Detection of elements of the staphylococcal cassette chromosome (SCC) in a methicillin-susceptible (mecA gene negative) homologue of a fucidin-resistant MRSA

John E. Corkill*, James J. Anson, Paul Griffiths and C. Anthony Hart

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


    Abstract
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Objectives: To determine the DNA relatedness of an outbreak of community-acquired fucidin-resistant methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolated from intravenous drug users (IVDUs).

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 Panton–Valentine 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 ~215–195 kb.

Keywords: MSSA , MRSA , SCCmec


    Introduction
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Staphylococcus aureus continues to be an important pathogen in both community- and hospital-acquired infection. Methicillin-resistant S. aureus (MRSA), which is associated with hospitals and long-term care facilities, is now being isolated in the community and debate continues as to whether these isolates reflect the spread of hospital strains or the emergence of new community-derived invasive clones. Many of the community-acquired strains carry staphylococcal cassette chromosome SCCmec type-IV and the Panton–Valentine leucocidin (PVL) locus.1

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.


    Material and methods
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Bacterial strains

During November 2002–May 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 1997–1998, 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 {gamma}-haemolysin

Genes encoding the extracellular proteins PVL and {gamma}-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 (1–4), 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.


    Results
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Fucidin resistance (MIC 4–8 mg/L) was present in the MRSA, MSSA and in MSSA476. The MSSA and MSSA476 were susceptible to methicillin (MICs < 4.0 mg/L) and PBP 2' negative by the Mastalex agglutination method. Clonal relatedness of the outbreak of MRSA (types A and A1) and MSSA (types A2, A3 and A4) was established by PFGE analysis of chromosome digested with Sma1, as isolates differed from each other by no more than three fragment bands (Figure 1). However, MSSA476 (ST1) differed by four bands from MRSA types A and A1, but by one, two and four bands from the MSSA subtypes (A2, A3 and A4) and therefore may only be possibly related. MSSA were associated with a change in PFGE pattern with a deletion in fragment size of approx. 215–195 kb (see Figure 1, subtypes A2, A3 and A4 of the outbreak clone). Relatedness was further supported by PCR analysis of agr, which produced an amplicon of 323 bp, typical of agr group 3 (Table 1). Polymorphisms in the agr operon were noted following digestion with both Alu1 (fragment sizes 772, 155 and 74 bp) and Rsa1 (fragment sizes 478, 287, 230 and 79 bp) restriction endonucleases. Such patterns are characteristic of the agr type R VI-A8 as described by Gilot et al.8



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Figure 1. Genomic typing of S. aureus. Chromosomal DNA digested with Sma1 (lanes 1–6) and separated by PFGE. Lanes 1 (genotype A) and 2 (genotype A1) show the profiles of fucidin-resistant MRSA and lanes 3 (genotype A2), 4 (genotype A3) and 5 (genotype A4) show the profiles of fucidin-resistant MSSA (mecA-negative). Lane 6 contains MSSA476 (genotype A5). Lane 7 is a DNA concatemer (size range 50–500 kb).

 

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Table 1. Clonal relatedness of fucidin-resistant Staphylococcus aureus isolated from blood of IVDUs and community-acquired MSSA476

 
PCR analysis of the MRSA clone identified two internal loci of SCCmec, the area downstream of the pls gene (495 bp, plasmin-sensitive surface protein) and dcs (342 bp, downstream constant region), the typical genetic organization of SCCmec type-I. This classification was confirmed by PCR detection of cassette chromosome recombinase genes ccrA1 and ccrB1.4 The allotypic form of the mec gene complex, type B, was detected (mecA-{Delta}mecR1(MS)-IS1272), which is normally carried in SCCmec types-I and -IV. The specificity of the PCR products was confirmed by nucleotide sequencing. However, analysis of the clonally related fucidin-resistant MSSA and MSSA476 (both mecA gene negative) unexpectedly gave positive signals for the dcs region and ccrA1 and ccrB1 genes, but not the area downstream of the pls gene. PVL encoding genes were not detected, but {gamma}-haemolysin genes were present in all isolates.


    Discussion
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
At the RLUH we have detected an increased prevalence of fucidin resistance in MRSA (5% in 2001, 7% in 2002 and 12% in the first 8 months of 2003), whereas in MSSA resistance to fucidin has remained constant at 13% over the same time period. This problem was highlighted by infections occurring in our IVDU patients. Molecular analysis of these isolates by both PFGE and polymorphism analysis of the agr gene showed a clonal relationship between the MRSA and MSSA.

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 1997–1998. 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.


    Footnotes
 
* Corresponding author. Tel: +44-151-706-4410 ext. 4421; Fax: +44-151-706-5849; Email: jecmm{at}liverpool.ac


    References
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 Introduction
 Material and methods
 Results
 Discussion
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1 . Vandenesch, F., Naimi, T., Enright, M. C. et al. (2003). Community-acquired methicillin-resistant Staphylococcus aureus carrying Panton-Valentine leukocidin genes: worldwide emergence. Emerging Infectious Diseases 9, 978–84.[ISI][Medline]

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4 . 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, 1323–36.[Abstract/Free Full Text]

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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, 1128–32.[CrossRef][ISI][Medline]

8 . 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, 4060–70.[Abstract/Free Full Text]

9 . 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, 2711–22.[Abstract/Free Full Text]

10 . 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, 3623–9.[Abstract/Free Full Text]