| Foodborne disease is a major public health problem that endangers human health,and salmonella is one of its most common foodborne pathogens.Salmonella is a common gram-negative facultative anaerobic bacteria,belonging to the enterobacteriaceae,which can infect humans through livestock meat,fruits,vegetables,and other ways,causing gastroenteritis,typhoid fever,bacteremia,and other diseases.Food poisoning caused by salmonella in our country ranks first in food poisoning caused by microorganisms every year.Contaminated raw livestock meat is the most important way of human infection with salmonella.Therefore,continuous monitoring of salmonella contamination levels in raw animal meat is necessary to control the transmission of the pathogen from food to humans.Polymyxin is a polypeptide antibacterial drug synthesized from bacillus polymyxins.Its clinical use was limited in the early days due to its renal and neurotoxicity.However,in recent years,due to the emergence of carbapenem-resistant enterobacter,polymyxin has been used again in clinical practice and is regarded as the last line of defense against gram-negative multi-drug-resistant bacteria infection.It was previously widely believed that the mechanism of polymyxin resistance was a chromosomal mutation.In 2015,Chinese scholars reported the plasmid-mediated mobile colistin resistance(MCR)mechanism and related gene mcr-1 globally for the first time.It was proved that the gene could be transmitted horizontally among different enterobacteriaceae bacteria.The re-use of polymyxin drugs in clinical practice and the discovery of new drug resistance mechanisms lead to a significant increase in the drug resistance rate,which brings a huge threat to human public health security.In recent years,more and more reports have been reported on the mcr family of colistin resistance genes.So far,10 mcr family members and 80 related mutants have been found.Escherichia coli is the most common type of bacteria in mcr related studies,while Salmonella carrying the mcr gene is relatively less reported,but the detection rate has been increasing year by year in recent years.Therefore,the long-term continuous monitoring of mcr gene should be strengthened to understand its epidemic characteristics and provide a theoretical basis for clinical treatment and policy formulation.OBJECTIVE:1.To investigate the contamination status and epidemiological characteristics of salmonella in retail chicken meat in Jinan from 2020 to 2021,to provide a scientific basis for prevention and control measures of salmonella in food.2.Serotype and molecular epidemiological characteristics of food-borne salmonella were analyzed based on whole genome sequencing.3.Plasmid-mediated colistin resistance gene mcr was screened for the isolates to study the resistance mechanism,transmission mechanism,and pathogenic characteristics of polymyxins in food-borne salmonella.METHODS:1.Samples collection and strains isolationFrom December 2020 to November 2021,20-30 chicken samples will be collected every month in Jinan City.The salmonella in the samples was tested according to the National Standard for Microbiology Test of Food Safety(GB 4789.4-2016).The standard strains of Salmonella enteritis(EQAS 2012S4)and Salmonella typhimurium(CMCC 50333)were used as positive controls in the whole process from pre-amplification to biochemical identification.2.Identification of salmonella speciesBiochemical tests and PCR methods were used to double-check and identify the isolated and cultured strains.For any strain whose test results were negative,biochemical identification was performed by VITEK 2 COMPACT automatic microbial analyzer,and salmonella isolates were obtained.3.Serotype screening of salmonella enteritidisA specific identification gene(ENT)was used by PCR to identify salmonella enteritidis dominant serotype in poultry food.4.Antimicrobial sensitivity test of salmonellaAccording to the sample information and serotype screening results of salmonella enteritis,50 representative strains of salmonella were selected,and the sensitivity of salmonella isolates to 16 antimicrobial drugs such as tetracycline and penicillin was determined by VITEK 2 COMPACT automatic microbial analyzer.5.Whole genome sequencing and analysisThe nucleic acid of salmonella isolates was extracted and its concentration and quality were determined.DNA that met the requirements was sent to Tianjin Novogene for whole genome sequencing to obtain its genome data,and then salmonella serotype analysis and multi locus sequence typing were performed on the genome data(MLST)and antimicrobial resistance gene analysis.6.mcr gene screeningThe polymyxin resistance genes mcr-1～mcr-9 were screened by PCR,and the strains with positive mcr genes were confirmed by triple check.7.mcr gene positive strain antimicrobial sensitivity test supplement testThe microbroth method was used to supplement the antimicrobial sensitivity test for the strains with mcr gene positive.There were 42 kinds of antibacterial drugs,including 12 classes such as ticarcillin and piperacillin.8.Study on virulence phenotype of mcr gene positive strainThe virulent salmonella Indiana MS1-1 and staphylococcus aureus SA were used as control strains,phosphate buffered saline was used as a negative control,and blank control was set up to establish a mellonella infection model.The death of mellonella larvae was recorded at intervals of 24 h,48 h,72 h,and 96 h,and the growth curve was drawn.The virulence phenotype of mcr gene positive strain was studied.9.Data analysisSPSS V25.0 software package was used to conduct χ2 test for the detection rate of salmonella,and the test level was α=0.05,P<0.05 indicated that the difference was statistically significant.RESULTS1.Salmonella detection in retail chicken meatAmong 260 chicken samples collected from December 2020 to November 2021,58 were positive,with a positive rate of 22.31%(58/260).A total of 100 strains of Salmonella were detected,among which 56 strains were Salmonella enteritidis,accounting for 56.00%of the total strains.In terms of sample collection time,the detection rate of salmonella was the highest(53.33%,32/60)in autumn(September-November),and the lowest(10.00%,6/60)in spring(March-May).There was a significant difference in the detection rate of salmonella in different seasons(χ2=44.110,P<0.01).In terms of sample collection locations,salmonella was detected in 24,2,21,and 14 samples collected from supermarkets,online e-commerce platforms,farmers’ markets,and retail stores,respectively.The detection rates were 22.92%(22/96),12.50%(2/16),19.44%(21/108),and 32.50%(13/40),respectively.There was no significant difference in the detection rate of salmonella in different sampling sites(χ2=3.817,P=0.282).In terms of sample types,Salmonella was detected in 52 samples of pre-packaged frozen chicken products and 9 samples of fresh whole chicken,with detection rates of 25.64%(50/195)and 12.31%(8/65),respectively.There was a significant difference in the detection rate of different samples(χ2=5.001,P=0.025).2.Antimicrobial sensitivity test resultsThe results of the salmonella antimicrobial susceptibility test showed that 43 of the 50 Salmonella strains were resistant to at least one antimicrobial drug,with a resistance rate of 86%(43/50),the resistance rate of doxycycline was the highest(48%),followed by mannan(42%),and the resistance rates of cefoperazone/sulbactam,amikacin,imipenem,and meropenem were all below 10%.No strains resistant to tigecycline and piperacillin/tazobactam were detected.In this study,there were 21 strains of multi-drug resistant bacteria,accounting for 48.83%(22/43)of the drug-resistant strains,among which 3 strains were resistant to 10 or more kinds of antimicrobials.3.Whole genome sequence analysis of salmonellaA total of 14 serotypes were detected in the 50 isolates,among which Salmonella enteritidis serotype was the highest(40%,20/50),followed by the Salmonella Kentucky serotype(22%,11/50).The MLST typing results showed that the dominant ST type was ST11,and the corresponding serotype was salmonella enteritidis.The MLST typing was closely related to the serotype.Among the 50 isolates of salmonella,44 carried drug-resistance genes,and 59 kinds of drug-resistance genes were detected in 11 classes.Among them,the β-lactam resistance gene blaTEM had the highest detection rate(52.27%,23/44).4.mcr gene screening resultsmcr-1 gene was detected in 2 of the 100 Salmonella strains,the detection rate was 2%(2/100),and mcr-2～mcr-9 gene was not detected.The two mcr-1 positive strains were obtained from two frozen chicken wing samples collected in January and April 2021,named S16 and S19,respectively.5.The antimicrobial sensitivity test of mcr gene positive strains supplemented the test resultsThe two mcr-1 positive strains were all multidrug-resistant strains.According to 42 kinds of drugs in 12 classes tested,strain S16 was resistant to 32 kinds of antibiotics in 11 classes,and strain S19 was resistant to 38 kinds of antibiotics in 12 classes.Both strains were resistant to colistin,and MIC values were 4mg/L.In addition,strain S19 was also resistant to the four carbapenems tested.6.Study on virulence phenotype of mcr gene positive strainsWhen the concentration of the bacterial solution was 1×10-7 CFU/mL,the larval survival rate of the MS1-1 experimental group was 0 after 24 h inoculation.The survival rate of the S19 test group was 30%.The staphylococcus aureus SA group had the highest larval survival rate.CONCLUSIONS1.From 2020 to 2021,the detection rate of salmonella in retail chicken meat in Jinan was 22.31%,indicating serious pollution.The contamination rate of samples collected in autumn was relatively high,with a detection rate of 53.33%.Salmonella enteritidis was the dominant serotype.The detection rate of the mcr-1 gene was 2%.2.The antimicrobial resistance of salmonella in retail chicken meat in Jinan is severe,with at least one antimicrobial resistance rate of 86%,and multi-drug resistance bacteria accounting for 52%.3.The two Salmonella strains carrying the mcr-1 gene not only showed severe multidrug resistance,but also one strain was even resistant to colistin and a variety of carbapenems at the same time,with strong virulence. |
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