| Background and objectivesAnimal breeding areas have become an important source of the occurrence and transmission of antibiotic resistant bacteria(ARB)and antibiotic resistant genes(ARGs)owing to the widespread use of veterinary antibiotics to promote animal growth,and prevent or treat diseases.Studies have shown that ARB and ARGs could be transmitted among animals,the environment and humans through various pathways,such as aerosols,drinking water,food and insects.In recent years,carbapenem-resistant Enterobacteriaceae(CRE)has attracted wide attention,and controlling the resistance and transmission of CRE has become an urgent challenge in the field of public health.According to the data of the China Antimicrobial Resistance Surveillance System,the resistance rate of Enterobacteriaceae bacteria to carbapenems in clinical settings is increasing in China.For example,the resistance rate of Klebsiella pneumoniae to meropenem has increased from 2.9%in 2005 to 24.4%in 2021.In addition to the unreasonable use of antibiotics in clinical settings,the occurrence of CRE is also closely related to animal breeding.CRE has been detected in various samples from animal farms,such as groundwater,aerosols,animal cloaca and animal feces.For example,one study reported that the detection rates of CRE were 5.3%and 12.8%in well water and animal feces in animal breeding areas,respectively.However,most CRE studies on animal farms in China focused on a single type of animal farms,and the impact of seasonal factors on the CRE detection rate are not considered.It cannot determine whether there are differences in the prevalence of CRE in different types of livestock farms and whether the detection rate of CRE in animal breeding areas is influenced by seasonal factors.Therefore,this study was conducted in an animal breeding area with a long breeding time and various types of livestock farms.The purpose is to investigate the prevalence characteristics of CRE in different types of farms in different months,and to explore the transmission pattern of CRE and its ARGs among animals,the environment and humans to provide data support for controlling the spread of CRE and protecting the occupationally exposed populations in animal breeding areas.MethodsThis study selected 60 animal farms,including chicken farms(n=20),pig farms(n=20)and cattle farms(n=20)in an animal breeding area in Shandong Province.In July 2019,263 samples were collected,including 107 worker feces,66 animal feces,37 wastewater and 53 well water samples.In November 2019,284 samples were collected from the same 60 farms,including 118 worker feces,71 animal feces,37 wastewater and 58 well water samples.Strains were screened by MacConkey agar supplemented with 2 mg/L meropenem.After strain identification,CRE were selected for further research.The resistant phenotype was analyzed using antimicrobial susceptibility testing.S1-nuclease PFGE was performed to evaluate the number and size of plasmids on strains,and Southern blot hybridization was used to identify the location of plasmids harboring carbapenemase genes.The horizontal transferability of the plasmids mediating carbapenem resistance was evaluated by filter mating.The ARGs,genetic environment of carbapenemase genes,virulence genes and genetic relationship of CRE were analyzed using second and third-generation genome sequencing.Results1.Detection of CRE:CRE was detected in 24 of 263 samples collected in July 2019,with a detection rate of 9.13%,including 3 chicken feces and 2 worker feces from chicken farms detected 2 different CRE.Thus,a total of 29 CRE were isolated,including Escherichia coli(n=18),Klebsiella pneumoniae(n=8),Enterobacteriaceae cloacae(n=2),and Citrobacter cerevisiae(n=1).The detection rates of CRE in different sample types were animal feces(6.06%,4/66),wastewater samples(5.41%,2/37),human feces(12.15%,13/107)and well water(9.43%,5/53).The detection rates of CRE in different types of farms were chicken farms(14.67%,11/75),pig farms(11.63%,10/86)and cattle farms(2.94%,3/104).The number of detections of CRE was significantly different in different types of farms(p<0.05),and chicken farms was significantly higher than bovine farms,whereas it was not significantly different between other types of livestock farms.CRE was detected in 12 of 284 samples collected in November 2019,with a detection rate of 4.23%,including 1 chicken feces detected 2 different CRE.Thus,13 CRE were isolated,including Escherichia coli(n=12)and Enterobacteriaceae cloacae(n=1).The detection rates of CRE in different sample types were animal feces(15.49%,11/71),wastewater samples(2.70%,1/37),human feces(0.00%,0/118),and well water(0.00%,0/58).The detection rates of CRE in different types of farms were chicken farms(12.16%.9/74),pig farms(0.93%,1/108)and cattle farms(1.96%,2/102).The number of detections of CRE was significantly different in different types of farms(p<0.05),and chicken farms was significantly higher than pig and bovine farms,whereas it was not significantly different between pig and bovine farms.The number of detections of CRE was significantly different in July and November(p<0.05).Notably,the detection rate of CRE of each type of livestock farms in July was higher than in November,and there was statistically significant difference in pig farms,whereas it was not significantly different in chicken farms and cattle farms.2.Resistance characteristics:All 42 CRE harbored blaNDM,and blaNDM-5(n=37)is the most common in chicken,pig and cattle farms,followed by blaNDM-4(n=2),blaNDM-9(n=2)and blaNDM-1(n=1).The AST results showed that all of CRE are multidrug-resistant bacteria and resistant to meropenem,imipenem,amoxicillin-clavoic acid,piperacillin/tazobactam,ceftazidime and tetracycline.3.blaNDM-carrying plasmids:The blaNDM of 1 CRE is located on chromosome and the blaNDM of other 41 CRE strains are located on plasmids,of which 35 are located on IncX3 plasmids with the size of 42-52 kb,2 are located on IncH12A plasmids with the size of 220 kb,1 is located on IncFIB plasmid with the size of 200 kb and the other 3 blaNDM-carrying plasmids cannot be determined.37 blaNDM-carrying plasmids were successfully transferred to Escherichia coli J53 by filter mating.4.The genetic environment of blaNDM-5:Highly similar and transferable blaNDM-5-carrying IncX3 plasmids were isolated from animal,environmental and human-related samples in chicken,pig and cattle farms,and the plasmids shared 99.99%-100%nucleotide identity.Tn3,IS3000 and IS5 are located upstream of blaNDM-5,while bleMBL,dsbD,IS26 and ISKox3 are located downstream.5.Virulence genes:all Escherichia coli and Klebsiella pneumoniae carry a variety of virulence genes related to bacterial adhesion,colonization and pathogenicity.6.Genetic relationship:Single nucleotide polymorphism(SNP)analysis revealed that 7 pairs of highly homologous CRE were isolated in July,including 4 pairs of clonal strains(SNPs<18)detected in worker feces samples from 3 pig and 1 cattle farms,and each pair of strains isolated from the same farm;2 pairs of clonal strains(SNPs≤7)detected in well water samples from 2 pig,1 chicken and 1 cattle farms;and 1 pair of clonal strains(SNPs=17)detected in worker feces and animal feces from 2 different chicken farms.Conclusions1.The detection of CRE varies among different types of livestock farms,and the detection rate of CRE in chicken farms is the highest,which is the key object of supervision and management in the breeding industry.2.In terms of seasonality,the detection of CRE in chicken and cattle farms is relatively stable,but the detection rate of CRE fluctuates greatly in pig farms,and the detection rate in July is significantly higher than in November,which may be related to the breeding patterns in different seasons.3.Among CRE strains,all Escherichia coli and Klebsiella pneumoniae carried virulence genes related to adhesion,colonization and pathogenicity,suggesting that the strains may pose a risk of colonization and infection to human and animal hosts.4.The horizontal transfer mediated by IncX3 plasmids and the clonal spread of CRE are important ways of transmission among animals,the environment and humans in animal breeding area. |
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