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Genetic Relationship between Methicillin Sensitive and Methicillin Resistant Staphylococcus aureus Strains from France and from International Sources: Delineation of Genomic Groups

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0095-1137/03/$08.00⫹0 DOI: 10.1128/JCM.41.7.2946–2951.2003

Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Genetic Relationship between Methicillin-Sensitive and

Methicillin-Resistant

Staphylococcus aureus

Strains from

France and from International Sources: Delineation

of Genomic Groups

Catherine Branger,* Carole Gardye, Jacques-Olivier Galdbart,†

Catherine Deschamps, and Nicole Lambert

Laboratoire de Microbiologie, Hoˆpital Beaujon, AP-HP, 92110 Clichy, Cedex, France

Received 17 January 2003/Returned for modification 6 March 2003/Accepted 7 April 2003

Cluster analysis of the SmaI patterns, generated by pulsed-field gel electrophoresis, of 44

methicillin-resistant (MRSA) and 118 methicillin-sensitive (MSSA) Staphylococcus aureus strains isolated in various

French hospitals and 61 MRSA and 48 MSSA strains from 20 other countries revealed 20 genomic groups distributed into four distantly related phylogenic branches. Eighty-three of the 105 MRSA strains (79%) were clustered in the six genomic groups of phylogenic branch I; and 154 of the 166 MSSA strains (92.8%) were clustered in the 14 genomic groups of phylogenic branches II, III, and IV. Agreement between genomic group and two other markers, esterase type and phage group, was obtained, emphasizing the clonal structure of the population. The genomic groups were delineated by esterase type. The distribution of the strains within the genomic groups was independent of their geographical origin; French strains were clustered with strains from

other countries. The three types of the staphylococcal cassette chromosome mec (SCCmec) complex were

distributed according to genomic groups. Most of the time, type I and type II SCCmeccomplexes were found

in the MRSA strains belonging to the same genomic groups. In contrast, the type III SCCmeccomplex was

specific to the MRSA strains belonging to the three genomic groups characterized by a common esterase type.

Staphylococcus aureusis a major human pathogen responsi-ble for a wide spectrum of diseases. Soon after methicillin was

introduced into clinical use, methicillin-resistant S. aureus

(MRSA) strains were reported. Since then, outbreaks of MRSA have been recorded worldwide (1, 9, 13) and the con-trol of MRSA has become a significant problem in hospitals (15, 26).

The methicillin resistance gene (mecA) is carried on a

mo-bile genetic element, the staphylococcal cassette chromosome

mec(SCCmec), which differs in size and genetic composition

among strains (17, 18). For movement, SCCmeccarries two

specific genes (ccrA and ccrB) which encode recombinases.

Three SCCmec complex elements, carrying a characteristic

combination of the ccr and mec gene complex, have been

identified in hospital-acquired MRSA strains (17). SCCmec

is integrated in the chromosome of MRSA at a unique site (attBscc), which is found inside an open reading frame (orfX) of unknown function.

Knowledge of the underlying genetic structure is important in understanding the epidemiological relationship between strains. Therefore, a number of epidemiological methods have

been used to identify and differentiate strains of S. aureus,

including phage typing, enzyme electrophoretic typing, pulsed-field gel electrophoresis (PFGE), randomly amplified

polymor-phic DNA analysis-PCR, ribotyping, and multilocus sequence typing (11, 14, 16, 19, 23, 27).

A high degree of diversity has been observed among

methi-cillin-sensitive S. aureus(MSSA) strains (3, 6, 10). However,

Schlichting et al. (27) noticed that epidemiologically unrelated MSSA strains with similar PFGE patterns were found in dif-ferent geographical regions, suggesting that some genotypes are ubiquitous. Multilocus enzyme electrophoresis

demon-strated the presence of clones in natural populations of S.

aureusand, in particular, the presence of a clone responsible for most cases of toxic shock syndrome (12, 22).

In contrast to MSSA, several studies showed that MRSA strains were represented by a limited number of genotypes (3, 10, 16, 23). There has been considerable speculation about the origin and evolution of the MRSA strains (19). According to

Fitzgerald et al. (12), themecgene has been horizontally

trans-ferred into distinctS. aureuschromosomal backgrounds,

dem-onstrating that methicillin-resistant strains have evolved sev-eral times independently.

In this study, the relationships between the genetic struc-tures of MRSA and MSSA strains isolated from various hos-pitals in France and of MRSA and MSSA strains from inter-national sources are described. We used PFGE, as it is an interesting tool not only for strain identification but also for estimation of interstrain genetic relatedness (7, 13, 21, 28). The Dice similarity coefficient and Ward’s algorithm (25, 30) were used for cluster analysis to define the degree of DNA related-ness between the strains. The correlation of the genomic group with two other markers, the esterase electrophoretic type and the phage type, was established. We also analyzed the

distri-bution of the type of SCCmeccomplex among MRSA strains.

* Corresponding author. Mailing address: Laboratoire de Microbi-ologie, Hoˆpital Beaujon, 100 Boulevard du Ge´ne´ral Leclerc, 92110 Clichy, Cedex, France. Phone: 33 1 40 87 56 05. Fax: 33 1 40 87 05 50. E-mail: Catherine.branger@bjn.ap-hop-paris.fr.

† Present address: AFFSAPS, F 93285 Saint Denis Cedex, France.

2946

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MATERIALS AND METHODS

Bacterial strains.A total of 271 strains ofS. aureusoriginating from diverse geographical origins were examined (Table 1). Two hundred twenty-six of the strains came from collections that have previously been analyzed by antibiotic typing, esterase electrophoretic typing, and phage typing (3–7, 27). Forty-five new strains were examined. Twenty-five strains isolated from bronchopulmonary infections were kindly provided by N. Hoiby (University of Copenhagen, Copen-hagen, Denmark), 11 strains were obtained from the collection of M. Fournier (Institut Pasteur, Paris, France), and 6 strains were isolated at our hospital between 1991 and 1998. Three reference strains were also included. The strains were selected according to their susceptibilities to methicillin and according to their geographical origins to maximize diversity. The strains were also selected to represent the most frequently encountered esterase electrophoretic types, as described in previous studies (4, 6). One hundred sixty-two strains were ered from French hospitals between 1980 and 1998, and 109 strains were recov-ered from 20 other countries. Except for strains of esterase type 1, each esterase type included strains from 2 to 16 countries (Table 1); esterase type 1 included only strains from France. MRSA strains were represented by 44 French strains and 61 strains from other countries. Resistance to methicillin was confirmed by detection ofmecAby PCR (24).

Esterase electrophoretic typing and phage typing.The electrophoretic mobil-ity patterns of three different esterases were investigated as described previously (3). Phage typing was performed at the routine test dilution (RTD) and 100 times the RTD with the international basic set at the Centre National de Reference des Staphylocoques, Institut Pasteur, Paris, France (N. El Solh).

PFGE of macrorestriction fragments and analysis of DNA relatedness.DNA fragment patterns were obtained as described previously (27) after digestion of genomic DNA with the restriction endonucleaseSmaI and subsequent PFGE.

The DNA patterns obtained by PFGE were analyzed with Gel Compar soft-ware (Applied Maths, Kortrijk, Belgium). A similarity matrix was created by using the band-based Dice similarity coefficient, and Ward’s algorithm was used to cluster the strains (25, 30).

SCCmeccomplex.The SCCmeccomplex type was determined by using PCR to detect a sequence overlapping the right SCCmec-chromosome junction (18). The primers used to detect type I and type II SCCmeccomplexes were designed on the basis of the type II SCCmecsequence (GenBank accession no. D86934) They were MDR1 (5⬘-TGCGTAAAATCTTCCAG-3⬘) and OR3 (5⬘-GTCTTACAA CGCAGTAACTA-3⬘). An amplification fragment of 877 bp was expected for

strains with complex type I, and a fragment of 979 bp was expected for strains with complex type II. The primers used to detect strains with the type III SCCmeccomplex were designed on the basis of the type III SCCmecsequence (GenBank accession no. AB047089). They were MRTc3 (5⬘-CCGTCGA TATC AATTGCTC-3⬘) and OR3. An amplification fragment of 562 bp was expected. The PCR mixture was as described previously (24). The following cycling con-ditions were used: 10 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, followed by 25 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 1 min.

RESULTS

PFGE pattern analysis.Among the 271 strains studied, 226

PFGE patterns that differed by at least one band were identi-fied. One hundred thirty-eight different patterns were obtained for MSSA strains, and 88 different patterns were obtained for MRSA strains. Eleven patterns were represented by MSSA strains recovered from more than one country, and two pat-terns were represented by MRSA strains recovered from more than one country. For example, esterase type 11 MSSA strains

from Denmark and France had the sameSmaI pattern (data

not shown).

Cluster analysis.After comparison of the Dice coefficients,

[image:2.603.44.548.81.355.2]

Ward’s algorithm was used to discern groups of isolates among the strains that have a defined level of similarity and to display relatedness between the groups. When clusters with more than 90% relatedness were considered, 20 genomic groups were defined. A dendrogram of all the genomic groups was con-structed on the basis of the levels of similarity (Fig. 1). Four major branches were identified. These branches were very di-vergent, with only 15 to 45% of relatedness. Branch I included six groups, branch II included seven groups, branch III in-cluded only two groups, and branch IV inin-cluded five groups.

TABLE 1. Repartition of the strains studied according to their geographical origin, esterase type, and resistance to methicillin

Geographical origin

No. of strains with the indicated esterase type:

Methicillin-susceptible strains Methicillin-resistant strains

15 16a 23 24 1 2 5 7 9a 10a 11 14 27 5 6 10a 11 14

Australia 2

Austria 1

Canada 1 1

Denmark 1 3 2 1 1 1 3 3 5 3 1 1

England 2 1 2 4 5 6

France 5 11 7 5 6 9 7 13 4 13 16 17 5 5 19 1 4 15

Germany 1 2 1 1 1 1 6

Hong Kong 1

Hungary 1

Ireland 1 1

Italy 1

Israel 1

The Netherlands 1 1

New Zealand 1

Norway 1

Portugal 1

Saudi Arabia 1

South Africa 1

Sweden 1 2 2

United States 2 1 1 4 4 5 7

Yugoslavia 1

Reference strains

NTCC8530 1

ATCC12599 1

ATCC25923 1

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Correlation between genomic group and esterase type. In most cases, a good correlation was obtained between genomic group and esterase type: strains within a given genomic group tended to be of the same esterase type (Table 2). This was true of all groups, except groups 1 and 2, which contained strains of two esterase types (type 5 and type 6). Strains of a given esterase type could be distributed into two or three groups. For example, strains of esterase type 11 were distributed in genomic groups 6a and 6b, but these groups are closely related and can be considered subgroups of a major group. This was also the case for strains of esterase type 7, which were distrib-uted in groups 12a and 12b. In contrast, strains of esterase type 14 were distributed, on the one hand, in the closely related

genomic groups 5a and 5b and, on the other, in a very diver-gent genomic group, group 17, which shares only 15% relat-edness with groups 5a and 5b.

Correlation between genomic group and phage type.When

the strains were typeable, a correlation was observed between the genomic group and the phage type (Table 2). Most of the MRSA strains (99 of 104) were lysed by phages of lytic group III. These strains belonged to genomic groups 1 to 6. Among the MSSA strains, phages of lytic group III preferentially lysed strains of genomic group 11. Strains of genomic groups 7, 9, 12, and 16 were lysed only by phages of lytic group II. Phages of lytic group I seemed to lyse preferentially strains of genomic group 17, and phages of lytic group V seemed to lyse prefer-entially strains of genomic group 14. Phage 95 lysed only strains of genomic group 13. Most of the strains from genomic groups 8, 10, and 15 were nontypeable.

Distribution of MRSA strains.Most of the MRSA strains

(83 of 105 strains) clustered in the six genomic groups of branch I (groups 1, 2, 3, 4, 5a, and 5b) (Fig. 1). Thus, these groups were called MRSA genomic groups. The groups were related by at least 68%. However, genomic groups 1, 2, and 3 also included 12 methicillin-sensitive strains, which did not

carry the mecA gene and which had none of the additional

antibiotic resistance markers usually associated with methicil-lin resistance. These 12 strains were fully sensitive to amino-glycosides, tetracyclines, sulfonamides, fluoroquinolones, and macrolides. In contrast, most of the methicillin-sensitive strains (154 of 166) were clustered in the 14 other genomic groups included in branches II, III, and IV (Fig. 1). Thus, these groups were called MSSA genomic groups. Four of these MSSA genomic groups included 22 MRSA strains. Sixteen of these MRSA strains were found in branch II, 11 were found in genomic group 6a, 5 were found in genomic group 6b, 5 were found in branch IV, 2 were found in genomic group 13, and 4 were found in genomic group 17. The MSSA genomic groups and the MRSA genomic groups were distantly related, as the coefficients of relatedness did not exceed 45%.

SCCmeccomplex analysis.The SCCmeccomplex types were

determined for 91 of the 105 MRSA strains. Type I and type II

SCCmeccomplexes share a common sequence at their right

ends, next to the junction with the chromosome. The type II

SCCmeccomplex differs from the type I SCCmeccomplex by

an additional 102 bp (17, 18). The type II SCCmeccomplex

includes SCCmectype II and type IV (20). The right sequence

of the type III SCCmeccomplex is different from those of the

type I and type II SCCmeccomplexes (18).

Primers specific for the type III SCCmeccomplex gave an

amplification product of about 562 bp for 37 strains (Table 3). All of these strains were of the same esterase type (type 14) and were distributed in the three corresponding genomic groups (groups 5a, 5b, and 17). Primers specific for the type I

and type II SCCmeccomplexes gave an amplification product

of about 877 bp, corresponding to the type I SCCmeccomplex,

for 33 strains and an amplification product of about 979 bp,

corresponding to the type II SCCmeccomplex, for 21 strains.

These strains belonged to genomic groups 1, 2, 3, 4, 6, and 13. Strains of esterase type 5 and type 6 from genomic groups 1, 2,

3, and 4 contained either the type I or the type II SCCmec

complex. Genomic groups 2 and 4 more frequently contained

the type I SCCmeccomplex (Table 3). Strains from genomic

FIG. 1. Dendrogram showing the estimated genetic relationships among 271 S. aureus strains clustered in 20 genomic groups. The dendrogram was generated by applying the Ward’s algorithm toSmaI patterns. R, resistant; S, sensitive.

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groups 6a and 6b (esterase type 11) could be differentiated by

their SCCmeccomplex types: all group 6a strains were of type

I, and all group 6b strains were of type II.

Comparison of the French strains with strains from other

countries.When the esterase type 1 strains, which clustered in

genomic group 15, were excluded (because only French strains of this esterase type were included in our collection), strains from France were found in all of the genomic groups, along with strains from 1 to 11 other countries. Strains from different geographical sources displayed similar or closely related pat-terns, differing by one to four bands, although there was no evidence of contact (Fig. 2).

The French MRSA strains were recovered in all the genomic groups that contained MRSA strains. However, 11 of the 19 French strains of esterase type 6, which was the most common type among the French MRSA strains, were clustered in genomic group 4, along with three strains from other countries (Ireland, England, and the United States). These strains

orig-inated from eight French hospitals in three distant areas and were isolated between 1984 and 1994. Two of the other French strains of esterase type 6 were found in genomic group 1, and six were found in genomic group 2. Strains of esterase type 14, which was the second largest group of French MRSA strains by esterase type, were found in genomic group 5a (4 strains), genomic group 5b (9 strains), and the distantly related genomic group 17 (2 strains).

DISCUSSION

[image:4.603.48.540.80.296.2]

In this study, we compared the genomic diversity of French strains of MSSA and MRSA with that of strains isolated in 21

[image:4.603.302.540.526.672.2]

FIG. 2. PFGE of SmaI-digested DNA from S. aureus strains of genomic group 13. Lanes 1, 8, 15, and 22, control strains; lanes 3, 4, 6, 7, 9 to 13, 16, 17, 21, and 23, strains from France; lanes 2, 5, and 18 to 20, strains from Denmark; lane 14, a strain from Germany; lane 24, a strain from England. The numbers on the left are in kilobases. TABLE 2. Correlation between esterase type, phage type, and genomic group

Genomic

group Esterase type

No. of strains

No. of strains in phage group:

I II III V III⫹OGa

I⫹OG 81 95 NTb

NDc

1 5 (5),d6 (5) 10 3 4 2 1

2 5 (2), 6 (11) 13 7 2 4

3 5 16 6 5 4 1

4 6 14 7 4 3

5a 14 21 1 7 2 6 5

5b 14 21 8 2 4 7

6a 11 13 1 3 1 1 2 5

6b 11 22 10 1 7 1 1 2

7 15 6 3 2 1

8 24 6 1 1 4

9 27 6 5 1

10 23 11 1 1 6 3

11 9a 7 7

12a 7 10 5 3 2

12b 7 9 5 2 4

13 10a 21 3 1 12 1 4

14 16a 17 17

15 1 6 1 1 3 1

16 2 15 12 1 2

17 14 27 8 1 5 4 7 2

aOG, other group. bNT, nontypeable. cND, not done.

dThe number of strains of each esterase type is given in parentheses.

TABLE 3. Distribution of the SCCmeccomplex among the genomic groups containing MRSA strains

Genomic group

No. of strains tested

No. of strains with SCCmeccomplex: Type I Type II Type III

1 7 2 5

2 10 8 2

3 7 3 4

4 12 9 3

5a 15 15

5b 18 18

6a 11 11

6b 5 5

13 2 2

17 4 4

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countries. In previous studies, typing ofS. aureusisolates re-covered from patients with cystic fibrosis showed concordance between the genome type obtained by PFGE and the types obtained by other typing methods such as esterase typing and phage typing (7, 27). As esterase electrophoretic typing ex-plores only a small part of the genome, it was less powerful

than PFGE for distinguishing between strains of S. aureus.

However, genotypes of the same esterase type were closely related and clustered in the same phylogenic branch (7). In contrast to PFGE, which detects events that occur randomly during chronic or recurrent infections, esterase electrophoretic typing detects variations that accumulate slowly and that there-fore could represent major phylogenic branches. To analyze

the clonal structure ofS.aureusfurther, a larger collection of

strains from various genetic backgrounds, as defined by ester-ase electrophoretic typing, and from diverse geographical ori-gins was studied. To obtain significant results, only strains of the most common esterase types were analyzed. PFGE has

been used to track the genetic development of naturalS.

au-reuspopulations (7, 21, 28). However, when strains from

var-ious origins are compared, the genetic variations indexed by

PFGE led to considerable differences inSmaI patterns.

There-fore, certain investigators do not consider this technique to be well suited for global epidemiological studies (11, 17). To de-fine strain relatedness accurately, computer analysis with Ward’s algorithm was chosen, because it minimizes overall variations. It discerned groups of strains that had some level of similarity and that were related to some extent. Twenty groups of strains were defined. In most cases, strains from the same genomic group or from a major group that included two sub-groups were represented by a unique esterase type, which identified the lineage. This was particularly true for the MSSA genomic groups. Conversely, strains of different esterase types (types 6 and 5) were found in two MRSA genomic groups (groups 1 and 2). It is noteworthy that these MRSA genomic groups were closely related to each other (90% similarity). Only strains of esterase type 14 were found in two very diver-gent groups, one with only MRSA strains and one with MSSA strains and a few MRSA strains. Thus, in some cases, strains of the same esterase type can be divergent. Either these two divergent lineages shared this trait or the technical conditions used were not able to reveal differences in the electrophoretic mobilities of the three enzymes tested.

Bacteriophage typing used to be the reference method for

epidemiological typing forS. aureus, but because of problems

with reproducibility and the nontypeability of strains, it was replaced by molecular typing methods, notably, PFGE (2, 29). As for esterase type, a correlation had previously been ob-served with PFGE type (7, 27). Our results revealed a rela-tionship between the genetic background of the strains and the lytic attack of the phages belonging to a given phage group. This was particularly noticeable for phage 95, which lysed only strains of genomic group 13. Group V phages (phages 94 and 95) almost exclusively lysed strains of genomic group 14. Witte (32) previously described strains lysed by group V phages as a complex containing strains that are possibly evolutionarily

re-lated clones that differ from otherS. aureus strains by

inde-pendent characteristics. In our study, although they presented

a certain degree of diversity in theirSmaI patterns, most of the

strains lysed by group V phages were closely related and very

divergent from the strains in other genomic groups. As ob-served many times during the characterization of epidemic MRSA strains, most of the MRSA strains tested were lysed by group III phages (8, 13). Epidemic MRSA strains that are lysed by phage groups other than group III appear to be very rare. Only one report has described an epidemic strain of phage group I (31). In our study, MRSA strains of esterase type 14 and genomic group 17 were lysed by group I phages.

MSSA strains were clustered in 17 different genomic groups. Although some of these groups were related, such as groups 7 to 11 and groups 15 and 16, the degree of interrelatedness was often very low (only 15%). These results are in agreement with previous estimates of genetic diversity (3, 6, 23); MSSA strains are polymorphic but are distributed in a limited number of major phylogenic branches. In contrast, MRSA strains were found to be clustered in a smaller number of genomic groups. Most of these groups, the MRSA groups, were closely related and constituted the major phylogenic branch, branch I. Only a few groups included in what we called MSSA groups were distantly related to the latter groups and to each other. Among these MSSA groups, only genomic group 6a included a large number of MRSA strains. MRSA strains appeared to be un-common in the other MSSA groups. Several studies have pro-vided evidence that MRSA strains evolved in a relatively small number of lineages that are clonally related and that some MRSA strains are present in distant lineages. This led some

investigators to the conclusion that the presence of the mec

gene in such widely divergent lineages is certainly the

conse-quence of the horizontal transfer of the mecregion into

dis-tantly relatedS. aureusstrains (11, 12).

The distribution of the SCCmeccomplex types among the

MRSA strains studied was interesting. The type I and type II

SCCmeccomplexes, on the one hand, and the type III SCCmec

complex, on the other, were found among strains from distinct

genetic backgrounds. The type III SCCmec complex was

re-covered exclusively from esterase type 14 strains clustered in major genomic group 5, and in distantly related genomic group

17. No other type of SCCmec complex was found in these

groups of strains. Although genomic groups 5 and 17 are dis-tantly related according to the PFGE results, they seem to have a common trait linked to the acquisition of the type III

SCC-mec complex element. Hiramatsu et al. (17) found that the

type III SCCmeccomplex is associated with a cluster of strains

defined by their ribotype, called clonotype III-A. Some of the strains of this clonotype were included in our study, such as the strains from Portugal, Hong Kong, Saudi Arabia, Yugoslavia, and Norway, and were clustered in major genomic group 5. In

contrast, the type I and type II SCCmeccomplexes were found

in strains belonging to the same genomic group, such as the strains of genomic groups 1, 2, 3, and 4. Enright et al. (11)

observed different SCCmeccomplex types among MRSA

iso-lates of the same multilocus sequence type and suggested that MRSA clones emerged on more than one occasion in the same genetic background. However, within genomic group 6

(ester-ase type 11), the type of SCCmeccomplex was clearly

associ-ated with the subgroup: strains from group 6a were of type I, and strains from group 6b were of type II.

S. aureus strains from France did not display specific genomic traits when they were compared to strains from other countries: the strains clustered in genomic groups with strains

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from other countries. As observed before (7, 27), MRSA and MSSA strains from different countries or from different areas of France were found to have identical or similar genome types, confirming that the degree of geographical spread is not correlated with the extent of genetic change detected by PFGE. This feature, which may be due to the slow genetic

development ofS.aureus, complicates epidemiological studies

involving the analysis of hospital isolates.

In conclusion, cluster analysis of the PFGE patterns of

pop-ulations of S. aureusstrains and correlation of the genomic

group with the esterase type and the phage type delineated 20 clonal groups. The distribution of the strains among the groups was independent of the geographical origin. The type III

SCCmeccomplex was strongly associated with three genomic

groups characterized by the same esterase type (type 14). Iden-tification of well-defined groups of strains with a common genetic background gives a basis for further understanding of the distribution of virulence genes or the dissemination of particular clones in the hospital environment.

ACKNOWLEDGMENTS

We gratefully acknowledge N. El Solh and A. Morvan from the Centre de Re´ference des Staphylocoques (Institut Pasteur) for help with phage typing. We thank N. Hoiby (University of Copenhagen) and M. Fournier (Institut Pasteur) for providing the strains, P. Ferron for technical assistance, and P. Pame for help typing the manuscript.

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Figure

TABLE 1. Repartition of the strains studied according to their geographical origin, esterase type, and resistance to methicillin
FIG. 2. PFGE of Smagenomic group 13. Lanes 1, 8, 15, and 22, control strains; lanes 3, 4, 6,7, 9 to 13, 16, 17, 21, and 23, strains from France; lanes 2, 5, and 18 to20, strains from Denmark; lane 14, a strain from Germany; lane 24, aI-digested DNA from S

References

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