Research On Drug-resistant Genes And Virulence Of Carbapenem-resistant Klebsiella Pneumoniae

Objective To investigate carbapenem resistant Klebsiella pneumoniae(CRKP) isolates from Tianjin Medical University General Hospital to all kinds of antibiotic sensitivity, and collect the clinical characteristics of the patients to assess the risk factors for development of CRKP infection; explore the relationship between resistance mechanism and carbapenems sensitivity,and detect the distribution of high virulence capsular serotypes and virulence genes.Methods A total of 20 non-repetitive CRKP were collected from General Hospital of Tianjin Medical University, between May 2014-May2015. The bacteria identification and the antimicrobial susceptibility test were performed by using VITEK-2 compact automatic system and confirmed with disk diffusion method for tigecycline and carbapenem. Retrospective analysis on the clinical characteristics of patients, including age, gender, department, basic disease, specimens source, ICU stay, invasive operation, antibiotic treatment and outcome. The modified Hodge test, EDTA double disk test, carbapenem inactivationd method(CIM)and Carba NP test were used to detection carbapenemases phenotypes. Polymerase chain reaction(PCR) was used to detect antimicrobial-resistant gene,including carbapenemases gene(KPC, IMP, VIM, NDM, SIM, OXA-48),ESBLs(TEM, SHV, CTX-M), Amp C ?-lactamases(DHA) and outer membrane encoding gene(ompk35, ompk36). The hypermucoviscosity, serum resistance, and ability to form biofilm were sought by phenotypic assays. PCR was used to investigate the presence of the genes specific for serotypes(K1, K2, K5, K20, K54, K57) and virulence genes(rmp A, aerobactin, fim H-1).The DNA sequencing were compared with those in the National Center Biotechnology Information database.Results All isolates exhibited resistance to cephalosporins and carbapenems, the resistant rate were equal or great than 80%,but to tigecycline ?amikacin and levofloxacin were lower than 30%. The patients were mainly from the cadre ward and pediatric ward, and sputum specimen were common. The source of infection were mainly from hospital acquired infection. The modified Hodge test detected carbapenemase was in11 strains(55%), EDTA double disk test detected metal enzyme was in 13 strains(65%), CIM test was positive in16 strains, Carba NP test detected A class of carbapenemase 11 strains, the class A and class B or class D carbapenemase 9 strains; The PCR showed that KPC and NDM gene, respectively, 7 strains(35%) and 8 strains(40%), SHV gene was the highest, 16 strains(80%) and DHA gene 2 strains(10%) and membrane pore protein coding gene ompk35 and ompk36 were lack of 8 strains(40%) and 13 strains(65%).The resistance genes combination modes were classified into 7 modes,the most important combination mode was carbapenemases ?ESBLs and porin deficiency,35%(7/20),the second mode was only ESBLs gene, 20%(4/20).The third mode was carbapenemases and porin deficiency,15%(3/20). The mode of carbapenemases and ESBLs or the mode of ESBLs and porin deficiency also occupied a certain position, both 10%(2/20).Else mode were 10%(2/20).The positive strains of virulence gene rmp A were 8 and all carried carbapenemases and porin deficiency at the same time,5 of KPC gene, 2 of NDM, one of KPC and NDM,5 strains also carried ESBLs genes. The positive strains of aerobactin were 6, 5 strain with rmp A and KPC genes simultaneously. All strains carried fim H-1. In total, 3 and 1 of the 20 strains of CRKP were belonged to K1 and K57 capsular serotype, 2 of K1 with rmp A and aerobactin genes at the same time and 1 of K1 and K57 only with rmp A and the four all carried KPC gene.Conclusion The study of clinical isolates of carbapenem resistant Klebsiella pneumoniae were lower resistant to tigecycline, amikacin, levofloxacin and ciprofloxacin,but cephalosporins, enzyme inhibitor class antimicrobial resistance rate is higher. Elderly patients and neonatal were mainly infected by CRKP in the hospital, invasive operation and environmental disinfection failure were important causes of nosocomial spread of epidemic strains. The KPC and NDM were the main mechanism leading to the drug resistance to carbapenems of klebsiella pneumoniae. Hyperproduction of ESBLs with deficiency of Omp gene were also the important resistant mechanism. The rate of K1 and K57 serotypes strains carried KPC was the highest in the world at present.