Molecular Evolution of Methicillin-Resistant Staphylococcus aureus in the Metropolitan Area of Cologne, Germany, from 1984 to 1998
http://www.100md.com
微生物临床杂志 2005年第11期
Institute for Medical Microbiology, Immunology and Hygiene, University of Cologne, Cologne, Germany
ABSTRACT
To investigate the molecular evolution of methicillin-resistant Staphylococcus aureus (MRSA) in a large metropolitan area in Germany, 398 nonrepetitive MRSA isolates recovered from patients from various teaching and nonteaching hospitals in Cologne between 1984 and 1998 were characterized by pulsed-field gel electrophoresis (PFGE). On this basis, 95 representative isolates were selected and further investigated by multilocus sequence typing (MLST), spa typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Overall, there were 9 MLST types and 16 spa types. The most prevalent sequence types (STs) were ST239 (38% of isolates), ST247 (29%), and ST228 (18%); the most prevalent spa types were 37 (32%) and 51 (29%). ST239 comprised five major PFGE types and various unique PFGE patterns, and ST5 comprised two PFGE types. While the same PFGE pattern was not observed among strains with different STs, spa type 37 was observed among strains representing two different STs (ST239 and ST241), and these belonged to the same clonal complex as single-locus variants. ST239 was the earliest predominant ST, with the highest prevalence from 1984 to 1988 (96%), followed by ST247 from 1989 to 1993 (83%) and ST228 from 1994 to 1998 (40%). Spa type 37 was the most prevalent from 1984 to 1988 (96%), spa type 51 was the most prevalent from 1989 to 1993 (83%), and spa types 1 and 458 were the most prevalent from 1994 to 1998 (26% and 14%, respectively). The prevalence of SCCmec type III decreased from 96% from 1984 to 1988 to 8% from 1989 to 1993, the prevalence of SCCmec type I increased from 4% from 1984 to 1988 to 97% from 1989 to 1993 and decreased to 62% from 1994 to 1998. While the genetic diversity of MRSA increased from 1984 to 1998, one prevalent ST usually accounted for most of the isolates in a given time period.
INTRODUCTION
Since the first discovery of methicillin-resistant Staphylococcus aureus (MRSA) in England and Denmark in the late 1960s, MRSA has become one of the most important nosocomial pathogens (5, 8, 19). In Germany, the first MRSA cases were observed in 1974 in the former Federal Republic of Germany (27) and in 1985 in the former German Democratic Republic (48). The prevalence of MRSA in Germany was below 5% until the early 1990s; it started to rise after 1995 and reached 20.7% in 2001 (24, 25). Large international surveillance studies have shown that only a limited number of MRSA clonal groups have been responsible for a disproportionately large fraction of MRSA outbreaks worldwide (4, 11, 20, 23, 33).
The resistance of staphylococci to oxacillin and other beta-lactam antibiotics is mediated by a specific penicillin-binding protein with a reduced affinity for beta-lactam antibiotics (PBP 2a) (18). PBP 2a is encoded by the mecA gene, which is carried on a large genetic element designated staphylococcal cassette chromosome mec (SCCmec). Five types of SCCmec elements have been identified in S. aureus. The elements range in size from 21 to 67 kb and are characterized by a unique combination of mec and ccr complexes.
Various molecular typing techniques, including pulsed-field gel electrophoresis (PFGE) (44), single-locus sequence typing of the spa gene repeat region (spa typing) (17, 22), SCCmec typing (31), and in particular, multilocus sequence typing (MLST) (12), have proven to be powerful tools for the epidemiological characterization of MRSA isolates. PFGE is largely used for the direct comparison of strains where high discriminatory power is needed, such as tracking the origins and routes of dissemination of MRSA within hospitals. The MLST typing scheme for S. aureus was developed in 2000 (12). It is based on the nucleotide sequences of approximately 450-bp internal fragments of seven housekeeping genes. For each gene fragment, the different sequences are assigned distinct alleles, and each isolate is defined by the resulting allelic profile or sequence type (ST) (11, 12, 43). MLST is particularly useful for the study of the population biology and long-term epidemiology of bacterial species, such as the spread of MRSA strains across different countries and continents (43). In addition, MLST, like other sequence-based typing methods, is highly portable; and MLST data are readily comparable between laboratories via web-based electronic data exchange (43). Spa typing is a single-locus sequence typing approach that combines the portability of sequence-based data with a high resolution comparable to that of PFGE (17, 22, 41). Several approaches to the typing of MRSA isolates on the basis of the SCCmec element have been published (31, 33). The SCCmec typing system has also turned out to be an important marker for distinguishing between health care-associated MRSA clones (SCCmec types I to III) and community-acquired MRSA clones (SCCmec type IV) (31).
This ongoing study is being conducted to investigate the temporal evolution of MRSA in the metropolitan area of Cologne, Germany. Here we report data obtained from the beginning of the MRSA epidemic in Germany from 1984 to 1998.
(This work was presented in part at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., 30 October 2004 to 11 November 2004 [B. Ewertz, H. Wisplinghoff, D. Stefanik, F. Perdreau-Remington, and H. Seifert, Abstr. 44th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C2-1508].)
MATERIALS AND METHODS
Facility description. The Institute for Medical Microbiology, Immunology and Hygiene of the University of Cologne manages all microbiological support for the Cologne University Hospital (a 1,380-bed, tertiary-care teaching hospital) and numerous other teaching and nonteaching hospitals in the Cologne metropolitan area in Germany.
Bacterial isolates. MRSA isolates have been collected prospectively at our institution since 1984, when MRSA was first observed in Cologne in patients hospitalized in the Cologne University Hospital as well as in other teaching and nonteaching hospitals in the metropolitan area of Cologne. The identification as S. aureus was confirmed by positive latex agglutination (Pastorex Staph Plus; Bio-Rad Laboratories, Munich, Germany) and the result of the tube coagulase test (BBL Coagulase Plasma Rabbit; Becton Dickinson, Heidelberg, Germany). Methicillin resistance was confirmed by a PBP 2a slide latex agglutination test (MRSA-Screen; Denka-Seiken, Tokyo, Japan) and by PCR of the mecA gene (21). For this analysis, all isolates recovered between 1 January 1984 and 31 December 1998 were selected, regardless of their mode of acquisition (i.e., nosocomial, health care associated, or community acquired), although true community-acquired MRSA isolates were not observed during the study period. Only one isolate per patient was included.
Epidemiological typing. For the provisional identification of major strain types, all isolates were initially typed by an IS256-based PCR, as described by Deplano et al. (9). These data were used to preselect isolates that shared a common IS256 PCR pattern so that they could be run on adjacent lanes in the PFGE gels to ease interpretation of the PFGE patterns.
PFGE was performed by the use of the Gene Path Group I reagent kit (Bio-Rad Laboratories), following the recommendations of the manufacturer, with SmaI used as the restriction endonuclease. The samples were run on a 1% agarose gel in 0.5% Tris-borate-EDTA buffer in a contour-clamped homogeneous electric field (CHEF DR-II apparatus; Bio-Rad Laboratories), as described elsewhere (28). The running parameters were as follows: 200 V (6 V/cm); temperature, 14°C; initial switch time, 5 s; final switch time, 30 s; and total run time, 20 h. The gels were stained with ethidium bromide and photographed under UV light. Fingerprint patterns were compared visually and by computer-aided analysis with use of Molecular Analyst software (Bio-Rad Laboratories). Analysis of the PFGE patterns was performed according to previously published criteria (45).
All isolates with a unique PFGE pattern (n = 31) as well as a representative sample of isolates that represented 1 of 13 common PFGE patterns and that were collected during different time periods and all isolates that shared up to six band differences from the respective common band pattern, i.e., up to 14 isolates per given PFGE pattern and 101 isolates in total, were selected for MLST, SCCmec typing, and spa typing.
MLST, spa typing, and SCCmec typing were performed as described previously (12, 17, 22, 31). Briefly, the genomic DNA of the S. aureus isolates was extracted with a Plasmid MiniKit (QIAGEN GmbH, Hilden, Germany), as recommended by the manufacturer, with the addition of lysostaphin at a final concentration of 50 μg/ml. PCR for MLST, spa typing, and SCCmec typing was performed by using the TaqPCR master mix kit (QIAGEN) with a 50-μl reaction volume in a GeneAmp 9600 thermal cycler (Perkin-Elmer, Boston, MA) with an initial 5 min of denaturation at 95°C, followed by 35 cycles of annealing at 50°C for 45 s, extension at 72°C for 2 min, and denaturation at 95°C for 45 s, followed by a final extension step at 72°C for 4 min. For MLST, the amplified products were processed by a QIAquick PCR purification kit (QIAGEN); and the sequences of both strands were determined with an ABI PRISM 310 DNA sequencer (Applied Biosystems, Foster City, CA) with ABI PRISM BigDye fluorescent terminators and the primers used in the initial PCR amplification. S. aureus strain NCTC 8325 was used as the reference strain for PFGE for normalization of the electrophoretic patterns across the gel; and S. aureus strains COL (SCCmec type I), N315 (SCCmec type II), ANS46 (SCCmec type III), and MW2 (SCCmec type IV) were used as SCCmec standards. For the analysis of the ST and SCCmec type distributions of the complete strain collection, it was assumed that isolates with identical PFGE patterns would also share the same ST, the same spa type, and the same SCCmec type.
Computer analysis of nucleotide sequences. All analyses of the sequences for MLST were carried out by using the Vector NTI Suite 7.1 software for Windows (InforMax, Bethesda, MD). To determine the MLST sequence types, sequences were compared to those in the S. aureus database by using the S. aureus interface at the MLST website (http://www.mlst.net). The correlation between strains was assessed by using the eBURST program (13). All programs are available from the MLST website (http://www.mlst.net). Sequence analysis and identification of spa types were performed by using the StaphType software package (RIDOM GmbH, Würzburg, Germany).
Nomenclature. For presentation of the results and discussion, we will use the nomenclature proposed by Enright et al. (11), designating a strain with its ST and SCCmec type, i.e., a strain with ST1 and SCCmec type IV is referred to as ST1-MRSA-IV. This nomenclature was agreed to by a subcommittee of the International Union of Microbiology Societies in Tokyo, Japan, in 2002 (37). The correlation of STs with other designations that have been frequently used in the literature are shown in Table 1. The terms clonal complex (CC), single-locus variant (SLV), and double-locus variant (DLV) are used as defined previously (http://www.mlst.net). Briefly, SLVs differ from their founding STs at only a single locus, and DLVs differ from their founding STs at two loci. Clonal complex refers to a cluster of closely related genotypes that are all descendants from one founding genotype.
RESULTS
The number of MRSA isolates (one isolate per patient) per year in the Cologne metropolitan area is displayed in Fig. 1. Among a total of 410 nonrepetitive MRSA isolates that were investigated by PFGE, 101 isolates were selected and subjected to MLST, spa typing, and SCCmec typing. The distribution of MLST sequence types is displayed in Table 1. A total of nine different STs were observed in the Cologne metropolitan area between 1984 and 1998. No novel STs were detected. Among the 101 isolates investigated, the STs correlated with the PFGE patterns for all isolates; i.e., isolates sharing a given PFGE pattern had the same ST. However, several of the STs included more than one PFGE type. ST239 comprised five major PFGE types and various unique patterns (Fig. 2), and ST5 comprised two PFGE types (Table 1). Based on the assumption that isolates with an identical PFGE pattern share the same ST and the same SCCmec type, clones ST239-III (38% of isolates), ST247-I (29%), and ST228-I (18%) were the most prevalent. Over time, ST239-III was the first predominant epidemic strain, with the highest prevalence from 1984 to 1988 (96% of isolates), followed by ST247-I from 1989 to 1993 (83%), and ST228-I from 1994 to 1998 (40%). Other clones were observed only sporadically. The distribution of the major MLST sequence types over time is shown in Fig. 3.
The relatedness of STs was assessed by using the eBURST program. Analysis of the Cologne isolates showed that seven of the nine STs clustered in two groups. Group one comprised five STs (ST239, ST241, ST247, ST250, and ST254), while group 2 contained two STs (ST5 and ST228, i.e., DLVs). STs 22 and 30 remained as singletons. All strains from this study were also analyzed together with all strains from the MLST database by using the eBURST program. A plot of the clonal complex 8 (CC8), in which the most prevalent STs from the Cologne metropolitan area clustered, is shown in Fig. 4. Figure 4 represents the evolutionary relations of STs within CC8, as determined by the eBURST algorithm, which links the prevalent Cologne isolates to other STs of CC8.
The most prevalent spa types observed during the study period were spa type 37 (which accounted for 32% of all isolates) and spa type 51 (29%). All spa types are detailed in Table 1. While the same PFGE pattern was not observed among strains representing different MLST types, spa type 37 was observed in strains of two different MLST types, ST239 and ST241, which belong to the same clonal complex as single-locus variants (Fig. 4). Likewise, several STs comprised different spa types, such as isolates representing ST228, which exhibited a single PFGE profile but two different spa types, types 1 and 458. Over time, spa type 37 was the most prevalent spa type between 1984 and 1988 (96%), spa type 51 was the most prevalent spa type between 1989 and 1993 (83%), and spa types 1 and 458 were the most prevalent spa types between 1994 and 1998 (26% and 14%, respectively).
Among the 101 isolates investigated by SCCmec typing, 6 isolates remained untypeable; all were ST254. The SCCmec types correlated with the PFGE types; i.e., only one SCCmec type was observed among isolates sharing the same PFGE type. However, different SCCmec types were found among isolates sharing the same ST. Therefore, the relation between STs and SCCmec types correlated to the data displayed for PFGE (Table 1). Again, assuming that isolates with an identical PFGE pattern share the same SCCmec type, the SCCmec types were distributed as follows: type I, 53%; type II, 4%; type III, 40%; type IV, 0.25%; and nontypeable, 1%. No subtypes were investigated. The prevalence of SCCmec type III decreased from 96% from 1984 to 1988 to 8% from 1989 to 1993, while SCCmec type I increased from 4% from 1984 to 1988 to 97% from 1989 to 1993 and decreased to 62% from 1994 to 1998. SCCmec type II was first observed in the Cologne metropolitan area in 1997; its prevalence was 16% from 1997 to 1998. A single SCCmec type IV strain (ST30-IV) was observed in 1998.
DISCUSSION
MRSA continues to be one of the major nosocomial pathogens. In a recent European study, one-quarter of all S. aureus isolates were MRSA, with higher rates in southern countries, such as Portugal (35%), Italy (41%), and Greece (44%), and lower rates in northern European countries, including Austria (9%), Denmark (0.6%), Sweden (0.8%), Switzerland (2%), and The Netherlands (0.6%) (16, 46). MRSA infections are associated with increased morbidity, mortality, and length of hospital stay and represent a major financial burden on health care services (7). For a decade it has been recognized that methicillin resistance has emerged multiple times from successful methicillin-susceptible S. aureus lineages (12, 15, 30, 38). In a recent study, a more complete picture of the evolutionary history of MRSA based on an MLST scheme augmented by an additional set of highly variable sas genes was proposed (37).
Several studies have analyzed the molecular epidemiology of MRSA in various parts of the world and indicated a considerable degree of geographic specificity in the spread of various MRSA lineages (6). For example, the so-called Iberian clone of MRSA (ST247-I, as described elsewhere [11]), which has been observed in Belgium and France since 1984 (10), became the predominant strain type in hospitals in southern Europe after 1989, when it was responsible for a major MRSA outbreak in Barcelona, Spain (32); and this strain was also the leading clone in the Cologne area from 1991 to 1993. Roberts and coworkers reported that in the late 1990s, between 42% and 92% of all MRSA isolates recovered from New York hospitals and the area of New Jersey, Pennsylvania, and Connecticut, respectively, belonged to the New York/Japan clone (ST5) (35, 36).
In contrast to previous studies that mainly analyzed strains from rather heterogeneous strain collections, our study is the first that almost completely analyzed the evolution of MRSA in a defined region from the beginning of the MSRA epidemic in 1984 over a period of 15 years. While the two peaks observed in 1984 and 1985 and in 1992 and 1993 correspond to two major MRSA outbreaks that occurred in a large community hospital, the steady increase of MRSA isolates after 1994 reflects the national trend observed in Germany (24). Our data demonstrate the interchanging predominance of certain MRSA clones (STs 239-III, 247-I, and 228-I), some of which, according to previously published data, belong to different clonal complexes (CC5 and CC8) and did not evolve from each other (37). In our survey, ST239-III was the earliest predominant MRSA clone in the Cologne area and accounted for approximately 96% of all MRSA isolates recovered between 1984 and 1988. These strains were also reported to be among the most prevalent clones in Portuguese hospitals (Portuguese clone, ST239-III) in the mid-1980s and early 1990s (2, 33). In contrast to other STs, whose isolates usually shared a common PFGE profile, ST239-III isolates comprised five major PFGE profiles and various unique patterns. This is consistent with findings reported in the literature that several epidemic MRSA (EMRSA) clones, including the EMRSA-1, EMRSA-4, EMRSA-7, EMRSA-9, EMRSA-11, Brazilian, Portuguese, and Vienna clones, which have different PFGE profiles, all share this ST (1, 2, 3, 11, 39), suggesting a unique degree of heterogeneity among isolates of ST239-III. Feil and Enright showed that ST239 evolved by the homologous transfer of a large chromosomal fragment of 557 kb that represented 20% of the S. aureus chromosome (14). According to Robinson and Enright, ST239 and its descendants represent a phylogenetically distinct and clinically important branch within CC8 (37). The ST239 mosaic chromosome contains this 557-kb fragment spanning the origin of replication (oriC) from an ancestral strain of ST30 and 2,220 kb spanning the terminus of replication (terC) from an ancestral strain of ST8 (37). Since neither ST8 nor ST30 carries SCCmec type III, the authors concluded that ST239 acquired its SCCmec element elsewhere. The evolution of the highly successful lineage ST239 shows that such a large recombination might have important long-term evolutionary implications, such as an acceleration of the rate of adaptive evolution (14).
The ST239-III clone was later replaced by ST247-I as the most prevalent clone in the Cologne metropolitan area, when it was also frequently observed in other European countries, such as Spain (40); Belgium and France (10); Poland (26); and Sweden, Italy, and Scotland (2, 32). This finding suggests that its predominance in our area paralleled its generally high prevalence in Europe in the mid-1980s and early 1990s. Also, ST228-I predominated in the Cologne area in the mid-1990s, when this clone was also reported with increasing frequency from other parts of Germany (47). In contrast, the "archaic" MRSA clone, ST250, was observed only sporadically in the late 1980s. While most isolates of a given ST exhibited a single PFGE pattern and corresponded to a single spa type, there were three main exceptions to this rule. Among isolates of ST239 and ST5, two main spa types were observed, spa types 30 and 37 and spa types 2 and 143, respectively, which corresponded to different PFGE profiles. Similarly, spa types 1 and 458 were observed among isolates identified as belonging to ST228; however, these isolates shared a single PFGE pattern. In addition, a few unique spa types that were each observed only once were found among ST239, ST250, and ST254 isolates. However, the spa repeat profiles among strains showing the same PFGE pattern or MLST but a different spa type were usually very similar, displaying insertions or deletions of repeats within the spa repeat region that did not necessarily affect the PFGE pattern (Table 1).
Of note, the strain from our collection that was identified as ST22 carried SCCmec type II, while the German Barnim strain and EMRSA-15 (also ST22) carry type IV. Therefore, our ST22 strain is probably not directly related to the epidemic strains. Similarly, we observed ST30-SCCmec type IV, which has been postulated to be a precursor strain of its SLV ST36-SCCmec type II, EMRSA-16 (37).
There are some limitations to our analysis. The complete set of typing data is restricted to a subset of 101 MRSA isolates, and these data were extrapolated to the complete strain collection based on the assumption that isolates with the same PFGE pattern belong to the same ST and share the same type of SCCmec element. We did not find any exception to this assumption, which is also supported by most recent studies (11, 38), which did not find any evidence that such a discrepancy may occur. In contrast to this experience, Peacock and colleagues reported that strains with the same ST may display not only different PFGE patterns but also vice versa (34). We did, however, observe that two different spa types were identified among ST228 isolates that had a common PFGE profile. Also, even though our institute managed the microbiological support for most hospitals in the Cologne metropolitan area during the study period, there are some hospitals whose specimens were processed elsewhere. Therefore, our data cover only about 80% of the patients seen at any hospital in the Cologne metropolitan area, and the possibility that some rarely occurring clones were missed cannot be ruled out. Finally, isolates of ST254 that were found to be untypeable by mec typing were not investigated for the presence of SCCmec type V.
In conclusion, we have described for the first time the long-term molecular evolution of MRSA in a large metropolitan area in central Europe, based on a comprehensive collection of prospectively collected isolates. While the genetic diversity of MRSA strains increased from 1984 to 1998, one highly prevalent clone usually accounted for most of the isolates in a given time period. Sequence types ST239, ST247, and ST228 were the most prevalent clonal strain types between 1984 and 1998. Some of the more prevalent spa types were shared by strains representing different MLST types, indicating a locally occurring change in ST rather than the replacement of one clone by another. The PFGE, spa typing, and MLST data correlated for most of the strains; but no single method alone is sufficient to perform a detailed analysis of a large collection of MRSA strains. One may follow our approach to start with PFGE or, alternatively, may use spa typing as the first method. Given the complexity of PFGE patterns, a computer-based analysis of the PFGE pattern is mandatory. To get a complete picture of the epidemiology of MRSA, the next step would be to perform MLST and either spa typing or PFGE, depending on which method was used for screening, of a representative subset of the major strain types and of all the unique strain types identified with the first method.
ACKNOWLEDGMENTS
We thank G. L. Archer for providing the SCCmec reference strains, A. E. Robinson for valuable discussion, and C. Haefs and A. Kündgen for excellent technical assistance.
This work was supported in part by the Maria Pesch Foundation.
Present address: Department of Medicine, Division of Infectious Diseases, San Francisco General Hospital, San Francisco, Calif.
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ABSTRACT
To investigate the molecular evolution of methicillin-resistant Staphylococcus aureus (MRSA) in a large metropolitan area in Germany, 398 nonrepetitive MRSA isolates recovered from patients from various teaching and nonteaching hospitals in Cologne between 1984 and 1998 were characterized by pulsed-field gel electrophoresis (PFGE). On this basis, 95 representative isolates were selected and further investigated by multilocus sequence typing (MLST), spa typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Overall, there were 9 MLST types and 16 spa types. The most prevalent sequence types (STs) were ST239 (38% of isolates), ST247 (29%), and ST228 (18%); the most prevalent spa types were 37 (32%) and 51 (29%). ST239 comprised five major PFGE types and various unique PFGE patterns, and ST5 comprised two PFGE types. While the same PFGE pattern was not observed among strains with different STs, spa type 37 was observed among strains representing two different STs (ST239 and ST241), and these belonged to the same clonal complex as single-locus variants. ST239 was the earliest predominant ST, with the highest prevalence from 1984 to 1988 (96%), followed by ST247 from 1989 to 1993 (83%) and ST228 from 1994 to 1998 (40%). Spa type 37 was the most prevalent from 1984 to 1988 (96%), spa type 51 was the most prevalent from 1989 to 1993 (83%), and spa types 1 and 458 were the most prevalent from 1994 to 1998 (26% and 14%, respectively). The prevalence of SCCmec type III decreased from 96% from 1984 to 1988 to 8% from 1989 to 1993, the prevalence of SCCmec type I increased from 4% from 1984 to 1988 to 97% from 1989 to 1993 and decreased to 62% from 1994 to 1998. While the genetic diversity of MRSA increased from 1984 to 1998, one prevalent ST usually accounted for most of the isolates in a given time period.
INTRODUCTION
Since the first discovery of methicillin-resistant Staphylococcus aureus (MRSA) in England and Denmark in the late 1960s, MRSA has become one of the most important nosocomial pathogens (5, 8, 19). In Germany, the first MRSA cases were observed in 1974 in the former Federal Republic of Germany (27) and in 1985 in the former German Democratic Republic (48). The prevalence of MRSA in Germany was below 5% until the early 1990s; it started to rise after 1995 and reached 20.7% in 2001 (24, 25). Large international surveillance studies have shown that only a limited number of MRSA clonal groups have been responsible for a disproportionately large fraction of MRSA outbreaks worldwide (4, 11, 20, 23, 33).
The resistance of staphylococci to oxacillin and other beta-lactam antibiotics is mediated by a specific penicillin-binding protein with a reduced affinity for beta-lactam antibiotics (PBP 2a) (18). PBP 2a is encoded by the mecA gene, which is carried on a large genetic element designated staphylococcal cassette chromosome mec (SCCmec). Five types of SCCmec elements have been identified in S. aureus. The elements range in size from 21 to 67 kb and are characterized by a unique combination of mec and ccr complexes.
Various molecular typing techniques, including pulsed-field gel electrophoresis (PFGE) (44), single-locus sequence typing of the spa gene repeat region (spa typing) (17, 22), SCCmec typing (31), and in particular, multilocus sequence typing (MLST) (12), have proven to be powerful tools for the epidemiological characterization of MRSA isolates. PFGE is largely used for the direct comparison of strains where high discriminatory power is needed, such as tracking the origins and routes of dissemination of MRSA within hospitals. The MLST typing scheme for S. aureus was developed in 2000 (12). It is based on the nucleotide sequences of approximately 450-bp internal fragments of seven housekeeping genes. For each gene fragment, the different sequences are assigned distinct alleles, and each isolate is defined by the resulting allelic profile or sequence type (ST) (11, 12, 43). MLST is particularly useful for the study of the population biology and long-term epidemiology of bacterial species, such as the spread of MRSA strains across different countries and continents (43). In addition, MLST, like other sequence-based typing methods, is highly portable; and MLST data are readily comparable between laboratories via web-based electronic data exchange (43). Spa typing is a single-locus sequence typing approach that combines the portability of sequence-based data with a high resolution comparable to that of PFGE (17, 22, 41). Several approaches to the typing of MRSA isolates on the basis of the SCCmec element have been published (31, 33). The SCCmec typing system has also turned out to be an important marker for distinguishing between health care-associated MRSA clones (SCCmec types I to III) and community-acquired MRSA clones (SCCmec type IV) (31).
This ongoing study is being conducted to investigate the temporal evolution of MRSA in the metropolitan area of Cologne, Germany. Here we report data obtained from the beginning of the MRSA epidemic in Germany from 1984 to 1998.
(This work was presented in part at the 44th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, D.C., 30 October 2004 to 11 November 2004 [B. Ewertz, H. Wisplinghoff, D. Stefanik, F. Perdreau-Remington, and H. Seifert, Abstr. 44th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C2-1508].)
MATERIALS AND METHODS
Facility description. The Institute for Medical Microbiology, Immunology and Hygiene of the University of Cologne manages all microbiological support for the Cologne University Hospital (a 1,380-bed, tertiary-care teaching hospital) and numerous other teaching and nonteaching hospitals in the Cologne metropolitan area in Germany.
Bacterial isolates. MRSA isolates have been collected prospectively at our institution since 1984, when MRSA was first observed in Cologne in patients hospitalized in the Cologne University Hospital as well as in other teaching and nonteaching hospitals in the metropolitan area of Cologne. The identification as S. aureus was confirmed by positive latex agglutination (Pastorex Staph Plus; Bio-Rad Laboratories, Munich, Germany) and the result of the tube coagulase test (BBL Coagulase Plasma Rabbit; Becton Dickinson, Heidelberg, Germany). Methicillin resistance was confirmed by a PBP 2a slide latex agglutination test (MRSA-Screen; Denka-Seiken, Tokyo, Japan) and by PCR of the mecA gene (21). For this analysis, all isolates recovered between 1 January 1984 and 31 December 1998 were selected, regardless of their mode of acquisition (i.e., nosocomial, health care associated, or community acquired), although true community-acquired MRSA isolates were not observed during the study period. Only one isolate per patient was included.
Epidemiological typing. For the provisional identification of major strain types, all isolates were initially typed by an IS256-based PCR, as described by Deplano et al. (9). These data were used to preselect isolates that shared a common IS256 PCR pattern so that they could be run on adjacent lanes in the PFGE gels to ease interpretation of the PFGE patterns.
PFGE was performed by the use of the Gene Path Group I reagent kit (Bio-Rad Laboratories), following the recommendations of the manufacturer, with SmaI used as the restriction endonuclease. The samples were run on a 1% agarose gel in 0.5% Tris-borate-EDTA buffer in a contour-clamped homogeneous electric field (CHEF DR-II apparatus; Bio-Rad Laboratories), as described elsewhere (28). The running parameters were as follows: 200 V (6 V/cm); temperature, 14°C; initial switch time, 5 s; final switch time, 30 s; and total run time, 20 h. The gels were stained with ethidium bromide and photographed under UV light. Fingerprint patterns were compared visually and by computer-aided analysis with use of Molecular Analyst software (Bio-Rad Laboratories). Analysis of the PFGE patterns was performed according to previously published criteria (45).
All isolates with a unique PFGE pattern (n = 31) as well as a representative sample of isolates that represented 1 of 13 common PFGE patterns and that were collected during different time periods and all isolates that shared up to six band differences from the respective common band pattern, i.e., up to 14 isolates per given PFGE pattern and 101 isolates in total, were selected for MLST, SCCmec typing, and spa typing.
MLST, spa typing, and SCCmec typing were performed as described previously (12, 17, 22, 31). Briefly, the genomic DNA of the S. aureus isolates was extracted with a Plasmid MiniKit (QIAGEN GmbH, Hilden, Germany), as recommended by the manufacturer, with the addition of lysostaphin at a final concentration of 50 μg/ml. PCR for MLST, spa typing, and SCCmec typing was performed by using the TaqPCR master mix kit (QIAGEN) with a 50-μl reaction volume in a GeneAmp 9600 thermal cycler (Perkin-Elmer, Boston, MA) with an initial 5 min of denaturation at 95°C, followed by 35 cycles of annealing at 50°C for 45 s, extension at 72°C for 2 min, and denaturation at 95°C for 45 s, followed by a final extension step at 72°C for 4 min. For MLST, the amplified products were processed by a QIAquick PCR purification kit (QIAGEN); and the sequences of both strands were determined with an ABI PRISM 310 DNA sequencer (Applied Biosystems, Foster City, CA) with ABI PRISM BigDye fluorescent terminators and the primers used in the initial PCR amplification. S. aureus strain NCTC 8325 was used as the reference strain for PFGE for normalization of the electrophoretic patterns across the gel; and S. aureus strains COL (SCCmec type I), N315 (SCCmec type II), ANS46 (SCCmec type III), and MW2 (SCCmec type IV) were used as SCCmec standards. For the analysis of the ST and SCCmec type distributions of the complete strain collection, it was assumed that isolates with identical PFGE patterns would also share the same ST, the same spa type, and the same SCCmec type.
Computer analysis of nucleotide sequences. All analyses of the sequences for MLST were carried out by using the Vector NTI Suite 7.1 software for Windows (InforMax, Bethesda, MD). To determine the MLST sequence types, sequences were compared to those in the S. aureus database by using the S. aureus interface at the MLST website (http://www.mlst.net). The correlation between strains was assessed by using the eBURST program (13). All programs are available from the MLST website (http://www.mlst.net). Sequence analysis and identification of spa types were performed by using the StaphType software package (RIDOM GmbH, Würzburg, Germany).
Nomenclature. For presentation of the results and discussion, we will use the nomenclature proposed by Enright et al. (11), designating a strain with its ST and SCCmec type, i.e., a strain with ST1 and SCCmec type IV is referred to as ST1-MRSA-IV. This nomenclature was agreed to by a subcommittee of the International Union of Microbiology Societies in Tokyo, Japan, in 2002 (37). The correlation of STs with other designations that have been frequently used in the literature are shown in Table 1. The terms clonal complex (CC), single-locus variant (SLV), and double-locus variant (DLV) are used as defined previously (http://www.mlst.net). Briefly, SLVs differ from their founding STs at only a single locus, and DLVs differ from their founding STs at two loci. Clonal complex refers to a cluster of closely related genotypes that are all descendants from one founding genotype.
RESULTS
The number of MRSA isolates (one isolate per patient) per year in the Cologne metropolitan area is displayed in Fig. 1. Among a total of 410 nonrepetitive MRSA isolates that were investigated by PFGE, 101 isolates were selected and subjected to MLST, spa typing, and SCCmec typing. The distribution of MLST sequence types is displayed in Table 1. A total of nine different STs were observed in the Cologne metropolitan area between 1984 and 1998. No novel STs were detected. Among the 101 isolates investigated, the STs correlated with the PFGE patterns for all isolates; i.e., isolates sharing a given PFGE pattern had the same ST. However, several of the STs included more than one PFGE type. ST239 comprised five major PFGE types and various unique patterns (Fig. 2), and ST5 comprised two PFGE types (Table 1). Based on the assumption that isolates with an identical PFGE pattern share the same ST and the same SCCmec type, clones ST239-III (38% of isolates), ST247-I (29%), and ST228-I (18%) were the most prevalent. Over time, ST239-III was the first predominant epidemic strain, with the highest prevalence from 1984 to 1988 (96% of isolates), followed by ST247-I from 1989 to 1993 (83%), and ST228-I from 1994 to 1998 (40%). Other clones were observed only sporadically. The distribution of the major MLST sequence types over time is shown in Fig. 3.
The relatedness of STs was assessed by using the eBURST program. Analysis of the Cologne isolates showed that seven of the nine STs clustered in two groups. Group one comprised five STs (ST239, ST241, ST247, ST250, and ST254), while group 2 contained two STs (ST5 and ST228, i.e., DLVs). STs 22 and 30 remained as singletons. All strains from this study were also analyzed together with all strains from the MLST database by using the eBURST program. A plot of the clonal complex 8 (CC8), in which the most prevalent STs from the Cologne metropolitan area clustered, is shown in Fig. 4. Figure 4 represents the evolutionary relations of STs within CC8, as determined by the eBURST algorithm, which links the prevalent Cologne isolates to other STs of CC8.
The most prevalent spa types observed during the study period were spa type 37 (which accounted for 32% of all isolates) and spa type 51 (29%). All spa types are detailed in Table 1. While the same PFGE pattern was not observed among strains representing different MLST types, spa type 37 was observed in strains of two different MLST types, ST239 and ST241, which belong to the same clonal complex as single-locus variants (Fig. 4). Likewise, several STs comprised different spa types, such as isolates representing ST228, which exhibited a single PFGE profile but two different spa types, types 1 and 458. Over time, spa type 37 was the most prevalent spa type between 1984 and 1988 (96%), spa type 51 was the most prevalent spa type between 1989 and 1993 (83%), and spa types 1 and 458 were the most prevalent spa types between 1994 and 1998 (26% and 14%, respectively).
Among the 101 isolates investigated by SCCmec typing, 6 isolates remained untypeable; all were ST254. The SCCmec types correlated with the PFGE types; i.e., only one SCCmec type was observed among isolates sharing the same PFGE type. However, different SCCmec types were found among isolates sharing the same ST. Therefore, the relation between STs and SCCmec types correlated to the data displayed for PFGE (Table 1). Again, assuming that isolates with an identical PFGE pattern share the same SCCmec type, the SCCmec types were distributed as follows: type I, 53%; type II, 4%; type III, 40%; type IV, 0.25%; and nontypeable, 1%. No subtypes were investigated. The prevalence of SCCmec type III decreased from 96% from 1984 to 1988 to 8% from 1989 to 1993, while SCCmec type I increased from 4% from 1984 to 1988 to 97% from 1989 to 1993 and decreased to 62% from 1994 to 1998. SCCmec type II was first observed in the Cologne metropolitan area in 1997; its prevalence was 16% from 1997 to 1998. A single SCCmec type IV strain (ST30-IV) was observed in 1998.
DISCUSSION
MRSA continues to be one of the major nosocomial pathogens. In a recent European study, one-quarter of all S. aureus isolates were MRSA, with higher rates in southern countries, such as Portugal (35%), Italy (41%), and Greece (44%), and lower rates in northern European countries, including Austria (9%), Denmark (0.6%), Sweden (0.8%), Switzerland (2%), and The Netherlands (0.6%) (16, 46). MRSA infections are associated with increased morbidity, mortality, and length of hospital stay and represent a major financial burden on health care services (7). For a decade it has been recognized that methicillin resistance has emerged multiple times from successful methicillin-susceptible S. aureus lineages (12, 15, 30, 38). In a recent study, a more complete picture of the evolutionary history of MRSA based on an MLST scheme augmented by an additional set of highly variable sas genes was proposed (37).
Several studies have analyzed the molecular epidemiology of MRSA in various parts of the world and indicated a considerable degree of geographic specificity in the spread of various MRSA lineages (6). For example, the so-called Iberian clone of MRSA (ST247-I, as described elsewhere [11]), which has been observed in Belgium and France since 1984 (10), became the predominant strain type in hospitals in southern Europe after 1989, when it was responsible for a major MRSA outbreak in Barcelona, Spain (32); and this strain was also the leading clone in the Cologne area from 1991 to 1993. Roberts and coworkers reported that in the late 1990s, between 42% and 92% of all MRSA isolates recovered from New York hospitals and the area of New Jersey, Pennsylvania, and Connecticut, respectively, belonged to the New York/Japan clone (ST5) (35, 36).
In contrast to previous studies that mainly analyzed strains from rather heterogeneous strain collections, our study is the first that almost completely analyzed the evolution of MRSA in a defined region from the beginning of the MSRA epidemic in 1984 over a period of 15 years. While the two peaks observed in 1984 and 1985 and in 1992 and 1993 correspond to two major MRSA outbreaks that occurred in a large community hospital, the steady increase of MRSA isolates after 1994 reflects the national trend observed in Germany (24). Our data demonstrate the interchanging predominance of certain MRSA clones (STs 239-III, 247-I, and 228-I), some of which, according to previously published data, belong to different clonal complexes (CC5 and CC8) and did not evolve from each other (37). In our survey, ST239-III was the earliest predominant MRSA clone in the Cologne area and accounted for approximately 96% of all MRSA isolates recovered between 1984 and 1988. These strains were also reported to be among the most prevalent clones in Portuguese hospitals (Portuguese clone, ST239-III) in the mid-1980s and early 1990s (2, 33). In contrast to other STs, whose isolates usually shared a common PFGE profile, ST239-III isolates comprised five major PFGE profiles and various unique patterns. This is consistent with findings reported in the literature that several epidemic MRSA (EMRSA) clones, including the EMRSA-1, EMRSA-4, EMRSA-7, EMRSA-9, EMRSA-11, Brazilian, Portuguese, and Vienna clones, which have different PFGE profiles, all share this ST (1, 2, 3, 11, 39), suggesting a unique degree of heterogeneity among isolates of ST239-III. Feil and Enright showed that ST239 evolved by the homologous transfer of a large chromosomal fragment of 557 kb that represented 20% of the S. aureus chromosome (14). According to Robinson and Enright, ST239 and its descendants represent a phylogenetically distinct and clinically important branch within CC8 (37). The ST239 mosaic chromosome contains this 557-kb fragment spanning the origin of replication (oriC) from an ancestral strain of ST30 and 2,220 kb spanning the terminus of replication (terC) from an ancestral strain of ST8 (37). Since neither ST8 nor ST30 carries SCCmec type III, the authors concluded that ST239 acquired its SCCmec element elsewhere. The evolution of the highly successful lineage ST239 shows that such a large recombination might have important long-term evolutionary implications, such as an acceleration of the rate of adaptive evolution (14).
The ST239-III clone was later replaced by ST247-I as the most prevalent clone in the Cologne metropolitan area, when it was also frequently observed in other European countries, such as Spain (40); Belgium and France (10); Poland (26); and Sweden, Italy, and Scotland (2, 32). This finding suggests that its predominance in our area paralleled its generally high prevalence in Europe in the mid-1980s and early 1990s. Also, ST228-I predominated in the Cologne area in the mid-1990s, when this clone was also reported with increasing frequency from other parts of Germany (47). In contrast, the "archaic" MRSA clone, ST250, was observed only sporadically in the late 1980s. While most isolates of a given ST exhibited a single PFGE pattern and corresponded to a single spa type, there were three main exceptions to this rule. Among isolates of ST239 and ST5, two main spa types were observed, spa types 30 and 37 and spa types 2 and 143, respectively, which corresponded to different PFGE profiles. Similarly, spa types 1 and 458 were observed among isolates identified as belonging to ST228; however, these isolates shared a single PFGE pattern. In addition, a few unique spa types that were each observed only once were found among ST239, ST250, and ST254 isolates. However, the spa repeat profiles among strains showing the same PFGE pattern or MLST but a different spa type were usually very similar, displaying insertions or deletions of repeats within the spa repeat region that did not necessarily affect the PFGE pattern (Table 1).
Of note, the strain from our collection that was identified as ST22 carried SCCmec type II, while the German Barnim strain and EMRSA-15 (also ST22) carry type IV. Therefore, our ST22 strain is probably not directly related to the epidemic strains. Similarly, we observed ST30-SCCmec type IV, which has been postulated to be a precursor strain of its SLV ST36-SCCmec type II, EMRSA-16 (37).
There are some limitations to our analysis. The complete set of typing data is restricted to a subset of 101 MRSA isolates, and these data were extrapolated to the complete strain collection based on the assumption that isolates with the same PFGE pattern belong to the same ST and share the same type of SCCmec element. We did not find any exception to this assumption, which is also supported by most recent studies (11, 38), which did not find any evidence that such a discrepancy may occur. In contrast to this experience, Peacock and colleagues reported that strains with the same ST may display not only different PFGE patterns but also vice versa (34). We did, however, observe that two different spa types were identified among ST228 isolates that had a common PFGE profile. Also, even though our institute managed the microbiological support for most hospitals in the Cologne metropolitan area during the study period, there are some hospitals whose specimens were processed elsewhere. Therefore, our data cover only about 80% of the patients seen at any hospital in the Cologne metropolitan area, and the possibility that some rarely occurring clones were missed cannot be ruled out. Finally, isolates of ST254 that were found to be untypeable by mec typing were not investigated for the presence of SCCmec type V.
In conclusion, we have described for the first time the long-term molecular evolution of MRSA in a large metropolitan area in central Europe, based on a comprehensive collection of prospectively collected isolates. While the genetic diversity of MRSA strains increased from 1984 to 1998, one highly prevalent clone usually accounted for most of the isolates in a given time period. Sequence types ST239, ST247, and ST228 were the most prevalent clonal strain types between 1984 and 1998. Some of the more prevalent spa types were shared by strains representing different MLST types, indicating a locally occurring change in ST rather than the replacement of one clone by another. The PFGE, spa typing, and MLST data correlated for most of the strains; but no single method alone is sufficient to perform a detailed analysis of a large collection of MRSA strains. One may follow our approach to start with PFGE or, alternatively, may use spa typing as the first method. Given the complexity of PFGE patterns, a computer-based analysis of the PFGE pattern is mandatory. To get a complete picture of the epidemiology of MRSA, the next step would be to perform MLST and either spa typing or PFGE, depending on which method was used for screening, of a representative subset of the major strain types and of all the unique strain types identified with the first method.
ACKNOWLEDGMENTS
We thank G. L. Archer for providing the SCCmec reference strains, A. E. Robinson for valuable discussion, and C. Haefs and A. Kündgen for excellent technical assistance.
This work was supported in part by the Maria Pesch Foundation.
Present address: Department of Medicine, Division of Infectious Diseases, San Francisco General Hospital, San Francisco, Calif.
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