a Bristol Centre for Antimicrobial Research and Evaluation, Southmead Health Services NHS Trust and University of Bristol, Department of Microbiology, Southmead Hospital, Bristol BS10 5NB b Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK
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Abstract |
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Introduction |
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A second type of vancomycin resistance which appears to be much more common than that described above has also been reported from Japan. Hiramatsu and colleagues have described strains of MRSA that, while they have an MIC below the breakpoint and are thus defined as sensitive by conventional testing, contain bacterial cells within their population that can grow in the presence of 49 mg/L of vancomycin.6 This heterogeneity in vancomycin susceptibility is similar to that seen in MRSA where cells expressing methicillin resistance may only constitute one-millionth of the total population of cells. Hetero-vancomycin-resistant S. aureus (hVRSA) have been detected in 9.3% of MRSA isolated from a group of university hospitals and 1.3% isolated from non-university hospitals in Japan.6 We have reported a strain of EMRSA-15 with similar characteristics to the Japanese hVRSA, which was isolated in Bristol from a patient who died of S. aureus septicaemia despite 22 days of vancomycin therapy.7
It has been suggested that hetero-resistance has been responsible for failure of vancomycin therapy. However, before the important questions regarding the prevalence and clinical relevance of hVRSA can be answered, a reliable method for their detection must be established. In this study we have tested the reliability of the method described by Hiramatsu et al.6 and investigated various modifications.
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Materials and methods |
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To establish the reproducibility of the method described by Hiramatsu et al.,1 the 100 MRSA strains, the VRSA and hVRSA strains and NCTC 6571 were tested on five separate occasions. After a 24 h incubation in tryptone soy broth (TSB) (CM129, Oxoid, Basingstoke, UK), each culture was adjusted to a turbidity equal to McFarland 0.5, and 10µL was spotted on to brain heart infusion (BHI) agar (CM225, Oxoid, Basingstoke, UK) incorporating 4 mg/L of vancomycin (Lilly, Basingstoke, UK). Growth was recorded following 24 and 48 h incubation at 37°C. Colonies growing at 48 h were subcultured on to BHI agar containing 8 mg/L of vancomycin. hVRSA status was confirmed if a strain produced a sub-clone with a vancomycin MIC of 8 mg/L which remained stably resistant for more than 9 days on drug-free medium. Any strains which grew on initial screening plates were subjected to this procedure.
To test the ability of S. aureus to express vancomycin resistance, a heavy inoculum of 20 isolates of MRSA, 10 isolates of MSSA and the control strain were subcultured on to BHI agar containing 32 mg/L of vancomycin.
The effect of pre-incubation with vancomycin on the expression of vancomycin resistance by VRSA and hVRSA was studied. Isolates were incubated overnight in TSB with or without 4 mg/L vancomycin and a 10µL aliquot of cells was inoculated on to agar plates containing a range of vancomycin concentrations.
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Results |
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Colonies of all clinical isolates tested (both MRSA and MSSA) grew after 24 h on BHI agar containing 32 mg/L vancomycin when a heavy inoculum was used. There was no growth of the control strain.
After pre-incubation of hVRSA and VRSA strains with vancomycin there was an increase in the expression of vancomycin resistance and colonies grew in the presence of 16 mg/L vancomycin (Table).
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Discussion |
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Although resistance to vancomycin in clinical isolates of S. aureus is new, the in-vitro selection of low-level resistance (e.g. MIC 16 mg/L) was reported as early as 1956.8 The method proposed for detection of hVRSA by Hiramatsu et al. is similar to the method used by Daum et al. to select S. aureus strains resistant to vancomycin.9 It remains unclear whether this method detects vancomycin resistance or selects for it.
The mechanism of vancomycin resistance in S. aureus remains unclear. Resistant strains have been shown to have thickened cell walls and it has been proposed that adsorption of vancomycin by the cell wall may be partly responsible for resistance. However, the genetic basis for this has not been elucidated. It remains possible that the ability to produce a thickened cell wall is a phenotypic adaptation to particular environmental circumstances which can be expressed by many (or most) strains of S. aureus. Our results suggest that colonies that will grow in the presence of 1632 mg/L of vancomycin are readily selected from most S. aureus strains by use of a heavy inoculum or prior vancomycin exposure. Resistance to vancomycin in these strains is not generally stable and thus they continue to test as sensitive using conventional methods. However, transmission electron microscopy of these strains shows that when grown in the presence of vancomycin they have a thickened cell wall similar to that described in VRSA.9 The fact that 30 strains of S. aureus grew on agar containing 32 mg/L of vancomycin when a heavy inoculum was used would tend to support the hypothesis that vancomycin is adsorbed by bacterial cells. Laboratory strains have been shown to remove vancomycin from culture medium. Sieradski and Tomasz, using a S. aureus mutant (VM) with a vancomycin MIC of 100 mg/L showed that cell wall suspensions of this strain could bind 500µg of vancomycin per mg cell wall in contrast to 235µg bound per mg of the parental cell wall.10 Thus it has been suggested that un-crosslinked D-alanyl-D-alanine of the thickened cell walls of VRSA may be binding sites which adsorb vancomycin before it is able to reach its target.
Studies have suggested that vancomycin is less effective than ß-lactam agents such as flucloxacillin in treating S. aureus infections. It is possible that therapeutic failures may be attributable to an unstable ability of sub-populations to survive in high vancomycin concentrations in vivo. Whether the method described in Japan detects or selects for vancomycin resistance, the ease with which colonies can be grown in the presence of vancomycin concentrations greater than the MIC suggests that these findings may have clinical relevance for treatment of serious MRSA infections. While the genetic basis for heterogeneous vancomycin resistance remains unknown, it remains possible that all strains of S. aureus have an ability to express unstable low-level resistance. The hVRSA reported from Japan may just represent one end of a spectrum.
Until hVRSA are better understood, and a reliable method discovered that will enable detection of those strains which are most likely to express resistance, it may be wise to use vancomcyin in combination with a second agent for serious MRSA infections.
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Notes |
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References |
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2 . British Society for Antimicrobial Chemotherapy. (1991). A Guide to Sensitivity Testing. Report of the Working Party on Antibiotic Sensitivity Testing of the British Society for Antimicrobial Chemotherapy. Academic Press, London.
3 . National Committee for Clinical Laboratory Standards. (1997). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow AerobicallyFourth Edition: Approved Standard M7-A4. NCCLS, Wayne, PA.
4 . Centers for Disease Control and Prevention. (1997). Staphylococcus aureus with reduced susceptibility to vancomycinUnited States. Morbidity and Mortality Weekly Report 46, 7656.[Medline]
5 . Ploy, M. C., Grelaud, C., Martin, C., de Lumley, L. & Denis, F. (1998). First clinical isolate of vancomycin-intermediate Staphylococcus aureusin a French hospital. Lancet 351,1212 .[ISI][Medline]
6 . Hiramatsu, K., Aritaka, N., Hanaki, H., Kawasaki, S., Hosoda, Y. & Hori, S. (1997). Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet 350, 16703.[ISI][Medline]
7 . Howe, R. A., Bowker, K. E., Walsh, T. R., Feest, T. G. & MacGowan, A. P. (1998) Vancomycin resistant Staphylococcus aureus. Lancet 351, 602.[ISI][Medline]
8 . Garrod, L. P. & Waterworth, P. M. (1956). Behaviour in vitro of some new antistaphylococcal antibiotics. British Medical Journal ii, 615.
9 . Daum, R. S., Gupta, S., Sabbagh, R. & Milewski, W. M. (1992). Characterization of Staphylococcus aureus isolates with decreased susceptibility to vancomycin and teicoplanin: isolation and purification of a constitutively produced protein associated with decreased susceptibility. Journal of Infectious Diseases 166, 106672.[ISI][Medline]
10 . Sieradzki, K. & Tomasz, A. (1997) Inhibition of cell wall turnover and autolysis by vancomycin in a highly vancomycin-resistant mutant of Staphylococcus aureus. Journal of Bacteriology 179, 255766.[Abstract]
Received 10 February 1999; returned 16 April 1999; revised 2 June 1999; accepted 24 June 1999