gene rpsA gene could work well as a target gene for

Mycobacterium species discrimination without ambiguousidentification The 16S rDNA gene has higher homologywithin the mycobacteria compared to the rpsA gene 943to 100 compared to 854 to 100 According to our workwhen used as sole marker rpsA had better resolution than 16srDNA but had similar resolution as ITS rpoB and hsp65

Due to the high resolution power of rpsA for speciesidentification we presume it has potential of clinical useFrom our own experience we recommend that when per-forming species identification by homologue DNA sequencecomparison one should start with 16s RNA gene plus atleast one of the other markers such as ITS rpoB hsp65and rpsA When conflicting outcome or dubious outcomesyield more markers should be added further Even though16s RNA gene is inferior to the other markers considering thecapacity it should be chosen firstly since 16s RNA gene has

8 BioMed Research International

the most robust sequence database which can help to avoidbig identification errors due to unreliable database of othermarkers [18]

Themain deterrents for the primary use of rpsA sequenc-ing as a routine means of identifying mycobacteria residein the need for a comprehensive database Therefore weconstructed our own rpsA sequence database by including asmany type strains as possible and integrating rpsA sequencesdeposited in GenBank such as M avium subspecies paratu-berculosis M ulcerans M vanbaalenii M abscessus sub-speciesmassiliense andM canettii Besides type strains rpsAsequences of confirmed clinical strains were also includedWe will constantly upgrade our database and the capabilityto identify the most recent described species

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HongfeiDuan andGuanLiu contributed equally to this study

Acknowledgments

The work was supported by the research funding fromInfectious Diseases Special Project Ministry of Health ofChina (2012ZX10003002-09) and Beijing Municipal Admin-istration of Hospitals Clinical Medicine Development ofSpecial Funding (ZYLX201304) All the type strains andclinical isolates used in this study were obtained from theldquoBeijing Bio-Bank of Clinical Resources on Tuberculosis(D09050704640000)rdquo Beijing Chest Hospital

References

[1] T Adekambi P Berger D Raoult and M DrancourtldquorpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacteriumbolletii sp nov Mycobacterium phocaicum sp nov andMycobacterium aubagnense sp novrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 1 ArticleID 63969 pp 133ndash143 2006

[2] B Ninet M Monod S Emler et al ldquoTwo different 16SrRNA genes in a mycobacterial strainrdquo Journal of ClinicalMicrobiology vol 34 no 10 pp 2531ndash2536 1996

[3] U Reischl K Feldmann L Naumann et al ldquo16S rRNAsequence diversity inMycobacterium celatum strains caused bypresence of two different copies of 16S rRNA generdquo Journal ofClinical Microbiology vol 36 no 6 pp 1761ndash1764 1998

[4] D E Griffith T Aksamit B A Brown-Elliott et al ldquoAn officialATSIDSA statement diagnosis treatment and prevention ofnontuberculous mycobacterial diseasesrdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 4 pp 367ndash416 2007

[5] W Shi X Zhang X Jiang et al ldquoPyrazinamide inhibits trans-translation inMycobacterium tuberculosisrdquo Science vol 333 no6049 pp 1630ndash1632 2011

[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998

[7] A Roth M Fischer M E Hamid S Michalke W Ludwig andH Mauch ldquoDifferentiation of phylogenetically related slowlygrowing mycobacteria based on 16S-23S rRNA gene internaltranscribed spacer sequencesrdquo Journal of Clinical Microbiologyvol 36 no 1 pp 139ndash147 1998

[8] H Ringuet A Honore A Varnerot et al ldquohsp65 sequencingfor identification of rapidly growing mycobacteriardquo Journal ofClinical Microbiology vol 37 no 3 pp 852ndash857 1999

[9] B-J Kim S-H Lee M-A Lyu et al ldquoIdentification ofmycobacterial species by comparative sequence analysis of theRNA polymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 37 no 6 pp 1714ndash1720 1999

[10] J Amita T Vandana R S Guleria and R K Verma ldquoQualita-tive evaluation of mycobacterial DNA extraction protocols forpolymerase chain reactionrdquoMolecular Biology Today vol 3 no2 pp 43ndash49 2002

[11] R J Pauls C Y Turenne J N Wolfe and A Kabani ldquoAhigh proportion of novel mycobacteria species identified by16S rDNA analysis among slowly growing AccuProbe-negativestrains in a clinical settingrdquo American Journal of ClinicalPathology vol 120 no 4 pp 560ndash566 2003

[12] G E Fox J D Wisotzkey and P Jurtshuk Jr ldquoHow close isclose 16S rRNA sequence identity may not be sufficient toguarantee species identityrdquo International Journal of SystematicBacteriology vol 42 no 1 pp 166ndash170 1992

[13] M Drancourt P Berger and D Raoult ldquoSystematic 16S rRNAgene sequencing of atypical clinical isolates identified 27 newbacterial species associated with humansrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2197ndash2202 2004

[14] P Kirschner E C Bottger D T Walton and M ValescoldquoMicroheterogeneity within rRNA of Mycobacterium gor-donaerdquo Journal of Clinical Microbiology vol 30 no 4 pp 1049ndash1050 1992

[15] C Taillard G Greub R Weber et al ldquoClinical implicationsofMycobacterium kansasii species heterogeneity swiss nationalsurveyrdquo Journal of Clinical Microbiology vol 41 no 3 pp 1240ndash1244 2003

[16] E Macheras A-L Roux F Ripoll et al ldquoInaccuracy of single-target sequencing for discriminating species of the Mycobac-terium abscessus grouprdquo Journal of Clinical Microbiology vol 47no 8 pp 2596ndash2600 2009

[17] L A Almeida and R Araujo ldquoHighlights on molecular iden-tification of closely related speciesrdquo Infection Genetics andEvolution vol 13 no 1 pp 67ndash75 2013

[18] J Dai Y Chen and M Lauzardo ldquoWeb-accessible databaseof hsp65 sequences from Mycobacterium reference strainsrdquoJournal of Clinical Microbiology vol 49 no 6 pp 2296ndash23032011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

Table 1 Continued

Species Type strain First match ()a Second match ()a

M avium subspeciesparatuberculosis K-10 M avium (994) M asiaticum (96)

M malmoense ATCC29571 M intracellulare (943) M szulgai (939)

M intracellulare ATCC13950 M avium (956) M avium subspecies paratuberculosis(954)

M scrofulaceum ATCC19981 M asiaticum (954) M szulgai (951)M marinum ATCC927 M ulcerans (996) M szulgai (960)M ulcerans Agy99 M marinum (996) M szulgai (960)M szulgai ATCC25799 M marinumM ulcerans (960) M asiaticum (956)M gordonae ATCC14470 M asiaticum (953) M kansasii (951)M asiaticum ATCC25276 M simiae (977) M kansasii (964)M kansasii ATCC12478 M asiaticum (963) M gastri (958)M gastri ATCC15754 M kansasii (958) M asiaticum (945)Rhodococcus equi 103S M smegmatis (903) M intracellulare (901)aBeside the queried type strain itself the first matched or the second matched type strain

33 Species Identification Outcomes of the Clinical IsolatesrpsA sequence and alignment efficiently identified clinicalisolates representing 8 mycobacterial species We foundthat a criterion of first distinct rpsA sequence match gt97confirmed the identification of 183 of 188 (973) clinicalisolates 20 M abscessus strains 2 M fortuitum strainsand 10 M avium strains were identified to the subspecieslevel Identification discrepancies between rpsA and othersequences were only encountered with 4M gordonae strainsand 1 M kansasii strain (see Table 3) No discrepancies werefound in the species submitted asM intracellulareM aviumM abscessus andM tuberculosis complex

The sequence divergence among MTC members was04 The intraspecies divergence among the 188 clinicalstrains ranged from 06 to 53 (Table 3) The sequencediversity among theM abscessuswas 06 while that amongthe M gordonae clinical isolates was 53 No M gordonaeclinical strain had an identical sequence with that of theM gordonae reference strain (ATCC14470) Interestingly therpsA sequence of one strain among the 23M kansasii isolatesexhibited a relatively low level of sequence similarity (956)to that of the reference strain (ATCC 12478) while those ofall the other M kansasii isolates were identical Since thestrain was confirmed as M kansasii by rpoB and hsp65 genealignment itmight suggest a distant variant or a new subtype

4 Discussion

16S rDNA gene sequence alignment has been used as thereference method for mycobacterial species identificationHowever it has been reported that by using the 16S rDNAgene alone for species identification of clinical NTM 37of such isolates remained unclassified which illustrates theneed for additional molecular tools for proper phylogeneticassignment and accurate NTM identification [11] The com-mon assumption that bacterial isolates belong to the samespecies if they have fewer than 5ndash15 bp differences within

the 16S rDNA gene sequence [12] or if they have more than97 16S rDNA gene sequence identities [13] may not beapplicable to genus of Mycobacterium whose members aremuch more closely related to each other Furthermore somemycobacterial strains shared uniform sequence of 16S rDNAgene such asM intracellulare andM aviumM kansasii andM gastri M abscessus and M chelonae and M szulgai andM malmoense

In this study we found that the rpsA gene is useful as acomplementary method to the 16S rDNA for mycobacterialspecies identification Among the 47 reference strains andtotally 1081 paired comparisons 1081 = [119899119886

1+ 119899(119899 minus 1)1198892]

1198861= 1 119899 = 46 119889 = 1 the rpsA gene alone can

differentiate 972 (1051 out of 1081) of them if using 97sequence homology as cutoff value When referring to thephylogenetic tree the rpsA resolution might increase Forthe most commonly seen pathogenic NTM species suchas M intracellulare M avium M abscessus M chelonaeand M xenopi rpsA gene sequence could differentiate thesespecies easily Furthermore 183 of the 188 (973) clinicalisolates representing 8mycobacterial species were identifiedcorrectly by rpsA gene blast Discrepancies of identificationwere only encountered with 4 M gordonae strains and 1M kansasii strain Both these species have reportedly moreintraspecies sequence divergence [9 14ndash16] Three out of the4 M gordonae strains with discrepant identification still hadthe first distinction asM gordonae by rpsA but the sequenceidentity was lower than 97 which means the identificationmight be correct when a more complete in-house database isbeing developed

rpsA alone cannot differentiate the following betweenmembers of the M tuberculosis complex between M sene-galense and M thermoresistibile between M parafortuitumand M trivial between M diernhoferi and M duvalii andbetween M austroafricanum and M terrae however thesespecies are also difficult to be separated by other markersalone such as 16s rDNA ITS rpoB and hsp65 Several

BioMed Research International 5Ta

ble2Sequ

ence

pairdistanceso

f47type

strainso

fmycob

acteria

and1o

ut-group

strain

determ

ined

byusingthemultip

lealignm

enta

lgorith

min

theMegAlignpackage(versio

n710

DNAST

AR

Madiso

nWI)

Percentidentity

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

2930

3132

3334

3536

3738

3940

4142

4344

4546

4748

1mdash

1000

987

981

960

953

954

964

962

945

945

951

945

945

920

939

941

943

945

945

928

956

937

947

932

924

888

892

911

911

911

911

911

913

915

939

941

913

930

928

926

926

924

934

934

924

928

892

1Mtherm

oresistibile

200

mdash987

981

960

953

954

964

962

945

945

951

945

945

920

939

941

943

945

945

928

956

937

947

932

924

888

892

911

911

911

911

911

913

915

939

941

913

930

928

926

926

924

934

934

924

928

892

2Msenegalense

313

13mdash

983

966

958

958

968

968

947

947

956

954

954

920

941

954

949

951

951

930

962

947

956

945

930

894

898

911

911

911

911

911

913

920

945

947

920

934

935

932

932

928

937

937

930

928

894

3Mp

orcin

um

419

1917

mdash962

958

958

962

962

941

941

954

945

945

920

943

945

943

945

945

939

962

949

949

937

937

901

905

909

909

909

909

909

911

920

947

949

913

932

932

930

934

928

935

937

928

930

892

4Mfortuitu

m

541

41

35

39

mdash943

954

964

960

939

939

934

943

943

915

943

943

945

947

947

915

943

941

949

928

911

898

903

901

901

901

901

901

903

911

935

937

913

934

930

935

935

930

937

934

930

920

896

5Mp

hlei

649

49

43

43

59

mdash951

953

941

937

937

947

941

941

913

937

935

934

935

935

932

937

937

937

930

924

886

890

907

907

907

907

907

909

915

943

941

907

932

920

918

922

915

930

932

918

915

880

6Mg

adium

747

47

43

43

47

51

mdash966

960

954

954

947

947

947

930

951

953

956

958

958

932

941

949

947

924

920

901

905

924

924

924

924

924

926

928

949

947

930

941

949

937

937

935

939

943

926

934

903

7Mfl

avescens

837

37

33

39

37

49

35

mdash966

966

966

953

962

962

920

962

962

964

962

962

935

947

945

945

939

915

905

909

911

911

911

911

911

913

922

937

935

920

943

935

932

932

930

934

935

924

922

909

8Msmegmatis

939

39

33

39

41

61

41

35

mdash966

966

951

968

968

924

958

956

962

964

964

930

956

949

947

924

920

915

918

909

909

909

909

909

911

922

943

945

924

939

928

934

934

932

939

935

928

928

892

9Mv

accae

1057

57

55

61

63

65

47

35

35

mdash1000

949

962

962

930

949

958

964

966

966

930

943

928

930

924

917

898

901

899

899

899

899

899

901

915

926

924

913

932

920

911

911

913

918

915

903

917

890

10Mp

arafortuitu

m

1157

57

55

61

63

65

47

35

35

00

mdash949

962

962

930

949

958

964

966

966

930

943

928

930

924

917

898

901

899

899

899

899

899

901

915

926

924

913

932

920

911

911

913

918

915

903

917

890

11Mtriv

iale

1251

51

45

47

7055

55

49

51

53

53

mdash964

964

918

945

953

951

953

953

941

951

928

922

926

930

903

907

899

899

899

899

899

901

913

932

928

899

920

920

917

917

911

918

924

909

899

882

12Mn

eoaurum

1357

57

47

57

59

61

55

39

33

39

39

37

mdash1000

926

953

958

956

958

958

941

949

941

937

928

922

913

917

894

894

894

894

894

896

907

920

918

911

930

918

915

915

913

917

918

913

905

898

13Md

iernh

oferi

1457

57

47

57

59

61

55

39

33

39

39

37

00

mdash926

953

958

956

958

958

941

949

941

937

928

922

913

917

894

894

894

894

894

896

907

920

918

911

930

918

915

915

913

917

918

913

905

898

14Md

uvalii

1584

84

84

84

9193

7485

80

7474

86

7878

mdash930

939

930

932

932

917

918

911

911

894

911

898

901

892

892

892

892

892

894

888

901

905

915

905

905

896

898

898

892

901

896

890

877

15Ma

urum

1663

63

61

59

59

65

51

39

43

53

53

57

49

49

74mdash

966

960

958

958

939

947

947

935

928

924

918

924

905

905

905

905

905

907

917

932

930

911

926

924

926

930

913

924

935

915

909

884

16Mc

hubu

ense

1761

61

47

57

59

68

49

39

45

43

43

49

43

43

63

35

mdash972

970

970

941

956

945

941

935

934

913

917

903

903

903

903

903

905

913

930

932

920

928

932

926

930

917

928

928

915

911

901

17Mg

ilvum

1859

59

53

59

57

7045

37

39

37

37

51

45

45

7441

29

mdash998

998

941

949

928

934

926

930

907

911

901

901

901

901

901

903

913

928

926

913

924

928

920

920

913

920

924

913

920

892

18Mv

anbaalenii

1957

57

51

57

55

68

43

39

37

35

35

49

43

43

7243

31

02

mdash1000

939

951

930

935

928

932

909

913

903

903

903

903

903

905

915

930

928

915

926

930

922

922

915

922

926

915

922

890

19Mterrae

2057

57

51

57

55

68

43

39

37

35

35

49

43

43

7243

31

02

00

mdash939

951

930

935

928

932

909

913

903

903

903

903

903

905

915

930

928

915

926

930

922

922

915

922

926

915

922

890

20Ma

ustro

afric

anum

2176

7674

63

9172

7267

7474

7461

61

61

89

63

61

61

63

63

mdash937

918

917

911

926

888

892

894

894

894

894

894

896

903

915

917

901

909

909

899

901

896

907

913

909

899

896

21Mrhodesia

e

2245

45

39

39

59

66

61

55

45

59

59

51

53

53

87

55

45

53

51

51

65

mdash937

945

934

941

903

907

903

903

903

903

903

905

905

937

939

903

926

928

922

922

918

926

930

917

909

892

22Mo

buense

2365

65

55

53

61

65

53

57

53

7676

7661

61

9555

57

7674

7486

65

mdash958

939

930

917

920

909

909

909

909

909

911

922

937

939

917

932

926

934

937

922

937

939

924

917

890

23Ma

ichien

se

2455

55

45

53

53

65

55

57

55

7474

82

65

65

9567

61

7067

67

89

57

43

mdash937

934

913

918

915

915

915

915

915

917

915

937

941

922

930

935

928

930

930

934

941

932

924

892

24Msphagni

2572

7257

65

7674

80

63

80

80

80

7876

76115

7667

7876

7695

7063

65

mdash920

888

894

896

896

896

896

896

898

905

945

945

907

930

928

918

922

920

924

934

922

915

882

25Mtokaiense

2680

80

7465

9580

84

9184

89

89

7482

82

9580

7074

7272

7861

7470

84

mdash915

918

896

896

896

896

896

898

898

922

926

894

905

911

907

911

898

920

917

907

903

871

26Mc

helona

e

27121

121

115

106

110

124

106

101

91

110

110

104

9393

110

86

9399

9797

121

104

89

93121

91mdash

987

867

867

867

867

867

869

884

884

888

884

886

894

896

896

884

901

898

882

871

871

27Ma

bscessu

s

28117

117

110

101

104

119

101

9786

106

106

9989

89

106

80

89

9593

93117

9984

86

115

87

13mdash

875

875

875

875

875

877

888

888

892

888

890

898

899

899

890

905

901

886

875

877

28Ma

bscessu

s

2995

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

mdash1000

1000

1000

1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

29Mtub

erculosis

3095

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

mdash1000

1000

1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

30Mtub

erculosis

3195

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

00

mdash1000

1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

31Mb

ovis

3295

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

00

00

mdash1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

32Ma

fricanu

m

3395

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

00

00

00

mdash998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

33Mb

ovis

3493

9393

95104

9778

9395

106

106

106

112

112

115

99102

104

102

102

112

102

9589

111

110

144

135

02

02

02

02

02

mdash911

918

922

907

918

924

918

917

918

926

932

920

917

880

34Mc

anettii

3591

9184

84

9591

7682

82

9191

9399

99122

89

9393

9191

104

102

82

91102

110

126

121

9898

9898

9895

mdash922

924

907

917

920

911

911

905

918

930

915

909

877

35Mx

enopi

3663

63

57

55

67

59

53

65

59

7878

7284

84

106

7274

7674

7491

65

65

65

57

82

126

121

89

89

89

89

89

87

82

mdash994

930

956

947

939

943

941

951

958

947

937

886

36Ma

vium

3761

61

55

53

65

61

55

67

57

80

80

7686

86

101

7472

7876

7689

63

63

61

57

78121

117

84

84

84

84

84

82

80

06

mdash935

954

951

945

949

945

954

960

953

939

888

37Ma

vium

3893

9384

9393

9974

84

80

9393

108

9595

9195

84

9391

91106

104

89

82

99115

126

121

9797

9797

97100

9974

67

mdash943

937

935

934

924

939

930

937

930

890

38Mm

almoense

3974

7470

7270

7261

59

63

7272

84

7474

102

7876

80

7878

9778

7274

74102

123

119

89

89

89

89

89

87

89

45

47

59

mdash945

941

941

934

951

947

943

943

907

39Mintracellu

lare

4076

7667

7274

84

53

67

7684

84

84

86

86

101

80

7276

7474

9776

7867

7695

115

110

82

82

82

82

82

80

84

55

51

65

57

mdash943

943

949

951

954

939

934

896

40Mscrofulaceum

4178

7872

7468

86

65

7270

9595

89

9191

112

7878

84

82

82

108

82

7076

87

99112

108

84

84

84

84

84

87

9563

57

68

61

59

mdash996

945

960

956

956

937

896

41Mm

arinum

4278

7872

7068

82

65

7270

9595

89

9191

110

7474

84

82

82

106

82

65

7482

95112

108

87

87

87

87

87

89

9559

53

7061

59

04

mdash943

960

956

954

939

894

42Mu

lcerans

4380

80

7676

7491

67

7472

9393

9593

93110

9389

9391

91112

86

82

7484

110

126

119

84

84

84

84

84

87

101

61

57

80

7053

57

59

mdash943

953

951

934

890

43Mg

ordona

e

4470

7065

67

65

7463

7063

86

86

86

89

89

117

80

7684

82

82

9978

65

7080

84

106

101

7676

7676

7678

86

51

47

63

51

51

41

41

59

mdash956

954

945

901

44Mszulga

i

4570

7065

65

7072

59

67

67

9191

80

86

86

106

67

7680

7878

9374

63

61

7089

110

106

7070

7070

7072

7443

41

7455

47

45

45

49

45

mdash964

947

888

45Ma

siaticum

4680

80

7476

7486

7880

76104

104

9793

93112

9191

9391

9197

89

80

7282

99128

123

82

82

82

82

82

85

9155

49

65

59

63

45

47

51

47

37

mdash958

888

46Mk

ansasii

4776

7676

7484

9170

82

7689

89

108

101

101

119

9795

84

82

82

108

9789

80

91104

142

137

87

87

87

87

87

89

9765

63

7459

7066

63

7057

55

43

mdash879

47Mg

astri

48117

117

115

117

112

130

104

97117

119

119

128

110

110

135

126

106

117

119

119

112

117

119

117

128

142

142

135

130

130

130

130

130

130

135

124

121

119

99112

112

115

119

106

121

121

132

mdash48

Rhodococcusequi

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

2930

3132

3334

3536

3738

3940

4142

4344

4546

4748

6 BioMed Research International

001

99

78

93

37

11

2

10

0

1

1

1

19

99

9 99

99

99

99

99

76

52

5

17

35

67

23

23

60

57

10

32

38

32

23

99

9

66

13

16

13

24

55

M thermoresistibileM senegalense

M porcinumM fortuitum

M phleiM gadium

M flavescensM smegmatisM vaccae

M parafortuitumM triviale

M neoaurumM diernhoferiM duvalii

M aurumM chubuense

M gilvumM vanbaalenii

M terraeM austroafricanum

M rhodesiaeM obuenseM aichienseM sphagni

M tokaienseM chelonae

M abscessusM abscessus subspecies massiliense

M tuberculosis H37RvM tuberculosis H37Ra

M bovisM bovis BCG

M canettiiM xenopi

M aviumM avium subspecies paratuberculosis

M malmoenseM intracellulareM scrofulaceum

M marinumM ulcerans

M szulgaiM gordonae

M asiaticumM kansassii

M gastri

M africanum

Rhodococcus equi

Figure 1 Phylogenetic tree based on rpsA gene sequences shows the relationship of the 47 type strains of mycobacteria and 1 outgroup strainThis tree was constructed by the neighbor-joiningmethod Topology was also evaluated by bootstrap analysis (MEGA program 1000 repeatswith R equi as the outgroup)The numerical values in the tree represent bootstrap results The distance between two strains is the sum of thebranch lengths between them

BioMed Research International 7

Table 3 Comparison of mycobacteria identified by 16S rDNAITShsp65rpoB gene sequencing and by rpsA sequencing among the 188clinical isolatesa

Primary identificationb (119899)Primary identificationb (119899)

First choice Second choiceIdentification (119899) match Identification (119899) match

M intracellulare (75)

M intracellulare (4) 100 M avium (4) 956M intracellulare (1) 998 M avium (1) 953M intracellulare (53) 989 M avium (53) 951M intracellulare (10) 987 M avium (10) 949M intracellulare (1) 985 M avium (1) 953M intracellulare (3) 981 M avium (3) 947M intracellulare (1) 975 M avium (1) 932M intracellulare (2) 971 M avium (2) 961

M abscessus (39)M abscessus (9) 100 M abscessus subspecies massiliense (9) 987M abscessus (10) 998 M abscessus subspecies massiliense (10) 985M abscessus subspecies massiliense (20) 994 M abscessus (20) 991

M kansasii (23) M kansasii (22) 100 M asiaticum (22) 963M szulgai (1) 966 M marinumM ulcerans (1) 960

M avium (10)M avium subspecies paratuberculosis (5) 996 M avium (5) 994M avium subspecies paratuberculosis (4) 998 M avium (4) 992M avium subspecies paratuberculosis (1) 975 M avium (1) 970

M fortuitum complex (17)

M fortuitum (2) 100 M senegalenseM thermoresistibile (2) 981M fortuitum (11) 998 M porcinum (11) 985M fortuitum (1) 996 M porcinum (1) 983M fortuitumM porcinum (1) 989 M senegalenseM thermoresistibile (1) 983M thermoresistibileM senegalense (2) 996 M fortuitum (2) 981

M gordonae (7)

M gordonae (1) 992 M asiaticum (1) 955M gordonae (2) 985 M asiaticum (2) 963M gordonae (3) 959 M asiaticum (3) 955M szulgai (1) 959 M gordonae (1) 947

M tuberculosis complex (16)M tuberculosis (2) 100 M canetti (2) 998M tuberculosis (12) 998 M canetti (12) 996M tuberculosis (2) 996 M canetti (2) 994

M neoaurum (1) M neoaurum (1) 987 M smegmatis (1) 954aNumbers in parentheses (119899) represent the numbers of isolates identified as a particular speciesbIdentification based on sequencing of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoB and hsp65 genes

reference strains had similar homology with two or morespecies which suggests inadequate taxonomy within thecurrently described species Based on nucleotide sequencesof rpoB hsp65 and sodA in clinical isolates of the Mycobac-terium abscessus group one-fourth of isolates had discordantidentification [16] Multilocus sequence typing and sequenceanalysis of several genes facilitate the identification of closelyrelated species or subspecies [17]

In our study we have demonstrated that the rpsA homol-ogy gene is a promising marker for mycobacterial speciesidentification for both fast- and slow-growing mycobacteriaTo be a good marker for species differentiation the targetgene should be stable and sequence variations should occurrandomly additionally an extremely conserved or highlyvariable gene may not be adequate As a single-copy house-keeping gene rpsA gene could work well as a target gene for

Mycobacterium species discrimination without ambiguousidentification The 16S rDNA gene has higher homologywithin the mycobacteria compared to the rpsA gene 943to 100 compared to 854 to 100 According to our workwhen used as sole marker rpsA had better resolution than 16srDNA but had similar resolution as ITS rpoB and hsp65

Due to the high resolution power of rpsA for speciesidentification we presume it has potential of clinical useFrom our own experience we recommend that when per-forming species identification by homologue DNA sequencecomparison one should start with 16s RNA gene plus atleast one of the other markers such as ITS rpoB hsp65and rpsA When conflicting outcome or dubious outcomesyield more markers should be added further Even though16s RNA gene is inferior to the other markers considering thecapacity it should be chosen firstly since 16s RNA gene has

8 BioMed Research International

the most robust sequence database which can help to avoidbig identification errors due to unreliable database of othermarkers [18]

Themain deterrents for the primary use of rpsA sequenc-ing as a routine means of identifying mycobacteria residein the need for a comprehensive database Therefore weconstructed our own rpsA sequence database by including asmany type strains as possible and integrating rpsA sequencesdeposited in GenBank such as M avium subspecies paratu-berculosis M ulcerans M vanbaalenii M abscessus sub-speciesmassiliense andM canettii Besides type strains rpsAsequences of confirmed clinical strains were also includedWe will constantly upgrade our database and the capabilityto identify the most recent described species

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HongfeiDuan andGuanLiu contributed equally to this study

Acknowledgments

The work was supported by the research funding fromInfectious Diseases Special Project Ministry of Health ofChina (2012ZX10003002-09) and Beijing Municipal Admin-istration of Hospitals Clinical Medicine Development ofSpecial Funding (ZYLX201304) All the type strains andclinical isolates used in this study were obtained from theldquoBeijing Bio-Bank of Clinical Resources on Tuberculosis(D09050704640000)rdquo Beijing Chest Hospital

References

[1] T Adekambi P Berger D Raoult and M DrancourtldquorpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacteriumbolletii sp nov Mycobacterium phocaicum sp nov andMycobacterium aubagnense sp novrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 1 ArticleID 63969 pp 133ndash143 2006

[2] B Ninet M Monod S Emler et al ldquoTwo different 16SrRNA genes in a mycobacterial strainrdquo Journal of ClinicalMicrobiology vol 34 no 10 pp 2531ndash2536 1996

[3] U Reischl K Feldmann L Naumann et al ldquo16S rRNAsequence diversity inMycobacterium celatum strains caused bypresence of two different copies of 16S rRNA generdquo Journal ofClinical Microbiology vol 36 no 6 pp 1761ndash1764 1998

[4] D E Griffith T Aksamit B A Brown-Elliott et al ldquoAn officialATSIDSA statement diagnosis treatment and prevention ofnontuberculous mycobacterial diseasesrdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 4 pp 367ndash416 2007

[5] W Shi X Zhang X Jiang et al ldquoPyrazinamide inhibits trans-translation inMycobacterium tuberculosisrdquo Science vol 333 no6049 pp 1630ndash1632 2011

[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998

[7] A Roth M Fischer M E Hamid S Michalke W Ludwig andH Mauch ldquoDifferentiation of phylogenetically related slowlygrowing mycobacteria based on 16S-23S rRNA gene internaltranscribed spacer sequencesrdquo Journal of Clinical Microbiologyvol 36 no 1 pp 139ndash147 1998

[8] H Ringuet A Honore A Varnerot et al ldquohsp65 sequencingfor identification of rapidly growing mycobacteriardquo Journal ofClinical Microbiology vol 37 no 3 pp 852ndash857 1999

[9] B-J Kim S-H Lee M-A Lyu et al ldquoIdentification ofmycobacterial species by comparative sequence analysis of theRNA polymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 37 no 6 pp 1714ndash1720 1999

[10] J Amita T Vandana R S Guleria and R K Verma ldquoQualita-tive evaluation of mycobacterial DNA extraction protocols forpolymerase chain reactionrdquoMolecular Biology Today vol 3 no2 pp 43ndash49 2002

[11] R J Pauls C Y Turenne J N Wolfe and A Kabani ldquoAhigh proportion of novel mycobacteria species identified by16S rDNA analysis among slowly growing AccuProbe-negativestrains in a clinical settingrdquo American Journal of ClinicalPathology vol 120 no 4 pp 560ndash566 2003

[12] G E Fox J D Wisotzkey and P Jurtshuk Jr ldquoHow close isclose 16S rRNA sequence identity may not be sufficient toguarantee species identityrdquo International Journal of SystematicBacteriology vol 42 no 1 pp 166ndash170 1992

[13] M Drancourt P Berger and D Raoult ldquoSystematic 16S rRNAgene sequencing of atypical clinical isolates identified 27 newbacterial species associated with humansrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2197ndash2202 2004

[14] P Kirschner E C Bottger D T Walton and M ValescoldquoMicroheterogeneity within rRNA of Mycobacterium gor-donaerdquo Journal of Clinical Microbiology vol 30 no 4 pp 1049ndash1050 1992

[15] C Taillard G Greub R Weber et al ldquoClinical implicationsofMycobacterium kansasii species heterogeneity swiss nationalsurveyrdquo Journal of Clinical Microbiology vol 41 no 3 pp 1240ndash1244 2003

[16] E Macheras A-L Roux F Ripoll et al ldquoInaccuracy of single-target sequencing for discriminating species of the Mycobac-terium abscessus grouprdquo Journal of Clinical Microbiology vol 47no 8 pp 2596ndash2600 2009

[17] L A Almeida and R Araujo ldquoHighlights on molecular iden-tification of closely related speciesrdquo Infection Genetics andEvolution vol 13 no 1 pp 67ndash75 2013

[18] J Dai Y Chen and M Lauzardo ldquoWeb-accessible databaseof hsp65 sequences from Mycobacterium reference strainsrdquoJournal of Clinical Microbiology vol 49 no 6 pp 2296ndash23032011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5Ta

ble2Sequ

ence

pairdistanceso

f47type

strainso

fmycob

acteria

and1o

ut-group

strain

determ

ined

byusingthemultip

lealignm

enta

lgorith

min

theMegAlignpackage(versio

n710

DNAST

AR

Madiso

nWI)

Percentidentity

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

2930

3132

3334

3536

3738

3940

4142

4344

4546

4748

1mdash

1000

987

981

960

953

954

964

962

945

945

951

945

945

920

939

941

943

945

945

928

956

937

947

932

924

888

892

911

911

911

911

911

913

915

939

941

913

930

928

926

926

924

934

934

924

928

892

1Mtherm

oresistibile

200

mdash987

981

960

953

954

964

962

945

945

951

945

945

920

939

941

943

945

945

928

956

937

947

932

924

888

892

911

911

911

911

911

913

915

939

941

913

930

928

926

926

924

934

934

924

928

892

2Msenegalense

313

13mdash

983

966

958

958

968

968

947

947

956

954

954

920

941

954

949

951

951

930

962

947

956

945

930

894

898

911

911

911

911

911

913

920

945

947

920

934

935

932

932

928

937

937

930

928

894

3Mp

orcin

um

419

1917

mdash962

958

958

962

962

941

941

954

945

945

920

943

945

943

945

945

939

962

949

949

937

937

901

905

909

909

909

909

909

911

920

947

949

913

932

932

930

934

928

935

937

928

930

892

4Mfortuitu

m

541

41

35

39

mdash943

954

964

960

939

939

934

943

943

915

943

943

945

947

947

915

943

941

949

928

911

898

903

901

901

901

901

901

903

911

935

937

913

934

930

935

935

930

937

934

930

920

896

5Mp

hlei

649

49

43

43

59

mdash951

953

941

937

937

947

941

941

913

937

935

934

935

935

932

937

937

937

930

924

886

890

907

907

907

907

907

909

915

943

941

907

932

920

918

922

915

930

932

918

915

880

6Mg

adium

747

47

43

43

47

51

mdash966

960

954

954

947

947

947

930

951

953

956

958

958

932

941

949

947

924

920

901

905

924

924

924

924

924

926

928

949

947

930

941

949

937

937

935

939

943

926

934

903

7Mfl

avescens

837

37

33

39

37

49

35

mdash966

966

966

953

962

962

920

962

962

964

962

962

935

947

945

945

939

915

905

909

911

911

911

911

911

913

922

937

935

920

943

935

932

932

930

934

935

924

922

909

8Msmegmatis

939

39

33

39

41

61

41

35

mdash966

966

951

968

968

924

958

956

962

964

964

930

956

949

947

924

920

915

918

909

909

909

909

909

911

922

943

945

924

939

928

934

934

932

939

935

928

928

892

9Mv

accae

1057

57

55

61

63

65

47

35

35

mdash1000

949

962

962

930

949

958

964

966

966

930

943

928

930

924

917

898

901

899

899

899

899

899

901

915

926

924

913

932

920

911

911

913

918

915

903

917

890

10Mp

arafortuitu

m

1157

57

55

61

63

65

47

35

35

00

mdash949

962

962

930

949

958

964

966

966

930

943

928

930

924

917

898

901

899

899

899

899

899

901

915

926

924

913

932

920

911

911

913

918

915

903

917

890

11Mtriv

iale

1251

51

45

47

7055

55

49

51

53

53

mdash964

964

918

945

953

951

953

953

941

951

928

922

926

930

903

907

899

899

899

899

899

901

913

932

928

899

920

920

917

917

911

918

924

909

899

882

12Mn

eoaurum

1357

57

47

57

59

61

55

39

33

39

39

37

mdash1000

926

953

958

956

958

958

941

949

941

937

928

922

913

917

894

894

894

894

894

896

907

920

918

911

930

918

915

915

913

917

918

913

905

898

13Md

iernh

oferi

1457

57

47

57

59

61

55

39

33

39

39

37

00

mdash926

953

958

956

958

958

941

949

941

937

928

922

913

917

894

894

894

894

894

896

907

920

918

911

930

918

915

915

913

917

918

913

905

898

14Md

uvalii

1584

84

84

84

9193

7485

80

7474

86

7878

mdash930

939

930

932

932

917

918

911

911

894

911

898

901

892

892

892

892

892

894

888

901

905

915

905

905

896

898

898

892

901

896

890

877

15Ma

urum

1663

63

61

59

59

65

51

39

43

53

53

57

49

49

74mdash

966

960

958

958

939

947

947

935

928

924

918

924

905

905

905

905

905

907

917

932

930

911

926

924

926

930

913

924

935

915

909

884

16Mc

hubu

ense

1761

61

47

57

59

68

49

39

45

43

43

49

43

43

63

35

mdash972

970

970

941

956

945

941

935

934

913

917

903

903

903

903

903

905

913

930

932

920

928

932

926

930

917

928

928

915

911

901

17Mg

ilvum

1859

59

53

59

57

7045

37

39

37

37

51

45

45

7441

29

mdash998

998

941

949

928

934

926

930

907

911

901

901

901

901

901

903

913

928

926

913

924

928

920

920

913

920

924

913

920

892

18Mv

anbaalenii

1957

57

51

57

55

68

43

39

37

35

35

49

43

43

7243

31

02

mdash1000

939

951

930

935

928

932

909

913

903

903

903

903

903

905

915

930

928

915

926

930

922

922

915

922

926

915

922

890

19Mterrae

2057

57

51

57

55

68

43

39

37

35

35

49

43

43

7243

31

02

00

mdash939

951

930

935

928

932

909

913

903

903

903

903

903

905

915

930

928

915

926

930

922

922

915

922

926

915

922

890

20Ma

ustro

afric

anum

2176

7674

63

9172

7267

7474

7461

61

61

89

63

61

61

63

63

mdash937

918

917

911

926

888

892

894

894

894

894

894

896

903

915

917

901

909

909

899

901

896

907

913

909

899

896

21Mrhodesia

e

2245

45

39

39

59

66

61

55

45

59

59

51

53

53

87

55

45

53

51

51

65

mdash937

945

934

941

903

907

903

903

903

903

903

905

905

937

939

903

926

928

922

922

918

926

930

917

909

892

22Mo

buense

2365

65

55

53

61

65

53

57

53

7676

7661

61

9555

57

7674

7486

65

mdash958

939

930

917

920

909

909

909

909

909

911

922

937

939

917

932

926

934

937

922

937

939

924

917

890

23Ma

ichien

se

2455

55

45

53

53

65

55

57

55

7474

82

65

65

9567

61

7067

67

89

57

43

mdash937

934

913

918

915

915

915

915

915

917

915

937

941

922

930

935

928

930

930

934

941

932

924

892

24Msphagni

2572

7257

65

7674

80

63

80

80

80

7876

76115

7667

7876

7695

7063

65

mdash920

888

894

896

896

896

896

896

898

905

945

945

907

930

928

918

922

920

924

934

922

915

882

25Mtokaiense

2680

80

7465

9580

84

9184

89

89

7482

82

9580

7074

7272

7861

7470

84

mdash915

918

896

896

896

896

896

898

898

922

926

894

905

911

907

911

898

920

917

907

903

871

26Mc

helona

e

27121

121

115

106

110

124

106

101

91

110

110

104

9393

110

86

9399

9797

121

104

89

93121

91mdash

987

867

867

867

867

867

869

884

884

888

884

886

894

896

896

884

901

898

882

871

871

27Ma

bscessu

s

28117

117

110

101

104

119

101

9786

106

106

9989

89

106

80

89

9593

93117

9984

86

115

87

13mdash

875

875

875

875

875

877

888

888

892

888

890

898

899

899

890

905

901

886

875

877

28Ma

bscessu

s

2995

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

mdash1000

1000

1000

1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

29Mtub

erculosis

3095

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

mdash1000

1000

1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

30Mtub

erculosis

3195

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

00

mdash1000

1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

31Mb

ovis

3295

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

00

00

mdash1000

998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

32Ma

fricanu

m

3395

9595

97106

100

80

9597

108

108

108

115

115

117

101

104

106

104

104

115

104

9791

113

113

146

137

00

00

00

00

mdash998

909

917

920

909

917

922

920

918

920

928

934

922

918

880

33Mb

ovis

3493

9393

95104

9778

9395

106

106

106

112

112

115

99102

104

102

102

112

102

9589

111

110

144

135

02

02

02

02

02

mdash911

918

922

907

918

924

918

917

918

926

932

920

917

880

34Mc

anettii

3591

9184

84

9591

7682

82

9191

9399

99122

89

9393

9191

104

102

82

91102

110

126

121

9898

9898

9895

mdash922

924

907

917

920

911

911

905

918

930

915

909

877

35Mx

enopi

3663

63

57

55

67

59

53

65

59

7878

7284

84

106

7274

7674

7491

65

65

65

57

82

126

121

89

89

89

89

89

87

82

mdash994

930

956

947

939

943

941

951

958

947

937

886

36Ma

vium

3761

61

55

53

65

61

55

67

57

80

80

7686

86

101

7472

7876

7689

63

63

61

57

78121

117

84

84

84

84

84

82

80

06

mdash935

954

951

945

949

945

954

960

953

939

888

37Ma

vium

3893

9384

9393

9974

84

80

9393

108

9595

9195

84

9391

91106

104

89

82

99115

126

121

9797

9797

97100

9974

67

mdash943

937

935

934

924

939

930

937

930

890

38Mm

almoense

3974

7470

7270

7261

59

63

7272

84

7474

102

7876

80

7878

9778

7274

74102

123

119

89

89

89

89

89

87

89

45

47

59

mdash945

941

941

934

951

947

943

943

907

39Mintracellu

lare

4076

7667

7274

84

53

67

7684

84

84

86

86

101

80

7276

7474

9776

7867

7695

115

110

82

82

82

82

82

80

84

55

51

65

57

mdash943

943

949

951

954

939

934

896

40Mscrofulaceum

4178

7872

7468

86

65

7270

9595

89

9191

112

7878

84

82

82

108

82

7076

87

99112

108

84

84

84

84

84

87

9563

57

68

61

59

mdash996

945

960

956

956

937

896

41Mm

arinum

4278

7872

7068

82

65

7270

9595

89

9191

110

7474

84

82

82

106

82

65

7482

95112

108

87

87

87

87

87

89

9559

53

7061

59

04

mdash943

960

956

954

939

894

42Mu

lcerans

4380

80

7676

7491

67

7472

9393

9593

93110

9389

9391

91112

86

82

7484

110

126

119

84

84

84

84

84

87

101

61

57

80

7053

57

59

mdash943

953

951

934

890

43Mg

ordona

e

4470

7065

67

65

7463

7063

86

86

86

89

89

117

80

7684

82

82

9978

65

7080

84

106

101

7676

7676

7678

86

51

47

63

51

51

41

41

59

mdash956

954

945

901

44Mszulga

i

4570

7065

65

7072

59

67

67

9191

80

86

86

106

67

7680

7878

9374

63

61

7089

110

106

7070

7070

7072

7443

41

7455

47

45

45

49

45

mdash964

947

888

45Ma

siaticum

4680

80

7476

7486

7880

76104

104

9793

93112

9191

9391

9197

89

80

7282

99128

123

82

82

82

82

82

85

9155

49

65

59

63

45

47

51

47

37

mdash958

888

46Mk

ansasii

4776

7676

7484

9170

82

7689

89

108

101

101

119

9795

84

82

82

108

9789

80

91104

142

137

87

87

87

87

87

89

9765

63

7459

7066

63

7057

55

43

mdash879

47Mg

astri

48117

117

115

117

112

130

104

97117

119

119

128

110

110

135

126

106

117

119

119

112

117

119

117

128

142

142

135

130

130

130

130

130

130

135

124

121

119

99112

112

115

119

106

121

121

132

mdash48

Rhodococcusequi

12

34

56

78

910

1112

1314

1516

1718

1920

2122

2324

2526

2728

2930

3132

3334

3536

3738

3940

4142

4344

4546

4748

6 BioMed Research International

001

99

78

93

37

11

2

10

0

1

1

1

19

99

9 99

99

99

99

99

76

52

5

17

35

67

23

23

60

57

10

32

38

32

23

99

9

66

13

16

13

24

55

M thermoresistibileM senegalense

M porcinumM fortuitum

M phleiM gadium

M flavescensM smegmatisM vaccae

M parafortuitumM triviale

M neoaurumM diernhoferiM duvalii

M aurumM chubuense

M gilvumM vanbaalenii

M terraeM austroafricanum

M rhodesiaeM obuenseM aichienseM sphagni

M tokaienseM chelonae

M abscessusM abscessus subspecies massiliense

M tuberculosis H37RvM tuberculosis H37Ra

M bovisM bovis BCG

M canettiiM xenopi

M aviumM avium subspecies paratuberculosis

M malmoenseM intracellulareM scrofulaceum

M marinumM ulcerans

M szulgaiM gordonae

M asiaticumM kansassii

M gastri

M africanum

Rhodococcus equi

Figure 1 Phylogenetic tree based on rpsA gene sequences shows the relationship of the 47 type strains of mycobacteria and 1 outgroup strainThis tree was constructed by the neighbor-joiningmethod Topology was also evaluated by bootstrap analysis (MEGA program 1000 repeatswith R equi as the outgroup)The numerical values in the tree represent bootstrap results The distance between two strains is the sum of thebranch lengths between them

BioMed Research International 7

Table 3 Comparison of mycobacteria identified by 16S rDNAITShsp65rpoB gene sequencing and by rpsA sequencing among the 188clinical isolatesa

Primary identificationb (119899)Primary identificationb (119899)

First choice Second choiceIdentification (119899) match Identification (119899) match

M intracellulare (75)

M intracellulare (4) 100 M avium (4) 956M intracellulare (1) 998 M avium (1) 953M intracellulare (53) 989 M avium (53) 951M intracellulare (10) 987 M avium (10) 949M intracellulare (1) 985 M avium (1) 953M intracellulare (3) 981 M avium (3) 947M intracellulare (1) 975 M avium (1) 932M intracellulare (2) 971 M avium (2) 961

M abscessus (39)M abscessus (9) 100 M abscessus subspecies massiliense (9) 987M abscessus (10) 998 M abscessus subspecies massiliense (10) 985M abscessus subspecies massiliense (20) 994 M abscessus (20) 991

M kansasii (23) M kansasii (22) 100 M asiaticum (22) 963M szulgai (1) 966 M marinumM ulcerans (1) 960

M avium (10)M avium subspecies paratuberculosis (5) 996 M avium (5) 994M avium subspecies paratuberculosis (4) 998 M avium (4) 992M avium subspecies paratuberculosis (1) 975 M avium (1) 970

M fortuitum complex (17)

M fortuitum (2) 100 M senegalenseM thermoresistibile (2) 981M fortuitum (11) 998 M porcinum (11) 985M fortuitum (1) 996 M porcinum (1) 983M fortuitumM porcinum (1) 989 M senegalenseM thermoresistibile (1) 983M thermoresistibileM senegalense (2) 996 M fortuitum (2) 981

M gordonae (7)

M gordonae (1) 992 M asiaticum (1) 955M gordonae (2) 985 M asiaticum (2) 963M gordonae (3) 959 M asiaticum (3) 955M szulgai (1) 959 M gordonae (1) 947

M tuberculosis complex (16)M tuberculosis (2) 100 M canetti (2) 998M tuberculosis (12) 998 M canetti (12) 996M tuberculosis (2) 996 M canetti (2) 994

M neoaurum (1) M neoaurum (1) 987 M smegmatis (1) 954aNumbers in parentheses (119899) represent the numbers of isolates identified as a particular speciesbIdentification based on sequencing of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoB and hsp65 genes

reference strains had similar homology with two or morespecies which suggests inadequate taxonomy within thecurrently described species Based on nucleotide sequencesof rpoB hsp65 and sodA in clinical isolates of the Mycobac-terium abscessus group one-fourth of isolates had discordantidentification [16] Multilocus sequence typing and sequenceanalysis of several genes facilitate the identification of closelyrelated species or subspecies [17]

In our study we have demonstrated that the rpsA homol-ogy gene is a promising marker for mycobacterial speciesidentification for both fast- and slow-growing mycobacteriaTo be a good marker for species differentiation the targetgene should be stable and sequence variations should occurrandomly additionally an extremely conserved or highlyvariable gene may not be adequate As a single-copy house-keeping gene rpsA gene could work well as a target gene for

Mycobacterium species discrimination without ambiguousidentification The 16S rDNA gene has higher homologywithin the mycobacteria compared to the rpsA gene 943to 100 compared to 854 to 100 According to our workwhen used as sole marker rpsA had better resolution than 16srDNA but had similar resolution as ITS rpoB and hsp65

Due to the high resolution power of rpsA for speciesidentification we presume it has potential of clinical useFrom our own experience we recommend that when per-forming species identification by homologue DNA sequencecomparison one should start with 16s RNA gene plus atleast one of the other markers such as ITS rpoB hsp65and rpsA When conflicting outcome or dubious outcomesyield more markers should be added further Even though16s RNA gene is inferior to the other markers considering thecapacity it should be chosen firstly since 16s RNA gene has

8 BioMed Research International

the most robust sequence database which can help to avoidbig identification errors due to unreliable database of othermarkers [18]

Themain deterrents for the primary use of rpsA sequenc-ing as a routine means of identifying mycobacteria residein the need for a comprehensive database Therefore weconstructed our own rpsA sequence database by including asmany type strains as possible and integrating rpsA sequencesdeposited in GenBank such as M avium subspecies paratu-berculosis M ulcerans M vanbaalenii M abscessus sub-speciesmassiliense andM canettii Besides type strains rpsAsequences of confirmed clinical strains were also includedWe will constantly upgrade our database and the capabilityto identify the most recent described species

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HongfeiDuan andGuanLiu contributed equally to this study

Acknowledgments

The work was supported by the research funding fromInfectious Diseases Special Project Ministry of Health ofChina (2012ZX10003002-09) and Beijing Municipal Admin-istration of Hospitals Clinical Medicine Development ofSpecial Funding (ZYLX201304) All the type strains andclinical isolates used in this study were obtained from theldquoBeijing Bio-Bank of Clinical Resources on Tuberculosis(D09050704640000)rdquo Beijing Chest Hospital

References

[1] T Adekambi P Berger D Raoult and M DrancourtldquorpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacteriumbolletii sp nov Mycobacterium phocaicum sp nov andMycobacterium aubagnense sp novrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 1 ArticleID 63969 pp 133ndash143 2006

[2] B Ninet M Monod S Emler et al ldquoTwo different 16SrRNA genes in a mycobacterial strainrdquo Journal of ClinicalMicrobiology vol 34 no 10 pp 2531ndash2536 1996

[3] U Reischl K Feldmann L Naumann et al ldquo16S rRNAsequence diversity inMycobacterium celatum strains caused bypresence of two different copies of 16S rRNA generdquo Journal ofClinical Microbiology vol 36 no 6 pp 1761ndash1764 1998

[4] D E Griffith T Aksamit B A Brown-Elliott et al ldquoAn officialATSIDSA statement diagnosis treatment and prevention ofnontuberculous mycobacterial diseasesrdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 4 pp 367ndash416 2007

[5] W Shi X Zhang X Jiang et al ldquoPyrazinamide inhibits trans-translation inMycobacterium tuberculosisrdquo Science vol 333 no6049 pp 1630ndash1632 2011

[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998

[7] A Roth M Fischer M E Hamid S Michalke W Ludwig andH Mauch ldquoDifferentiation of phylogenetically related slowlygrowing mycobacteria based on 16S-23S rRNA gene internaltranscribed spacer sequencesrdquo Journal of Clinical Microbiologyvol 36 no 1 pp 139ndash147 1998

[8] H Ringuet A Honore A Varnerot et al ldquohsp65 sequencingfor identification of rapidly growing mycobacteriardquo Journal ofClinical Microbiology vol 37 no 3 pp 852ndash857 1999

[9] B-J Kim S-H Lee M-A Lyu et al ldquoIdentification ofmycobacterial species by comparative sequence analysis of theRNA polymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 37 no 6 pp 1714ndash1720 1999

[10] J Amita T Vandana R S Guleria and R K Verma ldquoQualita-tive evaluation of mycobacterial DNA extraction protocols forpolymerase chain reactionrdquoMolecular Biology Today vol 3 no2 pp 43ndash49 2002

[11] R J Pauls C Y Turenne J N Wolfe and A Kabani ldquoAhigh proportion of novel mycobacteria species identified by16S rDNA analysis among slowly growing AccuProbe-negativestrains in a clinical settingrdquo American Journal of ClinicalPathology vol 120 no 4 pp 560ndash566 2003

[12] G E Fox J D Wisotzkey and P Jurtshuk Jr ldquoHow close isclose 16S rRNA sequence identity may not be sufficient toguarantee species identityrdquo International Journal of SystematicBacteriology vol 42 no 1 pp 166ndash170 1992

[13] M Drancourt P Berger and D Raoult ldquoSystematic 16S rRNAgene sequencing of atypical clinical isolates identified 27 newbacterial species associated with humansrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2197ndash2202 2004

[14] P Kirschner E C Bottger D T Walton and M ValescoldquoMicroheterogeneity within rRNA of Mycobacterium gor-donaerdquo Journal of Clinical Microbiology vol 30 no 4 pp 1049ndash1050 1992

[15] C Taillard G Greub R Weber et al ldquoClinical implicationsofMycobacterium kansasii species heterogeneity swiss nationalsurveyrdquo Journal of Clinical Microbiology vol 41 no 3 pp 1240ndash1244 2003

[16] E Macheras A-L Roux F Ripoll et al ldquoInaccuracy of single-target sequencing for discriminating species of the Mycobac-terium abscessus grouprdquo Journal of Clinical Microbiology vol 47no 8 pp 2596ndash2600 2009

[17] L A Almeida and R Araujo ldquoHighlights on molecular iden-tification of closely related speciesrdquo Infection Genetics andEvolution vol 13 no 1 pp 67ndash75 2013

[18] J Dai Y Chen and M Lauzardo ldquoWeb-accessible databaseof hsp65 sequences from Mycobacterium reference strainsrdquoJournal of Clinical Microbiology vol 49 no 6 pp 2296ndash23032011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 BioMed Research International

001

99

78

93

37

11

2

10

0

1

1

1

19

99

9 99

99

99

99

99

76

52

5

17

35

67

23

23

60

57

10

32

38

32

23

99

9

66

13

16

13

24

55

M thermoresistibileM senegalense

M porcinumM fortuitum

M phleiM gadium

M flavescensM smegmatisM vaccae

M parafortuitumM triviale

M neoaurumM diernhoferiM duvalii

M aurumM chubuense

M gilvumM vanbaalenii

M terraeM austroafricanum

M rhodesiaeM obuenseM aichienseM sphagni

M tokaienseM chelonae

M abscessusM abscessus subspecies massiliense

M tuberculosis H37RvM tuberculosis H37Ra

M bovisM bovis BCG

M canettiiM xenopi

M aviumM avium subspecies paratuberculosis

M malmoenseM intracellulareM scrofulaceum

M marinumM ulcerans

M szulgaiM gordonae

M asiaticumM kansassii

M gastri

M africanum

Rhodococcus equi

Figure 1 Phylogenetic tree based on rpsA gene sequences shows the relationship of the 47 type strains of mycobacteria and 1 outgroup strainThis tree was constructed by the neighbor-joiningmethod Topology was also evaluated by bootstrap analysis (MEGA program 1000 repeatswith R equi as the outgroup)The numerical values in the tree represent bootstrap results The distance between two strains is the sum of thebranch lengths between them

BioMed Research International 7

Table 3 Comparison of mycobacteria identified by 16S rDNAITShsp65rpoB gene sequencing and by rpsA sequencing among the 188clinical isolatesa

Primary identificationb (119899)Primary identificationb (119899)

First choice Second choiceIdentification (119899) match Identification (119899) match

M intracellulare (75)

M intracellulare (4) 100 M avium (4) 956M intracellulare (1) 998 M avium (1) 953M intracellulare (53) 989 M avium (53) 951M intracellulare (10) 987 M avium (10) 949M intracellulare (1) 985 M avium (1) 953M intracellulare (3) 981 M avium (3) 947M intracellulare (1) 975 M avium (1) 932M intracellulare (2) 971 M avium (2) 961

M abscessus (39)M abscessus (9) 100 M abscessus subspecies massiliense (9) 987M abscessus (10) 998 M abscessus subspecies massiliense (10) 985M abscessus subspecies massiliense (20) 994 M abscessus (20) 991

M kansasii (23) M kansasii (22) 100 M asiaticum (22) 963M szulgai (1) 966 M marinumM ulcerans (1) 960

M avium (10)M avium subspecies paratuberculosis (5) 996 M avium (5) 994M avium subspecies paratuberculosis (4) 998 M avium (4) 992M avium subspecies paratuberculosis (1) 975 M avium (1) 970

M fortuitum complex (17)

M fortuitum (2) 100 M senegalenseM thermoresistibile (2) 981M fortuitum (11) 998 M porcinum (11) 985M fortuitum (1) 996 M porcinum (1) 983M fortuitumM porcinum (1) 989 M senegalenseM thermoresistibile (1) 983M thermoresistibileM senegalense (2) 996 M fortuitum (2) 981

M gordonae (7)

M gordonae (1) 992 M asiaticum (1) 955M gordonae (2) 985 M asiaticum (2) 963M gordonae (3) 959 M asiaticum (3) 955M szulgai (1) 959 M gordonae (1) 947

M tuberculosis complex (16)M tuberculosis (2) 100 M canetti (2) 998M tuberculosis (12) 998 M canetti (12) 996M tuberculosis (2) 996 M canetti (2) 994

M neoaurum (1) M neoaurum (1) 987 M smegmatis (1) 954aNumbers in parentheses (119899) represent the numbers of isolates identified as a particular speciesbIdentification based on sequencing of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoB and hsp65 genes

reference strains had similar homology with two or morespecies which suggests inadequate taxonomy within thecurrently described species Based on nucleotide sequencesof rpoB hsp65 and sodA in clinical isolates of the Mycobac-terium abscessus group one-fourth of isolates had discordantidentification [16] Multilocus sequence typing and sequenceanalysis of several genes facilitate the identification of closelyrelated species or subspecies [17]

In our study we have demonstrated that the rpsA homol-ogy gene is a promising marker for mycobacterial speciesidentification for both fast- and slow-growing mycobacteriaTo be a good marker for species differentiation the targetgene should be stable and sequence variations should occurrandomly additionally an extremely conserved or highlyvariable gene may not be adequate As a single-copy house-keeping gene rpsA gene could work well as a target gene for

Mycobacterium species discrimination without ambiguousidentification The 16S rDNA gene has higher homologywithin the mycobacteria compared to the rpsA gene 943to 100 compared to 854 to 100 According to our workwhen used as sole marker rpsA had better resolution than 16srDNA but had similar resolution as ITS rpoB and hsp65

Due to the high resolution power of rpsA for speciesidentification we presume it has potential of clinical useFrom our own experience we recommend that when per-forming species identification by homologue DNA sequencecomparison one should start with 16s RNA gene plus atleast one of the other markers such as ITS rpoB hsp65and rpsA When conflicting outcome or dubious outcomesyield more markers should be added further Even though16s RNA gene is inferior to the other markers considering thecapacity it should be chosen firstly since 16s RNA gene has

8 BioMed Research International

the most robust sequence database which can help to avoidbig identification errors due to unreliable database of othermarkers [18]

Themain deterrents for the primary use of rpsA sequenc-ing as a routine means of identifying mycobacteria residein the need for a comprehensive database Therefore weconstructed our own rpsA sequence database by including asmany type strains as possible and integrating rpsA sequencesdeposited in GenBank such as M avium subspecies paratu-berculosis M ulcerans M vanbaalenii M abscessus sub-speciesmassiliense andM canettii Besides type strains rpsAsequences of confirmed clinical strains were also includedWe will constantly upgrade our database and the capabilityto identify the most recent described species

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HongfeiDuan andGuanLiu contributed equally to this study

Acknowledgments

The work was supported by the research funding fromInfectious Diseases Special Project Ministry of Health ofChina (2012ZX10003002-09) and Beijing Municipal Admin-istration of Hospitals Clinical Medicine Development ofSpecial Funding (ZYLX201304) All the type strains andclinical isolates used in this study were obtained from theldquoBeijing Bio-Bank of Clinical Resources on Tuberculosis(D09050704640000)rdquo Beijing Chest Hospital

References

[1] T Adekambi P Berger D Raoult and M DrancourtldquorpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacteriumbolletii sp nov Mycobacterium phocaicum sp nov andMycobacterium aubagnense sp novrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 1 ArticleID 63969 pp 133ndash143 2006

[2] B Ninet M Monod S Emler et al ldquoTwo different 16SrRNA genes in a mycobacterial strainrdquo Journal of ClinicalMicrobiology vol 34 no 10 pp 2531ndash2536 1996

[3] U Reischl K Feldmann L Naumann et al ldquo16S rRNAsequence diversity inMycobacterium celatum strains caused bypresence of two different copies of 16S rRNA generdquo Journal ofClinical Microbiology vol 36 no 6 pp 1761ndash1764 1998

[4] D E Griffith T Aksamit B A Brown-Elliott et al ldquoAn officialATSIDSA statement diagnosis treatment and prevention ofnontuberculous mycobacterial diseasesrdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 4 pp 367ndash416 2007

[5] W Shi X Zhang X Jiang et al ldquoPyrazinamide inhibits trans-translation inMycobacterium tuberculosisrdquo Science vol 333 no6049 pp 1630ndash1632 2011

[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998

[7] A Roth M Fischer M E Hamid S Michalke W Ludwig andH Mauch ldquoDifferentiation of phylogenetically related slowlygrowing mycobacteria based on 16S-23S rRNA gene internaltranscribed spacer sequencesrdquo Journal of Clinical Microbiologyvol 36 no 1 pp 139ndash147 1998

[8] H Ringuet A Honore A Varnerot et al ldquohsp65 sequencingfor identification of rapidly growing mycobacteriardquo Journal ofClinical Microbiology vol 37 no 3 pp 852ndash857 1999

[9] B-J Kim S-H Lee M-A Lyu et al ldquoIdentification ofmycobacterial species by comparative sequence analysis of theRNA polymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 37 no 6 pp 1714ndash1720 1999

[10] J Amita T Vandana R S Guleria and R K Verma ldquoQualita-tive evaluation of mycobacterial DNA extraction protocols forpolymerase chain reactionrdquoMolecular Biology Today vol 3 no2 pp 43ndash49 2002

[11] R J Pauls C Y Turenne J N Wolfe and A Kabani ldquoAhigh proportion of novel mycobacteria species identified by16S rDNA analysis among slowly growing AccuProbe-negativestrains in a clinical settingrdquo American Journal of ClinicalPathology vol 120 no 4 pp 560ndash566 2003

[12] G E Fox J D Wisotzkey and P Jurtshuk Jr ldquoHow close isclose 16S rRNA sequence identity may not be sufficient toguarantee species identityrdquo International Journal of SystematicBacteriology vol 42 no 1 pp 166ndash170 1992

[13] M Drancourt P Berger and D Raoult ldquoSystematic 16S rRNAgene sequencing of atypical clinical isolates identified 27 newbacterial species associated with humansrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2197ndash2202 2004

[14] P Kirschner E C Bottger D T Walton and M ValescoldquoMicroheterogeneity within rRNA of Mycobacterium gor-donaerdquo Journal of Clinical Microbiology vol 30 no 4 pp 1049ndash1050 1992

[15] C Taillard G Greub R Weber et al ldquoClinical implicationsofMycobacterium kansasii species heterogeneity swiss nationalsurveyrdquo Journal of Clinical Microbiology vol 41 no 3 pp 1240ndash1244 2003

[16] E Macheras A-L Roux F Ripoll et al ldquoInaccuracy of single-target sequencing for discriminating species of the Mycobac-terium abscessus grouprdquo Journal of Clinical Microbiology vol 47no 8 pp 2596ndash2600 2009

[17] L A Almeida and R Araujo ldquoHighlights on molecular iden-tification of closely related speciesrdquo Infection Genetics andEvolution vol 13 no 1 pp 67ndash75 2013

[18] J Dai Y Chen and M Lauzardo ldquoWeb-accessible databaseof hsp65 sequences from Mycobacterium reference strainsrdquoJournal of Clinical Microbiology vol 49 no 6 pp 2296ndash23032011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

Table 3 Comparison of mycobacteria identified by 16S rDNAITShsp65rpoB gene sequencing and by rpsA sequencing among the 188clinical isolatesa

Primary identificationb (119899)Primary identificationb (119899)

First choice Second choiceIdentification (119899) match Identification (119899) match

M intracellulare (75)

M intracellulare (4) 100 M avium (4) 956M intracellulare (1) 998 M avium (1) 953M intracellulare (53) 989 M avium (53) 951M intracellulare (10) 987 M avium (10) 949M intracellulare (1) 985 M avium (1) 953M intracellulare (3) 981 M avium (3) 947M intracellulare (1) 975 M avium (1) 932M intracellulare (2) 971 M avium (2) 961

M abscessus (39)M abscessus (9) 100 M abscessus subspecies massiliense (9) 987M abscessus (10) 998 M abscessus subspecies massiliense (10) 985M abscessus subspecies massiliense (20) 994 M abscessus (20) 991

M kansasii (23) M kansasii (22) 100 M asiaticum (22) 963M szulgai (1) 966 M marinumM ulcerans (1) 960

M avium (10)M avium subspecies paratuberculosis (5) 996 M avium (5) 994M avium subspecies paratuberculosis (4) 998 M avium (4) 992M avium subspecies paratuberculosis (1) 975 M avium (1) 970

M fortuitum complex (17)

M fortuitum (2) 100 M senegalenseM thermoresistibile (2) 981M fortuitum (11) 998 M porcinum (11) 985M fortuitum (1) 996 M porcinum (1) 983M fortuitumM porcinum (1) 989 M senegalenseM thermoresistibile (1) 983M thermoresistibileM senegalense (2) 996 M fortuitum (2) 981

M gordonae (7)

M gordonae (1) 992 M asiaticum (1) 955M gordonae (2) 985 M asiaticum (2) 963M gordonae (3) 959 M asiaticum (3) 955M szulgai (1) 959 M gordonae (1) 947

M tuberculosis complex (16)M tuberculosis (2) 100 M canetti (2) 998M tuberculosis (12) 998 M canetti (12) 996M tuberculosis (2) 996 M canetti (2) 994

M neoaurum (1) M neoaurum (1) 987 M smegmatis (1) 954aNumbers in parentheses (119899) represent the numbers of isolates identified as a particular speciesbIdentification based on sequencing of at least two of the following 16S rDNA 16-23S rRNA gene internal transcribed spacer (ITS) and rpoB and hsp65 genes

reference strains had similar homology with two or morespecies which suggests inadequate taxonomy within thecurrently described species Based on nucleotide sequencesof rpoB hsp65 and sodA in clinical isolates of the Mycobac-terium abscessus group one-fourth of isolates had discordantidentification [16] Multilocus sequence typing and sequenceanalysis of several genes facilitate the identification of closelyrelated species or subspecies [17]

In our study we have demonstrated that the rpsA homol-ogy gene is a promising marker for mycobacterial speciesidentification for both fast- and slow-growing mycobacteriaTo be a good marker for species differentiation the targetgene should be stable and sequence variations should occurrandomly additionally an extremely conserved or highlyvariable gene may not be adequate As a single-copy house-keeping gene rpsA gene could work well as a target gene for

Mycobacterium species discrimination without ambiguousidentification The 16S rDNA gene has higher homologywithin the mycobacteria compared to the rpsA gene 943to 100 compared to 854 to 100 According to our workwhen used as sole marker rpsA had better resolution than 16srDNA but had similar resolution as ITS rpoB and hsp65

Due to the high resolution power of rpsA for speciesidentification we presume it has potential of clinical useFrom our own experience we recommend that when per-forming species identification by homologue DNA sequencecomparison one should start with 16s RNA gene plus atleast one of the other markers such as ITS rpoB hsp65and rpsA When conflicting outcome or dubious outcomesyield more markers should be added further Even though16s RNA gene is inferior to the other markers considering thecapacity it should be chosen firstly since 16s RNA gene has

8 BioMed Research International

the most robust sequence database which can help to avoidbig identification errors due to unreliable database of othermarkers [18]

Themain deterrents for the primary use of rpsA sequenc-ing as a routine means of identifying mycobacteria residein the need for a comprehensive database Therefore weconstructed our own rpsA sequence database by including asmany type strains as possible and integrating rpsA sequencesdeposited in GenBank such as M avium subspecies paratu-berculosis M ulcerans M vanbaalenii M abscessus sub-speciesmassiliense andM canettii Besides type strains rpsAsequences of confirmed clinical strains were also includedWe will constantly upgrade our database and the capabilityto identify the most recent described species

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HongfeiDuan andGuanLiu contributed equally to this study

Acknowledgments

The work was supported by the research funding fromInfectious Diseases Special Project Ministry of Health ofChina (2012ZX10003002-09) and Beijing Municipal Admin-istration of Hospitals Clinical Medicine Development ofSpecial Funding (ZYLX201304) All the type strains andclinical isolates used in this study were obtained from theldquoBeijing Bio-Bank of Clinical Resources on Tuberculosis(D09050704640000)rdquo Beijing Chest Hospital

References

[1] T Adekambi P Berger D Raoult and M DrancourtldquorpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacteriumbolletii sp nov Mycobacterium phocaicum sp nov andMycobacterium aubagnense sp novrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 1 ArticleID 63969 pp 133ndash143 2006

[2] B Ninet M Monod S Emler et al ldquoTwo different 16SrRNA genes in a mycobacterial strainrdquo Journal of ClinicalMicrobiology vol 34 no 10 pp 2531ndash2536 1996

[3] U Reischl K Feldmann L Naumann et al ldquo16S rRNAsequence diversity inMycobacterium celatum strains caused bypresence of two different copies of 16S rRNA generdquo Journal ofClinical Microbiology vol 36 no 6 pp 1761ndash1764 1998

[4] D E Griffith T Aksamit B A Brown-Elliott et al ldquoAn officialATSIDSA statement diagnosis treatment and prevention ofnontuberculous mycobacterial diseasesrdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 4 pp 367ndash416 2007

[5] W Shi X Zhang X Jiang et al ldquoPyrazinamide inhibits trans-translation inMycobacterium tuberculosisrdquo Science vol 333 no6049 pp 1630ndash1632 2011

[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998

[7] A Roth M Fischer M E Hamid S Michalke W Ludwig andH Mauch ldquoDifferentiation of phylogenetically related slowlygrowing mycobacteria based on 16S-23S rRNA gene internaltranscribed spacer sequencesrdquo Journal of Clinical Microbiologyvol 36 no 1 pp 139ndash147 1998

[8] H Ringuet A Honore A Varnerot et al ldquohsp65 sequencingfor identification of rapidly growing mycobacteriardquo Journal ofClinical Microbiology vol 37 no 3 pp 852ndash857 1999

[9] B-J Kim S-H Lee M-A Lyu et al ldquoIdentification ofmycobacterial species by comparative sequence analysis of theRNA polymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 37 no 6 pp 1714ndash1720 1999

[10] J Amita T Vandana R S Guleria and R K Verma ldquoQualita-tive evaluation of mycobacterial DNA extraction protocols forpolymerase chain reactionrdquoMolecular Biology Today vol 3 no2 pp 43ndash49 2002

[11] R J Pauls C Y Turenne J N Wolfe and A Kabani ldquoAhigh proportion of novel mycobacteria species identified by16S rDNA analysis among slowly growing AccuProbe-negativestrains in a clinical settingrdquo American Journal of ClinicalPathology vol 120 no 4 pp 560ndash566 2003

[12] G E Fox J D Wisotzkey and P Jurtshuk Jr ldquoHow close isclose 16S rRNA sequence identity may not be sufficient toguarantee species identityrdquo International Journal of SystematicBacteriology vol 42 no 1 pp 166ndash170 1992

[13] M Drancourt P Berger and D Raoult ldquoSystematic 16S rRNAgene sequencing of atypical clinical isolates identified 27 newbacterial species associated with humansrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2197ndash2202 2004

[14] P Kirschner E C Bottger D T Walton and M ValescoldquoMicroheterogeneity within rRNA of Mycobacterium gor-donaerdquo Journal of Clinical Microbiology vol 30 no 4 pp 1049ndash1050 1992

[15] C Taillard G Greub R Weber et al ldquoClinical implicationsofMycobacterium kansasii species heterogeneity swiss nationalsurveyrdquo Journal of Clinical Microbiology vol 41 no 3 pp 1240ndash1244 2003

[16] E Macheras A-L Roux F Ripoll et al ldquoInaccuracy of single-target sequencing for discriminating species of the Mycobac-terium abscessus grouprdquo Journal of Clinical Microbiology vol 47no 8 pp 2596ndash2600 2009

[17] L A Almeida and R Araujo ldquoHighlights on molecular iden-tification of closely related speciesrdquo Infection Genetics andEvolution vol 13 no 1 pp 67ndash75 2013

[18] J Dai Y Chen and M Lauzardo ldquoWeb-accessible databaseof hsp65 sequences from Mycobacterium reference strainsrdquoJournal of Clinical Microbiology vol 49 no 6 pp 2296ndash23032011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 BioMed Research International

the most robust sequence database which can help to avoidbig identification errors due to unreliable database of othermarkers [18]

Themain deterrents for the primary use of rpsA sequenc-ing as a routine means of identifying mycobacteria residein the need for a comprehensive database Therefore weconstructed our own rpsA sequence database by including asmany type strains as possible and integrating rpsA sequencesdeposited in GenBank such as M avium subspecies paratu-berculosis M ulcerans M vanbaalenii M abscessus sub-speciesmassiliense andM canettii Besides type strains rpsAsequences of confirmed clinical strains were also includedWe will constantly upgrade our database and the capabilityto identify the most recent described species

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

HongfeiDuan andGuanLiu contributed equally to this study

Acknowledgments

The work was supported by the research funding fromInfectious Diseases Special Project Ministry of Health ofChina (2012ZX10003002-09) and Beijing Municipal Admin-istration of Hospitals Clinical Medicine Development ofSpecial Funding (ZYLX201304) All the type strains andclinical isolates used in this study were obtained from theldquoBeijing Bio-Bank of Clinical Resources on Tuberculosis(D09050704640000)rdquo Beijing Chest Hospital

References

[1] T Adekambi P Berger D Raoult and M DrancourtldquorpoB gene sequence-based characterization of emerging non-tuberculous mycobacteria with descriptions of Mycobacteriumbolletii sp nov Mycobacterium phocaicum sp nov andMycobacterium aubagnense sp novrdquo International Journal ofSystematic and Evolutionary Microbiology vol 56 no 1 ArticleID 63969 pp 133ndash143 2006

[2] B Ninet M Monod S Emler et al ldquoTwo different 16SrRNA genes in a mycobacterial strainrdquo Journal of ClinicalMicrobiology vol 34 no 10 pp 2531ndash2536 1996

[3] U Reischl K Feldmann L Naumann et al ldquo16S rRNAsequence diversity inMycobacterium celatum strains caused bypresence of two different copies of 16S rRNA generdquo Journal ofClinical Microbiology vol 36 no 6 pp 1761ndash1764 1998

[4] D E Griffith T Aksamit B A Brown-Elliott et al ldquoAn officialATSIDSA statement diagnosis treatment and prevention ofnontuberculous mycobacterial diseasesrdquo American Journal ofRespiratory and Critical Care Medicine vol 175 no 4 pp 367ndash416 2007

[5] W Shi X Zhang X Jiang et al ldquoPyrazinamide inhibits trans-translation inMycobacterium tuberculosisrdquo Science vol 333 no6049 pp 1630ndash1632 2011

[6] S T Cole R Brosch J Parkhill et al ldquoDeciphering the biologyof Mycobacterium tuberculosis from the complete genomesequencerdquo Nature vol 393 no 6685 pp 537ndash544 1998

[7] A Roth M Fischer M E Hamid S Michalke W Ludwig andH Mauch ldquoDifferentiation of phylogenetically related slowlygrowing mycobacteria based on 16S-23S rRNA gene internaltranscribed spacer sequencesrdquo Journal of Clinical Microbiologyvol 36 no 1 pp 139ndash147 1998

[8] H Ringuet A Honore A Varnerot et al ldquohsp65 sequencingfor identification of rapidly growing mycobacteriardquo Journal ofClinical Microbiology vol 37 no 3 pp 852ndash857 1999

[9] B-J Kim S-H Lee M-A Lyu et al ldquoIdentification ofmycobacterial species by comparative sequence analysis of theRNA polymerase gene (rpoB)rdquo Journal of Clinical Microbiologyvol 37 no 6 pp 1714ndash1720 1999

[10] J Amita T Vandana R S Guleria and R K Verma ldquoQualita-tive evaluation of mycobacterial DNA extraction protocols forpolymerase chain reactionrdquoMolecular Biology Today vol 3 no2 pp 43ndash49 2002

[11] R J Pauls C Y Turenne J N Wolfe and A Kabani ldquoAhigh proportion of novel mycobacteria species identified by16S rDNA analysis among slowly growing AccuProbe-negativestrains in a clinical settingrdquo American Journal of ClinicalPathology vol 120 no 4 pp 560ndash566 2003

[12] G E Fox J D Wisotzkey and P Jurtshuk Jr ldquoHow close isclose 16S rRNA sequence identity may not be sufficient toguarantee species identityrdquo International Journal of SystematicBacteriology vol 42 no 1 pp 166ndash170 1992

[13] M Drancourt P Berger and D Raoult ldquoSystematic 16S rRNAgene sequencing of atypical clinical isolates identified 27 newbacterial species associated with humansrdquo Journal of ClinicalMicrobiology vol 42 no 5 pp 2197ndash2202 2004

[14] P Kirschner E C Bottger D T Walton and M ValescoldquoMicroheterogeneity within rRNA of Mycobacterium gor-donaerdquo Journal of Clinical Microbiology vol 30 no 4 pp 1049ndash1050 1992

[15] C Taillard G Greub R Weber et al ldquoClinical implicationsofMycobacterium kansasii species heterogeneity swiss nationalsurveyrdquo Journal of Clinical Microbiology vol 41 no 3 pp 1240ndash1244 2003

[16] E Macheras A-L Roux F Ripoll et al ldquoInaccuracy of single-target sequencing for discriminating species of the Mycobac-terium abscessus grouprdquo Journal of Clinical Microbiology vol 47no 8 pp 2596ndash2600 2009

[17] L A Almeida and R Araujo ldquoHighlights on molecular iden-tification of closely related speciesrdquo Infection Genetics andEvolution vol 13 no 1 pp 67ndash75 2013

[18] J Dai Y Chen and M Lauzardo ldquoWeb-accessible databaseof hsp65 sequences from Mycobacterium reference strainsrdquoJournal of Clinical Microbiology vol 49 no 6 pp 2296ndash23032011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology