INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Staphylococcus sciuri was first described by Kloos and colleagues in 1976 (15) and is considered one of the most ancestral and dispersed staphylococcal species, with a wide range of habitats that includes the skin of several animals as well as environmental reservoirs, such as soil, sand, water, and furniture (14, 15, 16, 17). The impressive colonizing capacity of this species may result from its broad range of biochemical activities, which includes the ability to use inorganic nitrogen salts as the sole source of nitrogen. Traditionally described as a commensal species of rodents, marsupials, cetaceans, artiodactyls, and perissodactyls, S. sciuri has also been isolated from healthy and sick domestic and husbandry animals, including household cats (4, 12), domestic dogs (15), cattle, goats, poultry, sheep, horses, and pigs (3, 7, 8, 13, 27), and houseflies (9). Although S. sciuri is associated rarely with colonization or infection in humans (14), it has been occasionally isolated from human clinical samples (1, 3, 6, 10, 11, 17, 18, 21, 29, 31).

In an earlier report (3), we described a collection of 134 S. sciuri isolates, mainly of animal origin, all carrying a homologue of the mecA gene present in methicillin-resistant Staphylococcus aureus (MRSA) strains and other methicillin-resistant pathogenic staphylococci. The homology between the mecA sequences found in S. sciuri and MRSA (79.5% DNA sequence similarity and 87.7% amino acid sequence similarity) (32) and the ubiquitous presence of mecA sequences in the S. sciuri chromosome led to the proposal that mecA might be a native gene of S. sciuri and the ancestor of the mecA element carried by MRSA. In the same study and another study, we also described five S. sciuri isolates which carried, in addition to S. sciuri mecA, a second copy of mecA, identical to the one in MRSA (3, 33).

In the present work, we analyzed a new collection of 28 S. sciuri strains, all isolated from humans, including healthy and hospitalized individuals, with three major aims: first, to assay the genomic diversity of several S. sciuri isolates recovered from individuals sharing a common environment, in order to gather additional data on the main patterns of S. sciuri colonization and dissemination among humans; second, to assay the presence of both variants of the mecA gene among the isolates; and third, to search for any correlation between carriage or infection caused by strains with the MRSA mecA gene and antibiotic consumption.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Bacterial strains. The S. sciuri strains characterized in this study are listed in Table 1 and were from two distinct sources: healthy human carriers (adults and children) and hospitalized patients.

Media and growth conditions. All strains were aerobically grown at 37°C. S. aureus and S. sciuri strains were grown in tryptic soy broth or agar (Difco Laboratories, Detroit, Mich.). Luria-Bertani medium (26) supplemented with 50 µg of ampicillin (Sigma Chemical Company, St. Louis, Mo.) per ml was used to grow E. coli strain MC1061-1.

Species identification tests. Species identification tests included a catalase assay with 3% hydrogen peroxide to detect the presence of cytochrome oxidase; mannitol fermentation, by spreading overnight cultures with a sterile inoculation loop on mannitol salt agar (Difco) and incubating them at 37°C for 20 h; and coagulase production with the Bacto Coagulase Plasma test (Difco) according to the manufacturer's instructions. Detection of cytochrome c (modified oxidase test) was carried out with DrySlide Oxidase (BBL Microbiology Systems, Cockeysville, Md.) according to the manufacturer's instructions. Further biochemical profiles were determined with the ID32 STAPH system (bioMérieux Vitek Inc., Hazelwood, Mo.).

Antimicrobial susceptibility testing. Antibiotic susceptibilities were determined by the Kirby-Bauer technique with Muller-Hinton agar (Difco) according to National Committee for Clinical Laboratory Standards recommendations and definitions (22). The following antibiotic disks (BBL) were used: penicillin (10 µg), tetracycline (30 µg), erythromycin (15 µg), gentamicin (30 µg), clindamycin (12 µg), and novobiocin (5 µg). S. aureus ATCC 25923 and the type strains of the three S. sciuri subspecies were used as controls. The breakpoints used for novobiocin resistance were inhibition zones up to 12 mm for resistant strains and larger than 16 mm for susceptible strains (19).

Detection of beta-lactamase production. The beta-lactamase assay was carried out with DrySlide Nitrocefin (Difco) according to the manufacturer's instructions.

PAPs for oxacillin. Population analysis profiles (PAPs) were determined as recently described (24). Briefly, 10-µl drops of several dilutions (from 10⁰ to 10⁵ or 10⁷) of aerobically grown overnight cultures were spotted on the tops of Falcon Integrid square plates (100 by 15 mm) (BBL) containing tryptic soy agar with serial (twofold) dilutions of oxacillin (Sigma) at concentrations of 0 and 0.75 to 800 µg/ml. After inoculation, the plates were held vertically for a few seconds to allow the spread of the cultures across the surfaces of the plates. Colonies were counted after incubation for 48 h at 37°C. A graphic representation (PAP) was constructed by plotting the logarithm of colony counts against the concentration of oxacillin. The MIC was defined as the lowest concentration of antibiotic that inhibited the growth of 99.9% of cells.

Preparation of chromosomal DNAs for conventional and pulsed-field gel electrophoresis (PFGE). Chromosomal DNAs were prepared as previously described (5) with the following modifications for S. sciuri strains: EC buffer was supplemented with 0.5% Brij 58 (Sigma), and lysis took place for 5 h at 37°C.

Restriction digestion. Restriction digestion with Bsp106 (an isoschizomer of ClaI) and SmaI was performed according to manufacturer recommendations (Stratagene and New England Biolabs, respectively).

Conventional gel electrophoresis. Conventional gel electrophoresis was performed with 1% LE agarose (FMC BioProducts, Rockland, Maine) gels in Tris-acetate-EDTA (TAE) buffer (26) for 14 to 16 h at 1.5 V/cm.

PFGE. PFGE was carried out with a contour-clamped homogeneous electric field apparatus (CHEF-DRII; Bio-Rad, Hercules, Calif.) as previously described (25). Analysis of SmaI macrorestriction profiles was done by visual inspection, and PFGE patterns were assigned using the criteria proposed by Tenover and colleagues (30). Isolates with an identical PFGE pattern were included in the same type, designated by an uppercase letter. Isolates with PFGE types differing by up to six fragments were assigned to subtypes, identified by uppercase letters followed by numerical codes.

Hybridization with the mecA probe. ClaI and SmaI DNA fragments in conventional and PFGE gels were transferred by vacuum blotting as previously described (5). The mecA probe used was the 1.196-kb XbaI-PstI fragment from the mecA gene of the Australian MRSA strain ANS46 cloned in plasmid pTZ19 (20). For probe labeling and hybridization, an enhanced chemiluminescence nonradioactive labeling kit (RPN3040; Amersham Life Science, Arlington Heights, Ill.) was used according to the manufacturer's instructions.

Detection of the S. aureus mecA and mecI and S. sciuri mecA sequences. Detection of gene sequences was carried out by PCR amplification of chromosomal DNA with specific primers: for S. sciuri mecA, we used primers SAMECA358 (5'-ATCCATCAATATTGAACCA) and SAMECA1482 (5'-TATATCTTCACCAACACC); for S. aureus mecA, we used primers SAMECA349 (5'-GTTAAAGAAGATGGTATG) and SAMECA1482; and for S. aureus mecI, we used primers MECI1 (5'-GTATGAAATATCATCTGCAG) and MECI2 (5'-AACAGAGGAAATATTCAACG). Amplifications were carried out with the Perkin-Elmer Cetus (Norwalk, Conn.) PCR reagent kit according to the manufacturer's instructions and with the following amplification protocol: 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C for 25 cycles and a final extension at 72°C for 4 min. Amplification products were resolved in 0.8% LE agarose (FMC BioProducts) gels in TAE.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Twenty-eight S. sciuri isolates recovered from 27 individuals (20 draftees, 2 children, and 5 hospital patients) were included in this study (Table 1).

Biochemical profile. All S. sciuri isolates showed a similar biochemical profile, as determined by the ID32 STAPH system. Variations were observed in the fermentation of lactose, ribose, N-acetylglucosamine, turanose, and arabinose and the production of alkaline phosphatase. Some isolates produced beta-glucuronidase. Two isolates (SS-5 and SS-24) could not be identified with this system because they failed to reduce nitrates. However, their similarity to the other isolates, namely, the remaining biochemical profile and PFGE type (see below), led us to classify them as S. sciuri. Moreover, these two isolates resembled another S. sciuri strain, designated K2 or BT22 and described earlier (3, 17), which was also reported as unable to reduce nitrate to nitrite (17). Two other strains (SS-11 and SS-20) had subpopulations detected in the oxacillin disk assay. The colonies grown outside and inside the halo had different behaviors toward ribose fermentation according to the ID32 STAPH system; the more resistant subpopulations failed to produce ribose. These subpopulations also grew slowly on blood agar plates, producing only small colonies.

Antibiotic susceptibility testing. As expected, all 28 S. sciuri isolates were resistant to novobiocin, which is a characteristic of this species. Twenty-one isolates shared a common antibiotype that included additional resistance to penicillin, intermediate resistance to clindamycin, and susceptibility to tetracycline, erythromycin, and gentamicin. Exceptions to this pattern were found in seven isolates and are detailed in Table 2. The MICs of oxacillin for 17 isolates were low (0.75 to 6 µg/ml). The majority of these isolates (13 of 17) had subpopulations resistant to up to 3 to 6 µg/ml. The MICs for 11 of the 28 isolates were higher (12 to 25 µg/ml), and there were subpopulations resistant to up to 800 µg of oxacillin per ml. The majority of the isolates (79%) produced beta-lactamase.

Genotypic analysis. (i) PFGE patterns. The 28 S. sciuri isolates could be assigned to 16 PFGE types (Table 2 and Fig. 1A); four of them had two or more different subtypes (Table 2 and Fig. 1A). The most common PFGE patterns were A and B (each with five isolates), C (four isolates), and D (two isolates). The remaining 12 PFGE patterns (corresponding to 43% of the isolates) were represented by single isolates only. Although PFGE patterns A, B, and C had more than six band differences, according to the interpretation proposed by Tenover and colleagues (30), we should emphasize that their similarity suggests that isolates with these PFGE patterns have probably evolved from a single strain, as can be observed by visual inspection of the macrorestriction profiles (Fig. 1A).

View larger version (73K): [in this window] [in a new window]   FIG. 1.   (A) PFGE patterns of S. sciuri strains after SmaI digestion. (B) Hybridization of S. sciuri SmaI digests with an S. aureus mecA probe.

(ii) Localization of the mecA sequences in PFGE profiles. Hybridization of SmaI chromosomal digests with a DNA probe internal to the mecA gene of an MRSA strain showed that 13 isolates hybridized with this probe in two SmaI bands (Table 2 and Fig. 1B). The molecular size of the hybridizing SmaI bands ranged from 140 to 490 kb. Twelve of the 15 isolates with a single SmaI-mecA hybridization band carried the mecA copy in a high-molecular-size (374 to 520 kb) SmaI fragment. The other three isolates (SS-34, SS-37, and SS-41) hybridized with the mecA probe in smaller SmaI bands, ranging from 138 to 164 kb.

(iii) Analysis of the mecA copies. All isolates with two SmaI-mecA hybridization bands had three ClaI-mecA hybridization bands, corresponding to the two expected bands of MRSA mecA and the single band of S. sciuri mecA; this result is in accordance with our previous observation that unlike mecA of MRSA strains, S. sciuri mecA has no restriction sequence for ClaI (3, 32). On the other hand, isolates with only one SmaI-mecA hybridization band had a single ClaI-mecA hybridization band (Fig. 2), suggesting that mecA carried by these isolates is the S. sciuri native copy.

View larger version (89K): [in this window] [in a new window]   FIG. 2.   Polymorphisms in the S. sciuri mecA vicinity after hybridization of ClaI digests with an S. aureus mecA probe.

Expression of resistance to oxacillin. For all S. sciuri isolates with both copies of mecA, the MICs of oxacillin ranged from 3 to 25 µg/ml; the MICs for the majority (77%) were 12 or 25 µg/ml, with subpopulations able to grow in the presence of up to 800 µg of antibiotic per ml. On the other hand, for all but two isolates with a single copy of mecA, the MICs were lower (0.75 to 3 µg of oxacillin per ml), and no subpopulations were detected at antibiotic concentrations of higher than 6 µg/ml. The two exceptions found were strains SS-37 and SS-41. Although both showed single SmaI-mecA and ClaI-mecA hybridization bands, the MIC for strain SS-37 was 25 µg/ml, with subpopulations able to grow in the presence of up to 800 µg/ml, while for strain SS-41, the MIC was low (3 µg/ml), with subpopulations able to grow in the presence of up to 50 µg/ml (Fig. 3).

View larger version (37K): [in this window] [in a new window]   FIG. 3.   PAPs of analyzed S. sciuri strains with only one mecA copy (A) or two mecA copies (B). oxac., oxacillin.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this work, we characterized a group of 28 S. sciuri isolates from human origin, mostly from colonization sources. Twenty-three out of the 28 isolates described in this study were found among a collection of 252 staphylococcal isolates recovered from healthy carriersselected on the basis of their resistance to oxacillin in the disk diffusion method (22)and characterized in our laboratory (H. de Lencastre et al., unpublished data). Although the percentage of S. sciuri isolation relative to that of other staphylococci may vary with an increase in the number of isolates analyzed (or the use of new resistance breakpoints [23]), we speculate that S. sciuri may be more relevant as a human colonizer species than has been considered.

Some of the S. sciuri isolates recovered from different individuals (draftees) over a 3-year period were clonally related, as determined by PFGE analysis. The clonal dissemination of these strains within individuals in this barrack and their persistence in this environment over a 3-year period may be explained by the high survival skills of this bacterium, which allow its residence in an environmental niche that would represent a source for the continuous contamination of individuals. It has been claimed that the isolation of S. sciuri in humans is a result of close contact with animals. At the time of sampling, the population studied had no permanent contact with animals, and among individuals who did have sporadic contact, the majority carried strains with PFGE patterns already found in previous years. These results sustain our previous hypothesis that these S. sciuri strains were acquired in the barrack environment rather than from animals.

Another interesting result was the isolation of S. sciuri strains among clinical isolates, particularly from one infection source (blood). Two other reports also documented the isolation of this species from blood samples (11, 31), and other authors mentioned the isolation of S. sciuri strains from other clinical sources, such as infected wounds of hospital patients (18, 31) and umbilici of infants and the teats of their mothers (17). Furthermore, several strains of S. sciuri were reported to be adherence positive (10) or to produce slime (31), which is considered a potential factor for both colonization and virulence. Nevertheless, the paucity of reports of infections caused directly by S. sciuri indicates that it is probably a rare and opportunistic pathogen in humans. In fact, besides the study of Hedin and Widerström (11) that clearly identified S. sciuri as the bacterium responsible for an endocarditis case, all the other reports of clinical S. sciuri did not prove unequivocally that this was the agent responsible for the infections reported. Our own observation that S. sciuri may colonize humans more frequently than previously thought may explain the recovery of this bacterium from clinical samples.

Although the clinical significance of S. sciuri may remain controversial, the capacity of this species to carry resistance determinants is well established. It is known that S. sciuri strains may carry plasmids with antibiotic resistance markers (28), and some clinical isolates were found to be multiresistant (18, 31). Kawano and colleagues (13) described the isolation from healthy chickens of S. sciuri strains resistant to several classes of antibiotics; many of the strains studied by Kloos et al. (17), mostly isolated from wild animals, showed resistance patterns comparable to the ones described in this work, with the exception that most of the strains studied by those authors were susceptible to clindamycin. In our work, no obvious differences were seen among the resistance patterns of the strains isolated from healthy carriers or hospital patients, although the clinical strains showed some additional antibiotic markers.

All S. sciuri strains characterized in this study carried the S. sciuri mecA copy. This result confirms our earlier findings (3) and further supports the hypothesis that this is a native element of the S. sciuri chromosome. Furthermore, a high percentage of isolates (46%) also carried the MRSA mecA copy. This finding is even more striking if we consider that most isolates carrying MRSA mecA (11 out of 13) were isolated from healthy individuals. Of these, only one person (carrier of strain SS-26) had taken antibiotics, namely, amoxicillin-clavulanic acid, during the month previous to sampling. Therefore, no correlation was found between carriage of S. sciuri with MRSA mecA and antibiotic consumption. Similarly, it was not possible to find any correlation between the presence of these strains and attendance at hospitals, since only three individuals had been in a hospital recently and, of these, only one carried a strain (SS-20) with the MRSA mecA gene.

MRSA mecA was found in a heterogeneous chromosomal background, since five different strains carried this element. Four out of these five strains were isolated from healthy individuals. This is an interesting result, because it illustrates the in vivo dissemination of MRSA mecA in an antibiotic-free environment. In addition, all S. sciuri isolates carrying MRSA mecA also carried the mecI element. The simultaneous presence of both sequences strongly supports the hypothesis that the MRSA-like elements were recently acquired from an exogenous donor, probably a pathogenic species of staphylococci, followed by their spread within different S. sciuri strains. We had previously reported the presence of MRSA mecA in S. sciuri isolated from human samples (3); however, in the previous study, all the S. sciuri isolates carrying MRSA mecA were clonally related and were isolated from a single hospital ward, suggesting that mecA transfer from a pathogenic, MRSA strain had occurred once, followed by clonal dissemination of the S. sciuri strain carrying the newly acquired MRSA mecA gene. The results presented in this work seem to illustrate this event as well as the transfer of MRSA mecA among different S. sciuri strains.

In this same previous study, it was reported that S. sciuri mecA was not able to confer significant resistance to beta-lactam antibiotics (3). This observation was confirmed in the present study. Comparison between the profiles of resistance toward oxacillin of the S. sciuri strains with one or two mecA copies clearly indicated that only strains with the MRSA mecA copy are able to grow in the presence of this antibiotic. However, two exceptions were found, strains SS-37 and SS-41. Although both strains carried only S. sciuri mecA, their PAPs resembled those of strains with both mecA copies. Furthermore, both strains were resistant to penicillin but failed to produce beta-lactamase, indicating that the mecA copy present in their chromosomes conferred resistance to beta-lactams. This mecA copy could be amplified with primers specific for S. sciuri mecA but not with primers specific for MRSA mecA, thus excluding the hypothesis of the presence of a single MRSA mecA copy. Therefore, it seems that the mecA gene found in these strains is able to confer the same level and type of resistance as the copy carried by MRSA, a finding to be further analyzed in future work. Further studies should also focus on the presence of S. sciuri strains in human samples and their relationship to contacts with animal or environmental contamination, in order to establish the real risk factors and impact of human colonization by S. sciuri as well as to address the pathogenicity of this species.