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.
Wachino
Jun-ichi
J
Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Diseases, Musashi-Murayama, Tokyo, Japan.
Doi
Yohei
Y
Yamane
Kunikazu
K
Shibata
Naohiro
N
Yagi
Tetsuya
T
Kubota
Takako
T
Arakawa
Yoshichika
Y
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