Investigation On The Resistance Of Mcr-1 Positive Escherichia Coli In Part Areas Of Guangxi And The Preliminary Study On Phage Resistance Leads To Fitness Cost Of Escherichia Coli

Escherichia coli(E.coli)are common gram-negative bacteria.Some types of E.coli can infect humans and animals and cause serious diseases and economic losses.Multi-drug resistant E.coli seriously affect the therapeutic effect of clinical bacterial infections.At present,there are a variety of"antibiotic substitutes"for the aquaculture industry on the market,such as Chinese veterinary medicine preparations,probiotics and antibacterial peptides.As we all know,bacteriophages are natural killers that feed on bacteria.However,phage therapy has been hovering in the field of laboratory and clinical,and has not been successfully promoted to the breeding industry.So,this study carried out work from two perspectives.One was to investigate the drug resistance of mcr-1positive Escherichia coli(MCRPEC)from Guangxi pigs and the prevalence of drug resistance genes.As well as,the next-generation sequencing technology analyzed the genome of extensively drug-resistant E.coli in order to find out the reason why MCRPEC had evolved into super drug-resistant bacteria from pigs in Guangxi.The second was to use ATCC25922 as the host to isolate lysis phage.Based on the ATCC25922 and lysis phage,we studied the effect of phage resistant on fitness cost preliminarily.In this study,33 MCRPEC strains were isolated from different regions in Guangxi,which could be divided into seven unique ST types and the number of ST10 strains was at most.Six incompatible plasmids(Inc FI,Inc HI1,Inc Y,Inc N,Inc I1 and Inc X1)were found in 33 MCRPEC stains.The drug resistance test showed that 19 MCRPEC strains were resistant to the third and fourth generation cephalosporins,and 12 MCRPEC strains were resistant to carbapenem antibiotics.This study proved that 3 MCRPEC strains were extensively drug-resistant(XDR),while other strains were multi-drug resistant(MDR).The results of the investigation of drug resistance genes showed that a large number of acquired drug resistance genes coexisted with mcr-1.For example:?-lactam antibiotic resistance genes,including extended-spectrum?-lactamase genes(bla_CTX-M14,bla_CTX-M24,bla_CTX-M123,bla_OXA-1),p Amp C gene(bla_CMY-2),Carbapenemase gene(bla_NDM-5)and non-?-lactam antibiotic resistance genes(qnr A,qnr B,qnr S,aac(6')-Ib-cr,tet A,tet B,sul1,sul2,flo R,aad A).Through sequencing and analysising the genome of extensively drug-resistant E.coli GXEC-D6,in was found the mcr-1,bla_CTX-M-14and bla_NDM-5coexisted in it.At the same time,it was determined that this strain used movable genetic elements(MGEs)introduce mcr-1 and enterotoxin(stb).This study was consistent with most literature reports,confirming that ST224 isolates are an important pathogen with multi-drug resistance and carrying many drug-resistant genes.When ATCC25922 strain was as the host,a lytic phage was successfully isolated from the sewage of a pig farm in Guangxi and named v B?Ecn P?25922.The phage belonged to the subfamily of Autographivirinae,T7/T3-like virus.The optimal multiplicity of infection of the phage was 1×10^-3,the incubation period was 5 min,the outbreak period was 40 min,and the burst volume was 62.The phage exhibited stable activity at p H 6.0-9.0.The phage titer began to decrease at temperatures higher than 50°C.Once the temperature reacheed 80°C,the phage almost completely lost activity.The bacteriophage v B?Ecn P?25922 was ds DNA virus.The whole genome length was 38890 bp,the length of coding domain sequence(CDS)was 34809 bp(89.51%)and there were 41 CDS in the genome.There were no harmful genes(drug resistance genes and virulence factors).Based on the above study,we successfully observed that the phage v B?Ecn P?25922induced phage resistance and affected the proliferation and drug resistance of the host.According to the experimental results,50 phage resistant mutant strains had a significant down-regulation of the proliferation ability compared with the parent strain accounting for 66.7%(50/75).Fifty-three phage resistant mutant strains had a significantly lower minimum inhibitory concentration than the parent strain accounting for 70.7%(53/75),and the minimal inhibitory concentration was no increased.The q PCR detection results showed that the fitness cost of phage resistance was correlated with the differential expression of RND efflux pump family proteins and outer membrane proteins,which played a good role in confirming the results of the previous test.In summary,mcr-1 positive E.coli has seriously threatened for public health safety and animal production.From the perspective of phage resistance,this study provided a basis for using phage to prevent and treat drug-resistant E.coli infection,the study showed bacteriophages have good application prospects as a substitute of antibiotics.