| Dysentery caused by Shigella spp.is a continuous threat to global public health.Shigella spp.infect about 188 million people worldwide each year and cause about160,000 deaths,mostly in infants and young children.Shigella flexneri(S.flexneri)and Shigella Sonnei(S.sonnei)are the main types of Shigella prevalent in the world.In recent years,S.sonnei has gradually replaced S.flexneri that dominant in the past,becoming the dominant Shigella spp.in China and many other developing or developed countries.S.sonnei evolved from a common ancestor less than 500 years ago,and underwent evolution and divided into several different lineages.This differentiation process mainly derived from Europe,and then spread to other continents.Currently,there are five major lineages.After many years of evolution and spread,S.sonnei has evolved globally and produced a variety of environmental adaptive phenotypes.Many studies have reported phenotypes of strains from different countries and regions with different biological characteristics,including changes in virulence and the emergence of resistance to specific antibiotics.The evolution of antibiotic resistance is mainly through the acquisition of drug-resistant plasmids or mutations in specific genes,resulting in resistance to specific antibiotics.Evolution in virulence genes or other functional genes also affects the phenotype of strains through the acquisition and deletion of genes or mutations in specific genes.In conclusion,these S.sonnei have undergone evolution with different characteristics in many countries and regions,and developed different epidemic phenotypes to adapt to different survival pressures.In addition,domestic strains have also undergone evolution in drug resistance,and strains resistant to multiple antibiotics have emerged.It is worth noting that the co-resistant strains of cephalosporin and azithromycin caused many outbreaks.The results of Pulsed Field Gel Electrophoresis(PFGE)showed that the DNA sequence of S.sonnei isolated from the water source was highly similar to the DNA sequence of the strains isolated from the patients.These outbreak strains were most likely to be transmitted through water sources.The emergence of these outbreak strains poses a major threat to public health.It is urgent to study genome of these strains to explore the evolution between cephalosporin azithromycin co-resistant strains and other strains,which may give the strain the ability to spread in water and cause water-borne outbreaks.This is the first study to systematically study the phylogenetic evolution of outbreak strains of six outbreak and sporadic strains collected in different period and regions in China mainly by means of genomic data analysis,which providing important theoretical support and reference for the prevention and control of S.sonnei.The purpose of this study is to summarize the evolution law of domestic strains through phylogenetic study and analysis of the information of the region and collected age of strains.And then analyse divergence evolution direction of strains.By predicting drug resistance genes,combined with results of phylogeny,type of plasmids,insertors,integrons and plasmid structure,to explore the law of resistance spread and evolution of resistance of domestic strains.Through the study of functional gene annotation and related single nucleotide polymorphisms,combined with phylogenetic analysis,the possible evolutionary law of other functional genes and the role and significance of functional genes in evolution were explored.Through phylogenetic analysis of sequencing data of 743 strains collected by our research group,including 431 sporadic strains,156 outbreak strains and 156 strains in foreign studies,the results showed that 743 strains underwent evolution and formed four independent branches,and 700 strains distributed in the Lineage III branch,accounting for 94.21%of the total number,18 strains were distributed in Lineage I,accounting for2.42%of the total,25 strains were distributed in Lineage II,accounting for 3.36%of the total.Only one strain of Lineage IV branch existed.There were 578 Chinese strains distributed in Lineage III which also underwent evolution,accounting for 98.47%of the total strains.The latter half of Lineage III was mainly divided into two large subbranches,including 269 strains and 279 strains,which were named MCC I and MCC Ⅱ,respectively.The strains on MCC I were mainly from Guangxi,Anhui,Shanghai,Gansu and other regions,including the strains of five outbreaks in Guangxi and strains of one outbreak in Anhui,and the distribution of the outbreak strains was relatively clustered.The MCC Ⅱ strains mainly came from Shanghai,Henan,Xinjiang and other places,and the distribution was relatively dispersed,and the strains in the same area did not accumulate in a large range.Genotypic analysis showed that these strains belonged to seven epidemics,most of which belonged to Global III(n=633)epidemics.It was found that 3.7.6 genotype was the main genotype in China,and other genotypes included 1.5,3.7,3.7.18,3.7.25,3.7.29,etc.BEAST was used to construct the maximum branch trust tree to calculate the time of branch differentiation.The two Chinese clones of MCCI and MCCII appeared in the 1980s,and the common ancestor of the six outbreak strains appeared around 2010.In this study,we also found evolution in resistance,105 drug resistance genes corresponding to 9 classes of antibiotics and three QRDR mutation sites related to quinolone resistance were found in 743 strains through the prediction of drug resistance genes.Aminoglycoside resistance genes mainly include aac(3)-IId,aad A5,ant(3")-IIa,str A,str B,act-30,and sat-2 genes.Theβ-lactam resistance genes mainly include blaCTX-M-14,blaCTX-M-15,blaCTX-M-55,blaCTX-M-3,blaCMY-2,bla TEM-1,bla TEM-209,etc.mcr-1was the main resistance gene of polymyxin.The drug resistance genes of macrolides include mph A and erm B,and the drug resistance genes of sulfamides are sul1 and sul2.Tetracycline resistance genes mainly were tet(A)and tet R.Methyrimidine resistance genes were mainly dfr A1,dfr A12 and dfr A17.Quinolone resistance related QRDR sites included gyr A-S83L,gyr A-D87Y,gyr A-D87G,with gyr A-S83L as the main type.According to the statistics of the carrying rate of major genes,the detection rate of the resistance genes of the old generation antibiotics such as sat-2,dfr A1,aad A1,tet(A),str A,str B and sul2 were at a high level,reaching more than 70%.According to the statistics of the number of drug-resistance genes carried by strains,the average number of drug-resistance genes carried by strains between Lineage III and Lineage I,II and IV were significantly different.The number of drug resistance genes carried by MCC I,MCC Ⅱ and other strains of Lineage III also had significant differences(P<0.05).The analysis of plasmid types showed that the plasmids carried by the domestic strains mainly belonged to Inc B/O/K/Z,Inc FII,Inc I1-(Alpha)and Inc I2.In this study,12specific drug-resistant plasmids were obtained,including 4 Inc B/O/K/Z plasmids,3Inc FII plasmids,3 Inc I1-I(Alpha)plasmids and 2 Inc I2 plasmids.Inc B/O/K/Z plasmids were the main drug-resistant plasmids carried by domestic strains,and the plasmids on MCC I and MCC Ⅱ were Inc B/O/K/Z type plasmids with completely different structures.The Inc B/O/K/Z plasmid on MCC I carries blaCTX-M-14,mph A,aac(3)-IId and other genes,while the Inc B/O/K/Z plasmid on MCC Ⅱ mainly carries bla TEM-1,aac(3)-IId,dfr A12 and other genes.The Inc B/O/K/Z plasmid corresponding to MCC I was also the internal cause of the six outbreaks of cephalosporin and azithromycin resistance.The most popular ESBLs in China are TEM-1,CTX-M-14,CTX-M-15,CTX-M-55 and CMY-2,and the blaCTX-M-15 gene is carried by Inc FII plasmid.The blaCTX-M-55 gene is carried by two types of plasmids,Inc I1-(Alpha)and Inc I2,and blaCMY-2 is carried by Inc I1-(Alpha)plasmids.The popular azithromycin resistance genes in China are mainly mph A and erm B.mph A and blaCTX-M-14 genes are carried by the same plasmid,and their transfer is related to IS26 and ISEcp1,respectively.The erm B gene is carried by the Inc FII plasmid.In this study,we identified genes and SNPs associated with different clade that represent the evolution of each branch.A total of 16822 genes and 3213 core genes were identified in 743 strains by functional gene annotation,pan-genome study and genome association analysis.The 92 pangenes significantly related to MCC I mainly include transcription,translation,recombination,repair,cell wall biosynthesis and other functions.The MCC Ⅱ related pangene,whose function is known,is cea gene,which can encode coliform,and tns E gene is generally absent in MCCII strains.GWAS analysis filtered out SNPS related to the outbreak strain and cef~Razi~R strain respectively,and 10 were with P<1e-100,among which 7 were missense mutations and 3 were synonymous mutations.The significant sites obtained by the two groups only have differences in P values,and the sites are consistent.In order to further understand the differences of other significant sites,59 sites with P<1e-50 were statistically analyzed,and it was found that the outbreak strain had only one more significant site than the cef~Razi~R strains.It was a synonymous mutation located in the yim F gene.Among the sites with P<1e-50,55 were located in the coding region,of which 30 were missense mutations,24 were synonymous mutations,and 1 was nonsense mutation.These genes were mainly involved in energy generation and conversion,inorganic ion transport metabolism,carbohydrate transport metabolism and transcription.Sixty-six mutation sites(P<1e-50)were significantly associated with MCC Ⅱ,of which 61 were located in the CDS region of the coding genes and 5 were located in the intergene region.Sites in CDS of coding genes included 30 missense mutations,30 synonymous mutations,and one was a start codon mutation.The functions of related genes were mainly related to carbohydrate transport and metabolism,inorganic ion transport and metabolism,energy production and conversion,and transcription.The annotation results of virulence genes showed that there were significant differences in the T3SS gene in the strains,but no significant differences in the virulence genes of other functions.Besides the five ipa H genes in some strains,most other genes of T3SS were missing.In summary,this study comprehensively analyzed the diversity of the evolution of domestic S.sonnei,and indicated the different directions of the evolution of domestic S.sonnei,including the diversity of the spread and evolution of drug resistance,the diversity of drug-resistant plasmids,the diversity of mobile genetic elements and the diversity of other functional genes,and discussed the law of the evolution of domestic S.sonnei from multiple perspectives.These results have important guiding significance for the surveillance of bacillary dysentery in the future,and provide a reliable theory for the effective prevention of public health events caused by S.sonnei,and provide formulation of scientific and reasonable prevention and control strategies. |
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