一株副溶血弧菌噬菌体生物学特性、全基因组特征及其在食品中的应用

目的：分析1株副溶血性弧菌噬菌体474x1的生物学特性、全基因组及在食品中的抑菌效果。方法：以副溶血性弧菌474菌株为宿主菌，从海鲜市场基围虾分离噬菌体474x1，利用透射电镜观察其形态，并绘制一步生长曲线，分析474x1对温度及pH的敏感性。分析474x1全基因组序列，根据474x1末端酶大亚基构建系统进化树。通过测定菌落总数评价噬菌体对虾肉中副溶血弧菌的抑制效果。结果：分离到1株新型的副溶血弧菌噬菌体，命名为474x1，该噬菌体能够裂解23株副溶血弧菌中的19株（19/23=82.61%）。电镜观察474x1具有典型的短尾病毒科病毒形态特征。最佳感染复数（MOI）为0.01，一步生长曲线显示474x1的潜伏期为10 min，裂解量为115 PFU/cell。该噬菌体能够在较大的温度范围（30～60℃）和pH(4～11)范围内维持活性。474x1全基因组长47830 bp，包含69个开放阅读框（open reading frames,ORFs），其中14个具有特定功能的基因。比较基因组学分析474x1与短尾噬菌体科弧菌属噬菌体Vp41s3基因组具有较高同源性，进化分析表明474x1与...     

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。     

1 株金黄色葡萄球菌烈性噬菌体的生物学特性及其裂解效果

以耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus，MRSA）为宿主菌从奶牛养殖场污水中分离到1 株烈性噬菌体P65。透射电镜表明，噬菌体P65头部呈多面体结构，属于有尾噬菌体目长尾噬菌体科。噬菌体P65最佳感染复数为0.01，一步生长曲线显示，噬菌体P65潜伏期为10 min，裂解周期为90 min，裂解量为35.46 PFU/cell。在pH 4～10和温度30～45 ℃条件下具有稳定的裂解活性。紫外线照射70 min后，噬菌体P65效价由1.91×108 PFU/mL降至2.65×104 PFU/mL。经噬菌体处理48 h后，菌株MRSA 2和MRSA 24的生物被膜清除率分别为91.3%和92.2%。在25 ℃条件下，噬菌体P65在牛奶和牛肉细菌污染模型中有一定的抑菌效果。本研究表明噬菌体P65具有较宽的宿主谱和稳定的裂解杀菌能力，在清除生物被膜和食品抗菌方面有一定的运用潜能。     

J H耶氏菌噬菌体在小肠结肠炎耶尔森氏菌检测中的应用

小肠结肠炎耶氏菌噬菌体对耶氏菌的检测,在国内是一种新的尝试。J H 噬菌体具有较高的敏感性和特异性我们于1988～1989年利用 J H 噬菌体对耶氏菌进行了裂解性试验,发现 J H 噬菌体对人有致病性的菌株裂解性强,而对人无致病性或者致病性较弱的菌株裂解性弱,现将结果报告如下。1 材料与方法噬菌体:J H 系 JA HB 两种噬菌体的等量混合物,裂解效价为10~4～10~5RTK.由     

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。     

1 株裂解性短尾沙门氏菌噬菌体T139的生物学特性及其对牛奶和牛肉的抑菌作用

采用双层平板的方法以鼠伤寒沙门氏菌（ATCC 13311）为宿主菌,从污水中分离得到1 株裂解性噬菌体,命名为T139,研究其生物学特性及其在牛奶和牛肉样品中的抑菌作用。结果表明：噬菌体T139的噬菌斑透亮清晰;能裂解宿主菌及其他沙门氏菌,为宽宿主谱;电镜观察噬菌体T139属于短尾噬菌体科,头部直径为（43±1）nm,尾部长（11±0.6）nm;最佳感染复数（multiplicity of infection,MOI）为0.001;最佳吸附速率为66%;一步生长曲线结果显示潜伏期为5?min,爆发期为60?min,平均裂解量为54.54?PFU/cell;在30～50?℃和pH?4～12条件下稳定且对体外培养的鼠伤寒沙门氏菌有良好的裂解效果。噬菌体T139对牛奶中鼠伤寒沙门氏菌的抑菌效果为：在4?℃无显著效果,在25?℃条件下MOI=10和MOI=100时抑菌效果极其显著,宿主菌数量分别下降4.32（lg（CFU/mL））和4.27（lg（CFU/mL））;噬菌体T139对牛肉中鼠伤寒沙门氏菌的抑菌效果为：在4?℃条件下,MOI=10时无显著效果,在MOI=100时宿主菌数量下降0.66（lg（CFU/mL））,在25?℃条件下MOI=10和MOI=100时宿主菌数量分别下降了0.77（lg（CFU/mL））和1.16（lg（CFU/mL））,表明噬菌体T139对牛奶和牛肉中鼠伤寒沙门氏菌有良好抑制作用。     

陕西省典型冷冻食品中大肠杆菌的分子特征及耐药性检测

为了解冷冻食品中大肠杆菌污染情况,分析其潜在的食品安全问题,从陕西省4市（宝鸡、咸阳、西安和渭南）共收集冷冻食品样品360份（120份速冻水饺和240份冰淇淋）。通过选择培养和聚合酶链式反应（polymerase chain reaction,PCR）进行大肠杆菌分离鉴定。然后对分离株进行21种毒力基因、23种耐药基因、15种抗生素耐药性检测及种群分型。360份样品中大肠杆菌污染率为13.6%(49/360),其中5份样品（10.2%,5/49）大肠菌群计数超过100 CFU/g。21种被检毒力基因中有9种毒力基因被检出,以肠外致病性大肠杆菌相关基因FyuA和iss的检出率最高（均为14.3%,7/49）。药敏结果显示,菌株对β-内酰胺类抗生素和叶酸代谢抑制剂的耐药最为普遍（均为98.0%,48/49）,其次为四环素（20.4%,10/49）。其中β-内酰胺类主要编码基因为blaCTX,四环素主要编码基因为tetA。研究还发现1株分离株携带多黏菌素类抗性基因mcr-9。此外,20.4%的菌株为多重耐药菌株,最多可对13种抗生素耐药。所有分离株共有4种系统发育群（A、B1、C和F）,A群...     

1 株宽宿主谱的阪崎肠杆菌噬菌体生物特性分析及其在乳制品中的应用

以阪崎肠杆菌菌株ATCC25944为宿主菌从河涌水中分离1 株噬菌体TBC-1,具有较宽的阪崎肠杆菌宿主范围。电镜形态学及生物特性结果显示：此噬菌体衣壳为二十面体,具有可收缩性尾部;该噬菌体的最佳感染复数（multiplicity of infection,MOI）为0.001,一步生长曲线显示其潜伏期20 min,爆发期50 min;爆发量为100 PFU/cell;该噬菌体在温度区间40～50 ℃及pH 4～10之间可以保持其效价稳定;对阪崎肠杆菌有较好的裂解杀菌效果,并且MOI越高抑制效果越好。全基因组测序分析显示：该噬菌体的基因组为双链DNA,全基因组分子质量85 313 bp,平均GC含量40.65%,共有118 个蛋白编码区,24 个tRNA。应用结果显示,TBC-1对牛乳中2 种阪崎肠杆菌的抑制效果：室温（25 ℃）培育3 h ,在MOI为106时,能将102 CFU/mL菌抑制到检出限以下;TBC-1对乳粉中2 种阪崎肠杆菌的抑制效果：在25 ℃与37 ℃培育3 h,ATCCBAA894菌量均减少到检出限以下,在25 ℃培育3～12 h,ATCC25944菌量均在检出限以下。本研究表明噬菌体TBC-1具有宽宿主谱系特性、稳定的裂解杀菌以及在牛乳与乳粉中对不同的阪崎肠杆菌均有良好的抑制作用,有潜力成为阪崎肠杆菌生物抑制剂。     

鱼类水产品中耐冷性摩根菌Morganela psychrotolerans污染情况与分离株组胺生成能力

对市售水产品中耐冷性摩根菌Morganella psychrotolerans的污染情况进行调查,研究其分离株组胺生成。实验共调查了广东省市售100个鱼类样本,研究结果表明,M. psychrotolerans广泛分布于广东省市售中水产品中,在样品中的污染率达34%,在青皮红肉鱼中的污染率为35.9%,在白肉鱼中的污染率为27.3%,其中鲭鱼样本的污染率在总阳性样品中占比38.24%,金枪鱼样本的污染率占比29.41%。在呈现阳性的样品中共分离到16株M. psychrotolerans,对分离的16株M. psychrotolerans进行组胺生成能力研究发现,在20℃培养48 h后分离株组胺生成量均大于1 000 mg/L,其中分离株1的组胺生成量大于模式菌株。将分离株1与模式菌株在不同温度（4、20℃）条件下对比研究发现,分离株1与模式菌株的生长并未呈现明显差异,但组胺生成量呈现不同。分离株1在4℃培养10 d后,其组胺生成量显著高于模式菌株;在20℃培养60 h后,模式菌株组胺生成量显著高于离株1。本研究为冷链物流水产品中可能存在的组胺积累问题提供了理论数据支撑。     

产褐藻胶裂解酶菌株筛选与高产酶菌株的ARTP诱变选育

该研究采用平板酶解圈法从南海海域海藻、动物及沉积物样品中筛选产褐藻胶裂解酶细菌,对筛选菌株进行分子生物学鉴定,并利用常压室温等离子体（ARTP）诱变技术选育高产褐藻胶裂解酶菌株。结果表明,共分离筛选得到酶活较高的细菌14株,16S rDNA比对分析结果表明,它们隶属于3个门、5个纲、7个目、8个科、9个属,其中有5株菌（占35.7%）可能代表着新的分类单元。以高产褐藻胶裂解酶海藻类芽孢杆菌（Paenibacillus algicola）HB172198T作为原始菌株,利用ARTP诱变技术选育获得2株褐藻胶裂解酶活力明显提高的突变株（编号分别为30-19、80-6）,其酶活力分别比原始菌株提高了32.6%和21.6%,且连续6次传代培养诱变菌株的产酶遗传性状稳定。     

Phage inactivation of Staphylococcus aureus in fresh and hard-type cheeses

Bacteriophages are regarded as natural antibacterial agents in food since they are able to specifically infect and lyse food-borne pathogenic bacteria without disturbing the indigenous microbiota. Two Staphylococcus aureus obligately lytic bacteriophages (vB_SauS-phi-IPLA35 and vB_SauS-phi-SauS-IPLA88), previously isolated from the dairy environment, were evaluated for their potential as biocontrol agents against this pathogenic microorganism in both fresh and hard-type cheeses. Pasteurized milk was contaminated with S. aureus Sa9 (about 10(6)CFU/mL) and a cocktail of the two lytic phages (about 10(6)PFU/mL) was also added. For control purposes, cheeses were manufactured without addition of phages. In both types of cheeses, the presence of phages resulted in a notorious decrease of S. aureus viable counts during curdling. In test fresh cheeses, a reduction of 3.83log CFU/g of S. aureus occurred in 3h compared with control cheese, and viable counts were under the detection limits after 6h. The staphylococcal strain was undetected in both test and control cheeses at the end of the curdling process (24h) and, of note, no re-growth occurred during cold storage. In hard cheeses, the presence of phages resulted in a continuous reduction of staphylococcal counts. In curd, viable counts of S. aureus were reduced by 4.64log CFU/g compared with the control cheeses. At the end of ripening, 1.24log CFU/g of the staphylococcal strain was still detected in test cheeses whereas 6.73log CFU/g was present in control cheeses. Starter strains were not affected by the presence of phages in the cheese making processes and cheeses maintained their expected physico-chemical properties.     

Development of lytic Lactococcus lactis bacteriophages in a Cheddar cheese plant

The mixed TK5 starter culture was used in a Danish factory as the only starter for production of Cheddar cheese for more than 11 years before the factory experienced serious bacteriophage attacks with inhibition of the acid production in the curd. The cheese whey contained some phages from the beginning, and gradually new phages appeared able to infect an increasing number of isolates. Three bacteriophages jw30, jw31 and jw32 were isolated from the factory whey collected in 1989–94 and compared with lytic bacteriophages isolated in the period 1982–86 (Josephsen et al., 1994) DNA hybridisation showed that the type phage P008 had high homology to the new phages jw30, jw31 and jw32 as had the phages isolated in 1982–84. The new phages had broader host ranges and higher burst sizes than the previously isolated phages, showing that the lytic phages had become more virulent with time.     

Isolation and characterisation of lytic bacteriophages against Pseudomonas spp., a novel biological intervention for preventing spoilage of raw milk

Thirteen novel lytic bacteriophages against 13 Pseudomonas strains were isolated from local sewage and initially identified by morphology using a transmission electron microscope. PP1 and PP5 were identified as Pedoviridae and Cystoviridae, respectively; while the other 11 phages were identified as Leviviridae. Most phages showed high infectivity at either 4 °C or 25 °C, and the optimum pH range for phage infectivity was pH 5–7. A strong antimicrobial effect of the phage cocktail was evidenced by a 2-log reduction in Pseudomonas cell number of UHT milk inoculated with Pseudomonas, and a 1-log reduction in the psychrotrophic bacteria and total bacteria counts of raw milk at 4 °C over 5 d. A similar result was obtained at 25 °C over 8 h. Results indicated that the 13 phages with different morphological and physiological characteristics may have a potential application as biological preservative agent in raw milk.     

Use of bacteriophages as biocontrol agents to control Salmonella associated with seed sprouts

Two Salmonella bacteriophages (SSP5 and SSP6) were isolated and characterized based on their morphology and host range, and evaluated for their potential to control Salmonella Oranienburg in vitro and on experimentally contaminated alfalfa seeds. Phages SSP5 and SSP6 were classified as members of the Myoviridae and Siphoviridae families, respectively. Both phages had a broad host range of over 65% of the 41 Salmonella strains tested. During in vitro trials, the phages resulted in incomplete lysis of Salmonella cultures, in spite of high levels of phage remaining in the system. Phage SSP5 was more effective in reducing Salmonella populations. Addition of phage SSP6 to alfalfa seeds previously contaminated with S. Oranienburg caused an approximately 1 log(10) CFU g(-1) reduction of viable Salmonella, which was achieved 3 h after phage application. Thereafter the phage had no inhibitory effect on Salmonella population growth. A second addition of the same (SSP6) or different (SSP5) phage to a Salmonella culture treated with phage SSP6, did not affect Salmonella populations. It was further shown that development of Salmonella permanently resistant to phage was not evident in either seed or in vitro challenge trials, suggesting the existence of a temporary, acquired, non-specific phage resistance phenomenon. These factors may complicate the use of phages for biocontrol.     

冻藏黄花鱼微波解冻条件的研究

以冻藏黄花鱼为研究对象,考察了微波功率、挂冰厚度及鱼的层厚等条件对微波解冻过程的影响。结果表明:功率越大,解冻速度越快,同时解冻有效时间段(解冻有效时间段指-5～4℃)越短;挂冰可以延长解冻有效时间段,随着挂冰厚度的增加,解冻有效时间段越长;随着冻鱼层次的增多,鱼体解冻过程中达到有效时间点所用时间越长,有效解冻时间段也越长。对于5层厚度的冻鱼(10cm以上),2450MHz的微波炉达不到很好的解冻效果。     

The effects of environmental factors on the prevalence and diversity of bacteriophages lytic against the top six non-O157 Shiga toxin-producing Escherichia coli on an organic farm

Bacteriophages (or phages) specific to Shiga toxin-producing Escherichia coli (STEC) strains are frequently isolated from animal-associated environments primarily because ruminant animals are the natural reservoir of STEC. However, little is known about these phages in produce-growing environments. The objective of this study was to determine the effects of environmental factors on the prevalence of the phages lytic against O157 and the top six non-O157 STEC on an organic farm. A total of 370 samples were collected from an organic farm, containing animal-active and produce-growing areas, for 1 year. A bacterial cocktail, including nonpathogenic and pathogenic E. coli strains, was used for phage isolation. Meanwhile, culture methods and PCR were used to isolate STEC strains. Weather information was also collected for each sampling trip. Twenty-eight samples contained phages lytic against STEC (or STEC-infecting phages), of which 26 were collected from the animal-active area. Moreover, the winter season had a higher phage prevalence than other seasons, likely due to high rain precipitation. The phages belonging to the Myoviridae family and those lytic against STEC O103 were the most prevalent. One E. coli O103:H2 was isolated from a water sample where no STEC O103-infecting phages were found. Additionally, no STEC O103 was present in the samples containing STEC O103-infecting phages. The findings indicate that animal is the primary factor contributing to the prevalence of the STEC-infecting phages in the surrounding environment of the organic farm, and the presence of these phages contributes to a negative correlation with their STEC hosts.     

霍乱弧菌噬菌体在消除海产品中相关弧菌的潜在应用研究

噬菌体具有裂解病菌的作用,可用作生物净化因子.本实验以霍乱弧菌为宿主菌,采用双层平板法从鲍鱼养殖环境中分离到5株霍乱弧茵噬菌体.以23株弧菌为宿主菌,本文研究了霍乱弧菌噬菌体对海产品中常见弧茵的裂解消除能力,同时针对宽裂解谱噬菌体进行包括热失活、pH稳定性、紫外照射、不同镁离子浓度、不同柠檬酸钠浓度等理化因素对其裂解能力的影响,以探索其裂解消除弧茵的最优条件.实验结果表明噬菌体在消除霍乱弧菌方面有潜在的应用价值,紫外照射、柠檬酸钠对噬菌体裂解有不同程度的抑制作用,温度和pH值对噬茵体裂解也有不同程度的影响,而适度的镁离子则有促进作用.     

Bacteriophage-based enrichment coupled to immunochromatographic strip-based detection for the determination of Salmonella in meat and poultry

Immunochemical-based methods for the detection of Salmonella in food can be complicated by the presence of closely related, immunocrossreactive non-Salmonella species in the sample that may cause false-positive results. To circumvent this problem, specific bacteriophages against immunocrossreactive, non-Salmonella bacteria were used in the sample enrichment step to suppress their growth and improve the performance of an immunochromatographic strip-based detection method for Salmonella. Cross-reactive bacteria were isolated from various food sources and were characterized with a panel of Salmonella somatic O antigen-specific monoclonal antibodies. These cross-reactive bacteria were primarily Citrobacter spp. and Escherichia coli with serology shared with Salmonella serogroups B, D, and F. These bacteria were used as hosts for the isolation of specific lytic bacteriophages. When formulated with the primary enrichment, the bacteriophage cocktail significantly reduced false positives with a broadly reactive immunochromatographic test strip. This was demonstrated in both artificially and naturally contaminated meat. False positives in naturally contaminated beef samples were reduced from 32 of 115 samples tested to zero. In raw meat and poultry with a relatively high bioburden (>10(5) CFU/g), the use of the bacteriophage-based enrichment procedure gave improved recovery of Salmonella compared with the conventional culture-based reference method. This was observed when coupled to either test strip-based or selective agar-based detection. The use of specific bacteriophages for the control of immunocrossreactive and competitive microflora during the food sample enrichment step provides a new approach for enhancing the performance of both immunological- and cultural-based detection methods.     

Thermophilic lactic acid bacteria phages isolated from Argentinian dairy industries

Sixty-one natural phages (59 of Streptococcus thermophilus and 2 of Lactobacillus delbrueckii subsp. bulgaricus) were isolated from Argentinian dairy plants from November 1994 to July 2000. Specifically, 17 yogurt samples (18% of all samples) and 26 cheese samples (79%) contained phages lytic to S. thermophilus strains. The number of viral particles found in samples ranged from 10(2) to 10(9) PFU/ml. The phages belonged to Bradley's group B or the Siphoviridae family (morphotype B1). They showed high burst size values and remarkably short latent periods. The results of this study show that phages were found more frequently in cheesemaking processes than in yogurt-making processes. The commercial streptococcus strains appeared to propagate more phages, whereas the natural strains propagated fewer phage strains. These results suggest that the naturally occurring cultures are inherently more phage resistant.