| Background and ObjectivesAntibiotic resistance has become an increasingly urgent global public health problem,and the consequence is increased mortality and increased clinical medical expenditures caused by ARB infections.China is one of the countries with the most serious bacterial resistance problem in the world and fastest growing rates of bacterial resistance in the world,and all departments attach great importance to curbing the development of drug resistance.In 2016,14 government departments including the National Health and Family Planning Commission responded positively,emphasizing that multi-sectoral joint control of the spread of ARB and clarifying that bacterial resistance is not only a challenge faced by clinical and aquaculture industry but also for the environmental filed.Carbapenem antibiotics such as imipenem and meropenem have always been considered as a last resort in the treatment of multidrug-resistant bacterial infections.Carbapenem-resistant Enterobacteriaceae(CRE)are resistance to carbapenem antibiotics,which can cause clinical CRE infection treatment to fall into a "no medicine available" situation,has attracted widespread attention worldwide.In 2017,the World Health Organization(WHO)listed CRE as the highest priority pathogen for the development of new antibiotics.There is increasing evidence that the environment is known to play a key role in the transmission and evolution of ARB.However,most of the current studies on ARB in the field of environmental sanitation focuses on the breeding environment,while the prevalence of ARB in the vegetable growing environment and its potential public health risks have been ignored.In vegetable growing areas,large amounts of livestock manure is applied all year round,antibiotics and ARB in manure directly enter the greenhouse soil.The residual antibiotics in the soil,even at concentrations below the minimal inhibitory concentrations(MICs),can accelerate the emergence and spread of ARB through selection pressure.In addition,the vegetable greenhouse is a relatively closed environment.The ARB in the soil can enter the air through water evaporation or settle on the surface of the vegetable,and then enter the body through hand contact(picking),digestive tract and respiratory tract,and even colonize the human intestine.Therefore,more attention should be paid to the prevalence and health risks of environmental ARB in vegetable growing areas.Our aim in this study was to investigate the occurrence of CRE in greenhouse soils,well water,river water,river sediments,on vegetable and fruit surfaces,pickers’hands and among farm workers in an intensive vegetable cultivation area in China and to characterise these isolates in terms of antimicrobial susceptibility levels,carriage of antibiotic resistance genes,virulence genes and phylogenetic affiliation.In addition,we aimed to investigate the mobile genetic elements,along with their genetic relatedness and transfer pattern,which provided the basis for the spread of CRE in intensive vegetable areas.MethodsIn June 2019,the study was conductedin the largest intensive vegetable growing area.A total of 382 samples were collected from vegetables and melons(n=106),soil(n=87),well water(n=24),river water(n=20),river sediments(n=20),farmer hands(n=58),and farmer faeces(n=67).Carbapenem-resistant Enterobacteriaceae(CRE)was obtained by screening and identification of ARB.The drug resistance genotype and drug-resistance phenotype of the strain were determined by PCR and Antimicrobial susceptibility testing(AST).S1-nuclease PFGE was performed to estimate the number and sizes of plasmids harbored by isolates carrying carbapenemase genes.The determination of whether the carbapenemase genes are plasmid-mediated by Southern blot.The genome-specific phenotype,the type of carried virulence factor,the mobile element and the genetic environment of carbapenemase gene were obtained by whole genome sequencing analysis(WGS).Results1.A total of 838 carbapenems resistant strains were isolated from 382 samples,among which 9 strains were carbapenase gene positive CRE,with a positive rate of 2.4%.The 9 CRE strains include 3 Citrobacter sedlakii strains detected from soil samples,3 Escherichia coli and 1 Citrobacter freundii detected from human fecal samples,as well as 2 Klebsiella pneumoniae strains isolated from river water and river sediments respectively..No CRE isolates were detected on vegetable or melon surfaces,farmer hands or in well water.2.AST shows that all isolates are multidrug-resistant.All isolates were resistant to meropenem,imipenem,ciprofloxacin,ampicillin,cefotaxime,ceftazidime,and trimethoprim/sulfamethoxazole and showed high resistance to meropenem and imipenem,≥64 mg/1 and≥32 mg/l,respectively.3.S1-PFGE and Southern hybridisation reveals that blaNDM-5 is located on IncX3 plasmids of same size(～46 kb)in all isolates except Z5.Different Enterobacteriaceae bacteria carrying the IncX3 plasmid were detected in the same farmer,and the plasmid-mediated carbapenems gene could be transferred to the recipient E.coli J53 by conjugation.4.Whole genome sequencing showed that all nine CRE isolates carried the carbapenemase gene blaNDM-5 and genes encoding extended-spectrum β-lactamases(ESBLs)and other resistace genes.All NDM-5 genes are associated with mobile elements IS5 and bleMBL(IS5-blaNDM-5-bleMBL).The virulence gene analysis demonstrated that all strains not only carry a variety of resistance genes,but also carry adherence factors that could facilitate adhesion and colonisation.Conclusions1.Various CRE exist in both environment media and farm worker in vegetable growing area,and the environmental distribution of different types of CRE is different.2.Multiple ARGs were identified in CRE isolates,which may also carry virulence factors that can promote adhesion and colonization,suggesting the potential of these isolates to acquire adaptive phenotypes through mobile genetic elements and the risk of colonizing in human hosts and causing infection.3.Transmetastable IncX3 plasmid and highly conserved IS5-blaNDM-5-bleMBL structure indicated that NDM-5 had high transmissibility.Escherichia coli and Citrobacter sedlkii detected in the feces of healthy farmers contained blaNDM-5 carrying IncX3 plasmids similar to environmental CRE isolates,suggesting the transmission of NDM-5 among humans and environment.through horizontal gene transfer. |
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