| All around the world,Salmonellosis caused by Salmonella enterica serovar Enteritidis is a major foodborne infectious disease.S.Enteritidis can cause the outbreak and death of avain.After S.Enteritidis infected,chickens often appear as insidious carriers and do not develop.The bacteria can infect humans and many other animals.It is an important and harmful zoonoses bacteria.The use of antibacterial antimicrobials is one of the important means to prevent S.Enteritidis infection,and the emergence and spread of antimicrobial resistance of S.Enteritidis greatly restricts the effect of antibiotics.The emergence and spread of antimicrobial resistance genes is the molecular mechanism of S.Enteritidis resistance.The development and breakthrough of high-throughput sequencing technologies have provided great convenience for studying the molecular mechanisms of antimicrobial resistance.In this study,190 S.Enteritidis were resequenced to investigate the application of high-throughput sequencing technology in the study of S.Enteritidis resistance and analyze the antimicrobial resistance mechanism and evolution of S.Enteritidis in China.In this study,bioinformatics technology was used to quality control and splicing the whole genome sequencing raw reads of 190 isolated strains to obtain assembled long sequences.The final assembly file was used as a complete genome data material for subsequent analysis.1.Biometric identification of Salmonella EnteritidisBioinformatics technology was used to quality control and splicing the whole genome sequencing raw reads of 190 isolated strains to obtain assembled long sequences.The final assembly file was used as the whole genome data material for subsequent analysis.The species was first identified using the MicrobeTrakr online tool and the results showed that all 190 strains were Salmonella.Then the sistr_cmd software was used to identify 190 strains of the sequenced strains and the results showed that they were all S.Enteritidis.Finally,using the PubMLST tool to identify the ST-type,the ST-type except SAL0000131 and SAL0000132 two strains are new,the rest are ST11,consistent with the ST-type characteristics of 5.Enteritidis,and SAL0000131 and IS AL0000132 unknown aroC gene is the result of the two strain STs showed a novel cause.2.Drug resistance genes and drug resistance of Salmonella EnteritidisThe resistance phenotypes of 190 strains of S.Enteritidis were tested.The results showed that S.Enteritidis had the highest resistance to NAL(83.7%),and the resistance to AMP and AMC was very high,reaching 62.6%and 62.1%,CFZ(41.6%),STR(45.8%),SXT(29.5%)and TET(21.6%)were also high.The number of resistant strains of other antimicrobials was rare,and the strains did not show any resistance to AMK.Among the different hosts,the resistance of 3 geese-derived S.Enteritidis strains reached 100%,and the MIC50 and MIC90 were both>512?g/ml;The rate of antimicrobial resistance of AMP,AMC,STR and TET reached 66.7%.The resistance rate of CFZ was 33.3%.The MIC90 was 8 ?g/ml.No antimicrobial resistant strains appeared in other antimicrobials.The S.Enteritidis derived from human not only has a very broad spectrum of antimicrobial resistance,but also has a high level of antimicrobial resistance.The overall antimicrobial resistance situation is the most complex.The rate of multiantimicrobial resistance reaches 65.1%,and the multiantimicrobial resistant strains have a very broad spectrum of antimicrobial resistance.The resistance rate of NAL was the highest,reaching 81.4%;the resistance rates to AMP,AMC and CFZ were all over 50%,reaching 62,8%,65.1%and 53.5%respectively;the resistance rates to ATM and STR reached 23.3%and 37.2%respectively;resistance rates to KAN,GEN,TET,SXT,CHL and OLA also reached 16.3%,16.3%,16.3%,11.6%,11.6%and 9.3%respectively;the resistance rate of CIP and ENR was only 2.3%and 4.7%and MEM,AMK and NIT had no antimicrobial-resistant strains.The resistance spectrum of S.Enteritidis in chickens was wide,the resistance rate and resistance level were both high,and the overall antimicrobial resistance was more complicated.The resistant rate of NAL(94.3%)was the highest,and the MIC50 was 512 ?g/ml.The antimicrobial resistance rates of AMP,AMC,CFZ and STR were 69.9%,68.3%,42.3%and 52.0%respectively.SXT and TET were highly resistant,reaching 31.7%and 25.6%.The overall resistance of S.Enteritidis from duck was low,with the highest resistance to OLA(100%)and low resistance to AMP,AMC,STR,TET,and NAL(20%,20%,40%,20%and 20%respectively).No antimicrobial resistance was found in the remaining antimicrobials.The overall resistance of S.Enteritidis was low,and the resistance rate to AMP,AMC,CFZ,STR and NAL was only 20%;the resistance rate to TET and SXT was only 13.3%;the resistance to OLA was 6.7%;no antimicrobial-resistant strains were found in the remaining antimicrobials.The remaining host strains are small in number,and their resistance rates are much lower than those of human and chicken-derived S.enteritidis.Using blast software to compare assembly sequence with ResFinder database,a total of 50 resistance-associated genes(aadA1?aadA2?aadA5?aadA16?aadA22?aac(6')Ib-cr?aac(3)-Iva?aac(3)-?d?aph(3')-?a?aph(3')-Ia?aph(3')-Ic?aph(4)-Ia?aph(3)-Iva?arr-3?blaTEM-1?blaCTX-M-3?blaCTX-M-14?blaCTX-M-55?blaCTX-M-65?blaOXA-1?blaNDM-5?blaMIR-2?blaMIR-6?blaCMY-2?catA1?catB?catB3?cmlA1?dfrA1?dfrA12?dfrA14?dfrA17?dfrA27?ermB?fosA?floR?InuF?mefB?mphA?oqxA?oqxB?qnrA7?strA?strB?sul2?sull?sul3?tetA?tetB and tetC)were detected.Removal of genes with identity less than 80%and coverage less than 70%and related antimicrobials not included in the MIC test,resulted in phenotypic test-related resistance genes totaling 31 types,including seven beta-lactam resistance-related genes blaTEM-1,blaCTX-M-3,blaCTX-M-14,blaCTX-M-55,blaCTX-M-65,blaOXA-1 and blaCMY-2)and 13 aminoglycoside-resistance related genes(strA,strB,aadAl,aadA2,aadA5,aadA16,aadA22,aac(3)-?d,aph(3')-?a,aph(3')-Ia,aph(4)-Ia,aph(3)-Iva and aac(6')Ib-cr),three quinolone related genes(qnrA7,oqxA and oqxB),two tetracycline related genes(tet A and tetC),three sulfa resistance related genes(sull,sul2 and sul3),and three trimethoprim-related resistance genes(dfrAl,dfrA17 and dfrA27).The correlating relationship between genotype and phenotype was good.The correlating rates of blTEM-1 gene and penicillins AMP and AMC reached 96.1%and 95.1%.The correlating rate of strA gene and strB gene and aminoglycoside STR reached 90.9%.The correlating rate of tetA gene and tetracycline was 82.9%,the correlating rate of dfrA17 gene to sulfa antimicrobial compound sulfamethoxazole was 88.9%,and the correlatinon between catB3,catB,floR,cmlAl and amidohydrin antimicrobial CHL reached 100%.It shows excellent correlatinon,but there are still cases where the phenotype does not correspond to the genotype,indicating that there is still an unknown mechanism of antimicrobial resistance.During the detection and analysis of plasmids,it was found that IncXl plasmid carries many resistance genes,which may be closely related to the resistance epidemic of Salmonella Enteritidis.For the detection of antimicrobial resistance genes carried by different plasmids,we found that 95 IncXl plasmids carried 76 of 77 strA genes,76 of 77 strB genes,76 of 77 sul2 genes,and 31 of 31 tetA genes and 95 of the 103 blaTEM-1 genes,none of the rest of the plasmids carry antimicrobial resistance genes.This suggests that the IncXl type plasmid is a key mobile element for S.Enteritidis resistance.Studies of point mutations in genes suggest that gene point mutations can indeed affect antimicrobial resistance phenotypes,in which the A120C mutation in the blaTEM-1 gene may be the cause of the great phenotype of cefazolin,and numerous mutations in strB also affect the phenotype.The A124G of tetA may have a weakening function on the phenotype,G250A and G277A may have an enhanced effect on the phenotype,and G250A and G277A in the tetA may be able to compensate for the reduced phenotype caused by A124G,resistance to quinolone production and QRDR The point mutations are related and result in different antimicrobial resistance phenotypes and levels due to the interplay between each gene and each point mutation.3.The evolution and drug resistance of Salmonella EnteritidisUsing Parsnp software,190 strains of S.Enteritidis were used to make the core genome phylogenetic tree and analyzed with background information.The MIC phenotype,antimicrobial resistance genotype,and plasmid typing showed a significant phylogenetic difference in the evolutionary relationship.The different branches generated by the construction of the core genome phylogenetic trees were able to correspond accurately and clearly with the background information on antimicrobial resistance.A total of 208 strains of S.Enteritidis were constructed using Parsnp software at home and abroad.The results showed that the strains in the United States,Canada,South Korea,Moscow,Denmark,and Brazil can be clearly distinguished in the evolution tree,and the foreign strains are in independent branches,and can form a more definite lineage according to the host and age sources.The prevalence of S.Enteritidis in the country is composed of two parts,one is Salmonella Enteritidis,which is endemic in China,and the other is S.Enteritidis from North American countries the United States and Canada.According to the comparative analysis of the evolutionary relationship between strains domestically and abroad,the evolutionary structure of S.Enteritidis domestically is more complex than that of foreign strains.The evolutionary relationship between S.Enteritidis in each host and region in China is very similar,and the non-exotic strains show different lineages.This may mean that the prevalence of S.Enteritidis in the country is even greater,and the strains between the hosts are more frequently exchanged.Evolutionary analysis based on whole genome resequencing can predict and guide S.Enteritidis resistance to a certain extent. |
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