INTRODUCTION

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Infection is one of the most devastating complications of prosthetic joint surgery (3, 12, 14, 15, 17, 27, 33). Staphylococcus epidermidis, which can adhere to implants, has increasingly been identified as a cause of such infections (6, 16, 21, 23, 24, 26, 35). S. epidermidis is a normal commensal organism of the skin which may contaminate specimens during collection, and it is therefore difficult to assess the clinical significance of its detection, particularly when the bacterial colonies obtained have diverse aspects and drug resistance phenotypes. Detection of gram-positive cocci by direct inspection and/or repeated isolation of the same mixtures of strains from a series of specimens from the same patient provides good evidence of infection causality (17, 19, 36). The presence in pus and tissue specimens of multiple S. epidermidis strains, frequent in infections following implantation of joint prostheses (17), may be due either to the genomic instability of a single infectious clone or to an infection caused by a polyclonal mixture of strains such as the mixture of strains found in the skin flora. To examine these two possibilities, we compared the genomes of the various phenotypically distinct S. epidermidis strains detected in pus and tissue specimens from 14 patients with chronic prosthetic joint infections. Diverse S. epidermidis colonies isolated from the skin flora of eight healthy individuals were used for comparative analysis of the intraindividual colonies that were selected on the basis of their different colony morphologies and drug resistance phenotypes.

	MATERIALS AND METHODS

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Patients and healthy individuals. The 14 patients included in this study were infected following the implantation of joint prostheses (see Table 1). Joint replacements were carried out in diverse hospitals, and there was no epidemiological link between the patients. The files of all patients except patients B and N were consulted. The 12 patients whose files were available had pain without fever. The delay between surgery and pain was not regularly registered in the files; therefore, we report (see Table 1) the delay between the implantation of the prosthesis and its replacement (3 to 84 months). The replacement was not necessarily done immediately after the first signs of infection. Five of the 12 patients (patients A, E, I, L, and P) had abscesses, which had fistulized in patients A, L, and P, and 6 patients (patients E, F, I, L, O, and P) presented with edema. Bacteriological samples were obtained at the time of intraoperative assessment and surgery for excisional arthroplasty. No antibiotic was administered to any patient for at least 1 week before joint surgery. Patients whose infected specimens yielded only S. epidermidis were included in this study.

Bacterial strains and plasmids. A total of 82 S. epidermidis strains originating from 14 infected patients (54 strains from infected specimens and 5 strains from cutaneous samples of patient P) and from 8 healthy individuals (23 strains) were studied (see Tables 1 and 2, respectively). The infected specimens and the skin swabs were cultured on sheep blood agar. After at least 48 h of incubation at 37°C, the species of the colonies that were isolated from each sample and that had different morphologies (size, color which varied from white to grey, shape and outline, presence or absence of hemolytic activity, rough or smooth aspect) was determined as described previously (8), and the colonies were tested with the ID32 Staph system (BioMérieux, Marcy-l'Etoile, France). Patients whose infected specimens contained only S. epidermidis colonies were included in this study. The drug resistance pattern was determined for each different S. epidermidis colony isolated from the specimens of each patient. The strains that were distinguishable by at least one drug resistance marker were studied independently. Similarly, the S. epidermidis colonies isolated from skin flora were selected on the basis of their distinct morphologies and drug resistance patterns. Bacterial suspensions in brain heart infusion containing 30% glycerol were stored at 80°C before analysis.

Susceptibility to antimicrobial agents. The pattern of resistance to antimicrobial agents was determined by the disk diffusion method (4). The markers tested were those which enabled us to detect the drug resistance phenotypes described to date among staphylococci. Commercially available disks loaded with the following antibiotics were used: penicillin G, oxacillin, spectinomycin, streptomycin, kanamycin, neomycin, gentamicin, tobramycin, chloramphenicol, erythromycin, lincomycin, trimethoprim, sulfonamide, tetracycline, minocycline, pefloxacin, rifampin, fusidic acid, fosfomycin, and vancomycin (Diagnostics Pasteur, Marne-la-Coquette, France) and mupirocin (Mast Diagnostics, Mast Group Ltd., Merseyside, United Kingdom). Additional disks prepared in our laboratory contained 20 µg of pristinamycin IIA, 40 µg of pristinamycin IB, 0.2 µmol of cadmium acetate, 0.2 µmol of sodium arsenate, 0.2 µmol of mercuric nitrate, 200 µg of ethidium bromide, 200 µg of acriflavine, 200 µg of propamidine isethionate, or 10 µg of cetyltrimethylammonium bromide.

Ribotype determination. Cellular DNA was extracted, cleaved with HindIII and EcoRI (Pharmacia Biotech, Uppsala, Sweden) separately, electrophoresed, transferred onto Hybond N⁺ nylon membranes (Amersham International), and tested for hybridization under stringent conditions with pBA2 radiolabeled with [-³²P]dTCP (110 TBq/mmol) as described elsewhere (7, 8).

Pulsed-field gel electrophoresis of macrorestriction fragments and comparative analysis of banding patterns. The protocol used for the determination of SmaI or SstII restriction patterns was described previously (10). Concatameric bacteriophage lambda DNA molecules (48.5 kb; Bio-Rad) and the SmaI fragments of the cellular DNA from Staphylococcus aureus NCTC8325 were used as size standards. Macrorestriction fingerprints were compared visually and were scanned with GelCompar software (Applied Maths, Kortrijk, Belgium). A similarity matrix was created by using the band-based Dice similarity coefficient (11). The unweighted pair-group method with average linkages was used to cluster the strains on the basis of the patterns obtained with each of the two enzymes used.

	RESULTS

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Identification to the species level. Fifty-one of the 54 coagulase-negative staphylococci isolated from the infected specimens from patients were assigned to S. epidermidis by the ID32 Staph system. Analysis of the hybridization patterns with pBA2 enabled us to assign to S. epidermidis three strains, strains 95160, 96388, and 96390 (Table 1) not classified by the ID32 Staph system.

Drug resistance phenotype. The intrapatient strains had in common 1 to 14 markers and were distinguishable by 1 to 10 additional markers (Table 1). Among the strains from healthy individuals, the intraindividual strains had in common one to three resistance markers and differed by 1 to 13 additional markers (Table 2).

Analysis of the macrorestriction patterns. (i) Comparison of the SmaI and SstII patterns of patient strains isolated from infected specimens. The 54 isolates of S. epidermidis tested (Table 1) gave a total of 30 different SmaI patterns and 29 different SstII patterns. Each SmaI profile included 12 to 17 fragments (Fig. 1), and each SstII profile included 12 to 21 fragments (data not shown).

View larger version (41K): [in this window] [in a new window]   FIG. 1.   Classification and schematic representation of the SmaI patterns of the S. epidermidis strains isolated from 14 patients (patients A to F, H, I, K and P) and of the two variants of 94351, Rep1 and Rep2, obtained by subculture.

(ii) SmaI profiles of the S. epidermidis strains isolated from the skin flora of patient P. From patient P's flora, five strains with distinct phenotypes were distributed into five different SmaI genotypes which did not include any of the three strains isolated from the infected specimens (Table 1; Fig. 1). The three strains isolated from both nares, narR, narL1, and narL2, were possibly related because their SmaI patterns differed by four or five fragments.

(iii) SmaI patterns of the hand flora strains isolated from healthy individuals. Among the samples from the eight healthy individuals, only the hand swab of surgeon t gave monoclonal strains (Table 2). The SmaI patterns of strains t4 and t8 differed by only one SmaI fragment, whereas the pattern of the third strain (strain t5) from the same surgeon differed by at least eight fragments. For each of the seven other healthy individuals, the intraindividual colonies were considered unrelated, with at least seven SmaI band differences between the patterns (Table 2). Some of the S. epidermidis strains isolated from different surgeons working in the same operating room were clustered (Table 2), with percentages of similarity above 90 (data not shown). Indeed, the SmaI patterns of strains f1, l3, and j2 from surgeons f, l, and j, respectively, differed from each other by no more than two fragments, and those of strains w3 and m3 from surgeons w and m, respectively, differed from each other by three fragments. The related strains, which were resistant to oxacillin, were detected on the hands of several surgeons and were probably acquired in the hospital.

Analysis of HPs with pBA2 (ribotypes). The HindIII and EcoRI HPs obtained with pBA2 were determined for the three coagulase-negative staphylococci which could not be classified by the ID32 Staph system (strains 95160, 96388, and 96390) and for a strain representative of each of the genotypes and subtypes of strains from patients (Table 1). For the latter strains, the assignment to S. epidermidis by the ID32 Staph System was confirmed by the comparative analysis of the ribotypes with those of the validly classified staphylococci already in our database.

Analysis of the stability of strain 94351 from patient F. Although they were clustered in two genotypes, the six strains from patient F were scored as possibly related (fewer than six band differences). Strain 94351 had the largest number of drug resistance markers and was chosen for use in an evaluation of phenotypic and genomic stability after subculturing in brain heart infusion for 900 generations. Three hundred isolated colonies were studied, and two types of variants were detected: (i) those which had lost their oxacillin resistance, for example, strain Rep2, and (ii) those which had lost their resistance to oxacillin and to macrolides-lincosamides-streptogramin B, for example, Rep1 (Table 1). The loss of oxacillin resistance was associated with the loss of the mecA gene (data not shown). Neither of the two variants selected in vitro had a drug resistance pattern that was the same as those of any of the S. epidermidis strains isolated from the specimens from patient F. The SmaI patterns of strains Rep2 and 94351 were indistinguishable. The SmaI pattern of strain Rep1 differed from that of strain 94351 by five restriction fragments and from that of each of 94348, 94349, and 94350 by six fragments. The variant Rep2 strain was more closely related to the strains from patient F than to those from the 13 other patients (Fig. 1), suggesting that this variant was not a contaminant.

	DISCUSSION

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Variations in phenotypic characteristics, including virulence factors (2, 9, 32, 35) and drug resistance patterns (6, 13, 29), and in plasmid content (22) have often been reported for S. epidermidis strains. The detection of multiple S. epidermidis strains distinguishable by their drug resistance patterns in blood cultures, in pus, and in various other specimens from infected patients does not, however, result exclusively from the instability of phenotypic traits but results also from coinfection with unrelated S. epidermidis strains (1, 17, 20, 30, 34). Indeed, some cases of endocarditis following implantation of a prosthetic valve were recently shown to be attributable to polyclonal S. epidermidis populations (1, 30). Therefore, the detection in samples from the same patient of S. epidermidis strains with different antibiograms does not necessarily indicate contamination of the samples during collection. For the 14 patients in our study, contamination of the specimens by the polyclonal S. epidermidis strains of the skin flora is not likely, not only because most specimens were collected under the rigorously aseptic conditions required for surgery and in a sterile operating field but also because mixtures of phenotypically divergent colonies were detected in at least two specimens from the same patient. The proportion of each S. epidermidis strain in the mixtures could not be ascertained in our study because the drug resistance phenotype of every different strain was taken into consideration when the antibiotic therapy was chosen. This policy has probably contributed to the very high rate of successful eradication of infections following prosthesis replacements in the two hospitals that participated in the study.

The source of the delayed infections for the 14 patients in our study is not known. S. epidermidis of the skin flora or the environment may have been introduced into the operative wound. Alternatively, infection of the prosthesis may have been of blood origin and thus was not acquired during surgery. Collection of the S. epidermidis strains from the skin flora of the patient just before surgery, from the flora of the staff, and from the environment of the operating room would be required to trace the source of infection.

The comparative analysis of drug resistance patterns was insufficient to assess the degree of genomic relatedness of strains because some monoclonal strains in our study gave colonies that differed by up to nine drug resistance markers. Mapping of the genome regions carrying the drug resistance genes in the related strains from each patient would be required to elucidate the genetic changes responsible for the differences in drug resistance markers. When patients are infected with a single clone, the mutation, rearrangement, loss, or transposition of DNA may lead to the diversity of phenotypes (25). Genetic transfers, in particular, conjugation, may also occur when a polyclonal population is present at a single site. When phenotypic variations are not associated with detectable modifications of the macrorestriction profiles, it is likely that drug resistance phenotypic diversity results from divergence in plasmid content, but minor chromosomal modifications cannot be ruled out. This was probably the case for the Rep2 derivative whose SmaI pattern was indistinguishable from that of the parental strain, strain 94351, despite the loss of mecA, which is usually located in the chromosomes of staphylococci.

In our study, only 4 of the 14 patients appeared to be infected with a polyclonal population of strains. For the 10 other patients, all the intrapatient strains appeared to be closely or possibly related, despite the substantial diversity of the drug resistance patterns. Thus, for most patients, the multiple S. epidermidis strains found in the specimens were derivatives of a single clone. Such derivatives may have resulted from changes that occurred in situ during the infection process or may have preexisted in the intraoperative source of infection, which is often the skin flora. However, the latter possibility is not probable because closely related S. epidermidis colonies distinguishable by their morphologies and drug resistance patterns were detected in the skin flora of only one of the eight healthy individuals studied.

In conclusion, it is important to try to identify the largest number of colonies with distinct aspects in deep samples collected from patients whose prostheses are suspected of being infected so that antibiotic therapy can be directed at the most resistant isolates. Failure to eradicate these infections may, in some cases, be due to the failure to detect all the different S. epidermidis variants and clones present at the site of infection.