Effect of a previous heat shock on the thermal resistance of Listeria monocytogenes and Pseudomonas aeruginosa at different pHs

In this work we study the effect of heat shocks of various durations up to 60 min, at different temperatures between 35 and 45 degrees C, in media of pH 4.0, 5.5 and 7.4 on the heat resistance of Listeria monocytogenes and Pseudomonas aeruginosa. The pattern of survival curves after heat treatment did not change with the application of a previous heat shock. However, the kinetics of inactivation was different for the two microorganisms studied. Whereas the inactivation of L. monocytogenes was similar to an exponential function of heating time and therefore straight survival curves were obtained, survival curves corresponding to P. aeruginosa showed convex profiles. All survival curves obtained in this investigation were fitted to Weibull-based Mafart equation: log(10)S(t)=-(t / delta)(p). The magnitude of the heat shock induced thermotolerance increased with treatment medium pH. At pH 7.4 the increase in heat tolerance depended on the duration and temperature of the heat shock. On the contrary, at pH 5.5 and pH 4.0, the heat-shock temperature did not exert any effect. The observed maximum delta values increased 2.3, 4.0 and 9.3 fold for L. monocytogenes, and 1.3, 2.1 and 8.4 fold for P. aeruginosa, at pH 4.0, 5.5 and 7.4, respectively. This research has proven that Mafart equation allows studying and quantifying the effect of heat shocks on bacterial heat resistance.     

Heat and acid tolerance of Listeria monocytogenes after exposure to single and multiple sublethal stresses

The majority of published studies on the adaptive heat or acid tolerance response of Listeria monocytogenes have been performed with a single strain exposed to a single adaptation treatment; however, in food ecosystems, microorganisms commonly exist as multi-species communities and encounter multiple stresses, which may result in "stress hardening". Therefore, the present study evaluated the adaptive responses to heat (52, 57 and 63 degrees C) or lactic acid (pH 3.5) of a 10-strain composite of L. monocytogenes meat and human isolates at stationary phase, following exposure to combinations of osmotic (10% NaCl), acidic (pH 5.0 with HCl) and thermal (T; 46 degrees C) stresses, sequentially or simultaneously within 1.5h, in tryptic soy broth with 0.6% yeast extract (TSBYE). All treatments induced adaptive responses on L. monocytogenes at 57 degrees C, while no such cross-protection was observed at 52 and 63 degrees C. Survivor curves at 57 degrees C appeared convex with profound shoulders determined by a Weibull model. The highest thermotolerance was observed after combined exposure to acid and heat shock (pH-T), followed by exposure to osmotic shock, and by the combination of osmotic with heat shock (NaCl-T). Regarding acid tolerance, prior exposure to low pH, pH-T, or a combination of NaCl, pH and T resulted in a marked increase of resistance to pH 3.5, showing concave inactivation curves with tails at higher levels of survivors (log(10)CFU ml(-1)) than the control cultures. The sequence of exposure to sublethal stresses did not affect the thermotolerance of L. monocytogenes, whereas simultaneous exposure to most multiple stresses (e.g., NaCl-pH-T, NaCl-T and NaCl-pH) resulted in higher survivors of L. monocytogenes at pH 3.5 than exposure to the same stresses sequentially. The results indicate that combinations and sequences of sublethal hurdles may affect L. monocytogenes acid and heat tolerance, especially in acidic environments with mild heating or in low moisture environments.     

环境因素对单增李斯特菌inlB毒力基因表达的影响

针对单增李斯特菌毒力基因inl B设计五对引物,从中筛选出一对特异性强的引物,利用反转录荧光定量PCR技术来考察毒力基因表达的强弱。在不同环境因素条件下培养单增李斯特菌,分别提取菌体RNA,继而合成c DNA,以管家基因16S r RNA为内参基因,利用循环阈值(Ct)的变化计算inl B基因的相对表达量。结果表明,毒力基因inl B在温度为15℃、p H为6、氯化钠浓度为1.5%~2.0%、蔗糖浓度为0.75%~1.25%时能高效表达;温度为0℃和45℃、p H为4和9、氯化钠浓度为3.5%以上以及蔗糖浓度为0%~0.25%和2.25%以上时,对毒力基因inl B的表达起明显的抑制作用;在p H为6的条件下,酸的种类对毒力基因inl B的表达影响不大。      

The VIT technology for rapid detection of Listeria monocytogenes and other Listeria spp

Detection of Listeria monocytogenes is generally performed in a two-step cultural enrichment process and takes on average 1 week until the biochemical identification of a L. monocytogenes suspicious colony is completed. However, food processing companies depend increasingly on test methods, which attempt to generate results comparable to standard methods but in reduced time-frame and which allow to release produced batches dependent on such results. In the present study, the vermicon identification technology (VIT), a rapid commercial test system using fluorescently labelled gene probes, was compared to a cultural standard method. In total, 298 naturally contaminated samples were analysed. The sensitivity and the specificity of the VIT system were 100% for the detection of L. monocytogenes and 97.1% and 100%, respectively, for the detection of the genus Listeria.     

SigB plays a major role in Listeria monocytogenes tolerance to bile stress

The ability of Listeria monocytogenes to tolerate high levels of bile stress is critical to its successful infection and colonization in the human gastrointestinal tract. L. monocytogenes encodes bile salt hydrolase by a bsh gene which plays a significant role in hydrolyzing high concentrations of bile salt when L. monocytogenes grows under hypoxemic condition. As the bsh promoter contains consensus SigB and PrfA binding sites, we investigated the role of SigB (σ^B) and PrfA in L. monocytogenes tolerance against bile stress by comparing the survival of isogenic deletion mutants of L. monocytogenes EGD_ΔsigB, EGD_ΔprfA and EGD_ΔprfAΔsigB with their parent strain EGD at high levels of bile salt. Our results show that the sigB deletion significantly reduced the MICs of bile salt for EGD_ΔsigB and EGD_ΔprfAΔsigB (2.6% and 2.2% vs 3.5% in wild type strain EGD), while the growth rates of these two sigB deletion mutants (EGD_ΔsigB and EGD_ΔprfAΔsigB) were affected the most in the presence of 3% bile salt. Pre-exposure to alkali (pH 9.0) and osmotic (0.3 M NaCl) stresses for a short period of time (30 min) resulted in improved growth of L. monocytogenes as well as its prfA-sigB isogenic mutants even under sublethal concentrations of bile salt, while pre-exposure to acid pH (pH 4.5) failed to provide cross-protection against subsequent bile stress. Furthermore, the sigB gene had more remarkable influence than that of prfA on bsh expression, as much lower levels of bsh transciption were observed in EGD_ΔsigB and EGD_ΔprfAΔsigB. Meanwhile, bsh expression in the deletion mutants did not respond to elevated levels of bile salt. These data indicate that σ^B might play a crucial role in Listeria survival under bile salt environment in the gastrointestinal tract before its successful colonization, invasion and intracellular propagation.     

Predicting heat inactivation of Listeria monocytogenes under nonisothermal treatments

The aim of this study was to find a model that accurately predicts the heat inactivation of Listeria monocytogenes (ATCC 15313) at constantly rising heating rates (0.5 to 9 degrees C/min) in media of different pH values (4.0 to 7.4). Survival curves of L. monocytogenes obtained under isothermal treatments at any temperature were nearly linear. Estimations of survival curves under nonisothermal treatments obtained from heat resistance parameters of isothermal treatments adequately fit experimental values obtained at pH 4.0. On the contrary, survivors were much higher than estimations at pH 5.5 and 7.4. The slower the heating rate and the longer the treatment time, the greater the differences between the experimental and estimated values. An equation based on the Weibullian-like distribution, log S(t) = (t/delta)p, accurately described survival curves of L. monocytogenes obtained under nonisothermal conditions within the range of heating rates investigated. A nonlinear relationship was observed between the scale parameter (delta) and the heating rate, which allowed the development of an equation capable of predicting the inactivation rate of L. monocytogenes under nonisothermal treatments at pH 5.5 and 7.4. The model predictions were a good fit to the measured data independent of the magnitude of the thermotolerance increase. This work might contribute to the increase in safety of those food products that require long heating lag phases during the pasteurization process.     

Heat shock and cold shock treatments affect the survival of Listeria monocytogenes and Salmonella Typhimurium exposed to disinfectants

The foodborne pathogens Listeria monocytogenes and Salmonella Typhimurium were subjected to heat shock at 48°C for 10 and 30 min, respectively, and then cold shocked at 15°C for 3 h. The effect of these shocks on the viability of test organisms exposed to chlorine dioxide and quaternary ammonium compounds was then determined. After exposure to the disinfectants, the viable population of each test organism, regardless of heat shock or cold shock treatment, decreased as the exposure period was extended. Both heat shock and cold shock treatments reduced the susceptibility of L. monocytogenes to both disinfectants at 25°C. However, for Salmonella Typhimurium, exposure to the chlorine dioxide disinfectant or quaternary ammonium compounds at 25°C significantly reduced (P < 0.05) survival of heat-shocked cells but significantly increased (P < 0.05) survival of cold-shocked cells compared with control cells. Survival of both L. monocytogenes and Salmonella Typhimurium generally was reduced after exposure to disinfectants at 40°C compared with 25°C.     

Stress response of Listeria monocytogenes isolated from cheese and other foods

The responses to pH and sodium chloride of four strains of Listeria monocytogenes isolated from Portuguese cheese, with a sodium chloride concentration of about 2% (w/v) and a pH value from 5.1 to 6.2, were studied. Two isolates from meat and two clinical isolates related to food-borne listeriosis, in which the implicated food product had about 2-3.5% (w/v) sodium chloride, also were studied. The effect of temperature on pH and sodium chloride sensitivity was also determined. The results show that natural isolates vary in response to these stresses and the data were often at variance with previously published data. Strains varied in sensitivity to low pH and to high sodium chloride concentration but the cheese isolates tended to be more resistant. A lower temperature was associated with a decrease in resistance to low pH and to sodium chloride. All strains showed an acid tolerance response induction when grown at pH 5.5 and although the time required for maximum induction of the response varied between strains, 2 h of acid adaptation, at least, was necessary which is longer than previously reported. Some strains showed an osmotolerance response after incubation in 3.5% (w/v) sodium chloride. Osmoadaptation, in addition to inducing an osmotolerance response, also induced cross-protection against acid shock conditions (pH 3.5). The acid tolerance response also induced a cross-protection against osmotic shock conditions (20% (w/v) sodium chloride). In some cases there was a relationship between the degree of resistance and adaptation, but usually the behaviour of a particular strain was independent of the conditions from which it was isolated.     

单核细胞性李斯特菌基因dal的敲除及其生物学特性初步分析

在单核细胞性李斯特菌(Listeria monocytogenes)野生株EGDe act A及inl B双基因缺失株(EGDeΔact AΔinl B)的基础上,利用同源重组的方法进一步构建了缺失营养基因dal的菌株(EGDe Δact AΔinl BΔdal),并对该缺失菌株生长状态、毒力基因表达水平、生物被膜的形成量及细胞侵袭等方面作进一步分析。结果显示,37℃摇床培养6 h后,缺失株的菌浓度显著低于EGDe Δact AΔinl B(P0.001),培养基中补充D-丙氨酸的缺失株生长速率与亲本株相比无显著差异;实时荧光定量聚合酶链式反应结果显示,缺失株的sig B基因表达水平变化最明显(P0.01),约下调90%;缺失株生物被膜形成量显著增加(P0.05),培养基补充D-丙氨酸后缺失株生物被膜的生成量与亲本株相比无差异;对Coca-2细胞的侵袭无影响,表明该基因对细菌生长能力及生物被膜形成具有重要的调控作用,并不影响菌株对细胞的侵袭力。此缺失株的构建为进一步研究基因dal的功能提供了理论支持。     

Antimicrobial Effect of Pressurized Carbon Dioxide on Listeria monocytogenes

Listeria monocytogenes was inactivated by carbon dioxide at 35 and 45°C under pressures of 70.3 and 210.9 μg/cm². Inactivation rates were sensitive to temperature and pressure. Other factors such as pH, moisture content, and environmental conditions of cell growth also influenced the effectiveness of CO₂ treatment. Bacteria were more difficult to inactivate when they were suspended in the medium with fat or oil, which may have protected the cells from penetration by CO₂. Fat in growth medium where Listeria monocytogenes cells were inoculated apparantly increased their resistance. Several methods may be useful for increasing inactivation rates.     

Adaptive tolerance to phenolic biocides in bacteria from organic foods: Effects on antimicrobial susceptibility and tolerance to physical stresses

The aim of the present study was to analyze the effects of step-wise exposure of biocide-sensitive bacteria from organic foods to phenolic biocides triclosan (TC) and hexachlorophene [2,2′-methylenebis(3,4,6-trichlorophenol)] (CF). The analysis included changes in the tolerance to the biocide itself, the tolerance to other biocides, and cross-resistance to clinically important antibiotics. The involvement of efflux mechanisms was also studied as well as the possible implication of modifications in cytoplasmic membrane fluidity in the resistance mechanisms. The influence of biocide tolerance on growth capacity of the adapted strains and on subsequent resistance to other physical stresses has also been analyzed. Repeated exposure of bacteria from organic foods to phenolic biocides resulted in most cases in partially increased tolerance to the same biocide, to dissimilar biocides and other antimicrobial compounds. Nine TC-adapted strains and six CF-adapted strains were able to develop high levels of biocide tolerance, and these were stable in the absence of biocide selective pressure. Most strains adapted to TC and one CF-adapted strain showed significantly higher anisotropy values than their corresponding wildtype strains, suggesting that changes in membrane fluidity could be involved in biocide adaptation. Exposure to gradually increasing concentrations of CF induced a decrease in heat tolerance. Biocide adaptation had no significant effects of gastric acid or bile resistance, suggesting that biocide adaptation should not influence survival in the gastrointestinal tract.     

Incidence and susceptibility to antimicrobial agents of Listeria spp. and Listeria monocytogenes isolated from poultry carcasses in Porto,Portugal

The occurrence of Listeria spp. and Listeria monocytogenes in 63 samples of Portuguese poultry carcasses obtained from two local butcher shops and one canteen in the city of Porto, Portugal, and the susceptibility of these bacteria to antimicrobial agents allowed for use in human or animal therapeutics were evaluated. All poultry samples were contaminated with Listeria spp., and L. monocytogenes was isolated from 41% (26 of 63) of the samples. Other Listeria species, including L. innocua, L. welshimeri, and L. seeligeri, were also isolated from poultry samples. A multiplex polymerase chain reaction method was used for the identification of all of the Listeria isolates; this method showed total conformity with the conventional method of biochemical identification and proved to be more reliable, faster, and less arduous. In addition, high percentages of Listeria spp. (84%) and L. monocytogenes (73%) isolates were found to be resistant to one or more antimicrobial agents of different groups, and 12 different resistance profiles were recorded. The frequency of the resistance of L. monocytogenes isolates to enrofloxacin and clindamycin is notable. The results of this study suggest a high incidence of L. monocytogenes on Portuguese poultry products available for consumers and indicate that poultry could be a potential vehicle of foodborne infections due to strains of L. monocytogenes that are resistant to antimicrobial agents.     

Effect of sodium lactate on thermal inactivation of Listeria monocytogenes and Salmonella in ground chicken thigh and leg meat

The effect of sodium lactate on thermal inactivation D- and z-values of Listeria monocytogenes and Salmonella was determined for chicken thigh and leg meat. At 55 to 70 degrees C, the D-value of L. monocytogenes in ground chicken thigh and leg meat with the addition of 4.8% sodium lactate (4.8 g sodium lactate per 100 g of meat) was 53 to 75% higher than that in the meat without sodium lactate. No significant difference was found for the D-values of Salmonella at 55 to 70 degrees C between the meat with and that without sodium lactate (4.8%. wt/wt). The z-values of both L. monocytogenes and Salmonella were not affected by sodium lactate (4.8%). The results from this study are useful for predicting thermal process lethality of L. montocytogenes and Salmonella in formulated chicken thigh and leg meat products.     

硫醚类香料对金黄色葡萄球菌和单增李斯特菌的抑制作用

选取10种常见的硫醚类香料,通过测量其抑菌圈直径来研究各硫醚类香料对金黄色葡萄球菌和单增李斯特菌的抑制能力,并分析硫醚香料的结构与抑菌能力的关系。筛选出抑菌能力最强的硫醚类香料,利用反转录荧光定量聚合酶链式反应来考察其对金黄色葡萄球菌耐热核酸酶nuc基因表达水平的影响。结果显示:二烯丙基二硫醚(DADS)、甲基糠基二硫醚(MFDS)和二烯丙基硫醚(DAS)对两种革兰氏阳性菌的抑菌效果较好,且抑菌能力为DADS＞MFDS＞DAS。DADS相较于其它9种硫醚类香料抑菌效果最好,对金黄色葡萄球菌和单增李斯特菌的最低抑制浓度(MIC)分别为312.5 mmol/L和1.25 mol/L。当DADS的浓度为亚抑制浓度时(1/16MIC、1/8MIC、1/4MIC、1/2MIC),均能显著降低金黄色葡萄球菌nuc基因的表达水平(P＜0.01)。以上结果说明:烯丙基和二硫键的存在能够增强硫醚类香料的抑菌能力,并且对抑制金黄色葡萄球菌毒力发挥重要作用。     

融合魏斯氏菌对单增李斯特菌生物膜形成的影响

从东北传统酸菜的12株乳酸菌中筛选对单增李斯特菌有较强拮抗作用的低温生长菌株,并分析其对单增李斯特菌生物膜形成的影响.首先测定乳酸菌菌株的低温生长特征和抑菌活性;然后采用定性法和定量法分析乳酸菌菌株对单核细胞增生李斯特菌生物膜形成和形态结构的影响;最终通过生理生化和16S rDNA测序对乳酸菌菌株进行鉴定.结果表明:菌...     

Susceptibility of Vibrio parahaemolyticus to various environmental stresses after cold shock treatment

Vibrio parahaemolyticus was subjected to cold shock treatment at 20 or 15 degrees C for 2 or 4 h. The effect of cold shock on the survival of V. parahaemolyticus subjected to subsequent low temperature (5 and -18 degrees C) and other adverse conditions (47 degrees C, 6 ppm crystal violet, 1000 ppm H(2)O(2), 25 mM acetic acid and 25 mM lactic acid) was investigated. Regardless of the cold shock treatment, survival of V. parahaemolyticus increased when stored at 5 or -18 degrees C, while no increase in survival was noted for cells cold shocked in the presence of chloramphenicol. Cold shock treatment under the conditions tested, in general, enabled V. parahaemolyticus cells to survive better following subsequent challenge by crystal violet, while the cold-shocked organism was more susceptible to high temperature (47 degrees C), H(2)O(2) and organic acids (lactic and acetic acid) than the non-shocked cells. Furthermore, the temperature and time of the cold shock treatment affected the cold shock response of V. parahaemolyticus.     

A predictive model for heat inactivation of Listeria monocytogenes biofilm on stainless steel

Heat treatment of potential biofilm-forming sites is sometimes used for control of Listeria monocytogenes in food processing plants. However, little information is available on the heat treatment required to kill L. monocytogenes present in biofilms. The purpose of this study was to develop a predictive model for the heat inactivation of L. monocytogenes in monoculture biofilms (strains Scott A and 3990) and in biofilms with competing bacteria (Pseudomonas sp. and Pantoea agglomerans) formed on stainless steel in the presence of food-derived soil. Biofilms were produced on stainless steel coupons with diluted tryptic soy broth incubated for 48 h at 25 degrees C. Duplicate biofilm samples were heat treated for 1, 3, 5, and 15 min at 70, 72, 75, 77, and 80 degrees C and tested for survivors using enrichment culture. The experiment was repeated six times. A predictive model was developed using logistic regression analysis of the fraction negative data. Plots showing the probability of L. monocytogenes inactivation in biofilms after heat treatment were generated from the predictive equation. The predictive model revealed that hot water sanitation of stainless steel can be effective for inactivating L. monocytogenes in a biofilm on stainless steel if time and temperature are controlled. For example, to obtain a 75% probability of total inactivation of L. monocytogenes 3990 biofilm, a heat treatment of 80 degrees C for 11.7 min is required. The model provides processors with a risk management tool that provides predicted probabilities of L. monocytogenes inactivation and allows a choice of three heat resistance assumptions. The predictive model was validated using a five-strain cocktail of L. monocytogenes in the presence of food soil.     

Effect of temperature and growth media on the attachment of Listeria monocytogenes to stainless steel

Listeria monocytogenes ATCC 19111 cultivated in nutrient-rich medium (brain heart infusion, BHI) or starved in minimal medium (10% filter sterilized pond water and 90% sterilized distilled water) were investigated for their initial attachment to austenitic stainless steel No. 4 with satin finish at 4 °C, 20 °C, 30 °C, 37 °C, or 42 °C. A droplet (10 μl) containing  10⁷ CFU/ml of L. monocytogenes suspended in BHI or minimal medium was placed on the stainless steel surface. After holding in saturated humidity for 3 h at the desired temperature the surface was washed and prepared for scanning electron microscopy (SEM). Using SEM, attachment of L. monocytogenes was determined by counting cells remaining on the surface. When L. monocytogenes cultivated in BHI were used, with the exception of the number of attached cells being lower at 42 °C than at 37 °C and 30 °C, the number of attached cells increased with increasing temperature (P < 0.05). When L. monocytogenes starved in minimal medium were used, the number of attached cells also increased with increasing attachment temperature (P < 0.05), but the number of attached cells at 42 °C was lower than that at the other temperatures. The attachment of L. monocytogenes to stainless steel surface was greater when cultivated in rich medium of BHI vs starved in the minimal medium.