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Antimicrobial Agents and Chemotherapy, August 2005, p. 3492-3494, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3492-3494.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

First Detection of a Carbapenem-Hydrolyzing Metalloenzyme in Two Enterobacteriaceae Isolates in Spain

M. Teresa Tórtola,1 Susana Lavilla,1 Elisenda Miró,2 Juan José González,1 Nieves Larrosa,1 Montserrat Sabaté,1 Ferran Navarro,2 and Guillermo Prats1*

Department of Microbiology, Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain,1 Department of Microbiology, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain2

Received 31 March 2005/ Returned for modification 15 April 2005/ Accepted 25 April 2005


   ABSTRACT
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Two strains of Enterobacteriaceae, Escherichia coli and Klebsiella pneumoniae, producing VIM-1 were isolated for the first time in Spain. In both strains, blaVIM-1 was found to be carried on a gene cassette inserted into a class 1 integron. The blaVIM-1-containing integron was located on a transferable plasmid.


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The metallo-ß-lactamases (MBLs) are carbapenem-hydrolyzing ß-lactamases which belong to molecular class B of Ambler. The most frequent carbapenem-hydrolyzing ß-lactamases are IMP and VIM. These enzymes effectively hydrolyze not only carbapenems but also all ß-lactam antibiotics except aztreonam. Both are zinc dependent, so their activities are inhibited by EDTA, and they are often encoded by gene cassettes inserted into integrons, which are sometimes located on plasmids (1, 7, 14, 18). Although the overall prevalence of these enzymes among clinical isolates has remained low, they have been reported among isolates of various members of the family Enterobacteriaceae, Pseudomonas aeruginosa, and other nonfastidious gram-negative bacteria (1, 3, 5-7, 9, 11, 14, 18).

The aim of the present study was to evaluate the presence of these enzymes among the 4,345 clinically relevant Enterobacteriaceae strains isolated during 2003 in the Hospital Vall d'Hebron. Moreover, in order to detect fecal carriers of Enterobacteriaceae with MBLs, a total of 1,043 stool specimens submitted for bacterial culture were inoculated onto two MacConkey agars supplemented with 8 mg/liter of imipenem and 2 mg/liter of cefotaxime, respectively. These plates were incubated overnight and the isolates were submitted to disk diffusion susceptibility tests by using Neo-Sensitabs disks (Rosco, Denmark) (13). The antibiotics tested were ampicillin, amoxicillin-clavulanic acid, cefoxitin, cefotaxime, ceftazidime, cefepime, aztreonam, imipenem, and meropenem. The breakpoints were those defined by the Clinical and Laboratory Standards Institute (formerly NCCLS) for Enterobacteriaceae (13). Escherichia coli ATCC 25922 and E. coli ATCC 35218 were used as control strains.

Among all 4,345 clinical strains and the 2,398 strains isolated from stools, one E. coli isolate from a urinary tract infection and one Klebsiella pneumoniae isolate from a fecal carrier were resistant to ampicillin, amoxicillin-clavulanic acid, cefoxitin, cefotaxime, ceftazidime, and cefepime and susceptible to aztreonam. Although both strains were susceptible to imipenem, the inhibition zone diameters were 20 mm for E. coli and 22 mm for K. pneumoniae, smaller than the diameters expected for those species. No synergy of cefotaxime and ceftazidime with clavulanic acid was detected by Etest (AB Biodisk, Solna, Sweden) in either strain. This resistance pattern suggested the presence of a carbapenemase, although the strains were ostensibly susceptible to imipenem.

The MICs obtained by a microdilution method (MicroScan Dade Behring, Inc., W. Sacramento, Calif.) are shown in Table 1. MICs of imipenem, meropenem, and ertapenem for E. coli, determined by Etest, were 2 µg/ml, 1 µg/ml, and 2 µg/ml, respectively, and those for K. pneumoniae were 2 µg/ml, 0.5 µg/ml, and 0.75 µg/ml, respectively, all of them in the range of susceptibility.


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  		TABLE 1. MICs of 23 antimicrobial agents for Escherichia coli and Klebsiella pneumoniae by MicroScan

 
The synergy of imipenem and EDTA was difficult to interpret by Etest due to the relatively low MICs of the strains. By the disk test, the inhibition zone diameters for imipenem and for imipenem plus EDTA were, respectively, 20 and 32 mm for E. coli and 22 and 30 mm for K. pneumoniae, supporting our suspicion that the enzyme was a carbapenemase.

Conjugation experiments were done by the filter mating method (17). The resistance pattern of K. pneumoniae was transferred by using E. coli HB101 (Nalr) as a recipient strain. Transconjugant clones were selected in brain heart infusion agar containing nalidixic acid (50 µg/ml) plus cefotaxime (5 µg/ml). Negative results were obtained for E. coli. In this case, the plasmid was extracted with the High Pure plasmid isolation kit (Roche Applied Science, Penzberg, Germany) and was electroporated into E. coli DH5{alpha} (as specified by Bio-Rad Laboratories). Later on, this transformant strain was successfully conjugated with E. coli HB101 (Rifr), with the transconjugant clones being selected on brain heart infusion agar containing cefotaxime (5 µg/ml) plus rifampin (50 µg/ml). Transformants and transconjugants exhibited a resistance phenotype similar to that of the donor (data not shown).

Plasmid analysis was performed by nuclease S1 digestion and hybridization with a blaVIM-1 probe (12). Both isolates contained the same plasmid of about 40 kb carrying the VIM-1 carbapenemase (pMVH202).

Analytical isoelectric focusing (17), which was performed on both strains, showed two bands per strain; these had pIs of 5.4 and 5.1 for E. coli and pIs slightly lower than 7.6 and 5.1 for K. pneumoniae. All the bands were detected by nitrocefin (data not shown).

PCRs of blaVIM and blaIMP were performed as described previously (15). Direct sequencing of blaVIM amplicons, by use of a Beckman 8000 sequencer, showed 100% sequence identity with the blaVIM-1 in both strains (15, 16).

The transconjugants were also positive for the blaVIM by PCR. Subsequently, amplification for class 1 integrons was performed by PCR with a set of primers, including 5'-CS and 3'-CS (8), and yielded a 4-kb amplification product from E. coli and K. pneumoniae. In both strains, the sequence of the new integron (In113) showed the presence from 5' to 3' of the gene cassette blaVIM-1, aacA4, dfrII, aadA1, and catB2.

The ß-lactamase with a pI of 5.4 found in E. coli may correspond to the TEM family, because PCR with blaTEM primers was positive (17). Moreover, we want to note that the K. pneumoniae ß-lactamase with a pI slightly lower than 7.6 corresponds to the OKP family, as determined by specific PCR and sequencing results (data not shown) (4).

In this survey, the prevalence of carbapenemases among enterobacteria was very low. We found MBLs in only one strain of E. coli among 4,345 clinically relevant isolates and one K. pneumoniae strain among 2,398 isolates from stools.

The detection of MBLs based on resistance to imipenem is difficult in strains in which MICs of carbapenems are low, as was the case with the strains isolated in this study and as was the case in studies published by others (2). In both of the present MBL-positive strains, the overall susceptibility profiles provided the only suggestion that a MBL was present. In these strains, the results of the Etest were difficult to evaluate; however, the synergy detected by disk diffusion test was evident and suggestive of MBL production.

We have observed a rapid increase of extended-spectrum ß-lactamase (ESBL)-producing strains. The first ESBL in the Hospital Sant Pau was detected in an E. coli strain in 1994 (17), and in 2004, in our laboratories, we found that 4% of enterobacteria had ESBL (data not shown). Moreover, in 2002, about 7.5% of healthy people carried a strain from the family Enterobacteriaceae with an ESBL (10). Nevertheless, the situation seems to be different for MBLs, at least in our country. In 1996, we detected the first VIM-2 in Spain in a Pseudomonas aeruginosa strain (16). Since then, only one to three P. aeruginosa isolates with MBLs are being recovered per year in our laboratories (data not shown).

Although the enzyme VIM-1 has been found in E. coli and K. pneumoniae strains (2, 3, 5, 11) in other countries in Europe, the two strains with VIM-1 described in the present study are, to our knowledge, the first MBL-positive strains of enterobacteria described in Spain.

Looking to the future, it seems that the rate of diffusion of this kind of ß-lactamase through the enterobacterial population via genetic vectors or clonal expansion is fortunately low.

Nucleotide sequence accession numbers. The sequences for the new integron from E. coli and K. pneumoniae were submitted to GenBank under accession no. AY970968 and AY987853, respectively.


   ACKNOWLEDGMENTS
 
This work was supported by "Fondo de Investigación Sanitaria" grant FIS PI 020358 of the Ministerio de Sanidad of Spain and Spanish Network for the Research in Infectious Diseases Health Institute "Carlos III" (REIPI) C03/14, Madrid, Spain.


   FOOTNOTES
 
* Corresponding author. Mailing address: Servicio de Microbiología, Hospital Vall d'Hebron, Pg. Vall d'Hebron 119-129, 08035 Barcelona, Spain. Phone: 34-93-2746817. Fax: 34-93-2746801. E-mail: gprats{at}vhebron.net. Back


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Antimicrobial Agents and Chemotherapy, August 2005, p. 3492-3494, Vol. 49, No. 8
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.8.3492-3494.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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