Identification and Antimicrobial Susceptibility of <i>Yersinia enterocolitica</i>Found in Chitterlings, Raw Milk and Swine Fecal Samples

Foodborne illness is an escalating concern upon public health. The prevalence of Yersinia enterocolitica was assessed in chitterlings, raw milk and swine fecal from North Carolina. Uncleaned thirty chitterling samples procured from a local grocery store, forty-five swine fecal samples, and forty unpasteurized cow milk samples supplied by the University farm were evaluated for the presence of Y. enterocolitica. Isolates identified as presumptive positive were characterized as colonies with a pink or deep-red center on MacConkey and CIN agar, and verified further through polymerase chain reaction (PCR) for the presence of 16S rRNA gene for the Yersinia genera. Results showed that 4.4% swine fecal samples, 7.5% milk samples and 11.3% chitterling samples were presumptive positive for Y. enterocolitica by the direct plating method on selective agars. Of the thirty-chitterling samples examined by PCR for the 16S rRNA gene, 26% samples contained the identification gene for the bacteria of interest. After conducting virulence tests, the fecal samples were revealed as non-pathogenic. Only one of the milk samples were considered pathogenic and consisted of the following virulent genes: Yersinia heat-stable toxin (yst), invasion (inv), attachment invasion locus (ail), virulence regulon transcriptional activator (virF), Yersinia adehesin A (yadA), and the O:3 antigen gene (rfbC). Seven out of the eight (87.5%) chitterling samples were shown to be pathogenic. Disc diffusion was conducted to determine the antimicrobial susceptibility of the isolates. Over half (55.5%) of the antimicrobial agents were found effective, with isolates being completely susceptible to ciprofloxacin, kanamycin, trimethoprim, cefotaxime, and gentamycin. Ampicillin was determined to be least effective, where 84.6% of the samples presented resistance to the drug. Random amplified polymorphic DNA (RAPD) analysis and ERIC-PCR techniques were used to evaluate genetic similarity among the Yersinia isolates. There was approximately 85% similarity between two chitterlings and a fecal isolate during RAPD testing. With ERIC-PCR the largest similarity among all samples was at 95%, which was found between isolates from a chitterling and milk sample. Chitterling samples showed the highest prevalence of Y. enterocolitica compared to the other samples. Cross contamination at the farm level could be the root cause of this pathogen being prevalent in farm animal and food sources, which does pose a risk to public human health when food is improperly prepared.

Comparison of Lipopolysaccharide and Protein Immunogens from Pathogenic Yersinia enterocolitica Bio-serotype 1B/O:8 and 2/O:9 using SDS-PAGE

Objective Yersinia enterocolitica is an extracellular pathogen and its related antigens interact with the host immune system. We investigated the difference in immunological characteristics between a highly pathogenic and poorly pathogenic strain of Y. enterocolitico. Methods We used SDS-PAGE and western blotting to characterize lipopolysaccharide （LPS）, Yersinio outer membrane proteins （Yops）, membrane proteins, and whole-cell proteins from poorly pathogenic Y. enterocolitico bio-serotype 2/0：9, isolated from China, and highly pathogenic bio-serotype 1B/O：8, isolated from Japan. Results These two strains of Y. enterocolitica had different LPS immune response patterns. Comparison of their Yops also showed differences that could have accounted for their differences in pathogenicity. The membrane and whole-cell proteins of both strains were similar; immunoblottting showed that the 35 kD and perhaps the 10 kD proteins were immunogens in both strains. Conclusion The major antigens of the two strains e and membrane proteins, as shown by comparing preparations. citing the host immune protein samples with response were the LPS reference and purified

Pleiotropic Effect of tatC Mutation on Metabolism of Pathogen Yersinia enterocolitica

Objective To analyze the impact of depletion of the twin arginine translocation （TAT） system on virulence and physiology of Yersinia enterocolitica for a better understanding of its pathogenicity. Methods We constructed a △tatC：：Sp^R mutant of Yersinia enterocolitica by P1 phage mediated transduction using Escherichia coli K-12 △tatC：：Sp^R strain as a donor. Results A Pl-mediated genetic material transfer was found between the two species of enterobacteria, indicating a great potential of acquisition of antibiotic resistance in emergency of a new threatening pathogen by genetic material exchanges. Periplasmic trimethylamine N-oxidase reductase activity was detected in the wild type E enterocolitica strain and translocation of this enzyme was completely abolished by the △tatC：：Sp^R mutation. In addition, the △tatC：：Sp^R mutation showed a pleiotropic effect on the metabolism of E enterocolitica. However, the tat mutation did not seem to affect the mobility and virulence of Y. enterocolitica under the conditions used. Conclusion Unlike other pathogenic bacteria studied, the TAT system of E enterocolitica might play an important role in the pathogenic process, which is distinct from other pathogens, such as Pseudomonas aeruginosa and enterohemorrhagic E. coli O 157：H7.

Isolation and Pathogenicity Analyses on Yersinia enterocolitica from Pelteobagrus vachelli

Yersinia enterocolitica is an important zoonotic pathogen that can induce disease outbreaks in a wide host range. Strain YER6022 was isolated from Pelteobagrus vachelli and identified using bacterial morphology and 16S rDNA sequence analysis. Five virulence factors were detected, then artificial infection experiment and histopathological method were carried out. These results showed that strain YER6022 was one of Y. enterocolitica family members. In addition, ail, ystb, virF, yadA and HPIint were dectected. In artificial infection experiment, with 80% mortality and 100% morbidity, injected Pelteobagrus vachellis showed red swollen of the anus, abdomen swelling and fim bleeding. There existed serious hyperaemia and edema in kidney, spleen, intestine and liver at the light microscope. Ultrastructural observation indicated that mitochondria of the liver, kidney, spleen and intestine swelled and mitochondrial cristae broke. The data had further shed light on its pathogenicity in Pelteobagrus vachelli. It would benefit for further studies on pathogenesis ofPelteobagrus vachelli infected with Y. enterocolitica.

Behavioral Pattern of Native Food Isolates of <i>Yersinia</i><i>enterocolitica</i>and <i>Yersinia</i><i>intermedia</i>under Simulated Time-Temperature Combinations of the Food Chain

The public health significance of Yersinia spp. gives a new dimension to the prevailing food chain, wherein the foods do get exposed to heat and cold treatments. In this study, the effect of heat treatment on the native isolates of Yersinia enterocolitica CFR 2301 and Y. intermedia CFR 2303 revealed the D-values ranging from the lowest of 0.08 min at 65℃ in skim milk/beef gravy to the highest of 18.52 min at 50℃ in beef gravy. The heat sensitivity of both these cultures was in the order of Milli-Q water > 0.85% saline > skim milk > beef gravy. The z-values of the test cultures ranged from 7.55℃ for Y. intermedia to 12.08℃ for Y. enterocolitica. The heat sensitivity in Y. enterocolitica appeared to be related with growth incubation temperatures and also fatty acid profile of cell membrane. The effect of low temperature treatments (–20 ℃, 0℃ and 4℃ for 20 d) in water, saline and skim milk revealed the ability of Y. enterocolitica to survive more efficiently at –20℃, while Y. intermedia was more tolerant at 0℃. In packaged drinking water, Y. enterocolitica could survive and grow at 4℃ and 16℃, while at 30℃, inactivation was rapid. The findings did indicate that heat and cold treatments would not always ensure safety from Y. enterocolitica and Y. intermedia in the food chain.

Investigation of <i>Yersinia enterocolitica</i>Bioserotype 4/O:3 Clusters in Finland, 2017-2018

In December 2017, two suspected outbreaks of Yersinia enterocolitica 4/O:3 were notified in Finland. We analyzed the surveillance and outbreak investigation data and genotyped Y. enterocolitica strains in order to understand Y. enterocolitica epidemiology in Finland and to find out whether the notified outbreaks were related. A total of 13,344 Y. enterocolitica cases were reported in 1995-2018 to the National Infectious Diseases Register. The mean annual incidence ranged from 7.9 to 15.9/100,000 inhabitants. The highest incidence was observed in young adults (14/100,000). Incidence varied geographically. The incidence was higher in spring compared to other seasons. The most common pathogenic bioserotype was 4/O:3 but the information on bio/serotype was missing in 64% of the surveillance cases. For most of the cases, 87%, no travel history was reported. In the YE 4/O:3 outbreak investigation, whole genome sequencing was performed for isolates from 29/69 (42%) of the outbreak cases. The sequencing results showed that cases consisted of six independent clusters with 2 - 6 isolates in each cluster, and 5 sporadic isolates. No food item was found to be common to all of the clusters. In order to improve monitoring and facilitate the detection and investigation of Y. enterocolitica outbreaks, more comprehensive information on bio/serotype and on travel history is required in the surveillance.

Yersinia enterocolitica Infection of a Prosthetic Knee Joint.Case Report and Review of the Literature on Deep Sited Infections Caused by Y.enterocolitica

Prosthetic joint infection is a rare manifestation of Yersinia enterocolitica. We report a case of a patient presenting with fever and a purulent infection in his prosthetic knee joint caused by Y. enterocolitica. He had been operated in 1990 for arthrosis of the right knee. Re-operation was performed in 2007 for loosening of the prosthesis. Seven months later, following progressively increasing knee pain, he became acutely febrile and a purulent knee joint infection was diagnosed. Y. enterocolitica was isolated from the joint fluid. Serum antibodies against Y. enterocolitica were also positive. He was treated with debridement, replacement of the liner component of the prosthesis and a long course of intravenous antimicrobial therapy. The infection was thought to be in a chronic suppressive state. The final outcome after all therapy was good.

Public health implications of Yersinia enterocolitica investigation:an ecological modeling and molecular epidemiology study

Background Yersinia enterocolitica has been sporadically recovered from animals,foods,and human clinical samples in various regions of Ningxia,China.However,the ecological and molecular characteristics of Y.enterocolitica,as well as public health concerns about infection in the Ningxia Hui Autonomous Region,remain unclear.This study aims to analyze the ecological and molecular epidemiological characteristics of Y.enterocolitis in order to inform the public health intervention strategies for the contains of related diseases.Methods A total of 270 samples were collected for isolation[animals(n=208),food(n=49),and patients(n=13)],then suspect colonies were isolated and identified by the API20E biochemical identification system,serological tests,biotyping tests,and 16S rRNA-PCR.Then,we used an ecological epidemiological approach combined with machine learning algorithms(general linear model,random forest model,and eXtreme Gradient Boosting)to explore the associations between ecological factors and the pathogenicity of Y.enterocolitis.Furthermore,average nucleotide identity(ANI)estimation,single nucleotide polymorphism(SNP),and core gene multilocus sequence typing(cgMLST)were applied to characterize the molecular profile of isolates based on whole genome sequencing.The statistical test used single-factor analysis,Chi-square tests,t-tests/ANOVA-tests,Wilcoxon rank-sum tests,and Kruskal–Wallis tests.Results A total of 270 isolates of Yersinia were identified from poultry and livestock(n=191),food(n=49),diarrhoea patients(n=13),rats(n=15),and hamsters(n=2).The detection rates of samples from different hosts were statistically different(χ^(2)=22.636,P<0.001).According to the relatedness clustering results,270 isolates were divided into 12 species,and Y.enterocolitica(n=187)is a predominated species.Pathogenic isolates made up 52.4%(98/187),while non-pathogenic isolates made up 47.6%(89/187).Temperature and precipitation were strongly associated with the pathogenicity of the isolates(P<0.001).The random forest(RF)prediction model showed the best performance.The prediction result shows a high risk of pathogenicity Y.enterocolitica was located in the northern,northwestern,and southern of the Ningxia Hui Autonomous Region.The Y.enterocolitica isolates were classified into 54 sequence types(STs)and 125 cgMLST types(CTs),with 4/O:3 being the dominant bioserotype in Ningxia.The dominant STs and dominant CTs of pathogenic isolates in Ningxia were ST429 and HC100_2571,respectively.Conclusions The data indicated geographical variations in the distribution of STs and CTs of Y.enterocolitica isolates in Ningxia.Our work offered the first evidence that the pathogenicity of isolates was directly related to fluctuations in temperature and precipitation of the environment.CgMLST typing strategies showed that the isolates were transmitted to the population via pigs and food.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.

2020-2023年辽宁省小肠结肠炎耶尔森菌表型分布和分子特征研究

目的通过研究表型分布和分子特征的关联性,了解辽宁省小肠结肠炎耶尔森菌(Yersinia enterocolitica,YE)的流行趋势,为辽宁省YE病的监测和防控提供数据支撑。方法对2020-2023年辽宁省60株YE分离株进行血清分型、生物分型、毒力基因检测和PFGE、MLST分子分型研究。结果60株YE分离株可分为4个血清型、2个生物型、8个毒力基因型和29种ST型。2023年40株YE分离株分为8组PFGE优势带型A-G,A组为2023年沈阳市地区流行分子型。PFGE的分辨力(DI)为0.954,MLST分辨力(DI)为0.948。ST3、ST252位于树状图核心位置向四周发散。PFGE较MLST方法分辨力稍高。结论2020-2023年辽宁省YE分离株流行致病血清型是O∶3,毒力基因型和ST型均呈现多样化状态。沈阳市YE污染较严重。应重点加强对于生物1A型菌株的研究工作。猪是辽宁省地区重要的宿主和传染源。PFGE和MLST对YE分离株分型均是可行的,ST分型与PFGE分型具有一致性。

温州市食源性小肠结肠炎耶尔森氏菌毒力基因分布及遗传多样性研究

目的了解温州市食源性小肠结肠炎耶尔森氏菌的毒力基因分布及遗传多样性特征。方法对温州市食品及食源性疾病腹泻病人中分离得到的小肠结肠炎耶尔森氏菌进行生物型、血清型和耐药性分析,并基于全基因组测序进行菌株的毒力基因、多位点序列分型(MLST)、核心基因组多位点序列分型(cgMLST)分析。结果71株食源性小肠结肠炎耶尔森氏菌,94.4%(67/71)的分离株为生物1A型,生物血清型以1A/O∶5占比最高(29.6%,21/71)。分离株对14种抗菌药物的敏感率达到95.8%以上。共获得了16种126个毒力基因,其中2株菌株携带pYV质粒和染色体相关毒力基因。分离株以ST3型最常见(31.6%,12/38),cgMLST分析显示除具有相同ST型的菌株外,菌株无明显基因型簇形成。结论温州市食源性小肠结肠炎耶尔森氏菌存在致病性菌株,菌株呈高度遗传多态性,需加强此类菌株的监测,以预防其引起食源性疾病的暴发。

麝香草酚破坏小肠结肠炎耶尔森氏菌细胞膜并抑制其生物被膜的形成研究

小肠结肠炎耶尔森氏菌(Yersinia enterocolitica)是食品工业中常见的致病菌,其生物被膜是主要的污染源.为了阐明麝香草酚对Y.enterocolitica的抑制机制,本研究首先测定了最小抑制浓度(MIC)、最小抑制生物被膜浓度(MBIC)和最小清除生物被膜浓度(MBEC),随后检测了细胞膜电位以及细胞内ATP和pH值的变化,最后在激光共聚焦显微镜(CLSM)、场发射扫描电子显微镜(FEGSEM)和光学显微镜下,直接观察了麝香草酚引起的细胞膜损伤及其对生物被膜的抑制作用.结果显示,麝香草酚对Y.enterocolitica的MIC为0.156 25 mg/mL,MBIC仅为1/16 MIC,改变了细胞形态并导致胞内ATP降低、膜去极化和pH下降.旨在探索麝香草酚抑制Y.enterocolitica及其生物被膜的机理,从而为麝香草酚能否作为植物源天然防腐剂提供更多参考.

CpxRA双组分系统对小肠结肠炎耶尔森菌环境耐受性和抗生素耐药性的影响

目的探究CpxRA双组分系统对小肠结肠炎耶尔森菌环境耐受性和抗生素耐药性的影响。方法本研究选用Cat-SacB无标记基因敲除系统,利用同源重组双交换原理,构建了基因敲除菌株ΔcpxA和ΔcpxR,以及相应的回补菌株ΔcpxA-cpxA和ΔcpxR-cpxR,然后分别测定了野生型、突变型和回补型在酸性、碱性、高氧和高渗环境中的存活率,以及红霉素、罗红霉素、卡那霉素、链霉素的最小抑菌浓度值。结果cpxA和cpxR基因的缺失使小肠结肠炎耶尔森菌的在酸性环境下的存活率由55.1%分别降为45.9%和42.8%;使该菌在碱性环境下的存活率由43.4%分别降为38.9%和37.2%;在高渗环境下的存活率由44.1%分别降为36.3%和33.7%;在高氧环境下的存活率由20.8%分别降为15.1%和15.8%。cpxA基因的缺失增强了该菌对于氨基糖苷类抗生素:卡那霉素、链霉素的耐药性,而cpxR基因的缺失减弱了该菌对于大环内酯类抗生素:红霉素、罗红霉素的耐药性。结论CpxRA双组分系统参与调控小肠结肠炎耶尔森菌在不良环境中的存活能力以及抗生素耐药性。

小肠结肠炎耶尔森氏菌分子生物学检测技术研究进展

小肠结肠炎耶尔森氏菌(Yersinia enterocolitica,Y.enterocolitica)是一种重要的食源性致病菌,可以引起人类的耶尔森氏菌病,该病的典型症状为腹泻、回肠炎和肠系膜淋巴结炎等。由于该菌在特定食品(如肉与肉制品)中的检出率较高,因此对食品安全构成一定的威胁。就现有的检测方法而言,分离纯化和生理生化鉴定方法耗时且难以检测出复杂食品基质中低浓度的小肠结肠炎耶尔森氏菌。所以,研发能够快速、准确地检测食品中小肠结肠炎耶尔森氏菌的鉴别方法迫在眉睫。该研究将小肠结肠炎耶尔森氏菌现有的分子生物学检测方法分为两大类:变温扩增技术和等温扩增技术进行相关研究最新进展的总结,并对其优缺点和发展方向进行讨论,以期为小肠结肠炎耶尔森氏菌快速检测方法研究提供参考。

O∶3和O∶9小肠结肠炎耶尔森菌主要毒力基因分布调查

目的 了解我国常见致病性血清型毒力基因的分布情况。方法 参照国外发展成熟的聚合酶链反应(PCR)技术进行毒力基因检测。结果 实验证实在我国分离到的主要流行血清型O∶3和O∶9小肠结肠炎耶尔森菌毒力基因分布特征为 :ail+ 、ystA+ 、ystB-、yadA+ 、virF+ 占 5 6.2 % ;ail+ 、ystA+ 、ystB-、yadA-、virF-占 2 5 .6% ;其他型占18.2 %。结论 通过实验证实在我国引起感染暴发和流行的血清型O∶3和O∶9小肠结肠炎耶尔森菌毒力基因的分布主要为 :ail +、ystA+ 、yadA+ 、ystB-、virF+ 型 ,很少部分的ail+ 、ystA+ 、ystB-、yadA-、virF-根据以前的资料被认为是毒力质粒丢失的结果 ,其他型很有可能是非致病的菌株。

正交实验法在PCR反应条件优化中的应用

本研究以小肠结肠炎耶尔森氏菌为研究材料,将纯理论数学方法-正交设计实验法,应用于分子生物学技术PCR体系的优化之中.首先采用搜索性正交实验,在此实验结果基础上,进一步设计细调性正交实验来寻求PCR反应的最适条件.所得优化扩增体系稳定,重复性好,表明正交实验设计方法高效快速,科学性强,是PCR反应条件优化中值得推广使用的有效方法.

不同动物宿主小肠结肠炎耶尔森氏菌分离及结果分析

目的了解中国不同地区小肠结肠炎耶尔森氏菌在不同动物宿主中分布状况。方法采集我国不同地区的家畜家禽以及啮齿动物等标本进行小肠结肠炎耶尔森氏菌分离,并对分离到的菌株进行血清分型、生物分型及毒力基因检测。结果从3 386份标本中分离出134株小肠结炎耶尔森氏菌,分离率为3.96%;其中致病性菌株(ystA+ail+virF+yadA+ystB-)30株(22.39%),均为O∶3/3型。非致病性菌104株(ystA-ail-virF-yadA-ystB+或者ystB-)(77.61%)。结论结果表明,我国致病性小肠结肠炎耶尔森菌以O:3/3型为主,猪的分离率最高为6.28%;依次为旱獭、鼠,分别为4.05%、3.41%。

河南省2011-2013年小肠结肠炎耶尔森菌监测结果分析

目的了解河南省小肠结肠炎耶尔森菌的宿主分布、血清学及毒力基因情况。方法于2011—2013年采集腹泻病人粪便、各类家禽家畜粪便、苍蝇以及生熟肉制品涂抹标本,共6 057份,采用冷增菌方法进行目的菌分离。对不同宿主样本中分离到的小肠结肠炎耶尔森菌进行系统生化鉴定、血清学分型和毒力基因PCR检测。结果 6 057份样本中共分离出288株小肠结肠炎耶尔森菌,检出率为4.75%。所分离的小肠结肠炎耶尔森菌主要血清型是O∶8和O∶5,生物分型以1A为主。O∶3血清型多具有致病性,其中以猪分离株最多。结论河南省小肠结肠炎耶尔森菌在人群和各类动物中均有存在,猪是小肠结肠炎耶尔森菌致病性菌株的主要宿主。

2004-2005年我国部分地区小肠结肠炎耶尔森菌宿主分布调查

目的了解2004-2005年小肠结肠炎耶尔森菌宿主分布情况。方法对从不同宿主样品中分离到的小肠结肠炎耶尔森菌进行生物化学鉴定、血清学分型、毒力基因PCR检测。结果2004-2005年从8种常见动物宿主及食品中共分离到230株小肠结肠炎耶尔森菌,35.65%分离自猪,致病性菌株中51.52%来自猪,所占比例最高,而分离自腹泻病人、兔的菌株均为致病性菌株。结论我国小肠结肠炎耶尔森菌宿主分布广泛,猪是最主要的宿主动物。

河南省2007-2010年小肠结肠炎耶尔森菌监测分析

目的监测河南省小肠结肠炎耶尔森菌在腹泻病人和宿主动物中带菌情况,分析其血清学分布及毒力基因情况,探索疾病发生的原因。方法对2007-2010年河南省监测点的标本进行初步鉴定,鉴定后菌株送中国疾病预防控制中心传染病所进行最后鉴定,对部分菌株进行血清分型,并进行ail、ystA、ystB、yadA、verF、rfbC毒力基因检测。结果河南省2007-2010年共监测各类标本8 561份,检出小肠结肠炎耶尔森菌株273株,检出率为3.19%;各年份菌株检出率之间有统计学差异。血清分型中O∶5血清型占比最高(31.63%),其次为O∶8(17.35%),O∶3型占比也较高(11.22%),O∶9则未发现。猪的血清型分布最广,共涉及9个血清型,人的血清型主要为O∶3,O∶8,O∶10。毒力基因测定在病人、猪、牛、犬中检出致病菌株。结论河南省小肠结肠炎耶尔森菌在人群和各类动物中均有存在。人群中流行的血清型与全国流行情况一致。河南省最重要的动物宿主是猪、牛、犬,其在人类感染该病中可能会起到比较重要的作用。