2-酮基-D-葡萄糖酸产生菌荧光假单胞菌A46 抗噬菌体菌株的选育

从2-酮基-D-葡萄糖酸产生菌荧光假单胞菌(Pseudomonas fluorescens)A46的异常发酵液中分离到1种噬菌体，将其命名为KS461。透射电镜观察表明，KS461呈近球形，直径为52nm。以荧光假单胞菌A46为出发菌株，利用紫外诱变的方法，获得了4株对噬菌体KS461具有抗性的2-酮基-D-葡萄糖酸高产菌株，并研究了这些变异株的遗传稳定性。研究结果表明，经8次传代，这些变异株对噬菌体的抗性及其发酵产酸能力没有改变。     

噬菌体对荧光假单胞菌A46的感染及其防治的初步研究

利用透射电镜观察2- 酮基-D- 葡萄糖酸产生菌荧光假单胞菌A46 感染噬菌体KS461-2 后的菌体形态变化,测定该菌株耐噬菌体突变频率,考察不同pH 值对噬菌体增殖的影响,检测4 种常用药物对噬菌体的防治效果。结果表明,随着噬菌体感染时间的延长,菌体形态变化差异显著;该菌株耐噬菌体KS461-2 的突变频率在2.5 × 10-6左右;噬菌体在pH7.5～8.0 的条件下容易吸附于宿主而增殖;草酸铵能有效抑制该噬菌体的增殖,而对宿主菌的生长无明显影响,最佳添加浓度为0.7%。     

2-酮基-D-葡萄糖酸产生菌球状节杆菌K1022抗噬菌体菌株的选育

从 2 酮基 D 葡萄糖酸产生菌球状节杆菌K10 2 2的异常发酵液中分离到 3种噬菌体 ,分别将其命名为KS2 11、KS2 12和KS2 13。采用紫外诱变或自然选育的方法 ,获得了 6株具有抗噬菌体能力的 2 酮基 D 葡萄糖酸高产菌株。研究了抗株C2 2 4和K2 32的遗传稳定性 ,并在 5 0kL的发酵罐上进行了发酵生产试验。结果表明 ,经多次传代 ,菌株C2 2 4和K2 32对噬菌体的抗性及其发酵产酸能力没有改变 ,可以应用于工业生产中     

杀菌剂与无致病力烟草青枯菌对噬菌体存活的影响

了解不同噬菌体在不同环境中的存活情况,探索化学杀菌剂与噬菌体联合防治烟草青枯病的可行性。将P7-1、P1556-1及P1556-2等3种噬菌体与7种化学杀菌剂混合后,置于4℃和30℃培养箱,测定噬菌体效价随时间的变化规律。结果表明,3个噬菌体在7种杀菌剂中存活时间存在明显差异:3种噬菌体与氢氧化铜和王铜混合后1个月,其效价均为0;对其他5种杀菌剂的耐药由强到弱为P1556-2>P1556-1>P7-1,其中P1556-2在噻唑锌、中生菌素、春雷霉素、喹啉铜和辛菌胺醋酸盐中12个月测定的效价对数值分别为8.73、7.65、7.93、7.39和8.18。部分噬菌体在30℃可存活1~5个月,但在4℃下可存活6~12个月。噬菌体P1556-2和P1556-1在无致病力青枯菌Trp574中保存,其效价明显高于在无菌水中保存。可见,噬菌体在杀菌剂中的存活(效价)受时间、温度、药剂种类和噬菌体自身特性的影响,氢氧化铜与王铜制剂对噬菌体具有明显抑制作用,4℃(低温)下能够延长噬菌体的存活时间。无致病力青枯菌Trp574可用于噬菌体P1556-1和P1556-2的保存和大量培养。     

嗜热链球菌抗噬菌体菌株的选育

研究以酸奶生产菌株嗜热链球菌S2为出发菌株。采用自然选育的方法筛选出其抗噬菌体突变株。获得4株生产性能较好的抗噬菌株KS21、KS22、KS23、KS24,溶源性检测其为非溶源菌。对4株抗性菌株进行20次传代和酸奶发酵实验,结果表明抗性稳定,其中KS23发酵酸奶产酸达109°T,凝乳时间与出发菌株相当。细胞全蛋白SDS-PAGE电泳分析显示突变菌株与出发菌株在某些蛋白表达上有所差异,可能是抗性产生的原因。     

4株酸奶噬菌体的分离及其生物学特性

从上海市光明乳业股份有限公司车间发酵罐中分离出4株酸奶噬菌体，电镜下均呈蝌蚪形。1号噬菌体的裂解量为549，潜伏期小于15min；pH4～6、Ca^2＋浓度为0．04％时对其生长有利，60℃条件下处理30min时完全失活。2号噬菌体的裂解量为681，潜伏期小于15min；pH4～8、Ca^2＋浓度为0．04％时对其生长有利，50℃条件下处理40min时和60℃条件下处理20min时完全失活。3号噬菌体的裂解量为424，潜伏期小于15min;pH8～10、Ca^2＋浓度为0．06％时对其生长有利，60℃条件下处理10min时完全失活。4号噬菌体的裂解量为506，潜伏期小于15min；pH7～8、Ca^2＋浓度为0．04％时对其生长有利，60℃条件下处理40min时完全失活。     

抗噬菌体菌株荧光假单胞菌A46在D-异抗坏血酸钠工业生产中的应用研究

荧光假单胞菌A4 6是通过紫外线诱变K10 0 5菌株所得到的一株抗噬菌体菌株。在噬菌体存在的情况下 ,使用菌株A4 6和K10 0 5在 50kL的发酵罐上进行了 2 酮基 D 葡萄糖酸的发酵生产。结果表明 ,荧光假单胞菌A4 6对噬菌体的抗性稳定 ,其发酵周期短 ,发酵转化率高 ;经过多次传代 ,荧光假单胞菌A4 6对噬菌体的抗性及其发酵生产 2 酮基 D 葡萄糖酸的能力没有改变 ;该菌株的发酵产物可以用于D 异抗坏血酸钠的合成中     

一株地衣芽孢杆菌噬菌体的分离及其性质分析

从生产碱性蛋白酶的车间附近的下水道淤泥和后处理车间空气中分离得到了一株噬菌体。电镜观察表明该噬菌体有多面体的头部和细长的尾部组成,有着长尾病毒科噬菌体的形态特征。在供试菌种中,该噬菌体以地衣芽孢杆菌为宿主,对其生理特性研究发现,该噬菌体在pH 5-10之间有着很好的稳定性,在高温下有一定的稳定性,且金属离子可以促进其对宿主菌的侵染。根据实验结果绘制出噬菌体的一步生长曲线,可知感染宿主菌的潜伏期约为40 min,爆发期约为110 min。对噬菌体核酸性质的分析发现,其遗传物质为双链线状DNA,其分子量大约为36 kb。     

工业生产条件下噬菌体对2-酮基-D-葡萄糖酸发酵的影响

考察了工业生产条件下噬菌体对2-酮基-D-葡萄糖酸发酵的危害，并研究了噬菌体侵染后菌体形态、种子培养过程和发酵过程的变化。研究结果表明，噬菌体侵染导致了发酵转化率的下降、发酵周期的延长和发酵液过滤速率的降低。发酵前期、中期和后期的噬菌体侵染所造成的危害差异显著（p〈0．01），侵染时期愈早所造成的危害愈大。     

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。     

1株T1样噬菌体vB_EcoS_IME18的分离鉴定及其受体分析

以大肠杆菌BL21为指示菌分离得到1 株噬菌体vB_EcoS_IME18（IME18）,通过电镜观察噬菌体形态,并测定其最佳感染复数、一步生长曲线、裂解谱和体外杀菌效果;使用Illumina MiSeq测序仪完成噬菌体全基因组测序,利用比较基因组学分析噬菌体基因组特征;通过筛选噬菌体抗性株分析其耐受原因,利用受体基因沉默突变实验、噬菌体斑点实验和噬菌体吸附实验验证噬菌体受体。结果表明,噬菌体IME18具有二十面体头部和非收缩性尾部,属于长尾噬菌体科,Tunavirinae亚科,T1类噬菌体;噬菌体IME18的最佳感染复数为0.01,潜伏期为5 min,裂解期50 min,爆发量高达223 PFU/cell;在体外,噬菌体IME18能在90 min内有效杀死处于对数期的BL21;噬菌体IME18基因组全长50 354 bp,G＋C含量为45.6%;蛋白FhuA、TonB在噬菌体IME18的吸附和侵染过程中发挥重要作用。     

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。     

Comparison of lactococcal bacteriophage isolated in the United States and Argentina

Bacteriophage of Lactococcus lactis ssp. lactis and ssp. cremoris, isolated in the United States and Argentina, were compared with respect to host range, adsorption, latent period, burst size and immunological cross-reactivity. Only 1 out of 13 U.S. culture isolates was sensitive to Argentinian phage. Argentinian L. lactis ssp. lactis C2 mutants were resistant to 13 U.S. phage isolates (4 prolate and 9 isometric). While Argentinian phage Stl-3 multiplied on U.S. culture isolate 59-1, low adsorption (38%) and insignificant burst size and latent period data were evident. Antisera prepared against U.S. phage D59-1 (prolate) and F4-1 (isometric) neutralized the lytic activities of all Argentinian prolate phage although the F4-1 antiserum was less effective. The data suggest homology especially between U.S. phage D59-1 and the Argentinian phage.     

Isolation and characterization of a Lactobacillus plantarum bacteriophage,phiJL-1, from a cucumber fermentation

A virulent Lactobacillus plantarum bacteriophage, PhiJL-1, was isolated from a commercial cucumber fermentation. The phage was specific for two related strains of L. plantarum, BI7 and its mutant (deficient in malolactate fermenting ability) MU45, which have been evaluated as starter cultures for controlled cucumber fermentation and as biocontrol microorganisms for minimally processed vegetable products. The phage genome of PhiJL-1 was sequenced to reveal a linear, double-stranded DNA (36.7 kbp). Sodium dodecyl sulfate-polyacryamide gel electrophoresis (SDS-PAGE) profiles indicated that PhiJL-1 contains six structural proteins (28, 34, 45, 50, 61, and 76 kDa). Electron microscopy revealed that the phage has an isometric head (59 nm in diameter), a long non-contractile tail (182 nm in length and 11 nm in width), and a complex base plate. The phage belongs to the Bradley group B1 or Siphoviridae family. One-step growth kinetics of the phage showed that the latent period was 35 min, the rise period was 40 min, and the average burst size was 22 phage particles/infected cell. Phage particles (90%) adsorbed to the host cells 20 min after infection. Calcium supplementation (up to 30 mM CaCl(2)) in MRS media did not affect the first cycle of phage adsorption, but promoted rapid phage propagation and cell lysis in the infection cycle subsequent to adsorption. The D values of PhiJL-1 at pH 6.5 were estimated to be 2.7 min at 70 degrees C and 0.2 min at 80 degrees C by a thermal inactivation experiment. Knowledge of the properties of L. plantarum bacteriophage PhiJL-1 may be important for the development of controlled vegetable fermentations.     

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。     

Environmental factors for phage-induced fermentation problems: replication and adsorption of the Lactococcus lactis phage P008 as influenced by temperature and pH

The lactococcal phage P008 was investigated for its growth characteristics under certain environmental conditions. Phage growth was characterized by the latency period, the average burst size (determined in one-step growth experiments) and by the percentage of adsorption to the host cells after 1, 5, 10 and 15 min (determined in modified one-step growth experiments). The incubation conditions studied were temperatures from 20 to 40 degrees C and pH values of 4.8 and 6.4. The growth medium was ultrafiltration permeate of skim milk, which forms the water phase of milk. Both, the temperature and the pH influenced the growth parameters: Increased temperature as well as low pH led to a faster latency along with a higher average burst size. The percentage of adsorption was at maximum when the standard conditions of 30 degrees C and pH 6.4 were applied. Below pH 5, adsorption was reduced to below 10%. A decrease of temperature slightly reduced phage multiplication. Data were incorporated into a model to simulate phage multiplication under certain conditions, taking the percentage adsorption into account. The cell destruction of the host culture was calculated accordingly. The simulation (extrapolation) was validated by experimental growth curves of phages and phage-infected host cultures.     

烈性噬菌体vB_VpP_AC2的分离鉴定及生物学特性分析

该研究从水产市场污水中分离纯化副溶血性弧菌噬菌体，并对其中一株噬菌体vB_VpP_AC2（AC2）进行分离鉴定、基因组及部分生物学特性分析，探究该噬菌体作为副溶血性弧菌生防制剂的潜力。噬菌体AC2的噬菌斑透明且边缘清晰，透明部分的直径约为1.12 mm，无可见晕圈。噬菌体AC2的全基因组序列长44 270 bp，鸟嘌呤-胞嘧啶（GC）含量为49.30%，共预测到55个假定的开放阅读框，其中37个与编码已知功能蛋白质的基因相似（占67.27%），包括一个类似holin功能的蛋白（AC2_gp43，UTQ72417.1），该功能蛋白首次在Maculvirus属中被表征。经蛋白网络与末端酶系统发育树分析鉴定，噬菌体AC2属于自转录病毒科（Autograohiviridae），Maculvirus属，与噬菌体vB_VpaP_MGD1的ANIb值最高，为95.62%。在生物学特性方面，噬菌体AC2能够裂解30.77%的受测菌株，其最佳感染复数为0.001~0.1，一步生长曲线显示AC2的潜伏期为20 min，裂解期为35 min，裂解量为143 pfu/cell。总之，噬菌体AC2的分离鉴定不仅可为相关功能蛋白的研究提供参考，还可为水产食品安全提供较高应用价值的噬菌体资源。     

Characteristics of a phage effective for colibacillosis control in poultry

BACKGROUND: Colibacillosis is one of the main causes of economic loss in the poultry industry worldwide. Although antibiotics have been used to control this infection, the emergence of antibiotic‐resistant bacteria poses a threat to animal and human health. Phage therapy has been reported as one of the potential alternative methods to control bacterial infections. However, efficient phage therapy is highly dependent on the characteristics of the phage isolated. In the present study the characteristics of a lytic phage, ØEC1, which was found to be effective against the causative agent of colibacillosis in chickens in a previous in vivo study, are reported. RESULTS: Examination by transmission electron microscopy revealed that ØEC1 is a DNA phage belonging to the Podoviridae family. ØEC1 showed an optimum multiplicity of infection of 0.1‐1. The latent period of ØEC1 was 25 min, with a burst size of 200 particles per infected cell. Under the experimental conditions the maximum adsorption rate for ØEC1 was 99.9% within 8 min. ØEC1 demonstrated an optimum phage lytic activity at pH 6‐9 and 25–41 °C. CONCLUSION: These characteristics can serve as a guideline for selection of effective candidates for phage therapy, in this case for collibacillosis control in chickens. Copyright © 2012 Society of Chemical Industry