In vivo development of carbapenem resistance in clinical isolates of Enterobacter aerogenes producing multiple beta-lactamases.
Four clinical strains of extended-spectrum beta-lactamase- and AmpC-producing Enterobacter aerogenes were isolated successively from a liver transplantation patient. Isolates C(1) and C(2) were isolated prior to carbapenem therapy, whilst isolates C(3) and C(4) were recovered after 40 days of carbapenem therapy. The homology of these strains was analysed by pulsed-field gel electrophoresis (PFGE). beta-Lactamases were analysed by isoelectric focusing, polymerase chain reaction (PCR) and sequencing. Outer membrane proteins were analysed by PCR, sequencing, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blot. Disruption of OmpE36 in C(1) in vitro was also performed by homologous gene recombination. The isolates demonstrated an indistinguishable PFGE pattern. Molecular characterisation revealed that, in addition to the pre-existing multiple beta-lactamases (DHA-1, TEM-1, SHV-5, CTX-M-3 and CTX-M-14) found in C(1) and C(2), isolates C(3) and C(4) failed to express OmpE36 owing to insertional inactivation by an IS903-like insertion sequence. Other resistance mechanisms, such as production of carbapenem-hydrolysing enzymes or expression of chromosomal efflux, were apparently not involved. Completely replacing OmpE36 by the kanamycin resistance gene (kan) resulted in a significant increase in carbapenem minimum inhibitory concentrations of an ompE36 mutant. Thus, C(3) and C(4) were apparently derived from the previously imipenem-susceptible isolates C(1) and C(2). Following carbapenem exposure, depletion of OmpE36 expression resulted in the collateral effect of carbapenem resistance.
State Key Laboratory of Infectious Disease Diagnosis and Treatment, First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang, China.
Ya-Gang
YG
Chen
Ying
Y
Zhang
Yun-Song
YS
Yu
Ting-Ting
TT
Qu
Ze-Qing
ZQ
Wei
Ping
P
Shen
Lan-Juan
LJ
Li
D000328:Adult; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis; D015780:Carbapenems / Q000494:pharmacology*; D024901:Drug Resistance, Multiple, Bacterial; D021902:Enterobacter aerogenes / Q000187:drug effects* / Q000201:enzymology / Q000302:isolation & purification; D004756:Enterobacteriaceae Infections / Q000188:drug therapy* / Q000382:microbiology; D017809:Fatal Outcome; D006801:Humans; D015378:Imipenem / Q000494:pharmacology; D016031:Liver Transplantation / Q000009:adverse effects; D008297:Male; D000077731:Meropenem; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D013845:Thienamycins / Q000494:pharmacology; D018440:beta-Lactam Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000096:biosynthesis*
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D015780:Carbapenems; D013845:Thienamycins; D015378:Imipenem; C048795:AmpC beta-lactamases; D001618:beta-Lactamases; D000077731:Meropenem