Prevalence,Antimicrobial Resistance And Genomic Characterization Of Salmonella Recovered From Pig-Borne Food Chain In Henan

Salmonella is one of the main food-borne pathogens,which can lead to significant public health concerns,particularly with emerging antimicrobial-resistant(AMR)Salmonella clones.Most human Salmonella infections are usually associated with ingestion of contaminated animal-derived foods,including pork,beef,mutton,chicken,and so on.Pork or its products are the main sources of Salmonella infection in humans.Studies abroad have shown that outbreaks caused by Salmonella serotype 4,[5],12:i:-are associated with pork or pork products.Furthermore,antimicrobial-resistant Salmonella is one of the threats to human health because it limits the choice of effective antimicrobial agents,even leading to treatment failure of salmonellosis.Like Salmonella Typhimurium,Salmonella serotype 4,[5],12:i:-currently represents one of the most common serotypes among human cases in the world.According to a new survey based on the national surveillance system,Salmonella serotype 4,[5],12:i:-has overtaken Salmonella Typhimurium and ranked the second most frequently encountered serotype in patients in Henan,China,just following Salmonella Enteritidis.Henan Province is the second leading pork producer in China,contributing almost 10% of Chinese pork production.Does this mean that compared to Salmonella Typhimurium,Salmonella serotype 4,[5],12:i:-has a higher prevalence in the pig-borne food chain in Henan Province? However,no relevant reports were presented.To reduce the amount of pork contaminated by Salmonella and ensure the safety of pork products for consumers,samples were collected from pig farms,slaughterhouses,and retail markets in different regions of Henan Province to monitor the prevalence and antimicrobial resistance of Salmonella along the whole pig industry chain.Furthermore,whole-genome sequencing was used to explore genomic characteristics of virulence,antimicrobial resistance,and plasmid,as well as phylogenetic relationship of Salmonella Typhimurium and Salmonella serotype 4,[5],12:i:-strains from the pig production chain in Henan Province through the genomic analysis,in an attempt to explain the possible mechanism of selective advantages in the evolution of Salmonella serotype 4,[5],12:i:-strains.The main research contents are as follows:1.Prevalence and antimicrobial resistance of Salmonella recovered from farms and slaughterhouses in Henan ProvinceIn this study,we analyzed 1,732 pig fecal samples collected from March to October 2017 at Henan Province for evaluating the prevalence and resistance diversity of Salmonellae isolated from feces of asymptomatic,live and slaughtered pigs.The Salmonellae were isolated and identified by PCR.They were serotyped using commercial antisera and assayed for the MIC(Minimum inhibitory concentration,MIC)of 16 antibiotics by broth microdilution method.The average prevalence of Salmonella was 19.46%(95% CI: 17.6–21.4).Large farms(herd size ≥1,000)were found to have a higher prevalence as compared to the small-and medium-scale farms(p < 0.0001).The prevalence of salmonellae in samples collected from the farms [11.77%(95% CI: 10.1–13.6)] and the slaughterhouse [45.23%(95% CI: 40.3–50.30)] was statistically different(p < 0.0001).Uncommon serovars of Salmonella such as Agama and common serovars such as Derby and Typhimurium were isolated.High resistance(>80%)was recorded toward ampicillin(81.6%),amoxicillin/clavulanic acid(80.4%),ciprofloxacin(100%),tetracycline(99.4%),doxycycline(97%),and sulfamethoxazole(85.8%).Multidrug resistance(MDR)to four,five,and seven classes of antibiotics was recorded to be approximately 25% in the most prevalent serovar like Derby.In conclusion,the presence of alarmingly high resistance of Salmonella isolates,toward the critical antibiotics such as fluoroquinolones and beta-lactams in swine farms and slaughterhouse in Henan Province,should draw public attention.2.Prevalence and antimicrobial resistance of Salmonella recovered from pig-borne food products in Henan ProvinceFor investigating the prevalence,serovars,and antimicrobial resistance pattern of Salmonella isolated from pig-borne food in Henan Province,1441 pork and viscera pig products were collected from retail markets in 11 cities of Henan Province between March and November in 2019 for Salmonella isolation and culture.The Salmonella isolates identification were subjected to microbiological culture and PCR assay.Positive isolates were assayed for serotyping by slide agglutination test and antimicrobial resistance susceptibility by MIC test.We found that 219(15.20%)were positive for Salmonella isolates from liver(n = 83),bile(n = 19)and pork(n = 117).The highest prevalence of positive Salmonella isolates was observed 25.86% in liver(n = 83/321),followed by 21.84% in bile(n = 19/87),and 11.33% in pork(n = 117/1033).Seventeen different serovars were identified and classified into five serogroups.The most prevalent identified serovar was S.Derby(32.87%),followed by S.Rissen(25.57%)and S.Goldcoast(10.50%).We also found a high resistance rate against tetracycline(96.5%),sulfisoxazole(96.3%),followed by ampicillin(84.4%),streptomycin(83.7%),respectively.An additional investigation of multi-drug resistance pattern(MDR ≥3 antimicrobial classes)indicated that serovar Indiana(n = 13)showed the highest MDR(ACSSu T-AMC-CIP)prevalence(100%),followed by Goldcoast and Derby.Collectively,this study showed considerable diversity among Salmonella isolates in the pig-borne food and highlighted a high prevalence and emerging foodborne MDR clones that may increase the frequency of therapeutic failure in the clinical settings.3.Comparative Genomic Analysis of Salmonella Typhimurium and Salmonella Serotype 4,[5],12: i:-Strains in the pig production chain of Henan ProvinceTo investigate the phylogenetic relationships,drug resistance genes,virulence genes,and plasmid types of 91 Salmonella Typhimurium and Salmonella serotypes 4,[5],12:i:-isolates from the swine industry chain of Henan Province,the whole genome sequencing of these isolates was performed by next-generation sequencing using Illumina Nova Seq 6000 Platform.Based on the whole genome data of the isolates tested,the in silico serotyping was determined using the SISTRS tool,multilocus sequence typing(MLST)was carried out by online tools,and the phylogenetic tree was constructed by single nucleotide polymorphism analysis.The plasmid types,virulence genes,and drug resistance genes in 91 Salmonella strains were analyzed by the corresponding database analysis software.Serovar prediction by the SISTR tool and MLST typing indicated that fifty Salmonella strains belonged to serovar 4,[5],12:i:-/sequence type(ST)34,but the remaining 41 Salmonella strains belonged to serovar Typhimurium /ST34(n = 15)and serovar Typhimurium /ST19(n = 26).Compared with Salmonella Typhimurium,Salmonella serotype 4,[5],12:i:-has a slightly higher prevalence in the swine industry chain in Henan Province,especially in pork and viscera pig products from retail markets.This may explain why Salmonella serovar 4,[5],12:i:-exceeded Salmonella Typhimurium and ranked the second most frequently encountered serotype in patients in Henan,China.Compared with ST19 strains(n = 26),ST34 strains(n = 65)were the dominant sequence type strains in this area.Phylogenetic tree analysis showed that regardless of the sources of isolated samples,serotypes,and isolation sites,all ST19 strains or all ST34 strains were clustered together,respectively,forming two branches.However,whether among ST34 strains or ST19 strains,the Salmonella strains from different sources may be clustered together,indicating that the same Salmonella clones may spread with the breeding pig trade or through the pork food chain over long distances.Plasmid type analysis showed that ST34 strains or ST19 strains carried the corresponding preference plasmids.Antimicrobial resistance gene and virulence gene analysis showed that 91 isolates have the high resistance to various drugs such as ampicillin,tetracycline,and sulfamethoxazole,with a multi-drug resistance rate of 98.90%,of which the multi-drug resistance rate of Salmonella serotype 4,[5],12:i:-was 98%.Compared to Salmonella Typhimurium/ST34 and Salmonella Typhimurium/ST19,Salmonella serotype 4,[5],12:i:-/ST34 carried more types of resistance genes and virulence genes.In conclusion,Salmonella serotype 4,[5],2:i:-strains from the Henan pig industry chain carry more types of drug resistance genes and virulence genes,which may be one of the main reasons for the evolutionary success of this serotype.In addition,compared with S.Typhimurium/ST19 strains,S.Typhimurium/ST34 strains are more closely related to serotype 4,[5],12:i:-/ST34 strains and have some common preference plasmids,being the ancestor of serotype 4,[5],12:i:-/ST 34 strains.To sum up,this study found that Salmonella isolates from pig farms,slaughterhouses,or retail markets showed high resistance to some key antimicrobial drugs such as ampicillin,ciprofloxacin,etc.,but low resistance to cephalosporins,polymyxin,etc.Therefore,these results provide a reference for farmers or veterinarians in clinical drug use.At the same time,it reminds government management departments to continue to carry out antibiotic management planning in maintaining animal health and producing safe pork,and to guide animal producers to use some key antibiotics wisely,so as to avoid the emergence of antimicrobial-resistant strains because antimicrobial-resistant strains may pose a threat to human public health.Furthermore,this study firstly reported that compared to Salmonella Typhimurium,Salmonella serotype 4,[5],12:i:-has a higher prevalence in the pig production chain in Henan Province.In addition,this study has revealed some information on sequence types,drug resistance genes,plasmid types,and virulence genes in Salmonella serotype 4,[5],12:i:-strains,which lays a foundation for the prevention and control of this serotype infection in humans or animals.