Study On The Molecular Characteristic Of Carbapenem-non-susceptible Escherichia Coli And Fitness Cost Of NDM-1

Carbapenems are one of the most important antimicrobial agents that are effective in the treatment of infections caused by extended-spectrum β-lactamase(ESBL)- and/or Amp C β-lactamase producing Enterobacteriaceae. However, the widespread use of carbapenems has led to the emergence of carbapenem-non-susceptible or carbapenem-resistant Enterobacteriaceae worldwide. The most prevalent mechanism of carbapenem resistance among Enterobacteriaceae is the acquisition of carbapenemases, and both the Klebsiella pneumoniae carbapenemase(KPC) and the New Delhi metallo-β-lactamase(NDM) are the most prevalent carbapenemases in the world. Escherichia coli is one of the most popular Enterobacteriaceae in human clinical and veterinary clinical, and at the same time it is an opportunistic pathogens and commensal bacteria, it can cause serious infection. In recently years, carbapenem-resistant E. coli has been increased detection year by year. For many years, carbapenem resistant bacteria have been found mainly in medical settings and there are rarely researches about the epidemiology of carbapenem resistant bacteria isolated from animals and envirnonmental. Fitness cost refers that in order to adapt to changing environmental conditions and the bacteria should changes himself. There exist some relationship between resistance and fitness cost. It has been proved that the resistance bacteria can change the growth, virulence, and stability when compared with the susceptible one. If the resistance bacteria could not compare with the susceptible one, the resistance gene will be lost. So that, in this study, we studied the molecular characteristic of carbapenem-non-susceptible E. coli and we also detected the fitness cost of bla NDM-1 in E. coli.In the present study, a total of 488 E. coli strains isolated from pigs were collected from different districts of Heilongjiang province from 2009 to 2014, and we also collected 105 E. coli strains isolated from Majigou river across the Harbin city from 2013 to 2014. The total 593 E. coli strains antimicrobial susceptibility to 22 antimicrobial agents was determined by broth microdilution. The antimicrobial susceptibility test reveal that most of the E. coli isolated from pigs and rivers were m-ultidrug resistance, the resistance rates to amocicillin, tetracycline, sulfamethoxazole trimethoprim were exceed 90%. There are significant difference between pig isolates and river isolates on resistance rates to amocicillin/clavulanic acid, ceftiofur, ceftriaxone, cefotaxime, ceftazidime, aztreonam, imipenem, meropenem, ertapenem, florfenicol, ciprofloxacin, enrofloxacin, colistin, tigecycl- ine. A total of 40 carbapenem-non-susceptible(CNS) E. coli isolates were collected for further study. The E. coli isolated from river have a higher MIC50 and MIC90 compared to E. coli isolated f- rom pigs, it proved that there existed a serious antimicrobial resistance problem in river isolates.M HT and EDTA synergy test were used to detect the carbapenemase on the 40 CNS E. coli. 12 isolates were MHT positive and 5 isolates were EDTA synergy positive, the MHT-positive isolates might be the carbapenemase-producing one and the EDTA synergy positive might carry metal carbapenemase. Polymerase chain reaction(PCR) and sequencing were performed to identify genes for carbapenemase(bla BIC, bla KPC, bla IMP, bla VIM, bla NDM, bla GIM, bla SPM, bla SIM, bla AIM, bla DIM, and bla O- XA-48), extended spectrum β-lactamase(bla TEM, bla SHV, blaCTX-M, bla CTX-M-1-group, bla CTX-M-2-group, bl- a CTX-M-8-group, bla CTX-M-9-group, blaCTX-M-25-group, and bla OXA), Amp C lactamase(bla MOX, bla CMY, bla- DHA, bla ACC, bla EBC, and bla FOX), plasmid-encoded 16 S r RNA methylases(arm A, rmt A, rmt B, rmt C, rmt D, rmt E, and npm A), plasmid-mediated quinolone resistance(qnr A, qnr B, qnr C, qnr D, qnrS, q- ep A, oqx AB, and aac(6’)-Ib-cr). Overall, bla KPC gene was detected in 5 isolates, and bla NDM gene was also detected in 5 isolates, the gene subtype were bla KPC-2 and bla NDM-1 through sequencing. Other carbapenemase genes were not detected in all of the isolates. The ESBL genes were detected in 95%(38/40) isolates and 12 ESBL genes were as follows: bla TEM-52, bla SHV-11, bla SHV-12, bla CTX- M-3, bla CTX-M-15, bla CTX-M-55, bla CTX-M-64, bla CTX-M-14, bla CTX-M-27, bla CTX-M-65, bla CTX-M-125, and bla OXA-1. To the best of our knowledge, this is the first report of the bla TEM-52 gene in pig E. coli isolat- ates in China. The Amp C gene were detected in 11 isolates and the bla CMY gene was only detected. The gene subtype of bla CMY were bla CMY-2 and bla CMY-30. The plasmid-encoded 16 S r RNA methyla- ses were detected in 16 isolates, arm A and rmt B genes were detected in 9 and 7 isolates, respectiv- ly. The plasmid-mediated quinolone resistance genes were detected in 33 isolates, and detection ra- te of oqx AB, qnr S, aac(6’)-Ib-cr, and qep A were as follow: 55%, 40%, 37.5%, and 22.5%. Pulsed- field gel electrophoresis(PFGE) and Multilocus sequence typing(m LST) for 40 CNS E. coli were performed. PFGE was successfully performed with 19 colones as A~S colone types. Except the C- colone type, F-colone type, G-colone type, I-colone type, K-colone type, M-colone type, and R-co- lone type were the multi colone types, the other were single colone types. The KPC-2 producing CNS E. coli were seperated into five single colone types, and the NDM-1 producing CNS E. coli were seperated into one multi colone types and four single colone types. m LST was also success- fully performed with 15 ST types, and CC10, CC23, CC38, CC405 were the multi colone type,the others were all single colone type. The KPC-2 producing CNS E. coli were seperated into five ST types: ST69, ST131, ST405, ST410, and ST648, and the NDM-1 producing CNS E. coli were sepe- rated into four ST types: ST72, ST131, ST167, and ST410. Phylogenetic group of CNS E. coli was also performed, 24 isolates were assigned to low virulence A type and B1 type, the other 16 isol- ates were assigned to high virulence B2 and D type. Three KPC-2 producing CNS E. coli were ass- igned to high virulence type, two KPC-2 producing CNS E. coli were assigned to low virulence type. Four NDM-1 producing CNS E. coli were assigned to low virulence type. The strains keeping the high virulence and high antimicrobial resistance is a threat for human clinical. A total of 12 CNS E. coli isolates were successful transferred to E. coli J53 by conjugation. The KPC-2 prod- ucing isolates and NDM-1 producing isolates were also transferred the antimicrobial resistance genes, and it proved that the bla KPC-2 gene and bla NDM-1 gene were located on the plasmid. Inc N and Inc A/C plasmid replicon typing were detected in the KPC-2 producing isolates, and Inc A/C and Inc FII were detected in the NDM-1 producing isolates. PCR mapping approach was performed to identify the genetic environment surrounding the bla NDM-1 gene in all the five NDM-1 producing isolates. Sequence analysis of PCR amplicons revealed that three difference genetic environment surrounding the bla NDM-1 gene were detected. One of the three genetic environment found in the E. coli was the same as in the Acinetobacter baumannii, it revealed that the resistant plasmid could be transferred between Enterobacteriaceae and Acinetobacter baumannii. The gene sequence of blaNDM-1 containing the promoter were colone and transferred to DH5α, bacterial growth and vitro competition experiments were performed. There was no different on bacterial growth between positive one and negative one. The fitness cost constant of bla NDM-1 gene was 0.93 and it belonged a low fitness cost. If E. coli carry the bla NDM-1 gene,it can be existance for a long time.