Analysis Of Antimicrobial Resistance And Studies Of Resistance Mechanism On Some Commonly Used Antibiotics Against Pasteurella Multocida

One of the important pathogens of bovine respiratory disease(BRD) that seriously harm cattle in China and other countries is Pasteurella multocida(Pm) of Serogroup A. This pathogen was found in china in 2008, frequently isolated from cattle which had gone through long-distance transportation. Today, the disease caused by Pm is still serious, challenging china’s model of beef cattle fattening. A Pm infection is treated mainly by using antibiotics, but with the long time existence of drug pressure, resistance of the pathogen to antibiotics has gradually emerged. In Germany, a strain of a high level of drug resistance which was resistant to 12 normal ntibiotics was isolated, ringing alarm bells for resistance and resistance mechanism of Pm. The present study monitored in spots the cattle which had gone through long-distance transportation from the three provinces in Northeast China to the south. And we found there are risks associated with the use of fluoroquinolones and Molecular for Pm infections of cattle in China. However, fluoroquinolones and molecular resistance of Pm isolates has rarely been studied. In the present study, the manuscript researched, for the first time, the molecular mechanism of quinolone and molecular resistance in Pm of Serogroup A. The main results are as follows: 1. Antibiotic resistance and whole genome sequencing of a Pasteurella multocida.A total of 23 Pm strains originated from cattle herds located in 6 provinces in China were tested in this study. Samples from lungs and nasal swabs were collected from distinct outbreaks between 2011 and 2013. The results of the present study demonstrated that all of the isolates were resistant to sulfonamides, part of the isolates were resistant to amikacin and gentamycin. And we found there are risks associated with the use of fluoroquinolones and molecular for Pm. The isolates were only susceptible to florfenicol. The resistant isolate genome harbored a 2,443,368 bp circular chromosome and carried some resistance genes which gave resistance ability for Trimethoprim, vancomycin, kasugamycin, streptogramin. The genome carried three genes encoding efflux of Antibiotic, which gave resistance ability for quinolone and molecular. A total of 23 Pm also carried class Ⅰ integrons, but the gene cassettes of Ⅰ integrons were simplex, only including aac(6’)-Ib, and there was a close relationship between class I integrons and sulfonamides resistance of Pm. And at the same time, 23 Pm were divided into 7 different PFGE profiles, and the results of the study demonstrated that there are resistance risks associated with the cattle through long-distance transportation. 2. Target mutation analysis of fluoroquinolone and macrolide resistance of pasteurella multocida.The minimum inhibitory concentrations(MICs), mutation prevention concentrations(MPCs) and contribution of quinolone resistance-determining region(QRDR) mutations to fluoroquinolone(ciprofloxacin, enrofloxacin and orbifloxacin) susceptibility in 23 Pasteurella multocida(Pm) isolates were investigated. Fluoroquinolone-susceptible isolates(MICs≤0.25 μg/mL, 9 isolates) had no QRDR mutations, and their respective MPCs were low. Fluoroquinolone-intermediate isolates had QRDR mutations(Asp87 to Asn or Ala84 to Pro in gyrA), and their respective MPCs were high(4-32 μg/mL). First-step mutants(n=5) and laboratory-derived highly resistant fluoroquinolone mutants(n=5) also had QRDR mutations. Homologous modeling and molecular docking demonstrated that the mutation in gyr A(83) and parC(80) can lead to the disappearance of Hydrogen Bond Interaction. At the same time, the target mutation of 23 S rRNA was investigated, the macrolide-resistance isolates had mutations of 23 S Rrna in 2059, and the isolates had no mutations in L4, L22. 3. Analysis of gene expression of drug efflux of resistant isolates under subinhibitory concentration fluoroquinolone or macrolide.The MICs of fluoroquinolones for mutant-derived strains were decreased in the presence of efflux inhibitors(CCCP). The results indicated that the fluoroquinolone resistance of Pm is mainly due to multiple target gene mutations in gyrA and parC and the overexpression of efflux pump genes. Therefore, in this study, Real time fluorescent quantitative PCR was used to detect the expression of gene(mexv, maca, mdtk). At the same time, the MRM was used to detect the expression of protein(Mexv, Maca, Mdtk), to verify the results of expression of genes. The results of the present study demonstrated that the fluoroquinolone-resistance isolates under subinhibitory concentration fluoroquinolone can lead to higher level expression of mexv and Mexv than fluoroquinolone- usceptible isolates. 4. Transcriptome analyses of pasteurella multocida under subinhibitory concentration fluoroquinolone or macrolide.Transcriptome analyses were carried out on pasteurella multocida under subinhibitory concentration fluoroquinolone or macrolide. The results of the present study demonstrated that the resistance of fluoroquinolone and macrolide was closely involved in the regulation of gene expression of SOS. But there was a big difference with other bacteria, and the gene expression level of lexA, ssB, recO and ruvA is significant.