1 Centre Hospitalier Universitaire de Clermont-Ferrand, rue Montalembert, 63000 Clermont-Ferrand; 2 Centre Hospitalier de Vichy, Boulevard Denière, 03209 Vichy; 3 Centre Hospitalier de Roanne, rue de Charlieu, 42328 Roanne; 4 Centre Hospitalier de Thiers, Chemin Fau, 63300 Thiers; 5 Centre Hospitalier de Moulins, Avenue du Général de Gaulle, 03009 Moulins; 6 Centre Hospitalier de Montluçon, Avenue du 8 mai 1945, 03109 Montluçon; 7 Centre Hospitalier du Puy, Route Montredon, 43000 Le Puy; 8 Centre Hospitalier de Riom, Boulevard Etienne-Clémentel, 63204 Riom; 9 Centre Hospitalier d'Aurillac, Avenue de la République, 15000 Aurillac, France
Received 9 April 2004; returned 29 May 2004; revised 3 July 2004; accepted 6 July 2004
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Abstract |
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Methods: During 20012002, all the non-duplicate isolates of P. aeruginosa resistant to ceftazidime and of Enterobacteriaceae intermediate or resistant to ceftazidime and/or cefotaxime and/or aminoglycosides with an AAC(6') I phenotype were collected in nine hospitals of the area. ESBL isoelectric points were determined, bla genes were amplified and sequenced and epidemic isolates were genotyped with ERIC2-PCR.
Results: ESBLs were observed in 297 Enterobacteriaceae (0.8%). The most frequent were TEM-3 like (n=152; 51.2%) and TEM-24 (n=115; 38.7%). Four new enzymes were observed, TEM-112 (pI 5.4), TEM-113 (pI 6.3), TEM-114 (pI 5.9) and TEM-126 (pI 5.4). Other TEMs were TEM-8, TEM-12, TEM-16, TEM-19, TEM-20, TEM-21, TEM-29 and TEM-71. The other ESBLs were SHV-4, SHV-5 and SHV-12, CTX-M-1, CTX-M-3, CTX-M-14 and CTX-M-15. In 37 P. aeruginosa (0.7%) only one ESBL was observed, PER-1. Five epidemic strains were detected, Serratia marcescens TEM-3 and four observed in several hospitals, Enterobacter aerogenes TEM-24, Citrobacter koseri TEM-3, Proteus mirabilis TEM-3 and P. aeruginosa PER-1.
Conclusion: ESBL frequency was lower than in 1998, and CTX-M-type frequency higher (2.1% of ESBLs in 2001, 4.9% in 2002). This long-term survey detected new sporadic enzymes (TEM-112, TEM-113, TEM-114 and TEM-126) and interhospital epidemic strains while avoiding any overestimation of ESBL frequency that may otherwise have occurred because of acute epidemics.
Keywords: ESBLs , Enterobacteriaceae , Pseudomonas aeruginosa , epidemiology
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Introduction |
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Materials and methods |
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Identification of the strains was verified by Rapid ID 32 E for Enterobacteriaceae and ID 32 GN for P. aeruginosa (bioMérieux) and antibiotic susceptibility was checked by the disc diffusion method according to the recommendations of the Antibiogram Committee of the French Society for Microbiology (http://www.sfm.asso.fr/). From an overnight non-selective agar medium culture plate, a suspension in MuellerHinton broth equivalent to 0.5 McFarland standard (108 cfu/mL) was prepared and then diluted to 1/100 and seeded by swabbing onto MuellerHinton agar. Antibiotic discs (Bio-Rad, Marnes la Coquette, France) were applied and the inhibitory diameter was read after 18 h of incubation at 3537°C. Strains were screened for ESBL by the double-disc synergy test12 and with discs containing 30 µg of cefotaxime or ceftazidime alone and in combination with 10 µg of clavulanate (CDO2 and CDO3; Oxoid SA 69571 Dardilly France). ß-Lactamases were characterized by isoelectric focusing, and the genes blaTEM, blaSHV, blaCTX-M, blaPER were detected by PCR using specific primers as described previously (Table 1). 3 ESBL-producing isolates were classified according to year, centre, species, resistance phenotype, pI and ESBL type. For TEM-type enzymes with pI 6.3 (TEM-3 like) and pI 6.5 (TEM-24 like), and for PER-type enzymes, the bla PCR products obtained from the isolates (n=57) of the first sample of each class were selected for direct DNA sequencing to confirm identification of the enzyme. For the other isolates producing TEM-3 or TEM-24 the enzyme type was confirmed with allele-specific PCR (ASPCR) for up to four isolates per year and per centre (n=61).13 The bla genes of all the other isolates were sequenced.
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Results and discussion |
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In comparison with 1998 French data, the proportion of ESBL-producing isolates obtained during the survey was lower among Enterobacteriaceae (3.2% versus 0.8%, P < 107) (Table 2).7 The proportions were stable for E. coli (0.2%), increased slightly for Providencia stuartii (0.0% versus 0.5%), Klebsiella oxytoca (0.0% versus 0.7%), Serratia marcescens (0.0% versus 2.5%) and Citrobacter freundii (0.0% versus 0.8%) and decreased, but not significantly, for Citrobacter koseri (16.7% versus 12.4%) and Proteus mirabilis (3.7% versus 1.7%). In contrast, there was a significant decrease in K. pneumoniae (9.4% versus 0.9%; P < 107), E. aerogenes (53.5% versus 16.5%; P < 107) and Enterobacter cloacae (6.7% versus 0.1%; P < 107). Some ESBL producers with low-level resistance to ceftazidime and cefotaxime and susceptible to aminoglycosides may not have been detected in our study. Such isolates used to be rare and were observed mainly in P. mirabilis.16 In 1998, the number of E. aerogenes may have been disproportionately inflated by the fact that the survey was short (3 months) and because in some areas, during this period there had been outbreaks.7
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Among the SHV derivatives, three types were observed: SHV-5 (n=2), SHV-4 (n=1) and SHV-12 (n=1). The other non-TEM, non-SHV ESBLs observed were PER-1 like (n=37), CTX-M-3 (n=4), CTX-M-14 (n=3), CTX-M-15 (n=3) and CTX-M-1 (n=1). There was no evidence of the production of other ESBLs or of the presence of several ESBLs in the same strain.
In comparison with the 1998 survey,7 the distribution of ESBLs among Enterobacteriaceae observed during the 20012002 period showed an increase in TEM-3-like enzymes (36.7% versus 51.2%, P=0.02) and in CTX-M (1.3% versus 3.7%, not significant) and a decrease in TEM-24-like enzymes (50.6% versus 38.7%, not significant), and in SHV (5.1% versus 1.3%, P=0.04). The percentages of ESBLs varied considerably between centres, ranging from 0.2% to 3.6% for Enterobacteriaceae isolates and 0.0% to 0.7% for P. aeruginosa isolates. This variation was due mainly to local epidemics (documented by different centres) involving the following species and ß-lactamases: S. marcescens, C. koseri, E. coli and P. mirabilis producing TEM-3, E. aerogenes producing TEM-24 and P. aeruginosa producing PER-1.
After genotyping, a clonal relationship was confirmed in five species/enzyme groups responsible for epidemics.
ESBL frequency was considerably lower than in other parts of the world. In Clermont-Ferrand hospital they decreased from about 13 per month in 19881989 to four per month during the period of this study.11 This may have been due to a decrease in the use of rooms with several beds in intensive care units and the use of closed urinary drainage systems. Between the two periods, detection of digestive carriage of ESBL-producing bacteria was implemented, and specific daily alerts were given by the laboratory to the units to allow patients to be isolated when necessary. Antibiotic use was limited and treatment duration was shortened.
The main advantages of a long-term survey are to avoid any overestimation of one species or one enzyme that may otherwise have occurred because of acute epidemics and to detect new sporadic enzymes. On the other hand, because of its length, the survey is exposed to the risk of underestimating the total number of ESBLs, by missing some isolate inclusions. This study showed that, although the frequency of ESBLs is decreasing, these enzymes may still cause epidemics, especially in centres with long-stay care units.
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Acknowledgements |
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Footnotes |
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