蛭弧菌的生长特性及其培养条件的优化

为了将从海洋环境中分离得到的4株蛭弧茵发酵制备为微生态制剂,应用于水产食品安全生产,本实验研究了不同条件对蛭弧茵生长的影响.结果表明,4株蛭弧茵的最适pH为7.1～7.4;最适NaCl浓度为2.5%～3%;最适温度为30℃;二价金属离子Ca2+浓度的提高可有效增强蛭弧茵的生长及裂解能力;4株蛭弧菌对青霉素、土霉素、氯霉素和诺氟沙星均敏感;有机污染(苯酚和尿素)和重金属离子(Pb2+)污染对蛭弧菌的生长繁殖能力均有显著影响.并在单因素实验的基础上,运用响应面分析法确定了BDZ-100的最佳生长条件为:宿主菌浓度1xlO10 CFU/mL,,摇床速度250 r/min,培养液pH7,温度31℃,Nacl浓度3.1%,Ca2+浓度8.5mmol/L.从而为蛭弧菌微生态制剂的发酵制备和应用提供理论基础.     

李斯特菌噬菌体的分离鉴定及其裂解特性

以单核细胞增生性李斯特菌(Lm)为宿主菌,从畜禽养殖区污水中分离(Lm)特异的噬菌体,分析其生物学特性、裂解特性及其在食品中的灭菌效果。用双层平板法从污水样中分离Lm噬菌体,PEG/NaCl法沉淀纯化噬菌体,负染色后电镜观察。分析噬菌体对宿主菌的裂解特性及其对温度和pH值的敏感性,同时对该噬菌体进行宿主谱系分析。将噬菌体运用于即食性食品中,分析其潜在的灭菌效果。结果表明:分离的噬菌体为裂解性噬菌体,属长尾噬菌体科,命名为LipG2-5。体外能够高效裂解宿主菌,对温度及pH值亦有良好的耐受性。基因组酶切分析表明,LipG2-5为双链DNA噬菌体。噬菌体谱系分析表明,该噬菌体为1株宽宿谱噬菌体,在固态及液态即食性食品中能够高效杀灭Lm。     

蛭弧菌的分离鉴定及其对非O1霍乱弧菌的消除作用研究

从海洋环境中分离到2株蛭弧菌(A-1和A-2),研究了其对6株不同来源非O1霍乱弧菌的消除作用,以探索其对由非O1霍乱弧菌所引起的腹泻疾病的预防效果。实验结果表明,在20～35℃温度条件下,蛭弧菌均有较好的裂解效果,30℃时裂解能力最强;在pH6.5～8.1条件下,蛭弧菌均有较好的裂解能力,pH为7.2时,达到最佳裂解效果;A-1能裂解其中5株非O1霍乱弧菌,A-2能裂解其中4株非O1霍乱弧菌,而2株蛭弧菌协同作用,能全部裂解6株非O1霍乱弧菌。本研究结果揭示了蛭弧菌作为生物消除剂在预防由非O1霍乱弧菌所引起的腹泻疾病方面具有潜在应用价值。     

一株沙门氏菌噬菌体SalmpYZU27的生理学特性及其在牛奶和鸡肉中的抑菌作用研究

目的 分离鉴定沙门氏菌裂解性噬菌体,并研究其生理学特性及抑菌能力。方法 以沙门氏菌SalmonellaagonaCICC21586为宿主菌,采用点斑法从农贸市场污水中分离沙门氏菌噬菌体,研究其生理学特性,并测定其在牛奶和鸡肉中的抑菌效果。结果 分离到一株沙门氏菌裂解性噬菌体Salmp YZU27,其具有宽裂解谱,可裂解鼠伤寒沙门氏菌CICC 21483、丙型副伤寒沙门氏菌CICC 21512和肠沙门氏菌2011K-1440染色体血清型N16等12株不同血清型沙门氏菌;最佳感染复数为0.001; pH在4～11时保持高活性;潜伏期为20 min,爆发期为100 min,爆发量为89 PFU/cell;在40～70℃可保持较高活性;噬菌体全基因组测序未检出致病因子和抗性基因;不同感染复数下在LB肉汤中均可抑制宿主菌至少8h,且在牛奶和鸡肉片中对宿主菌均有一定的抑制效果。结论 本研究分离鉴定的沙门氏菌噬菌体SalmpYZU27裂解谱广,酸碱耐受性较好,耐热能力强,爆发量适中,为后续噬菌体抑菌剂的研制提供理论基础。     

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(RoutineTest Dilution,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.     

沙门氏菌裂解性噬菌体的分离鉴定及其生物学特性

从家禽屠宰场污水中,以肠炎沙门氏菌ATCC13076和禽沙门氏菌CVCC2184为宿主菌,分离到两株裂解性沙门氏菌噬菌体,分别命名为vB_SenM-PA13076（简称PA13076）和vB_SenM-PC2184（简称PC2184）。同时对这两株噬菌体进行了噬菌斑、噬菌谱系（含耐药菌）、形态学（透射电镜）、遗传物质、酸碱及热稳定性、最佳感染复数、一步生长曲线等生物学特性研究。结果表明：两株噬菌体的裂解空斑均透亮清晰;除对本身宿主菌产生强裂解作用,还裂解其他沙门氏菌,为宽噬菌谱,其中PA13076能强裂解一株耐氨苄青霉素ATCC13076转化菌株（A+13076）,PC2184则能裂解另一株耐氨苄青霉素CVCC2184转化菌株（A+2184）;电镜观察这两株噬菌体均为肌尾病毒科;遗传物质为dsDNA;pH值和温度耐受能力较好;PA13076、PC2184最佳感染复数分别为0.01、10,潜伏期分别为20、30 min,爆发期分别为70、60 min,平均爆发量分别为21、23。     

植物乳杆菌噬菌体Lpla的分离鉴定及抗噬菌体菌株的筛选

从泡菜中分离得到1 株以植物乳杆菌WCFS1为宿主菌的烈性噬菌体,命名为Lpla。一步生长曲线结果表明Lpla的潜伏期为15 min,裂解期为180 min,裂解量为43 PFU/cell。根据透射电镜观察的形态将其归为长尾病毒科,B1类。噬菌体的理化性质结果表明,Lpla对酸、碱的耐受性较强,对乙醇、温度和紫外射线的耐受性较低。吸附实验结果显示,Lpla在4 ℃时吸附率最大。Ca2＋、Mg2＋均可促进Lpla对宿主菌的吸附,热灭活宿主菌可使Lpla的吸附率下降20%。本研究筛选出6 株对噬菌体Lpla具有稳定抗性的植物乳杆菌,为制定出有效的噬菌体防治策略提供理论依据,并为进一步将抗性菌株应用于生产实践奠定基础。     

原料乳中假单胞菌噬菌体的分离及部分生物学特性分析

目的是分离原料乳中假单胞菌的噬菌体,以期利用噬菌体控制原料乳中这一主要嗜冷菌.以从原料乳中分离得到的10株假单胞菌(Pseudomonas spp.)为宿主菌,采用双层平板法从当地食品厂和医院的污水中分离并纯化假单胞菌噬菌体,通过透射电镜观察噬菌体的结构,最后研究了温度、pH、盐浓度等因素对噬菌体活性的影响.研究获得了3株噬菌体(PP6、PP8和PP10),发现均为裂菌性噬菌体,初步推断3株噬菌体均为轻小(光滑)噬菌体.噬菌体PP8和PP10在4℃下能保持较高活性,而噬菌体PP6则在37℃下活性较高;3株噬菌体在pH为5～7以及NaCl为1.5％～5.5％的范围内可保持较高活力.本实验研究结果为进一步利用噬菌体控制原料乳中的假单胞菌提供重要的理论基础.     

食源性大肠杆菌噬菌体的分离及其特性分析

目的从污水样品中分离致病性大肠杆菌的烈性噬菌体,分析其生物学特性,为食品中致病性大肠杆菌的控制提供参考。方法以标准菌株Escherichia coli EPEC(CICC10664)为宿主菌,采用双层平板法,从扬州市各地污水中分离纯化噬菌体,通过透射电镜观察其形态、测定最佳感染复数、一步生长曲线、裂解镨及其对宿主菌生物被膜的影响。结果分离到一株肠致病性大肠杆菌烈性噬菌体,命名为Ec.P01,电镜观察其为肌尾噬菌体,直径约为80 nm,最佳感染复数为0.01,一步生长曲线显示其噬菌体Ec.P 01的平均裂解量为48 PFU/m L,潜伏期约为10 min,裂解期约为90 min,热稳定性较好,对宿主菌生物被膜的形成有明显的抑制作用。结论本研究分离到一株肠致病性大肠杆菌烈性噬菌体,为治疗大肠杆菌感染疾病和治理食品及其环境污染提供新的思路与方法。     

一株广域pH耐受性微小噬菌体αα的分离、纯化及生理学特性分析

本研究采用双层平板法从污水中分离纯化出一株烈性沙门氏菌噬菌体αα。利用电镜观察其形态特征,通过琼脂糖凝胶电泳分析其核酸组成,并对其生理学特性进行分析,包括一步生长曲线、温度耐受性、pH耐受性、裂解谱、最佳离子浓度和最佳感染复数。结果表明,该烈性噬菌体αα有一个直径为20 nm的头部,无尾部结构,其核酸类型为单链DNA。由此推断,噬菌体αα属于微小噬菌体科。该噬菌体无明显潜伏期,裂解期为60 min,在pH2.10~11.45内和温度40~50 ℃内效价分别保持在106~108和108 pfu/mL左右,最佳感染复数值为10-4,钙镁离子均能促进该噬菌体的液体增殖,其中培养基中含10 mmol/L MgCl₂时促进效果最佳,与未添加离子的对照组相比提高了1.5个log。研究结果为噬菌体的多样性及其应用等方面的研究奠定了基础。     

产超广谱β-内酰胺酶霍乱弧菌耐药表型和基因型初步研究

目的S 对北京市朝阳区产超广谱β-内酰胺酶(ESBLs)霍乱弧菌耐药表型和基因型进行初步的研究。方法S 复苏2012-2019年北京市朝阳区保存的37株霍乱弧菌,应用肉汤微量稀释法进行产ESBLs菌株的鉴定及21种药物敏感实验,应用荧光 PCR 检测菌株的10种耐药基因：SHV、TEM-1/TEM-2、TCX-M1、TCX-M2、TCX-M8、TCX-M9、TCX-M25、OXA1,OXA2和OXA10。结果 试验菌株中,产ESBLs菌株检出率8.11%(3/37),这3株菌10种耐药基因均为阴性;ESBLs耐药基因携带率为16.22%(6/37),这6株菌均为非产ESBLs霍乱弧菌,其中TEM1/ TEM2携带率为16.22%(6/37),OXA2携带率为16.81%(4/37)。37株霍乱弧菌对21种抗生素耐药率为2.70%~94.59%,对多西环素不耐药;多重耐药率为35.13%(13/37),最多耐11种药。结论 霍乱弧菌中产ESBLs菌株与ESBLs耐药基因菌株一致性差;北京地区霍乱弧菌耐药严重且多重耐药趋势明显;应加强霍乱弧菌耐药机制、耐药表型与基因型关系的研究。     

北京进出口水产品中259株霍乱弧菌分离株的耐药性研究

目的对北京进出口水产品中259株霍乱弧菌分离株的耐药性进行研究。方法采用纸片扩散法对259株霍乱弧菌分离株进行药敏性试验,共选择10大类21种抗生素。结果所测试的259株菌株对庆大霉素、阿米卡星、诺氟沙星、妥布霉素、强力霉素及亚胺培南的敏感率高,均在95%以上;对除亚胺培南外20种抗生素都具有不同程度的耐药性,其中对链霉素和多粘菌素B耐药率较高,分别为36.3%和54.1%。所有菌株对21种抗生素均有不同程度的中介反应,其中链霉素、卡那霉素、多粘菌素B及红霉素中介率较高,分别为50.2%、44.0%、43.2%和83.4%;其中93株表现为多重耐药性。结论北京地区水产品中霍乱弧菌分离株对所测试的抗生素存在大量的中介和耐药情况,应加强对霍乱弧菌耐药性的监测力度。     

蛭弧菌消除海产品中潜在致病性弧菌的研究

噬菌蛭弧菌具有裂解病原菌的特殊功效,可以用作生物净化因子。本实验从海洋环境中分离到4株Bh04系列蛭弧菌(Bh04-4,Bh04-41a,Bh04-A+和Bh04-1f)。以41株弧菌为蛭弧菌的宿主菌,本文研究了蛭弧菌对海产品中常见弧菌的裂解消除能力,以探索其对由弧菌所引起的食物中毒等流行性传染疾病的预防效果。实验结果表明,41株弧菌中共有36菌株可被4株蛭弧菌(Bh04-4,Bh04-41a,Bh04-A+和Bh04-1f)中的任一株所裂解,占总试验菌株的87.8%;对副溶血弧菌、非01非0139群霍乱弧菌、溶藻胶弧菌这三种常见致病弧菌的裂解率分别达88.9%、83.3%、81.8%。本研究结果揭示了蛭弧菌作为生物消除剂在由弧菌所引起的食物中毒等流行性传染疾病的防治方面有潜在应用价值。     

Design and validation of a novel multiplex real-time PCR assay for Vibrio pathogen detection

Three species--Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus--account for the majority of vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species. Additionally, biochemical identification and confirmation require 2 or more days to complete. Rapid and sensitive molecular techniques for the detection of vibrio pathogens would be useful for the surveillance and management of outbreaks. To facilitate the identification of human-pathogenic species, we designed and validated a highly sensitive, specific, and robust multiplex real-time PCR assay to identify V. cholerae, V. parahaemolyticus, and V. vulnificus using a four-dye configuration in a convenient lyophilized format. Multiple Vibrio strains were sequenced to verify candidate target TaqMan sites. Several individual assays within the multiplex contain multiple primers or probes to ensure detection of polymorphic variants. V. cholerae, V. parahaemolyticus, and V. vulnificus were detected either individually or in mixtures at ≤30 genomic copies. V. cholerae was specifically detected in the presence or absence of Vibrio mimicus. The Vibrio multiplex assay showed 100% specificity to all targets analyzed and no detection of nearest neighbor strains. Each assay exhibited 100% ± 10% efficiency. Multiplex real-time PCR can simplify pathogen detection and reduce costs per test since three species can be analyzed in a single reaction tube. Rapid and accurate detection of pathogenic vibrios in shellfish or seawater samples will improve the microbiological safety of seafood for consumers.     

2016年江苏省淡水养殖环节常见致病性弧菌污染状况调查

目的 了解江苏省淡水动物性水产品养殖环节中常见弧菌(副溶血性弧菌、霍乱弧菌等)的污染程度。 方法 参照《2016年国家食品安全风险监测淡水动物性水产品养殖环节中常见弧菌专项监测工作手册》进行样品的采集和检测。 结果 从淡水养殖场中采集水产品、水体、水底沉积物样品共507份, 检出副溶血性弧菌17株(3.4%); 霍乱弧菌4株(0.8%); 溶藻弧菌及创伤弧菌均未检出。检出的副溶血性弧菌均不携带毒力基因(trh-;tdh-), 主要血清型为O5:H7(41.2%), 主要耐受的抗生素为头孢唑林(94.1%), 其中3株PFGE带型相似性系数100%; 4株霍乱弧菌均为非O1/O139群, 未发现耐药株。结论 江苏省淡水养殖环节中副溶血性弧菌环境污染程度低, 但在淡水养殖环节中检出致病性弧菌, 提示淡食用水产品依然可能存在引发食物中毒的风险。     

两种致病性弧菌二重LAMP-熔解曲线检测方法的建立

应用环介导等温扩增技术(Loop-mediated isothermal amplification,LAMP),建立了食品中产毒素性霍乱弧菌和副溶血性弧菌二重LAMP-熔解曲线快速检测方法。本方法针对产毒素性霍乱弧菌ctx A基因和副溶血性弧菌gyr B基因分别设计引物组进行二重LAMP扩增,并利用熔解曲线法分析扩增产物,从而判断DNA模板中所含目标菌。应用本方法对9株目标菌和17株非目标菌的检测结果与预期一致,并可通过熔解曲线的特征峰准确分析DNA模板中所含目标菌。对二重LAMP扩增产物的测序分析表明,扩增所得序列与目的基因序列吻合,从而进一步验证了该方法的特异性。经测试,本方法对两种目标菌的检测灵敏度均可达100 fg DNA/反应管。实验证明所建立的方法具有良好的特异性,并可为食品中两种致病性弧菌的快速检测提供一种重要技术手段。     

Risk of Vibrio transmission linked to the consumption of crustaceans in coastal towns of Côte d'Ivoire

The purpose of this study was to assess the risk of Vibrio spp. transmission from crustaceans to humans in two coastal towns of C?te d'Ivoire. Bacteriologic analysis was performed on 322 crustacean samples obtained from six markets in Abidjan and one in Dabou. Suspected Vibrio colonies were identified by morphological, cultural, biochemical, and molecular tests and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. PCR assays were used to further characterize Vibrio strains. A survey on consumption of crustaceans was conducted among 120 randomly selected households in Abidjan. Overall, Vibrio spp. were isolated from 7.8% of the crustacean samples studied, at levels as high as 6.3 log CFU/g. Of the Vibrio strains identified, 40% were V. alginolyticus, 36% were V. parahaemolyticus, and 24% were nontoxigenic V. cholerae; the latter two species can cause mild to severe forms of seafood-associated gastroenteritis. Among interviewed households, 11.7% reported daily consumption of crustaceans, confirming the high probability of exposure of human population to Vibrio spp., and 7.5% reported symptoms of food poisoning after consumption of crustaceans. The absence of genes encoding major virulence factors in the studied strains, i.e., cholera toxin (ctxA and ctxB) in V. cholerae and thermostable direct hemolysin (tdh) and thermostable direct hemolysin-related hemolysin (trh) in V. parahaemolyticus, does not exclude the possibility of exposure to pathogenic strains. However, human infections are not common because most households (96.7%) boil crustaceans, usually for at least 45 min (85.9% of households) before consumption.     

Sensitivity of Vibrio species in phosphate-buffered saline and in oysters to high-pressure processing

Multiple strains of Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio cholerae non-O1 were tested in phosphate-buffered saline for their sensitivity to high-pressure processing (HPP). Variability in sensitivity among strains was observed for all species; this variability decreased at higher pressures. V. vulnificus was the species that was most sensitive to treatment at 200 MPa (decimal reduction time [D] = 26 s), and V. cholerae was the species that was most resistant to treatment at 200 MPa (D = 149 s). The O3:K6 serotype of V. parahaemolyticus was more resistant to pressure than other serotypes of V. parahaemolyticus were. The results of studies involving V. vulnificus naturally occurring in oysters revealed that a pressure treatment of 250 MPa for 120 s achieved a > 5-log reduction in the levels of this bacterium. V. parahaemolyticus serotype O3:K6 in oysters required a pressure of 300 MPa for 180 s for a comparable 5-log reduction. When properly applied, HPP can be effective in improving the safety of shellfish with respect to Vibrio spp.     

非产毒霍乱弧菌基因组流行病学研究进展

非产毒霍乱弧菌指缺乏主要毒力基因ctxAB而不能产生霍乱毒素的霍乱弧菌。部分非产毒霍乱弧菌可以定植人类肠道,引起腹泻等疾病,但由于临床症状相对较轻而容易被忽视。基因组流行病学基于群体基因组学研究方法,结合传统流行病学调查结果,可以对食品和临床来源的非产毒霍乱弧菌进行溯源及传播分析。该学科的发展极大促进了对非产毒霍乱弧菌遗传多样性、致病机制及传播规律的认识。研究表明部分非产毒霍乱弧菌谱系已在全球多个国家流行,对食品质量安全形成风险。本文从基因组特征、遗传分型、重要遗传因子及国内外传播4个方面,归纳总结了非产毒霍乱弧菌基因组流行病学相关研究进展,以期为非产毒霍乱弧菌的检测、监测及其相关食源性疾病防控工作提供参考。     

Occurrence of Vibrio spp. in fish and shellfish collected from the Swiss market

The genus Vibrio includes gram-negative bacteria that inhabit estuarine ecosystems. V. cholerae, V. parahaemolyticus, and V. vulnificus pose a considerable public health threat as agents of sporadic and epidemic foodborne infections associated with the consumption of raw or undercooked contaminated fish or shellfish. In this study, we analyzed 138 fish and shellfish samples collected from the Swiss market (fish fillets [n = 102], bivalves [n = 34], and squid [n = 2]). Microbiological analysis was done according to International Organization for Standardization method 21872-1/21872-2:2007, using thiosulfate citrate bile sucrose agar and chromID Vibrio agar as selective agar. Presumptive-positive colonies on thiosulfate citrate bile sucrose agar or chromID Vibrio agar were picked and were identified by the API 20E and species-specific PCR systems. V. cholerae isolates were tested further by PCR for the presence of the cholera toxin A subunit gene (ctxA). V. parahaemolyticus isolates were tested by PCR for genes encoding for thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). V. cholerae was isolated from three samples and V. parahaemolyticus from eight samples. None of these strains harbored species-specific virulence factors. Further, V. alginolyticus was isolated from 40 samples, and V. fluvialis was isolated from 1 sample. Our study provides, for the first time, data for the assessment of exposure to Vibrio spp. in raw fish and bivalves consumed in Switzerland.