Mechanism Of Antibiotics Resistance And Molecular Epidemiology Of Carbapenem Non-susceptible Enterbacter Cloacae

Objective: To explore the characteristics of resistance phenotype and primary research the mechanism of antibiotics resistance in carbapenem-non-susceptible(CNS) Enterobacter cloacae(E. cloacae)isolates collected during 2012-2014 in a teaching hospital of Chongqing.Meanwhile molecular epidemiology of carbapenemase-producing E.Cloacae was also investigated.Methods: Carbapenem-non-susceptible E. cloacae isolates, indentified by VITEK2 Compact automated bacterial identification and susceptibility testing system, were collected from March 2012 to December 2014, and the antibiotic suscepbilities to carbapenem were validated by broth dilution susceptibility test. Carbapenemase genes, extended spectrum β-lactamases(ESBLs) genes, fluoroquinolones resistance determinant(QRDs) genes,aminoglycosides resistance determinant(ARDs) genes of these isolates were determined by PCR assay, and Class I integron gene Intl-1 as well as porin genes omp F and omp C were also identified. Carbapenemase phenotypes were tested by using Carba NP test and modified Hodge test,respectively. Pulsed Field Gel Electrophoresis(PFGE) and Multiple Locus Sequence Typing(MLST) were employed to investigate the molecular epidemiology.Results: 1. In the present study, a total of 101 non-duplicated CNS E.cloacae strains were collected from March 2012 to December 2014 in our hospital. Most of the strains were isolated from urology surgery(19.8%),liver and gallbladder surgery(15.8%), and neurology department(9.9%).The most frequent sites of recovery was the urine(27.7% of isolates).Other major sites of recovery included sputum(23.8% of isolates),secretions(15.8% of isolates) and bile(13.9% of isolates).2. Of all these isolates, 101(100%) were ETP non-susceptible, 88.12% of isolates were ETP resistant. 24(23.76%) were IPM non-susceptible, and25(24.75%) were MEM non-susceptible. 57.43%(58/101) of these strains were resistance to cefepime, and the rates of resistance to aminoglycosides and quinolones were 58.42% and 55.45%, respectively.3. Of all 101 CNS E. cloacae isolates, 14(13.86%) harbored carbapenemase genes: 6 harbored bla NDM-1, 3 harbored bla IMP-8, 2 harbored bla IMP-4 and 1 harbored bla KPC-2, a E. cloacae isolate co-haboring of bla NDM-1 and bla IMP-8. 62(61.39%) carried ESBLs genes: bla TEM(40/101,39.60%), bla SHV(28/101, 27.72%), bla CTX-M(29/101, 28.71%); and 64(63.37%) harbored quinolone genes: qnr A(11/101, 10.89%), qnr B(38/101,37.62%), qnr S(29/101, 28.71%), aac(6’)-Ib-cr(37/101, 36.63%); 43(42.57%) carried aminoglycoside genes: aac(6’)-Ib 43/101, 42.57%), arm A(15/101, 14.85%), rmt B(5/101, 4.95%). Intl-1 gene was detected in 49(48.51%) stains, and deletions in omp F or omp C were found in 29 strains.21(20.79%) omp C gene deficiency, 5(4.95%) omp F gene deficiency.Three strains was detected to possess deletions in the two porin genes simultaneously.4. Carba NP test could detect the results in a relatively short time, by modified Hodge test at least 16 to 24 hours. Carba NP test showed higher specificity and sensitivity than modified Hodge test(100% vs 85.71%).5. PFGE results showed that the 14 carbapenemase-producing E. cloacae isolates were divided into 12 different types. Type F and H had 2 subtypes,respectively. MLST results suggested that the 14 carbapenemase positive isolates were classified into 8 STs, and the results showed genetic relationship among some strains.Conclusion: 1. During the period 2012-2014, the Ertapenem non-susceptible rate of the CNS E. cloacae was more high, and most of the isolates were multiple drug resistance.2. The positive rate of carbapenemase genes was not very high,overexpression of ESBLs genes combined with porin genes lose or degrade might be the primary mechanisms of carbapenem non-susceptible in our hospital. Co-expression of multiple resistant genes and overexpression of Intl might closely relating to multiple drug resistance.3. Most of the strains isolated from urine and sputum, also most of the strains isolated from from urology surgery, liver and gallbladder surgery,and neurology department. Molecular epidemiology analysis revealed clonal diversity.4. Carba NP method is more easy than the modified Hodge test, the sensitivity and specificity are higher and can be used for rapid detection of carbapenemase-producing Enterobacteriaceae isolates.