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Alterations in phage-typing patterns in vancomycin-intermediate Staphylococcus aureus

John E. Gustafson¹, Frances G. O'Brien², Geoffrey W. Coombs², Mary J. Malkowski², Warren B. Grubb², Richard F. Pfeltz^3, and Brian J. Wilkinson³

¹Department of Biology, New Mexico State University, Las Cruces, NM 88003-8001, USA ²Gram-positive Bacteria Typing and Research Unit, Curtin University of Technology, and Royal Perth Hospital, Perth 6845, Western Australia ³Microbiology Group, Department of Biological Sciences, Illinois State University, Normal, IL 61790-4120, USA

Correspondence John E. Gustafson jgustafs{at}nmsu.edu

Received February 3, 2003
Accepted April 16, 2003

	Abstract

TOP Abstract Introduction Methods Results and Discussion Acknowledgements References

 
The ability of phage-typing and SmaI chromosomal RFLPs to conclude appropriate strain relatedness between a collection of 12 well-characterized in vitro-selected vancomycin-intermediate Staphylococcus aureus (VISA) strains and their seven vancomycin-susceptible parent strains is reported. Generally, no SmaI RFLP alterations were observed in VISA strains when they were compared with their respective parent strains, and clonal relationships between isogenic strains were clearly evident. Unlike the SmaI RFLP results, parent strains and VISA derivatives generally did not share similar phage-typing profiles. Depending on the phage set investigated, some VISA strains even became untypable by this method. Loss of phage infectivity is probably due to cell wall (phage receptor) alterations that are expressed by the VISA strains investigated. Collectively, these findings indicate that inappropriate relationships between VISA and vancomycin-susceptible parents might be drawn if only phage-typing and antibiotic susceptibility are utilized to determine epidemiological relationships.

Abbreviations: MRSA, methicillin-resistant S. aureus; VISA, vancomycin-intermediate S. aureus; VRSA, vancomycin-resistant S. aureus.

	Introduction

TOP Abstract Introduction Methods Results and Discussion Acknowledgements References

 
Analysis of SmaI chromosomal RFLPs produced using PFGE is very effective in tracking Staphylococcus aureus clones (Jorgensen et al., 1996; O'Brien et al., 1999; Simor et al., 2002). Phage typing is also used to track outbreak, epidemic and community strains of S. aureus effectively (Blair & Williams, 1961; O'Brien et al., 1999; O'Neill et al., 2001). While phage typing is often valuable in discerning strain relationships quickly and inexpensively, the technique is not always helpful, since non-typable S. aureus strains exist (Blair & Williams, 1961; Andrasevic et al., 1999; Tambic et al., 1997).

The glycopeptide antibiotic vancomycin is still the major drug of choice for treatment of severe disease caused by infection with methicillin-resistant S. aureus (MRSA). Since 1997, vancomycin-intermediate S. aureus (VISA) have become an emerging clinical reality (Hiramatsu et al., 1997a). Vancomycin-intermediate expression in S. aureus is mediated by chromosomal mutation(s) (Avison et al., 2002; Pfeltz et al., 2000; Sakoulas et al., 2002; Walsh & Howe, 2002) and is not due to horizontal gene transfer of vancomycin-resistance determinants (van) from organisms such as the vancomycin-resistant enterococci. VISA strains harbour a chromosomal mutation(s) that leads to alterations in cell-wall synthesis and structure, which are thought to impart resistance by sequestering glycopeptide molecules away from their target (for review, see Walsh & Howe, 2002). PFGE and SmaI chromosomal RFLP analysis have been used to demonstrate that VISA can disseminate in a clonal fashion (Chesneau et al., 2000; Hiramatsu et al. 1997b; Kim et al., 2002). Two reports have now appeared on high-level vancomycin-resistant S. aureus (VRSA) that have acquired the enterococcal vanA determinant (Miller et al., 2002; Sievert et al., 2002). Outbreaks with VISA or VRSA have not yet become a common occurrence.

We now report on the ability of phage typing and PFGE to conclude appropriate strain relatedness between a collection of 12 in vitro-selected VISA and their seven vancomycin-susceptible parents.

	Methods

TOP Abstract Introduction Methods Results and Discussion Acknowledgements References

 
Strains.

The strains analysed represent various genetic backgrounds and methicillin resistance levels and their isogenic in vitro step-selected VISA derivatives (Pfeltz et al., 2000). The subscript V in a strain designation refers to a vancomycin-intermediate derivative (e.g. BB270_V5) of a susceptible parent strain (e.g. BB270), and the number associated with the subscript V (such as V5) refers to the vancomycin concentration at which the strain was maintained directly following VISA phenotype selection (e.g. 5 µg ml^-1 for BB270_V5) and not the actual vancomycin MIC (Pfeltz et al., 2000). The MICs of the strains for vancomycin, teicoplanin and oxacillin and other characteristics of these strains were described by Pfeltz et al. (2000).

Phage typing and PFGE.

Phage typing, performed on strains passaged in antibiotic-free medium, and PFGE of SmaI chromosomal digests were performed as described previously (Blair & Williams, 1961; O'Brien et al., 1999). Chromosomal banding patterns were scanned with a Flour-S MultImager and analysed by Multi-Analyst/PC (Bio-Rad).

	Results and Discussion

TOP Abstract Introduction Methods Results and Discussion Acknowledgements References

 
SmaI chromosomal RFLPs of the isogenic parents (BB255, BB270, SH108, BB399, 13136p-m+, BB568 and COL) and in vitro-selected VISA strains (BB255_V3, BB270_V5, BB270_V15, SH108_V5, BB399_V5, BB399_V12, 13136p-m+_V5, 13136p- m+_V20, BB568_V5, BB568_V15, COL_V5 and COL_V10) and a dendrogram generated from these RFLPs are shown in Fig. 1. It should be noted that strain BB270 is a methicillin-resistant transductant of BB255, and strains BB568 and COL are isogenic (Pfeltz et al., 2000); therefore, as expected, these strain pairs cluster together (Fig. 1). SmaI restriction profiles also revealed that, with the exception of VISA strain BB399_V12, no significant alterations occurred in the SmaI chromosomal RFLPs of most strains (BB255, BB270, SH108, 13136p-m+, BB568 and COL) as they were step-selected to become vancomycin-intermediate. These data confirm that major genetic alterations are not required for S. aureus to develop the VISA phenotype. In addition, clonal relationships between the collection of in vitro-selected VISA mutants and isogenic parent strains are clearly evident. Other investigators have also demonstrated that in-vitro-selected VISA retain parental SmaI RFLP patterns (Schaaff et al., 2002). SmaI chromosomal RFLPs are also similar between VISA that arise from vancomycin-susceptible strains during vancomycin therapy in a single patient (Rotun et al., 1999; Sieradski et al., 1999a; Smith et al., 1999).

View larger version (34K): [in this window] [in a new window]   Fig. 1. Cartoon of SmaI-macrorestricted chromosomal RFLPs of S. aureus strains investigated and dendrogram to demonstrate percentage relatedness. , Band alterations between BB399 and BB399V12.

A 300 kb SmaI fragment present in parent strain BB399 has been replaced by a 275 kb fragment in BB399_V12 (Fig. 1), indicating the loss of 25 kb, which actually allowed BB399_V12 to cluster with COL and BB568. Altered SmaI RFLP patterns have been reported to occur within in vitro-selected VISA strains (Riepert et al., 2003). These alterations have been attributed to loss of the methicillin-resistance determinant (mec) as well as other genes (Riepert et al., 2003; Sieradzki et al., 1999b). BB399_V12, however, maintains high-level methicillin resistance, similar to parent BB399 (Pfeltz et al., 2000), indicating that the former has not lost mec. SmaI RFLP alterations can result from the loss of a prophage (Smeltzer et al., 1994) and, perhaps, during the selection for the VISA phenotype in BB399_V12, a prophage excised and was lost.

Unlike the SmaI RFLP results, parent strains and VISA derivatives generally did not share similar phage-typing profiles using the international basic phage set (IBPS), the international MRSA phage set (IMRSA) or the Australian MRSA phage set (AMRSA) (Table 1). When the IBPS was used, all VISA derivatives demonstrated altered phage types when compared with their respective parent strains. VISA mutant 13136p-m+_V5 even became non-typable by this phage set. Typing with IMRSA revealed that isogenic strain sets BB255 and BB255_V3 and BB568 and BB568_V5 had the same phage types, while strain 13136p-m+ and its VISA mutants were non-typable by this phage set. All other VISA strains exhibited altered IMRSA types compared with their respective parent strains, and SH108_V5 became untypable. Typing with AMRSA revealed that the BB255 and BB568 strain sets all had identical phage types, as did COL and its VISA derivative COL_V5. All other VISA mutants demonstrated altered AMRSA types compared with their respective parent strains, and BB399_V5 became untypable. Other studies have also shown alterations in phage-typing patterns following in vitro acquisition of a VISA phenotype in S. aureus (Daum et al., 1992; Schaaff et al., 2002).

Phage infection can be prevented in Gram-positive bacteria as a result of adsorption inhibition (Chatterjee, 1969; Tran et al., 1999; Wilkinson & Holmes, 1979), restriction modification systems (Kong & Josephsen, 2002), DNA injection-blocking systems (McGrath et al., 2002), lysogenization (Beard-Pegler & Vickery, 1985) and abortive infection systems (Dai et al., 2001). We showed that the in vitro VISA investigated demonstrated minimal to no alteration in SmaI RFLPs compared with parent strains; however, alterations in phage types were common. This latter finding is probably due to modifications of phage cell-wall receptors that result from alterations in cell-wall physiology that are expressed by the VISA strains investigated (Pfeltz et al., 2000). Collectively, these findings indicate that inappropriate relationships between VISA and vancomycin-susceptible parents might be drawn if only phage-typing is utilized to determine epidemiological relationships. This might be especially true in situations where VISA arise within a single infected patient being treated with vancomycin, as has previously been reported to occur (Rotun et al., 1999; Sieradski et al., 1999a; Smith et al., 1999). Recognizing vancomycin- susceptible parent strains may be of clinical importance, since some S. aureus strains can mutate to become VISA with greater ease compared with other strains (Pfeltz et al., 2000; Schaaff et al., 2002), and the presence of these strains may therefore indicate a unique threat.

	Acknowledgements

TOP Abstract Introduction Methods Results and Discussion Acknowledgements References

 
J. E. G. acknowledges support from NIH grant R15 AI99197–01A1 and B. J. W. acknowledges support from NIH grant R15 AI049964. W. B. G. acknowledges a grant from the Australian NHMRC.

	References

TOP Abstract Introduction Methods Results and Discussion Acknowledgements References

 

Wilkinson, B. J. & Holmes, K. M. (1979). Staphylococcus aureus cell surface: capsule as a barrier to bacteriophage adsorption. Infect Immun 23, 549–552.[Abstract/Free Full Text]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Gustafson, J. E. Articles by Wilkinson, B. J. Search for Related Content PubMed PubMed Citation Articles by Gustafson, J. E. Articles by Wilkinson, B. J. Agricola Articles by Gustafson, J. E. Articles by Wilkinson, B. J.

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