Molecular characterization of a cephamycin-hydrolyzing and inhibitor-resistant class A beta-lactamase, GES-4, possessing a single G170S substitution in the omega-loop.
The nosocomial spread of six genetically related Klebsiella pneumoniae strains producing GES-type beta-lactamases was found in a neonatal intensive care unit, and we previously reported that one of the six strains, strain KG525, produced a new beta-lactamase, GES-3. In the present study, the molecular mechanism of cephamycin resistance observed in strain KG502, one of the six strains described above, was investigated. This strain was found to produce a variant of GES-3, namely, GES-4, which was responsible for resistance to both cephamycins (cefoxitin MIC, >128 microg/ml) and beta-lactamase inhibitors (50% inhibitory concentration of clavulanic acid, 15.2 +/- 1.7 microM). The GES-4 enzyme had a single G170S substitution in the Omega-loop region compared with the GES-3 sequence. This single amino acid substitution was closely involved with the augmented hydrolysis of cephamycins and carbapenems and the decreased affinities of beta-lactamase inhibitors to GES-4. A cloning experiment and sequencing analysis revealed that strain KG502 possesses duplicate bla(GES-4) genes mediated by two distinct class 1 integrons with similar gene cassette configurations. Moreover, the genetic environments of the bla(GES-4) genes found in strain KG502 were almost identical to that of bla(GES-3) in strain KG525. From these findings, these two phenotypically different strains were suggested to belong to a clonal lineage. The bla(GES-4) gene found in strain KG502 might well emerge from a point mutation in the bla(GES-3) gene harbored by its ancestor strains, such as strain KG525, under heavy antibiotic stress in order to acquire extended properties of resistance to cephamycins and carbapenems.
Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, Musashi-Murayama, Tokyo, Japan.
Jun-ichi
J
Wachino
Yohei
Y
Doi
Kunikazu
K
Yamane
Naohiro
N
Shibata
Tetsuya
T
Yagi
Takako
T
Kubota
Yoshichika
Y
Arakawa
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D015780:Carbapenems / Q000494:pharmacology; D002513:Cephamycins / Q000378:metabolism* / Q000494:pharmacology; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D024881:Drug Resistance, Bacterial; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006868:Hydrolysis; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014169:Transformation, Bacterial; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism; D047090:beta-Lactams / Q000494:pharmacology
D015780:Carbapenems; D002513:Cephamycins; D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D047090:beta-Lactams; D001618:beta-Lactamases; C490566:beta-lactamase GES-4, Klebsiella pneumoniae