Real-Time Duplex Applications of Loop-Mediated AMPlification (LAMP) by Assimilating Probes

Isothermal nucleic-acid amplification methods such as Loop-Mediated isothermal AMPlification (LAMP) are increasingly appealing alternatives to PCR for use in portable diagnostic system due to the low cost, weight, and power requirements of the instrumentation. As such, interest in developing new probes and other functionality based on the LAMP reaction has been intense. Here, we report on the development of duplexed LAMP assays for pathogen detection using spectrally unique Assimilating Probes. As proof of principle, we used a reaction for Salmonella enterica as a model coupled with a reaction for λ-phage DNA as an internal control, as well as a duplexed assay to sub-type specific quarantine strains of the bacterial wilt pathogen Ralstonia solanacearum. Detection limits for bacterial DNA analyzed in individual reactions was less than 100 genomic equivalents in all cases, and increased by one to two orders of magnitude when reactions were coupled in duplexed formats. Even so, due to the more robust activity of newly available strand-displacing polymerases, the duplexed assays reported here were more powerful than analogous individual reactions reported only a few years ago, and represent a significant advance for incorporation of internal controls to validate assay results in the field.     

Survival of Salmonella typhimurium on Sterile Ground Chicken Breast Patties After Washing with Salt and Phosphates and During Refrigerated and Frozen Storage

ABSTRACT: This study investigated the effects of 10% (w/v) salt, trisodium phosphate (TSP), sodium tripolyphosphate (STPP), and tetrapotassium pyrophosphate (TKPP) washes on removal of attached Salmonella typhimurium from sterile chicken breast patties, as well as on their injury and survival in a refrigerator for 16 d, in a –20 °C freezer for 10 mo, and after 3 freeze-thaw cycles. S. typhimurium were grown on chicken patties at 20 °C for 20 h, washed, and enumerated by plating on selective and nonselective media. Salt and phosphates washing significantly lowered the survival populations of attached S. typhimurium on patties, but did not cause any significant sublethal injury of attached S. typhimurium , irrespective of storage treatments. The TSP washes showed superior effects of removing and inactivating S. typhimurium compared to other washing treatments.     

Attachment of Salmonella typhimurium to Poultry Skin as Related to Cell Viability

³H-labeled S. typhimurium cells inactivated by gamma-radiation (⁶⁰Co) and by formaldehyde (5%) were used to determine if cell viability affected attachment to poultry skin. Both gamma-irradiated and formal-dehyde-treated cells attached like live cells. There was no difference in attachment between cells grown in a chemically defined medium (SCDM) or in brain heart infusion broth (BHI). Live and gamma-irradiated cells and BHI-grown and SCDM-grown cells had similar surface hydrophobicity. Live and gamma-irradiated cells had similar cell surface charges, whereas BHI and SCDM-grown cells had different surface charges. Differences in cell surface charge did not affect attachment rate.     

Antimicrobial resistance and R-factor transfer of salmonellae isolated from chicken carcasses in Greek hospitals

A total of 62 salmonellae, belonging to six different serotypes, were isolated from 60 out of 87 (69.0%) chicken carcasses delivered to hospitals of Thesssaloniki, Greece. Salmonella enteritidis, Salmonella anatum and Salmonella bredeney were the most prevalent serovars. Isolates were examined for antibiotic resistance patterns and R-determinants. Resistance to at least one antibiotic was observed in 36 (58.1%) of them and 18 different resistant profiles were recorded. Nitrofurantoin-resistance was the most common (29.0%), followed by spectinomycin (21.0%), ampicillin (19.4%) and ticarcillin (19.4%). Fourteen (38.9%) of the resistant isolates possessed R-factors and resistance to ampicillin, ticarcillin, trimethoprim and kanamycin was easily self-transferable. However, nitrofurantoin- and spectinomycin-resistance although prevailing, was not found transferable even after mobilization. The high incidence of antibiotic resistant salmonellae among chicken carcasses in our hospital setting suggests the need for public health interventions and possible withdrawal of drug selective pressure.     

肠道杆菌的扫描电镜观察

我们用扫描电镜观察肠道杆菌种(Enterobacteriaceae)中的四种细菌,大肠埃希氏杆菌(Escherichiacoli),普通变形杆菌(Proteus vulgaris),伤寒沙门氏菌(Salmonella typhi)和福氏志贺氏菌(Shigella Flexneri)。未处理的菌落标本,菌落表面都可形成一层厚薄不同的表膜(Surface film)。四种细菌菌落的表膜,形状不一,千姿百态。适当处理后的菌落标本,则可显示菌体的本来面目。四种细菌菌体在菌落表面的分布和排列,也是各不相同,千差万别。本文根据扫描电镜的观察,对四种不同菌落进行了讨论。     

Recent Advances in the Detection of Antibiotic and Multi-Drug Resistant Salmonella: An Update

Antibiotic and multi-drug resistant (MDR) Salmonella poses a significant threat to public health due to its ability to colonize animals (cold and warm-blooded) and contaminate freshwater supplies. Monitoring antibiotic resistant Salmonella is traditionally costly, involving the application of phenotypic and genotypic tests over several days. However, with the introduction of cheaper semi-automated devices in the last decade, strain detection and identification times have significantly fallen. This, in turn, has led to efficiently regulated food production systems and further reductions in food safety hazards. This review highlights current and emerging technologies used in the detection of antibiotic resistant and MDR Salmonella.     

Apple,Carrot, and Hibiscus Edible Films Containing the Plant Antimicrobials Carvacrol and Cinnamaldehyde Inactivate Salmonella Newport on Organic Leafy Greens in Sealed Plastic Bags

The objective of this study was to investigate the antimicrobial effects of carvacrol and cinnamaldehyde incorporated into apple, carrot, and hibiscus‐based edible films against Salmonella Newport in bagged organic leafy greens. The leafy greens tested included organic Romaine and Iceberg lettuce, and mature and baby spinach. Each leafy green sample was washed, dip inoculated with S. Newport (10⁷ CFU/mL), and dried. Each sample was put into a Ziploc® bag. Edible films pieces were put into the Ziploc bag and mixed well. The bags were sealed and stored at 4 °C. Samples were taken at days 0, 3, and 7 for enumeration of survivors. On all leafy greens, 3% carvacrol films showed the best bactericidal effects against Salmonella. All 3 types of 3% carvacrol films reduced the Salmonella population by 5 log₁₀ CFU/g at day 0 and 1.5% carvacrol films reduced Salmonella by 1 to 4 log₁₀ CFU/g at day 7. The films with 3% cinnamaldehyde showed 0.5 to 3 log reductions on different leafy greens at day 7. The films with 0.5% and 1.5% cinnamaldehyde and 0.5% carvacrol also showed varied reductions on different types of leafy greens. Edible films were the most effective against Salmonella on Iceberg lettuce. This study demonstrates the potential of edible films incorporated with carvacrol and cinnamaldehyde to inactivate S. Newport on organic leafy greens.     

Transmission of Bacterial Zoonotic Pathogens between Pets and Humans: The Role of Pet Food

Recent Salmonella outbreaks associated with dry pet food and treats raised the level of concern for these products as vehicle of pathogen exposure for both pets and their owners. The need to characterize the microbiological and risk profiles of this class of products is currently not supported by sufficient specific data. This systematic review summarizes existing data on the main variables needed to support an ingredients-to-consumer quantitative risk model to (1) describe the microbial ecology of bacterial pathogens in the dry pet food production chain, (2) estimate pet exposure to pathogens through dry food consumption, and (3) assess human exposure and illness incidence due to contact with pet food and pets in the household. Risk models populated with the data here summarized will provide a tool to quantitatively address the emerging public health concerns associated with pet food and the effectiveness of mitigation measures. Results of such models can provide a basis for improvements in production processes, risk communication to consumers, and regulatory action.     

次氯酸钠对德尔卑沙门氏菌生物被膜的抑制作用及机制

目的：为控制德尔卑沙门氏菌（Salmonella Derby）在食品加工过程中的污染，研究不同体积分数次氯酸钠对S. Derby生物被膜的抑制作用及其机制。方法：采用结晶紫染色法和平板菌落计数法观察次氯酸钠对生物被膜的抑制和清除效果，进一步观察不同体积分数次氯酸钠作用下沙门氏菌泳动能力和生物被膜内细胞的代谢活性、胞外聚合物含量和微观形态的变化规律。结果：次氯酸钠的最小抑菌浓度（minimum inhibitory concentration，MIC）为0.08%（体积分数，下同），亚MIC（0.04%、0.02%、0.01%）的次氯酸钠可以抑制S. Derby生物被膜的形成，对成熟生物被膜也有一定的清除效果，且不同亚MIC次氯酸钠的作用效果差异显著（P＜0.05）；不同体积分数次氯酸钠处理能降低生物被膜内细胞代谢活性，抑制胞外多糖的合成，且不同体积分数次氯酸钠的作用效果差异显著（P＜0.05）。激光共聚焦扫描显微镜结果显示，与对照组相比，次氯酸钠处理使得生物被膜厚度减小、活菌数减少、死菌数增多、细菌由聚集状态变为分散状态。结论：不同体积分数次氯酸钠对S. Derby生物被膜的形成有明显的抑制作用，这一作用与干扰细菌生长、生物被膜内细胞代谢活性和胞外多糖的合成有关。