裂解性福氏志贺氏菌噬菌体SF-A2 的生物学特性及其在巴氏杀菌牛奶中的灭菌效果

分离裂解性福氏志贺氏菌(Shigella flexneri)噬菌体,鉴定其生物学特性,研究其在巴氏杀菌牛奶的灭菌效果。以福氏志贺氏菌为宿主菌,从污水中分离获得特异性针对福氏志贺氏菌的具有高裂解活性的噬菌体,命名为SF-A2。运用PEG/NaCl 法纯化SF-A2,经负染后电镜观察,该噬菌体为肌尾科噬菌体。生物学特性分析表明：该噬菌体特异性针对福氏志贺氏菌,且对温度及pH 值具有良好的耐受能力。将SF-A2 以高感染复数(103)用于即食性食品(牛奶)中的福氏志贺氏菌的生物防控实验。结果表明：SF-A2 能够有效灭菌,在加入48h 后,福氏志贺氏菌降低了3lg(CFU/mL);作用至72h,能够完全将福氏志贺氏菌灭活。从而表明,裂解性福氏志贺氏菌噬菌体SF-A2具有良好的杀菌能力,能够在食品中防控福氏志贺氏菌的污染,也为防治由福氏志贺氏菌引起的腹泻性疾病提供生物源性治疗源。     

志贺氏菌CRISPR位点的比较基因组学及与质粒数量的相关性分析

成簇规律间隔的短回文重复序列(CRISPR)在多数细菌和古生菌中广泛存在,该遗传结构可以有效防御外源DNA(如质粒、噬菌体等)的侵袭,从而降低外源基因的水平转移。本文的研究对象为志贺氏菌属中的鲍氏志贺氏菌、痢疾志贺氏菌、宋内氏志贺氏菌以及福氏志贺氏菌等40株菌。研究不同志贺氏菌种中CRISPR位点的结构差异,其与可移动质粒之间的关联。采用生物信息学的方法比较间隔序列与插入序列的同源性,判断CRISPR位点与RNA二级结构稳定性的关系。结果:40株志贺氏菌中均含CRISPR结构,共有241个CRISPR位点和6个确定位点。本研究发现cas3基因和重复序列均不可作为这4类细菌的分类依据。结论:CRISPR位点的数量和间隔区数量之间在统计学上无关。耐药基因、间隔序列整合子等移动遗传原件具有一定的同源性,表明志贺氏菌在进化过程中不断受到外源基因的侵袭。     

一种快速筛查志贺氏菌的荧光纳米免疫层析方法的建立

本文以建立志贺氏菌荧光纳米颗粒免疫层析法为目的。首先,制成志贺氏菌单抗-荧光纳米颗粒偶联物,以双抗体夹心模式制备志贺氏菌免疫层析试纸条。利用微型手持荧光检测仪读出质控线和检测线上的荧光信号,并利用荧光强度来半定量检测志贺氏菌。结果显示:用本研究建立的荧光纳米颗粒试纸条检测30株菌,对宋内志贺氏菌和福氏志贺氏菌呈阳性结果,其余菌株的呈阴性结果,试纸条灵敏度为9.0×104 CFU/m L。用志贺氏菌免疫层析试纸条和标准法检测60份样品,两种方法的总符合率为88.3%。结论:本研究成功建立了志贺氏菌的荧光纳米免疫层析检测方法,能够快速检测志贺氏菌,具有良好的特异性和灵敏性,便于开展现场快速检测。     

抗志贺氏菌IgY的提纯及建立间接ELISA检测志贺氏菌

采用水稀释法提纯抗志贺氏菌IgY,检测10mg/mL纯化抗志贺氏菌IgY的效价为1∶320,并以此IgY为基础建立间接ELISA检测志贺氏菌,测定志贺氏菌纯培养液的检出限为105～106cfu/mL。对蜡样芽孢杆菌等10株不同菌株的检测结果表明,该方法对志贺氏菌有明显的检测特异性,对所测定的其它菌株无交叉反应。     

大肠埃希O157:H7等致病菌多重PCR法快速检测试剂盒的研制

目的:研制一种能够同时快速检测大肠埃希O157:H7、志贺氏菌和致病性蜡样芽孢杆菌的三重PCR试剂盒.方法:以大肠埃希O157:H7的hlyAB基因、痢疾志贺氏菌的IpaH基因和致病性蜡样芽孢杆菌的hblA基因作为目的基因片段O157:H7、痢疾志贺氏菌和致病性蜡样芽孢杆菌的特异性抗原基因序列,分别设计1对引物,并对其反应条件进行优化,建立1种多重PCR检测试剂盒.结果:本试剂盒可准确检测出生肉和即食肉制品中的上述3种致病菌,O157:H7检出极限为19.8 cfu/mL,志贺氏菌检出极限为17 cfu/mL,蜡样芽孢杆菌检出极限为17.7 cfu/mL.可在5 h内完成全部反应过程,得出检测结果.结论:本试剂盒在理论和实际应用方面均具有优越性,能够同时检测上述3种病原菌,可用于食品及其原料的生物安全检测,也可用于兽医临床诊断.     

乳饮料中志贺氏菌的快速PCR检测技术研究

建立乳饮料中快速检测志贺氏菌的有效方法。根据GeneBank公布的志贺氏菌ipaH基因的保守序列设计特异性引物,筛选合适的DNA模板制备方法,采用快速常规PCR和定量实时PCR结合,对培养液中及乳饮料阳性样品中的志贺氏菌进行检测,菌液敏感度可达到2cfu／mL,检出时间小于20h。新建的PCR方法具有特异性好,灵敏度高等特点,适用于快速、准确检测乳饮料中志贺氏菌的需要。     

4种分离培养基检测志贺氏菌效果比较

志贺氏菌是一类具有高度传染性的肠道致病菌。文中基于GB 4789.5—2012《志贺氏菌检验》标准,比较了4种分离培养基检测志贺氏菌的效果。通过4种分离培养基对福氏志贺氏菌、宋内氏志贺氏菌的回收率、最低检出限及对不同样品检测效果的比较,对不同培养基的检测效果进行评价。结果显示4种分离培养基最低检出限相近,显色培养基01检测特异性优于其他3种;针对实际检测样品,应灵活选择培养条件,以提高检测的准确性及效率。     

进出口动物源性食品中志贺氏菌检验方法优化

为建立进出口动物源性食品中志贺氏菌检验方法,从检测条件入手,通过增菌方式、分离培养基特异性和生化鉴定优化方法,以不同种类食品检测和不同实验室检测验证方法。确定的检测条件为志贺氏菌增菌液中(41.5±1)℃厌氧培养16～20h作为志贺氏菌检测的增菌方式;在分离上,同时使用MAC、XLD和HE三种平板,以提高志贺氏菌的检出率;选择了微量生化管和API20E对志贺氏菌和其他肠杆菌进行了生化鉴定和比较,效果明显。以此方法检测人工污染食品均检出志贺氏菌,各实验室也均检出志贺氏菌。     

牛奶样品中志贺氏菌胶体金免疫试纸条检测方法的建立

通过用超声波破碎的宋内氏志贺氏菌和福氏志贺氏菌的菌体碎片为免疫原免疫BALB/c小鼠,获得4株能稳定分泌抗志贺氏菌单克隆抗体的杂交瘤细胞株,选取其中一株制备单抗,并用胶体金标记。同时抗志贺氏菌的兔多抗和驴抗鼠抗体(二抗)分别喷涂于硝酸纤维素膜作为检测线(T线)和质控线(C线),研制了志贺氏菌胶体金快速检测试纸条。结果显示试纸条的灵敏度为105mL-1,并只与两株志贺氏菌有阳性反应外,而与其它23株常见的细菌无交叉反应。同时在检测添加有阪崎肠杆菌和长双歧杆菌的牛奶样品中,结果显示试纸条的灵敏度为106mL-1。志贺氏菌免疫胶体金试纸条的建立,为加强食品中志贺氏菌的检测工作,建立一种快速、简便的筛查方法具有重要的意义。     

进出口动物源性食品中志贺氏菌检验方法优化

为建立进出口动物源性食品中志贺氏菌检验方法,从检测条件入手,通过增菌方式、分离培养基特异性和生化鉴定优化方法,以不同种类食品检测和不同实验室检测验证方法。确定的检测条件为志贺氏菌增菌液中(41.5±1)℃厌氧培养16～20h作为志贺氏菌检测的增菌方式;在分离上,同时使用MAC、XLD和HE三种平板,以提高志贺氏菌的检出率;选择了微量生化管和API20E对志贺氏菌和其他肠杆菌进行了生化鉴定和比较,效果明显。以此方法检测人工污染食品均检出志贺氏菌,各实验室也均检出志贺氏菌。      

金黄色葡萄球菌噬菌体的分离筛选

以金黄色葡萄球菌为宿主菌,采用富集法从污水、粪便样品中分离筛选噬菌体,纯化后观察噬菌斑特征,电镜形态,并测定其效价、RTD（RoutineTestDilution,常规实验稀释度）值,初步分析该噬菌体的宿主谱。结果获得一株对金黄色葡萄球菌敏感的噬菌体,该噬菌体在宿主菌平板上能形成直径1～2mm、边界清晰、圆形透明的噬菌斑,效价是3.2×1010PFU/mL,RTD值为10-3,电镜观察显示此噬菌体由直径约为30nm的多面体头部和约50nm的尾部组成,可以裂解宿主菌及其它2株金黄色葡萄球菌,不能裂解大肠杆菌,鼠伤寒沙门氏菌及痢疾志贺氏菌。本文为深入研究金黄色葡萄球菌噬菌体的生物学特性及其功能提供了依据,为进一步利用噬菌体检测食源性金黄色葡萄球菌、防治由金黄色葡萄球菌引起的疾病提供了理论基础。      

Comparison of chromogenic Shigella spp. plating medium with standard media for the recovery of Shigella boydii and Shigella sonnei from tomato surfaces

Isolation of Shigella spp. from food is difficult because of a lack of appropriate selective media and the presence of low numbers of shigellae relative to competitive microorganisms. Chromogenic Shigella spp. plating medium (CSPM) was evaluated for use with the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) enrichment procedure for isolation of artificially contaminated Shigella boydii UI02 and Shigella sonnei UI05 from tomato surfaces. Tomatoes were inoculated with various concentrations of S. boydii UI02 or S. sonnei UI05 and rinsed using a shake-rub-shake procedure. Tomato rinses were enriched overnight according to the BAM procedure and streaked for isolation on CSPM, Salmonella-Shigella agar (SSA), and MacConkey agar (MAC). To access the isolation of S. boydii UI02 and S. sonnei UI05 without competition from natural tomato microflora, experiments were repeated using rifampin-adapted inocula and enrichments supplemented with 50 microg/ml rifampin. Isolation of S. boydii UI02 and S. sonnei UI05 with or without natural tomato microflora was not significantly different (P > 0.05) on CSPM, MAC, or SSA. Colony color enhancements created by CSPM may ease differentiation of Shigella colonies from those of closely related competitors.     

Antimicrobial effect of spices and herbs on Shigella sonnei and Shigella flexneri

Of 17 spices and herbs tested at 1% (wt/vol) in Mueller-Hinton (MH) agar, only cloves, thyme, oregano, allspice, basil, rosemary, and marjoram showed antimicrobial effects on Shigella. The MICs of thyme, oregano, basil, and rosemary (as determined by the agar dilution method) ranged from 0.5 to 1% (wt/vol) depending on the Shigella strain used. With the use of various combinations of temperatures (12, 22, and 37 degrees C), pHs (5.0, 5.5, and 6.0), and NaCl concentrations (1, 2, 3, and 4%, wt/vol) and the inclusion or exclusion of thyme or basil at 1% (wt/vol) in an MH agar model system, it was established that basil or thyme can contribute to combination processing as a growth-inhibitory factor for Shigella spp. In the presence of basil and thyme, Shigella flexneri did not develop CFU during the 7-day incubation period for, respectively, 14 and 16 of the 18 tested combinations, while growth was noted in the corresponding temperature-pH-NaCl concentration combinations without basil or thyme. A growth-inhibitory effect on Shigella sonnei was also noted. The results of an orientation study involving the addition of basil and thyme to spaghetti sauce prior to autoclaving and S. sonnei inoculation indicated that basil and thyme contributed to the reduction of S. sonnei after 16 days at 12 degrees C but not at 4 degrees C.     

Application of bacteriophages during depuration reduces the load of Salmonella Typhimurium in cockles

As bivalve molluscs are filter feeder, often consumed raw or lightly cooked and are frequently cultivated in contaminated waters, they are implicated in food-borne disease transmission to human. The present study investigated the potential application of bacteriophage (or phage) phSE-2, phage phSE-5 and phage cocktail phSE-2/phSE-5 to decrease the concentration of Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) during the depuration of natural and artificially contaminated cockles (Cerastoderma edule). Cockles were artificially infected with 10⁵ and 10⁶ colony-forming units (CFU)/mL of S. Typhimurium in static seawater and infected group were treated with phages at four different MOI values: 0.1, 1, 10 and 100. Depuration in static seawater at multiplicity of infection (MOI) of 0.1 with single phage suspensions of phSE-2 and phSE-5 provided the best results, as it decreased by ~ 1.3 and 1.7 log CFU/g, respectively, the concentration of Salmonella spp. after a 4 h treatment. At a MOI of 0.1, the rate of inactivation with single phage suspensions was higher when compared with the results obtained using the phage cocktail. However, in naturally contaminated cockles treated in static seawater with single phage suspensions and phage cocktail phSE-2/phSE-5, similar decreases in cultivable bacteria concentration (~ 0.7–0.9 log CFU/g) were achieved after 6 h of treatment. When artificially contaminated cockles were depurated with phage phSE-5 in a recirculated seawater system (mimicking industrial depuration conditions), a 0.9 and 2.0 log CFU/g reduction of Salmonella spp. was reached after 4 and 6 h treatment. Once the depuration process was performed without phage, a 6 h treatment was needed to obtain a 1.1 log CFU/g reduction of Salmonella spp. Results indicated that combining phage biocontrol with depuration procedures enhance bivalve microbial safety for human consumption by improving decontamination efficiency, proving that this technology can be transposed to the bivalves industry. Moreover, this approach also displays the advantage of reducing the time required for depuration and consequently its associated costs.     

Molecular strategies for the detection, identification, and differentiation between enteroinvasive Escherichia coli and Shigella spp

A strategy for the detection, identification, and differentiation of enteroinvasive Escherichia coli (EIEC) and Shigella spp. has been developed. The strategy includes (i) a multiplex PCR for the amplification of two virulence genes, i.e., iuc (222 bp) and ipaH (629 bp); (ii) amplification of the ial gene (a 1,038-bp amplicon) located within a large plasmid; and (iii) restriction fragment length polymorphism (RFLP) of the ial gene amplicon. The multiplex PCR provided three patterns. Pattern 1 (iuc-/ ipaH+) was found in 10 (67%) of 15 EIEC strains tested, pattern 2 (iuc+/ipaH-) in only 2 (4.4%) of 46 non-EIEC isolates, whereas pattern 3 (iuc+/ipaH+) was observed in all Shigella spp. and also in 5 (33%) of 15 EIEC strains tested. The pattern 3 EIEC strains were all positive for the ial gene. The PCR-RFLP of the ial gene amplicon using the endonuclease AclI was used to differentiate Shigella spp. from the EIEC strains that belonged to pattern 3. The ial gene was present in 21 (38%) of 56 and 6 (40%) of 15 Shigella spp. and EIEC strains tested, respectively. The PCR-RFLP of the ial gene amplicon divided the strains in two types. Type 1 did not contain the restriction enzyme site and was found in 6 (100%) of 6 EIEC strains, 4 (80%) of 5 Shigella boydii, and 4 (100%) of 4 Shigella dysenteriae strains tested. Type 2, which gave two fragments of 286 and 752 bp, was observed in 5 (83%) of 6 Shigella fiexneri strains and 6 (100%) of 6 Shigella sonnei strains. Detection, identification, and differentiation of Shigella spp. and EIEC were achieved by analyses of the PCR patterns and RFLP types. To our knowledge, this is the first study to demonstrate a simple and rapid method for detecting, identifying, and differentiating, at the molecular level, Shigella spp. and EIEC strains. This method will have tremendous utility as an epidemiological tool and in helping to develop policies, risk assessments, and national and international methods for Shigella spp.     

Impact of acid on survival of Vibrio vulnificus and Vibrio vulnificus phage

Three strains of Vibrio vulnificus and V. vulnificus phages were tested for acid sensitivity at 21 degrees C. V. vulnificus strain 304 was more resistant to pH 4.0 than strains CVD-1 and A-9, whereas acid sensitivities of V. vulnificus strains at pH 3.0 and 2.0 were similar. V. vulnificus phage strain 110A-7 was more resistant to pH 4.0 than strain 153A-7, whereas acid sensitivities of phage strains at pH 3.5 and 3.0 were similar. Numbers of V. vulnificus and its phage were close to the limit of detection after 100 s at pH 2.0 and after 24 min at pH 3.0. Acid D-values at 21 degrees C decreased as pH decreased for both V. vulnificus and phages. D-values of phage strains at pH 3.5 were 10-fold greater than those of host strain at pH 4.0. D-values of phage strains were slightly greater than those of host strain at pH 3.0. These results suggest that V. vulnificus and its phage were very sensitive to pH of less than 3.0, although V. vulnificus phages were more resistant to acid than their host.     

Evaluation of a cocktail of three bacteriophages for the biocontrol of Salmonella of wastewater

Salmonella serovars are increasing in importance as significant pathogens of both human and animals. Although water and wastewater are treated to eliminate pathogenic microorganisms, they still play an important role in the transmission of Salmonella spp. In this study, bacteriophages infecting Salmonella spp. were isolated from wastewater and evaluated; for their potential to lyse environmental Salmonella strains in vitro at different MOIs and temperatures; and to control the wastewater bacterial community. Three distinct phages designated sww65, sww275, and sww297; as defined by plaque morphology, electron microscopy and host range; were obtained from wastewater. Challenge tests were performed at 37, and 30 °C with the infection of the Salmonella cultures with individual phage, a mixture of two phages, and cocktail of three phages at MOIs of 10⁰, 10², and 10⁴ PFU/CFU. At 30, and 37 °C, a cocktail of three phages reduced all of the Salmonella cultures tested. These results required a high multiplicity of infection. However, when infected with only one phage or a mixture of two phages at MOIs of 10⁰ or 10 ² PFU/CFU, an emergence of bacterial resistance was observed. The dynamic monitoring of wastewater enterobacterial community was conducted using Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR). The number of bands decreased gradually with the use of individual phage or phage cocktails. Moreover, the dynamic monitoring of Salmonella community during wastewater treatment was performed using PCR detection of virulence gene invA. The results correlated with the ERIC-PCR fingerprints, and suggested that Salmonella community was affected by the phage treatment. Indeed, in wastewater, bacteriophages are reducing Salmonella and other members of the Enterobacteriaceae. These results indicated that dynamic changes are closely related with the process of treatment. The introduction of wide host range bacteriophages in wastewater can have a potential impact on the dynamics of the microbial communities, manifested by the reduction or the elimination of microbial species.     

Survival of Shigella sonnei on smooth tomato surfaces, in potato salad and in raw ground beef

This study investigated the survival of a five-strain Shigella sonnei cocktail on smooth tomato surfaces, in potato salad and in raw ground beef. All inocula were resistant to the antibiotic rifampicin to allow simple detection of the target culture among the indigenous microflora of the food samples. Inoculated tomatoes were stored at 13 degrees C/85% relative humidity, the standard holding conditions for mature, green tomatoes. Inoculated potato salad and ground beef samples were stored at 2.5 degrees C and 8.0 degrees C to study the effects of varied refrigerated temperatures. Surviving populations were estimated using a three-tube most probable number (MPN) method. Tryptic soy broth tubes supplemented with 100 ppm rifampicin were inoculated with appropriate dilutions of the recovered inocula and scored for growth after overnight enrichment. S. sonnei populations declined rapidly to undetectable levels (2 days) when dried on smooth surfaces of tomatoes. S. sonnei populations did not decrease in potato salad and ground beef stored at 2.5 degrees C and 8.0 degrees C over the shelf-life of the products.