Isolation,Identification,Drug Resistance Phenotype And Whole Genome Sequencing Analysis Of Avian Salmonella From Some Areas Of Sichuan Province

Salmonellosis is an important zoonotic disease that causes great harm to animal and human health.Salmonella is widespread in farms,especially poultry farms;on poultry,Salmonella can not only spread horizontally,but also vertically spread through eggs,which seriously affects the development of the poultry industry.Salmonella is also a major food-borne pathogen.Humans eat infected eggs and poultry meat products,which lead to food poisoning.Salmonella takes the first place in bacterial food poisoning.In recent years,antibacterial drugs have been mainly used at home and abroad to prevent and control avian salmonellosis.However,due to the abuse of antibacterial drugs,its drug resistance has increased year by year,and the drug resistance spectrum has become wider and wider,which makes the prevention and control of salmonellosis more difficult.and pose a safety hazard to public health.Therefore,it is of great practical significance to strengthen the surveillance of Salmonella,especially to strengthen the research on the prevalence and drug resistance of drug-resistant Salmonella.In this study,from December 2020 to August 2021,poultry samples were collected from 7 districts in Panzhihua City,Jintang County,Xindu District,Longquanyi District,Shifang County,Wenchuan County,and Pengshan District,Sichuan.Cloacal cotton swabs,943 dead birds,dead embryos,etc.,were isolated from Salmonella,and the isolated bacteria were identified by Salmonella A-F multivalent serum and single-factor serum agglutination test,as well as inv A gene PCR amplification;further drug susceptibility testing of Salmonella isolates was carried out.The use of antibiotics in the farms in the sampling area provides a basis for the rational selection of antibiotics to control Salmonella infection in clinical practice;the pathogenicity and whole-genome sequencing analysis of the multidrug-resistant strains of dominant serotypes is performed to clarify their pathogenic mechanisms and drug resistance mechanisms Provide scientific evidence.The main results are as follows:1.Isolation,identification and drug resistance of Salmonella fowl（1）According to the GB4789.4-2016 method,a total of 100 strains of Salmonella were isolated and identified from 943 poultry samples,and the total isolation rate was10.60%（100/943）.The results of serotype identification by slide agglutination showed that 95 of the 100 Salmonella strains have been identified,belonging to 7 serotypes,among which,55 strains of Salmonella typhimurium,17 strains of Salmonella pullorum,and 10 strains of Salmonella enteritidis have been identified.The strain was the dominant serotype;Salmonella Harto and Bonn were 5 and 4 respectively.Salmonella Hato was isolated from duck source for the first time at home and abroad;Salmonella Lisen and Chester were both 2 strains.The above research results enriched the Salmonella resource bank and provided important materials for carrying out related research.（2）The K-B method was used to determine the resistance phenotype of Salmonella isolates.The results showed that the resistance rate of Salmonella isolates to sulfisoxazole was up to 83.2%;followed by streptomycin（80.4%）and polyisoxazole.Colistin B（75.7%）;para-trimoxazole（58.9%）,nalidixic acid（53.3%）,ampicillin（50.5%）,tetracycline（44%）,doxycycline（44.9%）,and Moxicillin（41%）was more resistant;resistance rates to cefazolin（15%）,ceftriaxone（14.7%）,chloramphenicol（10.3%）and cefotaxime（10.3%）were 10%to 15%,the drug resistance rate was low;the drug resistance rates for ciprofloxacin（8.5%）and norfloxacin（3%）were both lower than 10%.The results of the multi-drug resistance study found that the multi-drug resistance rate of 100 fowl salmonella isolates was as high as 86%,and one of the duck-derived isolates was resistant to all the 17 antibacterial drugs tested in this study.It is necessary to further study its drug resistance mechanism.According to the use of antibiotics in each farm,combined with the results of the resistance rate of Salmonella in this field,it can provide a reference for the rational selection of antibiotics for the farm.2.Pathogenicity and Whole Genome Sequencing Analysis of Two Predominant Serotype Multidrug-resistant Salmonella Isolates（1）The pathogenicity test was carried out on the two dominant serotype multidrug-resistant Salmonella isolates S19 and C46,and the bacterial counts of 1.0×10⁵～1.0×10⁹ CFU were selected to determine the LD₅₀ of Kunming mice.The results showed that the LD50 of S19 and C46 strains to Kunming mice were 6.30×10⁶ CFU and1.58×10⁶ CFU,respectively,indicating that the two dominant serotypes of multidrug-resistant Salmonella have certain pathogenicity.（2）The S19 and C46 strains were sequenced by Nanopore Prometh ION and Illumina Nova Seq PE150 methods.The sequencing results showed that the genomes of the S19 and C46 strains were 4974488 bp and 4974486 bp,respectively,and the GC content was 52.16%;A large plasmid with plasmid sequence sizes of 186949 bp and165552 bp,and GC contents of 47%and 46.54%,respectively;S19 and C46 strains predicted 5007 and 4984 coding genes and 108 non-coding genes,respectively.indivual.There were 14 gene islands in the genomes of the two Salmonella strains,and the total length of the gene islands were both 344765 bp;there were 12 prophages,and the total lengths of the prephage sequences were 777348 bp and 789041 bp,respectively.（3）The results of gene prediction showed that the S19 and C46 strains had 573and 571 virulence genes,respectively,among which the structural virulence genes were the most,with 173 genes,indicating the invasiveness of the two Salmonella strains and the defense mechanism against the host.Among them,there are 34 pili-related genes,mainly type I fimbriae（fimA,fimC,fimF,fi I,fimD,fimH,fimW,fimY,etc.）and type IV fimbriae（pil L,pil W,etc.）.Followed by the virulence genes on the virulence island,both Salmonella have 82;mainly SPI-1（inv,hil,iac,org,sip,sop,spa,etc.）and SPI-2（ssa,ssc,sse,ssp,etc.）toxicity island.There were 29 endotoxin-related virulence genes and 18 genes involved in iron synthesis,transport and uptake.ARGD predicted 38 drug resistance genes in S19 strains and 37 in C46 strains;CRDB predicted new drug resistance genes in recent years and found that S19 strains had 173 drug resistance genes and C46 had 171 drug resistance genes;CRDB Among the drug resistance genes predicted by ARGD and ARGD,the two isolates both had a large number of drug resistance genes to aminoglycosides,β-lactams and sulfonamides,and both isolates had resistance to multiple antibiotics.Drug gene;from the gene sequence analysis of the two Salmonella strains sequenced,the drug resistance gene is basically consistent with the drug resistance phenotype.