Investigation Of Carbapenem Resistance Mechanismsin Clinical Isolates Of Enterobacteriaceae

Objectives:Investigate the mechanisms of carbapenem resistance in 86 clinical isolates of Enterobacteriaceae by detection ofβ-lactamase, outer membrane proteins, and efflux pump. Investigate the transmission mechanisms of carbapenem resistance gene by analysis of genetic structures surrounding carbapenem resistance gene.Methods:Antibiotic susceptibilities were determined by agar dilution method. Pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) were performed to analyze the molecular epidemiology of isolates. Isoelectric focusing (IEF), PCRs, and DNA sequencing were carried out to confirm the genotype of P-lactamases. Conjugation experiments, elimination of plasmid, and southern blot were performed to confirm whether the carbapenem resistance gene was located on plasmid. Outer membrane proteins (OMPs) were extracted and examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Inhibition assays by carbonyl cyanide m-chlorophenylhydrazone (CCCP) were performed to confirm whether the presence of efflux pump. Typing the carbapenem resistance gene encoding plasmids on the basis of the restriction maps of EcoRI and HindⅢ. Two representative plasmids were sequenced and the DNA sequence surrounding carbapenem resistance gene were analyzed.Results:Eighty-six isolates exhibited various level of carbapenem resistance. Susceptibility rate to imipenem, meropenem, and ertapenem were 43.0%,44.2%, and 1.2%, respectively. Resistance rate and resistance level to otherβ-lactams and ciprofloxacin were high, whereas polymoxin B showed strong activity against carbapenem-non-susceptible Enterobacteriaceae with susceptibility rate of 92.9%, followed by amikacin (80.2%). Major isolates from the same hospital were indistinguishable or closely related to each other. Eighty-two isolates produced KPC-2 carbapenemase,5 produced IMP metallo-β-lactamase (3 of them produced KPC-2 simultaneously), and 2 did not produced any carbapenemase. Severalβ-lactamase genes of blaTEM, blaSHV-11 or-12, and blaCTX-M-14 or -3 were prevalent in these isolates, and majority produced more than oneβ-lactamases. Reduced carbapenem susceptibility can be transferred from most isolates to E. coli (EC600) by conjugation. Southern blot analysis confirmed that blaKPC-2 was located on a ca.50kb conjugative plasmid. Four K. pneumoniae (including two isolates which did not produce carbapenemase) failed to express OmpK36 because of nonsense mutations or insertional inactivation by an insertion sequence in ompK36 gene. An E. cloacae lack a 38 kDa OMP, and expression of two OMPs were decreased in a C. freundii. CCCP can not reduced the minimum inhibitory concentrations values (MICs) of imipenem in Enterobacteriaceae.Eighty-two blaKPC-2-encoding plasmids can be divided into 11 types (type I to XI) according to the restriction maps of EcoRI and HindⅢ. The type I plasmid predominant (62/82), and 8 isolates carried type V plasmid. Two plasmids of pKP1 (type I) and pKP20 (type V) were sequenced. A 7788bp DNA sequence from pKPl and a 6000bp from pKP20 were obtained. Sequence analysis revealed several transposon-associated elements surround blaKPC-2. pKP1 encoded Tn3 transposase, Tn3 resolvase,ISKpn8 transposase, KPC-2, and ISKpn6-like transposase, while pKP20 encoded IS26 transposase, truncated Tn3 resolvase,ISKpn8 transposase, KPC-2, truncated ISKpn6-like transposase, and IS26 transposase. DNA sequence for truncated Tn3 resolvase,ISKpn8 transposase, KPC-2, and truncated ISKpn6-like transposase were the common segment in two plasmids (total 3354bp). A series of primers were designed according to the sequence of pKP1 and pKP20 to amplify the blaKPC-2-surrounding sequence from other 80 plasmids. Sixty-nine plasmids and pKPl shared the same PCR results, and 8 type V plasmids and pKP20 have the same PCR results. All blaKPC-2-encoding plasmids except 3 contained the common 3354bp segment. Another 2 variants were detected in 2 plasmids.Conclusions:Carbapenem-resistant Enterobacteriaceae clonally spread within the same hospital. Carbapenem resistance in Enterobacteriaceae was mainly due to production of carbapenemases, especially KPC-2. Carbapenemases production combined with reduced amounts of porins contributed to high-level resistance to carbapenems. Combination of extended-spectrumβ-lactamase and OmpK36 deficiency resulted in ertapenem resistance, and reduced imipenem and meropenem susceptibility in K. pneumoniae. The same blaKPC-2-encoding plasmids wide disseminated among different species. Majority of the blaKPC-2 genes were located on the same transposon. Four transposon variants were detected in other blaKPC-2-encoding plasmids.