Characterization of blaTEM-3 and blaSHV-4 beta-lactamase-encoding genes in Citrobacter diversus

Citrobacter diversus (also called Citrobacter koseri) is an opportunistic pathogen which causes various infections (4). C. diversus is intrinsically sensitive to all antibiotics active against gram-negative bacilli, except for penicillins (7). Additional resistances, to b-lactam antibiotics in particular (2), have rarely been reported. Between 1989 and 1991, we isolated three clinical strains of C. diversus (Cd2, Cd3, and Cd4) resistant to most b-lactam agents, including expanded-spectrum cephalosporins and aztreonam (Table 1); b-lactamase inhibitors were highly synergistic with these antibiotics against the isolates. These isolates were also resistant to most aminoglycosides, chloramphenicol, sulphonamides, and trimethoprim. b-Lactam resistance of the three C. diversus strains was transferable to Escherichia coli by conjugation or transformation. Analytical isoelectric focusing on polyacrylamide gels indicated that the three C. diversus isolates produced two types of b-lactamases: one enzyme with a pI of ;5 (pI of 4.8 for Cd2 and Cd3 and pI of 5.4 for Cd4), likely to be the species-specific constitutive penicillinase (7), and one additional enzyme with a pI of 6.3 (Cd2 and Cd3) or 7.8 (Cd4), the single b-lactamase synthesized by the transformants or the transconjugants, expected to be an extended-spectrum b-lactamase (ESBL) (Table 1). Plasmid DNA extracted from the transformants in E. coli (pCd2, pCd3, and pCd4) demonstrated the presence of a single large plasmid. Plasmids pCd2 and pCd3 (;150 kb) gave the same patterns after restriction by BglII and SspI; an internal blaTEM-1 probe hybridized with the same BglII fragment (;20 kb). Plasmid pCd4 (between 40 and 60 kb) gave a distinct restriction pattern with both enzymes; an internal blaSHV-3 probe hybridized with an SspI fragment (2.7 kb). It is highly probable that the ESBL produced by Cd4 was an SHV-4 enzyme, as SHV-4 is the only SHV-derived enzyme with a pI of 7.8 described at present. However, the identity of the ESBL produced by Cd2 and Cd3 was more uncertain, since several TEM-derived ESBLs have a pI of 6.3 (TEM-3, TEM18, and TEM-22) (8). Consequently, the structural genes of the ESBLs synthesized by Cd2 and Cd4 were amplified by polymerase chain reaction by using a set of oligonucleotide primers complementary to both strands of the TEM-1 (10) and the SHV-1 (5) genes and directly sequenced. The nucleotide sequence of the blaTEM-encoding gene of Cd2 differed from that of the blaTEM-1 gene (10) at seven nucleotide positions: four of them were silent and the remaining three caused substitutions of amino acids (Gln 393Lys, Glu 1043Lys, Gly 2383Ser) (Table 1) as previously described for TEM-3 (9). The nucleotide sequence of the blaSHV-encoding gene of Cd4 differed from that of the blaSHV-1 gene by three base changes, all of which resulted in amino acid substitutions: Arg 2053Leu, Gly 2383Ser, and Glu 2403Lys; the blaSHVpCd4 sequence was identical to that of blaSHV-4 except for one silent mutation (G3A, Lys-240) (6) (Table 1). The present study is the first genetic characterization of ESBLs in C. diversus and the first report of an SHV-type ESBL in this species.