Efficacy of direct plating media for recovering Listeria monocytogenes from foods

Studies were done to evaluate 14 direct plating media for their suitability to recover Listeria monocytogenes strain Scott A from pasteurized whole milk, chocolate ice cream mix, Brie cheese and raw cabbage. Healthy cells were inoculated into foods to achieve viable populations of 10(2), 10(4), or 10(6) cells/ml(g). Bind's Acriflavine Agar, Trypaflavine Nalidixic Acid Serum Agar, Listeria Transport Enrichment Agar, Doyle and Schoeni Selective Enrichment Agar (DSSEA) and Modified DSSEA were not suitable for recovering L. monocytogenes from milk and Brie cheese and were therefore not evaluated as direct plating media for recovering the organism from ice cream mix and cabbage. McBride Listeria Agar (MLA), Gum Base Nalidixic Acid Tryptone Soya Agar (GBNTSA), Modified Despierres Agar (MDA) and Modified MLA (MMLA) performed best for recovering all inoculum populations from milk and ice cream mix. Enumeration of L. monocytogenes on several test media was complicated by the growth of large numbers of background microflora present in cabbage and Brie cheese, especially at the lowest test inoculum (10(2)/g). Generally, complete recovery of L. monocytogenes from Brie cheese and cabbage was attained on media when the inoculum population was greater than or equal to 10(4) cells/g. For Brie cheese, MLA, MDA, MMLA and Dominguez Rodriguez Isolation Agar were superior for recovering L. monocytogenes, while GBNTSA, DLEA, MDA and MMLA were best for recovering the organism from cabbage. Results of this experiment indicate that direct-plating procedures, without prior enrichment, can successfully be utilized for recovering L. monocytogenes from foods such as pasteurized milk and ice cream mix which contain low populations of background microflora. However, recovery of L. monocytogenes from foods such as raw cabbage and Brie cheese, which contain high populations of other microorganisms, was not satisfactory using the direct-plating procedures evaluated in this investigation.     

Consequences of the development of nisin-resistant Listeria monocytogenes in fermented dairy products

Wild Listeria isolates representing serovars found in artisanal cheeses commercialized in Asturias (northern Spain) were assessed for their susceptibility to several bacteriocins. Pediocin PA-1 was the most active bacteriocin followed by enterocin AS-48, nisin, and plantaricin C. However, some Listeria monocytogenes and Listeria innocua strains were already highly resistant to PA-1. Among the wild L. monocytogenes populations, the frequency of development of nisin resistance ranged from 10(-6) up to 10(-3), depending on the strain. Highly stable mutants with increased nisin resistance (two- to fourfold) were isolated and tested for potential cross-resistance to lysozyme, EDTA, and various NaCl concentrations and pH values. All mutants were cross-resistant to lysozyme but sensitive to EDTA. In contrast, no clear correlation could be established between nisin resistance and an altered susceptibility to NaCl or pH changes. Nisin-resistant variants were able to survive and even to multiply in milk fermented by a nisin-producing Lactococcus, but the growth of the wild-type strain was inhibited. The different phenotypes evaluated in this study are indicative of the unpredictability of the consequences of the development of nisin resistance in a dairy environment. This resistance should be considered when making a risk assessment of the long-term use of nisin to control L. monocytogenes.     

Inhibition of Listeria monocytogenes in dairy products using the bacteriocin-like peptide cerein 8A

The efficacy of the antimicrobial peptide cerein 8A to control the development of Listeria monocytogenes in milk and soft cheese was investigated. The addition of 160 AU ml(-1) cerein 8A to UHT milk resulted in a decrease of 3 log cycles in viable cells within the 14-day period at 4 degrees C. The viable counts of L. monocytogenes in pasteurized milk samples containing cerein 8A was lower than those observed in controls without bacteriocin. Addition of cerein 8A to Minas-type soft cheese caused a delay in the start of exponential growth phase, although similar counts were observed after day 6. When cerein 8A was used to control cheese surface contamination by L. monocytogenes, a decrease of 2 log cycles in viable counts of cerein-treated samples was observed during 30 days at 4 degrees C. This antimicrobial peptide shows potential use as a biopreservative for application in dairy products.     

Evaluation of dairy and food plant sanitizers against Salmonella typhimurium and Listeria monocytogenes

Six commonly used dairy and food plant sanitizers were evaluated against Salmonella typhimurium and Listeria monocytogenes. Of these six, two were acid anionic sanitizers, one contained a quaternary ammonium compound, one was based on active iodine, and two contained active chlorine. Of the last two, one contained hypochlorite and the other contained active chlorine in organic form. The chlorine-based sanitizers were effective at 100 ppm of available chlorine against both these organisms. The sanitizer containing iodine was effective at 12.5 and 25 ppm titratable iodine against L. monocytogenes and S. typhimurium, respectively. The acid anionic sanitizers were effective at 200 ppm of active agent against both the bacteria, and the quaternary ammonium-based sanitizer was effective at 100 and 200 ppm of active compound against L. monocytogenes and S. typhimurium, respectively. The sanitizer containing iodine at 12.5 and 25 ppm of titratable iodine showed activity equivalent to 50 and 200 ppm of available chlorine, respectively, against L. monocytogenes and 100 and 200 ppm of available chlorine, respectively, against S. typhimurium.     

Survival of Listeria monocytogenes in vanilla-flavored soy and dairy products stored at 8 degrees C

The survival of Listeria monocytogenes V37 in vanilla-flavored yogurt (low-fat and nonfat) and soy milk (low-fat and Plus) stored at 8 degrees C for 31 days was investigated. Commercial samples of yogurt and soy milk were used. These samples were inoculated with either 10(4) or 10(7) CFU of L. monocytogenes per ml. Sampling was carried out every 3 to 4 days initially and was then carried out weekly, for a total storage time of 31 days. Each time a sample was collected, the pH of the sample was measured. After 31 days, low-fat plain, low-fat vanilla, and nonfat plain yogurt samples inoculated with 10(4) CFU/ml showed 2.5-log reductions in viable cell populations, and nonfat vanilla yogurt showed a 3.5-log reduction. For yogurt inoculated with 10(7) CFU/ml, reductions of 2.5 log CFU/ml were observed for plain low-fat and nonfat yogurts, and reductions of 5 log CFU/ml were observed for vanilla-flavored low-fat and nonfat yogurts. In vanilla-flavored and plain low-fat and Plus soy milk samples, cell counts increased from 10(4) and 10(7) CFU/ml to 10(9) CFU/ml at 7 and 3 days of storage, respectively, at 8 degrees C. Coagulation in soy milk samples was observed when the cell population reached 10(9) CFU/ml. In soy milk, the L. monocytogenes population did not change for up to 31 days. Vanillin had an inhibitory effect on L. monocytogenes in yogurt but not in soy milk.     

乳制品常见致病菌选择性共增菌技术研究

以乳制品常见致病菌大肠杆菌、沙门氏菌和金黄色葡萄球菌为目标菌,研究6种基础培养基、2种培养模式、20种培养基添加剂对增菌的影响,并以无显著抑制效应的添加剂对背景微生物进行了抑菌效应研究,建立了一种选择性富集目标菌的ESS培养基。使用该培养基37℃静止培养16 h,可实现目标菌的共增菌,对可能影响目标菌检测的乳制品中其他致病菌有一定抑制效果。     

Modelling the competitive growth of Listeria monocytogenes and Listeria innocua in enrichment broths

The overgrowth of Listeria innocua in enrichment broths designed for the isolation of Listeria monocytogenes is believed to result from two factors: a selective growth advantage of L. innocua, and/or an inhibitory interspecies interaction. The generation times of 13 isolates of L. innocua and L. monocytogenes were determined in Brain Heart Infusion (BHI) and a variety of enrichment media. No significant differences were found in growth characteristics between either species in the various media, suggesting that the growth advantage of L. innocua in enrichment media was not as significant as previously described. Kinetic analysis of mixed cultures of L. monocytogenes and isolates of L. innocua producing a variety of inhibitory activities demonstrated the possibility of an inhibitory interaction between these two species resulting in the overgrowth of the enrichment culture with L. innocua. Modelling the evolution of the ratio between two populations in an enrichment process was used to analyze the impact of a selective growth advantage in L. innocua in an enrichment process for growth of L. monocytogenes. These findings support the widely held view that an overgrowth of L. innocua in the enrichment process can result from both a selective growth advantage as well as the production of inhibitory compounds. From a practical perspective, these interactions can result in an increase in false negatives.     

Comparison of sampling procedures for recovery of Listeria monocytogenes from stainless steel food contact surfaces

A number of techniques exist for microbiological sampling of food processing environments in food industries. In the present study the efficacies of nine sampling procedures for the recovery of Listeria monocytogenes from food contact surfaces, including a new sampling device consisting of a miniroller, were evaluated and compared. A stainless steel table was inoculated with L. monocytogenes strain 935 (serovar 4b, human origin) and L. monocytogenes strain 437/07 (serovar 1/2b, food origin), at 10(5) CFU/100 cm(2). L. monocytogenes strain 935 was best recovered with the minirollers (recovery of up to 6.27%), while poor recoveries (<0.30%) were obtained with the towel (one-ply composite tissue), alginate swab, metallic swab, and Petrifilm methods. In the case of L. monocytogenes strain 437/07 the replicate organism detection and counting (RODAC) ALOA contact plates yielded the best recoveries (4.15%), followed by the minirollers (up to 1.52%). Overall, recovery percentages with the minirollers were higher with stomacher homogenization than with Vibromatic agitation. The recovery percentages obtained for the Listeria strain of human origin were higher than those obtained with the food strain for all sampling procedures except Petrifilm and RODAC ALOA. With the miniroller device coated with wool fiber, the recovery of L. monocytogenes can be improved from 2 to 17 times over recoveries obtained with the sponge and cotton swab. This is the first report of a miniroller device for microbiological sampling in the available literature. The novel sampling procedure is convenient to apply on surfaces, is cost-effective, and results in better recovery of L. monocytogenes than do the conventional methods.     

Listeria monocytogenes: Its importance in the dairy industry

Recent outbreaks of listeriosis associated with consumption of milk products contaminated with Listeria monocytogenes have focused attention on the importance of food-borne transmission of this disease. This review outlines current knowledge of the incidence of L monocytogenes in raw milk and its incidence in, and ability to survive, the manufacturing process for other dairy products—notably soft cheeses. Discrepancies in results obtained by researchers concerning growth of the organism at refrigeration temperatures and the bacterium's ability to survive pasteurisation are discussed.     

Listeria monocytogenes isolated from cold-smoked fish products in Osaka City, Japan

Listeria monocytogenes contamination of ready-to-eat seafood products commercially available in Osaka was examined between 1999 and 2000. L. monocytogenes was isolated from 12 (13%) of the 95 products tested. All positive samples were from cold-smoked fish with 9 being obtained during the summer. Thirteen isolates of L. monocytogenes were typed by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction (PCR)-based typing methods. Isolates of the same serotype originating from the same manufacturer gave similar DNA profiles, irrespective of the type of sample or date of isolation. The finding suggest that persistent strains in each manufacturing facility proliferate during the summer and contaminate products during manufacturing processes.     

Characterisation of anti-Listeria monocytogenes bacteriocins from Enterococcus faecium strains isolated from dairy products

Bacteriocin-producing lactic acid bacteria were isolated from 34 samples of dairy products. Nine bacteriocin producers were phenotypically and genotypically identified as Enterococcus faecium . By means of PCR-techniques, enterocin A was characterised in all of the nine bacteriocin-producing Enterococcus isolates. Enterocin-producing lactic acid bacteria were the most abundant in dairy products collected from different areas in Iran. Maximum bacteriocin production by Enterococcus faecium strains was detected in the stationary phase of growth. Bacteriocins produced by all isolates were found to have anti-listerial activity in sterile milk. The purified bacteriocins were identified as <6.5 kDa peptide by sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE). The molecular weights of bacteriocins were found to be the same in all strains. This bacteriocin might be useful as a natural preservative.     

Comparison of growth kinetics for healthy and heat-injured Listeria monocytogenes in eight enrichment broths

Detection of Listeria in food products is often limited by performance of enrichment media used to support growth of Listeria to detectable levels. In this study, growth curves were generated using healthy and heat-injured Listeria monocytogenes strain F5069 in three nonselective and five selective enrichment broths. Nonselective enrichment media included the current Food and Drug Administration Bacteriological Analytical Manual Listeria enrichment broth base (BAM), Listeria repair broth (LRB), and Trypticase soy broth. Selective enrichment media included BAM with selective agents and LRB with selective agents, BCM L. monocytogenes preenrichment broth, Fraser broth, and UVM-modified Listeria enrichment broth. The Gompertz equation was used to model the growth of L. monocytogenes. Gompertz parameters were used to calculate exponential growth rate, lag-phase duration (LPD), generation time, maximum population density (MPD), and time required for repair of injured cells. Statistical differences (P < 0.05) in broth performance were noted for LPD and MPD when healthy and injured cells were inoculated into the broths. With the exception of Fraser broth, there were no significant differences in the time required for the repair of injured cells. Results indicate that the distinction between selective and nonselective broths in their ability to grow healthy Listeria and to repair sublethally injured cells is not solely an elementary issue of presence or absence of selective agents.     

A comparison of two procedures for the isolation of Listeria monocytogenes from raw chickens and soft cheese

A cold enrichment method and a modified FDA procedure were compared for the isolation of Listeria monocytogenes from raw chickens and soft cheese. L. monocytogenes was isolated from a total of 23 of 222 cheese and 70 of 160 chicken samples by either one or both methods. Neither method alone yielded all isolates from the two food types. Only 12 cheese and 13 chicken samples were shown to be positive by both methods, although the serotypes isolated were not always identical. On some occasions one method yielded L. monocytogenes while the other produced a different Listeria sp. Reasons for differences in the performance of the two procedures and various points of technical interest are discussed.     

单增李斯特菌鞭毛蛋白提取方法的研究

本实验对单增李斯特菌鞭毛蛋白提取方法进行研究,分别在23℃和37℃的条件下对单增李斯特菌(血清型IVb)进行扩增培养,发现23℃下单增李斯特菌产鞭毛的能力更强。运用酸解法处理单增李斯特菌后,分别通过超速离心法和硫酸铵沉淀法对鞭毛蛋白进行纯化,SDS-PAGE 电泳以及电镜的实验结果表明,超速离心较硫酸铵沉淀法简便、耗时短、纯度高、产量高,适用于单增李斯特菌鞭毛蛋白的提取。     

Assessment of different selective agar media for enumeration and isolation of Listeria from dairy products

Different selective agar media were compared for the recovery and isolation of five species of Listeria from raw milk and cheese. The selective media examined were Beerens medium, MacBride medium and that described by Dominguez et al. (1984) with 6 mg/l acriflavine, listeria selective agar medium (LSAM), and LSAM with 12 mg/l acriflavine (LSAM X 2A); a non-selective yeast glucose Lemco agar was included for comparison. When the difference between listeria and the natural microflora of raw milk and cheese was 10(2) cfu/ml, listeria could be isolated by direct plating on all media tested. When it was lower than 10(3)-10(4) cfu/ml, listeria were isolated by direct plating only on LSAM and LSAM X 2A. When the difference was greater than 10(4) cfu/ml, a previous enrichment was necessary to isolate them. LSAM and LSAM X 2A media performed better than the other media tested for isolating listeria by direct plating and improved their isolation from dairy products. This superior performance was evaluated by the ability of these media to support colony formation of different species of Listeria tested, the easy recognition of these colonies from those formed by other microorganisms and by their capacity to inhibit the natural microflora of these foods.     

散装熟肉制品中单增李斯特菌污染状况分析

目的分析散装熟肉制品中单增李斯特菌污染状况。方法根据GB 4789.30-2016《单核细胞增生李斯特氏菌检验》规定的方法对散装熟肉制品的加工用原料、生产环境、各加工环节中产品以及不同销售环境下产品中的单增李斯特菌进行定性和定量检测。结果原料肉的总体带菌率达到21%;生产环境中第三区(远离食品接触面的区域)检出率为2%,其余区域未检出;各加工环节中产品单增李斯特菌检测结果均小于10CFU/g;不同销售环境下产品中单增李斯特菌检测结果小于10 CFU/g的比例为93%,处于10～50 CFU/g之间的样本占比6%,大于50 CFU/g的占比为1%。结论原料散装熟肉制品中单增李斯特菌污染的主要来源,蒸煮等加工环节能有效地杀灭单增李斯特菌。销售环境也关系到散装熟肉制品单增李斯特菌的污染程度,对于未包装的产品,专卖店优于农产品市场。     

Evaluation of selective enrichment PCR procedures for Yersinia enterocolitica

Four enrichment PCR protocols for detecting unlysed cells of pathogenic Yersinia enterocolitica were studied. First, the probability of detecting Y. enterocolitica cells of known concentrations by a multiplex PCR assay was determined, and it was found to follow a logistic regression model. From this model, the probability of detecting Y enterocolitica at a specific concentration could be estimated; for example, the detection probability of 10(4) CFU/ml was estimated to be 85.4%. The protocols were evaluated on enrichment cultures inoculated with 10(2) CFU/ml Y. enterocolitica and 10(2)-10(6) CFU/ml of a defined background flora. For each protocol, the time for sample withdrawal and the presence of background flora were studied with respect to PCR detection. The optimal point in time of sample withdrawal was found to be different for each protocol employed. Early detection was favoured by concentrating the target cells, and the most rapid PCR detection of Y. enterocolitica was achieved with enrichment in Yersinia-PCR-compatible-enrichment (YPCE) medium for 3 h at 25 degrees C, followed by a centrifugation prior to PCR analysis. For detection of Y. enterocolitica in the presence of high concentrations (10(6) CFU/ml) of background flora, a long incubation time followed by density centrifugation and a dilution step was most successful. The protocol that gave the most reliable PCR detection in the presence of 10(6) CFU/ml background flora included 24 h incubation in Yersinia-selective-enrichment (YSE) broth at 25 degrees C, followed by Percoll density centrifugation, and a 100 times dilution prior to PCR analysis.     

Comparison of 3M Petrifilm environmental Listeria plates against standard enrichment methods for the detection of Listeria monocytogenes of epidemiological significance from environmental surfaces

ABSTRACT:  Environmental monitoring using sensitive methods for detection and elimination of harborage sites of Listeria monocytogenes is key to the control of this organism. The 3M™ Petrifilm™ Environmental Listeria (EL) Plate—a no enrichment method—was compared with the USDA/FSIS, modified USDA/FSIS (mUSDA), and ISO methods for detection/recovery of L. monocytogenes on 4 environmental surfaces (brick, dairy board, stainless steel, and epoxy resin). The efficacy of 3 sampling devices including the Microbial-Vac system^®, environmental sponge, and 3M Quick swab in recovering epidemiologically significant strains of uninjured and sublethally injured L. monocytogenes from environmental surfaces was evaluated. Environmental surfaces were inoculated with Listeria to obtain final cell densities of approximately 10 to 100 CFU/100 cm². The surfaces were then sampled and processed. For all methods, percent recovery (% samples where Listeria was detected) was significantly higher ( P < 0.05) for uninjured cells (75% to 100%) compared to injured cells (58.9% to 81.1%). The Petrifilm EL Plate method efficiently recovered both low level and injured Listeria populations from environmental test surfaces when used in conjunction with environmental sponge and the 3M Quick swab sampling. The mUSDA method was superior to all other methods for recovering both uninjured (100% recovery) and injured L. monocytogenes (80.8% to 81.1% recovery). Sponges and swabs were equally effective in recovering uninjured and injured Listeria and were significantly different ( P < 0.05) from the Microbial-Vac system. The findings indicate that both mUSDA and Petrifilm EL Plate methods can be used for the detection of potentially injured Listeria on food processing environmental surfaces.     

RapidChek检测食品中单增李斯特菌的方法评价

目的评价RapidChek法检测食品中单增李斯特菌的检测效果并验证。方法采用t检验,比较RapidChek方法与GB 4789.30-2016方法的培养基增菌效果。依据ISO 16140:2003《食品和动物饲料微生物学-可替代方法的验证方案》,比较RapidChek检测方法与GB 4789.30-2016检测方法的检测效果,涉及的性能指标有相对准确性、相对特异性和相对灵敏度、包含性和排他性。结果 RapidChek检测方法增菌培养基效果优于GB 4789.30-2016方法增菌培养基LB_1。根据RapidChek检测方法、GB 4789.30-2016培养基+RapidChek试纸条方法、RapidChek培养基+GB 4789.30分离鉴定方法的统计检验得出,3种方法与参照方法在相对准确性、相对特异性和相对灵敏度方面无显著性差异。在包含性和排他性方面,RapidChek检测方法与GB 4789.30-2016检测方法的检测结果均一致。结论在所验证的指标上,RapidChek检测方法(包括与GB4789.30-2016组合的方法)与GB 4789.30-2016方法无差异。