Bioprotective Leuconostoc strains against Listeria monocytogenes in fresh fruits and vegetables

Ten Leuconostoc mesenteroides and one Ln. citreum strains isolated from fresh fruit and vegetables were tested for their antagonistic capacity against Listeria monocytogenes. Genetic differences among strains were analyzed by Random Amplified Polymorphic DNA (RAPD). All the isolates clustered together and differed from the type strain Ln. mesenteroides ATCC 8293 as well as from Ln. fallax and Ln. citreum. Organic acids, hydrogen peroxide and bacteriocins were detected as main inhibition mechanisms. Characterization of culture supernatants from the bacteriocinogenic strains, CM135 and CM160 revealed a high resistance of antibacterial activity to temperature and pH, and a bactericidal mode of action against L. monocytogenes. Produced bacteriocins belonged to the Class IIa and sequencing of genes showed complete homology with mesentericin Y105. A study of the effect of the relative dose of pathogen and LAB on control of L. monocytogenes in wounds of Golden Delicious apples and Iceberg lettuce leaf cuts was performed. A comparison of the dose of bioprotective strain needed for a ten fold reduction of the viable pathogen concentration (ED(90)) revealed that strain CM160 was the most effective against L. monocytogenes. ED(90) values varied from 1.3.10(4) to 5.0.10(5) cfu.g(-1) or wound, at ranges of pathogen levels from 1.0.10(3) to 5.0.10(4) cfu.g(-1) of lettuce or wound of apple. The efficiency of the strains was also calculated as the ratio of the ED(90) value to the pathogen dose inoculated. The lowest ratio was found for strain CM160 at 5 to 50 cells of LAB per cell of pathogen. The strain offers potential application for prevention of the presence of L. monocytogenes in fresh fruit and vegetables.     

Genetic variation of Listeria monocytogenes isolates from domestic and imported foods in Japan

Phylogenetic analyses were carried out on a total of 118 Listeria monocytogenes isolates from foods or food processing environments, and 7 isolates from listeriosis patients in Japan to evaluate the genetic variation in the pathogen in this country. Isolates of serotypes 1/2a, 1/2b and 4b were mainly examined to assess the risk of exposure of humans to L. monocytogenes from foods in Japan. The nucleotide sequences of the part of the iap gene that contains the region encoding the threonine-asparagine repeat units were determined in order to construct phylogenetic trees of the isolates investigated. A phylogram showed high genetic diversity among lineage 2 isolates, while the lineage 1 isolates showed clonal characteristics. The results of the genetic analyses suggested the presence of rare putative lineage 3 isolates and epidemic clone I (ECI) isolates in foods in Japan. The results showed that ECI was also isolated from listeriosis patients. The genetic variation in L. monocytogenes in Japan reported here suggests the necessity of monitoring the pathogen in foods and environments in addition to surveillance of listeriosis patients.     

Potential of a nisin-containing bacterial cellulose film to inhibit Listeria monocytogenes on processed meats

A bacterially produced cellulose film containing nisin was developed and used in a proof-of-concept study to control Listeria monocytogenes and total aerobic bacteria on the surface of vacuum-packaged frankfurters. Bacterial cellulose pellicles were produced by Gluconacetobacter xylinus K3 in Corn Steep Liquor-Mannitol Medium and were subsequently purified before nisin was incorporated into them. Investigations into the effect of nisin concentrations and contact times on incorporation of nisin into cellulose films showed that the lowest nisin concentration and shortest time needed for production of an effective antimicrobial cellulose film were 625IUml(-1) and 6h, respectively. The active cellulose films produced under these conditions did not, however, significantly reduce L. monocytogenes populations on frankfurters (P>0.05) during refrigerated storage for 14 days as compared to the controls. Films produced using a higher concentration of nisin (2500IUml(-1)) with the same exposure time (6h) resulted in a significant (P<0.05) decrease in L. monocytogenes counts on frankfurters of approximately 2logCFUg(-1) after 14 days of storage as compared to the control. Both the above-mentioned films showed a similar effectiveness in reducing total aerobic bacterial populations as measured by total aerobic plate counts on frankfurters. For both films, total aerobic bacterial levels were significantly (P>0.05) reduced by approximately 3.3logCFUg(-1) after 14 days of storage as compared to control samples. Bacterial cellulose films were demonstrated in this study to have potential applicability as antimicrobial packaging films or inserts for processed meat products.     

Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target

A real-time PCR assay was designed to detect a 162-bp fragment of the ssrA gene in Listeria monocytogenes. The specificity of the assay for L. monocytogenes was confirmed against a panel of 6 Listeria species and 26 other bacterial species. A detection limit of 1-10 genome equivalents was determined for the assay. Application of the assay in natural and artificially contaminated culture enriched foods, including soft cheese, meat, milk, vegetables and fish, enabled detection of 1-5 CFU L. monocytogenes per 25g/ml of food sample in 30h. The performance of the assay was compared with the Roche Diagnostics 'LightCycler foodproof Listeria monocytogenes Detection Kit'. Both methods detected L. monocytogenes in all artificially contaminated retail samples (n=27) and L. monocytogenes was not detected by either system in 27 natural retail food samples. The method developed in this study has the potential to enable the specific detection of L. monocytogenes in a variety of food types in a time-frame considerably faster than current standard methods. The potential of the ssrA gene as a nucleic acid diagnostic (NAD) target has been demonstrated in L. monocytogenes. We are currently developing NAD tests based on the ssrA gene for a range of common foodborne and clinically relevant bacterial pathogens.     

Microbiological quality of retail cheeses made from raw, thermized or pasteurized milk in the UK

Two studies of retail fresh, ripened and semi-hard cheeses made from raw, thermized or pasteurized milk were undertaken in the UK during 2004 and 2005 to determine the microbiological quality of these products. Using microbiological criteria in European Commission Recommendations 2004/24/EC and 2005/175/EC, 2% of both raw, thermized (37/1819 samples) and pasteurized (51/2618 samples) milk cheeses were of unsatisfactory quality. Raw or thermized milk cheeses were of unsatisfactory quality due to levels of Staphylococcus aureus at 10(4)cfu g(-1), Escherichia coli at 10(5)cfu g(-1), and/or Listeria monocytogenes at 10(2)cfu g(-1), whereas pasteurized milk cheeses were of unsatisfactory quality due to S. aureus at 10(3)cfu g(-1) and/or E. coli at 10(3)cfu g(-1). Salmonella was not detected in any samples. Cheeses were of unsatisfactory quality more frequently when sampled from premises rated as having little or no confidence in management and control systems, and stored/displayed at above 8 degrees C. Raw or thermized milk cheeses were also more likely to be of unsatisfactory quality when they were unripened types, and pasteurized milk cheeses when they were: semi-hard types; from specialist cheese shops or delicatessens; cut to order. These results emphasize the need for applying and maintaining good hygiene practices throughout the food chain to prevent contamination and/or bacterial growth. Labelling of cheeses with clear information on whether the cheese was prepared from raw milk also requires improvement.     

单增李斯特菌感染RAW264.7细胞转录组差异表达分析

为了探讨单核细胞增生李斯特菌（Listeria monocytogenes,LM）感染前后RAW264.7细胞基因表达谱变化,筛选关键功能基因和通路,为探究其致病机制奠定基础,本研究以感染复数（MOI）10的LM 感染RAW264.7细胞6 h后,提取细胞RNA进行转录组测序,选取感染组与对照组相比差异倍数≥1.5且P<0.05的差异表达基因进行GO和KEGG富集分析,并通过RT-qPCR验证部分差异基因。转录组结果显示RAW264.7细胞在感染LM前后有1 782 个基因差异表达,其中上调为989,下调为793。GO分析结果主要涉及免疫系统过程、免疫反应、细胞程序性死亡调控等。KEGG分析结果显示,显著富集的通路有细胞因子-受体相互作用、肿瘤坏死因子信号通路、IL-17信号通路等。RT-qPCR验证试验结果显示,随机选择的11 个显著差异基因表达倍数趋势与测序结果一致。该研究为进一步深入研究LM的致病机制奠定了基础。     

基于磁性金属有机框架分离的纳米金比色法检测单核细胞增生李斯特菌

以磁性金属有机框架复合材料作为捕获探针,免疫功能化纳米金作为信号探针,建立一种纳米金比色法快速检测单核细胞增生李斯特菌的方法。结果表明：在最适条件下,单核细胞增生李斯特菌的可视化检出限为1.2×10³ CFU/m L,紫外光谱检测结果在1.2×10¹～1.2×10⁸ CFU/mL范围内有良好的线性关系(Y=0.519-0.043X,R²=0.978),检出限低至0.45 CFU/mL,各浓度梯度的批内变异系数在0.31%～1.30%之间。将单核细胞增生李斯特菌接种到鸡胸肉上进行检测,其加标回收率在91.1%～108.7%之间,变异系数不高于7.4%,且结果与平板计数法一致。本方法具有准确、灵敏、快速等特点,在食源性致病菌检测方面具有较好的应用前景。     

Modelling the cross‐contamination of Listeria monocytogenes in pork during bowl chopping

This study has developed a predictive model for the cross‐contamination of pork by Listeria monocytogenes during bowl chopping. The transfer rates of L. monocytogenes were measured in sixteen chopping scenarios based on practical work. Meanwhile, contaminated bowl chopper was cleaned with either a dry rag (DR), warm water (WW) or 70% ethanol + water (EW), respectively. It was showed that significant differences (P < 0.05) were observed among the three cleaning methods on the reduction of L. monocytogenes, the greatest log reduction being achieved by EW. Moreover, the model introduced by a previous study, predicting cross‐contamination of L. monocytogenes during meat slicing, was improved and validated in this study. Verification results showed that the improved model was acceptable for predicting L. monocytogenes cross‐contamination during pork chopping with coefficients of determination (R² > 0.82), accuracy factors (A_f < 1.44), bias factors (B_f < 1.42), and root mean square error (RMSE < 0.99). Furthermore, the modified model might provide an effective tool for assessing the risk of the cross‐contamination of meat products.     

Improved process for decontamination of whey by a continuous flow-through pulsed light system

The effect of a continuous flow-through pulsed light (PL) system on the inactivation of Listeria innocua was evaluated in different liquid substrates: distilled water, whey, diluted whey and skimmed whey. Reductions in L. innocua counts increased with number of pulses and total fluence. For similar total fluence, treatments consisting of a higher number of pulses but lower voltage were more effective in L. innocua inactivation due to a higher probability to expose cells to the incident light. Microbial inactivation by PL depended on the quantity of light transmitted in the range 230–290 nm by liquid substrates. Regardless of the limited effectiveness of PL for decontamination of whey, its shelf life during storage at 4 °C was extended by at least 7 days. In order to improve the antimicrobial effectiveness of PL, this treatment could be carried out at mild processing temperatures (60 °C), which was shown to exhibit a synergistic effect on the inactivation of L. innocua. This combined process appears as a promising technique to decontaminate complex substrates such as whey or to reduce the intensity of individual treatments to achieve the required level of microbial inactivation.