| Yersinia enterocolitica is a small gram-negative bacterium belonging to the genus Yersinia of the Enterobacteriaceae family.It is a human-animal pathogen that is widely distributed in nature.It naturally reside in a variety of animals,and is infected by the faecal-oral route through contaminated food and water,or through contact with infected animals.In addition to gastrointestinal symptoms like diarrhoea,the bacterium can also cause respiratory,cardiovascular,and skeletal connective tissue diseases,as well as sepsis,which can lead to death.Y.enterocolitica is heterogeneous and consists of six biotypes:1A,1B,2,3,4,and 5.Biotype 1B is regarded as highly pathogenic,and biotype 1A is regarded as being non-pathogenic in humans;other biotypes are considered to have low pathogenicity.Enteric yersiniosis has been reported globally,with the most severe epidemic in Europe,where it was the fourth most common zoonotic disease.There were two outbreaks of Y.enterocolitica in China in the mid-1980s,resulting in over 500 infections.Surveillance of the disease in China then ceased for nearly 20 years until 2016,when Y.enterocolitica was included in the national food contaminant surveillance network.Y.enterocolitica has been detected sporadically in animal,food and human clinical samples during 12 consecutive years of surveillance in Ningxia.Its ecology,molecular biology traits,and drug resistance status are still unknown,though.In this study,the whole genome sequencing(WGS)of 270 Yersinia isolates were performed using the Illumina Nova sequencing platform,and the species genus and evolutionary relationships of the isolates were determined by constructing phylogenetic trees and ANI analysis;the population structure,pan-genome,molecular typing,differentiation time,and virulence factors of Y.enterocolitica in Ningxia region were analysed by bioinformatics methods;the machine learning model was used to predict the risk areas for Y.enterocolitica infection in Ningxia;the broth dilution method was used to analyze the drug resistance of Y.enterocolitica in Ningxia;analysis of the genomic characteristics and associated pathways of the streptomycin-resistant isolate NX 12061 utilizing completion map sequencing analysis and transcriptome sequencing;the deletion strains of the cyd gene were constructed to study the relationship between drug resistance and pathogenicity of Y.enterocolitica.Results:(1)The detection rate of Y.enterocolitica in Ningxia ranged from 0.92%to 7.55%between 2007 and 2019,with statistically significant diferences in positivity rates between samples from different hosts(χ2=22.636,p<0.001).(2)Using WGS and ANI analysis,the results showed that the 270 isolates included Y.enterocolitica(187/270,69.26%),Y.intermedia(30/270,11.11%),Y.massiliensis(30/270,11.11%),Y.mollaretii(7/270,2.59%),Y.pekkanenii(5/270,1.85%),Y.proxima(4/270,1.48%),Y.alsatica(2/270,0.74%),Y.frederiksenii(1/270,0.37%),Y.kristensenii(1/270,0.37%),Y.hibernica(1/270,0.37%),Y.canariae(1/270,0.37%),and Y.rochesterensis(1/270,0.37%).(3)Phylogenetic trees based on single-copy core orthologous genes were directly related to biotypes.Pathogenic isolates from the Ningxia region were genetically more distant from the reference genome and regionally specific compared to pathogenic isolates from other countries.Isolates of biotype 1A had a wider range of host species and serotypes and showed no geographical variation.Biotype 4 isolates were associated with serotype O:3 and biotype 1A isolates with serotypes O:5,O:8 and O:9.187 Y.enterocolitica isolates were identified as 54 STs and 125 CTs.(4)Temperature,precipitation,elevation and NDVI of the isolate collection site were statistically significantly different from pathogenicity.Ecological factors at the collection site and pathogenicity of the isolates were statistically significantly different from the biotype,serotype,and host of the isolates.Precipitation at the collection site was not statistically different among isolates from different hosts.Predictive modeling is a powerful tool in public health and epidemiology for predicting the spread and dissemination of pathogenic bacteria based on external environmental factors and molecular characteristics,allowing for surveillance and early warning of relevant zoonotic diseases.(5)Y.enterocolitica isolates from the Ningxia region had the highest resistance level was 97.88%for cefazolin(CAZ),followed by ampicillin(AMP)(44.97%),ciprofloxacin(CIP)(25.40%),streptomycin(STR)(11.11%),and tetracycline(TET)(10.58%).Isolates emerged as chloramphenicol(CHL)and trimethoprim/sulfamethoxazole(SXT)resistant.The majority of drug-resistant strains in Ningxia were biotype 4/O:3/ST429 and the hosts were pigs and diarrhoea patients.The number of multidrug-resistant isolates reached 27.51%.(6)The transcriptome enrichment analysis of isolate NX 12061 showed that the highest number of genes and the most significant level of variation in cell composition were found in the GO enrichment analysis,with the most significant up-and down-regulation of genes responsible for the biosynthesis of organic nitrogen compounds in biological processes.The highest significance in the KEGG enrichment analysis was found for genes located in the ribosomal pathway,with the most significant up-and down-regulation of genes responsible for the nucleotide metabolism pathway.(7)Compared to the reference strain 52203,isolate NX 12061 had 11 genes deletions:astA,astB,astC,astD,astE,cbl,cydA’,cydB,narX,RS12350,and RS12365.The deletion strains of cydA’,cydB and RS12365 genes were constructed using the reference strain 52203 showing that the deletion of three genes did not contribute to the appearance of a streptomycin resistance phenotype;the biofilm-forming ability of the cydA’,cydB,and RS12365 gene deletion strains did not differ from that of the reference strain,but the adhesion rate and invasion rate to colon cancer cells were significantly lower compared to that of the reference strain.From the above studies,the following conclusions were drawn:(1)Yersinia presented a diversified epidemiological trend in Ningxia over a 12-year period,with a pattern of the diminished predominance of prevalent isolates and the coexistence of multiple subtypes.Pigs were the primary source of Yersinia in Ningxia.Therefore,strengthening health surveillance on pig farms in high-risk areas and focusing on testing food of pig origin are optional strategies to prevent disease outbreaks.(2)The phylogeny of Y.enterocolitica isolates in Ningxia was closely related to biotype.The distribution of STs and CTs correlated with biotype,serotype,host,and region.Bio-serotype 4/O:3 was the dominant Y.enterocolitica type in Ningxia.(3)The distribution of pathogenic isolates was correlated with environmental factors such as temperature and precipitation,and the machine learning model predicted that the risk areas for Y.enterocolitica infection in Ningxia were concentrated in northern,northwestern and southern Ningxia.(4)The level of drug resistance of Y.enterocolitica in different regions varied.Compared with non-pathogenic isolates,pathogenic isolates had more antimicrobial resistance genes,plasmids,insertion sequences,and transposons,were more likely to have an antimicrobial resistance phenotype and easier to develop into multidrug-resistant strains.(5)The absence of cydA’,cydB,and RS12365 did not lead to the appearance of a streptomycin resistance phenotype and the resistance of isolate NX 12061 to streptomycin may be triggered by interactions with other missing genes,or by other enzymes or other resistance mechanisms.Deletion of the cydA’,cydB,and RS12365 genes can affect the pathogenicity of isolates. |
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