Research Article
1 October 2006

Genetic Background and Antibiotic Resistance of Staphylococcus aureus Strains Isolated in the Republic of Georgia

ABSTRACT

The genetic composition and antibiotic sensitivities of 50 clinical isolates of Staphylococcus aureus obtained from various clinics in the Republic of Georgia were characterized. S. aureus strains ATCC 700699 and ATCC 29737 were included as reference standards in all analyses. All 52 strains had identical 16S rRNA profiles. In contrast, pulsed-field gel electrophoresis (PFGE) identified 20 distinct PFGE types among the 52 strains examined, which indicates that PFGE is more discriminating than is 16S rRNA sequence analysis for differentiating S. aureus strains. The results of our PFGE typing also suggest that multiple genetic subpopulations (related at the ca. 85% similarity level, based on their SmaI PFGE patterns) exist among the Georgian S. aureus strains. Twenty-two of the 50 Georgian strains were methicillin resistant and PCR positive for mecA, and 5 strains were methicillin sensitive even though they possessed mecA. None of the strains were vancomycin resistant or contained vanA. The nucleotide sequences of mecA fragments obtained from all mecA-containing strains were identical. Our data indicate that the population of S. aureus strains in Georgia is fairly homogeneous and that the prevalence of methicillin-resistant, mecA-positive strains is relatively high in that country.
Staphylococcus aureus is an important opportunistic pathogen responsible for a variety of diseases, ranging from minor skin infections to life-threatening systemic infections, including endocarditis and sepsis (31). It is a common cause of community- and nosocomially acquired septicemia, and approximately 260,000 of 2 million infections nosocomially acquired annually in the United States are associated with S. aureus (13). The bacterium is responsible for approximately one-half of all skin and connective tissue infections, and it is one of the most common causes of surgical site infections. The mortality rate for S. aureus septicemia ranges from 11 to 48% (33).
Penicillin was one of the antibiotics most commonly used to curtail S. aureus infections; however, the rapid emergence and spread of penicillin-resistant S. aureus strains has severely limited the use of that antibiotic, and methicillin rapidly became one of the first synthetic antibiotics developed to treat penicillin-resistant staphylococcal infections. However, after the initial emergence of methicillin-resistant S. aureus (MRSA) strains (which also are resistant to oxacillin and nafcillin) in the 1980s, their prevalence increased drastically in the United States and abroad (5, 23, 39). Methicillin resistance in S. aureus is mediated by a specific penicillin-binding protein (PBP 2A) with a decreased binding affinity for β-lactam antibiotics (17). This phenotype is conferred by the mecA gene, which encodes the 2A protein (7, 17). mecA is carried by a mobile genetic element, called the “staphylococcal-cassette chromosome mec” (SCCmec), which is localized in the S. aureus chromosome (22, 43). The evolution of SCCmec and the mechanisms responsible for the spread and acquisition of mecA are not fully understood, and their elucidation is important for designing proper intervention strategies and infection control measures to prevent the spread and continued emergence of MRSA strains. The glycopeptide antibiotics, particularly vancomycin, are effective for treating MRSA infections; however, although overt resistance to vancomycin has not yet been documented for clinical S. aureus isolates, strains with reduced sensitivity to vancomycin or strains that are resistant to vancomycin have been reported (8, 18, 46) in Japan, the United States, Europe (France, the United Kingdom, and Spain), and the Far East (Hong Kong and Korea). The emergence of vancomycin resistance in S. aureus is a serious public health concern and is likely to have a dramatic negative impact on many current medical practices.
Improving our understanding of the genetic structure of the global S. aureus population, and of the mechanisms responsible for the acquisition and spread of the major antibiotic resistance genes within the species, is of clear public health importance. However, while data concerning the prevalence and genetic composition of S. aureus in the United States (11, 25, 37) and in many other countries around the world (1, 6, 10, 38, 40) have been broadly reported, similar information is still very limited for the countries of the former Soviet Union (FSU) (32, 44). Indeed, there is a striking paucity of data in the English literature about the genetic background of S. aureus strains isolated in the FSU and about the genetic relationships among the various lineages. Furthermore, the prevalence and nature of the major antibiotic resistance genes in S. aureus isolates from the FSU have not been reported in peer-reviewed English-language journals. Therefore, the goals of the present study were to determine (i) the prevalence of S. aureus strains in major clinics in the country of Georgia (one of the republics of the FSU), (ii) the sensitivity of Georgian S. aureus strains to methicillin/oxacillin and vancomycin, (iii) the genetic background of, and the relationships among, Georgian S. aureus strains, and (iv) the prevalence and genetic composition of methicillin resistance genes in the Georgian S. aureus population. During the course of these studies, we also compared the abilities of 16S rRNA sequencing-based typing and pulsed-field gel electrophoresis (PFGE) typing to differentiate S. aureus strains.

MATERIALS AND METHODS

Bacterial strains, biochemical properties, growth conditions, and antibiotic sensitivity.

Our bacterial collection contained 50 S. aureus strains isolated from various clinical specimens in Georgia during 2002 to 2004 and 2 ATCC S. aureus strains included for comparative purposes (Table 1). The 50 Georgian S. aureus strains were collected as part of a comprehensive study designed to elucidate the molecular epidemiology and antibiotic resistance of bacterial infections in Georgia. The study specifically examined the prevalence and antibiotic susceptibility patterns of S. aureus, Pseudomonas aeruginosa, and Enterococcus spp. in Georgia. Samples and strains were collected from seven clinical centers involved in the study (Table 1). According to data from the Georgian National Center of Infectious Diseases and Statistics (NCIDS), >1,000 S. aureus strains were isolated from all clinics participating in the study during the project's 3-year duration. The 50 strains described in the present report were randomly selected from this large collection of strains, and there was no obvious association among the patients from whom the strains were isolated. The sources of the strains and other pertinent information are summarized in Table 1.
All of the strains were grown (37°C, overnight) on Luria-Bertani (LB) agar or in LB broth. The strains' biochemical properties were determined using the API Staph System (bioMérieux, Inc. Durham, N.C.), and their sensitivities to methicillin/oxacillin and vancomycin were determined by using Etest (AB Biodisk, North America, Inc., Piscataway, N.J.) according to the manufacturer's instructions.

PFGE.

Plugs were prepared essentially as described by Mulvey et al. (34). Briefly, LB agar plates inoculated with strains from frozen stocks were incubated (37Co, overnight), the bacterial growth was harvested with a sterile loop, and the bacteria were dispersed in aliquots (0.5 ml) of cell suspension buffer (10 mM Tris-HCl buffer [pH 7.2] supplemented with 20 mM NaCl and 50 mM EDTA). The suspension's cell density was adjusted to a McFarland standard possessing a theoretical optical density of 1.25, which corresponds to ca. 2 × 109 CFU/ml. After lysis, proteinase treatment, and washing, the DNA in the plugs was digested with SmaI (40 U/reaction mixture; incubation at 30°C for 16 h), and PFGE was performed with a CHEF Mapper apparatus (Bio-Rad Laboratories, Hercules, Calif.). The PFGE patterns were compared by the Dice coefficient, using GelCompar II software (Applied Maths, Inc., Austin, Tex.). Clustering of strains was based on the unweighted-pair group method using average linkages (UPGMA); bands less than ca. 135 bp were excluded from the analysis. Computer-assisted analyses were performed according to the manufacturer's instructions.

DNA extraction for 16S rRNA and mecA sequencing.

Bacterial genomic DNA required for PCR amplification and sequencing of the 16S rRNA gene was isolated from the plugs by using a QIAEX II gel extraction kit (QIAGEN, Inc., Valencia, Calif.) according to the manufacturer's instructions. Bacterial DNA for mecA amplification and sequencing was extracted from overnight cultures grown in LB broth by using a Promega (Madison, Wis.) genomic DNA extraction kit.

PCR amplification and DNA sequencing.

PCR amplification of the 16S rRNA locus (504 bp) was performed with a RoboCycler Gradient 96 machine (Stratagene Inc., La Jolla, Calif.) and with previously reported 16S rRNA-specific primers (49). An initial denaturation step (94°C, 2 min) was followed by 30 cycles of 94°C for 20 s, 55°C for 20 s, and 72°C for 45 s and then by a final extension step (72°C, 2 min). The PCR primers used to amplify the mecA and vanA loci were those proposed by Murakami et al. (35) and Patel et al. (41), respectively. Amplifications were performed under the following conditions: 30 cycles of 94°C for 20 s, 55°C for 30 s, and 72°C for 1 min, followed by incubation at 72°C for 5 min. Sequencing was performed with an ABI 3700 DNA analyzer (Applied Biosystems, Inc., Foster City, Calif.), and the ClustalW program from the European Bioinformatics Institute (http://www.ebi.ac.uk/clustalw/ ) was used to align the sequences. Sequence dissimilarities were converted to evolutionary distances according to the method of Jukes and Cantor (24).

Nucleotide sequence accession numbers.

DNA sequences have been deposited in GenBank under accession number DQ342242 (from strain 2793G) for the 16S rRNA locus and under accession number DQ320012 (from strain 2793G) for the mecA locus.

RESULTS

Biochemical characterization.

The biochemical properties of the 2 ATCC control isolates and of 45 Georgian strains were typical of S. aureus. The remaining five Georgian strains (4120G, 4544G, 5239G, 5251G, and 5282G) were pyruvate negative, which resulted in their classification as Staphylococcus chromogenes by the API Staph system. However, the five “atypical” strains had the same 16S rRNA type (G1), and they grouped together (in various PFGE types) with other API Staph system-confirmed S. aureus strains. For example, the pyruvate-negative strains 4120G and 5282G belonged to PFGE type P1, as did 17 pyruvate-positive, biochemically typical S. aureus strains.

PFGE typing.

Twenty SmaI-based PFGE types were identified among the 52 strains examined, including the 2 ATCC strains (Fig. 1); 18 of these PFGE types were identified among the 50 Georgian strains. Each of the two ATCC strains had a unique PFGE pattern not found in the Georgian strains (Table 1; Fig. 1). Eleven of the 18 PFGE types distinguished among the Georgian strains were unique, i.e., each contained only a single strain. PFGE type P1 was most common (19 [37%] of the strains), followed by PFGE types P2 and P3 (5 strains each), PFGE type P4 (4 strains), and PFGE types P5, P6, and P7 (2 strains each). The PFGE types containing four or more strains were not clearly associated with a specific patient population or clinic.
The dendrogram constructed on the basis of the strains' SmaI PFGE patterns identified two major clusters, A and B (Fig. 2). Cluster A contained five strains, including ATCC 29737. The remaining 47 strains, including ATCC 700699, were in cluster B. None of the strains in cluster A possessed mecA; however, approximately 60% of the strains in cluster B were mecA positive, including 17 of the 19 strains grouped in PFGE type P1 (Table 1). The remaining 11 mecA-positive strains were distributed among seven other PFGE types, but they all grouped within cluster B (Table 1; Fig. 2). Based on their SmaI PFGE patterns (Fig. 2), all strains (including the two ATCC isolates) were related at a similarity level of approximately 85%.

Antibiotic sensitivity and genetic markers for resistance.

Twenty-two (44%) of the 50 strains isolated from various Georgian clinics were resistant to methicillin/oxacillin (Table 1). ATCC strain 70069 was also methicillin/oxacillin resistant, as expected, and ATCC 29737 was methicillin/oxacillin sensitive. All strains were vancomycin sensitive and vanA negative. The primers for mecA (35) amplified the 533-bp locus in 28 of the 52 strains, including methicillin/oxacillin-resistant ATCC 700699. However, although all of the methicillin/oxacillin-resistant strains were mecA positive, five mecA-positive strains (4299G, 4544G, 5195G, 5227G, and 5246G) were methicillin/oxacillin sensitive (Table 1). The five mecA-positive, methicillin/oxacillin-sensitive strains were not associated with a specific cluster or PFGE type. However, the five PFGE type P2 strains (2565G, 4294G, 4489G, 5251G, and 5409G) and the two PFGE type P6 strains (2550G and 5422G) were mecA negative and methicillin/oxacillin sensitive.

16S rRNA and mecA sequences.

The amplified fragments of the 16S rRNA gene were identical (type G1) for all of the 52 strains examined. Similarly, all mecA sequences were identical for all mecA-positive strains, including ATCC 700699, i.e., all of the mecA-positive strains had the same mecA type, tentatively designated mecA1 (Table 1). Cluster A contained only mecA-negative strains, and all of the mecA-positive strains grouped in cluster B (Fig. 2).

DISCUSSION

To the best of our knowledge, this communication is the first peer-reviewed English-language publication concerning the prevalence, genetic composition, and carriage of major antibiotic-resistance genes in S. aureus strains isolated in Georgia. Phenotypically, most of the Georgian S. aureus strains examined during our study exhibited biochemical properties typical of S. aureus. Five exceptions (strains 4120G, 4544G, 5239G, 5251G, and 5282G) were pyruvate negative, which resulted in their initial classification as S. chromogenes by the API Staph system (bioMérieux). Biochemical reaction profiles have been reported (48) to not always correlate with other phenotypic and genetic characteristics of S. aureus strains (e.g., some MRSA strains lack urease or pyrrolidonylarylaminidase, which can lead to their being incorrectly identified as coagulase-negative Staphylococcus species). However, such strains usually can be differentiated based on their distinct 16S rRNA profiles or other genetic markers (48). The five pyruvate-negative Georgian strains examined during our study had the same 16S rRNA profiles as the biochemically typical S. aureus isolates, and they clustered together with other PFGE-typed S. aureus strains, which resulted in their final classification as S. aureus.
The 16S rRNA data we obtained suggest that the Georgian S. aureus strain population is homogeneous, at least in the locations from which samples were collected, i.e., all of the strains we examined had identical 16S rRNA sequences, irrespective of their origin or source (Table 1). Interestingly, the two non-Georgian (ATCC) strains had the same 16S rRNA sequences as the Georgian strains, strongly supporting the idea that the 16S rRNA gene is highly conserved in S. aureus. The population structure of S. aureus is considered to be largely clonal (15, 42), although recent evidence suggests that recombination has contributed to S. aureus evolution (27). Our 16S rRNA sequencing data support the idea of the clonal structure of S. aureus.
The results of our PFGE typing study revealed a small degree of genetic heterogeneity among the Georgian S. aureus strains, and they showed that multiple genetic subpopulations exist among Georgian S. aureus strains with identical 16S rRNA profiles. Eighteen distinct PFGE types were identified among the 50 Georgian strains we examined. The most common PFGE type (P1) contained 19 S. aureus strains and was not associated with a specific hospital or region of the country. This finding suggests that strains of S. aureus with PFGE type P1 may have an epidemiological advantage over other clones, which contributes to their spread among various Georgian hospitals.
All of the Georgian S. aureus strains were related at a similarity level of approximately 85% (Fig. 1 and 2), further supporting the idea that the overall population of S. aureus in Georgia is homogeneous. These data also demonstrate that PFGE is more discriminating than is 16S rRNA sequence analysis for differentiating S. aureus isolates. PFGE is currently considered the method of choice for typing of S. aureus (3) because it has been reported (2, 21, 45) to be superior to many other approaches used to type that bacterium, including restriction fragment length polymorphism of the penicillin-binding protein, phage typing, fluorescent amplified fragment length polymorphism analysis, sequence analysis of the repeat region within the gene encoding protein A (spa typing), and randomly amplified polymorphic DNA analysis. Our data are in agreement with that idea, and they suggest that PFGE will be more useful for studying S. aureus infections than 16S rRNA sequencing, at least in short-term epidemiological studies. Some authors (4) have indicated that PFGE may not be optimally suited for long-term epidemiological analysis of S. aureus strains.
The presence of mecA appeared to be cluster associated in the Georgian strains we examined, i.e., none of the strains in cluster A was mecA positive, but approximately 60% of the strains in cluster B were mecA positive (Table 1; Fig. 2). The origin and evolution of the mecA locus have been the subject of debate, and at least two theories have been advanced (26, 36) to explain the rapid emergence and spread of mecA-positive S. aureus strains. The single-clone theory proposes that all MRSA strains arose from a single progenitor strain that acquired the mecA determinant (26), and some experimental evidence supporting that idea has been presented (29). The multiclone theory suggests that horizontal transfer of mecA is primarily responsible for the worldwide emergence and spread of MRSA (36). The latter theory has been gaining increased recognition lately, with several investigators (16, 19) presenting data that support it. Also, mecA is fairly homogeneous in all S. aureus strains examined thus far, although small differences in the gene's nucleotide sequence have been described for some S. aureus strains, particularly for human and chicken isolates (30). Our finding that all of the mecA-positive strains we examined (including ATCC 700699) have identical mecA sequences confirms the gene's highly conserved nature. However, because of the relatively small number of strains we analyzed, it is difficult to state with certainty that the dissemination of mecA among the Georgian strains occurred via horizontal gene transfer. In that regard, mec was reported, more than 30 years ago, to be transferable among S. aureus strains by bacteriophage-mediated generalized transduction (9) but not by conjugation (28). More-recent data (14) confirm that horizontal transfer of mecA is relatively frequent within S. aureus. Thus, mecA may have been transferred horizontally among some of the Georgian strains (e.g., among the strains with the least relatedness according to PFGE analysis), but the spread of at least some MRSA strains in Georgia is likely to have been due to dissemination of the same clones (e.g., type P1 strains) within the country.
Five Georgian S. aureus strains (4299G, 4544G, 5195G, 5227G, and 5246G) possessing nucleotide sequences homologous to mecA were susceptible to methicillin/oxacillin in vitro. Some S. aureus strains have been reported (7, 35) to carry the genetic information for methicillin/oxacillin resistance, but only some of these actually expressed the resistant phenotype in vitro. Our data are in agreement with those observations, and they show that S. aureus strains that do not express mecA are also found in Georgia. The five mecA-positive, methicillin/oxacillin-susceptible strains were grouped in closely related PFGE types P2 and P6 (Table 1).
The emergence of multidrug-resistant S. aureus strains is of clear public health importance in many countries throughout the world, and it has become an increasing problem in Georgia. In this context, high rates of resistance to various antibiotics have been reported (20) for Georgian S. aureus strains. For example, 98% of the strains isolated in Georgia in 2002 were resistant to penicillin and ampicillin, and 67% of them were amoxicillin and azithromycin resistant. On the other hand, resistance to tetracycline, oxacillin, gentamicin, tobramycin, erythromycin, clindamycin, and trimethoprim was observed in <50% of the strains, and <10% of them were resistant to ciprofloxacin, nitrofurantoin, and imipenem (20). More-recent data from the NCIDS of Georgia suggest that resistance to some antibiotics has risen sharply in that country. For example, the prevalence of oxacillin-resistant S. aureus strains among hospitalized patients was ca. 50% during 2002 to 2003 (http://www.ncdc.ge/ ) and as high as 69% among patients in intensive-care units. Our data concerning methicillin/oxacillin resistance among the 50 Georgian strains analyzed during the present study are in agreement with those of the NCIDS report cited above, and they suggest that the prevalence of methicillin/oxacillin-resistant, mecA-positive strains is fairly high in Georgia.
Vancomycin resistance and the presence of vanA were not detected in the 50 Georgian strains examined during the present study; therefore, vancomycin resistance does not yet appear to have emerged in Georgia's S. aureus strain population. However, because that antibiotic is being used increasingly in Georgia, the potential emergence of such strains is of concern and should be closely monitored, while monitoring of the further emergence and spread of MRSA in that country should be continued. Specific recommendations for controlling the emergence of vancomycin-resistant staphylococci are available (12), and rigorous surveillance and infection control measures have been reported to be effective in controlling the spread of MRSA in some countries (47). Therefore, implementation in Georgia of the practices described previously (47) or of similar practices modified to fit the country's public health infrastructure should be seriously considered.
FIG. 1.
FIG. 1. PFGE patterns of SmaI-digested DNA of S. aureus strains. Lanes 1 and 24, MidRange II PFG marker (New England Biolabs, Ipswich, Mass.); lanes 2 and 23, λ ladder PFG marker (New England Biolabs); lanes 3 to 15, representative PFGE types from P1 to P13, respectively; lane 16, PFGE type P18; lanes 17 to 22, PFGE types P14, P15, P16, P17, P19, and P20, respectively.
FIG. 2.
FIG. 2. UPGMA dendrogram of S. aureus strains.
TABLE 1.
TABLE 1. Properties of S. aureus strains
StrainsSourceClinicaPFGE type16S rRNA typemecA typePresence or absenceb of: Result by Etestc 
      mecAvanAOXVA
ATCC 700699PusJapanP8G1mecA1+ResistantSusceptible
ATCC 29737UnknownUnknownP20G1 SusceptibleSusceptible
2528GThroat mucusOutpatientP1G1mecA1+ResistantSusceptible
2550GWound smearTCTIP6G1 SusceptibleSusceptible
2560GWound smearTCTIP1G1mecA1+ResistantSusceptible
2565GWound smearTMUCP2G1 SusceptibleSusceptible
2793GWound smearTCTIP1G1mecA1+ResistantSusceptible
2825GWound smearTCTIP1G1mecA1+ResistantSusceptible
2883GWound smearTCTIP1G1mecA1+ResistantSusceptible
2884GWound smearTCTIP1G1mecA1+ResistantSusceptible
4120GWound smearTCTIP1G1mecA1+ResistantSusceptible
4161GWound smearTCTIP3G1mecA1+ResistantSusceptible
4176GBloodCHP1G1mecA1+ResistantSusceptible
4244GWound smearTCTIP5G1 SusceptibleSusceptible
4294GThroat mucusCRHP2G1 SusceptibleSusceptible
4299GWound smearTMUCP9G1mecA1+SusceptibleSusceptible
4356GWound smearTCTIP10G1mecA1+ResistantSusceptible
4259GWound smearTCTIP3G1mecA1+ResistantSusceptible
4437GWound smearOnc. Ctr.P1G1 SusceptibleSusceptible
4469GPusBatumiP11G1 SusceptibleSusceptible
4489GWound smearTCTIP2G1 SusceptibleSusceptible
4544GWound smearTCTIP12G1mecA1+SusceptibleSusceptible
5190GWound smearTCTIP13G1mecA1+ResistantSusceptible
5192GWound smearTCTIP1G1 SusceptibleSusceptible
5195GWound smearTCTIP7G1mecA1+SusceptibleSusceptible
5291GNose mucusOutpatientP4G1 SusceptibleSusceptible
5227GWound smearOutpatientP1G1mecA1+SusceptibleSusceptible
5232GWound smearTCTIP14G1 SusceptibleSusceptible
5234GWound smearTCTIP7G1 SusceptibleSusceptible
5235GWound smearTCTIP3G1mecA1+ResistantSusceptible
5237GWound smearTCTIP1G1mecA1+ResistantSusceptible
5239GWound smearTCTIP4G1 SusceptibleSusceptible
5244GWound smearTMUCP1G1mecA1+ResistantSusceptible
5246GWound smearTCTIP3G1mecA1+SusceptibleSusceptible
5247GWound smearTCTIP15G1 SusceptibleSusceptible
5251GWound smearTCTIP2G1 SusceptibleSusceptible
5254GWound smearTCTIP16G1 SusceptibleSusceptible
5258GWound smearTCTIP4G1 SusceptibleSusceptible
5282GPusOutpatientP1G1mecA1+ResistantSusceptible
5284GNose mucusOutpatientP1G1mecA1+ResistantSusceptible
5290GBloodCHP1G1mecA1+ResistantSusceptible
5292GPusMestiaP1G1mecA1+ResistantSusceptible
5323GWound smearTCTIP17G1 SusceptibleSusceptible
5327GWound smearTCTIP1G1mecA1+ResistantSusceptible
5409GWound smearTCTIP2G1 SusceptibleSusceptible
5410GWound smearTCTIP3G1mecA1+ResistantSusceptible
5411GThroat mucusOutpatientP18G1 SusceptibleSusceptible
5422GWound smearTCTIP6G1 SusceptibleSusceptible
5425GThroat mucusOutpatientP5G1 SusceptibleSusceptible
5426GWound smearTCTIP1G1mecA1+ResistantSusceptible
5424GWound smearTCTIP19G1 SusceptibleSusceptible
5427GWound smearTCTIP4G1 SusceptibleSusceptible
a
CH, Tbilisi 2nd Children's Hospital, Georgian Ministry of Health; CRH, Children's Republican Hospital, Georgian Ministry of Health; Onc. Ctr., National Oncology Center, Georgian Ministry of Health; TCTI, Tbilisi Center of Thermal Injuries, Georgian Ministry of Health; TMUC, Tbilisi Medical University Clinic, Georgian Ministry of Education; Batumi, city in Western Georgia, on the Black Sea coast; Mestia, region in northwestern Georgia.
b
+, presence; −, absence.
c
OX, oxacillin; VA, vancomycin.

Acknowledgments

This study was supported by BTEP grant 10/ISTC G597 from the Biotechnology Engagement Program of the Department of Health and Human Services (to A.S.).
We thank William Conway and Jim Higgins for generous help with the GelCompar II software.

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cover image Journal of Clinical Microbiology
Journal of Clinical Microbiology
Volume 44Number 10October 2006
Pages: 3477 - 3483
PubMed: 17021070

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Received: 17 May 2006
Revision received: 3 July 2006
Accepted: 20 July 2006
Published online: 1 October 2006

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Tamara Revazishvili
Department of Epidemiology and Preventive Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201
Lela Bakanidze
National Center of Infectious Diseases and Statistics, Georgian Ministry of Health, Tbilisi, Georgia
Tsaro Gomelauri
National Center of Infectious Diseases and Statistics, Georgian Ministry of Health, Tbilisi, Georgia
Ekaterine Zhgenti
National Center of Infectious Diseases and Statistics, Georgian Ministry of Health, Tbilisi, Georgia
Gvantsa Chanturia
National Center of Infectious Diseases and Statistics, Georgian Ministry of Health, Tbilisi, Georgia
Merab Kekelidze
National Center of Infectious Diseases and Statistics, Georgian Ministry of Health, Tbilisi, Georgia
Chythanya Rajanna
Department of Epidemiology and Preventive Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201
Arnold Kreger
Department of Epidemiology and Preventive Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201
Alexander Sulakvelidze [email protected]
Department of Epidemiology and Preventive Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201

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