a Institut für Medizinische Mikrobiologie und Virologie, Universität Düsseldorf, Universitätsstrasse , Geb. 22.21, D-40225 Düsseldorf; b Bayer AG, PH-Research Antiinfectives, Wuppertal; c Robert Koch Institut, Nationales Referenzzentrum für Staphylokokken, Bereich Wernigerode, Germany
Sir,
The procedure used by us to detect subpopulations of strains of methicillin-resistant Staphylococcus aureus (MRSA) capable of growing in the presence of vancomycin at a concentration of 8 mg/L was that recommended by both Hiramatsu et al.1 and the Centers for Disease Control and Prevention.2 This method is also used routinely by the German National Reference Centre for Staphylococci and has been employed by us to evaluate 5120 MRSA isolates of different genotypes, of which only the strains described in our article were identified as heterogeneous vancomycin intermediate S. aureus (hetero-VISA).3 Finally, we have screened 203 MRSA isolates from 20 European university hospitals for VISA or hetero-VISA and neither of these phenotypes was detected.4 This indicates that the screening test for VISA used by us does not select simply for a few colonies of S. aureus with reduced susceptibilities to vancomycin, as suggested by Howe et al. Furthermore, the hetero-VISA status of the isolates described by us was confirmed by three independent laboratories. Our results were therefore consistent.
Howe et al. have proposed that the Hiramatsu method of detecting VISA strains actually selects for vancomycin resistance. However, the selection of vancomycin resistance in S. aureus strains, in contrast to coagulase-negative staphylococci, is difficult to achieve, even when antibiotics are used at sub-MIC concentrations, compared with the relatively high concentration of 4 mg/L used in the Hiramatsu method. In an article cited by Howe et al.,5 the authors reported that selection was possible only when serial subcultures were performed in the presence of vancomycin at low concentrations. While the selection of resistant strains can never be completely excluded, it is very unlikely that this happened under the test conditions used in our study.
Regarding the cell wall analysis, the experiments performed involved a strain grown on a medium that did not contain vancomycin. Moreover, isolation of the cell walls was preceded by culture in the absence of this antibiotic.
A large number of clinical isolates have been investigated in our laboratory and in only one case did the cell wall contain increased amounts of non-amidated muro-peptides. In contrast, we detected increased production of these muropeptides in several, albeit not all, confirmed VISA strains referred by investigators from around the world (unpublished data) and have linked this phenotypic characteristic to enhanced binding of vancomycin to the cell wall.6 We have also evaluated the effect of exposure to vancomycin on the composition of the cell wall of various strains and failed to detect an increase in the amounts of non-amidated muropeptides (unpublished data). The concerns of Howe et al. regarding the results of the cell wall analyses are therefore unwarranted.
We agree with the suggestion of Howe et al. that, for the purpose of screening a large number of isolates, which appears to be necessary in order to detect hetero-VISA strains early and at low frequencies, the population analysis method is too time-consuming. In the absence of other suitable screening techniques, the method of Hiramatsu et al. is both simple and effective in terms of detecting hetero-VISA isolates. The status of such strains can then be confirmed by other methods, such as population analysis. In our opinion, the inoculum density used by us (c. 107 cfu/spot) and the antibiotic concentration in the test plates are comparable to those used in the population analysis method employed by Howe et al. In view of the potential public health importance of hetero-VISA and VISA strains, any suggestion that their prevalences are increasing should be taken seriously.
Notes
* Corresponding author. Tel: +49-211-811-2490; Fax: +49-211-811-5323.
References
1 . Hiramatsu, K., Aritaka, N., Hanaki, H., Kawasaki, S., Hosoda, Y., Hori, S. et al. (1997). Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet 350, 16703.[ISI][Medline]
2 . Centers for Disease Control. (1997). Update: Staphylococcus aureus with reduced susceptibility to vancomycinUnited States. Journal of the American Medical Association 278, 11456.[ISI][Medline]
3
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Geisel, R., Schmitz, F.-J., Thomas, L., Berns, G., Zetsche, O., Ulrich, B. et al. (1999). Emergence of heterogeneous intermediate vancomycin resistance in Staphylococcus aureus isolates in the Düsseldorf area. Journal of Antimicrobial Chemotherapy 43, 8468.
4 . Schmitz, F.-J., Krey, A., Geisel, R., Verhoef, J., Heinz, H. P. & Fluit, A. C. (1999). Susceptibility of 302 methicillin-resistant Staphylo-coccus aureus isolates from 20 European university hospitals to vancomycin and alternative antistaphylococcal compounds. European Journal of Clinical Microbiology and Infectious Diseases 18, 52830.[ISI][Medline]
5 . 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]
6 . Hanaki, H., Labischinski, H., Inaba, Y., Kondo, N., Murakami, H. & Hiramatsu, K. (1998). Increase in glutamine-non-amidated muropeptides in the peptidoglycan of vancomycin-resistant Staphylococcus aureus strain Mu50. Journal of Antimicrobial Chemotherapy 42, 31520.[Abstract]