| Animal meat is the major way for human intake of protein.But in the animal breeding process,antibiotics such as cephalosporins and polymyxin and so on show a large number of and long-term usage,so increased bacterial resistance level of animal meat leads to the increase of human health risks.Salmonella is the main food-borne pathogen for human,and could cause treatment difficulty for Salmonella infections on account of its resistance in human.So the understanding of contamination and mechanism of drug resistance dissemination for Salmonella from animal meat,will have important significance for the transmission control of resistant Salmonella and the treatment of food-borne Salmonella diseases effectively.This paper mainly focuses on isolating methods of Salmonella from pork,beef,chicken and shrimp,the contamination situation and propagation mechanisms of CTX-M type ESBLs and mcr-1 resistance determinant of Salmonella bacteria from animal meat in Shenzhen.We hope these data will provide some theoretical basis for better isolation and detection for Salmonella and the use of antibiotics in animal husbandry,as well as prevention and treatment of food-borne Salmonella diseases in the clinical.The first part:Characterization of Salmonella spp.recovered from animal meat by different isolating methodsThe purpose of this study was to understand the prevalence and distribution of Salmonella from animal meat in Shenzhen currently and the effect of different isolation methods on isolation rates of Salmonella from animal meat,so that what we did could provide theoretical basis for the isolation and identification of Salmonella from animal meat as well as controlling the transmission of Salmonella from animal meat reasonably in Shenzhen.Firstly,we conducted the performance of isolating and identifying for Salmonella from 1055 livestock and poultry samples collected in Shenzhen.The results showed the total positive rate for samples was 21.71%(229/1055),and the isolation rates for four kinds of samples were 29.19%(61/209)in chicken,26.01%(148/569)in pork,15.00%(15/100)in beef and 2.82%(5/177)in shrimp.Morever,85%(195/229)of positive samples could be detected through TT enrichment,while 30%(69/229)through RV enrichment.Secondly,376 non-repeated strains were recovered in this study via MIC.The data released that 290(77.13%)could be got from samples enriched through TT broth,but only 86%(22.87%)from samples through RV;about 60%of Salmonella isolates were recovered from XLT4 without any antibiotics,followed by 31%from XLT4 containing CIP,7%from XLT4 containing CTX,1.6%from BGA plates.Thirdly,we conducted the identification and analysis for 376 non-repeated strains through serotyping technology.The data showed that 38 serotypes could be obtained and 329 strains were identified for specific serotypes.Derby,1,4,[5],12:i:-,Rissen,Agona and 8,20:z23,z4:-were among the most prevalent serotypes and 35 of 38 serotypes could be identified from TT enrichment broth,which was almost two times than those from RV broth(35/19),especially the major serotypes including Derby,Agona and Rissen.Above all,the prevalence of Salmonella from animal meat is serve in Shenzhen,and the most common serotypes contains Derby,1,4,[5],12:i:-,Rissen,Agona and 8,20:z23,z4:-.In addition,TT enrichment broth is more effective than RV broth as well as XLT4 plates than BGA plates in isolating Salmonella from animal meat,indicating the bonding of XLT4 and TT enrichment broth would perform better in Salmonella isolating.The second part Detection of CTX-M type ESBLs resistance gene and study of its prevalence and transmission mechanism in Salmonella from animal meatThe purpose of this study was to understand the contamination and drug resistance situation of Salmonella from animal meat in Shenzhen,including the carrying and epidemic mechanism of CTX-M ESBLs resistance gene against cephalosporin antibiotics,and to provide certain theoretical basis for reasonable use of antibiotics in farming enterprises and clinical,stopping the blacTX-M resistant gene in positive Salmonella from disseminating.Firstly,Salmonella isolating and drug resistance monitoring of Salmonella in animal meat samples in Shenzhen were done.217 positive samples were detected to be positive,and 336 non-repetitive Salmonella strains were isolated with 20.91%isolation rate,among which 26.4%Salmonella isolation rate of chicken samples was the highest.The antimicrobial susceptibility testing showed that the resistance of Salmonella from animal meat in Shenzhen to multiple drugs was serious,but to amikacin aminoglycoside and meropenem carbapenem was less,of course including certain resistance to cephalosporins.Secondly,the blaCTX-M gene detecting and PFGE genotyping of 336 Salmonella strains were done.The results showed 16 strains which only contained one of CTX-M-14 and CTX-M-15 two determinants were positive,in which Indiana and O4,O12:i:-were the predominant serotypes.PFGE analysis showed that the dissemination of CTX-M-14 and CTX-M-15 determinants of Salmonella from animal meat in Shenzhen was mainly through horizontal transmission,with clonal transmission in meat from the same source included.Finally,the conjugation testing were done for blaCTX-M positive strains and the S1-PFGE analysis was performed between the parent strains and corresponding transconjugants.The result indicated that the 7 Salmonella strains which transfered plasmids to J53 successfully were all from pork,and O4,O12:i:-was the dominant serotype in these 7 strains.S1-PFGE result showed that the plasmids transferable successfully ranged from?30kb to?250kb in sizes,inferring that the blaCTX-M determinants distributed widely in plasmids of different sizes.PFGE typing illustrate that the homology among 7 Salmonella strains was low,which indicated that the dissemination of blaCTX-M determinants in Salmonella from animal meat in Shenzhen was mainly through horizontal transmission.To sum up,the contamination of Salmonella from animal meat in Shenzhen is very serious,and multi-drug resistant phenomena are common.CTX-M-14 and CTX-M-15 are the major blaCTX-M determinants in animal meat.Indiana and O4,O12:i:-are the dominant serotypes in two blaCTX-M determinants.Conjugation phenomena exist in the plasmids containing CTX-M-14 or CTX-M-15 determinant in Salmonella.PFGE analysis results show that the dissemination of CTX-M-14 and CTX-M-15 determinants in Salmonella from animal meat in Shenzhen are mainly through horizontal transmission,with clonal transmission in meat from the same source.The third part Detection and epidemiological study on mcr-1 gene in Salmonella from animal meatThe purpose of this study aimed to understand the carrying situation of mcr-1 polymyxin resistance gene and the transferable ability along with mcr-1 plasmids in Salmonella from animal meat in Shenzhen currently,guiding rational use of polymyxin in livestock and poultry producing,and providing a theoretical basis for preventing polymyxin resistance from disseminating in the clinical.First of all,mcr-1 resistance determinant was screened among 830 non-repetitive Salmonella strains from Shenzhen and 230 non-repeated Salmonella strains from more than 20 provinces and cities nationwide,and then serotype identification were performed for mcr-1 positive strains.The isolation result showed 16 mcr-1 positive strains all from pork samples were obtained,and the isolation rate was 1.4%(12/830)and 1.7%(4/230),respectively.The serotyping results showed Derby(n=9)and Typhimurium(n=6)were main serotypes,with Weltevreden(n=1)a less rate.In addition,we also got that drug resistance spectrum of serotype Typhimurium was generally more extensive and multiple drug resistance was also more obvious,comparing with Derby and Weltevreden two serotypes.Secondly,drug susceptibility testing were performed among 13 kinds of antibiotics by agar dilution method CLSI recommended.The result showed that 16 strains were all resistant to polymyxin(8?g/mL-16?g/mL)to a high degree,and also all resistant to tetracycline and ampicillin.The rate of resistance to chloramphenicol(75%)was also high,and only 4 strains showed susceptibility.In addition,these 16 strains showed better susceptibility to other antibiotics,such as amoxicillin/clavulanic acid(12.5%)and ciprofloxacin(18.75%)antibiotics,with 100%susceptibility to azithromycin.The majority of polymyxin resistant Salmonella showed multidrug resistance,generally more than four kinds,even up to eight kinds.Thirdly,PFGE typing method was used to classify the mcr-1 positive Salmonella strains.The result showed that all the strains could be divided into 6 genotypes,so the genotypes were diversified.The similarity among 9 Derby strains from Shenzhen was 100%,as well as 2 Typhimurium strains from Shenzhen,but 3 Typhimurium strains and 1 Weltevreden strains from other provinces showed low homology,with only 1 Typhimurium strain 100%homologous to 2 Typhimurium strains from Shenzhen.In the end,all the mcr-1 positive Salmonella were performed by conjugation,with J53 used as the recipient bacterium.The result showed that all 16 strains could be transferable successfully,and the rate was 100%.The results of drug susceptibility testing between parent strains and transconjugants showed that all transconjugants obtained resistance ranging from 4?g/mL to 8?g/mL to polymyxin,and improved resistance to other antibiotics to a different degree.In addition,some transconjugants also got higher resistance to cefotaxime and ceftriaxone.In summary,the detection rate of mcr-1 positive Salmonella from animal meat in Shenzhen and even the nation are relatively low,but the vast majority of which are resistant to four kinds or more.The rate of successful conjugation is very high,and the serotypes of these Salmonella transferable successfully are mainly Typhimurium and Derby.The dissemination of mcr-1 resistant determinant in Salmonella from animal meat in Shenzhen is mainly clonal transmission,which is probably related to the sample collection scope,but horizontal transmission predominates across the country.The fourth part Study on the transmission mechanism of mcr-1 resistance gene in Salmonella from animal meatThe aim of this study was to investigate the transmission mechanism of mcr-1 resistance gene in Salmonella from animal meat.Firstly,plasmids analysis was performed between parental strains and transconjugants by S1-PFGE and Southern Blot.The result showed that mcr-1 gene mainly exists in?30kb and?244kb two kinds of plasmids,which were detected in 14 Salmonella strains and 2 strains Salmonella,respectively.And none strain containing both two mcr-1-bearing plasmids was found at the same time.Finally,the representative plasmids in transconjugants,SA258,SA618,SA655,were extracted,which were sequenced using plasmid sequencing subsequently.Then the obtained sequences were analyzed using plasmid analysis software,and compared with other reference sequences.The result showed that the?30kb plasmid was IncX4 type and the?244kb plasmid was IncHI2 type.Subsequently these two types of plasmids were compared with the reference sequences in GenBank.The result showed that these two plasmids all had a high similarity with the corresponding reference sequences,and all of them contained tra and so on responsible for transmission.In details,the?30kb plasmid had a above 99%similarity with pOW3El reference plasmid;the-244kb plasmid had a lack in the backbone area and also contained resistance determinants such as blaCTX-M and fosA3,comparing with pHNSHP45-2 plasmid,which explained the phenomena of resistance to cephalosporins and fosfomycin of Salmonella containing this kind of plasmid.In summary,this study shows that the dissemination of mcr-1 gene in Salmonella from animal meat is mainly through horizontal transmission,and the gene is in wide range;further studys of resistant plasmids show that mcr-1 gene in Salmonella from animal meat from different regions of the country mainly locates in IncX4 and IncHI2 two kinds of plasmids,which have high similarity with the corresponding plasmid sequences reported previously and more resistant determinants. |
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