Characterization Of Drug-susceptibility And Resistance Mechanism In Enterobacter Cloacae Among Patients With Clinical Infections

Objective The study was conducted to determine the drug susceptibility, prevalence of beta-Iactamases and integrons among isolates of Enterobacter cloacae. To analyse the molecular epidemiological characteristics of nosocomial resistant isolates, control spread of resistant isolates in the hospital.Methods A total of114non-repetitive E. cloace were isolated from January2008to June2011in the General Hospital of Tianjin Medical University. The antimicrobial susceptibility test was performed by using VITEK-2compact automatic system and microbroth dilution method. Isolates of E. cloace were screened for beta-lactamases by cefoxitin improved three-dimensional test, disk diffusion phenotypic confirmatory test, modified Hodge test, meropenem improved three-dimensional test and meropenem-EDTA synergy test. Isolates were then subjected to the touchdown PCR targeting genes encoding for4group beta-lactamases (AmpC, ESBLs, OXA-like, A/B type carbapenamases) and integrons. Single PCR was conducted to detect the insertion sequence ISEcpl, aac(6’)-Ib. Isolates harboring class I or class II integron genes were further investigated by PCR and sequencing of the variable regions. Conjugation experiments were carried out to detect the transferability of E. cloacae. To compare the similarity of the isolates, ERIC was used.Results1、Drug-susceptibility The multidrug resistance status of E. cloacae clinical isolates was severe. Imipenem and meropenem was the most active antimicrobial agent tested in this study (100%), amikacin and ertapenem were the second active agent (97.4%). All the114isolates exhibited resistant to cefoxitin, followed by cefotaxime (45.6%).2、Phenotype46.5%isolates were positive in cefoxitin modified three dimensional tests and14.9%positive in disk diffusion phenotypic confirmatory test.5.3%demonstrated positive in the modified Hodge test,12.3%were positive in the meropenem improved three-dimensional test. No isolate was positive in meropenem EDTA synergy test.3、Detection of touchdown PCR①the touchdown-PCR indicated that41.2%harbored AmpC beta-lactamase genes, mainly was MIR-type (40/47), and followed by DHA-1-type (12/47). No isolate carried with CIT-, FOX-, MOX-and ACC-type of AmpC beta-lactamase.29.8%possessed broad beta-lactamase gene,52.9%(18/34) carried with ESBLs genes, mainly was blaCTX-M (16/18). Analysis of the genetic context of blaCTX-M showed the presence of ISEcp1, while other type of ESBLs was not existed. And two isolates carried with OXA-1-type ESBLs. TEM-type (27/34) was the main non-ESBLs, and two isolates were SHV-type ESBLs producers. No isolate was carbapenemas producer in this study.②26isolates (22.8%) were carried with class1integron, and23strains owned the variable region while3didn’t. Two different length of variable region were detected with700bp (2isolates) and1000bp (21isolates), which harbored4gene cassettes. PCR amplification and sequencing revealed that2isolates contained a709bp gene cassette array with dfr A15. A1087bp PCR product was obtained from18isolates. And sequencing confirmed the presence of gene cassettes aadB-aadA2.3integron-positive isolates contained a1009bp gene cassette array with aadAl. No isolate harbored Int2.③Six isolates were aac(6’)-Ib producer.4、Sequencing Sequencing revealed that blaMIR、blaDHA、blaCTX-M、blaOXA-1、blaTEM、 blaSHV、aac(6’)-Ib were blaMIR-3、blaDHA-1、blaCTX-M-3/1、blaoxA-1、blaTEM-1、blaSHV-11and aac(6’)-Ib-cr, respectively.5、Fifteen to sixteen donors with CTX-M genes were successed in conjugation experiment.6、ERIC of63isolates showed11genotypes among E. cloacae with enzymes. The ERIC patterns were as followed:pattern A (26isolates), pattern B (9isolates), pattern C (8isolates), pattern D (8isolates), pattern E (2isolates), pattern F (2isolates), pattern G (2isolates, producing OXA-1), pattern H (2isolates), pattern I (2isolates), pattern J (1isolate), pattern K (1isolate).Conclusion (1)Multi-drug resistance was existed in E. cloacae with enzymes according to this study.(2)The phenotype tests could be used for screening beta-lactamases in E. cloacae. But it’s still necessary to carry out PCR assays designed for the detection of genes encoding these enzymes.(3)There was a widespread of AmpC beta-lactamases, broad beta-lactamases and class I integron in E. cloacae.(4)The resistance determinants ESBLs, AmpC β-lactamase had been rapidly becoming responsible for carbapenem reduced susceptibility or resistance.(5)ISEcpl could provide promoters and enhance the transferability of blaCTX-M.(6)Class1integron mainly mediated the resistance to aminoglycoside and trimethoprin.(7)aac(6’)-Ib-cr could mediate he resistance to aminoglycoside and fluoroquinolone.(8)Drug-resistant genes could transmite among bacteria by plasmids, which carried by E. cloacae.(9)And there were scattered transmission among E. cloacae with enzymes in the hospital environments.