| Background:Salmonella is one of the most common pathogens causing disease from microorganisms in foodborne illnesses worldwide.Salmonella is often detected in patients with diarrhea,healthy(non-diarrheal)people,poultry,and meat.Food workers can also be infected with Salmonella after processing and handling food contaminated with Salmonella,but do not show diarrhea,and there is a risk of becoming carriers and further contamination of food.If the population carries Salmonella and transmission,it should be of epidemiological significance in the link of contacting food,carrying pathogens or even drug-resistant pathogens to the people.Currently,the majority of studies reported on Salmonella detection by food workers focus on the comparison of detection rates,with less research on serotyping and antibiotic resistance of strains.On the one hand,the proliferation of pathogenic bacteria in the human intestine with infection is related to the pathogenicity of the pathogenic bacteria,on the one hand,the normal settled intestinal bacterial communities could also afect the intestinal proliferation of pathogenic bacteria through inter-flora rejection and competition for survival.The traditional bacterial isolation and culture of bacteria is based on the subjective screening of bacteria in selective medium,which has limitations for the isolation and analysis of unculturable or caustic bacteria.With the development of genome sequencing and microbiology research technology,culture-free genome sequencing technology could be used to analyze the human intestinal microbiome,pathogenic microorganisms and antibiotic resistance genes in a fine and comprehensive way and suggest the normal gut flora or strains that influence the survival of pathogenic microorganisms in the intestinal tract through the analysis of the reverse composition ratio of different bacteria in the microbiome,providing analytical support to explore the causes of pathogenic bacteria proliferation and the discovery of normal intestinal bacteria that inhibit pathogenic bacteria proliferation.Objectives:The aims of this study were to understand the carriage status of Salmonella in food workers,the serotype distribution and resistance phenotypes of the strains,and to investigate the genomic and resistance plasmid characteristics of Salmonella carrying high-grade resistance genes in food workers,so as to explain the significance of surveillance of pathogenic and resistant bacteria in healthy(non-diarrheal)food workers.The intestinal flora characteristics of Salmonella carriers were also analyzed to screen for flora that may be negatively associated with Salmonella intestinal proliferation by setting up a control group for comparison and microbiomes data analysis,providing candidates for further studies on intestinal bacteria that antagonize Salmonella proliferation.Materials and methods:From October 2021 to September 2022,the population of Yulin City who attended the health examination of food workers was selected.Rectal swab samples were collected from individuals without diarrhea,and duplicate samples were removed.The samples were collected by the Center for Disease Prevention and Control of Yulin,and Salmonella was isolated from the swabs,and serological identification and antibiotic phenotype determination were performed for the detected Salmonella.This study participated in the serotype identification and antibiotic resistance experimental analysis of some of the strains,and then collected all the data results for epidemiological statistical analysis.In this study,rectal swab samples of individuals with and without Salmonella detection were selected from food workers in the same period,matched for age and gender,and droplet digital PCR was performed,from which 32 healthy carriage of Salmonella and 56 healthy human rectal swab samples without Salmonella were selected for enrollment,forming the Salmonella carriage group and the Salmonella non-carriage group.A total of 88 samples were enrolled,and full-length sequencing of 16S rRNA gene was conducted.We analyzed and compared the composition of intestinal flora in the two groups,and screened for normal colonizing flora that were low in Salmonella carriers but high in Salmonella non-carriers.In addition,Salmonella and Klebsiella pneumoniae with high-grade drug(carbapenem antibiotic)resistance genes isolated from food workers were selected for second-and thirdgeneration sequencing of their genomes,and whole-genome completion maps of strains and plasmids were obtained.Sequence alignment analysis of strains was performed using core genomic and pan-genomic approaches,and conjugation experiments were performed to determine the transfer efficiency of plasmids.Results:(1)A total of 45,178 rectal swabs were collected from October 2021 to September 2022 in Yulin,and 859 Salmonella strains were detected,with a cumulative annual detection rate of 1.90%.There was no difference in the detection rate of Salmonella by gender.Using ten years as an age group,the lowest detection rate of Salmonella was 1.42%in the 18～age group,and the highest detection rate of Salmonella was 2.27%in the 50～age group,and there was a difference in the detection rate of Salmonella in different age groups,and the difference was statistically significant(P<0.05).A total of 56 serotypes were identified,among which Salmonella Typhimurium Monophasic variant was the dominant serotype,accounting for 17.81%,followed by Salmonella Rissen,accounting for 9.43%.Antimicrobial susceptibility testing showed that Salmonella had high resistance rates to tetracycline,chloramphenicol and cotrimoxazole,with 63.0%,49.1%and 37.5%,respectively.A total of 335 multi-drug resistant Salmonella strains were detected,accounting for 39.0%of the total number of isolated strains;there were 36 combinations of resistance profiles,among which tetracycline-cotrimoxazolechloramphenicol was the dominant multi-drug resistance profile.(2)The community composition of the intestinal bacteria was different between the Salmonella carriage group samples and the Salmonella non-carriage group samples.The relative abundance of the Actinobacteria phylum in the samples of the Salmonella carriage group was lower than that of the Salmonella non-carriage group,while the relative abundance of the Firmicutes phylum was higher than that of the Salmonella non-carriage group,and the difference was statistically significant(P<0.05).The species richness comparison of bacteria revealed that the relative abundance of Lactobacillus,Serratia,Cutibacterium acnes,Vibrio fluvialis and Actinobacillus in the intestinal bacteria of the samples from the Salmonella noncarriage group were higher than that of the Salmonella carriage group,and the differences were statistically significant(P<0.05).These five bacterial species were listed as candidates for possible inhibition of Salmonella proliferation in the human intestine.In fact,Lactobacillus and Serratia marcescens have been studied for their inhibitory effects on Salmonella proliferation.(3)Based on the analysis of the sequence complete map,the strain carrying the blaNDM-1 gene in this study was found to be the same clone as the clinically infected patient’s strain from the public database,suggesting that the clone can not only cause human infection but also colonize the healthy human intestine.The plasmid in which the blaNDM-1 gene is located is a hybrid plasmid,and pan-genomic analysis revealed that this type of plasmid could break the species barrier of the host bacterium for cross-host transmission.Conjugation assays showed that this plasmid could transfer and mediate the transmission of high levels of carbapenem antibiotic resistance.Based on the analysis of the sequence completion map,the strain carrying the blaNDM-1 gene in this study was found to be the same clone as the strain from the public database of clinically infected patients,suggesting that this clone can not only cause human infection but also colonize the intestine of healthy humans.Conclusions:By monitoring the Salmonella carriage of food workers in Yulin,it was found that the Salmonella serotypes carried by food workers were complex in composition and had serious antibiotic resistance,suggesting that food workers have a greater risk of carrying and transmitting Salmonella.The intestinal flora composition of Salmonella carriages and noncarriages was different,and some bacterial species in the non-carriages group might antagonize the intestinal proliferation of Salmonella,suggesting that the interaction of intestinal bacteria could bring the potential application in the prevention and treatment of Salmonella.Also,strains isolated from food workers carry the blaNDM-1 gene,and drug resistance genes can be hiddenly transmitted and spread across hosts through drug resistance plasmids,also showing that monitoring of enteric pathogens and drug resistance genes in healthy individuals is essential.Therefore,health surveillance of food workers and strict control of food hygiene should be strengthened.At the same time,reasonable and adequate preventive measures should be developed to reduce the risk of global transmission of enteropathogenic and drug-resistant strains. |
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