1 Centre Hospitalier A. Mignot, Versailles; 2 Institut Pasteur, Paris; 3 Collège de Bactériologie-Virologie Hygiène des Hôpitaux, France
Received 22 November 2002; returned 21 January 2003; revised 30 January 2003; accepted 9 February 2003
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Abstract |
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Keywords: bloodstream infection, antimicrobial susceptibility, VRSA, Streptococcus pneumoniae, France
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Introduction |
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Materials and methods |
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The Collège de Bactériologie-Virologie Hygiène des Hôpitaux (ColBVH study group) is a broad network of 105 non- teaching sentinel hospitals equally geographically distributed and representative of general hospitals in France. General data about size and activities of participating hospitals are listed in Table . One of the aims of the network is to monitor the predominant pathogens responsible for blood infections diagnosed in hospitals and their antimicrobial resistance patterns. All non-duplicate bloodstream strains isolated during a 1 month period (9 October 2000 to 4 November 2000) were sent to a central laboratory (Centre Hospitalier de Versailles). Only blood culture isolates judged to be clinically relevant by national guidelines which refer to the recommendation of Centers for Disease Control and Prevention were included.4 No patient age limitations were imposed.
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Blood culture isolates were identified at the participating institutions by the local routine methodology. Upon receipt at the central laboratory, species identification was carried out (detection of IS431 and femA genes by PCR for S. aureus, optochin susceptibility test for S. pneumoniae).5
Antimicrobial susceptibility testing
Data obtained by disc susceptibility tests according to the NCCLS guidelines and by the Etest method (AB Biodisk, Sweden) were collected.6,7 ESBL production was detected by means of double-disc synergy tests and phenotypic confirmatory tests as recommended by the NCCLS.7,8 At the central laboratory, methicillin resistance was confirmed by detection of the mecA gene and the MICs of various antibiotics for S. aureus and S. pneumoniae were determined by agar dilution according to NCCLS guidelines.5,9 The antibiotics were obtained from the manufacturers: penicillin G (Sigma, France), amoxicillin (GlaxoSmithKline, France), cefuroxime (GlaxoSmithKline, France), ceftriaxone (Roche, France), erythromycin (Abbott, France), ciprofloxacin (Bayer, France), gatifloxacin (Grünenthal, France), gentamicin (Schering-Plough, France), quinupristin/dalfopristin (Aventis, France), teicoplanin (Aventis, France) and vancomycin (Lilly, France). A screening test to detect glycopeptide reduced-susceptibility in methicillin-resistant S. aureus (MRSA) was carried out as follows: plates containing MuellerHinton agar supplemented with 5 µg/L teicoplanin or vancomycin were inoculated with 10 µL of bacterial suspension at approximately 108 cfu/mL. Isolates growing on the plates were studied further by population analysis.10,11 Quality control was carried out by testing: Escherichia coli ATCC 25922 and U2A 1298, S. aureus ATCC 25923, CIP6525 and glycopeptide-resistant strains Mu3 and Mu50, S. pneumoniae BM4203R3, BM4203R11 and U2A1449, Haemophilus influenzae ATCC 49766. The interpretative criteria were those of the NCCLS.9
Clonality of strains
The clonal relationship was studied by pulsed-field gel electrophoresis (PFGE) for MRSA strains and serotype determination by the Quellung reaction using type-specific rabbit antisera from the Statens serum Institut, Copenhagen, Denmark for pneumococci.12 Cross-reactivity of serotypes within the same serogroup was assumed in the analysis of the theoretical coverage by the vaccines.
Sequence determination of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC and parE
Portions of genes from pneumococcal strains highly resistant to fluoroquinolones (MIC of gatifloxacin 2 mg/L) were amplified by PCR as previously described.13 Independently obtained amplification products were purified with the QIAquick PCR purification kit (Qiagen) and sequenced on both strands by ABI Prism d-Rhodamine terminator cycle sequencing and an ABI 310 automated sequencer (Perkin-Elmer, Applied Biosystems, USA). Sequence analysis was carried out using BLAST software (www.ncbi.nlm.nih.gov).
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Results |
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The results of susceptibility testing and MIC determination for Enterobacteriaceae recovered from blood cultures are summarized in Table . Gentamicin, cefotaxime, ciprofloxacin and gatifloxacin susceptible strains represented 95% of the isolates. Ten isolates produced an extended spectrum ß-lactamase: Citrobacter freundii (n = 1), Enterobacter aerogenes (n = 4), Enterobacter cloacae (n = 1), Escherichia coli (n = 3) and Klebsiella pneumoniae (n = 1).
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Discussion |
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In this study, the distribution of bacterial species showed similarities with previous reports: the top three bacterial species were E. coli, S. aureus and coagulase-negative staphylococci.15,16,18 However, the ranking of S. pneumoniae seems to be higher in France than in other countries. S. pneumoniae was the fourth bacterial species isolated from blood cultures representing 7.7% of bloodstream strains whereas it was ranked eleventh (2.3% of blood cultures isolates) in the European SENTRY study and seventh (4.8%) in the USA, Canada and Latin America by the same network.15,16 This difference is surprising since the incidence of invasive pneumococcal diseases in young children is higher in North America than in western Europe.19 However, seasonal variation with a high incidence of S. pneumoniae bacteraemia during the winter months during the study period (OctoberNovember) could have artificially increased the frequency of pneumococcal bacteraemia.20 In contrast, and as already reported by several European networks, the ranking of enterococci was lower in this study than in North American reports.15,16
Coagulase-negative staphylococci ranked third, as in other European or American studies, however their role in bacteraemia remains controversial.15,16,21
Among S. aureus, the incidence of resistance to methicillin was 36%, similar to that observed by the Centers for Disease Control and a British network (34%) but higher than previous results from other European countries (23.8%).15,16,22 The MRSA were susceptible to gentamicin (88.5%), in contrast to 22.8% reported in Europe, but were resistant to fluoroquinolones as reported previously.22 Glycopeptides are the main antimicrobial agents for therapy of infections due to MRSA and emergence and spread of VRSA are thus of great concern; the vanA carrying strains have been recently detected.23,24 To the best of our knowledge, this work is the first national French prospective study that investigates glycopeptide susceptibility of bloodstream isolates of MRSA using a well-established screening test and confirming methods. The frequency of hetero-VRSA strains (8%) was higher than that reported in the UK and Wales (0%) but very similar to that in Germany (8.2%).25,26 Gentamicin-resistant strains appeared to be particularly common in French VRSA. The high percentage of VRSA susceptible to quinupristin/dalfopristin was also reported in a recent worldwide study.27
In the few outbreaks of VRSA reported in French hospitals, a low number of clones were implicated.28,29 However, the only national retrospective study carried out demonstrated clonal diversity in the French VRSA strains.30 In our work, a limited number of MRSA and VRSA clones seemed to have spread throughout the country. It will be of interest to follow prospectively the evolution of these clones. Moreover, comparison of the phenotypic and genotypic traits of the French clones with those of isolates in other neighbouring countries should provide indications of the extent of VRSA spread.
Lower respiratory tract infections are a major cause of morbidity and mortality. Among the bacterial species implicated, S. pneumoniae is the most life threatening in particular for patients with bacteraemic pneumonia.31 In this study, there was a high diversity of serotypes but the clinical importance of serotypes 1 (8.8%), 4 (6.9%) and 24-F (5.9%) has not been previously stressed in France.32 Type 1 serotype has a high capacity of spread. In Denmark, it caused 26.5% of all invasive pneumococcal infections in 1996 and in a recent study accounted for 50% of the cases of pneumococcal empyema in children, one of the most serious complications of pneumonia.33,34 The 7-valent conjugated vaccine covers 55.9% of the serotypes isolated in our study. The future 9-valent vaccine that includes serotypes 1 and 5 would cover 64.7% of the isolates and the 11-valent 73.5%. The consequences of lack of types 1 and 24-F in the recently available 7-valent vaccine should be investigated.
The rate of penicillin non-susceptible S. pneumoniae increased from 37.2% in 1999 to 43% in our study.35 The number of highly penicillin-resistant isolates doubled during this period (10.6% versus 23%). These results set France in the group of leading countries for penicillin-resistant pneumococci, next to Spain, although in our country there are no strains highly resistant to ceftriaxone, as opposed to 6.8% in Spain.18,36,37 The reasons for this difference remain unknown. The high number of erythromycin-resistant strains was not a surprise because of co-resistance to macrolides and the penicillins. Concerning the fluoroquinolones, the MIC90 of ciprofloxacin was low (2 mg/L), as already reported for Spain (2 mg/L) and the USA (1 mg/L).37,38 A single double mutant showed high-level resistance to fluoroquinolones and had mutations previously described in clinical isolates.39,40
As opposed to neighbouring countries, no vancomycin- resistant enterococci were detected.15,18
Imipenem was the most active antibiotic against P. aeruginosa (92% susceptible strains) followed by ceftazidime (87%) and ciprofloxacin (72%). Carbapenem-hydrolysing enzymes encoded by mobile elements have already spread in France, Italy and Greece.41 Gentamicin resistance was as frequent as in other European studies, representing about one-quarter of the isolates, but higher than the percentage established in North American studies. 15,16
Amongst Enterobacteriaceae, cefotaxime, gentamicin and ciprofloxacin showed the highest rates of in vitro efficiency and ciprofloxacin and gatifloxacin displayed similar in vitro activity. The incidence of ESBL-producing isolates was quite low, in particular for K. pneumoniae (1/66). In another European study, 16.7% of strains isolated during 19971998 putatively encoded such enzymes.15 The percentage of strains of Enterobacteriaceae susceptible to ciprofloxacin is stable and similar to other nationwide surveys.15,16
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Conclusions |
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Acknowledgements |
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Footnotes |
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The ColBVH study group participants are listed in the Acknowledgements.
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References |
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