Royal Liverpool University Hospital, Department of Medical Microbiology, Prescot Street, Liverpool L7 8XP, UK
Received 6 February 2004; returned 17 March 2004; revised 2 April 2004; accepted 17 April 2004
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Abstract |
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Materials and methods: Relatedness was determined by PFGE analysis of macro-restricted chromosome, together with a variety of PCR methods, to determine polymorphisms in the accessory gene regulator (agr) locus, the structure of the staphylococcal cassette chromosome (SCCmec) and the presence or absence of the gene encoding PantonValentine leucocidin (PVL).
Results: Clonality of the MRSA and MSSA was established by PFGE, a finding further supported by agr analysis. By PCR, the MRSA contained the typical genetic organization of SCCmec type-1. However, the MSSA, though mecA-negative, contained certain fragments of the SCC. Genes encoding PVL were not detected.
Conclusions: This outbreak involved a community-acquired fucidin-resistant MRSA and its methicillin-susceptible homologue. The MSSA did not contain the mecA gene but did contain elements of the mobile type-I SCC. The MSSA were associated with a change in PFGE pattern with a deletion in fragment size of 215195 kb.
Keywords: MSSA , MRSA , SCCmec
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Introduction |
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At the Royal Liverpool University Hospital (RLUH), we have experienced a cluster of invasive community-acquired fucidin-resistant MRSA in intravenous drug users (IVDUs), whose clinical presentations ranged from groin abscesses with or without septicaemia, to infective endocarditis. However, within the study group a number of patients infected with methicillin-susceptible (mecA gene negative) but fucidin-resistant S. aureus (MSSA) were found.
In this report, we have examined the clonal relatedness of the MRSA and MSSA and describe the staphylococcal cassette chromosome SCCmec present in the isolates.
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Material and methods |
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During November 2002May 2003, 18 IVDUs presented with bacteraemia due to S. aureus (14 MRSA, four MSSA), resistant to fucidin and erythromycin but susceptible to ciprofloxacin. However, two of the MRSA bacteraemic patients subsequently had fucidin-resistant MSSA isolated. A hyper-virulent community-acquired MSSA, strain 476 (MSSA476, sequence type ST1)2 isolated in the Oxford area of the UK between 19971998, was noted to have a similar PFGE profile to our isolates and was therefore included in our study for comparison (www.sanger.ac.uk/Projects/S_aureus).
Susceptibility testing
Disc susceptibility testing was performed according to BSAC guidelines on Iso-Sensitest agar (Oxoid Ltd, Basingstoke, UK).3 Methicillin and fucidin MICs were determined by the Etest method (AB Biodisk, Solna, Sweden). Production of penicillin binding protein 2' (PBP 2') was investigated using bacterial growth harvested from around cefoxitin (30 µg) discs (Mastalex; Mast Laboratories Ltd, Bootle, Merseyside, UK).
SCC typing
Total genomic DNA for PCR was extracted by suspending bacteria in 5% Chelex-100 resin slurry (Bio-Rad, Hemel Hempstead, UK) in injection grade water followed by boiling for 10 min. Samples were centrifuged (10 min at 13 400g) and used immediately or stored at 20°C. A number of different PCR methods were employed to identify internal loci of the SCCmec gene. Cassette chromosome recombinase genes ccrA1 and ccrB1, which are homologous to the DNA recombinases of the invertase/resolvase family and mobilize integration of mec into the S. aureus chromosome in the correct orientation, were detected with primers described by Ito et al.4 A multiplex PCR was used to detect eight other loci, an area located downstream of the pls gene, kdp operon, mecI gene, dcs region, and regions between plasmid pI258 and transposon Tn554, Tn554 and chromosomal right junction (orfX), IS431 and pU110, and IS431 and pT181.5 Genetic organization of the membrane-spanning (MS) and penicillin-binding domains of the mecA regulatory genes (mecR1 and mecI) was determined by the method described by Kobayashi et al.6 Sequence determination of amplicons was performed with a dideoxynucleotide-chain termination method using an automated DNA sequencer ABI PRISM 377 (Perkin Elmer, Warrington, UK). Sequence analysis was performed using commercial software (Lasergene; DNAStar Inc., Madison, WI, USA).
PVL and -haemolysin
Genes encoding the extracellular proteins PVL and -haemolysin were detected by PCR using primers and cycling times as described by Lina et al.7
Genomic analysis
Clonal relatedness was established by a combination of techniques. PFGE of whole chromosome Sma1 restriction digest fragments was performed on a CHEF DR III system (Bio-Rad) employing a recently harmonized international protocol. Polymorphisms in the accessory gene regulator (agr) locus have been used to indicate clonal relationships. A multiplex PCR of the accessory gene regulator was used to determine the agr group (14), and restriction endonuclease digestion (Rsa1 and Alu1) of the PCR amplicon of the variable region of the agr operon to determine agr type.8 Restriction products were separated by electrophoresis in 3% low-melting agarose (Metaphor; FMC Bio-Products, Flowgen, Staffordshire, UK) by PFGE in a non-ramping mode.
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Results |
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Discussion |
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Other workers have shown that loss of the mecA gene can give rise to methicillin-susceptible homologues of contemporaneously isolated MRSA, but none has commented on the presence of SCC in the susceptible isolates. We have also shown the absence of the mecA gene in our MSSA isolates and in the previously reported MSSA476, but surprisingly, we have detected the presence of SCC fragments. Current literature indicates that community-acquired MRSA strains from differing regions of the world are predominantly associated with type-IV SCCmec.1
SCCs without antibiotic resistance determinants have been detected in methicillin-susceptible Staphylococcus hominis GIFU12263 and have been shown to serve as a vehicle of transfer for various genetic markers between staphylococcal species.9 Other workers have demonstrated the presence of a degenerate form of the SCC element associated with the capI genes in S. aureus, which had the same right insertion junction, attC, as those of the SCCmec.10 To our knowledge, ours is the first description of the cassette chromosome recombinase genes ccrA1 and ccrB1 in MSSA. The cluster of infections at the RLUH involving both fucidin-resistant MRSA and MSSA emerged towards the end of 2002, but possibly similar MSSA isolates have been present in the UK at least since the isolation of SA476 (ST1) in the period 19971998. The emergence of MRSA from MSSA has been limited to a restricted number of lineages and it would be of value to determine the potential role of SCC for mediating gene movement in other MSSA.
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Footnotes |
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References |
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2
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Enright, M. C., Nicholas, P. J., Davies, C. E. et al. (2000). Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. Journal of Clinical Microbiology 38, 100815.
3
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Andrews J. M. for the BSAC Working Party on Susceptibility Testing (2001). BSAC standardized disc susceptibility testing method. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 4357.
4
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Ito, T., Katayama, Y., Asada, K. et al. (2001). Structural comparison of three types of staphylococcal cassette chromosome mec integrated in the chromosome in methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 45, 132336.
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Oliveira, D. C. & de Lencastre, H. (2002). Multiplex PCR strategy for rapid identification of structural types and variants of the mec element in methicillin-resistant Staphylococcus aureus. Antimicrobial Agents and Chemotherapy 46, 215561.
6 . Kobayashi, N., Taniguchi, K., Kojima, K. et al. (1996). Genomic diversity of mec regulator genes in methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis. Epidemiology and Infection 117, 28995.[ISI][Medline]
7 . Lina, G., Piemont, Y., Godail-Gamot, F. et al. (1999). Involvement of Panton-Valentine leukocidin-producing Staphylococcus aureus in primary skin infections and pneumonia. Clinical Infectious Diseases 29, 112832.[CrossRef][ISI][Medline]
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Gilot, P., Lina, G., Cochard, T. et al. (2002). Analysis of the genetic variability of genes encoding the RNA III-activating components Agr and TRAP in a population of Staphylococcus aureus strains isolated from cows with mastitis. Journal of Clinical Microbiology 40, 406070.
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Katayama, Y., Takeuchi, F., Ito, T. et al. (2003). Identification in methicillin-susceptible Staphylococcus hominis of an active primordial mobile genetic element for the staphylococcal cassette chromosome mec of methicillin-resistant Staphylococcus aureus. Journal of Bacteriology 185, 271122.
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Luong, T. T., Ouyang, S., Bush, K. et al. (2002). Type 1 capsule genes of Staphylococcus aureus are carried in a staphylococcal cassette chromosome genetic element. Journal of Bacteriology 184, 36239.