JAC Advance Access originally published online on December 9, 2005
Journal of Antimicrobial Chemotherapy 2006 57(2):331-334; doi:10.1093/jac/dki452
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Molecular characterization of methicillin-resistant Staphylococcus aureus bloodstream isolates from Croatia
1 Department of Clinical and Molecular Microbiology, Clinical Hospital Centre Zagreb, Croatia; 2 Maastricht Infection Center (MINC), Department of Medical Microbiology, University Hospital Maastricht, The Netherlands
* Corresponding author. Tel: +31-43-3874644; Fax: +31-43-3876643; E-mail: est{at}lmib.azm.nl
Received 3 May 2005; returned 15 September 2005; revised 7 November 2005; accepted 16 November 2005
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Abstract |
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Objectives: The objectives of this study were (i) to investigate the genetic background of methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates from Croatia and (ii) to monitor the prevalence of Panton-Valentine leucocidin (PVL) and toxic shock syndrome toxin-1 (TSST-1) among these isolates.
Methods: Eighty-two hospital-acquired MRSA bloodstream isolates, collected in 2001 and 2002 in Croatia, were characterized by PFGE, staphylococcal cassette chromosome mec (SCCmec) typing and multilocus sequence typing (MLST). The presence of genes encoding PVL and TSST-1 was investigated by real-time PCR.
Results: All strains were multiresistant and were distributed among 16 different similarity groups as determined by PFGE. Two of the groups, groups H and K, harboured the majority of the MRSA strains with 52 and 12%, respectively. The predominant SCCmec type found among the isolates was type I (89%). Eleven per cent of the strains harboured a modified SCCmec type III, which contained, in contrast to the regular type III, an additional dcs region. One strain harboured a novel SCCmec type, containing the ccrC gene in combination with the mecI gene, the dcs region, the locus between pI258 and Tn554 (locus E) and the locus between Tn554 and orfX (locus F). MLST showed the presence of ST111-MRSA-I and ST247-MRSA-I among Croatian MRSA isolates. All isolates were negative for both PVL and TSST-1.
Conclusions: These results indicate the emergence of ST111-MRSA-I and ST247-MRSA-I in Croatia among MRSA bloodstream isolates. The virulence factors PVL and TSST-1 were not present among these isolates.
Keywords: MRSA , PVL , SCCmec , TSST-1
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Introduction |
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The resistance of Staphylococcus aureus to ß-lactam antibiotics is associated with the expression of penicillin-binding protein 2a (PBP2a). This protein is encoded by the mecA gene, which is situated on a mobile genetic element, staphylococcal cassette chromosome mec (SCCmec). Five different SCCmec types have been identified in methicillin-resistant S. aureus (MRSA) strains. SCCmec types I, II and III are mainly found in hospital-acquired MSRA (HA-MRSA), whereas SCCmec types IV and V are mainly associated with community-acquired MRSA (CA-MRSA). SCCmec contains the mec complex (mecA and its regulators) and the ccr gene complex, which encodes site-specific recombinases, responsible for the mobility of SCCmec. Several different ccr genes have been identified: ccrA1 and ccrB1 in SCCmec type I, ccrA2 and ccrB2 in SCCmec types II and IV, ccrA3 and ccrB3 in SCCmec type III, ccrA4 and ccrB4 in SCCmec type IV and ccrC in SCCmec type V.1,2
S. aureus can produce a number of virulence factors. Panton-Valentine leucocidin (PVL) is predominantly associated with severe skin infections and necrotizing pneumonia. PVL, together with SCCmec type IV, is suggested to be a marker for CA-MRSA,2 although there are exceptions.3,4 Toxic shock syndrome toxin-1 (TSST-1) is a 29.1 kDa superantigen that is encoded by the tst gene. The release of TSST-1 into the bloodstream may give rise to a variety of severe clinical conditions, like TSS. TSST-1 is produced by many MRSA strains, particularly in Japanese hospitals.5
Croatia has a MRSA prevalence of 36.7% among bloodstream isolates.6 Information on the clonal distribution of MRSA in Croatia, however, is not available. Furthermore, nothing is known about the prevalence of PVL and TSST-1 among Croatian MRSA isolates. Therefore, the aim of this study was to determine the clonal distribution of MRSA among bloodstream isolates in Croatia isolated in 2001 and 2002 and to determine the presence of the virulence factors PVL and TSST-1.
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Materials and methods |
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Clinical isolates
Eighty-two MRSA bloodstream isolates were collected from 10 hospitals in seven Croatian cities during 2001 and 2002 as part of the European Antimicrobial Resistance Surveillance System (EARSS) project (Figure 1). All strains were hospital-acquired, i.e. isolated 
72 h after patient admission to the hospital. Reference strains S. aureus ATCC 25923 for PFGE, MRSA COL (SCCmec I), BK2464 (SCCmec II), ANS46 (SCCmec III), HDE288 (SCCmec IV) and WIS (SCCmec V) for SCCmec typing and ccr gene complex determination were used. MRSA Cluster 28 was used as a reference strain for PVL and S. aureus HT.2004.0349 as the reference strain for TSST-1.3,5 All strains were identified as S. aureus by catalase, coagulase and DNase production.
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Susceptibility testing
Susceptibility testing was performed by the broth microdilution method according to the guidelines of the CLSI (formerly NCCLS).7 The following antimicrobial drugs were tested: azithromycin, chloramphenicol, ciprofloxacin, clindamycin, erythromycin, gentamicin, linezolid, norfloxacin, oxacillin, penicillin, rifampicin, tetracycline, trimethoprim, trimethoprim/sulfamethoxazole and vancomycin.
SCCmec typing and detection of virulence factors
SCCmec typing and the detection of ccrAB3 and ccrC were performed as described previously.3 The presence of the genes encoding PVL and TSST-1 was investigated by using real-time PCR as described previously.3,5
PFGE and multilocus sequence typing
PFGE analyses and multilocus sequence typing (MLST) were performed as previously described.3
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Results |
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Susceptibility patterns
All 82 MRSA isolates had MIC values for oxacillin of 128 mg/L. All strains were resistant to gentamicin and ciprofloxacin and were susceptible to vancomycin and linezolid. Most of the strains (96%) were resistant to clindamycin, erythromycin and azithromycin. In addition, 36% of the strains were resistant to rifampicin, 32% were resistant to tetracycline, 9% were resistant to chloramphenicol and 0.2% were resistant to trimethoprim/sulfamethoxazole.
SCCmec typing
Seventy-three of the 82 (89%) MRSA strains were found to contain SCCmec type I. From nine (11%) strains, the SCCmec type was not typeable with the method used.3 SCCmec of these strains was found to harbour mecA and loci C, D, E and F. Therefore, these cassettes only differ from SCCmec type III in the presence of locus D. To investigate the nature of these cassettes in more detail, the ccr genes of these nine strains were characterized. In eight of the nine MRSA strains, the ccr gene was identified as ccrAB3. Consequently, these strains were classified as harbouring SCCmec type III. The other non-typeable strain, strain 79, harboured ccrC, which is normally only carried on SCCmec type V.
PFGE analyses
Three of the 82 isolates could not be typed using PFGE, either owing to difficulties with lysis of the bacteria or the digestion of the DNA. A total of 16 similarity groups (A to P) were found and two of these, H and K, were classified as major similarity groups (Figure 2). Similarity group H harboured 43 of the 82 (52%) isolates, whereas similarity group K harboured 11 of the 82 (13%) isolates. In both groups H and K, SCCmec type I was predominant (i.e. 95 and 82%, respectively). Furthermore, the strains from groups H and K had different susceptibility patterns. The majority (91%) of the strains from group K were resistant to rifampicin and tetracycline, whereas the majority of the strains from group H were susceptible to these antibiotics (84 and 91%, respectively). Strains harbouring SCCmec type III were classified into various similarity groups, i.e. groups D (2), H (2), K (2), Q (1) and NT (1).
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MLST analyses
Two representative strains from the major similarity groups H and K were characterized by MLST. As shown in Table 1, both strains from group H were identified as ST111, a single locus variant (SLV) of ST228 at the pta locus, whereas both strains from group K were classified as ST247. The distribution of these clones in Croatia is presented in Figure 1.
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PVL and TSST-1 analyses
The genes encoding virulence factors PVL and TSST-1 were not detected in any of the investigated strains.
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Discussion |
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In this study, the clonal distribution of MRSA isolates in Croatia was described. Most of the isolates (89%) were found to possess SCCmec type I. Eight of the nine strains that did not contain SCCmec type I carried a modified SCCmec type III. In contrast to a normal type III cassette, the cassette of these strains harboured locus D (dcs region), which is usually only found in SCCmec types I, II and IV.3 An MRSA strain with a similar SCCmec element (HSA10) has previously been described by Aires de Sousa and de Lencastre.4
The single remaining ‘non-SCCmec type I’ strain from this study (strain 79) was found to harbour ccrC, together with loci C (mecI, normally found in SCCmec II and III), D, E (a locus between pI258 and Tn554, which is specific for SCCmec type III) and F (a locus between Tn554 and orfX, which is specific for SCCmec type III).3 Since ccrC is usually only found in combination with locus E in SCCmec type V,1,3 MRSA strain 79 is likely to harbour a novel SCCmec element. Although this novel element shows many similarities with the SCCmec type III element, it lacks the type III-specific ccrAB3 gene. Further investigation into the exact structure of this element is currently underway. An encounter of two S. aureus strains harbouring different SCCmec elements could have led to the formation of a novel, not previously described, SCCmec element, probably through homologous recombination.1
Typing of the MRSA strains by PFGE revealed 16 similarity groups (A to P), two of which, H and K, were classified as major similarity groups. These results, together with the SCCmec typing, indicated that the distribution of particular MRSA genotypes was not restricted to specific hospitals or cities in Croatia. A total of 52% of the MRSA strains was classified within major similarity group H and 95% of these strains harboured SCCmec type I. ST111, found by MLST analyses of two representative strains from group H, was previously found in one strain (AB-903627/02) from Norway (http://www.mlst.net), and one strain harbouring SCCmec type I from the Czech Republic.8,9 ST111-MRSA-I could have evolved from ST228-MRSA-I, the Southern Germany clone, since ST111 is an SLV of ST228 at the pta locus. ST228-MRSA-I has been found in Belgium, Denmark, Germany, Italy, Slovenia and Spain.8 Thus far, ST111-MRSA-I has only been found in various European countries and not in other parts of the world.
The second major similarity group, group K, contained 13% of the MRSA isolates; 82% of the strains from this group harboured SCCmec type I. ST247-MRSA-I from group K is similar to the Iberian clone, one of the major MRSA clones currently isolated in European countries, such as the Czech Republic, Germany, Italy, Poland, Slovenia and Switzerland. ST247 is classified in clonal complex (CC) 8.2,8
PVL and TSST-1 were not detected in any of the isolates investigated. Similarly, a previous study reported only a single PVL-positive isolate among 86 bloodstream isolates.10 These results indicate that these virulence factors are not necessarily associated with MRSA strains causing MRSA bloodstream infections.
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None to declare.
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Acknowledgements |
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We thank Professor H. De Lencastre and Dr D. C. Oliveira from The Rockefeller University, New York, USA for providing the reference strains for SCCmec typing, Dr T. Ito from the Juntendo University, Tokyo, Japan for providing the reference strain (WIS) for SCCmec V and Dr W. Wannet from the National Institute of Public Health and Environment (RIVM) in Bilthoven, The Netherlands for MRSA Cluster 28 strain. Dr J. Etienne and Dr M. Bes from INSERM, Lyon, France are thanked for reference strain HT.2004.0349. C. Driessen is thanked for excellent technical assistance.
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References |
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1. Ito T, Ma X, Takeuchi F et al. Novel type V staphylococcal cassette chromosome mec driven by a novel cassette chromosome recombinase, ccrC. Antimicrob Agents Chemother 2004; 48: 2637–51.
2. Aires de Sousa M, de Lencastre H. Bridges from hospitals to the laboratory: genetic portraits of methicillin-resistant Staphylococcus aureus clones. FEMS Immunol Med Microbiol 2004; 40: 101–11.[CrossRef][Web of Science][Medline]
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Aires de Sousa M, de Lencastre H. Evolution of sporadic isolates of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals and their similarities to isolates of community-acquired MRSA. J Clin Microbiol 2003; 41: 3806–15.
5. Deurenberg RH, Nieuwenhuis RF, Driessen C et al. The prevalence of the Staphylococcus aureus tst gene among community- and hospital-acquired strains and isolates from Wegener's Granulomatosis patients. FEMS Microbiol Lett 2005; 245: 185–9.[CrossRef][Medline]
6. Tiemersma EW, Bronzwaer SL, Lyytikainen O et al. Methicillin-resistant Staphylococcus aureus in Europe, 1999–2002. Emerg Infect Dis 2004; 10: 1627–34.[Web of Science][Medline]
7. National Commitee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing—Ninth informational supplement M100-S9. NCCLS, Wayne, PA, USA, 1999.
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Robinson DA, Enright MC. Evolutionary models of the emergence of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2003; 47: 3926–34.
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Melter O, Aires de Sousa M, Urbaskova P et al. Update on the major clonal types of methicillin-resistant Staphylococcus aureus in the Czech Republic. J Clin Microbiol 2003; 41: 4998–5005.
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