| Salmonella is one of the important food-borne pathogens and causes harm to humans and animals,which seriously hinders the development of the livestock breeding industry.Pig farming is an important pillar industry in China,and pigs are one of the main hosts of Salmonella.Salmonella can use it as a carrier to spread,thereby causing pollution to food and the environment,The body ingests food contaminated with Salmonella or toxic and harmful pathogenic factors,which leads to diarrhea,vomiting and other symptoms in humans and animals.In severe cases,it can cause diseases such as bacteremia,typhoid fever,gastroenteritis,and even death.In this study,in order to investigate the contamination and drug resistance of Salmonella in pig slaughterhouses in different regions,samples were collected from five pig slaughterhouses of different scales in Henan,Shaanxi and Hebei,and then the isolated strains were tested for Multi-locus sequence typing(MLST)and drug susceptibility tests,and whole-genome sequencing of some strains.Isolation,identification and prevalence of Salmonella in pig slaughterhouses In this study,a total of 1455 samples were collected from five pig slaughterhouses of different scales in Henan,Shaanxi and Hebei.The samples were obtained from live pigs,during the slaughtering process(stunning,depilation,splitting,chilling)and the slaughtering workshop environment.336 Salmonella strains were isolated from the samples,The isolation conditions were 83 strains(live pigs),87 strains(stunned),64strains(depilation),39 strains(splitting),24 strains(chilling),14 strains(water),3strains(knife)and 17 strains(floor).And then all strains were subjected to multi-locus sequence typing(MLST).The results showed that among the 336 Salmonella isolates from the slaughterhouse samples,a total of 323 strains were identified as ST type,and13 strains could not be identified as ST type.A total of 11 ST types were obtained,among which ST40(n=119)was the dominant strain,followed by ST469(n=82)and ST34(n=46).The ST-type results of Salmonella isolated from 5 slaughterhouses(Field A,Field B,Field C,Field D,and Field E)totaled 7 batches of samples(where B and C were sampled twice each)The dominant Salmonella in A field,C1 field(The first batch of samples in field C)and C2 field(The second batch of samples in field C)are ST34,ST40 and ST19,respectively.However,the predominant ST type of Salmonella in B1 field(The first batch of samples in field B),B2 field(The first batch of samples in field B),D field and E field were all ST469 type.According to the distribution of Salmonella ST types in different regions,the predominant ST type of Salmonella in Hebei and Henan was the same ST469 type,while the predominant ST type of Salmonella in Shanxi was ST40.The distribution of ST-type Salmonella in different slaughtering stages is also different,mainly due to the contamination of ST40 and ST469 Salmonella.But the overlapping of Salmonella ST types in the three regions,such as ST19,ST34,ST40 and ST469 Salmonella were detected in three regions,indicating that the above four Salmonella ST types were widely present in pig slaughterhouses.The above research results showed that although the Salmonella isolated in this experiment is dominated by ST40 Salmonella,However,due to regional differences and environmental conditions of slaughterhouses,the prevalence of Salmonella dominant ST type in different slaughterhouses was different.The detection results of Salmonella in the slaughtering process and the environment found that there was Salmonella contamination in the slaughtering process and the environment of pig slaughterhouses.This shows that it is easy to spread through the air,water,knives and the floor of the slaughterhouse,and there is a risk of transmission along the slaughtering chain.For example,the ST40 strain in the C1 field and the ST469 strain in the E field exist in multiple slaughtering links.The ST40 and ST469 of Salmonella were mainly contaminated in pigs to be slaughtered and stunned,indicating that the contamination originated from the pigs themselves.ST40 and ST469 Salmonella were also detected in environmental samples of scalding water,floor and air,which also suggested that there was cross-contamination from the slaughter chain to the environment during the slaughtering process.Study on the drug resistance phenotype of Salmonella in pig slaughterhouses A total of 13 antibacterial antibiotics were selected in this study,and336 Salmonella were subjected to antibiotic resistance phenotype test by agar dilution method.Salmonella developed extensive drug resistance to the tested drugs,and90.77% of the strains were resistant to more than three antimicrobial drugs.All Salmonella were resistant to at least one antimicrobial drug.The most resistant strains were ceftazidime with a resistance rate of 99.40%,followed by trimethoprim with a resistance rate of 91.37%.All strains were sensitive to meropenem and amikacin.The data of MDR strains showed that the MDR strains were mainly resistant to 6antibiotics,accounting for 21.13%,followed by the number of strains resistant to 5antibiotics and 7 antibiotics,accounting for 17.56%.And it was found that one strain of Salmonella was resistant to 11 antibiotics.336 Salmonella strains produced a total of 65 antibiotic resistance spectrums,and the dominant antibiotic resistance profile was CAZ-AMP-DOX-CIP-TMP-FF(35/336,10.42%),and the Salmonella antibiotic resistance spectrums were different in different regions.In Henan Province,CAZAMP-DOX-CIP-TMP-FF(22/336,6.55%)was the dominant antibiotic resistance spectrum.Shanxi Province used CAZ-KAN-GEN-AMP-DOX-CIP-TMP-FF(21/336,6.25%)as the dominant antibiotic resistance spectrum,while Hebei Province used CAZ-AMP-DOX-TMP-FF(22/336,6.55%)as the dominant antibiotic resistance spectrum.There are differences in the resistance phenotypes of different ST types of Salmonella.Among them,ST34,ST469 and ST516 types of Salmonella are sensitive to amikacin(AK)and meropenem(MEM),and have different degrees of resistance to the other 11 antibacterial antibiotics.The results of the antibiotic susceptibility test also found that 11 different ST-type Salmonella were highly resistant to ceftazidime.Except for ST469 Salmonella,which had a resistance rate of 97.56% to this antibiotic,the other 10 ST-type Salmonella were highly resistant to ceftazidime,the resistance rate of bacteria to ceftazidime reached 100%.Whole Genome Sequencing of Salmonella in Pig Slaughterhouses Whole genome sequencing of 30 Salmonella strains from different slaughtering stages and environmental samples in C1 field.The sequencing results showed that 22 strains were S.Derby,7 strains were S.Typhimurium and 1 strain was S.Rissen.The multilocus sequence typing results showed that S.Derby are ST40 type,S.Typhimurium are ST34 type and S.Rissen are ST469 type,and a total of 33 antibiotic resistance genes were found,including quinolones,aminoglycosides,fosfomycin,sulfonamides,tetracyclines,chloramphenicol,beta lactams genes of lactams and rifampin to a variety of antibiotics,among which the aac(6’)-Iaa gene has the highest detection rate of 100%,the sul2 gene is the second with 93.33%,and the bla CARB-2 has the lowest detection rate,only 6.67%.And it was detected that some strains contain the qac E gene that is resistant to quaternary ammonium salts,so Salmonella carrying this gene may be resistant to quaternary ammonium compound disinfectants;the resistance genes carried by different ST-type Salmonella are also different,such as ST40 Salmonella contains aac(6’)-Ib-cr,oqx A,oqx B,par C,qnr S2 and other genes,but ST34 Salmonella does not carry the above several antibiotic resistance genes.The prediction results of virulence genes showed that Salmonella contains a large number of virulence genes,including 287 species,involving invasion,adhesion,secretion system and toxins,etc.The virulence genes of strains 7,9 and 18 are more than other strains,mainly related to acid resistance,invasion,immune evasion,endotoxin,flagella and iron uptake,etc.In addition,the Rcs B/Rcs A system contained in Klebsiella was also detected,This system can regulate the expression of capsular polysaccharides and help them resist external environmental stress.The phylogenetic analysis of the strains showed that there is a certain genetic relationship between Salmonella from different sources,it was found that both ST40 Salmonella and ST34 Salmonella existed in the slaughtering process of pigs,and each was divided into a branch,and the ST34 Salmonella isolated from the air sample had a similar genetic evolution relationship with the ST34 Salmonella in the slaughtering process,which proves that Salmonella can be transmitted along the slaughter chain during the slaughtering process in this slaughterhouse,and there is cross-contamination of Salmonella from environment-slaughtering processing.In this study,the multi-locus sequence typing(MLST),antibiotic susceptibility testing and whole genome resequencing were used to explore Salmonella in swine slaughterhouses in different regions,so as to explore the antibiotic resistance and epidemic spread of Salmonella.In order to formulate a reasonable clinical medication plan and trace its source,establish a key risk control point for Salmonella in the slaughtering process of live pigs,so as to achieve effective prevention and control and monitoring of Salmonella. |
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