French National Reference Centre for Staphylococci, Unité des Staphylocoques, Institut Pasteur, 28, rue du Docteur Roux, 75724 Paris Cedex 15, France
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Abstract |
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Introduction |
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Therapeutic failures with vancomycin, either in patients or in animal models, have been reported whenever the vancomycin MICs for the strains were > 4 mg/L.25 These strains have been designated vancomycin-intermediate S. aureus (VISA). In contrast, there are conflicting data about the effectiveness of vancomycin therapy for strains showing vancomycin resistance where most of the cell population is inhibited by 24 mg/L vancomycin but a minority of cells can grow at vancomycin concentrations of 49 mg/ L.26 These strains, named hetero-VISA, are a potential source of VISA subclones in patients receiving prolonged vancomycin administration. The differentiation of such strains from fully susceptible ones (VSSA) is an issue of great concern for infection control practice.
Hetero-VISA strains are not detected either by disc diffusion or by standard MIC methods. Alternative methods have therefore been proposed.2,7 We report in this study the use of four non-conventional methods to screen for hetero-VISA strains among nosocomial S. aureus strains representative of those that are known to have spread in French hospitals.
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Materials and methods |
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Twenty-six S. aureus strains from 11 hospitals in nine cities dispersed throughout France were studied (Table). Among them, only one, 98141, from a patient who had never been treated with glycopeptides, was sent to the French National Reference Centre for vancomycin susceptibility testing. The 25 other strains were chosen, without any information about their vancomycin susceptibility, as representative of strains prevalent in France between 1987 and 1997. Comparative analysis of the SmaI restriction patterns indicated that these strains comprise diverse clones.8 Antibiotyping classified the 26 strains into three main categories: 11 gentamicin-resistant (Ge-R) MRSA strains, 10 gentamicin-susceptible (Ge-S) MRSA strains and five methicillinsusceptible S. aureus (MSSA) strains. Control strains included the two Japanese strains Mu3 (hetero-VISA) and Mu50 (VISA) and two reference strains, ATCC25923 and FDA209P.
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Brainheart infusion (BHI) medium (Difco Laboratories, Detroit, MI, USA) was used both for growth of the staphylococci and for vancomycin susceptibility testing. Mu3 agar plates which consist of BHI agar (BHIA) supplemented with 4 mg/L vancomycin and specific amino acids, were supplied by Becton Dickinson (Tokyo, Japan). Etest strips came from AB Biodisk (Solna, Sweden). Vancomycin powder was kindly provided by Lilly France (Saint-Cloud, France). ß-Lactam discs were obtained from Sanofi Diagnostics Pasteur (Marnes-la-Coquette, France).
Vancomycin susceptibility tests
Staphylococcal strains were removed from storage, streaked on to BHIA plates, and incubated under aerobic conditions at 37°C for 16 h. Ten colonies of each strain were transferred into tubes containing 10 mL BHI and incubated at 37°C for 16 h. Vancomycin susceptibility was tested by various methods. The MIC of vancomycin was determined using the agar dilution method with 106 cfu/spot and 1 mg/L increments of the antibiotic in the range 110 mg/L. Population analysis was carried out by spreading 108 cfu across the surface of BHIA plates containing 4 mg/L vancomycin. The Etest strips were deposited on to the BHIA surface which had been overlaid with cell suspensions calibrated as 2 McFarland (c. 5 x 108 cfu/mL). The Mu3 agar plates were inoculated with cell suspensions calibrated as 1 McFarland (c. 108 cfu/mL). For all methods, all plates were incubated for 36 h at 37°C.
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Results and discussion |
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For 27 of the 30 strains tested, the Etest MICs were slightly higher than those obtained by agar dilution (Table). When evaluated by the Etest, MICs for Mu3 and Mu50 were the same. This result may be attributed to the detection in Mu3 of a few isolated colonies inside the ellipse of growth inhibition around 46 mg/L vancomycin. According to the supplier's guidelines, the Etest reading must take into consideration any isolated colonies growing near the strip. These criteria resulted in eight Ge-R MRSA strains being regarded as possible VISA or hetero-VISA (MICs = 58 mg/L).
The number of colonies that grew on in-house-prepared BHIA plates containing 4 mg/L vancomycin was evaluated in five independent experiments for each strain. Mu3 and Mu50 yielded between 30 and 1000 colonies, with Mu50 showing high variation (Table). We suspect that subculturing Mu50 without vancomycin since its first isolation may have resulted in a decline in resistance. Of the eight strains whose vancomycin MICs were > 4 mg/L by Etest, six yielded more than 25 colonies by population analysis (Table
). The other strains did not yield more than two colonies. In view of these results, we considered therefore only six Ge-R MRSA strains as possible VISA or hetero-VISA strains. Their teicoplanin MICs were all equal to or greater than 16 mg/L.
The use of commercial Mu3 agar plates containing 4 mg/L vancomycin was recently proposed for the detection of hetero-VISA strains.1 The ability to detect hetero-VISA strains is based on the antagonism of vancomycin and diverse ß-lactams. We used discs containing 10 µg cefixime and 30 µg aztreonam (Figure). In these tests, Mu50 produced confluent growth across the whole surface of the plates while Mu3 produced a typical passage from isolated colonies to confluent growth around the discs containing each of the two ß-lactams (hetero-VISA). Fewer than 10 colonies grew on each plate for the two VSSA strains and for all but six of the 26 French S. aureus strains. The growth aspects of the six Ge-R MRSA strains previously screened by population analysis were various: only one, 98141, had the same behaviour as Mu50; the other five were similar to Mu3 in terms of the number of colonies per plate. However, in contrast to Mu3, no increased growth was observed around the two ß-lactam discs for these five strains (Figure
). There was therefore no detectable antagonism between vancomycin and ß-lactams for the French hetero-VISA strains. This characteristic differentiated them from the hetero-VISA strains isolated recently in Japan and in the United Kingdom.9
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Screening for hetero-VISA strains is essential to evaluate their incidence and the frequency of selection of VISA subclones following vancomycin administration. Population analysis on BHIA containing 4 mg/L vancomycin seems the most appropriate technique for detecting such strains. Repeated subcultures without vancomycin may result in a decrease in the proportion of VISA cells growing at 4 mg/L vancomycin. Nevertheless, we demonstrated that the number of colonies remains significantly higher than those detected for VSSA strains.
Prospective surveillance of vancomycin susceptibility levels in MRSA strains should be conducted in all European countries. Hetero-VISA strains are already present in the United Kingdom, Greece, Germany and France.6,9,10 The German hetero-VISA strains emerged in 1998 and all of them had identical SmaI patterns. In France, where they have been present since 1993, five distinct clones have been detected. Screening for heterogeneous and low-level resistance to vancomycin in MRSA strains will soon become a necessary part of infection control practices when glycopeptides are used.
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Acknowledgments |
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Notes |
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References |
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2 . 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]
3 . Ploy, M. C., Grélaud, C., Martin, C., de Lumley, L. & Denis, F. (1998). First clinical isolate of vancomycin-intermediate Staphylococcus aureus in a French hospital. Lancet 351, 1212.[ISI][Medline]
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Received 29 September 1999; returned 23 November 1999; revised 15 December 1999; accepted 1 February 2000