Prevalence Characteristics Of Escherichia Coli Carrying Mcr-1 In Multi-environment Of Family Farming Area

Background and objectivesAntibiotic resistance has become a major issue that is of common concern and urgently to be solved globally.China is one of the countries with unreasonable application of antibacterial drugs and serious bacterial resistance.Faced with the health threats caused by drug-resistant bacteria,it is urgent to strengthen multi-field collaborative planning and joint response.In a "One Health "study,the environment is at the center of the "animal-environment-human" transmission chain.The environment plays an important role in the transmission of antibiotic resistance by serving as a repository of antibiotic resistance genes and by facilitating dissemination of antibiotic-resistant bacteria and antibiotic resistance genes via environmental routes.In the environment,current research on drug-resistant bacteria mostly focuses on single environmental media,and lacks the holistic study of multi-media transmission.Due to the lack of processing facilities for livestock and poultry manurein animal breeding areas,especially in rural,animal manure is directly discharged into the environment.Improper disposal of livestock and poultry waste will lead to the emergence of drug-resistant bacteria in the surrounding environment.And its possible propagation in environmental multi-media has become an urgent need to clarify the current research on environmentally resistant bacteria.Polymyxin has been considered as the last line of defense against a series of Gram-negative infections such as Pseudomonas aeruginosa,Klebsiella pneumoniae,Acinetobacter baumannii,but with the abuse of antibiotics,a variety of bacteria can carry the colistin resistance gene mcr-1,which makes this view seriously affected.Although the current study found that mcr-1 only caused moderate to low levels of colistin resistance,it was not confirmed that it did not continue to develop until the risk of high levels of resistance.Therefore,in order to understand and evaluate the prevalence of Escherichia coli carrying mcr-1 in rural aquaculture areas,we focused on mcr-1 in environmental media(water,soil,rivers,etc.)in rural family farming areas in Shandong Province.The prevalence characteristics of Escherichia coli were investigated,to provide a theoretical basis for guiding agricultural,aquaculture and clinical rational drug use,and finally to prevent and control bacterial resistance.MethodsThe study was conducted in July 2015 and collected environmental samples from 12 villages in a family-based farming area in Shandong Province.Sample types include pig manure,wastewater,sewage sediments,drinking water,river water,river sediments,soil,vegetables;ESBL-producing Escherichia coli carrying mcr-1 gene was isolated by screening and identification of drug-resistant bacteria;drug-resistant phenotype was determined by antibiotic sensitivity test;pulsed-field gel electrophoresis(PFGE)and multilocus sequence typing(MLST)were conducted for homology analysis;determination of whether the mcr-1 gene is plasmid-mediated by S1 pulsed-field gel electrophoresis(S1-PFGE)and Southern blot;The incompatibility type of plasmids carrying mcr-1 were determined by PBRT;antibiotic resistant gene,virulence factors,and the genetic environment of mcr-1 were obtained by whole genome sequencing analysis(WGS).Results1.A total of 89 ESBL-producing Escherichia coli were isolated,of which 17 strains carrying mcr-1 gene,with a positive rate of 19.1%.Except for soil samples,mcr-1 positive strains were isolated from other environmental medias,including pig manure,waste water,drinking water,river water,river sediments,vegetables,and outlet sediments.Most of mcr-1 positive strains are resistant to colistin,amoxicillin/clavulanic acid,piperacillin/tazobactam,cefotaxime,tetracycline and florfenicol,and sensitive to meropenem,tigecycline.All strains are multi-drug resistant and carry multiple drug resistance genes.2.MLST results showed that 17 strains belonged to 12 ST types.Except for new ST types,other ST types were reported in humans,animals and environment.The PFGE results showed that two pairs of strains showed high homology from different geographical locations.3.Results of S1-PFGE and Southern blot confirmed that mcr-1 gene was plasmid-mediated in most of strains,and the plasmid size ranged from 33 to 240kb.IncHI2,IncI2 and IncX4 are dominant.The plasmid-mediated mcr-1 gene can be transferred to the recipient strain E.coli J53 by conjugation experiment,and the resistance of the recipient strain to colistin is increased or even exceeded resistant level.4.The results of whole genome sequencing showed that downstream of mcr-1 gene has an open reading frame encoding PAP2 family proteins,and some strains have an ISAPI1 structure related to transposon formation upstream and/or downstream of mcr-1.Not only a variety of resistance genes,but some strains also carry virulence factors associated with enteroaggregative Escherichia coli(EAEC)and enteropathogenic Escherichia coli(EPEC).Conclusions1.ESBL-producing Escherichia coli carrying mcr-1 gene was widely detected in different environmental media and locations,includes well water and ready-to-eat vegetable surfaces that people are in direct contact with.In addition to human factors(such as animal husbandry),meteorological and hydrological factors may be important factors on mcr-1 transmission.2.Most of resistant bacteria carried various antibiotic resistance genes and virulence factors which have important effects on human disease,highlighting the diversity of the genetic composition of these strains and the potential risk that environmental media as hot spots which contain antibiotic resistancegenes and virulence genes.3.The mcr-1 positive strain isolated from multi-environmental media in family culture area is highly specific,and plasmid-mediatedis the main mode of mcr-1.The plasmid carrying mcr-1 in terms of the type of incompatibility group and the genetic environment of mcr-1 is highly diverse..