Extended-Spectrum Beta-Lactamases among Enterobacter Isolates Obtained in Tel Aviv, Israel

Abstract

The extended-spectrum beta-lactamase (ESBL)-producing phenotype is frequent among Enterobacter isolates at the Tel Aviv Sourasky Medical Center, Tel Aviv, Israel. We examined the clonal relatedness and characterized the ESBLs of a collection of these strains. Clonal relatedness was determined by pulsed-field gel electrophoresis. Isoelectric focusing (IEF) and transconjugation experiments were performed. ESBL gene families were screened by colony hybridization and PCR for bla TEM , bla SHV , bla CTX-M , bla IBC , bla PER , bla OXA , bla VEB , and bla SFO ; and the PCR products were sequenced. The 17 Enterobacter isolates studied comprised 15 distinct genotypes. All isolates showed at least one IEF band (range, one to five bands) whose appearance was suppressed by addition of clavulanate; pIs ranged from 5.4 to >8.2. Colony hybridization identified at least one family of beta-lactamase genes in 11 isolates: 10 harbored bla TEM and 9 harbored bla SHV . PCR screening and sequence analysis of the PCR products for bla TEM , bla SHV , and bla CTX-M identified TEM-1 in 11 isolates, SHV-12 in 7 isolates, SHV-1 in 1 isolate, a CTX-M-2-like gene in 2 isolates, and CTX-M-26 in 1 isolate. In transconjugation experiments with four isolates harboring bla TEM-1 and bla SHV-12 , both genes were simultaneously transferred to the recipient strain Escherichia coli HB101. Plasmid mapping, PCR, and Southern analysis with TEM-and SHV-specific probes demonstrated that a single transferred plasmid carried both the TEM-1 and the SHV-12 genes. The widespread presence of ESBLs among Enterobacter isolates in Tel Aviv is likely due not to clonal spread but, rather, to plasmid-mediated transfer, at times simultaneously, of genes encoding several types of enzymes. The dominant ESBL identified was SHV-12.Enterobacter spp. are leading nosocomial pathogens (26) that commonly cause pneumonia (25) and that are the most frequent gram-negative organisms causing bloodstream infections in intensive care units (14, 33). More than one-third of the Enterobacter sp. isolates in intensive care units reporting to the National Nosocomial Infection Surveillance System (26) are resistant to extended-spectrum cephalosporins. Moreover, treatment with extended-spectrum cephalosporins may lead to the emergence of resistance to these antimicrobial agents among susceptible strains of Enterobacter spp. (8,19,35). Emergence of resistance results in increased rates of mortality, longer hospital stays, and higher hospital charges (9, 10).The resistance of Enterobacter spp. to beta-lactam antibiotics is most frequently mediated by hyperproduction of chromosomal AmpC beta-lactamase, caused either by induction or, more likely, by selection of derepressed mutants (1). In the last decade, the production of plasmid-mediated extended-spectrum beta-lactamases (ESBLs) has been recognized as an additional important emerging mechanism of resistance among members of the family Enterobacteriaceae (3, 17), including clinical isolates of Enterobacter spp. (31). The most common ESBLs fo...

Utility of the VITEK 2 Advanced Expert System for Identification of Extended-Spectrum β-Lactamase Production in Enterobacter spp

“…These epidemiologically unrelated isolates were identified phenotypically as ESBL producers via the disk diffusion method established for E. coli, Klebsiella spp., and P. mirabilis (2, 3), using 30-g cefotaxime-, ceftazidime-, and cefepime-impregnated disks with and without 10 g clavulanic acid (cefotaxime-and ceftazidime-containing disks made by Oxoid [Basingstoke, Hampshire, England]; cefepime-containing disks were prepared in-house). Although CLSI does not recommend the disk diffusion method for Enterobacter, we and others have previously noted a strong correlation between demonstration of a clavulanic acid effect and the presence of an ESBL gene (3,12,15). E. coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were used as negative and positive controls for ESBL production, respectively.…”

“…by earlier automated systems, including VITEK, was hampered by the production of AmpC ␤-lactamase (17), the more recently developed AES has not been evaluated for Enterobacter specifically. Given the high proportion of ESBL production among Enterobacter isolates at our institution and elsewhere (8,12) and the fact that the AES includes ESBL production among the resistance mechanisms it suggests for Enterobacter isolates, we sought to determine the accuracy of the AES in ESBL detection among clinical isolates of Enterobacter.…”

“…While guidelines exist for ESBL detection in Escherichia coli, Klebsiella spp., and Proteus mirabilis isolates only (2), these enzymes are produced by a wide variety of other gram-negative organisms as well, including Enterobacter spp. (3,8,12,13).…”

“…by earlier automated systems, including VITEK, was hampered by the production of AmpC ␤-lactamase (17), the more recently developed AES has not been evaluated for Enterobacter specifically. Given the high proportion of ESBL production among Enterobacter isolates at our institution and elsewhere (8,12) and the fact that the AES includes ESBL production among the resistance mechanisms it suggests for Enterobacter isolates, we sought to determine the accuracy of the AES in ESBL detection among clinical isolates of Enterobacter.Forty unique-patient isolates of Enterobacter were included (30 E. cloacae isolates and 10 E. aerogenes isolates). These epidemiologically unrelated isolates were identified phenotypically as ESBL producers via the disk diffusion method established for E. coli, Klebsiella spp., and P. mirabilis (2, 3), using 30-g cefotaxime-, ceftazidime-, and cefepime-impregnated disks with and without 10 g clavulanic acid (cefotaxime-and ceftazidime-containing disks made by Oxoid [Basingstoke, Hampshire, England]; cefepime-containing disks were prepared in-house).…”

“…While guidelines exist for ESBL detection in Escherichia coli, Klebsiella spp., and Proteus mirabilis isolates only (2), these enzymes are produced by a wide variety of other gram-negative organisms as well, including Enterobacter spp. (3,8,12,13).The VITEK 2 Advanced Expert System (AES; bioMérieux, Durham, NC) is an automated system that uses the antimicrobial susceptibility data generated to suggest the phenotype of the tested isolate and thereby determine susceptibility or resistance to antibiotics not tested (10). It has been used successfully to determine ESBL presence in E. coli and Klebsiella spp.…”