Detection of bacteria using foreign DNA: the development of a bacteriophage reagent for Salmonella

A phage-based reagent was developed for the detection of Salmonella in food samples. The parental phage was Felix 01, which lyses practically all Salmonella. Using data obtained about the molecular biology of the phage, a recombinant phage that carried the bacterial genes specifying luciferase was produced. The method involved the isolation of amber nonsense mutations and subsequent crosses to render doubly mutant phage with a very low reversion rate on strains lacking an amber suppressor. A plasmid was constructed that contained a segment of Felix 01 DNA with two adjacent genes, one dispensable and the other essential, and their flanking sequences. Recombinant DNA technology was used to remove the two genes and the luxA and luxB genes for luciferase, and a gene specifying a tRNA that recognizes amber codons (supF=tyrT) was put in their stead. This region could be transferred into the genome of the phage by homologous recombination. The recombinant phage cannot grow because it lacks an essential gene. However, it can grow in a host that synthesizes the missing protein. This technique allows the construction of "locked" recombinant phages that carry foreign DNA but which cannot propagate themselves in nature.     

Suppression of Salmonella growth by wild-type and large-plaque variants of bacteriophage Felix O1 in liquid culture and on chicken frankfurters

The bacteriophage Felix O1, a member of Myoviridae, is specific for, and possesses a broad host range within, the genus Salmonella. This work explores a Felix O1 phage-based intervention for Salmonella enterica serotype Typhimurium DT104 that is potentially applicable at several stages of animal production and processing. A variant of Felix O1 was obtained that produces a larger, clearer plaque phenotype (LP) on Salmonella Typhi than wild-type Felix O1 (WT) does, not unlike r mutants of phage T4. LP exhibited slightly more extensive overall suppression of Salmonella Typhi in brain heart infusion (BHI) broth, as ascertained on the basis of culture turbidity (optical density at 600 nm). Both phage variants suppressed log phase BHI broth cultures containing 8.2 x 10(6) CFU of Salmonella Typhimurium DT104 per ml. A PFU/CFU ratio of 1.0 was effective for WT and LP, whereas increasing the PFU/CFU ratio to 5.0 did not increase suppression. Untreated Salmonella-contaminated frankfurters were compared with treated samples (PFU/CFU ratio, 1.9 x 10(4)) to test WT and LP for their ability to suppress Salmonella growth on chicken frankfurters contaminated with 300 CFU of Salmonella Typhimurium DT104. Suppression levels of 1.8 and 2.1 log units were achieved with WT and LP, respectively (P = 0.0001), but no difference was found between the performances of the two variants (P = 0.5088).     

噬菌体对鸡肉表面耐药沙门氏菌的抑制作用

目的 探究裂解性噬菌体对鸡肉表面耐药沙门氏菌的抑制效果.方法 以实验室前期筛选得到的沙门氏菌噬菌体D1-2和Pu20为研究材料,采用单独使用和1:1混合的方式对生鸡胸肉表面多重耐药沙门氏菌的抑制效果进行研究.结果 与单一噬菌体相比,噬菌体混合制剂对生鸡胸肉表面多重耐药沙门氏菌抑菌效果更显著.在4℃时,采用感染复数(mu...     

乳制品中沙门氏菌分子检测方法的验证研究

目的验证沙门氏菌、非沙门菌及阳性核酸模板对MICROFAST?沙门氏菌核酸检测试剂盒(PCR-探针法)的特异性和稳定性,同时比对试剂盒法与GB 4789.4—2016培养法定性检测结果的一致性。方法用实验室保存的30株沙门氏菌和15株非沙门氏菌菌株验证MICROFAST?沙门氏菌核酸检测试剂盒(PCR-探针法)的特异性。通过人工添加不同浓度沙门氏菌对30个乳制品样本,采用国家标准法和试剂盒法同时检验,探究方法的一致性。选择制备好的5份阳性核酸模板,每个模板分别使用3个批次的试剂盒进行检测,对实验结果进行重复性和显著性分析,确定不同试剂盒批次间是否存在显著性差异。结果 30株沙门氏菌菌株和15株非沙门氏菌菌株的特异性检测结果表明,MICROFAST?沙门氏菌核酸检测试剂盒(PCR-探针法)对沙门氏菌的特异性符合预期。人工添加的阳性样本检测结果表明,在乳制品样本范围内,试剂盒的假阳性率与假阴性率为0。3个批次的试剂盒对5份阳性模板检测结果之间没有显著性差异,变异系数CV均小于1%。结论该试剂盒方法与国家标准法相比,具有操作简单、快速等优势,适合乳制品加工过程微生物快速检测。     

分子生物学方法检测沙门氏菌的研究进展

沙门氏菌是一种能引起人畜共患病的病原菌,由其引起的食物中毒事件屡居首位,在食品致病菌的检测中,沙门氏菌的检测是尤为重要的。本文针对目前检测食品中的沙门氏菌常用的分子生物学方法,包括三种PCR技术、基因芯片技术、环介导等温技术和焦磷酸测序技术进行了综述,对分子生物学快速检测技术与其他学科技术的联合使用做简单的介绍,并对这些技术的未来发展进行展望。      

分子马达传感器对沙门氏菌快速检测方法的初步研究

利用F0F1-ATP合酶的旋转特性及对H+敏感的荧光物质F-DHPE作为指示剂来检测样本中有无目标物。通过生物素-亲和素系统将特异性invA核酸探针连接在F0F1-ATPase的ε亚基上构建生物传感器;将待测样品和阴性对照分别与生物传感器结合后,比较其催化ATP合成30min后的ATP产生量,依此对样品中的沙门氏菌DNA进行检测。结果表明,该方法对沙门氏菌DNA的检测时间为1h,检出限为10ng/mL。从食品样本分离得到的30株细菌,利用分子马达生物传感器的检测结果与PCR检测的结果一致。      

Application of a bacteriophage cocktail to reduce Salmonella Typhimurium U288 contamination on pig skin

Multidrug-resistant Salmonella Typhimurium U288 is a significant pathogen of pigs, accounting for over half of all outbreaks on UK pig production premises. The potential of this serovar, and other salmonellae, to enter the food chain during the slaughtering process requires that efforts be made to reduce the prevalence of these bacteria at both the pre- and post-harvest stages of production. A bacteriophage cocktail (PC1) capable of lysing various Salmonella enterica serovars was designed using the broad host-range phage Felix 01, and three phages isolated from sewage. PC1 applied to pig skin experimentally-contaminated with U288 achieved significant reductions (P < 0.05) in Salmonella counts when stored at 4 °C over 96 h. Reductions of > 1 log₁₀ unit were observed when the ratio of phage applied was in excess of the bacterial concentration. The treatment was found to be effective at a multiplicity of infection (MOI) of 10 or above, with no significant reductions taking place when the MOI was less than 10. Under these conditions U288 counts of log₁₀ 4.1-4.3 CFU were reduced to undetectable levels following the application of PC1 to pig skin (> 99% reduction). These data suggest phage cocktails could be employed post-slaughter as a means to reduce Salmonella contamination of pig carcasses.     

Comparison of four molecular typing methods for the differentiation of Salmonella spp

A total of 57 strains of Salmonella spp. were differentiated by random amplification of polymorphic DNA (RAPD) fingerprinting using three different primers (OPL-03, primer 1, and primer A); by Enterobacterial Repetitive Intergenic Consensus (ERIC) fingerprinting; by ribotyping-PCR; and by Single Strand Conformation Polymorphism (SSCP). From the 57 strains, RAPD fingerprinting with primers OPL-03, 1, and A produced 42, 51, and 54 fingerprint patterns respectively. ERIC fingerprinting produced 50 patterns; ribotyping-PCR produced four patterns, and SSCP produced 11 patterns. Combinations of two different typing methods generally increased the discrimination of Salmonella strains. A combination of two different RAPDs or a combination of RAPD and ERIC was better than the other combinations. Discrimination using a combination of RAPD (primer 1 or primer A) and ERIC, which could differentiate all 57 Salmonella strains, was better than the combination of two RAPDs. This study indicated that the use of a combination of RAPD (primer 1 or primer A) and ERIC should be useful for the differentiation of field-isolated Salmonella strains and epidemiological studies.     

沙门氏菌传统血清凝集分型和分子血清分型试剂盒方法比较

目的 验证沙门氏菌血清分型试剂盒的适用性, 考核本实验室传统沙门氏菌血清分型技术, 扩充沙门氏菌的分型方法, 寻找更有效、更快捷的沙门氏菌分型方法。方法 保存的样品菌株库中随意挑选33株沙门氏菌和6株标准菌株, 首先确证为沙门氏菌, 然后分别采用传统的玻片血清凝集方法和分子血清分型试剂盒对其进行分型, 最后采用16S rRNA系统发育树对试验菌株进行聚类分析。结果 2种分型手段的匹配率高达94.9%, 其中YP 281 Salmonella havana和YP 639 Salmonella liverpool没有匹配到, 这2株菌在试剂盒数据库中不存在, 但本实验利用传统血清分型手段可以对这2株菌进行准确分型, 其中YP 281是本实验室对GB 4789.4-2016《食品安全国家标准 食品微生物学检验 沙门氏菌检验》表B.1外成功分型的沙门氏菌。结论 本实验室传统血清凝集分型技术合格。沙门分子血清分型试剂盒具有较好的适用性, 能更快速、更方便对沙门氏菌进行分型。     

Optimization and validation of a simple method using P22::luxAB bacteriophage for rapid detection of Salmonella enterica serotypes A, B, and D in poultry samples

A simple method was developed for the fast and inexpensive detection of Salmonella Typhimurium using a recombinant P22::luxAB phage. All the steps from phage production to detection were considered. A strain of Salmonella Typhimurium harboring the prophage P22::luxAB was grown in batch culture to produce spontaneously the recombinant bacteriophage. Batch production to stationary phase was better for propagation of the phage and led to a total population of 4.3 x 10(9) (+/-4.3 x 10(9)) PFU/ml of P22, including only 1.4 x 10(6) (+/-1 x 10(6)) PFU/ml harboring the luxAB genes. After preenrichment, a simple four-step bioassay was tested and optimized for several parameters. The detection limit of the luminometer was only 5 x 10(2) (+/-1.75 x 10(2)) CFU Salmonella Typhimurium per ml, but increased to 1.5 x 10(4) (+/-1.17 x 10(4)) CFU Salmonella Typhimurium per ml when the cells were in a complex matrix. The detection limit after the preenrichment was 6.5 x 10(3) (+/-1.5 x 10(3)) CFU Salmonella Typhimurium per ml, but the detection limit after the preenrichment also increased markedly to 1.65 x 10(5) (+/-0.15 x 10(5)) CFU Salmonella Typhimurium per ml when Salmonella Typhimurium was in a complex matrix. Finally, the bioassay was applied to the detection of Salmonella Typhimurium LT2 in 14 different feed and environmental samples (including duck feed, litters, and feces) spiked either before or after the preenrichment process. It was possible to detect Salmonella Typhimurium LT2 in all samples within 16 h.     

Effectiveness of bacteriophage JN01 incorporated in gelatin film with protocatechuic acid on biocontrol of Escherichia coli O157:H7 in beef

A new gelatin-protocatechuic acid (PCA) film with Escherichia coli O157:H7 phage JN01 was developed and characterised. After incorporated with JN01, swelling value, water vapour permeability, water solubility and elongation at break of gelatin-PCA film were not significantly different. The addition of JN01 increased b value and transparency of film, while it decreased L value, a value and tensile strength of film. Moreover, the gelatin-PCA-JN01 film presented antioxidant activity of 60.07%. Furthermore, JN01 could be steadily released from gelatin-JN01 and gelatin-PCA-JN01 films in aqueous solution, and their release rates were 7.56% and 0.12% after 11 h, respectively. The microstructure analysis showed that JN01 particles were clustered and uniformly distributed in film, and the aggregation would be attenuated in the presence of PCA. Meanwhile, E. coli O157:H7 counts were 1.13 log₁₀CFU mL⁻¹ and 0.45 log₁₀CFU mL⁻¹ lower in gelatin-JN01 and gelatin-PCA-JN01 films compared with pure gelatin film in vitro at 4 °C for 24 h, respectively. After stored at 4 °C for 7 days, Escherichia coli O157:H7 counts were 1.00 log₁₀CFU g⁻¹ and 0.80 log₁₀CFU g⁻¹ lower in beef packed with these two gelatin films, compared with beef without gelatin film, respectively. In conclusion, the developed gelatin-PCA-JN01 film has potential application in food preservation.     

Biosensors for rapid detection of Salmonella in food: A review

Salmonella is one of the main causes of foodborne infectious diseases, posing a serious threat to public health. It can enter the food supply chain at various stages of production, processing, distribution, and marketing. High prevalence of Salmonella necessitates efficient and effective approaches for its identification, detection, and monitoring at an early stage. Because conventional methods based on plate counting and real-time polymerase chain reaction are time-consuming and laborious, novel rapid detection methods are urgently needed for in-field and on-line applications. Biosensors provide many advantages over conventional laboratory assays in terms of sensitivity, specificity, and accuracy, and show superiority in rapid response and potential portability. They are now recognized as promising alternative tools and one of the most on-site applicable and end user–accessible methods for rapid detection. In recent years, we have witnessed a flourishing of studies in the development of robust and elaborate biosensors for detection of Salmonella in food. This review aims to provide a comprehensive overview on Salmonella biosensors by highlighting different signal-transducing mechanisms (optical, electrochemical, piezoelectric, etc.) and critically analyzing its recent trends, particularly in combination with nanomaterials, microfluidics, portable instruments, and smartphones. Furthermore, current challenges are emphasized and future perspectives are discussed.     

Response surface studies on cholesterol reduction in egg yolk using #ps01β-cyclodextrin

The effects of β-cyclodextrin (BCD) and reaction time on the recovery of cholesterol from egg yolk were studied at 25°C based on response surface methodology. The developed response model is of the form: Y = 5.585 + 429.77 X _c + 0.534 X _t ², where Y is the predicted cholesterol recovery (%), X _c is the BCD/egg yolk ratio (kg/kg) and X _t is the reaction time (h). Cholesterol recovery increased with the both BCD/egg yolk ratio and reaction time and there was no optimum value. The model predicted a cholesterol recovery of 94.5% at a BCD/egg yolk ratio of 0.163 over a reaction time of 6 h whereas experiments showed a cholesterol recovery of 87.7%.     

Response surface studies on cholesterol reduction in egg yolk using #ps01β-cyclodextrin

 The effects of β-cyclodextrin (BCD) and reaction time on the recovery of cholesterol from egg yolk were studied at 25°C based on response surface methodology. The developed response model is of the form: Y = 5.585 + 429.77 X _c + 0.534 X _t ², where Y is the predicted cholesterol recovery (%), X _c is the BCD/egg yolk ratio (kg/kg) and X _t is the reaction time (h). Cholesterol recovery increased with the both BCD/egg yolk ratio and reaction time and there was no optimum value. The model predicted a cholesterol recovery of 94.5% at a BCD/egg yolk ratio of 0.163 over a reaction time of 6 h whereas experiments showed a cholesterol recovery of 87.7%. Received: 2 May 1997 / Revised version: 8 September 1997     

Combined effect of bacteriophage and antibiotic on the inhibition of the development of antibiotic resistance in Salmonella typhimurium

Food Science and Biotechnology - This study was designed to evaluate the combined effects of bacteriophage and antibiotic on the reduction of the development of antibiotic-resistance in Salmonella...     

Effects of penconazole on two yeast strains: growth kinetics and molecular studies

The aim of this study consisted to evaluate the impact of a pesticide (penconazole) on the growth kinetics and genotoxicity on two yeast strains (Saccharomyces cerevisiae and Metschnikowia pulcherrima). When the penconazole was added at different phases of the growth of M. pulcherrima, no effect was noticed on the kinetics of yeast growth but DNA adducts were observed when penconazole was added in the exponential phase. Increasing doses (1-15 maximum residue limit) of the pesticide added at the beginning of the fermentation did not induce DNA adducts while kinetics were affected.     

20株食源性科瓦利斯沙门菌药物敏感试验和分子分型分析

目的了解2016年河南省食源性沙门菌优势血清型(科瓦利斯沙门菌)药物敏感性和分子分型特征。方法食品中沙门菌的检测和血清分型参照2016年国家食品污染物和有害因素风险监测工作手册、脉冲场凝胶电泳(pulsed field gel electrophoresis, PFGE)分子分型分析和药物敏感试验参照2016年食源性疾病监测工作手册和国家食源性疾病分子溯源网络(TraNetChina)使用手册。结果采用微量最低抑菌浓度法对20株食源性科瓦利斯沙门菌进行15种抗生素的药物敏感试验,20株菌对四环素、红霉素100%耐药,对环丙沙星、萘啶酸耐药率分别为80.00%、45.00%,对头孢西丁、头孢唑啉产生中度耐药,中度耐药率分别为15.00%、10.00%,对头孢噻圬、头孢他啶、亚胺培南、庆大霉素、氨苄西林、氨苄西林/舒巴坦、氯霉素、甲氧苄氨嘧啶/磺胺甲噁唑、阿奇霉素全部敏感。有8株菌出现多重耐药现象,最多对4类4种抗生素产生耐药。20株科瓦利斯沙门菌用XbaⅠ酶切、经脉冲场凝胶电泳后,获得9种带型,每种带型包括1～6株菌,相似度61.7%～100%,以SCVX01.HA0008和SCVX01.HA0009为主要优势带型。结论河南省食源性科瓦利斯沙门菌对第三代头孢类抗生素全部敏感,对喹诺酮类和氟喹诺酮类出现不同程度耐药,且带型存在地区聚集性提示存在共同来源或者交叉污染的情况,相关部门应加强监测,提早防范。     

A New Procedure for the Differentiation of 01 Vibrio cholerae and Non-01 V. cholerae Recovered From Oysters

Live oysters were inoculated with 01 V. cholerae or non-01 V. cholerae and the liquor from the oysters was spread onto thiosulfate citrate bile salts agar plates. After incubation the colonies with yellow centers were picked and streaked onto sheep blood agar plates. Biochemical tests were performed on all colonies. Isolates exhibiting characteristic V. cholerae reactions were checked for serological identity and inoculated into a vibrio medium. After incubation, the isolates were further analyzed for lactic and succinic acids by gas-liquid chromatography. Peak height ratios of lactic/succinic acid provided a testing parameter to distinguish all isolates of 01 V. cholerae from non-01 V. cholerae isolates recovered from the oysters.     

Salmonella Enteritidis Risk Assessment: A Kinetic Analysis

ABSTRACT: Egg and egg preparations are important vehicles for Salmonella enteritidis infections. The influence of time-temperature becomes important when the presence of this microorganism is found in commercial shell eggs, particularly in countries where refrigeration is not mandatory as in Chile. The objective of this research was to develop a mathematical model to analyze the Salmonella enteritidis risk under variable ambient temperatures. Breakdown of vitelline egg membrane was assumed to be required for initiation of bacterial growth. When the critical factor concerning safety is the vitelline membrane breakdown, 15 °C was found to be the storage threshold temperature for a 30-d shelf life. This computer based tool can be used as a contribution in current regulation adjustments or modifications.     

Cell Surface Display of Salmonella Outer Membrane Protein A on Lactobacillus salivarius: A First Step Towards Food-Grade Live Vaccine Against Salmonella Infections

As one of the most important pathogens in both humans and animals, Salmonella is a significant health problem, especially in the developing countries. Outer membrane protein A (OmpA) is among the major outer membrane proteins of Salmonella with strong immunogenicity. Vaccination is an effective tool for the prevention of Salmonella infections. Lactic acid bacteria are the normal flora of gastrointestinal (GI) tract. In this research, lactobacilli from different parts of chicken GI tract were isolated and evaluated for bile salts and acidic pH tolerance. One strain isolated from the small intestine was identified as Lactobacillus salivarius by carbohydrate fermentation test and 16SrRNA partial sequencing. To address the need for safe oral vaccine to control Salmonella infection, we engineered this natural chicken isolate of L. salivarius to express OmpA under promotion of Lactate dehydrogenase (ldh) promoter. The surface displayed OmpA reacted with anti-OmpA in ELISA, Immunoblotting and flow cytometry assays, suggesting that the expressed protein adopted a native conformation. Strong agglutination reaction of the recombinant lactobacilli with sera of the naturally Salmonella infected chickens showed the multivalency and overexpression of the OmpA on the surface of L. salivarius. Experimental infection assay confirmed that the engineered commensal bacteria could be an attractive candidate as food-grade live vaccine against Salmonella infections.