基于酸处理的产志贺毒素大肠埃希菌选择性增菌方法研究

目的开发一套基于酸处理的产志贺毒素大肠埃希菌(Shiga toxin-producing Escherichia coli,STEC)选择性增菌方法,提高食品中STEC菌株的检出率。方法 选择12株不同血清型STEC菌株以及13株常见干扰菌,评价其对不同酸处理条件(pH2.0、2.5、3.0)的耐受性;考察筛选的酸处理条件对营养胁迫和冷冻胁迫状态STEC菌株生长活性的影响;比较研究酸水解酪蛋白、酵母提取物和丙酮酸钠等成分对胁迫状态STEC菌株的促生长作用,优化酸处理后中和/促生长培养基配方;比较增菌前与增菌后两种酸处理方式对STEC菌株分离效果的影响;最后通过人工污染样品,评价本研究建立的STEC酸处理选择性增菌方法的检测效果。结果 本研究建立了一种不依赖于抑菌成分的STEC选择性增菌方法,样品在增菌前经酸处理培养基室温处理2 h,降低背景杂菌干扰,再通过中和/促生长培养基(TSB-1.5%Tris-0.1%丙酮酸钠)进行目标菌的增菌培养;人工污染试验结果表明,本方法与我国现行GB4789.36-2016标准方法相比,能够有效减少背景杂菌的干扰、获得更高的STEC分离效率。结论 本研究建立的基于酸处理的STEC选择性增菌方法能够有效提高食品中STEC菌株的检测效率和检测准确性。     

检测产志贺毒素大肠杆菌的菌落PCR方法

针对产志贺毒素大肠杆菌的毒力基因stx 设计特异性引物,并建立一种菌落PCR 方法。菌落PCR 模拟实验证实,该方法特异性强,能良好的扩增出O157 的stx1 和stx2 基因,而普通大肠杆菌、蜡样芽孢杆菌、金黄色葡萄球菌则无PCR 扩增产物。应用分子检测初筛、选择性培养、菌落PCR 相结合的方法,检测实际食品样品,分离检测到一株携带 stx1 的产志贺毒素大肠杆菌。本实验建立的菌落PCR 方法可应用于食品检验。     

伊犁地区肉牛源产志贺毒素大肠杆菌的血清型和ERIC-PCR基因型研究

目的:了解产志贺毒素大肠杆菌在伊犁地区肉牛养殖环境和加工环节中的污染状况及其遗传多样性,为产业链中食源性致病性大肠杆菌的风险监测和控制提供基础数据。方法:采用传统方法和PCR方法对养殖环节的饲草料和粪便及屠宰环节的553份样品进行产志贺毒素大肠杆菌的污染调查,对分离鉴定的产志贺毒素大肠杆菌进行7种常见血清型（O145、O157、O45、O103、O111、O26、O121）的PCR检测和ERIC-PCR的基因分型。结果:检测553份样品中有39株编码志贺毒素基因,产志贺毒素大肠杆菌（STEC）的检测率是7.1%。常见血清型PCR检测中血清型O111有2株菌,检出率是5.1%;O145有5株菌,检出率是12.8%。ERIC-PCR基因分型产志贺毒素大肠杆菌有10种基因亚型,分成3簇,A簇有23株菌,相似性在59%¹00%,表明这些菌株之间的亲缘关系较近。结论:伊犁地区肉牛粪便是产志贺毒素大肠杆菌的污染源,这些菌株的亲缘关系较近。      

可视化环介导等温扩增法检测牛奶中产志贺毒素大肠埃希氏菌

为建立一种快速、简单、灵敏的产志贺毒素大肠埃希氏菌（Shiga toxin-producing Escherichia coli,STEC）检测方法,本研究以stx基因作为检测靶基因和利用羟基萘酚蓝（hydroxy naphthol blue,HNB）指示剂建立了一种STEC的可视化环介导等温扩增（Loop Mediated Isothermal Amplification, LAMP）方法。根据GenBank公布的STEC菌株stx1和stx2基因核苷酸序列为靶序列,分别设计并合成五组特异性LAMP引物,优化反应条件和体系,进行特异性和灵敏度试验。同时,将建立好的LAMP方法与聚合酶链式反应（polymerase chain reaction, PCR）检测方法相比较,并对人工污染STEC的脱脂乳进行检测。结果表明,建立的可视化LAMP方法在64℃反应50 min后可凭肉眼观察（紫罗兰色到天蓝色）判定结果。该方法特异性良好,与大肠埃希菌、沙门菌、金黄色葡萄球菌等均无交叉反应,可以准确区分STEC与非STEC菌。灵敏度的检测结果表明,stx1-LAMP和stx2-LAMP检测方法的检测限分别为3.54×10-4 ng/μL和3.54×10-5 ng/μL,而常规PCR的检测限stx1和stx2分别为3.54×10-3 ng/μL和3.54×10-2 ng/μL,即stx1-LAMP检测方法的灵敏度是stx1-PCR的10倍,stx2-LAMP检测方法的灵敏度是stx2-PCR的1000倍。对人工污染STEC菌牛奶样品进行检测,stx1-LAMP和stx2-LAMP分别可检测到细菌浓度为103 CFU/mL和102 CFU/mL的加标样品。本研究为STEC菌株提供了一种特异性强、灵敏度高、成本低,适用于现场和基层检测的可视化LAMP 检测方法。     

食用农产品中产志贺毒素大肠埃希菌污染与家庭厨房食物安全现状分析及防控措施

本文通过对食用农产品中产志贺毒素大肠埃希菌(STEC)来源与传播途径分析,阐述了STEC污染与家庭厨房食物安全之间的关系,并对当前世界各国食品中STEC的监管情况进行阐述,从而提出我国控制食用农产品中STEC进入家庭厨房的解决方案。     

Prevalence of Shiga toxin-producing Escherichia coli in beef

Over the past two decades, many human illness outbreaks were attributed to consumption of undercooked beef products containing Shiga toxin-producing Escherichia coli (STEC). The illnesses included mild or bloody diarrhea, hemorrhagic colitis, and the life-threatening hemolytic uremic syndrome (HUS). Tracing these outbreaks to O157 and an increasing number of non-O157 STEC strains suggests that beef safety concerns will continue to rise and may negatively affect the beef industry. To effectively address these concerns, it is critical to evaluate the role of beef in STEC infections. In this review, published reports on beef contamination were evaluated to assess prevalence rates and health risks of STEC isolates. Global testing of beef showed wide ranges of prevalence rates of O157 (from 0.01% to 54.2%) and non-O157 (from 1.7% to 62.5%) STEC. Of the 155 STEC serotypes found in beef, 31 and 25 are known to cause HUS and/or other illnesses, respectively.     

Host-dependent activation of IS1203v excision in Shiga toxin-producing Escherichia coli

IS1203v is an insertion sequence (IS) which is identical to the most abundant IS elements in the genome of Escherichia coli O157:H7. However, there is no sequence homologous to IS1203v in the genome of E. coli K-12. We constructed a system to analyze the excision frequency of IS1203v, and demonstrated that the frequency in E. coli O157:H7 was approximately 10(5) times higher than that in E. coli K-12. We also investigated the excision frequencies of IS1203v in various E. coli isolates, and showed that the excision frequencies of IS1203v-possessing strains were approximately 10(3) times higher than those of IS1203v-nonpossessing strains. The results suggest that the IS1203v-possessing strains use a common system to enhance IS1203v excision.     

Prevalence and characteristics of Shiga toxin-producing Escherichia coli in Swiss raw milk cheeses collected at producer level

The aim of this study was to describe the prevalence, serotypes, and virulence genes of Shiga toxin-producing Escherichia coli (STEC) isolated from raw milk cheese samples collected at the producer level with the purpose of determining whether raw milk cheeses in Switzer-land represent a potential source of STEC pathogenic for humans. Raw milk cheese samples (soft cheese, n = 52; semihard and hard cheese, n = 744; all produced from Swiss cows’, goats’, and sheep's milk) collected at the producer level throughout Switzerland within the national sampling plan during the period of March 2006 to December 2007 were analyzed. Of the 432 cheese samples obtained in the year 2006 and the 364 samples obtained in the year 2007, 16 (3.7%) and 23 (6.3%), respectively, were found to be stx positive. By colony dot-blot hybridization, non-O157 STEC strains were isolated from 16 samples. Of the 16 strains, 11 were typed into 7 E. coli O groups (O2, O15, O22, O91, O109, O113, O174), whereas 5 strains were nontypeable (ONT). Among the 16 STEC strains analyzed, stx₁ and stx₂ variants were detected in 1 and 15 strains, respectively. Out of the 15 strains with genes encoding for the Stx2 group, 4 strains were positive for stx₂, 6 strains for stx_2d2, 2 strains for stx_2-O118, 1 strain for stx_2-06, 1 strain for stx_2g, 1 strain for stx₂ and stx_2d2, and 1 strain for stx₂ and stx_2g. Furthermore, 3 STEC strains harbored E-hlyA as a further putative virulence factor. None of the strains tested positive for eae (intimin). Results obtained in this work reinforce the suggestion that semihard raw milk cheese may be a potential vehicle for transmission of pathogenic STEC to humans.     

Mathematical modeling of growth of non-O157 Shiga toxin-producing Escherichia coli in raw ground beef

Abstract: The objective of this study was to investigate the growth of Shiga toxin‐producing Escherichia coli (STEC, including serogroups O45, O103, O111, O121, and O145) in raw ground beef and to develop mathematical models to describe the bacterial growth under different temperature conditions. Three primary growth models were evaluated, including the Baranyi model, the Huang 2008 model, and a new growth model that is based on the communication of messenger signals during bacterial growth. A 5 strain cocktail of freshly prepared STEC was inoculated to raw ground beef samples and incubated at temperatures ranging from 10 to 35 °C at 5 °C increments. Minimum relative growth (<1 log₁₀ cfu/g) was observed at 10 °C, whereas at other temperatures, all 3 phases of growth were observed. Analytical results showed that all 3 models were equally suitable for describing the bacterial growth under constant temperatures. The maximum cell density of STEC in raw ground beef increased exponentially with temperature, but reached a maximum of 8.53 log₁₀ cfu/g of ground beef. The specific growth rates estimated by the 3 primary models were practically identical and can be evaluated by either the Ratkowsky square‐root model or a Bělehrádek‐type model. The temperature dependence of lag phase development for all 3 primary models was also developed. The results of this study can be used to estimate the growth of STEC in raw ground beef at temperatures between 10 and 35 °C. Practical Application: Incidents of foodborne infections caused by non‐O157 Shiga toxin‐producing Escherichia coli (STEC) have increased in recent years. This study reports the growth kinetics and mathematical modeling of STEC in ground beef. The mathematical models can be used in risk assessment of STEC in ground beef.     

O26等产志贺毒素的6 种血清型大肠杆菌的暴发流行情况及检测研究现状

近年来,产志贺毒素而非O157的大肠杆菌,尤其是被美国农业部称为“the Big Six”的6 种大肠杆菌在诸多国家及地区暴发流行,严重威胁人类的健康,越来越受到世界各国的关注。“the Big Six”包括大肠杆菌O26、O45、O103、O111、O121、O145等6 种血清型。目前,在国内,关于“the Big Six”的报道及研究相对较少。文中介绍“the Big Six”的暴发流行情况,并对各种检测方法进行综述。     

Penicillium camemberti and Penicillium roqueforti enhance the growth and survival of Shiga toxin-producing Escherichia coli O157 under mild acidic conditions

The effects of secondary starter molds of common mold-ripened cheeses on the Shiga toxin-producing Escherichia coli (STEC) O157 were assessed in 3 model systems. In the 1st model, 8 STEC O157 strains were incubated in the spent culture of Penicillium camemberti or Penicillium roqueforti under mild acidic conditions at 25 °C. In the spent cultures of the mold at pH 4.8 to 5.0, the lag times of STEC O157 growth were significantly shorter than those observed in fresh medium. Analyses of the spent culture of P. camemberti showed that the causative agents of the growth enhancement were produced by the mold in response to an acidic environment and were not fully inactivated in heat treatment. In the 2nd model, P. camemberti and STEC O157 were cocultured in acidified milk at 25 °C. The population of STEC O157 reached 10(8) CFU/mL in the presence of the mold, whereas the population steadily declined in the absence of the mold. Although this growth enhancement was partially attributable to alkalization by the mold, it was observed even when the pH of this model was stabilized. In the 3rd model, 2 STEC O157 strains were incubated in the spent cultures of molds at pH 4.5 at 10 °C. In the spent culture, proportions of injured cells were significantly lower and D values were significantly higher than those in control, except one STEC O157 strain in the spent culture of P. camemberti. These results showed that the molds could enhance the growth and survival of STEC O157 by changing the environment. Practical Application: This study demonstrated that molds in foods can improve the growth and survival of the Shiga toxin-producing Escherichia coli O157. Because microbial interactions are ubiquitous in food, our results provide an important insight for understanding the behavior of microorganisms in food.     

Lactic acid resistance of Shiga toxin-producing Escherichia coli and multidrug-resistant and susceptible Salmonella Typhimurium and Salmonella Newport in meat homogenate

This study compared lactic acid resistance of individual strains of wild-type and rifampicin-resistant non-O157 Shiga toxin-producing Escherichia coli (STEC) and of susceptible and multidrug-resistant (MDR) and/or MDR with acquired ampC gene (MDR-AmpC) Salmonella against E. coli O157:H7. After inoculation of sterile 10% beef homogenate, lactic acid was added to a target concentration of 5%. Before acid addition (control), after acid addition (within 2 s, i.e. time-0), and 2, 4, 6 and 8 min after addition of acid, aliquots were removed, neutralized, and analyzed for survivors. Of wild-type and of rifampicin-resistant non-O157 STEC strains, irrespective of serogroup, 85.7% (30 out of 35 strains) and 82.9% (29 out of 35 strains), respectively, reached the detection limit within 0–6 min. Of Salmonella strains, 87.9% (29 out of 33 isolates) reached the detection limit within 0–4 min, irrespective of antibiotic resistance phenotype. Analysis of non-log-linear microbial survivor curves indicated that non-O157 STEC serogroups and MDR and susceptible Salmonella strains required less time for 4D-reduction compared to E. coli O157:H7. Overall, for nearly all strains and time intervals, individual strains of wild-type and rifampicin-resistant non-O157 STEC and Salmonella were less (P < 0.05) acid tolerant than E. coli O157:H7.     

A rapid method for the discrimination of genes encoding classical Shiga toxin (Stx) 1 and its variants, Stx1c and Stx1d, in Escherichia coli

Subtyping of Shiga toxin (Stx)-encoding genes by conventional polymerase chain reaction (PCR) is time-consuming. We developed a single step real-time fluorescence PCR with melting curve analysis to distinguish rapidly stx1 from its variants, stx1c and stx1d. Melting temperatures (Tm) of 206 Stx-producing Escherichia coli (STEC) identified to harbor stx1 or stx1c were analyzed using a specific hybridization probe over the variable region. 170 of 171 stx1-harboring STEC displayed Tm of 69 degrees C to 70 degrees C, whereas 34 of 35 strains containing stx1c had Tm of 65 degrees C-66 degrees C. This constant and reproducible difference of 4 degrees C demonstrated that melting curve analysis is a reliable technique to differentiate stx1 from stx1c. Two isolates displayed atypical Tm. Sequence analysis showed that one of them was 100% identical to stx1d within a 511 bp DNA stretch. Our data demonstrate that real-time PCR is a rapid and reliable tool to differentiate stx1 from stx1c and stx1d and to detect new stx1 variants. Because stx1-harboring STEC cause diarrhoea and hemolytic-uremic syndrome, whereas those containing stx1c are often shed asymptomatically, a rapid differentiation between stx1 and its variants using the procedure developed here has both clinical implications and a direct significance for the risk assessment analysis of STEC isolated from foods.     

Characteristics of Shiga toxin-producing Escherichia coli from meat and milk products of different origins and association with food producing animals as main contamination sources

Shiga toxin-producing strains of Escherichia coli (STEC) cause diarrhoea and haemorrhagic colitis in humans. Most human infections are attributed to consumption of STEC contaminated foodstuff. Food producing animals constitute important reservoirs of STEC and serve as source of food contamination. In this study, we have analyzed 593 foodborne STEC strains for their serotypes and for nine virulence genes (stx1, stx1c, stx1d, stx2, stx2b, stx2e, stx2g, E-hly and eae). The 593 STEC strains grouped into 215 serotypes, and 123 serotypes (57.2%) were represented each by only one STEC isolate. Fifteen serotypes (7.0%) were attributed to 198 (33.3%) of the 593 STEC strains. The foodborne STEC were grouped into different categories in relation to the species of the food producing animal (cattle, pigs, sheep, goats, red deer, wild-boar and hare). Univariate and multivariate statistical analyses revealed significant similarities between the animal origin of the food and the virulence markers of foodborne STEC. Significant associations (p < 0.001) were found for stx1 and for stx2 with bovine meat and milk products. The stx2e gene was significantly (p < 0.001) associated with STEC from pork and wild boar meat. Stx1c and stx2b genes were significantly (p < 0.001) more frequent in STEC from deer meat, as well as from meat and milk products derived from sheep and goats. Using logistic regression models we detected significant (p < 0.01) combinations between stx1, stx2 and E-hly genes and STEC from bovine meat. The combination of stx1c and stx2b genes was significant (p < 0.001) for STEC derived from red deer, sheep and goat products. The properties of foodborne STEC were compared with published data on faecal STEC from food producing animals. Virulence profiles and serotypes of STEC from food showed remarkable similarities to those of faecal STEC that were from the same animal species. The findings from our study clearly indicate that the food producing animals represent the most important source for the entry of STEC in the food chain. Sound hygiene measures implemented at critical stages of food production (milking, slaughtering, and evisceration) should be most effective in reducing the frequency of STEC contamination of food derived from domestic and wildlife animals.     

二烯丙基二硫醚对产志贺毒性大肠杆菌毒力基因表达及生物学特性的影响

以二烯丙基二硫醚(DADS)为原料,以非O157产志贺毒性大肠杆菌CICC10670为供试菌,通过测定最低抑菌浓度探究DADS对大肠杆菌的抑菌活性,利用实时荧光定量PCR技术测定大肠杆菌stx2、eaeA和ehxA基因表达的影响,DADS作为抑制剂探究其对大肠杆菌游动能力的影响。结果表明:DADS对大肠杆菌有较好的抑菌能力,其最低抑菌浓度为2 mmol/L。亚抑菌浓度下DADS降低大肠杆菌毒力基因stx2、eaeA和ehxA的表达,相对表达量降低85%以上,并呈现浓度依赖性。DADS以剂量依赖的方式在亚抑菌浓度下抑制大肠杆菌游动能力。综上所述,二烯丙基二硫醚对产志贺毒性大肠杆菌有良好的抑菌作用,并且较低浓度下就可抑制相关毒力基因的表达,可作为潜在的天然防腐剂,防止食物中毒,延长食品的货架期。     

A systematized review and qualitative synthesis of potential risk factors associated with the occurrence of non-O157 Shiga toxin-producing Escherichia coli (STEC) in the primary production of cattle

Human infection with Shiga toxin-producing Escherichia coli (STEC) causes an estimated 2.8 million cases of acute illness worldwide each year. Serogroup O157 is the most commonly diagnosed STEC in humans, but cases linked to non-O157 STEC serogroups have increased recently due to increased surveillance and improvements to detection methods. Cattle are an important reservoir for STEC O157 and the same may be true for non-O157 STEC; therefore, reducing the occurrence of these pathogens in cattle could mitigate human infection risk. A systematized literature review of articles published within the Scopus database since 2010 (employing a partially systematic approach) was therefore conducted followed by qualitative synthesis of evidence to provide a structured overview of potential risk factors for non-O157 STEC in primary cattle production. Overall, few relevant studies were identified (n = 22), highlighting that more studies are needed. Consistently significant associations were only identified with respect to cattle age (broadly higher rate of isolation from young animals compared to adults) and season of sampling (generally increased isolation of non-O157 STEC in summer). However, wide variation in study designs, including notable differences in laboratory detection methods, means drawing more general conclusions is currently not possible based on the results of this review. However, it is likely that the development of more sensitive methods for non-O157 STEC detection in potential livestock reservoirs and increased standardization across statistically sound epidemiological investigations are required to identify pertinent risk factors.     

大肠杆菌生物膜形成特性及控制措施的研究进展

大肠杆菌既是人体内正常菌群的一部分,也是能够引起较高发病率和死亡率的重要病原体。大肠杆菌可以在生物或非生物表面形成生物膜,从而增强其在不利环境下的生存率（特别是对各种消毒剂、洗涤剂和抗生素的耐受力）。本文主要综述了目前对大肠杆菌形成生物膜研究所取得的重要进展,包括大肠杆菌形成生物膜的主要过程、特征、影响因素、分子调控机制、控制措施,并提出了今后可能的研究方向,为大肠杆菌生物膜的防控提供依据。     

Survival and Reduction of Shiga Toxin‐Producing Escherichia coli in a Fresh Cold‐Pressed Juice Treated with Antimicrobial Plant Extracts

This study was conducted to evaluate the survival of 7 Shiga‐toxigenic Escherichia coli (STEC) in fresh cold‐pressed juice and the antimicrobial efficacy of 4 essential oils (EO: achillea, rosemary, sage, and thyme). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of each EO was determined using microdilution assays evaluated at pH levels 4 and 7; as well as at 4 and 25 °C; daily for up to 5 d. Results indicated that 5 of 7 serotypes survived well in cold‐pressed raw juice for at least 4 d at 4 °C and pH 3.5 with no significant (P > 0.05) reduction in viability. The EO showed varying degrees of antimicrobial activity against the 7 STEC. The MIC and MBCs were lowest for thyme (2 μg/L) and highest for sage (15 to 25 μg/L). The antimicrobial activity was enhanced at low pH and temperature. Data showed that although the top 7 STEC could survive low pH and temperature in vitro and in cold‐pressed juices, EO, especially from thyme and rosemary, reduced STEC to an undetectable level at 4 °C, suggesting that they could be used as natural antimicrobials in juice.