1 Laboratoire de Bactériologie-Virologie and 2 Laboratoire de Parasitologie, Centre Hospitalier Universitaire, 35033 Rennes cedex 9, France
Received 1 August 2003; returned 10 November 2003; revised 6 February 2004; accepted 13 February 2004
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Abstract |
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Methods: Antibiotic susceptibilities were determined by agar diffusion. SCC typing was performed using PCR. PFGE demonstrated that isolates were phylogenetically related.
Results: Fourteen profiles of antibiotic resistance were defined among MRSA isolates. For each resistance profile, only one SCC type was associated: four patterns corresponded to SCC type I or IA, nine to SCC type IV or IVA, and none to types II and III. One was not typed. PFGE indicated that isolates with SCC type I or IA belong to a single genetic lineage, which also includes most of the epidemic isolates, which carry SCC type IVA. In contrast to type I or IA, isolates with SCC type IV or IVA were found to be associated with several different PFGE clusters, although not all of these represent epidemic isolates.
Conclusions: During the course of the study, the spectrum of antibiotic resistance in MRSA isolates decreased. This occurred due to the emergence of strains with SCC type IV or IVA, which are susceptible to more antibiotics than type I or IA strains. The greater prevalence of such isolates could not be linked conclusively to the presence of SCC type IV or IVA, or to one particular PFGE cluster.
Keywords: S. aureus, MRSA, mecA, SCC, resistance profiles
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Introduction |
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In the Centre Hospitalier Universitaire, Rennes, France, surveillance of overall antibiotic resistance in MRSA isolates has demonstrated a significant change during the 1990s: gentamicin-resistant, multi-resistant strains have almost disappeared since 1997 and have been replaced by gentamicin-susceptible, less resistant MRSA isolates.11 Gentamicin-susceptible MRSA strains have been epidemic in most hospitals in France since 1992.1214 The aims of this study were to type representative isolates expressing the most frequently encountered MRSA resistance profiles seen during 19922002 and to describe how these resistance profiles have changed over time. Furthermore, we set out to determine whether there is a link between resistance profile and the SCC types carried by these MRSA isolates.
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Materials and methods |
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The University Hospital of Rennes is a 1978 bed teaching hospital. For the period 19922002, inclusive, the mean annual incidence of methicillin resistance among S. aureus isolates was 36% ± 2%. During this period, 20 619 S. aureus isolates were recovered from samples from patients hospitalized in four of five facilities. Isolates were identified as S. aureus by their ability to produce acid on Chapman agar, and by their production of catalase and coagulase.
Changes in antibiotic resistance profiles among MRSA isolates
Antimicrobial susceptibility was measured using the agar diffusion method (Bio-Rad, Marnes-la-Coquette, France) on MuellerHinton agar (Oxoid, Dardilly, France) according to the recommendations of the Antibiogram Committee of the French Microbiology Society (CA-SFM), except that isolates with a zone inhibition diameter for fosfomycin >14 mm and <23 mm were categorized as intermediately resistant to this antibiotic. Patient information (sex, age, clinical specimen) and antibiogram results were collected from the laboratory information system and stored in a specific database. If several isolates with the same antibiogram were recovered at different times from one patient, only the first isolate was retained. In total, 4080 MRSA isolates were retained for further analysis. Temporal changes in antibiotic resistance profiles among these MRSA isolates were studied by use of the EPILOG software (Medasys, Gif-sur-Yvette, France).
Definition of resistance profiles
MRSA isolates were grouped by overall resistance profile, as reported previously.11 A resistance profile was characterized by a combination of results (S, susceptible and R, intermediate or resistant) for the following antibiotics: neomycin, tobramycin, gentamicin, erythromycin, lincomycin, sulfamethoxazole, pefloxacin, rifampicin and fosfomycin. An identical resistance profile, found in 10 or more MRSA during the same year, was considered as a frequent MRSA resistance profile in that year.
PFGE
For MRSAs isolated before 1998, PFGE was performed with a Gene Navigator apparatus (Amersham Pharmacia, Orsay, France), and for strains isolated after 1998, PFGE was performed with a CHEF-II system (Bio-Rad, Ivry-sur-Seine, France). Gel Compar software (Applied Maths, Sint-Martens-Latem, Belgium) was used to calculate Dice similarity indices and to perform cluster analysis by un-weighted pair group matching analysis (tolerance 2.0%). Two isolates were considered as genetically related if their Dice similarity index was equal to 85% or more.
Typing of staphylococcal chromosome cassette
Two, three or four isolates expressing each MRSA resistance profile were selected according to year of isolation. Since isolates with resistance profile V were no longer available, this profile has been excluded from further study. Bacterial DNA was extracted using a QIAamps tissue kit from Qiagen (Courtaboeuf, France). SCC typing was performed with a multiplex-PCR method according to Oliveira & de Lencastre.8
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Results |
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Discussion |
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The data presented here show that since 1995 major changes (i.e. typically a decrease in the spectrum of resistance) observed in the antibiotic resistance of MRSA isolates have been directly linked to the emergence of SCC type IV or IVA. SCC type IV is a shorter and more mobile genetic element than other SCC types4,8,9 and it has been found to be associated with a wide variety of phylogenetic backgrounds of S. aureus.6,8,10,20 Since 1992, SCC type IVA has been the type most frequently observed among MRSAs isolated in our hospital. It is found not only in gentamicin-susceptible and tobramycin-resistant MRSAs but also in two profiles of resistance (II and III) that include gentamicin resistance and were frequently isolated before 1997. Because of this observation, it is unlikely that the gentamicin-resistance determinant is encoded on the SCC IV element. Accordingly, these data imply transfer of SCC IVA into the gentamicin-resistant MRSA background. The finding of SCC IVA in isolates with varying degrees of prevalence (e.g. isolates expressing resistance profiles VII and XII) indicates that acquisition of SCC IVA does not itself confer a particular advantage in causing epidemics. Nonetheless these strains seem widely disseminated in hospitals in Europe; large outbreaks due to EMRSA-15, an SCC type IV strain, have been reported in England, Wales and Germany.8,18,21
In our study, SCC type I isolates were found to correspond to antibiotic resistance profile I, which was predominant during 19881991. SCC type I is specific for the Archaic clone.6 Archaic and Iberian MRSA clones are both characterized by SCC type I, but the Iberian SCC type (called IA) differs by the presence of a linearized pUB110 plasmid, which codes for the nucleotidyltransferase enzyme ANT4, inactivating kanamycin, neomycin, amikacin and tobramycin.7,8,19 This plasmid is also present in the SCC type IVA but not in type IV. This implies that resistance profiles corresponding to SCC types IA and IVA include resistance to neomycin, and those corresponding to SCC types I and IV do not, which has been confirmed in this study (Table 1). Consequently, a neomycin-susceptible MRSA isolate should be expected to have an SCC type I if it is resistant to gentamicin and an SCC type IV if it is susceptible to this drug. Other than neomycin and gentamicin, no other antibiotic or combination of antibiotics can be used to predict the SCC type carried by an isolate. Nonetheless, in this study, we have confirmedas general rulethat isolates carrying SCC type IV or IVA are susceptible to more antibiotics than others (three antibiotics at least), as described previously for HA-MRSA isolates related to the Paediatric clone in Lisbon, HA-MRSA in German hospitals or CA-MRSA in the USA.17,18,22
MRSA isolates with antibiotic resistance profiles IV, VI and XIII carry SCC type IA and so are presumed to belong to the Iberian clone. All resistance profile XIII isolates have teicoplanin MICs equal to 4 mg/L or more and must be classified as glycopeptide-intermediate S. aureus (GISA) according to the CA-SFM recommendations. It has been reported that GISA outbreaks occurring since 1996 in many French hospitals were due to strains related to this clone.23
Most of HA-MRSA from the USA and Japan have SCC type II or III.7,8 In Europe, two previous studies have reported such strains: EMRSA-16, the most epidemic MRSA in the UK, carries SCC type II, and isolates related to the BrazilianHungarian clone from Portugal and Hungary carry SCC type III,6,8 but in this study, no MRSA antibiotic-resistance profile was found to be associated with these SCC types.
There was a good concordance between antibiotic-resistance profile and PFGE profile (i.e. isolates with the same resistance profile belong to the same PFGE cluster), other than for isolates expressing resistance profiles X, XI and XII. These results suggest that, in our hands, results of antibiotyping fit well with molecular typing methods used to delineate epidemic strains.
Comparisons of results from the two different PFGE systems used for MRSA surveillance in our hospital must be interpreted cautiously. However, since all MRSA isolates with resistance profile VII, which has become very prevalent, were located by one method in cluster N1 and by another in cluster C1, we assume that all isolates in these two PFGE clusters are genetically related. All MRSAs with SCC types I or IA belong to this genetic background. However, we have shown that this same genetic background contains SCC type IVA strains, but, unlike SCC types I or IA, SCC types IV and IVA are not restricted to this genetic background. Among MRSA isolated before 1998, gentamicin-resistant isolates with SCC type IVA were all grouped in the well-separated cluster N2; SCC type IVA strains isolated since 1998 are scattered about clusters C1 and C2 and also represent single isolates not related to these clusters.
In France, genetic relatedness between recently isolated gentamicin-susceptible and some multi-resistant, gentamicin-resistant MRSA have been reported previously.13 A common genetic background has also been described in an evolutionary model for MRSA clonal complex 8 (CC8), which includes the Archaic, Iberian and Brazilian clones and several European MRSA clones with SCC type IV or IVA.10 Interestingly, MRSA isolates with resistance profile VII and SCC type IVA have a multilocus sequence type 8 (H. de Lencastre and D. Oliveira, personal communication). The diversity of genetic backgrounds of MRSAs with SCC types IV or IVA has been underlined previously for HA-MRSA as well as CA-MRSA isolates.6,10,24 Our results from a single hospital fully agree with these reports.
In conclusion, since 1997 the epidemiological background of MRSA in our hospital has been characterized by an increased prevalence of SCC type IV or IVA HA-MRSA isolates. Although the greater ability of isolates expressing antibiotic resistance profile VII to cause epidemics cannot be conclusively linked to their acquisition of SCC type IVA, nonetheless, this SCC type is present in the majority of recent epidemic MRSA strains. Since 1997, types IV and IVA MRSA, expressing less resistance to other antibiotics than to ß-lactams, have emerged independently from several different genetic lineages of S. aureus.
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Footnotes |
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References |
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2
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