硫醚类香料对金黄色葡萄球菌和单增李斯特菌的抑制作用

选取10种常见的硫醚类香料,通过测量其抑菌圈直径来研究各硫醚类香料对金黄色葡萄球菌和单增李斯特菌的抑制能力,并分析硫醚香料的结构与抑菌能力的关系。筛选出抑菌能力最强的硫醚类香料,利用反转录荧光定量聚合酶链式反应来考察其对金黄色葡萄球菌耐热核酸酶nuc基因表达水平的影响。结果显示:二烯丙基二硫醚(DADS)、甲基糠基二硫醚(MFDS)和二烯丙基硫醚(DAS)对两种革兰氏阳性菌的抑菌效果较好,且抑菌能力为DADS＞MFDS＞DAS。DADS相较于其它9种硫醚类香料抑菌效果最好,对金黄色葡萄球菌和单增李斯特菌的最低抑制浓度(MIC)分别为312.5 mmol/L和1.25 mol/L。当DADS的浓度为亚抑制浓度时(1/16MIC、1/8MIC、1/4MIC、1/2MIC),均能显著降低金黄色葡萄球菌nuc基因的表达水平(P＜0.01)。以上结果说明:烯丙基和二硫键的存在能够增强硫醚类香料的抑菌能力,并且对抑制金黄色葡萄球菌毒力发挥重要作用。     

二烯丙基二硫醚对单增李斯特菌毒力基因表达的影响

二烯丙基二硫醚(DADS)是一种典型的硫醚类香料。该类香料在抗菌消炎,提高机体免疫力,预防和治疗心血管疾病等保健及药理作用方面具有显著效果。而对其在食源性致病菌中的抑制作用及相关机理研究较少。四大食源性致病菌之一单增李斯特菌(Listeria monocytogenes,LM)容易感染食物而引发脑膜炎、败血症及孕妇流产等一系列恶性疾病。本研究以如何高效抑制单增李斯特的生长为出发点,以DADS为研究对象,采用微量肉汤稀释法初步筛选出其对单增能李斯特菌的最小抑菌浓度(MIC),利用RT-qPCR技术考察其对单增李斯特菌毒力基因inlA、hlyA、prfA表达的影响。结果表明:亚抑菌浓度下DADS均可显著抑制毒力基因inlA、hlyA、prfA的表达,且抑制作用呈现一定的浓度依赖性,hlyA及prfA对其作用更敏感。     

硫醚类香料对沙门氏菌和副溶血性弧菌的抑制作用

研究硫醚类香料对沙门氏菌和副溶血性弧菌两种常见食源性致病菌的抑制作用,探讨该香料抑菌活性与其结构间的关系。采用滤纸片法测定抑菌圈直径,倍比肉汤稀释法确定不同香料的最小抑菌浓度(MIC),并用波长600 nm下菌液的吸光值绘制生长曲线。采用qRT-PCR法研究硫醚类香料对副溶血性弧菌特异性毒力基因tdh基因的调控作用。结果表明,这些硫醚类香料对两种菌的作用效果不同,其中甲基丙基二硫醚、甲基糠基二硫醚、糠基异丙基硫醚、二烯丙基硫醚、二烯丙基二硫醚对两种菌都有抑制效果。二烯丙基二硫醚抑制作用最显著,对沙门氏菌的MIC为19.5 mmol/L,对副溶血性弧菌为4.88 mmol/L。二烯丙基二硫醚对副溶血性弧菌毒力基因tdh的表达存在明显的抑制作用并与浓度呈正相关。硫醚类香料中硫原子个数和烯丙基可促进其抑菌效果,其中二烯丙基二硫醚抑菌效果最好。本研究结果为硫醚类香料在食品中的应用提供参考。     

食源性产志贺毒素大肠杆菌的分离及菌株特征分析

了解不同食品中产志贺毒素大肠杆菌的流行情况、菌株特征及潜在致病性。方法 对我国不同地区采集的355份食品样品进行产志贺毒素大肠杆菌分离鉴定,对菌株进行stx1/stx2基因分型、eae等毒力基因检测,并对菌株进行多位点序列分型(MLST)分析。结果 355份样品中44份stx2基因阳性,共分离出11株非O157 产志贺毒素大肠杆菌,其中3株携带stx2a亚型,3株携带stx2e亚型,1株携带stx2b亚型,4株不能分型;5株携带ehxA、saa毒力基因,2株携带subA基因,1株携带katP基因;MLST将11株菌分为7个不同的ST型,存在与溶血性尿毒综合症患者肠出血性大肠杆菌分离株(HUS-associated enterohemorrhagic E.coli,HUSEC)及主要流行血清群产志贺毒素大肠杆菌亲缘关系较近的ST型别。结论 我国食品中存在一定程度的非O157产志贺毒素大肠杆菌污染,部分菌株具有潜在致病性,应加强对食品中STEC的监测。     

武汉肉类食品中大肠杆菌O157∶H7分离株stx亚型和毒力特征分析

从22 家市场销售的117 份肉类食品中分离出4 株大肠杆菌O157∶H7菌株,经PCR检测这4 株菌的stx、hly、 eae毒力基因均为阳性。采用聚合酶链式反应（polymerase chain reaction,PCR）法对这4 株菌的stx亚型进行鉴定。 3 株菌同时携带stx1、stx2基因,且均为stx1a、stx2a亚型。菌株EC5.11仅携带stx2基因,但在所有的stx2分型PCR反 应中都为阴性。用PCR扩增该菌株stx2基因全长并克隆测序,序列分析结果表明EC5.11志贺毒素基因为stx2c亚型。 采用Vero细胞毒性实验检测这4 株菌产志贺毒素的状况,结果显示这些菌株都具有一定的Vero细胞毒性。     

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。     

产志贺毒素突变株的毒力分析

对2株食物中毒病人体内分离的产志贺毒素突变株EC130和EC169进行毒力分析。EC130和EC169携带stx基因但不能正常表达志贺毒素,具有eae基因和hly基因,仍具有一定毒力。初步探讨了产志贺毒素突变株不能正常表达志贺毒素的机理,高产志贺毒素的对照株携带Q933基因,而EC130和EC169不携带Q933基因。结果表明,单一采用志贺毒素作为检测靶标,容易造成产志贺毒素突变株漏检,今后在检测食品中产志贺毒素株时应增加检验eae基因和hly基因。     

几种硫醚类香料抑菌活性的研究

选取八种硫醚类香料,通过测量抑菌圈直径考察其抑菌活性,并根据显著性分析结果探讨结构对其抑菌活性的影响。结果表明:对于大肠杆菌、金黄色葡萄球菌、沙门氏菌、福氏志贺氏菌及枯草芽孢杆菌,二甲基硫醚(DMS)、二乙基硫醚(DES)及二丁基硫醚(DBS)均无抑菌作用,而二烯丙基硫醚(DAS)则具有一定的抑菌作用;以二烯丙基二硫醚(DADS)为阳性对照,选取的硫醚类香料对五种供试细菌抑制作用大小为DADS>二丙基二硫醚(DPDS)>或≈二甲基二硫醚(DMDS),二甲基三硫醚(DMTS)>DMDS>DMS及DADS>DAS。这些结果说明二烯丙基对单硫醚类香料的抑菌作用有一定贡献,双键的存在对二硫醚类香料的抑菌作用有重要影响。对于具有相同取代基团的硫醚类香料,硫原子数越多,其抑菌效果越强。     

温州市食品中肠出血性大肠杆菌O157∶H7污染状况调查

目的调查温州市食品中肠出血性大肠杆菌O157∶H7的污染状况,了解肠出血性大肠杆菌O157∶H7毒力因子的携带与耐药情况。方法采用分层随机抽样的方法,对2008—2011年抽取的231份食品样品,用免疫磁珠富集法对大肠杆菌O157∶H7进行分离,对该分离株进行生化、血清学鉴定,以纸片法(K-B法)进行药敏试验;采用PCR方法检测O、H抗原基因和stx1、stx2、eaeA、hlyA等4种毒力基因。结果 231份样品中,分离出1株肠出血性大肠杆菌O157∶H7,检出率为0.43%;O157抗原和H7抗原的核酸检测结果阳性,但未检测到stx1、stx2、eaeA、hlyA等4种毒力基因,菌株对红霉素、利福平、150μg/片和10μg/片两种浓度的0/129(二氨基二异丙基喋啶磷酸盐)耐药。结论温州市食品中存在大肠杆菌O157∶H7的污染,检出率较低,但提示要加强主动监测,通过有效干预,防范肠出血性大肠杆菌O157∶H7所致食源性疾病的发生。     

大蒜中硫代亚磺酸酯提取工艺及其稳定性研究

采用乙醇溶液提取大蒜中的硫代亚磺酸酯,研究了乙醇浓度、提取温度、提取时间、料液比对硫代亚磺酸酯提取量的影响,在单因素实验的基础上采用响应面法确定了最佳提取条件:乙醇浓度为64.89%,提取温度为19.06℃,提取时间为1.48 h,料液比为1∶4.48(g∶mL),硫代亚磺酸酯含量可达4.17 mmol/100 g。对硫代亚磺酸酯的稳定性研究表明:随着温度的升高,硫代亚磺酸酯稳定性下降,提取液在贮存过程中,随着硫代亚磺酸酯含量的降低,二烯丙基二硫醚(DADS)与二烯丙基三硫醚(DATS)的含量会上升。     

Detection, occurrence, and characterization of Escherichia coli O157:H7 from raw ewe's milk in Spain

A study was carried out in the Castilla y León region of Spain to investigate the presence of Escherichia coli O157:H7 in raw ewe's milk samples collected from several cheese factories during 1 year. All specimens were examined for E. coli O157:H7 by selective enrichment at 41.5 +/- 1.0 degrees C, after both 6 and 22 h of incubation, and then immunomagnetically separated and plated on cefixime-potassium tellurite-sorbitol MacConkey agar. No growth was obtained in the enrichment broth after a 6-h incubation. Presumptive colonies obtained after 22 h of incubation were screened by a multiplex PCR assay for the presence of rfbO157 and fliCH7 genes. Of all the ewe's milk samples studied, three were positive for E. coli O157:H7. The E. coli O157:H7 strains that were positive for the rfbO157 and fliCH7 genes were then analyzed by multiplex PCR for the presence of virulence genes (stx1, stx2, ehxA, and eaeA). All E. coli O157:H7 isolates were Shiga toxigenic and harbored additional genes related to virulence (ehxA and eaeA). The predominant Stx toxin type was stx2. These results demonstrate that raw ewe's milk used in cheesemaking may be sporadically contaminated with E. coli O157:H7 strains that are potentially pathogenic for humans.     

Occurrence of Shiga toxin-producing Escherichia coli in a Spanish raw ewe's milk cheese

The aim of the present study was to investigate the occurrence of Escherichia coli O157:H7 and other Shiga toxin-producing E. coli (STEC) in 'Castellano' cheese, a non-cooked and hard or semi-hard Spanish cheese made from ewe's milk. A total of 83 raw milk cheese samples with different ripening times (2.5, 6 and 12 months) were taken at 30 cheese factories. Samples were examined for the presence of STEC using in the first stage the Association of Official Analytical Chemists (AOAC) official method number 997.11, and then, in the second stage, isolates were tested for virulence genes using genotypic (PCR) methods. Three STEC strains were detected in two samples (2.4%) of 'Castellano' cheese, one with 2.5 and the other one with 12 month-ripening period. From those STEC isolates, two were identified as E. coli O14 and the third presented an O-specific polysaccharide not-groupable serologically (ONG). PCR showed that all isolates were characterized by harbouring the Shiga toxin (stx) stx1 gene and by the absence of the genes for stx2, eaeA, and ehxA virulence factors. This study revealed the potential of STEC to survive in long-ripened-hard cheeses.     

Prevalence and characteristics of Escherichia coli O26 and O111 from cattle in Korea

Enterohemorrhagic Escherichia coli (EHEC) is an important cause of diarrhea, hemorrhagic colitis and hemolytic uremic syndrome worldwide. E. coli O26 and O111 are the serotypes most frequently isolated from human EHEC infections in Korea. Cattle are considered to be the major sources of E. coli O26 and O111. This study investigated the prevalence of E. coli O26 and O111 in fecal samples from cattle in Korea from April 2002 to March 2004. Out of 809 samples, 54 (6.67%), 37 (4.57%), and 16 (1.98%) tested positive for O26, O111, and both O26 and O111, respectively. Most of the E. coli O26 and O111 strains were isolated from May to October of each year. PCR analysis of the EHEC virulence markers revealed that most of the E. coli O26 and O111 isolates were positive for ehxA, eaeA and stx1 and/or stx2. These results suggest that the majority of Korean E. coli O26 and O111 isolates from cattle can cause serious diseases in humans.     

Effect of modified atmosphere packaging on the persistence and expression of virulence factors of Escherichia coli O157:H7 on shredded iceberg lettuce

Fresh-cut leafy greens contaminated with Escherichia coli O157:H7 have caused foodborne outbreaks. Packaging conditions, coupled with abusive storage temperatures of contaminated lettuce, were evaluated for their effect on the potential virulence of E. coli O157:H7. Shredded lettuce was inoculated with 5.58 and 3.98 log CFU E. coli O157:H7 per g and stored at 4 and 15°C, respectively, for up to 10 days. Lettuce was packaged under treatment A (modified atmosphere packaging conditions used for commercial fresh-cut produce, in gas-permeable film with N(2)), treatment B (near-ambient air atmospheric conditions in a gas-permeable film with microperforations), and treatment C (high-CO(2) and low-O(2) conditions in a gas-impermeable film). E. coli O157:H7 populations from each treatment were determined by enumeration of numbers on MacConkey agar containing nalidixic acid. RNA was extracted from packaged lettuce for analysis of expression of virulence factor genes stx(2), eae, ehxA, iha, and rfbE. E. coli O157:H7 populations on lettuce at 4°C under all treatments decreased, but most considerably so under treatment B over 10 days. At 15°C, E. coli O157:H7 populations increased by at least 2.76 log CFU/g under all treatments. At 15°C, expression of eae and iha was significantly greater under treatment B than it was under treatments A and C on day 3. Similarly, treatment B promoted significantly higher expression of stx(2), eae, ehxA, and rfbE genes on day 10, compared with treatments A and C at 15°C. Results indicate that storage under near-ambient air atmospheric conditions can promote higher expression levels of O157 virulence factors on lettuce, and could affect the severity of E. coli O157:H7 infections associated with leafy greens.     

Escherichia coli O157 and non-O157 Shiga toxin-producing Escherichia coli in fecal samples of finished pigs at slaughter in Switzerland

Fecal samples from 630 slaughtered finisher pigs were examined by PCR to assess the shedding of Escherichia coli O157 (rfbE) and Shiga toxin-producing E. coli (STEC, stx). The proportion of positive samples was 7.5% for rfbE and 22% for stx. By colony hybridization, 31 E. coli O157 and 45 STEC strains were isolated, and these strains were further characterized by phenotypic and genotypic traits. Among E. coli O157 strains, 30 were sorbitol positive, 30 had an H type other than H7, and none harbored stx genes. Intimin (eae), enterohemolysin (ehxA), EAST1 (astA), and porcine A/E-associated protein (paa) were present in 10, 3, 26, and 6% of strains. Among them, one eae-gamma1-positive O157:H7 strain testing positive for ehxA and astA and two eae-alpha1-positive O157:H45 strains were classified as enteropathogenic E. coli (EPEC). The O157:H45 EPEC harbored the EAF plasmid and the bfpA gene, factors characteristic for typical EPEC. The isolated STEC strains (43 sorbitol positive) belonged to 11 O:H serotypes, including three previously reported in human STEC causing hemolytic uremic syndrome (O9:H-, O26:H-, and O103:H2). All but one strain harbored stx2e. The eae and ehxA genes, which are strongly correlated with human disease, were present in only one O103:H2 strain positive for stx1 and paa, whereas the astA gene was found more frequently (14 strains). High prevalence of STEC was found among finisher pigs, but according to the virulence factors the majority of these strains seem to be of low virulence.     

Occurrence of non-O157 shiga toxin-producing Escherichia coli in ready-to-eat food from supermarkets in Argentina

Between June 2000 and December 2001, 500 food samples were collected from supermarkets and shops selling ready-to-eat food in Rosario, Argentina, and examined for Escherichia coli. Forty-nine E. coli isolates from food samples were further characterized for virulence genes by multiplex polymerase chain reaction (PCR) targeting the stx1, stx2, stx2e, eaeA, CNF1, CNF2, Einv, LTI, STI, and STII genes in four groups. Out of 49 E. coli isolates screened by multiplex PCR, only 10 possessed Shiga toxin genes, stx1 and stx2 genes and none possessed the other genes. The Shiga toxin positive E. coli strains (STEC) were isolated from soft, cottage cheeses, chicken with sauce and vegetables mayonase. These E. coli isolates were serogrouped and belonged to O18 (two strains), O8, O57w, O79, O44, and O128; three strains were untypeable. Pulsed-field gel electrophoresis (PFGE) with XbaI generated a unique profile for each, having 10-15 bands ranging from 50 to 500 kb, except that strain ARG 20 generated small bands and was partly degraded. These strains are potential foodborne pathogens and their presence in ready-to-eat food illustrates the need to keep a careful watch for the source of pathogens and then develop methods to control them.     

Impact of antibiotic stress on acid and heat tolerance and virulence factor expression of Escherichia coli O157:H7

This study was conducted to determine the effect of antibiotic stress on the virulence factor expression, simulated gastric fluid (SGF; pH 1.5) survival, and heat tolerance (56 degrees C) of Escherichia coli O157:H7. The MIC for three antibiotics (trimethoprim, ampicillin, and ofloxacin) was determined for two E. coli O157:H7 strains (ATCC 43895 [raw hamburger isolate] and ATCC 43890 [fecal isolate]) by the dilution series method. Subsequently, cells were stressed at the MIC of each antibiotic for 4 h, and poststress tolerance and virulence factor production were evaluated. Heat tolerance (56 degrees C) was determined by the capillary tube method, and SGF (pH 1.5) survival was used to assess acid tolerance. Virulence factor expression (stx, hlyA, and eaeA) was evaluated by the creation of lacZ gene fusions and then use of the Miller assay (a beta-galactosidase assay). Stressed and control cells were evaluated in triplicate. The MIC for trimethoprim was 0.26 mg/liter for both strains; for ampicillin, it was 2.05 mg/liter for both strains; and for ofloxacin, it was 0.0256 and 0.045 mg/liter for each strain. Heat tolerance and SGF survival following antibiotic stress decreased when compared with control cells (P < 0.05). Exposure to ofloxacin increased stx and eaeA expression (P < 0.05). Exposure to ampicillin or trimethoprim increased eaeA expression (P < 0.05). hly expression increased following trimethoprim stress (P < 0.05). Antibiotics can increase E. coli O157:H7 virulence factor production, but they do not produce a cross-protective response to heat or decreased pH.     

大肠杆菌O157:H7与假单胞菌混合菌膜形成时交互作用及其对毒力基因表达的影响

以大肠杆菌O157:H7和食品中常见腐败菌假单胞菌为对象,研究两种菌混合培养时菌体泳动能力和混合菌膜形成时的交互作用,进一步采用反转录实时定量聚合酶链式反应分析大肠杆菌O157:H7代表菌株在混合菌膜形成时毒力基因（stx1、stx2、hly和eae）表达变化。菌体泳动性结果显示,3 株大肠杆菌O157:H7泳动性均低于4 株假单胞菌（P＜0.05）,两种菌混合培养时假单胞菌的泳动能力受到显著抑制。选择性培养计数发现混合菌膜形成72 h时,两种菌未发生相互促进,其中4 株假单胞菌的菌膜形成均受到3 株大肠杆菌O157:H7显著抑制（P＜0.05）。选取大肠杆菌O157:H7 CICC21530菌株分析毒力基因表达,结果发现混合菌液单位体积、混合菌膜单位面积4 种毒力基因表达分别低于单种菌液、单种菌膜（P＜0.05）,进一步的单位菌数结果亦然,表明混合培养和混合菌膜形成时假单胞菌抑制了大肠杆菌O157:H7毒力基因表达;单位菌数结果还发现菌膜中4 种毒力基因表达均高于浮游菌（P＜0.05）,表明形成菌膜后菌体毒力增强。本研究可为揭示食源性致病菌和腐败菌混合菌膜形成的交互作用及进一步风险评估和风险防控提供科学依据。