Magnetic nano-beads based separation combined with propidium monoazide treatment and multiplex PCR assay for simultaneous detection of viable Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes in food products

We developed a rapid and reliable technique for simultaneous detection of Salmonella Typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes that can be used in food products. Magnetic nano-beads (MNBs) based immunomagnetic separation (IMS) was used to separate the target bacterial cells while multiplex PCR (mPCR) was used to amplify the target genes. To detect only the viable bacteria, propidium monoazide (PMA) was applied to selectively suppress the DNA detection from dead cells. The results showed the detection limit of IMS-PMA-mPCR assay was about 10² CFU/ml (1.2 × 10² CFU/ml for S. Typhimurium, 4.0 × 10² CFU/ml for E. coli O157:H7 and 5.4 × 10² CFU/ml for L. monocytogenes) in pure culture and 10³ CFU/g (5.1 × 10³ CFU/g for S. Typhimurium, 7.5 × 10³ CFU/g for E. coli O157:H7 and 8.4 × 10³ CFU/g for L. monocytogenes) in spiking food products samples (lettuce, tomato and ground beef). This report has demonstrated for the first time, the effective use of rapid and reliable IMS combined with PMA treatment and mPCR assay for simultaneous detection of viable S. Typhimurium, E. coli O157:H7 and L. monocytogenes in spiked food samples. It is anticipated that the present approach will be applicable to simultaneous detection of the three target microorganisms for practical use.     

Development of oscillation method for reducing foodborne pathogens on lettuce and spinach

In this study, the efficacy of an oscillator for reducing the numbers of foodborne pathogens on lettuce and spinach was tested. A cocktail of three strains each of Salmonella typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes cells and of Bacillus cereus spores was inoculated onto lettuce and spinach leaves and followed by oscillation at 10 Hz and 20 Hz for up to 30 s. After treatment of inoculated lettuce leaf with an oscillator at 20 Hz for 30 s, 2.58, 2.82, 2.21 and 2.22 Log₁₀ CFU/g reductions were obtained with the cells of S. typhimurium, E. coli O157:H7 and L. monocytogenes and the spores of B. cereus, respectively. In the case of the oscillation treatment of spinach leaf, 2.89, 3.73, 2.46 and 2.25 Log₁₀ CFU/g reductions of those pathogens were achieved under the same condition. Statistically significant reductions were observed after oscillation treatment at 20 Hz for 5-10 s. The oscillation treatment at 10 Hz led to slightly less reductions of the pathogens tested as compared to the treatment at 20 Hz. In conclusion, the oscillation method developed shows to be highly efficacious in reducing foodborne pathogens on lettuce and spinach leaves.     

Screen-printed integrated microsystem for the electrochemical detection of pathogens

Fast, reliable, selective and high sensitive pathogen detection from different samples is a necessary requirement in current legislation; besides today's society demands a decrease in the time to detect the presence of pathogens. A low-cost, easy-to-fabricate microsystem, where the electrodes are both structural and functional elements, capable of electrochemically detecting those microorganisms has been manufactured by screen-printing techniques. Capturing of the microorganisms in solution is carried out by paramagnetic immunoparticles that are then electrophoretically immobilized inside the microsystem. Immobilization, lysis and detection of the microorganism are operations that have been integrated inside the microsystem and are constantly electrochemically monitored. The electrochemical detection has been investigated by electrical and electrochemical impedance and by amperometric methods. The electrochemical detection platform shows here concentrations of 10² and 10⁸ CFU/mL have been detected in less than 30-min time since the sample is introduced. The microsystem has a tunable volume down to 10 μL and the whole microsystem has dimensions that open a great window for future applications and implementation in portable pathogen control devices.     

Removal of native coliphages and coliform bacteria from municipal wastewater by various wastewater treatment processes: implications to water reuse

The efficacy of a conventional activated sludge wastewater treatment process and the membrane bioreactor technology in removing microbial pathogens was investigated. Total and fecal coliforms and somatic and F-specific coliphages were used as indicators of pathogenic bacteria and viruses. Up to 5.7 logs removal of coliforms and 5.5 logs of coliphages were observed in the conventional treatment process with advanced tertiary treatment. Addition of chemical coagulants seemed to improve the efficacy of primary and secondary treatment for microorganism removal. Complete removal of fecal coliforms and up to 5.8 logs removal of coliphages was observed in the MBR system. It was shown that the MBR system was capable of high removal of coliphages despite the variation in feed coliphage concentrations. The results of this study indicated that the MBR system can achieve better microbial removal in far fewer steps than the conventional activated sludge process with advanced tertiary treatment. The final effluent from either treatment processes can be potentially reused.     

Validation of hollow fiber ultrafiltration and real-time PCR using bacteriophage PP7 as surrogate for the quantification of viruses from water samples

A quantitative real-time TaqMan PCR system for Pseudomonas aeruginosa bacteriophage PP7 was designed to detect PP7 as surrogate in performance tests of 2 hollow fiber ultrafiltration systems in series. Fifty-six storm water samples from 21 sites representing agricultural, urban and highway locations in California were collected. The optimized procedure gave recoveries of spiked PP7 of 64+/-4.8% (mean+/-SEM). The PP7 assay was validated over 5 orders of magnitude with an assay limit of detection of 5 gene copies per reaction volume. Sample-dependent variables like enzymatic inhibition during PCR analysis, filtration recovery and extraction efficiency were quantified and incorporated to calculate a specific sample limit of detection (S(LOD)) for the spiked surrogate PP7. S(LOD) values were highly variable among samples; they were independent of physicochemical parameters including conductivity, turbidity, total suspended solids and pH but strongly correlated with the dilution factor required to relieve enzymatic inhibition during PCR analysis. To determine actual gene copies of PP7, a dilution approach was developed that involves assaying several dilutions within a range where inhibitors do not affect the efficiency of amplification and linear regression to determine the theoretical C(t) value when there is no inhibition. For the detection of viral pathogens, an internal standard like PP7 can be used to calculate filtration recoveries when quantifying pathogens and to determine whether filtration or inhibitor concentration affect nucleic acid extraction efficiency. Additionally, by defining S(LOD) values per sample and pathogenic organism analyzed, it should be possible to critically investigate the absence of detects for a particular pathogen and determine probabilities of risk associated with a specific sample limit of detection.     

Application of caffeine, 1,3,7-trimethylxanthine,to control Escherichia coli O157:H7

The objective of this research was to determine the effectiveness of caffeine on inactivation of Escherichia coli O157:H7 in brain heart infusion (BHI) broth. Overnight samples of five E. coli O157:H7 strains of (E0019, F4546, H1730, 944 and Cider) were used in this study. These strains were individually inoculated at an initial inoculum level of 2 log CFU/ml into BHI broth containing caffeine at different concentrations (0.00%, 0.25%, 0.50%, 0.75%, 1.00%, 1.25%, 1.50%, 1.75%, and 2.00%). Samples were then incubated at 37 °C for 24 h. Bacterial growth was monitored at different time intervals by measuring turbidity at 610 nm using a spectrophotometer. Results revealed that the addition of caffeine inhibited the growth of E. coli O157:H7. Significant growth inhibition was observed with concentration levels of 0.50% and higher. These results indicate that caffeine has potential as an antimicrobial agent for the treatment of E. coli O157:H7 infection and should be investigated further as a food additive to increase biosafety of consumable food products.     

食源性致病菌检测方法研究进展——Ⅱ.分子生物学检测方法

为方便对食品生产和流通的整个过程进行卫生监督和管理,就以核酸为分析对象的各种分子生物学食品微生物快速检测方法进行了综述。     

Depuration of live oysters to reduce Vibrio parahaemolyticus and Vibrio vulnificus: A review of ecology and processing parameters

Consumption of raw oysters, whether wild-caught or aquacultured, may increase health risks for humans. Vibrio vulnificus and Vibrio parahaemolyticus are two potentially pathogenic bacteria that can be concentrated in oysters during filter feeding. As Vibrio abundance increases in coastal waters worldwide, ingesting raw oysters contaminated with V. vulnificus and V. parahaemolyticus can possibly result in human illness and death in susceptible individuals. Depuration is a postharvest processing method that maintains oyster viability while they filter clean salt water that either continuously flows through a holding tank or is recirculated and replenished periodically. This process can reduce endogenous bacteria, including coliforms, thus providing a safer, live oyster product for human consumption; however, depuration of Vibrios has presented challenges. When considering the difficulty of removing endogenous Vibrios in oysters, a more standardized framework of effective depuration parameters is needed. Understanding Vibrio ecology and its relation to certain depuration parameters could help optimize the process for the reduction of Vibrio. In the past, researchers have manipulated key depuration parameters like depuration processing time, water salinity, water temperature, and water flow rate and explored the use of processing additives to enhance disinfection in oysters. In summation, depuration processing from 4 to 6 days, low temperature, high salinity, and flowing water effectively reduced V. vulnificus and V. parahaemolyticus in live oysters. This review aims to emphasize trends among the results of these past works and provide suggestions for future oyster depuration studies.     

原料奶中病原菌的调查及代表性病原菌的研究

对榨乳过程进行了采样分析，确定了原料奶中的主要致病菌为金黄色葡萄球菌，并分析了原料奶在不同温度储存时金黄色葡萄球菌的生长情况以及金黄色葡萄球菌在酸奶发酵和冷藏过程中的存活情况。研究发现，酸奶的低酸性环境并不能有效的抑制金黄色葡萄球菌的生长，所以为了确保产品的安全性，要对奶牛的乳房炎和隐性乳房炎进行有效防治，并严格控制榨乳的卫生条件及原料奶的储存温度。