Molecular characterization of a ceftazidime-resistant Morganella morganii isolate producing a TEM-10 beta-lactamase.

Abstract

Among the various pathogens responsible for urinary infections can be found Morganella morganii. The different strains of this species are usually resistant to ampicillin, to the amoxicillin-clavulanic acid combination, and to cephalothin, and usually they are susceptible to other antibiotics active against gram-negative bacilli (7). The occurrence of extended-spectrum β-lactamases (ESBL) in M. morganii has been reported (3), although until now only one has been characterized at the molecular level (11). A strain of M. morganii (FFLM15), showing resistance to ceftazidime and aztreonam and reduced susceptibility to cefotaxime by the disk diffusion method, was isolated from the urine of a neonate hospitalized in the neonatology unit of Hospital de Santa Maria in Lisbon, Portugal. The double disk test for synergy between expanded-spectrum cephalosporins and clavulanic acid was performed, and the positive result indicated the presence of an ESBL producer. This isolate was also resistant to netilmicin, gentamicin, and amoxicillin. Transconjugants of a nalidixic acid-resistant mutant of Escherichia coli C600 were obtained on agar containing nalidixic acid (50 μg/ml) and ceftazidime (30 μg/ml) by the plate-mating method (4). These transconjugants showed the same pattern of resistance as that of FFLM15. The MICs for FFLM15 and its transconjugant were determined by the E test (AB Biodisk). The values obtained showed that the E. coli transconjugant was more resistant to aztreonam than FFLM15 (Table ​(Table1).1). Isoelectric focusing of crude β-lactamase extracts was performed on polyacrylamide gels containing ampholines (Pharmacia) with a pH range of 3 to 9, and the expression was revealed by nitrocefin. Both FFLM15 and its transconjugant expressed a single β-lactamase with an estimated isoelectric point (pI) of 5.6. From both FFLM15 and its transconjugant, we isolated a plasmid estimated to be 50 kb, which showed the same restriction fragment patterns after enzymatic restriction. As the pI obtained was identical to those of TEM-10 and TEM-26, it was expected that a TEM enzyme was present. PCR of the DNA of the transconjugant was performed with primers specific for TEM genes (5′-GAAAGGGCCTCGTGATACGC-3′ and 5′-GGAGTCAGGCAACTATGGATGA-3′, corresponding to nucleotides 12 to 31 and 996 to 1017, respectively) (5). A PCR product of 1,006 bp was cloned in the phage M13 single strand and sequenced by the dideoxy-chain termination method. Two additional oligonucleotides (5′-GAAAAGCATCTTACGGATGGC-3′ and 5′-CCATAACCATGAGTGATAACACTGC-3′, corresponding to nucleotides 519 to 539 and 568 to 592, respectively) (this study) were used to obtain the complete sequences of both strands. The nucleotide sequence analysis showed that the gene encoded a TEM-10 β-lactamase. Compared with ancestral TEM-1 it shows two nucleotide mutations at positions 682 (C→A) and 906 (G→A) (numbering system of Sutcliffe [10]) that lead to amino acid replacements at positions 164 and 240 from Arg to Ser and from Glu to Lys, respectively (numbering system of Ambler et al. [1]), substitutions already described for TEM-10 (2, 8, 9). The sequence was confirmed, a second time, with a BamHI-EcoRV restriction fragment obtained from the same plasmid. TABLE 1 MICs for M. morganii clinical isolate FFLM15 and its E. coli transconjugant TEM and SHV enzymes are most common in Klebsiella but also occur in other enterobacteria (6). A TEM-10 enzyme has been detected in a member of Proteae. In 1995 a strain of Proteus mirabilis synthesizing a TEM-10 was isolated in an American hospital (8). M. morganii FFLM15 also synthesized a TEM-10. This enzyme is common in the United States but has not been frequently identified in European centers where other ESBL have been reported (2). The MIC of aztreonam for M. morganii FFLM15 (3 μg/ml) was lower than the MIC of P. mirabilis harboring TEM-10 (>32 μg/ml) (8), although the MIC for the corresponding transconjugant of FFLM15 was higher (>256 μg/ml). Like other TEM-10 enzymes reported (2, 8, 9), the TEM-10 synthesized by FFLM15 hydrolyzed ceftazidime more rapidly than cefotaxime and aztreonam. The enzyme TEM-10 has already been isolated from various strains of Klebsiella in this hospital; however, this is the first identification of a TEM-10 in M. morganii. It is not known whether this blaTEM-10 gene evolved directly in M. morganii or was acquired from a strain of Klebsiella. This is the first report describing a clinical isolate of M. morganii producing a TEM-10. Nucleotide sequence accession number. The sequence of the 1,006-bp PCR product has been deposited in GenBank under accession no. {type:entrez-nucleotide,attrs:{text:AF093512,term_id:3851587}}AF093512.