Impact of multiple hurdles on Listeria monocytogenes dispersion of survivors.

Pathogen exposure to multiple hurdles could result in variation in the number of survivors, which needs to be carefully considered using appropriate regression models for dealing with survivor dispersion. The aim of this study was to evaluate the impact of the hurdles on the random component of the measured variation and on its unexplained part (over or under-dispersion) representing the departure from randomness, i.e. non-randomness, in survivors of a multi-strain mixture of L. monocytogenes. The pathogen inactivation curves were fitted to the Weibull model within the Conway-Maxwell-Poisson process. In all the 20 hurdle combinations, the surviving cells, whether they showed an upward curvature or linear kinetics, displayed the randomness revealed by the degree of dispersion of the inactivation parameters (-b and p). In 15 combinations, a significant dispersion coefficient (c0), which reflected the non-random component of variation was evident, denoting either over-dispersion (c0 > 0 in 13 combinations) or under-dispersion (c0 < 0 in 2 combinations). The observed dependence of the under- and over-dispersion conditions on the inactivation rate was confirmed by a Monte Carlo simulation based on the inactivation parameter -b. Including both randomness and non-randomness provides a more accurate estimation of survivors, which certainly impacts on intervention practices.

Assessment of growth and survival of Listeria monocytogenes in raw milk butter by durability tests.

Butter is a complex matrix characterized by a high fat content. Existing publications on the behavior of Listeria monocytogenes in this type of food reported contrasted results. This study was performed to provide further information and data about raw milk butter's ability to support survival or growth of L. monocytogenes. Durability tests were performed on naturally contaminated samples of raw milk butter with various physico-chemical characteristics. At the end of shelf life, no growth of L. monocytogenes was observed in the studied butters, regardless of their physico-chemical characteristics (pH, aw, water dispersion index and salt concentration) and the initial level of contamination. The number of positive samples and the colony counts of L. monocytogenes were even decreased at the end of the storage period.

Assessment of the Incubation Period for Invasive Listeriosis.

We characterized incubation periods among outbreak-associated listeriosis cases, using a simulation model to account for patients with multiple exposure dates. The median was 11 days; 90% of cases occurred within 28 days, and incubation periods varied by clinical manifestation.

Effect of glucose on Listeria monocytogenes biofilm formation, and assessment of the biofilm's sanitation tolerance.

Listeria monocytogenes is an important cause of human foodborne infections and its ability to form biofilms is a serious concern to the food industry. To reveal the effect of glucose conditions on biofilm formation of L. monocytogenes, 20 strains were investigated under three glucose conditions (0.1, 1.0, and 2.0% w v(-1)) by quantifying the number of cells in the biofilm and observing the biofilm structure after incubation for 24, 72, and 168 h. In addition, the biofilms were examined for their sensitivity to sodium hypochlorite. It was found that high concentrations of glucose reduced the number of viable cells in the biofilms and increased extracellular polymeric substance production. Moreover, biofilms formed at a glucose concentration of 1.0 or 2.0% were more resistant to sodium hypochlorite than those formed at a glucose concentration of 0.1%. This knowledge can be used to help design the most appropriate sanitation strategy.

The antimicrobial action of resveratrol against Listeria monocytogenes in food-based models and its antibiofilm properties.

BACKGROUND: Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural phytoalexin synthesized by plants in response to stress. This compound has several beneficial documented properties, namely anti-inflammatory, antioxidant, neuroprotective and antimicrobial activities. In this study the antimicrobial activity of resveratrol against Listeria monocytogenes and Listeria innocua was investigated. RESULTS: Resveratrol had a minimum inhibitory concentration of 200 µg mL(-1) for the tested strains, with time-kill curves demonstrating bacteriostatic activity. Inhibition of biofilm formation was also assessed, with resveratrol strongly inhibiting biofilm formation by both species even at subinhibitory concentrations. Overall, resveratrol showed antimicrobial properties on planktonic cells and on biofilm formation ability. Considering the potential use of resveratrol as a food preservative, the antimicrobial efficacy of resveratrol in food was studied in milk, lettuce leaf model and chicken juice. Resveratrol retained greater efficacy in both lettuce leaf model and chicken juice, but milk had a negative impact on its antilisterial activity, indicating a possible reduction of resveratrol availability in milk. CONCLUSION: This study reinforces resveratrol as an antimicrobial agent, pointing out its antibiofilm activity and its potential use as preservative in some food matrices. © 2016 Society of Chemical Industry.

Comparison of desiccation tolerance among Listeria monocytogenes, Escherichia coli O157:H7, Salmonella enterica, and Cronobacter sakazakii in powdered infant formula.

Bacterial pathogens such as Listeria monocytogenes, Escherichia coli O157:H7, Salmonella enterica, and Cronobacter sakazakii have demonstrated long-term survival in/on dry or low-water activity (aw) foods. However, there have been few comparative studies on the desiccation tolerance among these bacterial pathogens separately in a same food matrix. In the present study, the survival kinetics of the four bacterial pathogens separately inoculated onto powdered infant formula as a model low-aw food was compared during storage at 5, 22, and 35°C. No significant differences in the survival kinetics between E. coli O157:H7 and L. monocytogenes were observed. Salmonella showed significantly higher desiccation tolerance than these pathogens, and C. sakazakii demonstrated significantly higher desiccation tolerance than all other three bacteria studied. Thus, the desiccation tolerance was represented as C. sakazakii > Salmonella > E. coli O157:H7 = L. monocytogenes. The survival kinetics of each bacterium was mathematically analyzed, and the observed kinetics was successfully described using the Weibull model. To evaluate the variability of the inactivation kinetics of the tested bacterial pathogens, the Monte Carlo simulation was performed using assumed probability distribution of the estimated fitted parameters. The simulation results showed that the storage temperature significantly influenced survival of each bacterium under the dry environment, where the bacterial inactivation became faster with increasing storage temperature. Furthermore, the fitted rate and shape parameters of the Weibull model were successfully modelled as a function of temperature. The numerical simulation of the bacterial inactivation was realized using the functions of the parameters under arbitrary fluctuating temperature conditions.

Persistent and transient Listeria monocytogenes strains from retail deli environments vary in their ability to adhere and form biofilms and rarely have inlA premature stop codons.

Based on recent risk assessments, up to 83% of listeriosis cases from deli meat in the United States are predicted to be from ready-to-eat deli meats contaminated during processing at retail grocery stores. Listeria monocytogenes is known to use sanitizer tolerance and biofilm formation to survive, but interplay of these mechanisms along with virulence potential and persistence mechanisms specific to deli environments had yet to be elucidated. In this study, 442 isolates from food and nonfood contact surfaces in 30 retail delis over 9 months were tested for inlA premature stop codons (PMSCs); inlA encodes InlA, which is necessary to cause listeriosis. A total of 96 isolates, composed of 23 persistent and 73 transient strains, were tested for adhesion and biofilm-forming ability and sanitizer tolerance. Only 10/442 isolates had inlA PMSCs (p<0.001). Strains with PMSCs were not persistent, even in delis with other persistent strains. Most (7/10) PMSC-containing isolates were collected from food contact surfaces (p<0.001); 6/10 PMSC-containing isolates were found in moderate prevalence delis (p<0.05). Persistent strains had enhanced adhesion on day 1 of a 5-day adhesion-biofilm formation assay. However, there was no significant difference in sanitizer tolerance between persistent and transient strains. Results suggest that foods contaminated with persistent L. monocytogenes strains from the retail environment are (1) likely to have wild-type virulence potential and (2) may persist due to increased adhesion and biofilm formation capacity rather than sanitizer tolerance, thus posing a significant public health risk.

In vitro assessment of the susceptibility of planktonic and attached cells of foodborne pathogens to bacteriophage p22-mediated salmonella lysates.

This study was designed to evaluate the lytic activity of bacteriophage P22 against Salmonella Typhimurium ATCC 19585 (Salmonella Typhimurium P22(-)) at various multiplicities of infections (MOIs), the susceptibility of preattached Salmonella cells against bacteriophage P22, and the effect of P22-mediated bacterial lysates (extracellular DNA) on the attachment ability of Listeria monocytogenes ATCC 7644 and enterohemorrhagic Escherichia coli ATCC 700927 to surfaces. The numbers of attached Salmonella Typhimurium P22(-) cells were effectively reduced to below the detection limit (1 log CFU/ml) at the fixed inoculum levels of 3 × 10(-) CFU/ml (MOI = 3.12) and 3 × 10(3) CFU/ml (MOI = 4.12) by bacteriophage P22. The attached Salmonella Typhimurium P22(-) cells remained more than 2 log CFU/ml, with increasing inoculum levels from 3 × 10(4) to 3 × 10(7) CFU/ml infected with 4 × 10(8) PFU/ml of P22. The number of preattached Salmonella Typhimurium P22(-) cells was noticeably reduced by 2.72 log in the presence of P22. The highest specific attachment ability values for Salmonella Typhimurium P22(-), Salmonella Typhimurium ATCC 23555 carrying P22 prophage (Salmonella Typhimurium P22(+)), L. monocytogenes, and enterohemorrhagic E. coli were 2.09, 1.06, 1.86, and 1.08, respectively, in the bacteriophage-mediated cell-free supernatants (CFS) containing high amounts of extracellular DNA. These results suggest that bacteriophages could potentially be used to effectively eliminate planktonic and preattached Salmonella Typhimurium P22(-) cells with increasing MOI. However, further research is needed to understand the role of bacteriophage-induced lysates in bacterial attachment, which can provide useful information for the therapeutic use of bacteriophage in the food system.

Diversity assessment of Listeria monocytogenes biofilm formation: impact of growth condition, serotype and strain origin.

The foodborne pathogen Listeria monocytogenes has the ability to produce biofilms in food-processing environments and then contaminate food products, which is a major concern for food safety. The biofilm forming behavior of 143 L. monocytogenes strains was determined in four different media that were rich, moderate or poor in nutrients at 12°C, 20°C, 30°C and 37°C. The biofilm formation was mostly influenced by temperature, resulting in decreased biofilm formation with decreasing temperature. Biofilm formation was enhanced in nutrient-poor medium rather than in nutrient-rich medium, and especially in nutrient-poor medium significantly enhanced biofilm production was observed early in biofilm maturation underlining the effect of medium on biofilm formation rate. Also serotype had a significant effect on biofilm formation and was influenced by medium used because strains from both serotype 1/2b and 1/2a formed more biofilm than serotype 4b strains in nutrient-rich medium at 20°C, 30°C and 37°C, whereas in nutrient-poor medium the biofilm production levels of serotype 1/2a and 4b strains were rather similar and lower than serotype 1/2b strains. The strains used originated from various origins, including dairy, meat, industrial environment, human and animal, and the level of biofilm formation was not significantly affected by the origin of isolation, irrespective of medium used and temperature tested. A linear model was used to correlate crystal violet staining of biofilm production to the number of viable cells within the biofilm. This showed that crystal violet staining was poorly correlated to the number of viable cells in nutrient-poor medium, and LIVE/DEAD staining and DNase I treatment revealed that this could be attributed to the presence of non-viable cells and extracellular DNA in the biofilm matrix. The significant impact of intrinsic and extrinsic factors on biofilm production of L. monocytogenes underlined that niche-specific features determine the levels of biofilm produced, and insights in biofilm formation characteristics will allow us to further optimize strategies to control the biofilm formation of L. monocytogenes.

Assessment of the microbial safety and quality of cooked chilled foods and their production process.

Refrigerated processed foods of extended durability (REPFEDs) are a heterogeneous group of food products. This study assesses the microbial safety and quality along the production process in five REPFED companies. Samples were taken of raw materials (n=123), intermediate products (n=123), end products at production day (n=45) and at end of shelf life (n=90), food contact surfaces (n=226) and worker's hands/gloves (n=92). Samples are analysed for total psychrotrophic aerobic count, aerobic spore count, sulphite reducing Clostridia, Bacillus cereus and Listeria monocytogenes. Both L. monocytogenes and B. cereus were detected on the raw materials. Nine of 72 raw materials tested were positive (in 25g) for L. monocytogenes and all but one of these raw materials were raw or minimally processed animal products. Three of 123 raw materials contained high counts (>4log CFU/g) of B. cereus, all of these samples were dried herbs. During production both food contact surfaces (90/226) and gloves (43/92) contained increased levels of total psychrotrophic aerobic counts (≥3log CFU/25cm(2)). This points out a potential source of bacterial recontamination. However, only a four and six of 223 food contact surfaces were positive (per 25cm(2)) for L. monocytogenes and B. cereus respectively. None of the gloves sampled contained L. monocytogenes and only 2 sets of gloves were positive for B. cereus. Of the 123 intermediate products tested twelve tested positive for L. monocytogenes (in 25g) and 5 showed elevated counts of B. cereus (ca. 2.5log CFU/g). Despite the presence of L. monocytogenes in the raw materials, the production area and in some of the intermediate products, none of the end products were positive for L. monocytogenes and only 9 of 135 samples (6.7%) showed to have low numbers of B. cereus (<2.7log CFU/g). This results show that the current pasteurization processes and the food safety management system are adequate to guarantee the production of microbiologically safe foods but that some improvements can still be made with regard to supplier selection, cleaning and disinfection, hygiene training and setting the shelf life duration.

Survival of Listeria monocytogenes on a conveyor belt material with or without antimicrobial additives.

Survival of Listeria monocytogenes on a conveyor belt material with or without antimicrobial additives, in the absence or presence of food debris from meat, fish and vegetables and at temperatures of 10, 25 and 37 degrees C was investigated. The pathogen survived best at 10 degrees C, and better at 25 degrees C than at 37 degrees C on both conveyor belt materials. The reduction in the numbers of the pathogen on belt material with antimicrobial additives in the first 6h at 10 degrees C was 0.6 log unit, which was significantly higher (P<0.05) than the reduction of 0.2 log unit on belt material without additives. Reductions were significantly less (P<0.05) in the presence of food residue. At 37 degrees C and 20% relative humidity, large decreases in the numbers of the pathogen on both conveyor belt materials during the first 6h were observed. Under these conditions, there was no obvious effect of the antimicrobial substances. However, at 25 degrees C and 10 degrees C and high humidity (60-75% rh), a rapid decrease in bacterial numbers on the belt material with antimicrobial substances was observed. Apparently the reduction in numbers of L. monocytogenes on belt material with antimicrobial additives was greater than on belt material without additives only when the surfaces were wet. Moreover, the presence of food debris neutralized the effect of the antimicrobials. The results suggest that the antimicrobial additives in conveyor belt material could help to reduce numbers of microorganisms on belts at low temperatures when food residues are absent and belts are not rapidly dried.

Assessment of survival of Listeria monocytogenes, Salmonella Infantis and Enterococcus faecalis artificially inoculated into experimental waste or compost.

AIMS: To evaluate survival of pathogenic strains, Listeria monocytogenes and Salmonella Infantis and a sanitation indicator Enterococcus faecalis in composts at different stages of the composting process and during storage. METHODS AND RESULTS: The studied pathogenic and indicator strains, originally isolated from compost, were inoculated into compost samples from the various stages of the composting process. During incubation, indigenous microflora diversity was monitored with DGGE analysis. After 90 days of incubation, strain survival was observed in compost sampled before the beginning of the cooling phase, and DGGE analysis demonstrated an increase of microbial diversity up to the cooling phase. However, inoculated strains were not detected in composts after 30, 60 or 90 days of incubation in compost sampled after the start of the cooling phase. Microbial diversity also became stable, and DGGE profiles reached a maximum number of bands at this stage. CONCLUSIONS: Strain survival was not observed in stabilized composts. The cooling phase seems to be the turning point for pathogen survival and at this stage the indigenous microflora appeared to play a significant role in suppression. SIGNIFICANCE AND IMPACT OF THE STUDY: The importance of indigenous microflora in the survival of pathogens in four different composts was demonstrated. Stabilized composts were recommended for spreading on land.

Assessment of the roles of LuxS, S-ribosyl homocysteine, and autoinducer 2 in cell attachment during biofilm formation by Listeria monocytogenes EGD-e.

LuxS is responsible for the production of autoinducer 2 (AI-2), which is involved in the quorum-sensing response of Vibrio harveyi. AI-2 is found in several other gram-negative and gram-positive bacteria and is therefore considered a good candidate for an interspecies communication signal molecule. In order to determine if this system is functional in the gastrointestinal pathogen Listeria monocytogenes EGD-e, an AI-2 bioassay was performed with culture supernatants. The results indicated that this bacterium produces AI-2 like molecules. A potential ortholog of V. harveyi luxS, lmo1288, was found by performing sequence similarity searches and complementation experiments with Escherichia coli DH5alpha, a luxS null strain. lmo1288 was found to be a functional luxS ortholog involved in AI-2 synthesis. Indeed, interruption of lmo1288 resulted in loss of the AI-2 signal. Although no significant differences were observed between Lux1 and EGD-e with regard to planktonic growth (at 10 degrees C, 15 degrees C, 25 degrees C, and 42 degrees C), swimming motility, and phospholipase and hemolytic activity, biofilm culture experiments showed that under batch conditions between 25% and 58% more Lux1 cells than EGD-e cells were attached to the surface depending on the incubation time. During biofilm growth in continuous conditions after 48 h of culture, Lux1 biofilms were 17 times denser than EGD-e biofilms. Finally, our results showed that Lux1 accumulates more S-adenosyl homocysteine (SAH) and S-ribosyl homocysteine (SRH) in culture supernatant than the parental strain accumulates and that SRH, but not SAH or AI-2, is able to modify the number of attached cells.

Listeria monocytogenes sigma B regulates stress response and virulence functions.

While the stress-responsive alternative sigma factor sigma(B) has been identified in different species of Bacillus, Listeria, and Staphylococcus, the sigma(B) regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify sigma(B)-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate sigma(B)-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted sigma(B)-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and Delta sigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant sigma(B)-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting sigma(B)-dependent expression, 54 were preceded by a sequence resembling the sigma(B) promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the sigma(B)-dependent nature of a subset of eight selected promoter regions. Notably, the sigma(B)-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, sigma(B) also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that sigma(B) contributes to L. monocytogenes gene expression during infection.

Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation.

Listeria monocytogenes has the ability to form biofilms on food-processing surfaces, potentially leading to food product contamination. The objective of this research was to standardize a polyvinyl chloride (PVC) microtiter plate assay to compare the ability of L. monocytogenes strains to form biofilms. A total of 31 coded L. monocytogenes strains were grown in defined medium (modified Welshimer's broth) at 32 degrees C for 20 and 40 h in PVC microtiter plate wells. Biofilm formation was indirectly assessed by staining with 1% crystal violet and measuring crystal violet absorbance, using destaining solution. Cellular growth rates and final cell densities did not correlate with biofilm formation, indicating that differences in biofilm formation under the same environmental conditions were not due to growth rate differences. The mean biofilm production of lineage I strains was significantly greater than that observed for lineage II and lineage III strains. The results from the standardized microtiter plate biofilm assay were also compared to biofilm formation on PVC and stainless steel as assayed by quantitative epifluorescence microscopy. Results showed similar trends for the microscopic and microtiter plate assays, indicating that the PVC microtiter plate assay can be used as a rapid, simple method to screen for differences in biofilm production between strains or growth conditions prior to performing labor-intensive microscopic analyses.

Assessment of the pathogenic potential of two Listeria monocytogenes human faecal carriage isolates.

Two human faeces carriage isolates of Listeria monocytogenes (H1 and H2) were compared to reference strains (ScottA and LO28) with regard to their lethality in 14-day-old chick embryos, their haemolytic and phospholipase (phosphatidylcholine-phospholipase C and phosphatidylinositol-phospholipase C) activities and their invasiveness towards Caco-2 cells. Experimental infection of chick embryos allowed discrimination of the strains into those exhibiting high virulence (ScottA and H2), those exhibiting slightly attenuated virulence (LO28) and those exhibiting low virulence (H1). A similar percentage mortality and time to death for embryos was observed when they were infected with H2 as was seen with infection by the reference strain ScottA. Therefore, human carriage strain H2 was considered potentially pathogenic. In contrast to H2 and ScottA, H1 exhibited low virulence. Using the tissue-culture cell-line model, it was found that carriage strain H1 was unable to enter Caco-2 cells efficiently, even though it was similar to the virulent strains in terms of the enzymic activities involved in pathogenicity. Detection of the internalins InlA and InlB, involved in the internalization of L. monocytogenes in the host cells, by immunoblot indicated that a truncated form of InlA was produced by H1. Taken together, these data provide a starting point for the study of the behaviour of two types of human faeces carriage strains and their characterization.

Assessment of the virulence of Listeria monocytogenes: agreement between a plaque-forming assay with HT-29 cells and infection of immunocompetent mice.

Some Listeria monocytogenes strains not related to clinical cases have been found to exhibit a low virulence level in mice as well as in an in vitro test using Caco-2 cells. The purpose of this study was to validate a new in vitro test of virulence based on a plaque-forming assay (PFA) using a HT-29 cell monolayer with 118 Listeria strains. The use of HT-29 cells in 96-well tissue culture plates allowed the testing of 30 strains per day and providing results in 24 h. In addition. statistical analyses demonstrated the reproducibility and repeatability of the PFA. No quantitative relationship was observed between the virulence of the strains and the hemolytic titer or the cytotoxic effects on HT-29 cells. In contrast, good agreement was observed between virulence assessed after subcutaneous (SC) infection and virulence obtained by PFA. Three groups of L. monocytogenes strains (avirulent, hypovirulent and fully virulent) were established by comparison of the clinical origin of the strains, the number of immunocompetent contaminated mice and the numbers of Listeria strains recovered in the spleen after SC infection. With one exception, i.e. a clinical case of L. seeligeri (sensitivity 0.98), the PFA successfully detected the virulent strains only (specificity 1). Decision-tree algorithms performed by SAS and S-Plus demonstrated that this tissue culture assay discriminated between the avirulent and hypovirulent strains and the virulent strains. This test could therefore be an alternative to in vivo tests, allowing grading of virulence.

In vivo and in vitro assessment of the virulence of Listeria monocytogenes strains.

To evaluate whether the in vitro model (invasion and intracellular growth in Caco-2 cells) for determining virulence is a suitable alternative to the in vivo model (50% lethal dose), we compared the levels of virulence obtained with the two models. We tested L. monocytogenes strains isolated from food and clinical samples during three episodes of listeriosis occurring in Italy in the period 1993-1995. We also tested L. monocytogenes strains isolated from food during official control activities. The results obtained from the tested strains varied according to the experimental method adopted: the L. monocytogenes strains featuring the same genetic pattern showed a greater uniformity of response in vivo than in vitro. We can conclude that the in vitro model may be used as an alternative to the animal model to determine Listeria spp pathogenicity, though it cannot distinguish levels of virulence within the L. monocytogenes species.

Luminol-enhanced chemiluminescence by Listeria monocytogenes: a test for rapid assessment of antimicrobial agents.

Listeria monocytogenes produces chemiluminescence in brain heart infusion broth at 37 degrees C in the presence of carbonate ions and acetaldehyde. This phenomenon can be enhanced by the use of luminol rather than acetaldehyde. Furthermore, there is direct relationship between the extent of growth and the level of luminescence which culminates at the end of the exponential growth. This property was used to study the susceptibility of this bacterium to two antiseptics, cetrimonium bromide and chlorhexidine, and to two antibiotics, ampicillin and chloramphenicol. Inhibition of chemiluminescence was proportional to the antimicrobial agents' concentrations and was complete at their minimal inhibitory concentrations.