Characteristics of French methicillin-resistant Staphylococcus aureus isolates with decreased susceptibility or resistance to glycopeptides

Névine El Solh1,*, Marilyne Davi1, Anne Morvan1, Hélène Aubry Damon2, Nicole Marty3 and the other members of the GISA group, RAISIN subgroup§

1 Unité des staphylocoques, Institut Pasteur, 75724 Paris Cedex; 2 Département des maladies infectieuses, Institut de veille sanitaire, 94415 Saint-Maurice Cedex; 3 Laboratoire de Bactériologie–Virologie–Hygiène, CHU Rangueil, 31403 Toulouse Cedex 4, France

Received 31 March 2003; returned 18 May 2003; revised 27 June 2003; accepted 2 July 2003


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
According to the French Society of Microbiology, Staphylococcus aureus isolates are suspected to have decreased susceptibility to glycopeptide(s) when at least one colony is able to grow from an inoculum of 10 µL of 2 McFarland bacterial suspension plated on Mueller–Hinton agar containing 5 mg/L teicoplanin and incubated for 48 h at 35–37°C. We analysed 89 methicillin-resistant S. aureus isolates (MRSA), collected in 2000–2001 from 24 hospitals located in 18 French cities, which were able to grow on this selective medium. These isolates were distributed into six groups on the basis of their glycopeptide resistance phenotypes: (A) glycopeptide susceptible (GSSA, 21 isolates); (B) heterogeneous teicoplanin intermediately resistant (hetero-TISA, 24 isolates); (C) heterogeneous and intermediately resistant to both glycopeptides, teicoplanin and vancomycin (hetero-GISA, six isolates); (D) heterogeneous vancomycin intermediately resistant/teicoplanin intermediately resistant (hetero-VISA/TISA, 30 isolates); (E) GISA (four isolates); (F) TISA (four isolates). Despite the persistent decrease in gentamicin-resistant MRSA isolates in French hospitals since 1993, their prevalence is very high in groups D, E and F. Moreover, most of the group C, D and E isolates exhibiting decreased susceptibility to both glycopeptides belong to the same major SmaI genotype, which has been detected in Europe since at least 1989.

Keywords: MRSA, S. aureus, teicoplanin, vancomycin


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The first Staphylococcus aureus isolates with reduced susceptibility to vancomycin (VISA and hetero-VISA) were described by Hiramatsu.1 Polyclonal and epidemic MRSA isolates exhibiting this phenotype were also detected in additional countries.2 Whereas VISA isolates appear to be difficult to eradicate, there are conflicting data about the effectiveness of vancomycin therapy for hetero-VISA isolates, detection of which is difficult to standardize.2 The first case of a patient infected with a glycopeptide-resistant S. aureus isolate carrying the vanA gene was reported in 2002.3

The French Microbiology Society (SFM) has proposed a screening test to detect S. aureus isolates suspected to have decreased susceptibility to glycopeptide(s). Such isolates were defined when at least one colony is able to grow on Mueller–Hinton agar (MHA) + 5 mg/L teicoplanin inoculated with 10 µL of 2 McFarland bacterial suspension and incubated for 48 h at 35–37°C. An epidemiological survey was carried out in 2000–2001 in 45 French hospitals. During 1 month, the first MRSA isolate from each clinical sample was investigated for its ability to grow on this screening medium. Of 2066 isolates tested, 248 (12%) were able to grow on this medium. MIC determinations on MHA with 0.5 McFarland bacterial suspensions revealed that 45 of the screened isolates were teicoplanin-intermediate or -resistant (2.2%); three isolates were also intermediately resistant to vancomycin with MICs >4 mg/L (0.2%).4 The aim of this study was to type and determine the glycopeptide resistance phenotypes of 89 of the 248 isolates growing on the teicoplanin screening media.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial isolates

We included in this study 89 MRSA clinical isolates that grew on the teicoplanin screening media and were sent to the Unité des staphylocoques, Institut Pasteur. These isolates were isolated in 2000–2001 from 89 patients in 24 hospitals located in 18 French cities. The three S. aureus isolates used as controls (Table 1) were: ATCC 25923 (susceptible to glycopeptides), the hetero-VISA MRSA isolate Mu3,1 and the VISA MRSA isolate 98141 (CIP106757).5


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Table 1. Distribution of the 89 MRSA isolates selected on MHA + 5 mg/L teicoplanin according to their phenotypes of resistance or susceptibility to vancomycin and teicoplanin
 
Antimicrobial susceptibility testing

Vancomycin and teicoplanin MICs were determined by two Etest protocols (AB Biodisk, Solna, Sweden). In one protocol,5 the isolates removed from storage at –80°C were streaked on to Brain Heart Infusion agar (BHIA, Difco Laboratories, Detroit, MI, USA) plates, and incubated under aerobic conditions at 37°C for 24 h. Ten colonies of each isolate were transferred into tubes containing 10 mL BHI broth and incubated at 37°C for 24 h. The Etest strips were deposited on to the BHIA surface, which had been overlaid with cell suspensions calibrated as 2 McFarland. In the second protocol, BHIA and BHI broth were replaced by Mueller–Hinton (MH, Bio-Rad, Hercules, CA, USA) agar or broth and the Etest strips were deposited on the MHA plates inoculated with swabs dipped in bacterial suspensions diluted with MH broth to achieve a turbidity of 0.5 McFarland. The plates were incubated for 48 h at 37°C. The MIC breakpoints proposed by the SFM were used: <=4 mg/L for susceptible isolates, >16 mg/L for resistant isolates, and 8–16 mg/L for intermediate isolates.

Colony counting was carried out by spreading 108 cfu across the surface of BHIA plates containing either 4 mg/L vancomycin or 8 mg/L teicoplanin. The number of colonies that grew on these media was evaluated after 24 and 48 h incubation at 37°C. All the results reported here were obtained using the same batch of BHI or MH powder and the same media preparation. When no more than 8 cfu (maximal value for control ATCC 25923) were detected after 48 h incubation on BHIA + 4 mg/L vancomycin, the isolate was considered to be susceptible to this glycopeptide. We considered hetero-VISA to be isolates that yielded growth ranging from at least 25 cfu to sub-confluent growth (~103–104 cfu). When confluent growth was observed, heterogeneity was not detectable and, since for these isolates vancomycin MICs never exceeded 16 mg/L, the isolates were considered to be VISA. The colony counts were determined twice with independent cultures stored at –80°C for all isolates that yielded >8 and <25 cfu. The three isolates giving 10 and 11 cfu yielded 7–9 cfu in the further experiments whereas those giving at least 14 cfu yielded 21–29 cfu. Therefore, when no more than 11 cfu were detected, the isolate was considered to be susceptible to vancomycin.

Pulsed-field gel electrophoresis (PFGE)

Macrorestriction with SmaI and PFGE analysis were carried out. The SmaI fragments of the cellular DNA from S. aureus NCTC 8325 were used as size standards. Macrorestriction fingerprints were scanned with the GelCompar II software (Applied Maths, Sint-Martens-Latem, Belgium). A similarity matrix was created by use of the band-based Dice similarity coefficient (tolerance 1% and optimization 2%). When dendrograms revealed clusters that included isolates with similarity of at least 80%, the patterns of the isolates in the same cluster were compared visually on the same gel. The isolates were clustered according to the criteria proposed by Tenover et al.6 Major genotypes were designated by Roman numerals. Isolates with undistinguishable patterns were classified within the same subtype. Subtypes were designated by Roman numerals with suffixes in Arabic numerals.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Distribution of the 89 MRSA clinical isolates on the basis of their glycopeptide resistance phenotypes

The 89 MRSA were distributed into six groups (A–F) based upon their growth on BHIA plates containing either 4 mg/L vancomycin or 8 mg/L teicoplanin (Table 1). The 21 MRSA isolates associated with 0 to 10 cfu on BHIA + 8 mg/L of teicoplanin were placed into group A, considered susceptible, and were clearly distinct from those giving growth between 20 cfu and sub-confluent growth. The group B and C isolates with 20 cfu to subconfluent growth were considered to be heterogeneously and intermediately resistant to teicoplanin with MICs <=16 mg/L (hetero-TISA). Heterogeneity was not detectable for group D, E and F isolates giving confluent growth. These isolates were considered to be teicoplanin-intermediate or -resistant, depending on the MICs.

Antibiotic susceptibilities of isolates in groups A–F

Group A isolates were distinguishable from the other isolates by the absence, or the low frequency, of resistance to gentamicin, streptomycin, tetracycline, minocycline and rifampicin (P < 0.001, {chi}2 test); fusidic acid (P = 0.01); and fosfomycin (P = 0.04). Group B + F isolates could be distinguished from group C + D + E isolates by a lower occurrence of resistance to gentamicin, streptomycin and fusidic acid.

The highest prevalence of gentamicin resistance was detected among the isolates belonging to groups D, E and F (36/38 isolates). Lower prevalences were observed among the group B and C isolates (9/30 isolates) and isolates of group A (1/21 isolates).

SmaI genotypes of the group A–F isolates (data not shown)

Forty-four PFGE patterns were detected among the 89 MRSA isolates tested. Twenty-nine isolates distributed between the groups A–F, and isolated in 12 distinct hospitals, had the same pattern, I1, as that of the VISA control isolate, 98141. The I1 subtype included gentamicin-resistant and -susceptible isolates (24 and five isolates, respectively). Pattern I1 was predominant among isolates exhibiting decreased susceptibility to both vancomycin and teicoplanin (18/40 isolates); the prevalence of this pattern was significantly lower among isolates with decreased susceptibility or resistance only to teicoplanin (8/28 isolates), or among group A isolates that were susceptible to both glycopeptides (2/21 isolates) (P = 0.0033).

Fifteen PFGE patterns (I2–I16) differed from sub-type I1 by no more than three SmaI fragments. The 47 isolates belonging to the I major genotype were isolated in 19 hospitals and included 37 gentamicin-resistant isolates. The SmaI patterns of 17 other isolates were distributed into four major genotypes (V*, VI*, VII* and IX*) and differed from pattern I1 by four to six bands; therefore these isolates were considered to be ‘possibly-related’. Twenty-five other isolates, distributed into 13 major genotypes, were considered different from I1 isolates.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The use of the teicoplanin screening media proposed by the SFM enabled not only the detection of teicoplanin-intermediate or -resistant isolates (38 of the 89 tested isolates), but also of heterogeneous and intermediate resistance to teicoplanin (hetero-TISA: 30 isolates). However, only 40 of the 89 MRSA isolates screened exhibited decreased susceptibility to vancomycin, and 21 isolates were susceptible to both glycopeptides.

Despite the persistent decrease in gentamicin-resistant MRSA isolates observed in French hospitals since 1993,7 their prevalence was very high among isolates belonging to groups D, E and F. The occurrence of decreased susceptibility to glycopeptides among gentamicin-resistant MRSA isolates resistant to several other antibiotics is concerning from the perspective of therapy failure.

The SmaI pattern I1 of French vancomycin-intermediate, gentamicin-resistant MRSA isolate, 98141, was shown to be identical to the SmaI pattern of a hetero-vancomycin-intermediate, gentamicin-resistant MRSA, 93291, isolated in a French Hospital in 1993.8 This pattern was also identical to the predominant pattern found among the epidemic hetero-vancomycin-intermediate, gentamicin-resistant MRSA spread in a Parisian hospital9 and among those isolated in French hospitals and sent to our laboratory since 1998 (N. El Solh, data not shown). In this study, pattern I1 isolates and related isolates belonging to major genotype I were predominant among the MRSA isolates exhibiting decreased susceptibility to both glycopeptides found in 19 hospitals. It has already been reported that VISA and hetero-VISA isolates belong to a restricted range of epidemic or endemic MRSA genotypes.10


    Acknowledgements
 
We thank Iain Old for his help in reviewing this article and Beatrice Gallais for secretarial help. The GISA group, RAISIN subgroup (Réseau d’Alerte, d’Investigation et de Surveillance des Infections Nosocomiales) participants are: Vincent Jarlier, Anne Carbonne, Odile Bajolet-Laudinat, Daniel Talon, Alain Ros, Nathalie van der Mee Marquet, Roland Leclerq, and Jean Claude Désenclos.


    Footnotes
 
* Corresponding author. Tel: +33-1-45688363; Fax: +33-1-40613163; E-mail: nelsolh{at}pasteur.fr Back

§ GISA group, RAISIN subgroup participants are listed in the Acknowledgements. Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Hiramatsu, K. (1998). Vancomycin resistance in staphylococci. Drug Resistance Update 1, 135–50.[ISI]

2 . Walsh, T. R. & Howe, R. A. (2002). The prevalence and mechanisms of vancomycin resistance in Staphylococcus aureus. Annual Review of Microbiology 56, 657–75.[CrossRef][ISI][Medline]

3 . Anon. (2002). First U.S. case of Vancomycin-Resistant Staphylococcus aureus infection reported; patient has chronic renal failure. Dialysis and Transplantation September, 602–3.

4 . Aubry-Damon, H., Marty, A. C. N., Bajolet-Laudinat, O. et al. (2002). Le point sur la situation épidémiologique actuelle de Staphylococcus aureus de sensibilité diminuée aux glycopeptides (vancomycine et teicoplanine) en France. Hygiène S. 10, 324–9.

5 . Chesneau, O., Morvan, A. & El Solh, N. (2000). Retrospective screening for heterogeneous vancomycin resistance in diverse Staphylococcus aureus clones disseminated in French hospitals. Journal of Antimicrobial Chemotherapy 45, 887–90.[Abstract/Free Full Text]

6 . Tenover, F. C., Arbeit, R. D., Goering, R. V. et al. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. Journal of Clinical Microbiology 33, 2233–9.[Free Full Text]

7 . Galdbart, J.-O., Morvan, A. & El Solh, N. (2000). Phenotypic and molecular typing of nosocomial methicillin-resistant Staphylococcus aureus strains susceptible to gentamicin isolated in France from 1995 to 1997. Journal of Clinical Microbiology 38, 185–90.[Abstract/Free Full Text]

8 . Morvan, A., Aubert, S., Godard, C. et al. (1997). Contribution of a typing method based on IS256-probing of SmaI-digested cellular DNA to discrimination of European phage-type 77 methicillin-resistant Staphylococcus aureus strains. Journal of Clinical Microbiology 35, 1415–23.[Abstract]

9 . Guerin, F., Buu-Hoi, A., Mainardi, J. L. et al. (2000). Outbreak of methicillin-resistant Staphylococcus aureus with reduced susceptibility to glycopeptides in a Parisian hospital. Journal of Clinical Microbiology 38, 2985–8.[Abstract/Free Full Text]

10 . Denis, O., Nonhoff, C., Baudoin, B. et al. (2002). Emergence of vancomycin-intermediate Staphylococcus aureus in a Belgian hospital: microbiological and clinical features. Journal of Antimicrobial Chemotherapy 50, 383–91.[Abstract/Free Full Text]