pyrolysis mass spectrometry (pyms) and 16s23s rdna spacer region fingerprinting suggests the...

Pyrolysis Mass Spectrometry (PyMS) and 16S–23S rDNA SpacerRegion Fingerprinting Suggests the Presence of NovelAcinetobacters in Activated Sludge

EMMA CARR1, ALAN WARD2, VOLKER GURTLER3 and ROBERT J. SEVIOUR1

1Biotechnology Research Centre, La Trobe University Bendigo, Victoria, Australia2 Dept of Agricultural Science, University of Newcastle upon Tyne, Newcastle, UK3 Microbiology Dept. Austin and Repatriation Hospital, Melbourne, Victoria, Australia

Received May 25, 2001

Summary

Screening of large numbers of Acinetobacter spp. from activated sludge systems with Pyrolysis MassSpectrometry (PyMS) showed that many did not cluster tightly with the currently described genomicspecies which have been obtained mainly from clinical sources. Selected isolates were then genotypicallyfingerprinted using their 16S–23S rDNA spacer region, and again the data revealed considerable differ-ences in the genomic fingerprints of many of these activated sludge isolates to the predominantly clinicalgenomic species. In fact, few could be identified from them. The possibility that the current speciationwithin this genus is not adequate to encompass all these environmental isolates is addressed in relation tothe methods used to study the population dynamics of Acinetobacter in activated sludge.

Key words: activated sludge – Acinetobacter – 16S–23S spacer region – pyrolysis mass spectrometry – biological phosphorus removal

Introduction

The taxonomic status of the genus Acinetobacter hascaused much confusion since it was first proposed byBRISOU and PREVOT (1954). Now, organisms can be unam-biguously assigned to this genus with the transformationassay of JUNI (1972), but delineating species of Acineto-bacter is still problematic. Currently, 21 genomic speciesof Acinetobacter have been described. Most of these areclinical isolates, and only seven have been validly namedso far. BOUVET and GRIMONT delineated 12 genomicspecies of Acinetobacter (BG1–12) using DNA-DNA hy-bridization data (BOUVET & GRIMONT, 1986). Later,TJERNBERG and URSING (1989) described an additionalthree genomic species (TU13–15) also based on DNA-DNA hybridizations, and their DNA groups generallyagreed with those proposed by BOUVET and GRIMONT

(1986). An additional 5 genomic species were later de-scribed by BOUVET and JEANJEAN (1989), named BJ13–17,and their genomic species 13 was indistinguishable fromgenomic species 14 of TJERNBERG and URSING (1989).NISHIMURA et al. (1987) isolated a radiation resistantstrain of Acinetobacter which TJERNBERG and URSING

found belonged to the DNA group 12 of BOUVET and GRI-MONT (1986). More recently, GERNER-SMIDT et al. (1993)described two more genomic species, ‘between BG1 andBG3’ (represented by strains 10095 and 10169) and ‘closeto TU13’ (represented by strain 10090). These are mostclosely related to A. calcoaceticus, A. baumannii, genomicspecies 3 (BG) and genomic species 13 (TU), commonlyreferred to as the A. calcoaceticus-A. baumannii complex(TJERNBERG and URSING 1989).

Members of the genus Acinetobacter are found in awide range of habitats, including activated sludge (FUHS

& CHEN, 1975; BUCHAN, 1983; CLOETE et al., 1985;BEACHAM et al., 1990; KNIGHT et al., 1993). Acinetobac-ter spp. were once thought to be the bacteria primarily re-sponsible for the process of enhanced biological phospho-rus removal (EBPR) in activated sludge, but culture-inde-pendent methods of enumeration such as Fluorescence InSitu Hybridization (FISH) and clone library studies havequestioned their role as important phosphorus accumu-lating bacteria (PAB) (WAGNER et al., 1994; BOND et al.,1995, 1998).

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Pyrolysis Mass Spectrometry (PyMS) 431

Comparatively little taxonomic work has been carriedout on these activated sludge isolates, and many of thephenotypic identification schemes developed for the clini-cal isolates of Acinetobacter perform poorly when appliedto them (e.g. SODDELL et al. 1993). If identifiable at all,many emerge as A. junii (BG5), A. johnsonii (BG7) and A.lwoffii (BG 8/9) (BEACHAM et al., 1990; KIM et al., 1997;GUARDABASSI et al., 1999). Furthermore, genomic finger-prints of environmental isolates obtained using RAPD-PCR (CARR et al., 2001) differed markedly from those ofthe known genomic species of Acinetobacter and suggestedthat previously undescribed species of Acinetobacter mayexist in activated sludge. One of the main arguments ques-tioning the role of Acinetobacter as a PAB comes fromdata obtained from FISH studies using 16S rRNA-targetedprobes predominantly based on sequence data of clinicalisolates (WAGNER et al., 1994). However, SNAIDR et al.(1997) have shown that two environmental Acinetobacter-related sequences failed to hybridize with the Acinetobac-ter probe used in these FISH studies (WAGNER et al., 1994)which were designed using the then currently recognizedgenomic species. Thus, the diversity of acinetobacters inactivated sludge may be currently underestimated.

It was thus decided to examine a group of Acinetobac-ter isolates obtained from several activated sludge plantsin Australia for their diversity. Many of these isolatescould not be identified to species level in earlier studies(KNIGHT et al., 1993; BEACHAM et al., 1990). These werefirst screened using Curie-Point Pyrolysis Mass Spectrom-etry (PyMS), a physicochemical whole-cell fingerprintingmethod which has been valuable in rapidly screening andgrouping bacteria isolated from environmental samples(DALGAARD et al., 1997). PyMS is highly discriminatoryand has the ability to detect phenotypic differences inclosely related bacteria (TIMMINS et al., 1998; MAGEE etal., 1997) which are difficult to differentiate using con-ventional taxonomic methods. Good agreement has beendemonstrated between PyMS and numerical taxonomicdata with deep-sea actinomycetes (COLQUHOUN et al.,1998). Its highly discriminatory nature and ability tohandle large numbers of samples make it an ideal tech-nique for large-scale screening (BULL et al., 2000).

After PyMS analysis, appropriate strains were selectedfor genomic fingerprinting using the 16S–23S rDNAspacer region. This region is known to be variable in bothsize and sequence even within closely related taxonomicgroups (GURTLER & STANISICH, 1996), making it ideal forsuch taxonomic purposes. The 16S–23S intergenic spacerregion can be PCR amplified using primers from highlyconserved flanking regions on the 16S and 23S genes, re-spectively, and the polymorphisms present in the PCRproducts have been used to recognize genera and speciesin many bacteria (JENSEN et al., 1993). Fingerprinting ofthe 16S–23S spacer region using restriction enzymes hasbeen successful in discriminating between closely relatedbacteria and yeasts (RIFFARD et al., 1998; AAKRA et al.,1999; GRANCHI et al., 1999; DOIGNON-BOURCIER et al.,2000), including Acinetobacter, where all clinical strainsexamined were correctly identified at the DNA grouplevel (GARCIA-ARATA et al., 1997).

Materials and Methods

Bacterial StrainsThe 239 environmental strains used in this study were previ-

ously isolated from different wastewater treatment plants in Vic-toria, Australia (KNIGHT et al. 1993; BEACHAM et al., 1990) andare held in the La Trobe University Bendigo culture collection.All these environmental isolates were confirmed as belonging tothe genus Acinetobacter using the transformation assay of Juni(1972), and many could not be identified with Biolog (SODDELL

et al., 1993; KNIGHT et al., 1993). Strains representing the cur-rently recognised genomic species of Acinetobacter were also in-cluded in this study (Table 1). An additional 10 unidentified en-vironmental Acinetobacter strains from Ballarat (C), Albury (A),Wodonga (D) and Bendigo (B) wastewater treatment plantswere also included in the 16S–23S rDNA spacer region workwithout prior PyMS screening as RAPD-PCR fingerprinting(CARR et al., 2001) suggested they may be novel isolates. Allstrains used in the study were stored at –80 °C.

Curie-point Pyrolysis Mass Spectrometry (PyMS)All strains were cultured on an acetate defined mineral medi-

um (DEINEMA et al., 1980) and incubated at 30 ºC for 48 h. It isessential that all growth conditions are standardized when usingPyMS, as growth-related changes may influence the outcome ofthe results and affect reproducibility (MAGEE, 1993). Therefore,the same batch of medium was used for a complete PyMS run,and growth conditions were meticulously standardized for allruns and duplicated strains showed excellent congruence. Cellbiomass was evenly applied to clean iron-nickel foils partiallyinserted into clean pyrolysis tubes (Horizon Instruments Ltd.,Heathfield, UK) using a sterile plastic loop. Each sample wasrun in triplicate. Samples were then air-dried overnight. Prior topyrolysis, the foils were pushed into the pyrolysis tubes using astainless steel depth gauge and the preparations were oven-driedat 80 °C for 5 min to ensure the samples were completely dry.Finally Viton ‘O’- rings (Horizon Instruments) were placed ca.1mm from the mouth of each tube before the samples wereloaded onto the carousel of the PyMS machine. Each batch ofsamples was processed on a Horizon Instruments RAPyD 200Xpyrolysis mass spectrometer. Curie-point pyrolysis was at530 ºC for 3 s, under vacuum, with a temperature rise time of0.6 s. Data were collected over the m/Z range 51–200 and nor-malized to the total ion count to remove any influence of samplesize. The time required for each sample to be pyrolysed was ca.2 min.

To eliminate any influence of distorting statistical effects dueto poor ion reproducibility, mass ion counts of less than 5 × 105

and greater than 6 × 106 were removed from the data set. Dupli-cate triplicates were included in each PyMS run to verify repro-ducibility between separately grown and pyrolysed samples ofthe same organism. The PyMS data were subjected to principlecomponent analysis using the GENSTAT package (NELDER,1979), and principal components (PC) accounting for over0.1% of the variance were used as input data for canonical vari-ates analysis which separates the samples into groups on thebasis of the retained PC and knowledge of the triplicate groups.A percentage similarity matrix was constructed for the data andthis was used for hierarchical cluster analysis using the UPGMAalgorthim and the results presented as 3D ordination diagramsand dendrograms.

DNA Isolation Chromosomal DNA was extracted from overnight cultures

of the Acinetobacter strains using the Promega Wizard GenomicDNA purification kit (Promega, Melbourne, Australia) accord-

432 E. CARR et al.

ing to the manufacturer’s instructions. The resulting DNA wasresuspended in distilled water and electrophoresed on anagarose gel to determine its integrity, and then stored at –20 °Cuntil used.

PCR Amplification of the 16S–23S rDNA spacer regionThe PCR amplification of the 16S-23S ISR was carried out

using two universal primers complementary to conserved re-gions on the 16S and 23S ribosomal genes recommended byGURTLER and STANISICH (1996). The forward and reverse primersequences are 5′ TTG TAC ACA CCG CCC GTC 3′ (located atnucleotide position 1390–1407 of the 16S rRNA of Escherichiacoli, Region 2 of GURTLER & STANISICH 1996), and 5′ GGT ACTTAG ATG TTT CAG TTC 3′ (located at nucleotide positions188–208 of the 23S rRNA gene, Region 7 of GURTLER &STANISICH 1996), respectively. These primers have been used suc-cessfully in earlier studies to detect strain differences solely onthe basis of spacer-length variation (e.g. KOSTMAN et al., 1995).DNA templates were amplified in a total reaction volume of50 µl containing 2U of Amplitaq gold thermostable polymerase(Applied Biosystems), 0.5 µM of each primer, 200 µM of eachdeoxynucleotide, 1.5 mM MgCl2 , 10 mM Tris HCl (pH 8.3),and 50 mM KCl. Amplification was carried out in a GeneAmp2400 thermal cycler (Applied Biosystems) for 30 cycles accord-ing to the following program: Initial denaturation at 94 ˚C for10 min, followed by 94 ˚C for 1min, 52 ˚C for 1min, 72 ˚C for4min and a final extension of 10min at 72 ˚C to complete partialpolymerizations. The products were analyzed on a 2% Nusieveagarose (FMC Bioproducts, Edwards Instrument Company,NSW, Australia) gel, stained with ethidium bromide and viewedon a UV transilluminator.

Restriction Enzyme AnalysisThe PCR products were digested with 5 different restriction

enzymes: RsaI, Sau3AI (Roche Diagnostics, Castle Hill, Aus-tralia), Tsp509I, Taq(I and MseI (New England Biolabs, Gene-search, Arundel, Australia). These enzymes were selected on thebasis of polymorphisms found in the 16S–23S ISR sequence dataof the A. calcoaceticus-A. baumannii complex (LAGATOLLA etal., 1998). Restriction Mate software (Roche Diagnostics, Syd-ney, Australia) was used to select enzymes with the appropriatecut sites. Restriction digests were carried out according to themanufacturer’s instructions, and incubated for 4 h at the appro-priate temperature. Then 10 µl of the resulting digestion prod-ucts were resolved on a 3.5% NuSieve agarose (FMC BioProd-ucts) gel stained with ethidium bromide and electrophoresed at90 V for 1.5 h. The gel was viewed under UV light and imagescaptured using 665 Polaroid film. The DNA fragment sizes weredetermined with a 100 bp molecular weight marker (Roche Di-agnostics).

Data AnalysisThe combined 16S–23S rDNA fingerprints from all five en-

zymes were analyzed using Gel-Pro Analyzer (Media Cybernet-ics, Maryland, USA) and all images were normalized against theDNA molecular weight markers. Bands were assigned on a pres-ence-absence basis. A data matrix was constructed for all strainswith MacClade 3.04 (Sinauer Associates Inc., Massachusetts,USA) where each character was an individual band in the pat-tern from each strain. Numerical taxonomic analysis of the datawas performed using NTSYS-PC version 1.80 (Exeter Software,New York, USA), and the simple matching coefficient (Ssm) wascalculated for each isolate. Cluster analysis of the numericaldata was performed using the unweighted pair group methodusing arithmetic averages (UPGMA algorithm) and all possiblesolutions were generated. From these a consensus dendrogramwas produced.

Results

Screening of Acinetobacter isolates with PyMS

A selection of 20 strains representing the known ge-nomic species of Acinetobacter were initially analysed byPyMS to ensure conditions of pyrolysis were suitable forAcinetobacter strains. The dendrogram resulting fromthis analysis is shown in Fig. 1. BG3 and BG6 were omit-ted from the analysis of this run as the total mass ioncounts for the triplicates of these strains was very low.Strains BG2 and TU15 were run in duplicate triplicates toassess reproducibility within the run, and these replicateswere very closely related. BG1 and BG2 were most close-ly related to one another as were BG8 and BG12, BG7and BG5, BG10 and BG11, BJ13 and BJ16 and BG14and BG17.

The 239 activated sludge isolates of Acinetobacterwere organized into three groups for PyMS analyses (RunA, B and C) based on their earlier phenotypic identifica-tions using Biolog (KNIGHT et al., 1993; BEACHAM et al.,1990). Run A contained isolates all thought to be A.johnsonii (BG7), in addition to all the currently recog-nized genomic species. Run B contained genomic speciesBG8, BG9, BG1, 2, 3, 10, 11 and 12, and run C consisted

Fig. 1. Dendrogram of known genomic species of Acinetobacterusing PyMS data (Details of analysis given in the text).* duplicates

Pyrolysis Mass Spectrometry (PyMS) 433

of A. junii (BG5) and all the strains which were unidenti-fiable with Biolog. Analysis of the PyMS data revealedmost of the strains clustered tightly, with a few clearlyseparated from the main cluster. Triplicates of each strainwere closely grouped, demonstrating the high repro-ducibility of this technique. Data from Run A are present-ed as a dendrogram in Fig. 2, more clearly showing theclose relationships between the strains in the main clusterand the positions of the outliers. All known genomicspecies of Acinetobacter except TU15, BJ17, BG14, BG6,BG1, BG2, BG4 and BJ15, which apart from BG1 aremostly clinical isolates rarely found in activated sludge,occur in the main cluster. All environmental isolates out-side of the main cluster, as well as a random selection ofstrains from within the primary cluster, were then select-ed for further genotypic characterization. The data fromthe remaining two PyMS runs (data not shown) werehandled in the same way and selections made using thesame criteria. All strains from the PyMS work selected forfurther work are listed in Table 2, together with theiroriginal sites of isolation.

Amplification of the 16S–23S rDNA spacer region ofselected Acinetobacter isolates

A total of 93 isolates were analysed by this technique,including representatives of the known genomic speciesof Acinetobacter. Initially, the selected primers were usedto amplify the 16S–23S rDNA spacer region of 9 environ-mental isolates chosen at random to assess the degree ofpolymorphism in the target region of these Acinetobacterisolates. One, or in some cases, two bands very close to-

gether were found in all, ca. 800–1200 bases in size. Al-though some minor variation was evident in the fragmentsize generated, the degree of polymorphism was insuffi-cient to differentiate between these strains on this basisalone.

Fingerprinting of the 16S–23S rDNA spacer regionusing restriction enzymes

For better discrimination, restriction polymorphismanalysis using RsaI, Sau3A1, MseI, TaqaI and Tsp509Iwas performed on all 93 isolates. This analysis was per-formed twice on the known genomic species to ensurethe band patterns obtained were reproducible. A total of126 characters (i.e. individual bands) were generatedusing the five enzymes. Restriction with MseI, Sau3AIand Tsp509I yielded more fragments than the other twoenzymes. Fig. 3 shows the digestion patterns obtainedwith MseI for the representatives of the known genomicspecies of Acinetobacter. The patterns shown are quitediverse. Similar patterns are observed between lanes 2,3, 4, 25, 26 and 27, and this is reflected in the dendro-gram based on patterns for the known genomic specieswith all five restriction enzymes (Fig. 4). These six or-ganisms (BG1, BG2, BG3, 10090, 10095 and 10169)form a distinct cluster with BG1 and BG2 and 10095and 10169 being most similar to one another. Thesestrains are all members of the A. calcoaceticus-A. bau-mannii complex, which are known to be genotypicallyvery similar to one another (TJERNBERG & URSING,1989; GERNER-SMIDT & TJERNBERG, 1993). TU13 is alsoconsidered to belong to this group, but according to the

Table 1. Known genomic species of Acinetobacter and their sources used in this study.

Abbreviation used in study Species name Origin/Culture Collection Numbers

BG1 Acinetobacter calcoaceticus ATCC 23055T/ CIP 81.08T BG2 Acinetobacter baumannii ATCC 19606T/ CIP 70.34TBG3 Acinetobacter sp. 3 ATCC 19004/ CIP 70.29BG4 Acinetoabcter haemolyticus ATCC 17906T/ CIP 64.3TBG5 Acinetobacter junii ATCC 17908T/ CIP 64.5TBG6 Acinetobacter sp. 6 ATCC 17979/ CIP A165BG7 Acinetobacter johnsonii ATCC17909T/ CIP 64.6TBG8 Acinetobacter lwoffii NCTC 5866T/ CIP 64.10TBG9 Acinetobacter sp. 9 ATCC 9957/ CIP 70.31BG10 Acinetobacter sp. 10 ATCC 17924/ CIP 70.12BG11 Acinetobacter sp. 11 ATCC 11171/ CIP 63.46BG12 Acinetobacter radioresistens SEIP 12.81BJ13 Acinetobacter sp. 13 (BJ) ATCC 17905/ CIP 64.2BJ14 Acinetobacter sp. 14 (BJ) K.Irino 105/85BJ15 Acinetobacter sp. 15 (BJ) M.M. Adam Ac606 180:40 vaBJ16 Acinetobacter sp. 16 (BJ) ATCC 17988/ CIP 70.18BJ17 Acinetobacter sp. 17 (BJ) SEIP Ac87.314TU13 (ATTC) Acinetobacter sp 13 (TU) ATCC 17903TU13 (165) Acinetobacter sp. 13 (TU) Lund UniversityTU14 Acinetobacter sp. 14 (TU) ATCC 151a1TU15 Acinetobacter sp. 15 (TU) ATCC 7110090 “close to TU13” Statens Serum Institut (P. Gerner-Smidt)10095 “between BG1 &BG3” Statens Serum Institut (P. Gerner-Smidt)10169 “between BG1 & BG3” Statens Serum Institut (P. Gerner-Smidt)

434 E. CARR et al.

Table 2. Environmental Acinetobacter strains selected fromPyMS analysis for genomic fingerprinting.

Environmental Wastewater TreatmentAcinetobacter Strain Plant of origin

12A02 Bendigo3A02 Bendigo5N03 Bendigo4A03 Bendigo6A05 Bendigo9A01 Bendigo10A01 Bendigo5B02 Bendigo2N01 Bendigo10A02 BendigoN13/31 BendigoAB2110 BendigoAB1112 BendigoAB1141 BendigoN16/34 BendigoJ22/15 Bendigo26B02 Bendigo22B02 Bendigo17A02 Bendigo17A04 Bendigo5N13 Bendigo21B02 BendigoAB1030 Bendigo4N07 Bendigo26N03 Bendigo4B02 Bendigo22N11 BendigoAB1168 Bendigo13B02 Bendigo7B02 BendigoM10/15 Bendigo4N13 BendigoAB2010 BendigoRA3129 Lower PlentyAB2104 BendigoN14/49 BendigoAB3316 BendigoAB1160 BendigoRA3036 Ballarat7N16 Bendigo1A08 Bendigo11N04 Bendigo4B01 Bendigo25A01 Bendigo15M06 Bendigo2B07 Bendigo27N01 Bendigo6N03 Bendigo11A04 BendigoAB1110 Bendigo6A02 Bendigo8A01 Bendigo1A01 Bendigo9B03 Bendigo6N01 Bendigo19A01 BendigoAB1025 Bendigo1SR06 Bendigo6A07 Bendigo Fig. 2. Dendrogram of PyMS data for Acinetobacter from Run A

(see text for details). * duplicates

Pyrolysis Mass Spectrometry (PyMS) 435

Fig. 3. Gel patterns of the PCR amplified 16S–23S rDNA spacer region of the known genomic species of Acinetobacter after diges-tion with the restriction enzyme Mse I.Lanes 1, 12, 23 – 100bp DNA markerLanes 2–11 are isolates BG1, BG2, BG3, BG4, BG5, BG6, BG7, BG8, BG9 and BG10, respectively.Lanes 13–22 are isolates BG11, BG12, BJ13, BJ14, BJ15, BJ16, BJ17, TU13(165), TU13(ATCC) and TU14, respectively. Lanes 24–27 are isolates TU15, 10090, 10095, and 10169, respectively.

Fig. 4. Dendrogram of known ge-nomic species of Acinetobacterbased on numerical analysis of fin-gerprints of the 16S–23S rDNAspacer region with all restrictionendonucleases used in this study.

data (Fig. 4), the two strains of TU13 used here clusterinstead with TU14 and BJ13, which are considered thesame genomic species (TJERNBERG & URSING, 1989).BG8 is linked most closely with BG10 rather than withBG9, which was not expected (BOUVET & GRIMONT,1986), and BG9 seems clearly separate from all theother strains. Most of the proteolytic genomic species(BJ15, BJ16 and BJ17) cluster together.

A total of 159 different bands were obtained when allof the environmental Acinetobacter strains were digested

with the five enzymes. The gel patterns for the same 20environmental strains generated with each of the five en-zymes are presented in Fig 5. Lanes B and K show similarpatterns with all five enzymes used as do lanes C, E, F Iand J, with C appearing the most different to the others.Lanes U and V exhibited identical patterns to each otherwith all the enzymes used. These visual relationships arereflected in the dendrogram based on all 159 characters(Fig. 6). All isolates clustered at a similarity value of ca.0.63. Seven main clusters were identified, labelled Groups

436 E. CARR et al.

Fig. 5A. Gel patterns of the PCR amplified 16S–23S rDNA spacer region of environmental isolates of Acinetobacter after digestionwith the restriction enzyme Mse I.Lanes A & L – 100bp DNA LadderLanes B – K are isolates D11, C5, A23, B2, A7, A28, B3, C2, C1 and D5, respectively.Lanes M – V are isolates N16/31, AB2110, AB1112, AB1141, N16/34, J22/15, 26B02, 22B02, 17A02 and 17A04, respectively.

A

B

C

Pyrolysis Mass Spectrometry (PyMS) 437

D

E

Fig. 5D. Gel patterns of the PCR amplified 16S–23S rDNA spacer region of environmental isolates of Acinetobacter after digestionwith the restriction enzyme Taq_ I.Lanes A & L – 100bp DNA LadderLanes B – K are isolates D11, C5, A23, B2, A7, A28, B3, C2, C1 and D5, respectively.Lanes M – V are isolates N16/31, AB2110, AB1112, AB1141, N16/34, J22/15, 26B02, 22B02, 17A02 and 17A04, respectively.

Fig. 5E. Gel patterns of the PCR amplified 16S–23S rDNA spacer region of environmental isolates of Acinetobacter after digestionwith the restriction enzyme Tsp 509 I.Lanes A & L – 100bp DNA LadderLanes B – K are isolates D11, C5, A23, B2, A7, A28, B3, C2, C1 and D5, respectively.Lanes M – V are isolates N16/31, AB2110, AB1112, AB1141, N16/34, J22/15, 26B02, 22B02, 17A02 and 17A04, respectively.

Fig. 5C. Gel patterns of the PCR amplified 16S–23S rDNA spacer region of environmental isolates of Acinetobacter after digestionwith the restriction enzyme Sau 3AI.Lanes A & L – 100bp DNA LadderLanes B – K are isolates D11, C5, A23, B2, A7, A28, B3, C2, C1 and D5, respectively.Lanes M – V are isolates N16/31, AB2110, AB1112, AB1141, N16/34, J22/15, 26B02, 22B02, 17A02 and 17A04, respectively.

Fig. 5B. Gel patterns of the PCR amplified 16S-23S rDNA spacer region of environmental isolates of Acinetobacter after digestionwith the restriction enzyme Rsa I.Lanes A & L – 100bp DNA LadderLanes B – K are isolates D11, C5, A23, B2, A7, A28, B3, C2, C1 and D5, respectively.Lanes M – V are isolates N16/31, AB2110, AB1112, AB1141, N16/34, J22/15, 26B02, 22B02, 17A02 and 17A04, respectively.

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Fig. 6. Dendrogram of environmental and known genomic species of Acinetobacter based on numerical analysis of band patternsgenerated with all restriction endonucleases used in this study.

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gether with a few strains not identified with Biolog.Group G forms a coherent cluster, distinct from all otherstrains included in this study. None of the strains in thisgroup could be identified with Biolog and appear to bequite unrelated to any of the known genomic species. The20 strains at the bottom of the dendrogram are all dis-tinct and do not form any obvious clusters. Of these 20strains, four are known genomic species (i.e. BG4, BG14,BG6 and BG9) and eight not identified by Biolog. The re-lationships which emerged from PyMS for both theknown genomic species and the environmental strainswere quite different to those seen with the 16S–23SrDNA fingerprints. For example, the proteolytic genomicspecies mostly clustered together by the genomic finger-prints but were more widely separated based on thePyMS data. Similarly, BG10 and BG11 linked closely to-gether based on PyMS data but not according to the16S–23S spacer region fingerprints. PyMS was successfulin resolving strains distinct from the known genomic

A–G with Group B containing two subclusters, B1 andB2, respectively. The isolates contained in each group arelisted in Table 3 together with their Biolog identifications.Group A contains an equal number of known genomicspecies and environmental strains. The closely groupedA23, A28, D11 and D5 link most closely with BG11 butare more similar to one another than any other strain.Group B contains a large number of environmental iso-lates not identifiable with the Biolog system. The knowngenomic species occurring in this group are mostlygrouped together, with the exception of BG5, BJ13 andTU15, which are separated from other known genomicspecies by at least one environmental isolate. Group C,apart from two strains phenotypically identified as group5 (AB2104, N14/49), is made up of strains all phenotypi-cally identified as belonging to DNA group 7 (A. john-sonii). Group D also contains environmental strains iden-tified as belonging to DNA group 7. Strains in Groups Eand F belong to several of the known genomic species, to-

Group A BG1 NDBG2 NDBG3 NDJ22/15 BG10BG8 NDBG10 NDBG11 NDA23 UNA28 UND11 UND5 UN

Group BB1 BG5 ND

25A01 BG7BJ13 ND27N01 BG710A01 BG121B02 UNBJ15 NDBJ16 ND6N01 BG7TU15 ND12A02 UN4N13 UN4A03 UNAB3316 BG5RA3036 BG8/97N16 UN10A02 BG17B02 UN

B2 BJ17 NDTU13 (165) NDTU14 NDTU13 (ATTC) NDBG12 ND6A05 UN

Group Strain Biolog IdentificationGroup Strain Biolog Identification

3A02 UN10090 ND10095 ND10169 ND5N03 BG2

Group C AB2104 BG5N14/49 BG52B07 BG71A08 BG715M06 BG711A04 BG78A01 BG79B03 BG71A01 BG76N03 BG7AB1025 BG711N04 BG74B01 BG7

Group D AB1110 BG76A02 BG719A01 BG76A07 BG7

Group E AB1112 BG8/9AB1141 BG8/922B02 BG1217A02 BG317A04 UN

Group F BG7 NDAB2110 BG115B02 BG9

Group G C5 UNB2 UNA7 UNC2 UNC1 UN

Table 3. The Acinetobacter strains forming Groups A-G constructed on the basis of analysis of the 16S–23S rDNA spacer regionfingerprints. UN – unidentified, ND – not done

440 E. CARR et al.

species as many of the strains chosen for genomic finger-printing based on the PyMS data were unable to be iden-tified with Biolog.

Discussion

Twenty-one genomic species have been described so farfor the genus Acinetobacter, but the taxonomic status ofmany other isolates remains unclear due to inconsistenciesobtained with all the different taxonomic methods appliedto these organisms. The results from this study supportthose of other groups (WIEDMANN-AL-AHMAD et al., 1994;MASZENAN et al., 1997; CARR et al., 2001) in that the pre-sent speciation of Acinetobacter does not encompass allisolates of this ubiquitous genus. The data also adds fur-ther doubts to the claims of GUARDABASSI et al. (1999) thatthe current taxonomic groups of Acinetobacter encom-pass most or all of the species that occur in aquatic envi-ronments. In fact, studies with clinical isolates have alsodetected many unidentifiable Acinetobacter strains whichstill remain ungrouped (BOUVET & GRIMONT, 1986;TJERNBERG & URSING, 1989; DIJKSHOORN et al., 2000).

PyMS is ideally suited to rapidly screen and group or-ganisms from environmental samples (DALGAARD et al.,1997; COLQUHOUN et al., 1998). It has several advantagesover conventional identification methods, which includesimple sample preparation, rapid analysis time (ca. 2 minper sample), high automated throughput and general ap-plicability to almost all cultivable microorganisms (BULL

et al., 2000). PyMS yields highly discriminatory data re-flecting overall cell composition, which is difficult, expen-sive and slow to obtain using traditional techniques. Inother similar studies, data from PyMS has shown a highcorrelation with both genotypic and numerical taxonom-ic data (TIMMINS et al., 1998; COLQUHOUN et al., 1998),making it an important adjunct to other taxonomic meth-ods in polyphasic bacterial systematics. However, the in-terspecies relationships of the known genomic species ofAcinetobacter revealed by PyMS in this study correlatedpoorly with those determined with DNA-DNA hybridiza-tion studies (BOUVET & GRIMONT, 1986; TJERNBERG &URSING, 1989; BOUVET & JEANJEAN, 1989), although notall the described genomic species were included in thePyMS analysis. For example, BG8 and BG9 have beenconsidered to be closely related (TJERNBERG & URSING,1989), yet they emerged as very different to each otheraccording to the PyMS data. On the other hand, PyMSdata suggested a close relationship between BG10 andBG11 which agrees with DNA-DNA hybridization data.Similar results were obtained for BG1 and BG2, and BG7and BG9. The differences between the PyMs data andthat from DNA-DNA hybridization studies is not surpris-ing. Many different techniques have been used in an at-tempt to clarify the interspecies relationships of theknown genomic species of Acinetobacter, but most differfrom those resolved by DNA pairing studies (e.g. WIED-MANN-AL-AHMAD et al., 1994; VANEECHOUTTE et al.,1995; EHRENSTEIN et al., 1996; YAMAMOTO et al., 1999),including even 16S rDNA sequence analysis (IBRAHIM et

al., 1997). Again, this is not unexpected since the sugges-tion has been made that comparisons of 16S rDNA se-quences may be limited in their ability to resolve closelyrelated bacterial strains such as those belonging to thesame genus (ASH et al., 1991; FOX et al., 1992; STACKE-BRANDT & GOEBEL, 1994).

For these reasons, 16S–23S rDNA spacer region finger-printing has been recommended for the typing and identi-fication of bacteria at the species and strain level (BARRY

et al., 1991; GURTLER & STANISICH, 1996). This region ischaracterized by variations in both its length and se-quence, in contrast to the genes themselves which are re-markably well conserved (WOESE 1987). Such polymor-phisms in both the size and sequence of this spacer regioncan be exploited for taxonomic use as an important aid to16S rRNA sequencing and DNA-DNA hybridizationstudies (JENSEN et al., 1993). Several studies have finger-printed this region to differentiate known genomic speciesof Acinetobacter (NOWAK et al., 1995; DOLZANI et al.,1995; GARCIA-ARATA et al., 1997), and these studies alsofound that amplifying the 16S–23S rDNA spacer regionalone could not differentiate between the known genomicspecies. NOWAK et al. (1995) tried this method on the 17then described genomic species of Acinetobacter. Thetechnique appeared less discriminatory than in later workby GARCIA-ARATA et al. (1997), where all strains testedwere correctly identified to the DNA group level, andcomplete agreement with the classification previously ob-tained by conventional ribotyping and DNA-DNA hy-bridization studies was observed. Critical for successfuldetection of spacer length variation is the choice of PCRprimers (GURTLER & STANISICH, 1996). The primer basedon the 23S rRNA gene chosen by NOWAK et al. (1995) isknown to have limited success in detecting spacer varia-tion (GURTLER & STANISICH, 1996), unlike the primers se-lected by GARCIA-ARATA et al. (1997) which were success-ful in an earlier study with members of the Acb complex(IBRAHIM et al., 1996).

Interspecies relationships between all the known ge-nomic species of Acinetobacter were determined in thepresent study using this fingerprinting method and rea-sonably good correlation was observed between thesedata and the results of DNA-DNA hybridization studies(BOUVET & GRIMONT, 1986; BOUVET & JEANJEAN, 1989;TJERNBERG & URSING, 1989). All members of the Acbcomplex, with the exception of both strains of TU13,clustered together, as did the proteolytic strains, apartfrom BG14. Where there were multiple strains belongingto the same DNA group, these too clustered together (i.e.TU13, ‘Between 1 & 3’).

The different patterns from the activated sludge strainsresulting after enzyme digestion of this region suggestshigh diversity among the isolates analysed. The majority ofthe environmental isolates are grouped separately from theknown genomic species, which generally clustered witheach other. To some extent, the groupings generated afteranalysis of the 16S–23S spacer fingerprints support the Bi-olog identifications, which is interesting as Biolog per-formed poorly in identifying genomic species of Acineto-bacter in earlier studies (KNIGHT et al., 1993; EHRENSTEIN

Pyrolysis Mass Spectrometry (PyMS) 441

et al., 1996). Of the strains not phenotypically identified,the cluster of isolates constituting Group G is of particularinterest as their separation from the rest of the strains mayindicate the existence of new genomic species.

While the diversity of these fingerprints suggests theexistence of new genomic species of Acinetobacter amongthese isolates from activated sludge, caution must be ex-ercised when interpreting this kind of data. The numberof ribosomal operons can vary markedly in bacteria andare known to range from between 1 and 11 (KOSTMAN etal., 1992; GURTLER & STANISICH, 1996). Spacer regionsfound within these multiple operons can also show con-siderable variation in both their length and sequences.This diversity is often due to variations in the numbersand types of tRNA sequences found within the spacer re-gions (LOUGHNEY et al., 1982). DOLZANI et al. (1995)showed that members of the Acb complex had five or sixspacer sequences per genome, respectively, while GRAL-TON et al. (1997) found that Acinetobacter sp. strainADP1 contained 7 copies of the rRNA operon. Examin-ing sequence variability of all multiple operons within anorganism, some of which might be close or identical in se-quence, is difficult without the sequence of the wholegenome (NAGPAL et al., 1998). The sequence variability ofthe multiple operons in Acinetobacter is currently un-known and it is possible that variations in their sequenceswithin the genomic species of Acinetobacter may be sogreat as to make differentiation of these closely relatedstrains very difficult with this technique. Further work isrequired to examine the numbers of operons in theknown genomic species of Acinetobacter and investigatethe sequence variability between them.

However, even allowing for these possibilities, on thebasis of the data presented in this study it would appearthat the biodiversity of Acinetobacters in activated sludgeis much greater than that suggested from the currently de-scribed DNA groups of Acinetobacter. This is supportedby the findings that current phenotypic identificationschemes suitable for clinical Acinetobacter strains are notsatisfactory for identification of activated sludge isolates(SODDELL et al., 1993). If molecular techniques like FISHare to be used to elucidate the population dynamics ofAcinetobacter spp. and their role in activated sludge, thenthese techniques should be able to detect all isolates ofthis genus. If not, then the data may be misleadeading.

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Corresponding author:EMMA CARR, La Trobe University Bendigo, Biological Sciences,Edwards Rd, Bendigo 3550, Australiae-mail: emma.carr@bendigo.latrobe.edu.au