Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in Zürich, Switzerland (2003): Prevalence of Type IV SCCmec and a New
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微生物临床杂志 2005年第10期
Department of Medical Microbiology, University of Zürich, CH-8006 Zürich, Switzerland
Gram-Positive Bacteria Typing and Research Unit and Molecular Genetics Research Unit, School of Biomedical Sciences, Curtin University of Technology, Perth, Australia
Hospital Epidemiology Unit, Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zürich, CH-8091 Zürich, Switzerland
ABSTRACT
The majority of methicillin-resistant Staphylococcus aureus (MRSA) isolates, recovered in 2003 at the Department of Medical Microbiology in Zürich, Switzerland, belonged to major clones that are circulating worldwide. Staphylococcal cassette chromosome mec type IV (SCCmec-IV), harbored by half of the isolates, was found in sequence type 217 (ST217), which is an allelic variant of epidemic MRSA-15 (designated EMRSA-15), in a new local ST617 descending from clonal complex CC8 and in low-level oxacillin-resistant strains of multiple genetic lineages characteristic of community-onset MRSA. SCCmec-I, SCCmec-II, and SCCmec-III were in the minority, and four MRSA isolates had complex, rearranged SCCmec elements. A novel SCCmec-N1 of approximately 30 kb, associated with a dfrA gene and a ccr4-related recombinase complex, was identified in a large number of low-level oxacillin-resistant isolates, which descended from the successful clonal complex CC45 and are spreading among intraveneous drug users. In contrast, the SCCmec types of oxacillin-resistant coagulase-negative staphylococci (MRCNS) were of completely different composition. SCCmec type I (SCCmec-I) and SCCmec-II were more frequent than in the MRSA, while fewer contained SCCmec-IV. The other MRCNS displayed 11 different, complex patterns, suggesting frequent recombination between different SCCmec elements. With one ccr-negative exception, these strains amplified between one and three different ccr products, indicating either new varied complexes or multiple ccr loci. This suggests the presence of novel SCCmec types in MRCNS and no extensive interspecies SCCmec transfer between MRSA and MRCNS.
INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections and has also recently established itself as a significant community-acquired pathogen (7, 9). Community-onset MRSA (cMRSA) differs from nosocomial MRSA in that it does not generally belong to the major clonal groups of epidemic MRSA, is susceptible to most non--lactam antibiotics, contains the type IV SCCmec (for staphylococcal cassette chromosome mec, the mobile genetic element encoding methicillin resistance), and frequently carries genes responsible for the production of Panton-Valentine leukocidin (PVL) (13, 22, 26). In contrast, nosocomial MRSAs are generally multidrug resistant and contain SCCmec types I, II, or III.
Besides the five major allelic types of SCCmec elements, epidemiological studies paired with molecular characterizations suggest the existence of additional, different SCCmecs (16, 28, 36). The origin of the SCCmec element is still unclear, with the closest mecA homolog found in Staphylococcus sciuri (43). Methicillin-resistant coagulase-negative staphylococci, which are more frequently carriers of SCCmec than S. aureus, are postulated to be the reservoir for the transfer of methicillin resistance to S. aureus (2). SCCmec has the attributes of a mobile element, such as the ccr genes, encoding recombinases that were shown in vitro to be responsible for the precise excision and integration of SCCmec into the chromosome. However, the type 1 and type 3 ccrA and ccrB genes are dysfunctional. This, plus the larger size of nosocomial SCCmec types I to III, may have been the reason that they have spread only into a restricted number of genetic lineages, whereas the smaller type IV SCCmec seems to be more mobile and to be associated with more diverse strain lineages (29, 32).
The ability of MRSAs to segregate a highly resistant subpopulation in the presence of -lactams makes them resistant to virtually all -lactams and their derivatives. The level of oxacillin resistance reached is strain specific and can vary over a 1,000-fold concentration range, depending upon the genetic background of the strain. On the one hand, it depends upon the ability of MRSA to rapidly induce the synthesis of penicillin-binding protein 2a (PBP2a), which is indispensable for resistance; on the other hand, it is the genetic background of the strain into which the SCCmec element has entered which determines the final resistance level (for a review, see reference 5). Some clinical MRSA isolates have such a low level of resistance to oxacillin that they are difficult to identify phenotypically. Interestingly, an inverse relationship between resistance levels and growth rate has been observed (10).
The recent appearance of vancomycin-intermediate-resistant MRSA (VISA), which is due to alterations in gene expression caused by gene induction and/or the accumulation of multiple mutations which finally lead to intermediate levels of glycopeptide resistance (17), is a further challenge for the diagnostic laboratory. VISAs may go undetected by conventional resistance tests, since they produce only a small number of cells within a culture that express resistance. VISAs are still regarded as a rare cause of clinically relevant infections (35), but the evolution of their prevalence needs to be monitored.
The aim of this study was to examine the SCCmec types, the associated resistances, and clonal composition of MRSA isolates in the Zürich area and to search for a correlation between low-level oxacillin-resistant strains, which are increasingly being isolated, and the genetic background and/or SCCmec type. A comparison of the SCCmec elements occurring in methicillin-resistant coagulase-negative staphylococci (MRCNS) with those in MRSA should show if the distribution of SCCmec in MRSA is a reflection of that in MRCNS.
MATERIALS AND METHODS
Bacterial strains. Ninety independent MRSA isolates and 88 randomly selected coagulase-negative, methicillin-resistant staphylococcal isolates (MRCNS) sampled between November 2002 and January 2004 at the Department of Medical Microbiology of the University of Zürich, Zürich, Switzerland, which serves the university hospitals of Zürich, were analyzed. All strains were unique isolates from different patients. Identification of S. aureus and of the coagulase-negative staphylococci was by standard methods: colony morphology, Gram staining, catalase test, and confirmation with Staphaurex (Murex Diagnostics) and API Staph (bioMerieux), where required. Oxacillin resistance was confirmed by mecA PCR and with the MRSA screen from Denka Seiken (Japan) using -lactam-induced bacteria growing around the amoxicillin-clavulanic acid inhibition zone for the detection of PBP2a (PBP2') by latex agglutination (44). The strains were stored in skim milk at –80°C. Reference strains were S. aureus NCTC10442 for SCCmec type I, S. aureus N315 for SCCmec type II, S. aureus 85/3907 for SCCmec type III (19), S. aureus WSPP for SCCmec type IV (1, 25), and S. aureus WBG8404 for SCCmec type V (20). The Panton-Valentine leukocidin-positive WSPP strain and Swiss strain 497 (22) were used as references for lukS-PV. Mu3, a hetero-VISA (hVISA) strain, and susceptible strain N315 (18) were used as comparisons for glycopeptide intermediate resistance in population analysis profiles. The quality control strains for antibiotic resistance testing were S. aureus ATCC 29212 and Enterococcus faecalis ATCC 29213. Curing of SCCmec from clinical isolates was done by ccr overexpression, as described by Ito et al. (19).
Susceptibility testing. Oxacillin, cefoxitin, tetracycline, gentamicin, ciprofloxacin, erythromycin, and clindamycin susceptibility were determined by disk diffusion according to CLSI (formerly NCCLS) (7a) on Mueller-Hinton agar (Difco). Inducible macrolide-lincosamide-streptogramin B (MLSB) resistance was identified as a D-shaped inhibition zone by the clindamycin-erythromycin double-disk test (21). Oxacillin and linezolid MICs were determined by Etest (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar (Difco) with an inoculum of 0.5 McFarland standard as recommended. Rifampin, cotrimoxazole, and fosomycin MICs were determined by agar dilution according to the CLSI (7a). Vancomycin and teicoplanin resistance were determined by macro Etest on brain heart infusion plates (BBL), using a inoculum consisting of a 2 McFarland standard as recommended by the manufacturer (AB Biodisk, Solna, Sweden). Production of a penicillinase was shown qualitatively by nitrocefin hydrolysis from -lactam-induced cells growing around an amoxicillin-clavulanate disk. The hVISA phenotype was confirmed by population analysis profile, by plating aliquots of an overnight culture on brain heart infusion agar containing increasing concentrations of either vancomycin or teicoplanin and reading the CFU after a 48-h incubation, using strain Mu3 as a control (15). The growth temperature was 35°C.
Molecular typing. (i) PFGE. The MRSAs were genotyped by pulsed-field gel electrophoresis (PFGE) of SmaI-digested chromosomal DNA, following the protocol of Wada et al. (39). The banding patterns were analyzed visually, by scanning with a Fluor-S MultiImager, and by digital analysis with a Multianalys/PC (Bio-Rad) (28).
(ii) MLST. Multilocus sequence typing (MLST) was performed with selected isolates as specified by Enright et al. (11). The sequences obtained were compared with the sequences at the MLST website (http://www.mlst.net/) to assign a sequence type (ST).
(iii) SCCmec typing. Determination of SCCmec types I to IV was done by multiplex PCR (30). Untypable strains were further analyzed by ccr typing using a PCR screen with primers to identify ccr types 1, 2, and 3 (19); the SCCmec type V ccr complex ccrC gene (20); and primers C1 and C2 (23) to detect the ccr4 locus of the pediatric clone (31).
(iv) PVL. The primers lukSF (5'-ACAGAAGATACAAGTAGCGA-3') and lukSR (5'-TAATTCATTGTCTGGCACAA-3') were used to detect the presence of the lukS-PV gene, which is specific for Panton-Valentine leukocidin, by PCR.
(v) Colocalization of dfrA-mecA. Sequential Southern hybridizations (4) of SmaI-digested chromosomal DNA separated by PFGE with a mecA (30) or dfrA probe, amplified with the primer pair dfrAF-Tn4003 (5'-AATAGACGTAACGTCGTACT-3') and dfrAR-Tn4003 (5'-AAGAATGTATGCGGTATAGT-3'), showed whether dfrA mapped in the same SmaI band as mecA.
RESULTS AND DISCUSSION
Oxacillin resistance and -lactamase production. The University Hospital of Zürich, a 920-bed hospital, had an incidence of 1.1 cases of MRSA per 1,000 admissions in 2003. Ninety independent isolates were collected from November 2002 through January 2004 and characterized to determine their resistance profile, clonal distribution, PFGE pattern, and SCCmec types (Table 1). For comparison, 88 MRCNS clinical isolates were sampled randomly during the same time span to see if there was any correlation between the SCCmec types of MRCNS and MRSA. Methicillin resistance was confirmed in all strains by mecA PCR. The frequency of nonmultiresistant MRSA with low-level oxacillin resistance was rather high in this collection. Thirty percent of all MRSA strains had an oxacillin MIC below the breakpoint of 4 mg/ml (Fig. 1). In disk diffusion tests, the 30-mg cefoxitin disk was found to be superior and easier to interpret than the oxacillin disk and correctly identified all low-level resistant MRSA, with only one strain displaying intermediate resistance. The PBP2a agglutination by the MRSA latex-screening test using induced bacteria was as reliable as the mecA PCR, even for the phenotypically oxacillin-susceptible MRSA.
While MRSAs displayed oxacillin MICs over the entire range measured, the MRCNS formed two distinct clusters, consisting of 39 strains with oxacillin MICs of 32 mg/ml and 49 isolates with oxacillin MICs of 256 mg/ml. One MRCNS had an oxacillin MIC below the breakpoint for coagulase negative staphylococci of 0.5 mg/liter. Interestingly, penicillinase production was more frequent in MRSAs (90%) than in MRCNS (74%).
PFGE and SCCmec typing of MRSAs. PFGE indicated that there were four epidemiologically dominant genetic lineages and a number of sporadic isolates of MRSAs isolated from the University Hospital of Zürich (Fig. 2). The genetic backgrounds of representatives of each of the larger groups were investigated by MLST and determined to be ST217-MRSA-IV, a single-locus variant (SLV) of the pandemic epidemic United Kingdom strain epidemic MRSA-15 (designated EMRSA-15); ST225-MRSA-II, an SLV of the Japanese/America clone; ST613-MRSA-IV, a new ST so far reported only in Zürich; and ST45-MRSA-N1, the drug clone, so far predomantly found in isolates from intraveneous drug users and their contacts in Zürich. The ST217 and ST255 isolates have the characteristics of nosocomial MRSAs, while ST613 and ST45 have the characteristics of cMRSA (Table 1). The predominance of SLVs of pandemic isolates, the new ST, and the unique drug clone suggest that these MRSAs have evolved locally and are disseminating in the geographical region.
The classic nosocomial SCCmec types I, II, and III formed a minority in this MRSA strain collection with only 3, 9, and 12 isolates, respectively, representing 26% of all isolates. Two MRSAs produced a composite pattern consisting of a partial SCCmec type III lacking region C but with region D, otherwise found in types I, II, or IV SCCmec elements (Table 2). These two strains contained the ccr type 3 recombinase allele and clustered in the PFGE-based dendrogram within the SCCmec type III clusters (Fig. 2). They may therefore represent MRSAs with a new, composite SCCmec element, listed here as SCCmec type N4. Forty-one MRSAs carried SCCmec type IV and could be divided according to their PFGE pattern into three major groups (Fig. 2). The predominant clone, consisting of 18 strains with generally high levels of oxacillin and ciprofloxacin resistance, belonged to ST217 of CC22, differing from the ancestral clone EMRSA-15, a strain epidemic in the United Kingdom (12), at the tpi locus. The second major group of 10 strains belonged to a new sequence type, ST613, so far only found in Zürich and originating from clonal complex CC8. The remaining type IV strains formed a heterogeneous group with various PFGE patterns, which is common for the small community-onset-type IV SCCmec elements with enhanced mobility. PVL, reported to be associated with cMRSAs, was not frequent in this MRSA collection; the lukS-PV gene was present only in five SCCmec type IV strains. Interestingly, one of the PVL-producing strains was found during an outbreak in a dermatological ward.
Characterization of the "drug clone." Twenty-four MRSAs amplified only the mecA band with the SCCmec multiplex PCR. All isolates, except for three, carried none of the ccrAB alleles types 1, 2, or 3 or the ccrC gene, but with the primers specific for the ccr4 complex they yielded a band very closely related to that of the pediatric clone reported by Oliveira et al. (31; M. Ender, unpublished results). Curing the SCCmec determinant from a representative of these strains showed, according to the SmaI PFGE banding pattern, that the element had a size of approximately 30 kb. Interestingly, the SmaI band which carried the mecA gene also harbored a copy of the Tn4003-associated dfrA gene, which was lost upon SCCmec curing. PCR mapping and sequencing showed that the dfrA gene was integrated into the SCCmec determinant (Ender, unpublished). The association of dfrA with the SCCmec, the lack of any characteristic bands of type I to IV SCCmecs other than mecA by multiplex PCR, and the presence of a ccr4-like ccr complex suggested that this was a new SCCmec element, termed here SCCmec type N1. All of these strains belonged to an MRSA clone, which is spreading among isolates from injection drug users (14) and which is referred to as the "drug clone." It is characterized phenotypically by a very low level of oxacillin resistance, the presence of a penicillinase, and a high maximal growth rate of 1.8 h–1 in LB broth. All drug clones were trimethoprim resistant and generally either sulfomethoxazole or ciprofloxacin resistant. The drug clone appeared in 1994 and peaked in 2001 (Fig. 3), but it still represents a substantial part of the MRSA isolates in the Zürich area. It has also spread in a few cases to isolates from non-drug users, probably as a result of nosocomial transmission. MLST showed that the genetic background of the drug clone belongs to allelic profile ST45. The drug clone is thus similar to the epidemic Berlin MRSA clone, with low-level resistance to oxacillin (42), except for carrying the novel SCCmec type N1 element.
Two isolates, that like the drug clone only amplified the mecA band in the multipex PCR, had novel SCCmecs, N2 and N3, that were otherwise unrelated to N1 of the drug clone. They both lacked the dfrA gene; moreover, the strain containing SCCmec type N2 amplified products specific for the ccr2 and the ccr5 complex, whereas the strain containing SCCmec type N3 amplified a ccr2 complex. The strain containing SCCmec type N2 was highly oxacillin resistant and also gentamicin and rifampin resistant, very unlike the drug clone. The strain containing SCCmec type N3, a ciprofloxacin-resistant strain of low-level oxacillin resistance, was one of a cluster of ST45 isolates that harbored at least three different SCCmecs (types N1, N3, and IV) (Fig. 2), indicating that there have been three genetic events during which ST45 isolates found in this region have acquired methicillin resistance.
SCCmec typing of MRCNS. The distribution of SCCmec types in the MRCNS showed a completely different pattern. Among the MRCNS, we identified 14 strains with SCCmec type I, 12 strains with type II, none with type III, 8 strains with type IV, and a large number of untypable variants, which produced 11 new patterns by multiplex SCCmec and ccr typing. None of the MRCNS strains was a carrier of the new SCCmec type N1 found in the drug clone, since isolates amplifying mecA alone carry no ccr4 complex and no dfrA gene, suggesting that MRCNS were unlikely to have been the donors of the SCCmec type N1 for the drug clone. The marked difference in the distribution of SCCmec profiles in MRSAs and MRCNS suggests that there has not been extensive interspecies SCCmec transfer. However, it would be interesting to search for and analyze commensal MRCNS in isolates from drug addicts who are carriers of the drug clone to see if there is SCCmec type N1 transfer in that collective of patients.
The degree of multiresistance was clearly higher in MRCNS than in MRSAs (Fig. 4), when resistance to trimethoprim-sulfamethoxazole (SXT), tetracycline, chloramphenicol, gentamicin, ciprofloxacin, erythromycin, and rifampin was determined. The high number of MRSA isolates with no or only one additional resistance determinant was mainly due to the high proportion of SCCmec type IV and drug clone MRSA isolates in our strain collection.
Antibiotic resistance in MRSA. (i) SXT. The SXT combination blocks the synthesis of folate derivatives (33). Resistance to trimethoprim in S. aureus is formed by mutations in the chromosomal gene for dihydrofolate reductase or by acquisition of the transposon Tn4003-borne dfrA gene (8, 34). Sulfonamides, competitive inhibitors of dihydropteroate synthase, block folate biosynthesis. Resistance to sulfonamides in staphylococci is due to mutations in the chromosomal dihydropteroate synthase gene (38). The use of the inexpensive SXT combination in the treatment of infections in intravenous drug users may have been one of the driving forces for the association of dfrA with the SCCmec type N1 element. Although all drug clones amplified the dfrA gene, only approximately half of them were resistant to the SXT combination (Table 1). Interestingly, the SXT-susceptible drug clones were generally ciprofloxacin resistant instead, with two exceptions: one isolate was susceptible to both drugs, and one isolate was resistant to both drugs.
The integration of the dfrA gene was unique for type N1 SCCmec and has not been found in any of the other SCCmec types of MRSA and MRSCN analyzed so far. Two STX-resistant isolates with SCCmec type IV carried a dfrA gene unlinked to SCCmec, and 10 SXT-resistant MRSAs did not amplify any dfrA gene, suggesting that their SXT resistance was due to chromosomal mutations.
(ii) Glycopeptides. Glycopeptides sterically inhibit cross-linking and polymerization of the cell wall peptidoglycan by binding to the D-Ala-D-Ala of the nascent peptidoglycan precursor at the cell membrane. VISAs have been found to be the cause for glycopeptide therapy failure in several instances, especially in infections with high bacterial load, but their frequency and relevance have been questioned (35, 41). All MRSAs were screened for glycopeptide resistance upon isolation from patients and prior to storage at –80°C, by using the macro-Etest method that is indicative for potential hVISA (40). A few strains showed elevated teicoplanin and/or vancomycin MICs of >4 mg/ml. However, upon retesting after some months' storage at –80°C, resistance values had dropped, suggesting that glycopeptide resistance was unstable or that lower-resistance variants survived storage at low temperatures better. Only three strains out of all potential hVISAs could be confirmed by population analysis with vancomycin to be similar to that of strain Mu3 (data not shown). One of them belonged to the epidemic ST217 clone, one belonged to a multiresistant type IV isolate, and one belonged to the multiresistant SCCmec type N4 isolate.
(iii) Fosfomycin. Fosfomycin inhibits the MurA enzyme, preventing the formation of N-acetylmuramic acid, a precursor of the cell wall peptidoglycan. All MRSAs were susceptible to fosfomycin with MICs below the lower fosfomycin breakpoint of 16 mg/ml (24). However, the distribution of fosfomycin MICs suggested the presence of two populations of fosfomycin-susceptible strains in the MRSA collection, namely, isolates with a fosfomycin MIC of around 0.5 and a slightly more resistant population of strains with an MIC of 4 (Fig. 5). The increased fosfomycin MIC correlated with elevated initial teicoplanin MICs (Kendall's rank correlation coefficients: k-tau-a = 0.3346 and k-tau-b of 0.4083; P < 0.001). This may be due to upregulation of the murA gene, which upon overexpression can confer some fosfomycin resistance. Whether the fosfomycin MIC can be used as indication for upregulated cell wall synthesis in hVISA has to be analyzed further.
(iv) MLS. The structurally different, but functionally similar MLSB class of drugs binds to the 50S ribosomal subunit, blocking protein synthesis (37). The erm-encoded methylases are the most frequent resistance mechanisms against macrolides in staphylococi (3). The inability of lincosamides to induce MLSB resistance results in clindamycin susceptibility, while constitutive expression of erm genes confers resistance to all MLSB antibiotics. Over 30% (34/90) of the MRSAs had an intermediate level of erythromycin resistance. Inducible MLSB resistance was found in 17/91 isolates, and constitutive expression was found in 20/91 MRSA isolates.
(v) Linezolid. Linezolid is the first representative of oxazolidinones, a new class of antibiotics which inhibits the assembly of a functional initiation complex for bacterial protein synthesis. It shows no cross-resistance with existing antibiotic agents with the same target (6); in our collection, all MRSAs (MIC at which 50% of the isolates tested are inhibited [MIC50] = 0.5; MIC at which 90% of the isolates tested are inhibited [MIC90] = 1) and MRCNS (MIC50 = 0.75; MIC90 = 1) were susceptible to linezolid.
CONCLUSIONS
The rather high prevalence of the drug clone, a phenotypically oxacillin-susceptible MRSA, in the Zürich area is challenging, especially since it belongs to the same genetic background as the successfully spreading Berlin clone (42). Because of its low MIC, the drug clone is difficult to detect. The evolution and spread of this clone have to be monitored closely to prevent the clone's escape into other patient populations, where it may pick up other resistance determinants and increase its resistance spectrum. ST45 can harbor different SCCmec types. Here, it has acquired a new SCCmec type N1, which differs from those previously reported (type II and type IV) to be associated with ST45. The core genetic background of the drug clone is related to the epidemic Berlin clone. Two of its interesting characteristics are its rapid growth rate and the extremely low level of oxacillin resistance, the cause of which is under investigation. Interestingly, we found in this small survey no MRCNS with an SCCmec type N1 like that of the drug clone, suggesting that there may be not such a high rate of SCCmec exchange between MRCNS and S. aureus. Unexpectedly, the MRCNS seemed to harbor multiple new types of SCCmec and were more likely to amplify more than one representative of different ccr complexes, suggesting that the MRCNS may nevertheless be the breeding ground for new SCCmec elements.
ACKNOWLEDGMENTS
This study was supported by Swiss National Research Foundation grant NRP49 63201 and the Hartmann Müller Stiftung. W.Q. was supported by National Natural Science Foundation of China grant 30370080.
We thank T. Ito and N. Liassine for the reference strains, R. Zbinden for the clinical isolates, and P. Huynh for technical help.
Present address: Institute of Infectious Diseases, The Second Teaching Hospital, Tianjin Medical University, Tianjin 300211, People's Republic of China.
These two authors contributed equally to this study.
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Gram-Positive Bacteria Typing and Research Unit and Molecular Genetics Research Unit, School of Biomedical Sciences, Curtin University of Technology, Perth, Australia
Hospital Epidemiology Unit, Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zürich, CH-8091 Zürich, Switzerland
ABSTRACT
The majority of methicillin-resistant Staphylococcus aureus (MRSA) isolates, recovered in 2003 at the Department of Medical Microbiology in Zürich, Switzerland, belonged to major clones that are circulating worldwide. Staphylococcal cassette chromosome mec type IV (SCCmec-IV), harbored by half of the isolates, was found in sequence type 217 (ST217), which is an allelic variant of epidemic MRSA-15 (designated EMRSA-15), in a new local ST617 descending from clonal complex CC8 and in low-level oxacillin-resistant strains of multiple genetic lineages characteristic of community-onset MRSA. SCCmec-I, SCCmec-II, and SCCmec-III were in the minority, and four MRSA isolates had complex, rearranged SCCmec elements. A novel SCCmec-N1 of approximately 30 kb, associated with a dfrA gene and a ccr4-related recombinase complex, was identified in a large number of low-level oxacillin-resistant isolates, which descended from the successful clonal complex CC45 and are spreading among intraveneous drug users. In contrast, the SCCmec types of oxacillin-resistant coagulase-negative staphylococci (MRCNS) were of completely different composition. SCCmec type I (SCCmec-I) and SCCmec-II were more frequent than in the MRSA, while fewer contained SCCmec-IV. The other MRCNS displayed 11 different, complex patterns, suggesting frequent recombination between different SCCmec elements. With one ccr-negative exception, these strains amplified between one and three different ccr products, indicating either new varied complexes or multiple ccr loci. This suggests the presence of novel SCCmec types in MRCNS and no extensive interspecies SCCmec transfer between MRSA and MRCNS.
INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections and has also recently established itself as a significant community-acquired pathogen (7, 9). Community-onset MRSA (cMRSA) differs from nosocomial MRSA in that it does not generally belong to the major clonal groups of epidemic MRSA, is susceptible to most non--lactam antibiotics, contains the type IV SCCmec (for staphylococcal cassette chromosome mec, the mobile genetic element encoding methicillin resistance), and frequently carries genes responsible for the production of Panton-Valentine leukocidin (PVL) (13, 22, 26). In contrast, nosocomial MRSAs are generally multidrug resistant and contain SCCmec types I, II, or III.
Besides the five major allelic types of SCCmec elements, epidemiological studies paired with molecular characterizations suggest the existence of additional, different SCCmecs (16, 28, 36). The origin of the SCCmec element is still unclear, with the closest mecA homolog found in Staphylococcus sciuri (43). Methicillin-resistant coagulase-negative staphylococci, which are more frequently carriers of SCCmec than S. aureus, are postulated to be the reservoir for the transfer of methicillin resistance to S. aureus (2). SCCmec has the attributes of a mobile element, such as the ccr genes, encoding recombinases that were shown in vitro to be responsible for the precise excision and integration of SCCmec into the chromosome. However, the type 1 and type 3 ccrA and ccrB genes are dysfunctional. This, plus the larger size of nosocomial SCCmec types I to III, may have been the reason that they have spread only into a restricted number of genetic lineages, whereas the smaller type IV SCCmec seems to be more mobile and to be associated with more diverse strain lineages (29, 32).
The ability of MRSAs to segregate a highly resistant subpopulation in the presence of -lactams makes them resistant to virtually all -lactams and their derivatives. The level of oxacillin resistance reached is strain specific and can vary over a 1,000-fold concentration range, depending upon the genetic background of the strain. On the one hand, it depends upon the ability of MRSA to rapidly induce the synthesis of penicillin-binding protein 2a (PBP2a), which is indispensable for resistance; on the other hand, it is the genetic background of the strain into which the SCCmec element has entered which determines the final resistance level (for a review, see reference 5). Some clinical MRSA isolates have such a low level of resistance to oxacillin that they are difficult to identify phenotypically. Interestingly, an inverse relationship between resistance levels and growth rate has been observed (10).
The recent appearance of vancomycin-intermediate-resistant MRSA (VISA), which is due to alterations in gene expression caused by gene induction and/or the accumulation of multiple mutations which finally lead to intermediate levels of glycopeptide resistance (17), is a further challenge for the diagnostic laboratory. VISAs may go undetected by conventional resistance tests, since they produce only a small number of cells within a culture that express resistance. VISAs are still regarded as a rare cause of clinically relevant infections (35), but the evolution of their prevalence needs to be monitored.
The aim of this study was to examine the SCCmec types, the associated resistances, and clonal composition of MRSA isolates in the Zürich area and to search for a correlation between low-level oxacillin-resistant strains, which are increasingly being isolated, and the genetic background and/or SCCmec type. A comparison of the SCCmec elements occurring in methicillin-resistant coagulase-negative staphylococci (MRCNS) with those in MRSA should show if the distribution of SCCmec in MRSA is a reflection of that in MRCNS.
MATERIALS AND METHODS
Bacterial strains. Ninety independent MRSA isolates and 88 randomly selected coagulase-negative, methicillin-resistant staphylococcal isolates (MRCNS) sampled between November 2002 and January 2004 at the Department of Medical Microbiology of the University of Zürich, Zürich, Switzerland, which serves the university hospitals of Zürich, were analyzed. All strains were unique isolates from different patients. Identification of S. aureus and of the coagulase-negative staphylococci was by standard methods: colony morphology, Gram staining, catalase test, and confirmation with Staphaurex (Murex Diagnostics) and API Staph (bioMerieux), where required. Oxacillin resistance was confirmed by mecA PCR and with the MRSA screen from Denka Seiken (Japan) using -lactam-induced bacteria growing around the amoxicillin-clavulanic acid inhibition zone for the detection of PBP2a (PBP2') by latex agglutination (44). The strains were stored in skim milk at –80°C. Reference strains were S. aureus NCTC10442 for SCCmec type I, S. aureus N315 for SCCmec type II, S. aureus 85/3907 for SCCmec type III (19), S. aureus WSPP for SCCmec type IV (1, 25), and S. aureus WBG8404 for SCCmec type V (20). The Panton-Valentine leukocidin-positive WSPP strain and Swiss strain 497 (22) were used as references for lukS-PV. Mu3, a hetero-VISA (hVISA) strain, and susceptible strain N315 (18) were used as comparisons for glycopeptide intermediate resistance in population analysis profiles. The quality control strains for antibiotic resistance testing were S. aureus ATCC 29212 and Enterococcus faecalis ATCC 29213. Curing of SCCmec from clinical isolates was done by ccr overexpression, as described by Ito et al. (19).
Susceptibility testing. Oxacillin, cefoxitin, tetracycline, gentamicin, ciprofloxacin, erythromycin, and clindamycin susceptibility were determined by disk diffusion according to CLSI (formerly NCCLS) (7a) on Mueller-Hinton agar (Difco). Inducible macrolide-lincosamide-streptogramin B (MLSB) resistance was identified as a D-shaped inhibition zone by the clindamycin-erythromycin double-disk test (21). Oxacillin and linezolid MICs were determined by Etest (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar (Difco) with an inoculum of 0.5 McFarland standard as recommended. Rifampin, cotrimoxazole, and fosomycin MICs were determined by agar dilution according to the CLSI (7a). Vancomycin and teicoplanin resistance were determined by macro Etest on brain heart infusion plates (BBL), using a inoculum consisting of a 2 McFarland standard as recommended by the manufacturer (AB Biodisk, Solna, Sweden). Production of a penicillinase was shown qualitatively by nitrocefin hydrolysis from -lactam-induced cells growing around an amoxicillin-clavulanate disk. The hVISA phenotype was confirmed by population analysis profile, by plating aliquots of an overnight culture on brain heart infusion agar containing increasing concentrations of either vancomycin or teicoplanin and reading the CFU after a 48-h incubation, using strain Mu3 as a control (15). The growth temperature was 35°C.
Molecular typing. (i) PFGE. The MRSAs were genotyped by pulsed-field gel electrophoresis (PFGE) of SmaI-digested chromosomal DNA, following the protocol of Wada et al. (39). The banding patterns were analyzed visually, by scanning with a Fluor-S MultiImager, and by digital analysis with a Multianalys/PC (Bio-Rad) (28).
(ii) MLST. Multilocus sequence typing (MLST) was performed with selected isolates as specified by Enright et al. (11). The sequences obtained were compared with the sequences at the MLST website (http://www.mlst.net/) to assign a sequence type (ST).
(iii) SCCmec typing. Determination of SCCmec types I to IV was done by multiplex PCR (30). Untypable strains were further analyzed by ccr typing using a PCR screen with primers to identify ccr types 1, 2, and 3 (19); the SCCmec type V ccr complex ccrC gene (20); and primers C1 and C2 (23) to detect the ccr4 locus of the pediatric clone (31).
(iv) PVL. The primers lukSF (5'-ACAGAAGATACAAGTAGCGA-3') and lukSR (5'-TAATTCATTGTCTGGCACAA-3') were used to detect the presence of the lukS-PV gene, which is specific for Panton-Valentine leukocidin, by PCR.
(v) Colocalization of dfrA-mecA. Sequential Southern hybridizations (4) of SmaI-digested chromosomal DNA separated by PFGE with a mecA (30) or dfrA probe, amplified with the primer pair dfrAF-Tn4003 (5'-AATAGACGTAACGTCGTACT-3') and dfrAR-Tn4003 (5'-AAGAATGTATGCGGTATAGT-3'), showed whether dfrA mapped in the same SmaI band as mecA.
RESULTS AND DISCUSSION
Oxacillin resistance and -lactamase production. The University Hospital of Zürich, a 920-bed hospital, had an incidence of 1.1 cases of MRSA per 1,000 admissions in 2003. Ninety independent isolates were collected from November 2002 through January 2004 and characterized to determine their resistance profile, clonal distribution, PFGE pattern, and SCCmec types (Table 1). For comparison, 88 MRCNS clinical isolates were sampled randomly during the same time span to see if there was any correlation between the SCCmec types of MRCNS and MRSA. Methicillin resistance was confirmed in all strains by mecA PCR. The frequency of nonmultiresistant MRSA with low-level oxacillin resistance was rather high in this collection. Thirty percent of all MRSA strains had an oxacillin MIC below the breakpoint of 4 mg/ml (Fig. 1). In disk diffusion tests, the 30-mg cefoxitin disk was found to be superior and easier to interpret than the oxacillin disk and correctly identified all low-level resistant MRSA, with only one strain displaying intermediate resistance. The PBP2a agglutination by the MRSA latex-screening test using induced bacteria was as reliable as the mecA PCR, even for the phenotypically oxacillin-susceptible MRSA.
While MRSAs displayed oxacillin MICs over the entire range measured, the MRCNS formed two distinct clusters, consisting of 39 strains with oxacillin MICs of 32 mg/ml and 49 isolates with oxacillin MICs of 256 mg/ml. One MRCNS had an oxacillin MIC below the breakpoint for coagulase negative staphylococci of 0.5 mg/liter. Interestingly, penicillinase production was more frequent in MRSAs (90%) than in MRCNS (74%).
PFGE and SCCmec typing of MRSAs. PFGE indicated that there were four epidemiologically dominant genetic lineages and a number of sporadic isolates of MRSAs isolated from the University Hospital of Zürich (Fig. 2). The genetic backgrounds of representatives of each of the larger groups were investigated by MLST and determined to be ST217-MRSA-IV, a single-locus variant (SLV) of the pandemic epidemic United Kingdom strain epidemic MRSA-15 (designated EMRSA-15); ST225-MRSA-II, an SLV of the Japanese/America clone; ST613-MRSA-IV, a new ST so far reported only in Zürich; and ST45-MRSA-N1, the drug clone, so far predomantly found in isolates from intraveneous drug users and their contacts in Zürich. The ST217 and ST255 isolates have the characteristics of nosocomial MRSAs, while ST613 and ST45 have the characteristics of cMRSA (Table 1). The predominance of SLVs of pandemic isolates, the new ST, and the unique drug clone suggest that these MRSAs have evolved locally and are disseminating in the geographical region.
The classic nosocomial SCCmec types I, II, and III formed a minority in this MRSA strain collection with only 3, 9, and 12 isolates, respectively, representing 26% of all isolates. Two MRSAs produced a composite pattern consisting of a partial SCCmec type III lacking region C but with region D, otherwise found in types I, II, or IV SCCmec elements (Table 2). These two strains contained the ccr type 3 recombinase allele and clustered in the PFGE-based dendrogram within the SCCmec type III clusters (Fig. 2). They may therefore represent MRSAs with a new, composite SCCmec element, listed here as SCCmec type N4. Forty-one MRSAs carried SCCmec type IV and could be divided according to their PFGE pattern into three major groups (Fig. 2). The predominant clone, consisting of 18 strains with generally high levels of oxacillin and ciprofloxacin resistance, belonged to ST217 of CC22, differing from the ancestral clone EMRSA-15, a strain epidemic in the United Kingdom (12), at the tpi locus. The second major group of 10 strains belonged to a new sequence type, ST613, so far only found in Zürich and originating from clonal complex CC8. The remaining type IV strains formed a heterogeneous group with various PFGE patterns, which is common for the small community-onset-type IV SCCmec elements with enhanced mobility. PVL, reported to be associated with cMRSAs, was not frequent in this MRSA collection; the lukS-PV gene was present only in five SCCmec type IV strains. Interestingly, one of the PVL-producing strains was found during an outbreak in a dermatological ward.
Characterization of the "drug clone." Twenty-four MRSAs amplified only the mecA band with the SCCmec multiplex PCR. All isolates, except for three, carried none of the ccrAB alleles types 1, 2, or 3 or the ccrC gene, but with the primers specific for the ccr4 complex they yielded a band very closely related to that of the pediatric clone reported by Oliveira et al. (31; M. Ender, unpublished results). Curing the SCCmec determinant from a representative of these strains showed, according to the SmaI PFGE banding pattern, that the element had a size of approximately 30 kb. Interestingly, the SmaI band which carried the mecA gene also harbored a copy of the Tn4003-associated dfrA gene, which was lost upon SCCmec curing. PCR mapping and sequencing showed that the dfrA gene was integrated into the SCCmec determinant (Ender, unpublished). The association of dfrA with the SCCmec, the lack of any characteristic bands of type I to IV SCCmecs other than mecA by multiplex PCR, and the presence of a ccr4-like ccr complex suggested that this was a new SCCmec element, termed here SCCmec type N1. All of these strains belonged to an MRSA clone, which is spreading among isolates from injection drug users (14) and which is referred to as the "drug clone." It is characterized phenotypically by a very low level of oxacillin resistance, the presence of a penicillinase, and a high maximal growth rate of 1.8 h–1 in LB broth. All drug clones were trimethoprim resistant and generally either sulfomethoxazole or ciprofloxacin resistant. The drug clone appeared in 1994 and peaked in 2001 (Fig. 3), but it still represents a substantial part of the MRSA isolates in the Zürich area. It has also spread in a few cases to isolates from non-drug users, probably as a result of nosocomial transmission. MLST showed that the genetic background of the drug clone belongs to allelic profile ST45. The drug clone is thus similar to the epidemic Berlin MRSA clone, with low-level resistance to oxacillin (42), except for carrying the novel SCCmec type N1 element.
Two isolates, that like the drug clone only amplified the mecA band in the multipex PCR, had novel SCCmecs, N2 and N3, that were otherwise unrelated to N1 of the drug clone. They both lacked the dfrA gene; moreover, the strain containing SCCmec type N2 amplified products specific for the ccr2 and the ccr5 complex, whereas the strain containing SCCmec type N3 amplified a ccr2 complex. The strain containing SCCmec type N2 was highly oxacillin resistant and also gentamicin and rifampin resistant, very unlike the drug clone. The strain containing SCCmec type N3, a ciprofloxacin-resistant strain of low-level oxacillin resistance, was one of a cluster of ST45 isolates that harbored at least three different SCCmecs (types N1, N3, and IV) (Fig. 2), indicating that there have been three genetic events during which ST45 isolates found in this region have acquired methicillin resistance.
SCCmec typing of MRCNS. The distribution of SCCmec types in the MRCNS showed a completely different pattern. Among the MRCNS, we identified 14 strains with SCCmec type I, 12 strains with type II, none with type III, 8 strains with type IV, and a large number of untypable variants, which produced 11 new patterns by multiplex SCCmec and ccr typing. None of the MRCNS strains was a carrier of the new SCCmec type N1 found in the drug clone, since isolates amplifying mecA alone carry no ccr4 complex and no dfrA gene, suggesting that MRCNS were unlikely to have been the donors of the SCCmec type N1 for the drug clone. The marked difference in the distribution of SCCmec profiles in MRSAs and MRCNS suggests that there has not been extensive interspecies SCCmec transfer. However, it would be interesting to search for and analyze commensal MRCNS in isolates from drug addicts who are carriers of the drug clone to see if there is SCCmec type N1 transfer in that collective of patients.
The degree of multiresistance was clearly higher in MRCNS than in MRSAs (Fig. 4), when resistance to trimethoprim-sulfamethoxazole (SXT), tetracycline, chloramphenicol, gentamicin, ciprofloxacin, erythromycin, and rifampin was determined. The high number of MRSA isolates with no or only one additional resistance determinant was mainly due to the high proportion of SCCmec type IV and drug clone MRSA isolates in our strain collection.
Antibiotic resistance in MRSA. (i) SXT. The SXT combination blocks the synthesis of folate derivatives (33). Resistance to trimethoprim in S. aureus is formed by mutations in the chromosomal gene for dihydrofolate reductase or by acquisition of the transposon Tn4003-borne dfrA gene (8, 34). Sulfonamides, competitive inhibitors of dihydropteroate synthase, block folate biosynthesis. Resistance to sulfonamides in staphylococci is due to mutations in the chromosomal dihydropteroate synthase gene (38). The use of the inexpensive SXT combination in the treatment of infections in intravenous drug users may have been one of the driving forces for the association of dfrA with the SCCmec type N1 element. Although all drug clones amplified the dfrA gene, only approximately half of them were resistant to the SXT combination (Table 1). Interestingly, the SXT-susceptible drug clones were generally ciprofloxacin resistant instead, with two exceptions: one isolate was susceptible to both drugs, and one isolate was resistant to both drugs.
The integration of the dfrA gene was unique for type N1 SCCmec and has not been found in any of the other SCCmec types of MRSA and MRSCN analyzed so far. Two STX-resistant isolates with SCCmec type IV carried a dfrA gene unlinked to SCCmec, and 10 SXT-resistant MRSAs did not amplify any dfrA gene, suggesting that their SXT resistance was due to chromosomal mutations.
(ii) Glycopeptides. Glycopeptides sterically inhibit cross-linking and polymerization of the cell wall peptidoglycan by binding to the D-Ala-D-Ala of the nascent peptidoglycan precursor at the cell membrane. VISAs have been found to be the cause for glycopeptide therapy failure in several instances, especially in infections with high bacterial load, but their frequency and relevance have been questioned (35, 41). All MRSAs were screened for glycopeptide resistance upon isolation from patients and prior to storage at –80°C, by using the macro-Etest method that is indicative for potential hVISA (40). A few strains showed elevated teicoplanin and/or vancomycin MICs of >4 mg/ml. However, upon retesting after some months' storage at –80°C, resistance values had dropped, suggesting that glycopeptide resistance was unstable or that lower-resistance variants survived storage at low temperatures better. Only three strains out of all potential hVISAs could be confirmed by population analysis with vancomycin to be similar to that of strain Mu3 (data not shown). One of them belonged to the epidemic ST217 clone, one belonged to a multiresistant type IV isolate, and one belonged to the multiresistant SCCmec type N4 isolate.
(iii) Fosfomycin. Fosfomycin inhibits the MurA enzyme, preventing the formation of N-acetylmuramic acid, a precursor of the cell wall peptidoglycan. All MRSAs were susceptible to fosfomycin with MICs below the lower fosfomycin breakpoint of 16 mg/ml (24). However, the distribution of fosfomycin MICs suggested the presence of two populations of fosfomycin-susceptible strains in the MRSA collection, namely, isolates with a fosfomycin MIC of around 0.5 and a slightly more resistant population of strains with an MIC of 4 (Fig. 5). The increased fosfomycin MIC correlated with elevated initial teicoplanin MICs (Kendall's rank correlation coefficients: k-tau-a = 0.3346 and k-tau-b of 0.4083; P < 0.001). This may be due to upregulation of the murA gene, which upon overexpression can confer some fosfomycin resistance. Whether the fosfomycin MIC can be used as indication for upregulated cell wall synthesis in hVISA has to be analyzed further.
(iv) MLS. The structurally different, but functionally similar MLSB class of drugs binds to the 50S ribosomal subunit, blocking protein synthesis (37). The erm-encoded methylases are the most frequent resistance mechanisms against macrolides in staphylococi (3). The inability of lincosamides to induce MLSB resistance results in clindamycin susceptibility, while constitutive expression of erm genes confers resistance to all MLSB antibiotics. Over 30% (34/90) of the MRSAs had an intermediate level of erythromycin resistance. Inducible MLSB resistance was found in 17/91 isolates, and constitutive expression was found in 20/91 MRSA isolates.
(v) Linezolid. Linezolid is the first representative of oxazolidinones, a new class of antibiotics which inhibits the assembly of a functional initiation complex for bacterial protein synthesis. It shows no cross-resistance with existing antibiotic agents with the same target (6); in our collection, all MRSAs (MIC at which 50% of the isolates tested are inhibited [MIC50] = 0.5; MIC at which 90% of the isolates tested are inhibited [MIC90] = 1) and MRCNS (MIC50 = 0.75; MIC90 = 1) were susceptible to linezolid.
CONCLUSIONS
The rather high prevalence of the drug clone, a phenotypically oxacillin-susceptible MRSA, in the Zürich area is challenging, especially since it belongs to the same genetic background as the successfully spreading Berlin clone (42). Because of its low MIC, the drug clone is difficult to detect. The evolution and spread of this clone have to be monitored closely to prevent the clone's escape into other patient populations, where it may pick up other resistance determinants and increase its resistance spectrum. ST45 can harbor different SCCmec types. Here, it has acquired a new SCCmec type N1, which differs from those previously reported (type II and type IV) to be associated with ST45. The core genetic background of the drug clone is related to the epidemic Berlin clone. Two of its interesting characteristics are its rapid growth rate and the extremely low level of oxacillin resistance, the cause of which is under investigation. Interestingly, we found in this small survey no MRCNS with an SCCmec type N1 like that of the drug clone, suggesting that there may be not such a high rate of SCCmec exchange between MRCNS and S. aureus. Unexpectedly, the MRCNS seemed to harbor multiple new types of SCCmec and were more likely to amplify more than one representative of different ccr complexes, suggesting that the MRCNS may nevertheless be the breeding ground for new SCCmec elements.
ACKNOWLEDGMENTS
This study was supported by Swiss National Research Foundation grant NRP49 63201 and the Hartmann Müller Stiftung. W.Q. was supported by National Natural Science Foundation of China grant 30370080.
We thank T. Ito and N. Liassine for the reference strains, R. Zbinden for the clinical isolates, and P. Huynh for technical help.
Present address: Institute of Infectious Diseases, The Second Teaching Hospital, Tianjin Medical University, Tianjin 300211, People's Republic of China.
These two authors contributed equally to this study.
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