| In the world,China is the largest producer and consumer of meat products.With the continuous expansion and intensification of the industrial scale,antibiotics are widely used in pig breeding and epidemic prevention,mainly for promoting pig growth and preventing various bacterial diseases.However,the widespread use and even abuse of antibiotics have led to the continuous generation of bacterial resistance and drug resistance genes,which are spread along the pork production chain,causing serious harm to human health.This project aims to study the distribution of drug-resistant bacteria in large pig slaughterhouses in Zhejiang Province through Metagenomics and understand the structural differences of bacterial populations in different regions during the slaughter process,as well as the distribution characteristics of resistance genes and potential hosts.The main research results are as follows:The overall microbial flora species,distribution characteristics,and functional characteristics in the pig slaughterhouse in Zhejiang Province were analyzed using metagenome sequencing by collecting samples of sewage mixture,pig tissue-related samples,personnel saliva samples,and samples of flies and transport vehicles that intersected with the external environment including waiting area,slaughterhouse,visceral area,and cleaning area.From the perspective of the diversity index,the overall microbial diversity was high,with the highest diversity of flora in the effluent of the waiting area.The main bacterial species in the four areas of the waiting area,slaughter area,visceral area,and washing area were similar to some extent,and all of them were dominated by Proteobacteria,Firmicutes,Bacteroidetes,and Actinobacteria.However,their relative abundance varied widely.In the effluent samples,the slaughter area,visceral area,and wash area were dominated by the phylum Proteobacteria,while the waiting area was dominated by the Firmicutes.In the pig tissue samples,the Firmicutes were dominant in the feces of the waiting area and the intestinal contents of the viscera area,while the Proteobacteria were dominant in the tissue samples of the slaughter area.At the genus level,the predominant dominant genera in the samples from different sources were very different.In the effluent of the visceral zone,the relative abundance of Acinetobacter was much higher than the other genera,47.96%,and Acinetobacter was one of the main dominant genera.Overall,there were significant intergroup differences between saliva samples and other samples,and Streptococcus was most able to highlight the differences in microbial communities between saliva samples and other samples.The distribution of antibiotic resistance genes(ARGs),distribution of mobile elements(MGEs),abundance and species of virulence genes(VGs),antimicrobial and metal resistance genes(BMRGs),and potentially resistant host bacteria in microorganisms in pig slaughterhouses in Zhejiang Province were analyzed by metagenome sequencing.A total of436 ARGs were obtained,and in terms of their conferred bacterial resistance,the largest number of fluoroquinolone resistance genes were detected,with 96,followed by peptide and rifamycin resistance genes,with 48 and 32 detected,respectively;mupirocin resistance genes has the least number,with only one.And the overall abundance was higher for tetracycline resistance genes and multidrug resistance genes.According to the resistance mechanisms,the highest number of ARGs(about 85.28% of all resistance genes)was found under three resistance mechanisms: efflux pump mechanism(138 ARGs),antibiotic target site change mechanism(124 ARGs),and antibiotic inactivation mechanism(132 ARGs).In addition,19 classes of MGEs-related genes were detected,with the highest percentage of transposase genes detected,accounting for 74% of the total number of all MGEs genes.A total of 250 classes of virulence genes were detected,provided by 246 subspecies of pathogenic bacteria.A total of 88 classes of BMRGs were detected,including 23 metal resistance genes.Resistance genes associated with antimicrobial biocides mainly belonged to Sodium dodecyl sulfate(SDS),followed by Acriflavine and Ethidium Bromide.The highest relative abundance of Iron(Fe)resistance genes was detected in the saliva samples.In terms of correlation,there were significant correlations between ARGs and most subtypes of bacterial genera,MGEs,VGs,and BMRGs.The mobile elements with the highest correlation with resistance genes were transposase and IS91-related sequences,and in terms of co-occurrence of resistance genes with hosts,at the genus level,the potential hosts carrying resistance genes were Acinetobacter,Psychrobacter,and Myroides;to further validate the slaughter To further verify the resistance trend in different areas of the slaughterhouse,drug-resistant strains were extracted from samples collected in four areas of the slaughterhouse,and phenotypic and genotypic experiments were conducted.The sulfonamides,macrolides,and tetracycline antibiotics commonly used in pig breeding have high antibiotic resistance rates in the four regions.At the same time,the resistance genes mph A(53.75%)、sul2(71.15%),and tet A(71.94%)which corresponded to these antibiotics,had a detection rate of over 50%,and they also had the highest detection rate among all drug resistance genes.It means that the resistance to these antibiotics is widely distributed in different stages of the slaughtering process,which should be paid attention to.In addition,the resistance rate of quinolone antibiotics has decreased significantly compared to the early years,which is related to the prohibition of the use of four types of quinolones in animal feeding in the early years.Integron detection experiments found that the detection rate of type Ⅰ integrase int I1 was78.26%,which was much higher than that of type Ⅱ integrase and type Ⅲ integrase.The types of antibiotic-resistant genes contained in the detected gene cassettes include trimethoprim,sulfonamides,chloramphenicol,tetracyclines,and aminoglycosides.The overall detection rate of integrase genes was high,but the detection rate of gene cassettes was low.This indicates that there may be a large number of integrons that have not formed a complete structure in pig slaughterhouses,and drug-resistant bacteria can acquire more drug-resistant genes through integrons,which poses a great risk to human health.In summary,there was some similarity in the genus composition of different processing areas in large pig slaughterhouses in Zhejiang Province,and there were significant intergroup differences between saliva samples and other samples.Among the antibiotic resistance genes,more fluoroquinolone resistance genes were detected,and the abundance of tetracycline and multi-drug resistance genes was higher.The potential hosts of antibiotic resistance genes are mainly Acinetobacter,Psychrobacter,and Myrides.The apparent resistance rate of strains in the four regions of the slaughter process was suppressed to some extent through the slaughter chain,while sulfonamide,macrolide,and tetracycline antibiotics commonly used in swine farming had high resistance rates in all four regions.This topic provides theoretical data on the changing characteristics of bacterial resistance during the pig slaughter chain and provides a reference basis for the rational application and safety monitoring of antibiotics in the pork supply chain. |
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