Identification of TEM-10 beta-lactamase in a Kluyvera sp. and other Enterobacteriaceae at a Portuguese hospital.

Abstract

Among the Enterobacteriaceae, extended-spectrum β-lactamases (ESBLs) have been found mainly in Klebsiella spp. and Escherichia coli but have been reported also in other genera (1, 2, 3, 4, 7, 9). In the case of Kluyvera ascorbata cefotaxime (CTX)-M type β-lactamases were found (5); however, the present work is the first report of a clinical isolate of a Kluyvera sp. producing a TEM-10 β-lactamase, isolated from the urine of a patient infected with human immunodeficiency virus (HIV) and hospitalized in the intensive care unit. At Hospital de Santa Maria (HSM) plasmid-mediated TEM-10 β-lactamases were identified first in a Morganella morganii isolate (1) and 5 years later in Klebsiella pneumoniae isolates (2). Between February 1999 and April 2001, 33 ESBL-producing isolates were selected from 108 clinical isolates of Enterobacteriaceae, including 24 isolates of Klebsiella pneumoniae, 6 isolates of E. coli, 1 isolate of Enterobacter cloacae, 1 isolate of Enterobacter agglomerans, and 1 isolate of a Kluyvera sp. These isolates were recovered from inpatients in different wards (9 from intensive care units, 6 from surgery wards, and 18 from medicine wards) and were isolated from the following specimens (number of specimens in parentheses): urine (18), blood (3), sputum (7), wound (3), catheter (1), and feces (1). All the strains were identified by the API System 20E. The BBL Crystal E/NF ID system and cellobiose fermentation also confirmed the identification of the Kluyvera sp. The isolates selected for the study were those that, according to the antibiogram obtained by disk diffusion (6), showed resistance to ceftazidime (CAZ) and aztreonam (ATM) and reduced susceptibility to CTX. The positive synergy test with clavulanic acid (CLA) suggested the presence of an ESBL producer. The conjugation assay was performed by broth mating using E. coli C600 (Nalr) as the recipient strain. Plasmid DNA analyses showed that clinical isolates and transconjugants contained a plasmid of ca. 50 kb, although clinical isolates presented various smaller plasmids. An extract from the culture of each isolate was subjected to analytical isoeletric focusing as described elsewhere (2), showing a β-lactamase with a pI of 5.6. The MICs of β-lactam antibiotics were determined with E-test strips (AB Biodisk, Solna, Sweden). For Kluyvera sp. the MICs of amoxicillin (AMX)-CLA and CAZ were the same as those observed for its transconjugants (4 and 64 μg/ml, respectively). The MICs of ATM (8 μg/ml) and CTX (0,38 μg/ml) were lower than those observed for its transconjungant (64 and 1 μg/ml, respectively). The other clinical isolates and respective transconjugants presented for CAZ MICs of >32 μg/ml and MIC ranges of (in μg/ml) 6.0 to >256 for ATM, 0.38 to 3.0 for CTX, and 3 to 8 for AMX-CLA. The presence of ESBLs was confirmed by the ≥8.0 ratios of the MIC of CAZ over that of CAZ plus CLA (AB Biodisk). PCR experiments were performed with primers specific for detecting TEM genes (1) with plasmid DNA extracts (8) of clinical isolates and transconjugants as templates. A 1,052-bp amplification product was obtained from all the isolates. The deduced amino acid sequence was 100% identical to that of TEM-10 (1). ESBLs are usually encoded by genes within plasmids that are easily transmitted among different members of the Enterobacteriaceae (7). At HSM an emergence of K. pneumoniae isolates harboring high-stability plasmids containing the blaTEM-10 gene has been reported (2), and the blaTEM-10 gene has been disseminated within conjugative plasmids among different Enterobacteriaceae species and was also disseminated among different wards. The spread of plasmids is no longer exceptional (7) and can involve different species for which TEM-10 had not yet been described, like Kluyvera sp.