辽宁省婴幼儿食品中肠集聚性大肠埃希菌的病原学特征及耐药谱研究

目的 对2018—2020年辽宁省婴幼儿食品中致泻大肠埃希菌开展持续监测,了解和掌握本省内致泻大肠埃希菌的病原学特征、药物敏感性特征,为本省致泻大肠埃希菌流行病学研究奠定坚实基础,为可能导致的食源性疾病合理用药提供依据。方法 采集辽宁省共208份婴幼儿食品,分离肠杆菌科细菌,并进行16S rRNA基因测序分析与生化鉴定。对鉴定得到的大肠埃希菌进行毒力基因检测,收集分离的肠集聚性大肠埃希菌（EAEC）进一步进行血清学分型和耐药谱研究。结果 16S rRNA基因测序通过对肠杆菌科进行种鉴定,发现与生化结果一致。其中EAEC的检出率较高,且EAEC的毒力基因pic携带率高。毒力基因分型和血清分型具有一致性。共检测出25株EAEC,其中40%菌株（血清型O134:H9）携带毒力基因pic,28%菌株（血清型O3:H2）同时携带毒力基因aggR和astA,16%菌株（血清型O9:H6）携带毒力基因aggR,16%菌株（血清型O62:H7）携带毒力基因astA。78株大肠埃希菌中EAEC毒力基因的携带率为32.1%。25株EAEC为多重耐药株,主要对β内酰胺类、大环内酯类、喹诺酮类、四环素类抗菌药...     

产CTX-M-55型超广谱β-内酰胺酶大肠埃希菌全基因组序列耐药和毒力特征研究

目的基于全基因组序列,对2株食源性产CTX-M-55型超广谱β-内酰胺酶(ESBLs)大肠埃希菌的耐药和毒力分子机制进行研究。方法采用微量肉汤稀释法对大肠埃希菌食品分离株进行药敏试验,设计产ESBLs基因引物并通过聚合酶链式反应(PCR)扩增筛选出2株产CTX-M-55型ESBLs大肠埃希菌(编号为EC001和EC002)进行全基因组测序,并进行序列型(ST)、质粒复制子类型、血清型、耐药基因和毒力因子识别。结果 2株大肠埃希菌均为头孢类和喹诺酮类抗生素双重耐药ESBLs菌株;全基因组序列分析结果显示,2株菌的血清型均为肠道致病性大肠埃希菌(EPEC)O119∶H8,ST型分别为ST21(EC001)和ST342(EC002),EC001菌株含有Inc FII、IncX1、IncY、Col156和IncI2 5个不相容群质粒,EC002菌株含有IncFII和IncX1 2个不相容群质粒,2株菌株染色体中均携带bla_(CTX-M-55)和bla_(TEM-141)2种ESBLs基因。此外,EC001菌株还携带sul2/3、tet(A)/(B)、dfrA12、strA/B、aph(3')-IIa、cml/cmlA1、floR和oqxA/B耐药相关基因,其染色体喹诺酮耐药决定区gyr A基因存在2个突变位点(S83L,D87H)、parC基因存在1个突变位点(S80I),多粘菌素耐药相关基因pmr E基因存在3个突变位点(D73Y,M185T,S225T);而EC002菌株携带fosA和qnrS1耐药相关基因。2株菌均携带致黏附与脱落(A/E)损伤的肠细胞脱落位点(LEE)毒力岛基因esc、esp、eae A和tir,且EC001菌株还携带增强菌株在血清中存活能力的iss基因。结论对2株食源性产CTX-M-55型ESBLs大肠埃希菌进行全基因组测序以及耐药和毒力的分子机制的研究,其结果可为后续开展大肠埃希菌耐药和毒力表型预测、指导食用畜禽养殖过程中抗生素合理使用及耐药菌株防控提供依据。     

2018—2020年湖州市腹泻人群致泻大肠埃希菌流行特征及病原分析

目的 了解湖州市腹泻人群中致泻大肠埃希菌(DEC)的感染状况、主要毒力基因以及耐药情况。方法 对2018—2020年各哨点医院腹泻患者粪便标本进行分离,采用多重荧光定量聚合酶链式反应(PCR)法对DEC分离株进行毒力基因检测,用微量肉汤稀释法进行药敏试验。结果 共检出DEC阳性病例293例,分离菌株294株。肠集聚性大肠埃希菌(EAEC)占55.4%(163/294),肠产毒性大肠埃希氏菌(ETEC)占29.6%(87/294),肠致病性大肠埃希氏菌(EPEC)占11.2%(33/294),肠侵袭性大肠埃希氏菌(EIEC)占3.4%(10/294),肠出血性大肠埃希氏菌(EHEC)占0.3%(1/294)。随机选取117株DEC对氨苄西林、萘啶酸、氨苄西林-舒巴坦、四环素进行耐药性检测,耐药率分别为70.9%、60.7%、53.8%、51.3%。多重耐药率达到55.6%。对亚胺培南、头孢他啶、阿莫西林-克拉维酸、头孢西丁的敏感率为99.1%、92.3%、92.3%和91.5%。DEC引起的腹泻发病高峰在7—8月,20～39岁为主要感染人群。结论 湖州市腹泻人群感染DEC基因型别以EAEC和ETEC为主,存在多重抗生素耐药,病例分布呈现明显的季节性,感染人群以青壮年为主。     

荧光PCR法检测原料乳中大肠埃希氏菌喹诺酮类的耐药基因

目的 建立喹诺酮类抗生素主要耐药基因的TaqMan荧光PCR检测方法,为原料乳中耐药大肠埃希氏菌的监测提供技术方案。方法 利用Chelex 100法快速获取细菌基因组DNA,通过对aac(6’)-Ib-cr和qepA基因的序列分析,利用Primer Express 3.0软件设计特异性引物和探针,建立单重和双重TaqMan荧光PCR方法。通过耐药基因阳性菌株和原料乳中分离大肠埃希氏菌菌株,对方法进行评估。结果 以Chelex 100法快速提取耐药基因阳性菌株细菌基因组DNA为模板,建立的aac(6’)-Ib-cr和qepA基因的单重和双重荧光PCR反应均有特异性曲线扩增,方法灵敏度为菌液浓度10_^-4 CFU/mL。北京地区52份原料乳中共分离出32株大肠埃希氏菌,利用建立的双重荧光PCR方法检测aac(6’)-Ib-cr和qepA基因结果为阴性,和环丙沙星肉汤稀释法药敏结果一致。结论 本方法的建立为原料乳中大肠埃希氏菌常见喹诺酮类耐药基因的快速检测提供了新的技术手段。     

食品和腹泻患者分离耐环丙沙星大肠埃希菌耐药特征及耐药机制的比较研究

目的从食品和腹泻患者中分离耐环丙沙星大肠埃希菌进行耐药性及相关分子特征的研究。方法从645株食品和腹泻患者来源的大肠埃希菌中确定21株(3.3%)环丙沙星耐药菌株,并对分离株进行药敏试验,采用聚合酶链式反应和核苷酸序列分析技术对环丙沙星耐药菌株进行喹诺酮类染色体、质粒编码耐药机制、超广谱β-内酰胺酶耐药机制及系统发育分型研究。结果 21株环丙沙星耐药菌株均为多重耐药菌株,所有菌株分别在gyrA、parC和parE发生1~4个点突变,其中16株菌携带了质粒介导的喹诺酮耐药基因,包括oqx A、oqxB、qnrS、aac(6')-Ib-cr和qepA,同时所有分离株均携带bla基因。结论本研究提示食品和腹泻患者来源的耐环丙沙星大肠埃希菌耐药机制具有多样性的特点,编码耐药基因质粒存在潜在的传播可能,对公众健康产生巨大威胁。需进一步开展此类耐药菌株的监测,为研究此类菌株在食品和人群中的可能传播和评估其对人群的健康风险提供基础数据。     

海产源多重耐药共生菌的耐药表型及耐药基因分析

目的 对市售牡蛎和大黄鱼中分离的多重耐药共生菌进行耐药性分析、全基因组测序及耐药基因分析。方法 采用药敏纸片法进行耐药性分析, 采用二代测序技术进行基因组框架图测序, 通过与CARD数据库比对进行耐药基因分析。结果 菌株呈现高度的多重耐药性, 对10种或10种以上药物耐药, 多重耐药系数大于或等于0.48, 所有菌株对头孢唑啉、萘啶酸、四环素、复方新诺明及氯霉素耐药。分离菌株为水产品中常见共生菌, 基因组差异显著。菌株含有β-内酰胺类、氨基糖苷类、喹诺酮类、四环素类、磺胺类、氯霉素类、消毒剂类等药物的耐药基因, 与耐药表型基本一致, 部分菌含有对同一类药物具有不同耐药机制的多个基因。结论 牡蛎和大黄鱼中存在多重耐药共生菌, 耐药基因丰富, 如果耐药基因传播到致病菌会造成临床治疗失效, 具有潜在的危害。     

沙门氏菌耐药谱及质粒耐药基因的筛查

目的:分析不同来源沙门氏菌耐药性及其质粒耐药基因携带情况,从而揭示质粒耐药基因与菌株耐药性的对应关系。方法:对226株食源性及医源性的沙门氏菌分离株用试卡法测试其耐药性,按2012年美国实验室标准委员会(CLSI)指导原则的标准计算细菌对抗菌药物的耐药率(R),中介率(I),和敏感率(S),并对具有耐药表型和中介表型的非敏感菌株进行质粒耐药基因的筛查。结果:针对青霉素类抗生素进行筛查,耐氨苄青霉素的菌株达到15.9%(36/226),耐舒巴坦/氨苄青霉素达到12.8%(29/226);针对氨基糖苷类抗生素,耐妥布霉素的菌株达到6.2%(14/226),耐庆大霉素5.8%(13/226);针对喹诺酮类抗生素,耐环丙沙星的菌株达到4.0%(9/226),耐左氧氟沙星1.8%(4/226);针对头孢菌素类抗生素,耐头孢唑啉4.4%(10/226),耐头孢他啶和头孢曲松分别为2.2%(5/226),耐头孢替坦0.9%(2/226)。针对碳青霉烯类和除青霉素类、头孢菌素类以外的β-内酰胺类的筛查结果均为敏感。β-内酰胺类抗生素非敏感菌株中,质粒上β-内酰胺类耐药基因携带率为59.5%(50/84),其中CMY基因携带率为10.7%(9/84),OXA基因携带率为27.3%(23/84),TEM基因携带率为39.2%(33/84),PSE基因携带率为1.2%(1/84)。喹诺酮类抗生素非敏感菌株中,质粒上喹诺酮类耐药基因携带率为14.7%(5/34),其中qnrA基因携带率为11.8%(4/34);qnrS基因携带率为2.9%(1/34)。对喹诺酮类和氨基糖苷类抗生素非敏感菌株中,质粒上acc(6’)-Ib-cr基因的携带率为17.1%(7/41)。仅CMY基因在食源性和医源性分离株中的分布具有统计学差异(χ2=5.480,P0.05)。结论:本研究涉及的沙门氏菌分离株中,对青霉素类抗生素耐药性较强,其次是氨基糖苷类、头孢菌素类和喹诺酮类。大多数耐β-内酰胺类抗生素的沙门氏菌菌株都携带相对应的质粒耐药基因,而耐喹诺酮类以及氨基糖苷类抗生素的沙门氏菌携带相对应的质粒耐药基因较少。     

密云区致泻大肠埃希菌耐药性及耐药基因研究

目的建立密云地区腹泻患者致泻大肠埃希氏菌的毒力基因及耐药基因数据库。方法采集腹泻患者粪便样本,通过实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)方法鉴定致泻大肠埃希氏菌并确定毒力基因,采用微量肉汤稀释法确定耐药性,采用普通PCR方法对耐药基因进行检测。结果 2112份粪便样本中检出致泻大肠埃希氏菌114株,肠聚集性大肠埃希氏菌(enteroaggregative Escherichia coli,EAEC)占57.9%,毒力基因以astA+pic、eae为主,萘啶酸(nalidixic acid, NAL)、氨苄西林(ampicillin, AMP)、四环素(tetracycline, TET)、磺胺异恶唑(sulfamethoxazole, SUL)耐药率均超过30%, 41株菌株多重耐药,主要流行的耐药因子为GyrA、GyrB、blaTEM、ParC、aadA1、tetA、tetB、SUL2、floR、aac(3′)-IIa。结论密云地区腹泻患者致泻大肠埃希氏菌多重耐药性及耐药基因的存在情况较为普遍,应开展长期的主动监测。     

2019年福建省哨点医院腹泻患者中致泻大肠埃希菌感染状况及病原学特征分析

目的 了解2019年福建省哨点医院腹泻患者中致泻大肠埃希菌(DEC)感染状况、毒力基因携带、分子分型及耐药情况。方法 采集腹泻患者210份粪便标本按照GB 4789.6—2016《食品安全国家标准 食品微生物学检验 致泻大肠埃希氏菌检验》方法分离大肠埃希菌后进行荧光聚合酶链式反应(PCR)确认,并对分离出的DEC进行脉冲场凝胶电泳(PFGE)分子分型及药敏试验。结果 共检出阳性菌株32株,检出率为15.2%(32/210),其中肠道致病性大肠埃希菌(EPEC)占37.5%(12/32),肠道集聚性大肠埃希菌(EAEC)占37.5%(12/32),产肠毒素大肠埃希菌(ETEC)占25.0%(8/32)。药敏试验结果表明,32株菌对氨苄西林耐药程度最高,耐药率为78.1%(25/32),其次是四环素和甲氧苄啶/磺胺甲噁唑,耐药率分别为62.5%(20/32)和59.4%(19/32)。多重耐药(MDR)率为50.0%(16/32)。PFGE结果表明,32株菌共分为28种PFGE带型,其中12株EPEC共分为10种带型,12株EAEC共分为10种带型,8株ETEC共分为8种带型,其中EPEC聚类分析结果有两组菌株具有100.0%相似带型,EAEC有一组菌株具有100.0%相似带型,ETEC聚类分析没有完全一致的带型。结论 2019年福建省哨点医院腹泻患者DEC感染主要以EPEC、EAEC为主,菌株分型带型存在多样性,菌株耐药状况严重,且存在较高的多重耐药率。     

一株黄瓜源肠集聚性大肠埃希氏菌耐药及生长预测模型分析

目的 研究源自常见生食蔬菜黄瓜中肠集聚性大肠埃希氏菌(enteroaggregative Escherichia coli, EAEC)的分布和耐药情况, 并建立其生长预测模型。方法 将采集的黄瓜样品进行大肠埃希氏菌的分离和鉴定, 通过PCR对EAEC特征性毒力基因进行确认, 并通过VITEK 2 Compact全自动微生物药敏系统进行18种抗生素的耐药性分析; 以分离鉴定的EAEC为研究对象, 监测其在4、15、25、35℃贮藏条件下在黄瓜上的生长数据, 绘制生长曲线, 采用Slogistic模型进行拟合, 建立黄瓜中EAEC生长预测模型。结果 自黄瓜中分离到一株EAEC, 携带aggR和astA毒力基因, 对磺胺类药物复方新诺明高度耐药。4个不同温度条件下EAEC在黄瓜上的生长用Slogistic模型拟合的相关系数都大于0.95,该模型可以有效地反应EAEC在黄瓜上的生长情况 。结论 黄瓜上存在EAEC污染的风险, EAEC的耐药性进一步加剧生食黄瓜的安全风险。通过Slogistic方程可以有效的建立不同温度条件下EAEC在黄瓜上的生长预测模型, 对生食黄瓜的加工、供餐等过程中食源性致病菌风险控制提供参考依据。     

Occurrence of foodborne pathogens in Irish farmhouse cheese

Food safety is a critical factor in the production of farmhouse cheese. In Ireland the varieties of farmhouse cheese produced reflect a much broader range than those produced commercially and some of these cheese varieties are associated with greater microbiological risk. These include cheese produced from unpasteurised milk and soft ripened cheese such as mould or smear-ripened cheeses which have high pH and relatively short ripening times. The aim of this study was to determine the microbiological quality of farmhouse cheeses in Ireland. Three hundred and fifty one cheese samples, from 15 cheese producers, were analysed for microbiological quality on a monthly basis throughout the year. The analyses included enumeration of Escherichia coli, Staphylococcus aureus and Listeria monocytogenes (using the relevant agars) and enrichment for L. monocytogenes. The cheeses selected were produced from ovine, caprine and bovine milk. Both unpasteurised and pasteurised milk cheeses were sampled and these included hard, semi-hard and soft cheeses, internal/external mould-ripened and smear-ripened cheeses and the cheeses represented different geographic regions. Of the cheeses tested, 94% were free of L. monocytogenes, all were within the EU limits for E. coli and only one cheese variety had S. aureus levels above the recommended numbers for the first 6 months of the year. Due to a modified production process the numbers were within the guidelines for the second six months. The results indicate that Irish farmhouse cheeses are of a high microbiological quality.     

The effects of acid adaptation on Escherichia coli inactivation using power ultrasound

Inactivation of Escherichia coli in liquids was carried out using power ultrasound. Parameters examined included amplitude levels (0.4 µm, 7.5 µm, 37.5 µm), treatment time, cell condition (non-adapted cells, acid adapted cells), liquid media (TSB, model orange juice and model apple juice) and E. coli strain (ATCC 25922, NCTC 12900). The efficacy of ultrasound treatment was found to be a function of amplitude level, treatment time and media (p < 0.05). The kinetics of inactivation followed zero order kinetics (R > 0.95), with the highest inactivation achieved using an amplitude of 37.5 µm. The D-values of E. coli 25922 at all amplitudes in model orange juice were not significantly different than in TSB media. However, at 0.4 µm and 37.5 µm amplitude D-values of E. coli 12900 were significantly different in model orange juice compared to TSB media. When efficacy of ultrasound was assessed in model apple juice and phosphate buffered saline treatment times were significantly reduced by comparison with TSB. Inactivation of E. coli was found to be influenced by strain, prior acid adaptation and suspension liquid, but the effect was negated at the higher amplitude levels.

Industrial relevance

To facilitate the preservation of unstable nutrients many juice processors have investigated alternatives to thermal pasteurisation, including un-pasteurised short shelf life juices with high retail value. This trend has continued within the European Union. However within the US recent regulations by the FDA have required processors to achieve a 5-log reduction in the numbers of the most resistant pathogens in their finished products. This rule comes after a rise in the number of food borne illness outbreaks and consumer illnesses associated with consumption of untreated juice products. Pathogenic E. coli may survive in acid environments such as fruit juices for long periods. Ultrasound has been identified as one possible non-thermal technology to meet the required microbial log reduction. However it is important to determine if conditions such as acid adaptation and pathogen strain influence ultrasound efficacy, if the technology is to be adopted by industry.     

Comparison of a real-time PCR and an IMS/culture method to detect Escherichia coli O26 and O111 in minced beef in the Republic of Ireland

This study compared an immuno-magnetic separation (IMS)/culture method and a real-time PCR method to detect Verocytotoxigenic Escherichia coli (VTEC) serovar O26 and/or O111 in minced beef. A total of 65 samples were examined, 40 of which were frozen beef samples previously established as containing E. coli O157, and 25 were samples of fresh minced beef, purchased from butcher shops in the Dublin area. After selective enrichment, all samples were (a) subjected to IMS, plated on differential media and identified as E. coli O26 or O111 using biochemical and immuno-logical methods; and (b) subjected to DNA extraction and real-time PCR analysis using primers and probes against E. coli O111 and O26 serovar specific genes, and verotoxin genes. Overall, from the 65 minced beef samples collected, three were positive for E. coli O26 by real-time PCR, with only one of these samples positive for E. coli O26 by the culture method. One sample was positive for E. coli O111 by both real-time PCR and the culture method. The two samples found positive for E. coli O26 by real-time PCR method but not by culture method belonged to the group of frozen beef samples, indicating that the previously developed culture method for the detection of E. coli O26 may not be suitable for the detection of freeze injured cells. In conclusion, this study highlights the role of beef meat in the transmission of non-O157 VTEC. The results of the study emphasize that the analyses for emergent pathogens should be included in food safety surveillance systems and that the development of standard methods for the detection of E. coli O26 and O111 in routine food testing is needed in order to reduce the consumer exposure to contaminated food.     

Characterization of Escherichia coli from raw poultry in Belgium and impact on the detection of Campylobacter jejuni using Bolton broth

A comparative study examining Bolton broth and Preston broth for enrichment and reliable detection of Campylobacter jejuni (both healthy and freeze stressed cells) was performed. Tested as pure cultures, Bolton broth enabled faster resuscitation and growth of C. jejuni compared to Preston broth. When C. jejuni was co-incubated with extended-spectrum-beta-lactamase (ESBL) producing Escherichia coli isolated from Belgian poultry meat preparations, the latter dominated in the Bolton enrichment broth and crowded the mCCDA plates. This resulted in the inability to recover C. jejuni by ISO 10272-1:2006 standard method. Preston broth did not support the growth of the ESBL E. coli isolates, but showed longer detection time of C. jejuni compared to Bolton broth. The use of the same antibiotic (sodium cefoperazone) in Bolton broth and in mCCDA plates may explain the problems encountered for detection of C. jejuni, as high numbers of ESBL E. coli present after enrichment in Bolton broth, also caused overgrowth and masked the few C. jejuni colonies present on the mCCDA plates. The use of Campylobacter spp. specific real-time PCR circumvented these problems and enabled rapid detection of the pathogen after 24 h enrichment in both Bolton and Preston broth, for both healthy and freeze stressed cells.     

Detection of virulence determinants by real time PCR in E. coli isolated from mutton

Ninety three Escherichia coli isolates belonging to 35 serotypes isolated from market mutton were tested to find out the prevalence of virulence determinants, Verotoxin 1 (VT1), Verotoxin 2 (VT2), Intimin (eae) genes and enterohemolysin production. Real Time PCR based detection was carried out for virulence genes using SYBR green format and amplicons were confirmed by melt curve analysis. Prevalence of VT1 gene in these isolates was much higher (38.70%) on the other hand, that of VT2 gene was nil (0%) while that of eae was very low (3.22%). Enterohemolysin production was found in 31.18% isolates when tested on washed sheep blood agar supplemented with CaCl₂. All enterohemolysin producing isolates were also positive for the VT1 gene.     

UV-C inactivation of Escherichia coli at different temperatures

The influence of treatment parameters (dose and temperature), treatment medium characteristics (absorption coefficient, pH and water activity) and microbiological factors (strain, growth phase and UV damage and repair capacity) on Escherichia coli UV-C resistance has been investigated. UV-C doses to inactivate at 25 °C 99.99% of the initial population (4D) of five strains of E. coli in McIlvaine buffer of pH 7.0 with tartrazine added (absorption coefficient of 10.77 cm⁻¹) were 16.60, 14.36, 14.36, 13.22, 11.18 J/mL for strains E. coli STCC 4201, STCC 471, STCC 27325, O157:H7 and ATCC 25922, respectively. The entrance in the stationary growth phase increased the 4D value of the most resistant strain, E. coli STCC 4201, from 13.09 to 17.23 J/mL. Survivors to UV treatments showed neither oxidative damages nor injuries in cell envelopes. On the contrary, the photoreactivation by the incubation of plates for 60 min below visible light (11.15 klx) increased the dose to 18.97 J/mL. The pH and the water activity of the treatment medium did not affect the UV tolerance of E. coli STCC 4201, but the lethal effect of the treatments decreased exponentially (Log₁₀4D = − 0.0628α + 0.624) by increasing the absorption coefficient (α). A treatment of 16.94 J/mL reached 6.35, 4.35, 2.64, 1.93, 1.63, 1.20, 1.02 and 0.74 Log₁₀ cycles of inactivation with absorption coefficients of 8.56, 10.77, 12.88, 14.80, 17.12, 18.51, 20.81 and 22.28 cm⁻¹. The temperature barely changed the UV resistance up to 50.0 °C. Above this threshold, inactivation rates due to the combined process synergistically increased with the temperature. The magnitude of the synergism decreased over 57.5 °C. An UV treatment of 16.94 J/mL in media with an absorption coefficient of 22.28 cm⁻¹ reached 1.23, 1.64, 2.36, 4.01 and 6.22 Log₁₀ cycles of inactivation of E. coli STCC 4201 at 50.0, 52.5, 55.5, 57.5 and 60.0 °C, respectively.

Industrial relevance

Results obtained in this investigation show that UV light applied at mild temperatures (57.5 to 60 °C) could be an alternative to heat treatments for 5-Log₁₀ reductions of E. coli in liquid foods. Since microbial resistance to UV-C light did not depend on the pH and water activity (a_w) of the treatment media, eventual advantages of UV light for pasteurization purposes will be higher in low a_w foods. E. coli STCC 4201 could be considered as a target when UV light processing of foods.     

The prevalence of Listeria, Salmonella, Escherichia coli and E. coli O157:H7 on bison carcasses during processing

Bison meat is a relatively new, emerging meat species gaining increased popularity in the US and European meat markets, but little is known of its microflora or pathogens that may be present. This study was carried out to determine the incidence of the foodborne pathogens Listeria, Salmonella, Escherichia coli/E. coli O157:H7 on slaughtered bison and to evaluate the bison slaughter process. Bison carcass sampling was carried out at monthly intervals over a period of 1 year at a Bison processing facility in the Midwestern United States. A total of 355 Bison carcasses were sampled by surface swabbing the carcasses at five points on the production line: pre-dehiding, post-evisceration, post-USDA inspection, post-washing and 24 h chilled carcass. Overall, the prevalence of Listeria spp., Salmonella spp., E. coli and E. coli O157:H7 was 18.3%, 3.94%, 38.3% and 1.13%, respectively. The prevalence of Listeria spp. at each sampling point tested was 42.24%, 18.1%, 6.03%, 1.72% and 3.77% while the prevalence of E. coli at each sampling point was: 88.79%, 73.28%, 52.59%, 56.89% and 11.3%, respectively. The data obtained suggests that current antimicrobial intervention strategies used at the plant are relatively effective in reducing Listeria and E. coli contamination on bison carcasses to some extent, however further study is required to determine the influence of current slaughter practices on carcass contamination. The data reported in this study to the authors’ knowledge is some of the first information reporting on the bacteriological status of Bison, and provides some useful baseline information for future research.     

大肠杆菌四氢嘧啶合成途径的构建与优化

为了构建重组工程菌株发酵合成四氢嘧啶,解决野生型菌株对高盐环境的依赖,作者克隆了来自伸长盐单胞菌(Halomonas elongata ATCC 33173)的四氢嘧啶合成相关基因簇ectABC并在大肠杆菌(Escherichia coli,E. coli)中重构了四氢嘧啶的合成途径。通过宿主比较发现,与E. coli W3110、E. coli DH5α和高产天冬氨酸的大肠杆菌(命名为E. coli(asp))相比,E. coli BL21(DE3)更适用于四氢嘧啶的合成(185.23 mg/L)。进一步分析了不同拷贝数的表达系统对四氢嘧啶合成的影响,发现高拷贝的pRSFDuet-1为载体时产四氢嘧啶量最高,达267.3mg/L。在此基础上,采用核糖体结合位点(RBS)优化策略,对四氢嘧啶合成途径3个酶EctA、EctB与EctC进行了组合优化表达,四氢嘧啶产量提高至521.24 mg/L。为强化前体天冬氨酸和天冬氨酸β-半醛的供给,对天冬氨酸激酶、天冬氨酸半醛脱氢酶、磷酸烯醇丙酮酸羧化酶、天冬氨酸转氨酶和天冬氨酸裂解酶进行了过量表达。研究表明,单独强化天冬氨酸激酶的表达更有利于四氢嘧啶的合成(551.24 mg/L),为构建高产四氢嘧啶的工程菌种提供了新的策略。     

The DNA Sequence of the Escherichia coli O22 O-Antigen Gene Cluster and Detection of Pathogenic Strains Belonging to E. coli Serogroups O22 and O91 by Multiplex PCR Assays Targeting Virulence Genes and Genes in the Respective O-Antigen Gene Clusters

Multiplex polymerase chain reaction (PCR) assays were developed for detection of pathogenic strains belonging to Escherichia coli serogroups O22 and O91. The O-antigen gene cluster of E. coli O22 was sequenced to identify genes that could be employed as targets for serogroup-specific PCR assays. The wzx and wzy genes in the O-antigen gene clusters of E. coli O22 and E. coli O91 were selected as target genes. The assays were serogroup-specific when tested against 72 E. coli O22 strains and 57 E. coli O91 strains isolated from food, humans, and animals, representative strains belonging to 168 E. coli O serogroups and non-E. coli bacteria. Furthermore, 72 E. coli O22 strains and 57 E. coli O91 strains isolated from food, water, animals, and humans were tested by the PCR for the presence of six and 19 virulence genes, respectively, associated with pathogenic E. coli strains. Based on the PCR screening results, multiplex PCR assays targeting the O22 wzy gene and the cnf-1 and sfa genes in E. coli O22 and the O91 wzy gene, conserved sequences of stx ₁ and stx ₂ genes, and the astA and cdt-III genes in E. coli O91 were developed to detect and identify pathogenic strains belonging to serogroups O22 and O91. Furthermore, E. coli O22 and O91 were detected by multiplex PCR assays targeting the wzx or wzy genes and conserved sequences of the stx ₁ and stx ₂ genes in ground beef samples inoculated with approximately two colony-forming units (CFU)/25 g after 18-h enrichment. The results demonstrate that the E. coli O22 and O91 wzx and wzy gene sequences were specific for the respective serogroups and can be used as diagnostic markers for rapid identification of these serogroups as an alternative to serotyping. The multiplex PCR assays targeting the O22 and O91 wzx and wzy genes and virulence genes can be used to identify and to detect pathogenic strains of these serogroups in food and fecal samples. Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.