ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Nov. 2000, p. 3177–3179 0066-4804/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Vol. 44, No. 11

A 1998 Survey of Extended-Spectrum ␤-Lactamases in Enterobacteriaceae in France C. DE CHAMPS,* D. SIROT, C. CHANAL, R. BONNET, J. SIROT, AND THE FRENCH STUDY GROUP† Laboratoire de Bactériologie, Faculté de Médecine, Université d’Auvergne, 63001 Clermont-Ferrand Cedex, France Received 24 January 2000/Returned for modification 8 May 2000/Accepted 8 August 2000

In a 3-month period in 1998, 79 consecutive isolates of Enterobacteriaceae producing an extended-spectrum ␤-lactamase (ESBL) were collected. ESBLs were predominantly TEM derivatives (74 of 79): TEM-24-like (40 isolates), TEM-3-like (29 isolates), TEM-21 (3 isolates), and TEM-4 and TEM-52 (1 isolate each). Four isolates produced SHV derivatives SHV-4 (three isolates) and SHV-5 (one isolate), and one strain produced a CTX-M-3 enzyme. The high proportion of TEM-24-like-producing Enterobacter aerogenes isolates (36 of 79) suggests the occurrence of an epidemic strain in France. Extended-spectrum ␤-lactamases (ESBL) are now observed in all species of Enterobacteriaceae. Many of these enzymes are TEM or SHV derivatives and other newly emerging class A enzymes such as PER-1 and CTX-M. To evaluate the frequency and the diversity of ESBL produced by strains of Enterobacteriaceae in France, a survey was conducted from April to June 1998 in 14 French hospitals, 7 of 29 metropolitan university hospitals, and 7 nonteaching hospitals in 11 different regions including Ile-de-France, the Paris region (Fig. 1). The first 180 consecutive nonduplicate isolates of each laboratory were screened for ESBL by the double-disk synergy test. They were identified using automated systems. In all laboratories, antibiotic susceptibility testing was performed on Mueller-Hinton agar and with antibiotic discs from Diagnostic Pasteur (Marnes la Coquette, France), placed at defined positions. Strains were collected if the synergy test was positive. Isolates from superficial wounds, stools, ear, nose, and throat, those not involved in infections according to the Centers for Disease Control and Prevention criteria (8), and those from swab samples were excluded. Once weekly, Escherichia coli ATCC 25922 and E. coli C600/pCFF04 (TEM-3) were tested as quality controls. The ␤-lactamases were characterized by isolectric focusing. blaTEM genes were then detected by PCR using primers A (5⬘TAA-AAT-TCT-TGA-AGA-CG-3⬘) and B (5⬘TTA-CCAATG-CTT-AAT-CA-3⬘), and known mutations at positions 39, 104, 164, and 238 were detected by allele-specific PCR (ASPCR) (16). blaSHV genes were detected with primers OS5 (5⬘TTA-TCT-CCC-TGT-TAG-CCA-3⬘) and OS6 (5⬘GAT-TT G-CTG-ATT-TCG-CTC-3⬘). The ESBL that was neither a TEM nor a SHV derivative was identified by direct sequencing of the PCR product obtained with specific primers chosen according to the isoelectric point value (8.4) and the resistance phenotype (high-level resistance to cefotaxime), both of which point to a CTX-M-3 enzyme (9). For each ESBL TEM type in each species and each region (n ⫽ 23), direct DNA sequencing of PCR products was performed to confirm the mutations

observed by ASPCR or to detect any mutation hitherto unseen. For the SHV genes, sequences were obtained for one SHV-4 gene and one SHV-5 gene after plasmid transfer in E. coli. To study the clonal diversity of the strains, ribotyping was performed for the 36 TEM-24-like ESBL- and 7 TEM-3-like ESBL-producing Enterobacter aerogenes isolates and 11 TEM3-like ESBL-producing Klebsiella pneumoniae isolates with a cold-labeled probe obtained from E. coli ribosomal 16S and 23S RNA (Boehringer GmbH, Mannheim, Germany). Of the 2,506 Enterobacteriaceae isolated, 79 strains produced ESBL, a higher proportion (3.2%) than in 1990 (1.5%) and 1991 (0.9%) (10, 15). For E. coli species, this proportion did not increase from 1990 (1.5%) to 1991 (0.9%) to 1996 (0.1%) to 1998 (0.2%). The increase in the proportion of ESBL-producing Proteus mirabilis strains (0% in 1990 versus 3.7% in 1998) may have been underestimated. Frequencies close to 6% were observed in Clermont-Ferrand from 1996 to 1998 (6, 7). In P. mirabilis the synergy between extended-spectrum cephalosporins and clavulanate can be difficult to detect by disk diffusion. For K. pneumoniae the percentage in 1998 (9.4%) did not differ significantly from that in 1991 (9.6%). The increases observed in 1994 (22.2%) and in 1996 (17.7%) were apparently transient (6; M. H. Nicholas-Chanoine, H. Chardon, and the Multicenter Group, Abstr. 39th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 2253, 1999). The proportion of ESBL-producing E. aerogenes isolates rose from ⬍2.5% in 1990 to 53.5% in 1998 (15). This high occurrence may also have been transient and due to an epidemic strain, as reported in a survey in which the prevalence decreased from 20.2% in 1990 to 5.2% in 1994 (6). Without the 43 epidemic E. aerogenes isolates, the ESBL rate was 1.5%. The proportion of ESBL-producing strains among Enterobacteriaceae varied from 1.6% in hematooncology units to 7.1% in intensive care units to 7.7% in rehabilitation units. The proportions of Enterobacteriaceae isolated from the different samples were as follows: 4 of 29 (13.8%) from intravascular catheters, 4 of 38 (10.5%) from stools, 10 of 187 (5.3%) from the respiratory tract, 49 of 1,681 (2.9%) from urine, 5 of 219 (2.3%) from blood cultures, and 7 of 352 (2.0%) from wounds and serous effusions. Eight different ESBL were characterized: TEM-24-like (40 isolates, 50.62%), TEM-3-like (29 isolates, 36.70%), TEM-21 and SHV-4 (3 isolates each, 3.80%), and TEM-4, TEM-52, SHV-5, and CTX-M-3 (1 isolate each, 1.27%) (Table 1). Their

* Corresponding author. Mailing address: Laboratoire de Bactériologie, Faculté de Médecine, Université d’Auvergne, 28 Place Henri Dunant, 63001 Clermont-Ferrand Cedex, France. Phone: 33 (0)4 73 60 80 18. Fax: 33 (0)4 73 27 74 94. E-mail: Christophe.DECHAMPS @u-clermont1.fr. † Members are listed in the Appendix. 3177

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ANTIMICROB. AGENTS CHEMOTHER.

FIG. 1. Distribution of ESBL-producing strains among regions and centers. ●, TEM-24-like; Œ, TEM-3-like; f, others (TEM-21, SHV-4-like, TEM-52, SHV-5, CTX-M-3, TEM-4). Numbers in parentheses are numbers of isolates in which enzymes were found. Regions (cities where study hospitals are located are in parentheses [see Appendix for respective French Study Group members]): R1, Nord and Pas-de-Calais (Lille and Valenciennes); R2, Ile-de-France (Cre´teil and Versailles); R3, Champagne and Ardennes (Troyes); R4, Alsace-Lorraine (Mulhouse); R5, Brittany (Rennes); R6, Pays-de-Loire (Le Mans); R7, Auvergne (Clermont-Ferrand); Rhone-Alps (Lyon); R9 Midi-Pyrenees (Toulouse); R10, Languedoc-Roussillon (Perpignan); R11, Provence, Alps, and Co ˆte d’Azur (Nice and Aix-en-Provence).

geographical distribution is shown in Fig. 1. In three centers, Rennes, Le Mans, and Troyes, no ESBL were detected. TEM24-like ESBL- and TEM-3-like ESBL-producing strains were observed in seven and eight centers, respectively. The TEM24-like ESBL was the most frequent in E. aerogenes isolates (36 of 46; 78.3%). The TEM-24-like ESBL- and TEM-3-like ESBLproducing E. aerogenes isolates revealed only two different ribotypes according to their ESBL. This is suggestive of the spread of two clonal strains. TEM-24-like ESBL-producing strain outbreaks were reported elsewhere (4, 13), and all TEM3-like ESBL-producing E. aerogenes strains were isolated in the same region. TEM-3-like enzyme was observed in 11 of 13 TABLE 1. Distribution of ESBL according to species No. of isolates with ESBL:

Species

Total no. of isolates

E. coli P. mirabilis K. pneumoniae E. aerogenes Salmonella sp. E. cloacae Citrobacter koseri Othersa

1,606 215 l38 86 32 30 30 369

4 (0.2) 8 (3.7) l3 (9.4) 46 (53.5) 1 (3.1) 2 (6.7) 5 (16.7) 0 (0.0)

3 0 1 36 0 0 0 0

0 5 11 7 0 1 5 0

Total

2,506

79 (3.2)

40

29

a

Total (%)

TEM-24- TEM-3like like

Others (ESBL type)

1 (TEM-52) 3 (TEM-21) 1 (SHV-5) 3 (SHV-4-like) 1 (TEM-4) 1 (CTX-M-3) 0 0 10

Citrobacter freundii, Enterobacter spp., Hafnia alvei, Klebsiella oxytoca, Proteus spp., Providencia spp., Yersinia enterocolitica.

(84.6%) K. pneumoniae isolates belonging to five different ribotypes. When ESBL appeared, they were predominantly TEM-type enzymes (10), but in Europe since the mid-1990s the SHV-type ESBL is the most frequent (1). The greater incidence of the SHV-type ESBL seems related to the predominance of K. pneumoniae among the ESBL-producing strains (5, 6, 7, 11). In the present survey, the TEM-type ESBL was again the most frequent and concomitantly E. aerogenes was the most frequent of the ESBL-producing Enterobacteriaceae. This may be because TEM-24 is one of the most efficient ESBL owing to the combination of four amino acid substitutions at positions 104, 164, 237, 240, which are known to enhance the hydrolysis of oxyimino-amino thiazole cephalosporins (12). In addition the presence of T at position 32 in the blaTEM-24 gene promoter leads to an increase in transcriptional level. Further studies are needed to understand why TEM-24-producing E. aerogenes is currently the predominant ESBL-producing species of the Enterobacteriaceae in France. In this survey a CTX-M enzyme (CTX-M-3) was observed in Enterobacter cloacae in a patient residing in France and admitted to Versailles hospital. CTXM-3 was reported in Poland in 1996 (9). The class A cefotaxime-hydrolyzing enzymes (CTX-M) do not derive from TEM and SHV. Most of these enymes have been observed in South America, Israel, Japan, and eastern Europe (3). The differences in the frequencies of ESBL-producing strains between regions and from year to year are in part due to epidemic strains. In Europe an increasing proportion of ESBL-producing strains among Enterobacteriaceae has been reported (1). New mutant TEM or SHV and non-TEM, nonSHV class A enzymes are still being reported and are spreading worldwide to all species of Enterobacteriaceae and other genera such as Pseudomonas (2, 3, 11, 14). This underlines the need for regular surveillance of the prevalence of these enzymes and for continuing vigilance in prevention. We thank Marlène Jan, Rolande Perroux, and Dominique Rubio for their technical assistance. This study was conducted with the support of Hoechst Marion Roussel.

APPENDIX Participating members of the French Study Group: J. L. Avril (Rennes), C. Cattoen (Valenciennes), H. Chardon (Aix en Provence), J. L. Croix (Troyes), H. Dabernat (Toulouse), T. Fosse (Nice), J. C. Ghnassia (Versailles), E. Lecaillon (Perpignan), A. Marmonier (Le Mans), M. H. Nicolas-Chanoine (Boulogne), M. Roussel-Delvallez (Lille), J. Sirot (Clermont-Ferrand), C. J. Soussy (Créteil), A. Trevoux (Mulhouse), F. Vandenesch (Lyon). REFERENCES 1. Babini, G. S., and D. M. Livermore. 2000. Antimicrobial resistance amongst Klebsiella spp. collected from intensive care units in southern and western Europe in 1997–1999. J. Antimicrob. Chemother. 45:183–189. 2. Bonnet, R., C. De Champs, D. Sirot, C. Chanal, R. Labia, and J. Sirot. 1999. Diversity of TEM mutants in Proteus mirabilis. Antimicrob. Agents Chemother. 43:2671–2677. 3. Bonnet, R., J. L. M. Sampaio, R. Labia, C. De Champs, D. Sirot, C. Chanal, and J. Sirot. 2000. A novel CTX-M ␤-lactamase (CTX-M-8) in cefotaximeresistant Enterobacteriaceae isolated in Brazil. Antimicrob. Agents Chemother. 44:1936–1942. 4. Bosi, C., A. Davin-Regli, C. Bornet, M. Mallea, J. M. Pages, and C. Bollet. 1999. Most Enterobacter aerogenes strains in France belong to a prevalent clone. J. Clin. Microbiol. 37:2165–2169. 5. Buré, A., P. Legrand, G. Arlet, V. Jarlier, G. Paul, and A. Philippon. 1988. Dissemination in five French hospitals of Klebsiella pneumoniae serotype K25 harbouring a new transferable enzymatic resistance to third generation cephalosporins and aztreonam. Eur. J. Clin. Microbiol. Infect. Dis. 7:780– 782.

VOL. 44, 2000 6. Chanal, C., D. Sirot, J. P. Romaszko, L. Bret, and J. Sirot. 1996. Survey of prevalence of extended spectrum ␤-lactamases among Enterobacteriaceae. J. Antimicrob. Chemother. 38:127–132. 7. De Champs, C., D. Sirot, C. Chanal, M. C. Poupart, M. P. Dumas, and J. Sirot. 1991. Concomitant dissemination of three extended-spectrum ␤-lactamases among different Enterobacteriaceae isolated in a French hospital. J. Antimicrob. Chemother. 27:441–457. 8. Garner, J. S., W. R. Jarvis, T. G. Emori, T. C. Horan, and J. M. Hughes. 1988. CDC definitions for nosocomial infections, 1988. Am. J. Infect. Control 16:128–139. 9. Gniadkowski, M., I. Schneider, A. Palucha, R. Jungwirth, B. Mikiewicz, and A. Bauernfeind. 1998. Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX-M-3 cefotaximehydrolyzing ␤-lactamase that is closely related to the CTX-M-1/MEN-1 enzyme. Antimicrob. Agents Chemother. 42:827–832. 10. Goldstein, F. W., Y. Péan, A. Rosato, J. Gertner, L. Gutmann, and the Vigils’ Roc Study Group. 1993. Characterization of ceftriaxone-resistant Enterobacteriaceae: a multicentre study in 26 French hospitals. J. Antimicrob. Chemother. 32:595–603. 11. Heritage, J., F. H. M’Zali, D. Gascoyne-Binzi, and P. M. Hawkey. 1999.

NOTES

12. 13.

14. 15.

16.

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