stability of clonally related dna fingerprints and cell ... · peptides generated by enzymatic...

Original Report

Stability of Clonally Related DNA Fingerprints and Cell-Wall Peptide Patterns in Geographic Isolates of Multiresistant Epidemic Clones of Streptococcus pneumolziae Mirjana Nesin, MD;*+ Anatoly Severin, PhD;* and AlexanderTomasz, PhD*

ABSTRACT

Objective: To determine the stability of multidrug-resistant epidemic clones of Streptococcus pneumoniae.

Methods: Polymerase chain reaction (PCR) was used to amplify the penicillin-binding protein (PBP) genes IA, 2X, and 2B, fol- lowed by fingerprinting after restriction digestion of the amplified molecules. Cell walls were isolated, digested by the autolytic amidase of pneumococcus, and the family of stem peptides was separated by high-pressure liquid chromatography.

Results: Twenty-eight isolates of multidrug-resistant capsular type 6B S. pneumoniae recovered in Iceland between 1990 and 1992 and ten isolates of the multidrug-resistant capsular type 23F S. pneumoniae recovered in the United States, Por- tugal, Croatia, and South Korea between 1989 and 1996 were compared for restriction fragmentation length polymorphism (RFLP) of their PBP genes IA, 2X, and 2B, using the PCR for amplification. Five isolates each of the multidrug-resistant capsular type 6B pneumococci from Iceland and the capsular type 23F isolates from Cleveland, Ohio, were also compared for their muropeptide profiles, using a high-performance liquid chromatography (HPLC) system to separate the family of stem peptides generated by enzymatic hydrolysis. The isolates rep- resenting the two major and internationally spread epidemic clones have retained the clone-specific RFLP fingerprints of their PBP genes and also reproduced distinct cell-wall stem peptide patterns characteristic of the two pneumococcal clones.

Conclusions: The data further document the considerable stability of clonal types of the multiresistant epidemic clones of S. pneumoniae over time and over a wide range of geographic isolation sites.

*The Rockefeller University, New York, New York; +Perinatology Center, Cornell University Medical College, New York, New York.

Supported in part by grant ROI AI37275 from the National Institutes of Health, by the Aaron Diamond Foundation, and by the Bodman/ Achelis Fund.

Received: July 23,1997; Accepted: August 22,1997.

Address correspondence to Dr. AlexanderTomasz,The Rockefeller Uni- versity, 123OYork Avenue, NewYork, NY 10021-6399.

Key Words: cell-wall peptides, DNA probes for penicillin- binding proteins, multiresistant clones, Streptococcus pneumoniae

Int J Infect Dis 1998; 2:91-98.

Of the very large number of penicillin-resistant clones of Streptococcus pneumoniae, only a few have been shown to spread extensively over large geographic distances. One of these, a multidrug-resistant clone sometimes referred to as the Spanish/USA clone usually expressing a capsular type 23F,’ was documented to have spread extensively in Europe,lm9 the United States,1,10-12 South Africa,13J* South Korea,15J6 and South America.” Another distinct multidrug-resistant lineage, referred to as the Spanish/Icelandic clone and expressing capsular type GB, was spread extensively in Spain, the United Kingdom, and Iceland,ls where these bacteria have reached a sur- prisingly high prevalence and have been shown to be responsible for up to 20% of all invasive pneumococcal disease between 1993 and 1996.19

The microbial factors that may be involved with the epidemicity of these two clones are not known at the present time.To understand the factors that contribute to the extensive geographic spread of these clones, it was important to test the stability of the clone-specific properties of these bacteria. Earlier studies have already established the uniformity of the multidrug-resistant Icelandic isolates with regard to their multilocus enzyme profiles and pulsed-field gel electrophoretic (PFGE) profiles.‘* Evidence was also obtained for the similarity of PFGE patterns in isolates belonging to the Spanish/USA clone from South Korea,‘j Croatia,* PortugaJ6 France,2 and the United States.‘O Recent work also has established that isolates of this capsular type 23F clone recovered in South Africa and Germany share PBP mosaic genes of comparable fingerprint patterns.3s16 The ultimate purpose of this line of investigations is to obtain some insight into microbial factors involved with the remarkable epidemicity of these pneumococcal clones. In the investigations described here, the characterization of pneumococcal isolates belonging to the Spanish/USA and Spanish/Icelandic clones were extended by deter- mining their PBP fingerprint patterns and by comparing the stem peptide composition of their peptidoglycans.

91

92 International Journal of Infectious Diseases / Volume 2, Number 2, October-December 1997

MATERIAL AND METHODS

Bacterial Strains

Twenty-eight serotype 6B isolates were selected from the Icelandic strains, previously characterized by Soares et a1.18 Most of these isolates already had been shown to share an identical and unique pulsed-field gel electrophoresis (PFGE) pattern and multilocus enzyme profile, and they exhibited identical patterns of multidrug resistance to six antibacterial agents.lx Ten serotype 23F multiresistant isolates from different geographic locations

(two each from New York City, North Carolina, Croatia, Portugal, and South Korea) and four penicillin-susceptible isolates of the same serotype from North Carolina, were from the S.pneumoniae strain collection at Rockefeller UniversityThe penicillin-resistant 23F isolates shared similar PFGE patterns and multilocus enzyme profiles.’ Some relevant properties of these isolates are summarized in Table 1. Antibiotic susceptibilities were determined by disk diffusion following recommendations and breakpoint definitions of the National Committee for Clinical Labo- ratory Standards (NCCLS).20

Source

Table 1. Some Relevant Properties of the Multiresistant Streptococcus pneumoniae Strains

Antibiotic Resistance PFGE

Gene Profile

Strain Serotype Pen M/C Cam Tel Ery Pattern PBP 1A PBP 2x PBP 28

Iceland Ic 23 Ic 27 Ic 28 Ic 30 Ic 33 Ic 34 Ic 35 Ic 36 Ic 39 Ic 40 Ic 41 Ic 42 Ic 43 Ic 44 Ic 45 Ic 46 lc 48 Ic 49 Ic 50 Ic 51 Ic 52 Ic 53 Ic 54 Ic 55 Ic 56 Ic 57 Ic 58 Ic 60

Spain Md 1238 Sp 647 S&652

Alaska Ala1 Ala31

New York City Alb North Carolina 230

231 Portugal 1013

1118 Croatia 28

35 South Korea 2

4 North Carolina 30

119 157

6B 2 6B 1 6B 1 6B I 6B 1 6B 2 6B 1 6B 1 6B 1 6B 1 6B 2 6B 1 6B 1 6B 1 6B 1 6B 1 6B 0.5 6B 0.5 6B 0.5 6B 1 6B 0.5 6B 0.5 6B 0.5 6B 0.5 6B 1 6B 2 6B 2 6B 0.5 6B 2 6B 2 6B 2 6B 0.1 6B 0.1 23F R 23F R 23F R 23F R 23F R 23F R 23F R 23F R 23F R 23F S 23F S 23F S

R I R R I

R I I I I I I

R R R R R R S R R I I R I

R R - - S S R R R R R R R R R S S S

R R R R R R R R R R R R R R R R R R R R R R R R R

R R

S S R R R R R R R R R S S S

R I R R R R I

R R R I R R R S R R R R I

R R R R R R R R R - - S I

S R R S S S S R R S S S

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 - - - 1 1 I 1 1 1 1 1 1 1 1 2 2 2

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I 1 1 1 1 1 1 1 1 1 1 1 1 I+2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 I+3 1 I+3 2 4 1 4 3 5 3 5 3 5 3 5 3 5 3 5 3 5 3 5 3 5 4 6 4 6

4+5 6

*Minor variants of PFGE types OL and 4. PFGE = pulsed-field gel electrophoresis; Pen = penicillin: Cam = chloramphenicol; Tet = tetracycline; Ery = etythromycin. Susceptibilites defined according to NCCLS.20

PBP Gene and Cell-Wall Fingerprints in S.pneumoniae Clones / Nesin et al 93

Isolation of the Penicillin-Binding Protein Genes lA, 2B, and 2X

The fragments of genes coding for the penicillin-binding domains of PBPs lA, 2B, and 2X were amplified from the chromosomal DNA by PCR.The protocol used was a modification of the method described by Zhang et al.‘l Pneu- moccocal isolates were grown in a casein-hydrolysate- based and yeast extract supplemented (C + Y) medium until ODGOO = 0.7 to 0.9 was reached.22 Three microliters of the cultures were diluted in 147 microliters of water and lysed by immersing in a boiling water bath for 2 minutes. Cell lysates were used directly for the PCR.The gene fragments of PBP 2X and 2B were each amplified in a 50 FL reaction mixture containing 35 ~.LL of cell lysate, 2 mM MgCl,, 1 PM oligonucleotide primers, 0.2 mM deoxynucleoside triphosphates, 1.25 ~J,L of Taq DNA polymerase, and 5 yL of 10X PCR buffer (Gene Amp PCR Core Reagents, Perkin Elmer, Branchville, NJ). The gene fragments of PBP 1A were amplified in the 100 FL reaction mixture. The composition of the reaction mixture was the same as that used for the amplification of PBP 2X and PBP 2B gene fragments.The 1.5-kb PBP 2B gene fragments were amplified using the primers Pn 28 up and J+z 28 down as described by Dowson et a1.23 The 2-kb PBP 2X gene fragments were amplified using primers Pn 2X up and Pn 2X down as described by Munoz et al.‘* The gene coding PBP 1A was amplified as a 2.2-kb fragment with the oligonucleotides Pn la and Pn la rev, as described by McDougal et aLz5 The PCR cycling con- ditions were: denaturation at 95°C for 5 minutes and 72°C for 10 minutes; 30 cycles of 90°C for 30 seconds, 50°C for 30 seconds, and 72°C for 3 minutes, and a final exten- sion period of 72°C for 7 minutes. Amplified fragments were purified by preparative electrophoresis from the 5% polyacrylamide gel as described by Maxam and Gilbert.z6

DNA Fingerprinting of Penicillin-Binding Protein Genes lA, 2B, and 2X

The original method described by Zhang et al was used according to the modifications introduced by Munoz et a1.21,24 The amplified fragments of PBP lA, 2X, and 2B genes were digested with Hi@ and StyLThe PBP 2X fragments also were digested with MseI and DdeI. Of these restriction nucleases, Hinfl and Sty1 proved to have the best resolving power, and therefore, only fingerprints obtained with these enzymes are shown in the figures. Restriction fragments were end-labelled: [35S]dCTP was used for Sty1 generated fragments and [35SldATP for Hinfl, or MseI and DdeI generated fragments. Fragments were separated on an 8% nondenaturing polyacrylamide gel (25 X 20cm)The samples were electrophoresed at 150 V, until a bromphenol blue marker dye reached 5 mm from

the bottom of the gel, transferred to filter paper, dried under vacuum, and autoradiographed.

Plasmid DNA pBR322 digested with MspI and labelled with [35S]dCTP was used as a size marker. Frag- ment sizes are: 622,527,404,307,242, -200, -180, -160, -150,120,110,90,76,67, and 34 base pairs from the bottom to the top, respectively.

Cell-Wall Stem Peptide Fingerprints

Procedures for the isolation and enzymatic digestion of cell walls and the separation of the family of stem peptides by reverse phase high-performance liquid chromatography (HPLC) have been described.27,28

RESULTS

The pneumococcal isolates examined here have already been examined by PFGE and multilocus enzyme analysis (MLEA).The apparent stability of the multiresistant Span- ish/USA and Spanish/Icelandic clones over large geographic distances and considerable time intervals between dates of isolation is surprising in view of the capacity of S. pneumoniae to undergo spontaneous genetic transformations in vivo. Indeed, determinants encoding the capacity of pneumococci to develop competence for genetic transformation were recently shown to be ubiquitous among natural isolates of this bacterial species.29 The stability of these two widely spread multiresistant clones may be related to the presence of some microbial factors involved with the superior epidemic&y of the two clones. In an attempt to obtain some further insight into this question, we undertook to examine the clone-specific fingerprints of the PBP genes in represen- tatives of the 23F and 6B pneumococcal clones, and also examine the composition of their cell-wall peptidoglycan. Uniformity of PFGE and MLEA patterns in these isolates does not exclude the possibility of modification or replacement of the original clone-specific PBP genes among these isolates. In vivo acquisition and exchange of resistant PBP genes and genes involved with the synthe- sis of the polysaccharide capsule without effect on the chromosomal macrorestriction pattern have been documented.30,31 Determination of the composition of the pep tidoglycan was of particular interest, since the radically altered cell-wall composition in some penicillin-resistant pneumococci has raised the possibility that such modification of cell-wall composition may be a frequent or even obligatory consequence of the alteration of penicillin-binding proteins in resistant pneumococci.

DNA Fingerprints of Icelandic Isolates

Twenty-eight isolates of serotype 6B multidrug-resistant S. pneumoniae causing clinical infections in Iceland between April 1989 and September 1991 were analyzed


Figure 1. DNA fingerprint patterns of the PBP genes 1 A, 2X, and 28 amplified from multidrug-resistant capsular type 6B isolates from Ice- land. A, (HirVl) and B, (Styl) DNA fingerprints of PBP 1A from a group of Icelandic isolates identified through strain numbers marking the lanes in the gels. C, PBP 2X fingerprints generated by Hinfl digestion; D, (Styl) and E (H&l) PBP 28 fingerprints of a select group of Icelandic isolates. Other properties of these strains are presented in Table 1.

(see Table 1). An earlier study had already established the similarity of genetic backgrounds in these strains, using PFGE of SmaI macrorestriction digests and MLEA.” In the present study, characterization was extended to the comparison of the PBP mosaic gene fingerprints of these isolates. Figure 1 illustrates the PBP 1A DNA fingerprints obtained by the use of Hin. (Figure 1, A) and Sty1

PBP 28 (Hinf) Iceland Ala Sp --

h 54 55 56 57 58 ,60 1 31 647

Figure 2. DNA fingerprints of the PBP 2B gene amplified from capsular type 6B penicillin-resistant isolates originating in Iceland, Alaska, and Spain. Other proper-ties of these strains are shown in Table 1.

(Figure 1, B) enzymes, and fingerprints for PBP 2X are shown in Figure 1, C. The similarity of fingerprint patterns is apparent.

Figure 3. DNA fingerprints of PBP genes IA and 2X amplified from multidrug-resistant capsular type 23F S. pneumoniae recovered at a vari- ety of geographic locations, Penicillin-binding protein genes are amplified using the restriction enzyme Hinfl for PBP IA (A) and for PBP 2X (B). The origin of these strains is indicated.


Figure 4. DNA fingerprints of the PBP 28 gene amplified from multidrug-resistant capsular type 23F S. pneumoniae recovered at different geographic locations. The PBP 2B fingerprints were generated by the Hinfl restriction endonuclease.

The DNA fingerprints of the PBP 2B gene for a select group of Icelandic isolates is shown in Figure 1, D. Again, similarity of fingerprints is apparent with the exception of a single isolate, Ic 45, in which one of the two restriction nucleases (Hi@) was able to detect a differential fingerprint pattern. Interestingly, Ic 45 previously had been shown to be distinguishable from the other Ice- landic isolates through a small change in PFGE pattern and through change in one of the alleles used in the MLEA.18 Icelandic isolate 45, unlike most of the other Icelandic strains, was found to be susceptible to erythromycin.

Figure 2 shows Hi@ fmgerprints of the PBP 2B gene from a group of additional serotype 6B isolates from Ice- land and also from a Spanish and from two Alaskan isolates. The Spanish isolate, Sp 647, and the two Alaskan isolates, Ala1 and Ala 31, each belonged to two distinct

I I I 25

I 50

I I I 75 25 50 75

Time (min)

Figure 5. High-performance liquid chromatography elution profiles of stem peptides generated by enzymatic hydrolysis of cell walls of four S. pneumoniae strains: strain R36A, a penicillin-susceptible laboratory strain showing the species-specific stem peptide profile of S. pneumoniae; strain Pen 6, a penicillin-resistant transformant derivative of a South African multiresistant strain of fneumococcus; Ic 24 is representative of the capsular type 6B Spanish/Icelandic clone, and Ohio 7, of the capsular type 23F Spanish/USA clone. Roman and Arabic numerals refer to different stem peptide species, the structures of which are shown in Figure 6.

genetic lineages characterized through PFGE and MLFA.** Figure 2 shows that the PBP 2B fmgerprints of these three distinct penicillin-resistant clones also reflected their dif- ferences: common fingerprints were shared by the Ice- landic isolates; a distinct fingerprint was produced by Sp 647 and a third, different, fingerprint is shown by the two Alaskan strains.

DNA Fingerprints of Multiresistant Capsular ‘Qpe 23F Isolates of S. pneumoniae Recovered at Distant Geographic Locations

Ten clonally related serotype 23F isolates were analyzed: two each from NewYork City, North Carolina,iz Portugal6 Croatia,* and South Korea.15 These isolates were consid- ered to be clonally related since they have shown the same antibiotic susceptibilities and the same PFGE patterns.The DNA fingerprint patterns generated by Hi@ of the PBP 1A and 2X genes (Figure 3) and those of the PBP 2B genes (Figure 4) were identicalThe use of restriction nuclease Sty1 confirmed the identity of these patterns.

Whereas the DNA fingerprints of the PBP genes 2X and 28 were each unique for the two particular multiresistant clones, the PBP 1A fingerprints of the Icelandic and Spanish/USA clonal isolates were similar and did not allow distinction between these two lineages.This finding confirmed the observation already noted by Martin et a1,32

96 International Journal ofInfectious Diseases / Volume 2, Number 2, October-December 1997

R36A

1 2 3

Ala Ala Ala I I I

iGln iGln iGln

L:s L:s

A!,

Ala -SW-L:s

A!a

4 5

Ala Ala Ala Ala I I

iGln iGln I I

iGln iGln

LA L:s L:S I

Ala’ A/a - p\”

*&YS

6A

Ala 1

iGln

Ala

iGh

60

Ala Ala I I

iGln iGln I

Ala -SW-LY”, , L:s

7

Ala Ala I I

iGln iGln I

,AYS

8

Ala I

iGln

Ala Ala I I

iGln iGln

L:S

Pen 6

I II III

Ala Ala Ala I I I

iGln iGln iGln

Ala -Ala -Lbs Ala -Ser -Lh

A!a

Ala -Ala -Lh

A\a

A\a Ala

VI V

Ala Ala Ala Ala I I I

iGln iGln iGln I

iGln

Ala -Ala -L& I I

c pwLys Ala -Ala -L:s

Ala * Ha A/a * 5e’LYS

p\a

IV

Ala Ala I I

iGln iGln

Ala -SW-L:s I

Ala . p,\a’ NefLYS

VII

Ala Ala Ala I I I

iGln iGln iGln

Ala-Ser-L:s I I

&YS JYS /jla,NaaN Aia,flaMva

VIII

Ala Ala Ala I I I

iGln iGln iGln

Ala-Ala-L:s I I

LYS A~a~~a*se’+~~rp-fla

IX

Ala Ala Ala I I I

iGln iGln iGln

Figure 6. Structures of stem peptides identified in strains R36A and Pen 6 of S. pneumoniae. Identification of the structure of these stem peptides was done in previous studies.Z7,*8

who showed that the DNA fiigerprints of PBP IA may not be useful markers for molecular epidemiology.

Three penicillin-susceptible 23F serotype isolates also were compared for their corresponding PBP fingerprints.These isolates shared among themselves common patterns that were different from those of either the capsular type 6B or the 23F multiresistant isolates (see Table 1).

Cell-Wall Stem Peptide Patterns in Representative Isolates of the Spanish/Icelandic and the Spanish/ USA Clones of Streptococcus pneumoniae

Penicillin-resistant clinical isolates of S. pneumoniae often show an abnormal stem peptide composition in

their peptidoglycans, which deviate from the common and species-specific peptidoglycan pattern common to penicillin-susceptible strains.z7 Cell walls were isolated from five capsular type 6B Icelandic isolates (Ic 24, 25, 26,3 1, and 32) and five capsular type 23F isolates (Ohio 3-7) and the muropeptide patterns were determined after digestion with pneumococcal autolytic amidase fol- lowed by separation on reverse-phase HPLC columns. Figure 5 shows the stem peptide profiles of one of the Icelandic isolates (Ic 24) and one of the Spanish/USA isolates (Ohio 7). Also shown for comparison are the stem peptide patterns of strain R36A (the prototype of the species-specific stem peptide composition) and the grossly distorted stem peptide composition shown by strain Pen 6, which is a genetic transformant constructed


Table 2. Cell-Wall Stem Peptide Composition of Penicillin-Resistant Pneumococcal Isolates belonging to the Icelandic Serotype 6B Clone

C/one Iceland Serotype 66

Ic 24 Ic 25 Ic 26 Ic 31 Ic 32 Mean R36A

Pep tide Material (%) Dimer Total Peptide (%)

Recovery Monomer and Linear Branched 1 2 3 I II 4 111 5 6 7 IV v 8 VI 9 W) (%) Trimer (%) (L) (B)

26.8 2.8 4.0 4.3 0.5 26.2 0.1 11.8 13.0 0.9 0.8 1.0 3.5 3.0 1.3 74 38 62 67 33 28.3 3.0 6.0 3.0 0.4 25.7 0.1 14.6 9.0 2.3 0.7 0.8 3.4 1.4 1.3 75 41 59 6% 32 27.5 2.0 4.8 3.1 0.5 29.8 0.2 11.7 9.9 1.8 0.3 0.8 3.7 3.1 0.8 76 38 62 69 31 28.9 1.2 6.0 3.6 0.4 27.1 0.2 12.8 9.5 2.1 0.6 0.6 3.6 1.8 1.7 74 40 60 68 32 29.9 1.2 4.1 3.6 0.4 31.0 0.2 9.2 9.4 2.4 0.6 0.8 3.5 2.3 1.3 76 39 61 71 29 28.3 2.0 5.0 3.5 0.4 28.0 0.2 12.0 10.2 1.9 0.6 0.8 3.5 2.3 1.9 39 61 69 31 22.3 4.5 3.7 1.5 - 31.2 2.2 14.4 5.7 4.5 0.7 0.7 5.7 - 2.2 34 66 70 30

B:L

0.5 0.5 0.45 0.5 0.4 0.5 0.4

by crossing DNA from the highly penicillin-resistant South African strain into strain R36A, and selecting for donor level penicillin resistance.33 The numbers assigned to the peptide peaks represent differently structured stem peptides, the chemical formulae of which are shown in Figure 6.

The quantitative composition of the peptidoglycans resolved to 15 different stem peptides is shown inTable 2, for the five Icelandic isolates, and inTable 3, for the five Spanish/USA serotype 23F isolates. Comparison of the means of representation of the different stem peptide species in the two different multiresistant clones to those of strain R36A shows that these particular multiresistant clones had similar composition to that of the species-specific peptidoglycan of S.pneumoniae.**The main differ- ences were in the ratio of the major monomeric (peptide 1) and dimeric (peptide 4) components: whereas in the species-specific pattern (R36A) there was an abundance of peptide 4 (31.2%) over peptide 1 (22.3%) the ratio of these two peptides was close to unity in the Icelandic isolates (28% each) and the monomeric peptide 1 (24.8%) was more abundant than peptide 4 (19.6%) in members of the multiresistant serotype 23F clone (see Table 3). Additional, significant changes were apparent in some of the less abundant peptide components (for instance, the reduced amounts in peptide III in the Icelandic isolates or the increase of peptide VI in both the Icelandic and the 23F isolates over the representation of these stem peptides in the peptidoglycan of strain R36A (see Tables 2 and 3).

SUMMARY

The DNA fingerprinting and cell-wall profiling data described here confirm the stability of the Icelandic and Spanish/USA clones over different isolation dates and dif- ferences in geographic sites of isolationThe continued worldwide spread of these multiresistant clones makes it important to better understand microbial factors that may be associated with the epidemicity of these pneumococcal genetic lineages. It is conceivable that the relative closeness of the cell-wall composition of these two clones to that of the species-specific composition of cell wall conserved among antibiotic susceptible S. pneumoniae isolates contributes to the “fitness” and the stability of these multiresistant lineages, as evidenced by their abil- ity for massive geographic expansion.

REFERENCES

1. Munoz R, Coffey TJ, Daniels M, et al. Intercontinental spread of a multiresistant clone of serotype 23I; Streptococcuspneu- moniae.J Infect Dis 1991; 164:302-306.

2. Ferroni A, Nguyen L, Gehanno P Boucot I, Berche P Clonal distribution of penicillin-resistant Streptococcus pneumoniae 23F in France. J Clin Microbial 1996; 34:2707-2712.

3. Reichmann P Varon E, Gunther E, et al. Penicillin-resistant Streptococcus pneumoniae in Germany: genetic relation- ship to clones from other European countries. J Med Micro- biol 1995; 43:377-385.

4. Setchanova L,Tomasz A. Unique features of penicillin-resistant Streptococcus pneumoniae isolates from Bulgaria

Table 3. Cell-Wall Stem Peptide Composition of Penicillin-Resistant Pneumococcal Isolates belonging to the Spanish/USA Serotype 23F Clone

Clone Spanish/USA Peptide Material (%) Dimer

Total Peptide (%)

Serotype Recovery Monomer and Linear Branched 23F 1 2 3 I II 4 Ill 5 6 7 IV v 8 VI 9 (%) (%) Trimer (%) (L) (B) B:L

Ohio 3 23.4 2.1 7.5 5.2 0.7 18.4 1.8 11.0 8.6 6.9 1.7 3.3 4.2 2.1 3.2 67 41 59 55 45 0.8 Ohio 4 26.1 0.4 6.8 3.9 0.1 22.6 1.3 11.7 11.8 5.1 2.0 1.4 3.8 1.3 1.6 70 39 61 61 39 0.6 Ohio 5 23.9 2.0 6.8 3.7 0.3 19.8 2.2 11.7 8.5 7.1 1.6 3.0 4.9 1.8 2.7 68 39 61 57 43 0.75 Ohio 6 26.1 2.9 8.1 4.3 0.6 18.8 1.2 12.5 8.3 6.2 1.4 2.9 5.0 - 1.7 67 43 57 58 42 0.7 Ohio 7 24.4 1.6 9.4 3.0 0.6 18.3 1.0 17.9 8.2 6.4 1.2 1.5 3.0 1.1 2.6 77 40 60 58 42 0.7 Mean 24.8 1.8 7.7 4.0 0.5 19.6 1.5 13.0 9.1 6.3 1.6 2.4 4.2 1.6 2.4 40 60 58 42 0.7 R36A 22.3 4.5 3.7 1.5 - 31.2 2.2 14.4 5.7 4.5 0.7 0.7 5.7 - 2.2 34 66 70 30 0.4


[Abstract]. 37th Annual Meeting of the Interscience Con- ference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, September 28-October 1, 1997. Washing- ton, DC: American Society for Microbiology, 1997:54.

5. Marchese A, Schito GC, Tomasz A. Preponderance of multidrug-resistant clones among penicillin-resistant isolates of Streptococcus pneumoniae from Italy [Abstract]. 37th Annual Meeting of the Interscience Conference on Antimi- crobial Agents and Chemotherapy, Toronto, Canada, Sep- tember 28-October 1, 1997. Washington, DC: American Society for Microbiology, 1997.

6. Vaz Pato MV, de Carvalho CB, Tomasz A. The Multicenter Study Group. Antibiotic susceptibility of Streptococcus pneumoniae isolates in Portugal. A multicenter study between 1989 and 1993. Microb Drug Resist 1995; 1:59-69.

7. Lefevre JC, Fauqon G, Sicard AM, Gasc AM. DNA fingerprinting of Streptococcus pneumoniae strains by pulsed- field gel electrophoresis. J Clin Microbial 1993; 31: 2724-2728.

8. Tarasi A, Sterk-Kuzmanovic N, Sieradzki K, Schoenwald S, Austrian R,Tomasz A. Penicillin-resistant and multidrug-resistant Streptococcus pneumoniae in a pediatric hospital in Zagreb, Croatia. Microb Drug Resist 1995; 1:169-176.

9. Figueiredo AMS, Austrian R, Urbaskova P,Teixeira LA,Tomasz A. Novel penicillin-resistant clones of Streptococcus pneumoniae in the Czech Republic and in Slovakia. Microb Drug Resist 1995; 1:71-78.

10. McDougal LK, Facklam R, Reeves M, et al. Analysis of multiply antimicrobial-resistant isolates of Streptococcus pneumoniae from the United States. Antimicrob Agents Chemother 1992; 36:2176-2184.

11, Versalovic J, Kapur V, Mason EO, et al. Penicillin-resistant Streptococcus pneumoniae strains recovered in Houston: identification and molecular characterization of multiple clones. J Infect Dis 1993; 167:850-856.

12. Barnes DM, Whittier S, Gilligan PH, Soares S,Tomasz A, Hen- derson FWTransmission of multidrug-resistant serotype 23F Streptococcus pneumoniae in group day care: evidence sug- gesting capsular transformation of the resistant strain in vivo. J Infect Dis 1995; 171:890-896.

13. Klugman Kp Coffey TJ, Smith A, Wasas A, Meyers M, Spratt BG. Cluster of an erythromycin-resistant variant of the Span- ish multiply resistant 23F clone of Streptococcus pneumoniae in South Africa. Eur J Clin Microbial Infect Dis 1994; 13: 171-174.

14. Sibold C, Wang J, Henrichsen J, Hakenbeck R. Genetic rela- tionships of penicillin-susceptible and -resistant Streptococ- cus pneumoniae strains isolated on different continents. Infect Immun 1992; 60:4119-4126.

15. Tarasi A, Chong Lee YK, Tomasz A. Spread of the serotype 23F multidrug-resistant Streptococcus pneumoniae clone to South Korea. Microb Drug Resist 1997; 3:105-109.

16. McGee L, Klugman KI: Friedland D, Lee H-J. Spread of the Spanish multiresistant serotype 23F clone of Streptococcus pneumoniae to Seoul, Korea. Microb Drug Resist 1997; 3:253-262.

17. Di Fabio JE, Homma A, de Quadros C, et al. Pan American Health Organization epidemiological surveillance network for Streptococcus pneumoniae. Microb Drug Resist 1997; 3:131-164.

18. Soares S, Kristinsson KG, Musser JM,Tomasz A. Evidence for the introduction of a multiresistant clone of serotype 6B Streptococcus pneumoniae from Spain to Iceland in the late 1980s. J Infect Dis 1993; 168:158-163.

19. Kristinsson KG, Hjalmarsdottir MA, Steingrimsson 0. Increas- ing penicillin resistance in pneumococci in Iceland. Lancet 1992; 339:1606-1607.

20. National Committee for Clinical Laboratory Standards (NCCLS). Performance standards for antimicrobial disk susceptibility tests. 5th ed. Approved standards: M2-A5. Vilanova, PA: NCCLS, 1993.

2 1. Zhang QY, Jones DM, Saez-Nieto JA, Perez-Trallero E, Spratt BG. Genetic diversity of penicillin-binding protein 2 genes of penicillin-resistant strains of Neisseria meningitidis revealed by fingerprinting of amplified DNA. Antimicrob Agents Chemother 1990; 34:1523-1528.

22. Lacks S, Hot&kiss RD. A study of the genetic material deter- mining an enzyme activity in Pneumococcus. Biochim Bio- phys Acta 1960; 39:508-518.

23. Dowson CG, Hutchison A, Spratt BG. Extensive remodelling of the transpeptidase domain of penicillin-binding protein 2B of a penicillin-resistant South African isolate of Strepto- coccuspneumoniae. Mol Microbial 1989; 3:95-102.

24. Munoz R, Musser JM, Crain M, et al. Geographic distribution of penicillin-resistant clones of Streptococcuspneumoniae: characterization by penicillin-binding protein profile, sur- face protein A typing and multilocus enzyme analysis. Clin Infect Dis 1992; 15:112-118.

25. McDougal LK, Rasheed JK, Biddle m,Tenover FC. Identifi cation of multiple clones of extended-spectrum cephalo- sporin-resistant Streptococcus pneumoniae isolates in the United States. Antimicrob Agents Chemother 1995; 39: 2282-2288.

26. Maxam AM, Gilbert W DNA sequencing. Methods Enzymol 1980; 65:499-510.

27. Garcia-Bustos JF, Chait BT,Tomasz A. Structure of the peptide network of pneumococcal peptidoglycan. J Biol Chem 1987; 262:15400-15405.

28. Severin A,Tomasz A. Naturally occurring peptidoglycan variants of Streptococcus pneumoniae. J Bacterial 1996; 178:168-174.

29. Ramirez M, Morrison DA,Tomasz A. Ubiquitous distribution of the competence-related genes comA and comC among isolates of Streptococcuspneumoniae. Microb Drug Resist 1997; 3:39-52.

30. Coffey TJ, Dowson CG, Daniels M, et al. Horizontal transfer of multiple penicillin-binding protein genes, and capsular biosynthetic genes, in natural populations of Streptococcus pneumoniae. Mol Microbial 1991; 5:2255-2260.

31. Nesin M, Ramirez M,Tomasz A. Capsular transformation of a multidrug-resistant Streptococcus pneumoniae in vivo. J Infect Dis 1998 (in press).

32. Martin C, Sibold C, Hakenbeck R. Relatedness of penicillin- binding protein 1A genes from different clones of penicillin- resistant Streptococcuspneumoniae isolated in South Africa and Spain. EMBO J 1992; 11:3831-3836.

33. Zighelboim S, Tomasz A. Penicillin-binding proteins of the multiply antibiotic-resistant South African strains of Strep- tococcuspneumoniae. Antimicrob Agents Chemother 1980; 17:434-442.

stability of clonally related dna fingerprints and cell ... · peptides generated by enzymatic...

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