Journal of Antimicrobial Chemotherapy (2000) 45, 887-890
© 2000 The British Society for Antimicrobial Chemotherapy
Brief reports |
Retrospective screening for heterogeneous vancomycin resistance in diverse Staphylococcus aureus clones disseminated in French hospitals
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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Twenty-five different Staphylococcus aureus strains that are widespread in France were screened by various methods for heterogeneous and low-level resistance to vancomycin. Population analysis on brain–heart infusion agar containing 4 mg/L of the drug detected resistant cells at frequencies of 10–7 to 10–6 in five multiply resistant strains. There was no antagonism between vancomycin and ß-lactam antibiotics. One of the five strains, isolated in 1993, was considered a putative progenitor of a French nosocomial S. aureus strain isolated in 1998 and for which the vancomycin MIC was 8 mg/L.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Vancomycin resistance testing should be rigorous because infections due to methicillin-resistant Staphylococcus aureus (MRSA) strains resistant to other antibiotics, including teicoplanin, can be treated successfully with vancomycin.1
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.2–5 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 2–4 mg/L vancomycin but a minority of cells can grow at vancomycin concentrations of 4–9 mg/ L.2–6 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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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Bacterial strains
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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Media and antibiotics
Brain–heart 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 1–10 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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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Determination of vancomycin MICs using the modified agar dilution method allowed discrimination between the two Japanese VISA strains, Mu3 and Mu50 (MICs 4 and 5 mg/L, respectively), and the two VSSA strains ATCC25923 and FDA209P (MICs 2 and 1 mg/L, respectively). According to the internationally accepted breakpoint for vancomycin susceptibility (MIC
4 mg/L), Mu3 should be considered to be a VSSA strain. The MIC values for the 26 French strains were from 2 to 5 mg/L (Table
). Those for the 11 Ge-R MRSA strains were the highest: six exhibited MICs 
4 mg/L by agar dilution, whereas in the other categories, MICs were 3 mg/L.
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 4–6 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 = 5–8 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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Pulse-field gel electrophoresis (PFGE) of SmaI digests of total DNA demonstrated the clonal diversity of the five French hetero-VISA strains in contrast to those detected in Germany which were derivatives of the same clone.6 The clonal diversity of the French strains may be due either to independent selection of resistant variants derived from distinct parental clones or to genomic modifications occurring in a single hetero-VISA parental clone which emerged in France before 1993. However, genetic relatedness of the VISA strain 98141 to the hetero-VISA strain 93291 was demonstrated by PFGE analysis and we suggest that 98141 may be a derivative of 93291, which had been isolated 5 years earlier. The parental strain may be useful for testing the efficacy of glycopeptide therapy in animal models.
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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We thank Keiichi Hiramatsu for providing us with the Japanese VISA strains Mu3 and Mu50, Jacques-Olivier Galdbart for his critical reading of the manuscript and Catherine Tran for her secretarial assistance.
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Notes |
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* Corresponding author. Tel: +33-1-45688363; Fax: +33-1-40613163.
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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1 . Hiramatsu, K. (1998). Vancomycin resistance in staphylococci. Drug Resistance Updates 1, 135–50.
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, 1670–3.[Web of Science][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.[Web of Science][Medline]
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Received 29 September 1999; returned 23 November 1999; revised 15 December 1999; accepted 1 February 2000
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