Frequency and diversity of Class A extended-spectrum ß-lactamases in hospitals of the Auvergne, France: a 2 year prospective study

C. De Champs1,*, C. Chanal1, D. Sirot1, R. Baraduc1, J. P. Romaszko1, R. Bonnet1, A. Plaidy2, M. Boyer3, E. Carroy4, M. C. Gbadamassi5, S. Laluque6, O. Oules7, M. C. Poupart8, M. Villemain9 and J. Sirot1

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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
Objectives: To evaluate the frequency and diversity of extended-spectrum ß-lactamases (ESBLs) produced by Enterobacteriaceae and Pseudomonas aeruginosa in one French region.

Methods: During 2001–2002, 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


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
During the mid-1980s, extended-spectrum ß-lactamases (ESBLs) derived from TEM and SHV appeared in Klebsiella pneumoniae and Escherichia coli.1,2 The numerous mutations which occurred led to a great diversity of TEMs (>130) and SHVs (>50). The ESBLs disseminated not only to opportunistic species but also to specific pathogens. During the 1990s, new non-TEM non-SHV ESBL types appeared, the most frequent being CTX-M and PER-1, which were responsible for epidemics.310 Despite this great diversity, French epidemiological studies showed the persistence and predominance of a restricted number of enzymes such as TEM-3, TEM-24 or SHV-4.7 The last French multicentre survey, conducted over 3 months in 1998 in 14 hospitals of 11 regions, showed that ESBL-producing strains among Enterobacteriaceae had increased (3.2%), mainly due to the high prevalence of TEM-24-producing Enterobacter aerogenes (53.5%), and that the first CTX-M-3-producing strain had appeared in one centre.7 To evaluate the frequency and the diversity of ESBLs produced by strains of Enterobacteriaceae and Pseudomonas aeruginosa in the French region of the Auvergne, a 2 year survey was conducted at the university hospital, the only centre to have identified the ESBLs, and the seven largest non-teaching hospitals. Together, they constituted a regional surveillance network for detecting new ESBLs and known ESBLs that had not been observed in the Auvergne before.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
The Auvergne is a largely mountainous region in central France with a population of 1.4 million. In seven hospitals antibiotic susceptibility testing was performed on Rapid ATB E and ATB PSE (bioMérieux, Marcy l'Étoile, France) and in one laboratory by the disc diffusion method. From 1 January 2001 to 31 December 2002, all the non-duplicate isolates of P. aeruginosa resistant to ceftazidime (MIC > 32 mg/L) and of Enterobacteriaceae intermediate or resistant to ceftazidime and/or cefotaxime (MICs > 4 mg/L) and/or aminoglycosides with an AAC(6') I phenotype, i.e. amikacin (MIC > 8 mg/L), kanamycin (MIC > 8 mg/L) and tobramycin (MIC > 4 mg/L), because ESBLs were often associated with an AAC(6'),11 were collected and sent every 3 months to the laboratory of the teaching hospital in Clermont-Ferrand. All isolates from inpatients and outpatients were included, irrespective of where they were sampled or of their implication in infection. Every 6 months, two strains, some of them producing an ESBL, were sent to each laboratory as quality controls. On nine occasions, ESBLs failed to be detected, especially PER-1 produced by a P. aeruginosa strain, but all laboratories detected the decrease in antibiotic susceptibility which was the main criterion for the inclusion of the isolate in the study.

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 Mueller–Hinton broth equivalent to 0.5 McFarland standard (108 cfu/mL) was prepared and then diluted to 1/100 and seeded by swabbing onto Mueller–Hinton agar. Antibiotic discs (Bio-Rad, Marnes la Coquette, France) were applied and the inhibitory diameter was read after 18 h of incubation at 35–37°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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Table 1. Oligonucleotides used as primers for amplification and/or sequencing

 
When more than four isolates per year of the same species producing the same ESBL were observed in the same centre, the isolates were genotyped by ERIC2-PCR to study their clonal diversity.14 E. aerogenes isolates were ribotyped according to the procedure of Bingen et al.,15 and in addition they were compared with the clonal strain reported previously.7,11 When isolates related to the same clonal strain produced an ESBL of the same type and same pI the bla genes were sequenced for one isolate. For the other isolates, enzymes were designated TEM-3 like or TEM-24 like. The prevalence of ESBLs was calculated using the number of non-duplicate isolates in each species and each centre.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
During this 2 year survey, a total of 36361 strains of Enterobacteriaceae and 5304 P. aeruginosa were isolated. Of the 1829 resistant strains included in the study, 297 Enterobacteriaceae (0.8%) and 37 P. aeruginosa (0.7%) produced an ESBL.

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 < 10–7) (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 < 10–7), E. aerogenes (53.5% versus 16.5%; P < 10–7) and Enterobacter cloacae (6.7% versus 0.1%; P < 10–7). 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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Table 2. Evolution of the distribution of ESBLs according to species [no. ESBL/no. isolates (%)]

 
During the period 2001–2002 (Table 3), the most frequent ESBLs in Enterobacteriaceae were TEM-3 like (n=152/297; 51.2%) and TEM-24 like (n=115/297; 38.7%). Other previously reported TEM-types were TEM-8 (n=1), TEM-12 (n=1), TEM-16 (n=1), TEM-19 (n=1), TEM-20 (n=1), TEM-21 (n=2), TEM-29 (n=2) and TEM-71 (n=2). In addition, four new TEM types were characterized (Table 4): TEM-112 (pI 5.4; GenBank accession number AY589493), which differs from TEM-1 by the amino acid changes His-153->Arg and Gly-238->Ser. Residue 153 is often a basic residue, not conserved in class A ß-lactamases, and located at the end of helix H4. Previous reports on TEM-18 (Lys-39, Lys-104) and TEM-56 (Lys-39, Lys-104, Arg-153)17 suggest that Arg-153 does not modify the catalytic activity of the enzyme. In addition, mutation Thr-265->Met, which is frequently observed in TEM-derivative ESBLs, has no effect on the hydrolytic activity of the enzyme. The resistance level to broad-spectrum cephalosporins conferred by TEM-112 was similar to that observed with TEM-25. TEM-113 (pI 6.3; GenBank accession number AY589494), differs from TEM-3 by the substitution Met-182->Thr. This substitution is common in ESBLs (e.g. TEM-20, TEM-43, TEM-52, TEM-63, TEM-72, TEM-87; TEM-91–TEM-95, TEM-106). Molecular modelling has shown that the active site may be stabilized by a new hydrogen bond between the hydroxyl of Thr-182 and the carbonyl of the amide bond of Glu-64.18 Several studies of crystal structure suggested that the substitution Met-182->Thr has little effect on enzyme activity but restores stability lost by substitutions near the active site.1921 TEM-114 (pI 5.9; GenBank accession number AY589495), was derived from TEM-2 (Lys-39) and harboured the same substitutions, Arg-164->Ser, Ala-237->Thr and Glu-240->Lys, as TEM-5, which was derived from TEM-1. TEM-126 (pI 5.4; GenBank accession number AY628199), derives from TEM-1 by two substitutions. This is the first ESBL mutant of TEM harbouring the substitution Asp-179->Glu (in addition to the substitution Met-182->Thr). A substitution at position 179 was reported in SHV-1-derivative ESBLs (SHV-6, SHV-8 and SHV-24). In these enzymes aspartic acid 179 is replaced by a neutral residue (alanine, asparagine, glycine), which disrupts the salt bridge, and Arg-164 increases the flexibility of the omega loop. In the TEM-126 mutant, the disruption of the salt bridge probably did not occur since the aspartic residue was replaced by a glutamic acid residue. Kinetic analysis and molecular modelling are in progress to explain the extended-spectrum activity of this novel mutant.


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Table 3. ESBL distribution among various species [% in 2001–2002 (% in 19987)]

 

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Table 4. Isoelectric point and amino acid substitutions of the four new TEM types and related enzymes

 
TEM-3-like enzymes were produced by 10 different species isolates: C. koseri (n=60), P. mirabilis (n=47), E. coli (n=15), S. marcescens (n=10), K. pneumoniae (n=7), K. oxytoca (n=5), C. freundii (n=3), E. aerogenes (n=3), E. cloacae (n=1) and P. stuartii (n=1). Most TEM-24-like enzymes (87.8%) were produced by E. aerogenes isolates (n=101), and most ESBLs produced by E. aerogenes isolates (96.2%) were TEM-24 like. The other isolates producing TEM-24-like enzymes were P. mirabilis (n=5), E. coli (n=4), K. pneumoniae (n=3) and K. oxytoca (n=2), which lends support to the previously reported findings of interspecies dissemination of the enzyme.22

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 2001–2002 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.

  1. S. marcescens TEM-3 was limited to one centre. ERIC2-PCR patterns were identical for all the strains.
  2. E. aerogenes TEM-24 was observed in five centres in 2001 and six in 2002. ERIC2-PCR and ribotype patterns were the same for all the strains and identical to those observed in 1998. The isolates were far more frequent in one centre than in the others because of the persistence of the strain in a long-stay care unit.
  3. C. koseri TEM-3 was observed in six centres in 2001 and four in 2002. The epidemic was concentrated in one centre, which was in the east of the region, near Lyon, where an epidemic had occurred 3 years earlier (data not shown). ERIC2-PCR patterns were identical or closely related for all the isolates.
  4. P. aeruginosa PER-1 was observed in two centres in 2001, and one in 2002, where it was epidemic. All the strains had the same or a related genotype profile. P. aeruginosa PER-1 isolates are widespread in Turkey10 and the outbreak may have been related to the immigration of Turkish nationals, since the first strain of our study was isolated from a patient previously hospitalized in Strasbourg (Eastern France), where he might have been in contact with Turkish patients.6 This multiresistant strain has been difficult to eradicate.
  5. P. mirabilis TEM-3 was observed in four centres in 2001 and five in 2002. ERIC2 profiles were either identical or differed by less than three segments and were considered to be closely related.
  6. E. coli TEM-3 isolates, observed in six centres in 2001 and one in 2002, harboured different genotypes. CTX-M-type enzymes are emerging in the region; they increased from 2.1% in 2001 to 4.9% in 2002, and could give rise to epidemics, as in other countries.8

 ESBL frequency was considerably lower than in other parts of the world. In Clermont-Ferrand hospital they decreased from about 13 per month in 1988–1989 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.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
We thank Marlène Jan, Rolande Perroux and Dominique Rubio for their technical assistance. This work was supported by grants from the Centre Hospitalier Régional Universitaire de Clermont-Ferrand, France and the Ministère de la Santé, de la Famille et des Personnes Handicapées France (Projet Hospitalier de Recherche Clinique).


    Footnotes
 
* Correspondence address. Laboratoire de Bacteriologie, Virologie, Hygiène, CHU Reims, Avenue du Général Koenig, 51092 Reims Cedex, France. Tel: +33-326-789-251; Fax: +33-326-784-134; Email: cdechamps{at}chu-reims.fr


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Acknowledgements
 References
 
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