1 Laboratoire de Référence MRSAStaphylocoques, Department of Microbiology, Hôpital Erasme, Université Libre de Bruxelles, Brussels, Belgium; 2 Microbiology, Onze Lieve Vrouw Ziekenhuis, Aalst, Belgium; 3 Bacteriology, CHR Namur, Belgium; 4 Bacteriology, UCLMont Godinne, Yvoir, Belgium; 5 Infectious DiseasesHUDERF, Université Libre de Bruxelles, Brussels, Belgium
Received 9 August 2005; returned 8 September 2005; revised and accepted 19 September 2005
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Abstract |
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Methods: Clinical MRSA isolates (n = 41) collected from 2002 to 2004 from Belgian patients were investigated for the PVL gene by PCR. PVL-positive isolates were genotyped by PFGE, staphylococcal cassette chromosome mec (SCCmec) typing, spa sequence typing, accessory gene regulator (agr) polymorphism and multi-locus sequence typing (MLST). Susceptibility to 14 antimicrobials was determined by the disc diffusion method. Genes encoding resistance to tetracyclines, aminoglycosides and macrolide-lincosamide-streptogramin were determined by PCR.
Results: Sixteen isolates carried lukS-lukF genes that encode the PVL toxin. All but one isolate were community-acquired. Three patients reported recent travel to North Africa and South America. They were associated with skin or soft tissue infections, bacteraemia and peritonitis. By molecular typing, they belonged to five genotypes: ST80-SCCmec IV, ST8-SCCmec IV, ST30-SCCmec IV, ST153-SCCmec IV and ST88-SCCmec IV. They belonged to the agr type 3 except for ST8 strains, which showed agr type 1. All isolates were susceptible to fluoroquinolones. Approximately, half of them were resistant to tetracycline, fusidic acid and kanamycin. Tetracycline-resistant strains harboured the tet(K) gene and resistance to kanamycin was associated with the aph(3')-IIIa gene. The single erythromycin-resistant isolate harboured msr(A/B) genes conferring the M resistance phenotype.
Conclusions: These results indicate the recent emergence and sporadic importation into Belgium of PVL-positive community-associated MRSA strains belonging to five distinct clones.
Keywords: MRSA , PVL , molecular characteristics , MLST
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Introduction |
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Materials and methods |
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In 20022003, the Belgian Reference Laboratory for Staphylococci issued alert messages to microbiology laboratories about the need to refer MRSA isolates from community-acquired infection for detection of exotoxin genes. From 2002 to 2004, the Belgian Reference Laboratory for Staphylococci received 41 MRSA isolates referred for characterization of toxin production. S. aureus identification was confirmed by using the coagulase test with human plasma. Oxacillin susceptibility was tested by cefoxitin disc (60 µg) (Rosco Neo-Sensitabs, Taastrup, Denmark) according to the CLSI.4
PCR for toxin detection
A multiplex PCR assay was performed for the detection of 16S rRNA and mecA and nuc genes as previously described.5 The presence of PVL, TSST-1, and exfoliatin A and exfoliatin B genes was tested by multiplex PCR assays as previously described.2 S. aureus ATCC 49775 control strain harbouring PVL genes and S. aureus FRI913 control strain harbouring TSST-1 genes were included in each run.
Antimicrobial susceptibility testing
PVL-positive isolates were tested by the disc diffusion method (Rosco Neo-Sensitabs, Taastrup, Denmark) for susceptibility to 14 antimicrobial agents, including erythromycin, clindamycin, pristinamycin, ciprofloxacin, gentamicin, kanamycin, tobramycin, minocycline, tetracycline, rifampicin, trimethoprim/sulfamethoxazole, fusidic acid, linezolid and mupirocin. A double disc diffusion method with erythromycin and clindamycin discs was used to differentiate the MLS resistance phenotypes. CLSI susceptibility breakpoints4 were used for interpretation of inhibition zone diameters except for fusidic acid and mupirocin, which were interpreted according to the criteria of the Committee for Antimicrobial Testing of the French Society of Microbiology (CA-SFM).6 Resistance to vancomycin was tested by vancomycin screen with Brain Heart Infusion (BHI) agar (6 mg/L) (Becton-Dickinson, USA).4
Molecular typing
All PVL-positive MRSA isolates were genotyped by the SmaI macrorestriction analysis of genomic DNA resolved by PFGE, determination of staphylococcal cassette chromosome mec (SCCmec) type and multi-locus sequence typing (MLST) (http://www.mlst.net) as previously described.5 PFGE patterns were classified in groups according to the previously described nomenclature.5 The accessory gene regulator (agr) polymorphism was determined by PCR.7 DNA sequence analysis of the polymorphic repeat region of the protein A gene (spa typing) was performed as previously described.8
PCR for tetracycline, aminoglycoside and MLS resistance gene detection
Tetracycline resistance genes encoding efflux pump system tet(K) or for ribosomal protection protein tet(M),9 the aminoglycoside modifying enzymes (AME) encoded by aac(6')-Ie + aph(2''), ant(4')-Ia and aph(3')-IIIa genes,10 the ribosomal methylase encoded by erm(A) and erm(C) and the macrolide efflux pumps encoded by msr(A) and msr(B) genes11 were tested by PCR as previously described.
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Results and discussion |
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All PVL-positive isolates carried the SCCmec IV element (Table 1). They belonged to the agr type 3 except for PFGE group A strains, which showed agr type 1. They were clustered into four PFGE groups. Results of spa typing and MLST were in full agreement with the PFGE classification, except for two isolates which presented single repeat allele polymorphism by spa or MLST typing: one spa t131 isolate differed by a single deletion from t044 and one ST153 isolate was a single allele variant of ST80. All PVL-positive isolates were susceptible to ciprofloxacin, gentamicin and tobramycin. Another striking feature was that strains belonging to the PFGE X-ST80-SCCmec IV clone were resistant to fusidic acid, tetracycline and kanamycin (Table 1). A majority of PVL-positive isolates were resistant to tetracycline (56%), fusidic acid (56%) and kanamycin (56%) but showed only very occasionally resistance to erythromycin. Tetracycline-resistant isolates harboured the tet(K) gene in contrast with CA-MRSA strains from Germany that carry the tet(M) gene.12 Resistance to kanamycin was associated with the aph(3')-IIIa gene. The single erythromycin-resistant isolate harboured msr(A/B) genes conferring the M resistance phenotype. This mechanism of resistance is found in <1% of nosocomial MRSA isolates in Belgium.5
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Outbreaks of PVL-positive MRSA infections have been reported in many communities around the world.15,16 No such outbreak has been reported yet in Belgium. In this series, except for a cluster of two cases in the same family, cases occurred sporadically in different regions of the country. Three imported cases were associated with recent travel to North Africa (n = 2) (ST80-SCCmec IV) and Ecuador (n = 1) (ST8-SCCmec IV), suggesting the possibility of importing the infection from these countries into Belgium.
In conclusion, we report the emergence of PVL-positive CA-MRSA strains belonging to five different genetic lineages in Belgium, with a predominance of the ST80-SCCmec IV clone, which is widely disseminated in Europe. The presence of PVL genes in diverse genetic backgrounds suggests that their horizontal transfer into resident S. aureus has occurred repeatedly, sometimes affecting strains that are closely related to endemic nosocomial MRSA strains. In addition, travel-associated cases of these CA-MRSA infections illustrate the propensity of MRSA clones to spread over large geographical areas, underlying the potential for a pandemic of CA-MRSA disease.
Owing to the passive reporting design of this study, the data reported here are likely to underestimate the true occurrence of CA-MRSA infections in Belgium. As reported in previous surveys, CA-MRSA strains were clinically associated with severe skin and soft tissue infections. The emergence of hyper-virulent MRSA strains in the community highlights the importance of their rapid identification in infected patients to adapt antimicrobial therapy and implement appropriate infection control practices. Further study is required to monitor the incidence of CA-MRSA infections in the Belgian population.
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Transparency declarations |
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Acknowledgements |
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References |
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