Inhibition of Listeria monocytogenes Growth, Adherence and Invasion in Caco-2 Cells by Potential Probiotic Lactic Acid Bacteria Isolated from Fecal Samples of Healthy Neonates.

Lactic acid bacteria (LAB) isolated from healthy humans may prove an effective tool against pathogen growth, adherence and invasion in intestinal epithelial cells. This study aimed to evaluate the antilisterial properties of LAB isolated from fecal samples of healthy neonates. Forty-five LAB strains were tested for their antimicrobial activity against ten Listeria monocytogenes strains with spot-on-lawn and agar-well diffusion assays, and ten lactobacilli strains were further assessed for their inhibitory effect against adherence and invasion of Caco-2 cells by L. monocytogenes EGDe. Inhibition was estimated in competition, exclusion or displacement assays, where lactobacilli and L. monocytogenes were added to Caco-2 monolayers simultaneously or 1 h apart from each other. Inhibition of L. monocytogenes growth was only displayed with the spot-on-lawn assay; cell-free supernatants of lactobacilli were not effective against the pathogen. Lactobacillus (L.) paragasseri LDD-C1 and L. crispatus LCR-A21 were able to adhere to Caco-2 cells at significantly higher levels than the reference strain L. rhamnosus GG. The adherence of L. monocytogenes to Caco-2 cells was reduced by 20.8% to 62.1% and invasion by 33.5% to 63.1% during competition, which was more effective compared to the exclusion and displacement assays. These findings demonstrate that lactobacilli isolated from neonatal feces could be considered a good candidate against L. monocytogenes.

Influence of temperature on regulation of key virulence and stress response genes in Listeria monocytogenes biofilms.

Temperature is a major determinant of Listeria (L.) monocytogenes adherence and biofilm formation on abiotic surfaces. However, its role on gene regulation of L. monocytogenes mature biofilms has not been investigated. In the present study, we aimed to evaluate the impact of temperature up- and down-shift on L. monocytogenes biofilms gene transcription. L. monocytogenes strain EGD-e biofilms were first developed on stainless steel surfaces in Brain Heart Infusion broth at 20 °C for 48 h. Then, nutrient broth was renewed, and mature biofilms were exposed to 10 °C, 20 °C or 37 °C for 24 h. Biofilm cells were harvested and RNA levels of plcA, prfA, hly, mpl, plcB, sigB, bapL, fbpA, fbpB, lmo2178, lmo0880, lmo0160, lmo1115, lmo 2089, lmo2576, lmo0159 and lmo0627 were evaluated by quantitative RT-PCR. The results revealed an over-expression of all genes tested in biofilm cells compared to planktonic cells. When biofilms were further allowed to proliferate at 20 °C for 24 h, the transcription levels of key virulence, stress response and putative binding proteins genes plcA, sigB, fbpA, fbpB, lmo1115, lmo0880 and lmo2089 decreased. A temperature-dependent transcription for sigB, plcA, hly, and lmo2089 genes was observed after biofilm proliferation at 10 °C or 37 °C. Our findings suggest that temperature differentially affects gene regulation of L. monocytogenes mature biofilms, thus modulating attributes such as virulence, stress response and pathogenesis.

In Vitro Virulence Potential, Surface Attachment, and Transcriptional Response of Sublethally Injured Listeria monocytogenes following Exposure to Peracetic Acid.

The disinfectant peracetic acid (PAA) can cause high levels of sublethal injury to Listeria monocytogenes. This study aims to evaluate phenotypic and transcriptional characteristics concerning the surface attachment and virulence potential of sublethally injured L. monocytogenes ScottA and EGDe after exposure to 0.75 ppm PAA for 90 min at 4°C and subsequent incubation in tryptic soy broth supplemented with yeast extract (TSBY) at 4°C. The results showed that injured L. monocytogenes cells (99% of the total population) were able to attach (after 2 and 24 h) to stainless steel coupons at 4°C and 20°C. In vitro virulence assays using human intestinal epithelial Caco-2 cells showed that injured L. monocytogenes could invade host cells but could not proliferate intracellularly. The in vitro virulence response was strain dependent; injured ScottA was more invasive than EGDe. Assessment of PAA injury at the transcriptional level showed the upregulation of genes (motB and flaA) involved in flagellum motility and surface attachment. The transcriptional responses of L. monocytogenes EGDe and ScottA were different: only injured ScottA demonstrated upregulation of the virulence genes inlA and plcA. Downregulation of the stress-related genes fri and kat and upregulation of lmo0669 were observed in injured ScottA. The obtained results indicate that sublethally injured L. monocytogenes cells may retain part of their virulence properties as well as their ability to adhere to food-processing surfaces. Transmission to food products and the introduction of these cells into the food chain are therefore plausible scenarios that are worth taking into consideration in terms of risk assessment. IMPORTANCE L. monocytogenes is the causative agent of listeriosis, a serious foodborne illness. Antimicrobial practices such as disinfectants used for the elimination of this pathogen in the food industry can produce a sublethally injured population fraction. Injured cells of this pathogen that may survive antimicrobial treatment may pose a food safety risk. Nevertheless, knowledge regarding how sublethal injury may impact important cellular traits and phenotypic responses of this pathogen is limited. This work suggests that sublethally injured L. monocytogenes cells maintain virulence and surface attachment potential and highlights the importance of the occurrence of sublethally injured cells regarding food safety.

Assessing the survival and sublethal injury kinetics of Listeria monocytogenes under different food processing-related stresses.

The foodborne pathogen L. monocytogenes can be present in food processing environments where it is exposed to various stressors. These antimicrobial factors, which aim to eliminate the pathogen, can induce sub-lethal injury to the bacterial cells. In the present study, we investigated the efficacy of different treatments (stresses) relevant to food processing and preservation as well as sanitation methods, in generating sub-lethal injury at 4 °C and 20 °C to two L. monocytogenes strains, ScottA and EGDe. Additionally, we evaluated the survival and extent of L. monocytogenes injury after exposure to commonly used disinfectants (peracetic acid and benzalkonium chloride), following habituation in nutrient-deprived, high-salinity medium. Each stress had a different impact on the survival and injury kinetics of L. monocytogenes. The highest injury levels were caused by peracetic acid which, at 4 °C, generated high populations of injured cells without loss of viability. Other injury-inducing stresses were lactic acid and heating. Long-term habituation in nutrient-limited and high salinity medium (4 °C) and subsequent exposure to disinfectants resulted in higher survival and injury in benzalkonium chloride and increased survival, yet with lower injury levels, in peracetic acid at 20 °C. Taken together, these results highlight the potential food safety risk emerging from the occurrence of injured cells by commonly used food processing methods. Consequently, in order to accurately assess the impact of an antimicrobial method, its potential of inducing sublethal injury needs to be considered along with lethality.

Control of Listeria monocytogenes Biofilms in a Simulated Food-Processing Environment.

Biofilm-forming ability may vary significantly among different Listeria (L.) monocytogenes strains. This interstrain variation is also observed in L. monocytogenes biofilm resistance to antimicrobial compounds commonly used in the food-processing environment. The screening of a large set of L. monocytogenes strains with specific characteristics, such as serotype, MLST type, and other genetic characteristics under various environmental conditions, may lead to a better understanding of the mechanisms underlying the establishment of the pathogen on food contact surfaces. In this chapter, traditional methods for L. monocytogenes strains characterization with regard to biofilm formation and novel biofilm control methods will be described.

Differential Modulation of Listeria monocytogenes Fitness, In Vitro Virulence, and Transcription of Virulence-Associated Genes in Response to the Presence of Different Microorganisms.

Interactions between Listeria monocytogenes and food-associated or environmental bacteria are critical not only for the growth but also for a number of key biological processes of the microorganism. In this regard, limited information exists on the impact of other microorganisms on the virulence of L. monocytogenes In this study, the growth of L. monocytogenes was evaluated in a single culture or in coculture with L. innocua, Bacillus subtilis, Lactobacillus plantarum, or Pseudomonas aeruginosa in tryptic soy broth (10°C/10 days and 37°C/24 h). Transcriptional levels of 9 key virulence genes (inlA, inlB, inlC, inlJ, sigB, prfA, hly, plcA, and plcB) and invasion efficiency and intracellular growth in Caco-2 cells were determined for L. monocytogenes following growth in mono- or coculture for 3 days at 10°C or 9 h at 37°C. The growth of L. monocytogenes was negatively affected by the presence of L. innocua and B. subtilis, while the effect of cell-to-cell contact on L. monocytogenes growth was dependent on the competing microorganism. Cocultivation affected the in vitro virulence properties of L. monocytogenes in a microorganism-specific manner, with L. innocua mainly enhancing and B. subtilis reducing the invasion of the pathogen in Caco-2 cells. Assessment of the mRNA levels of L. monocytogenes virulence genes in the presence of the four tested bacteria revealed a complex pattern in which the observed up- or downregulation was only partially correlated with growth or in vitro virulence and mainly suggested that L. monocytogenes may display a microorganism-specific transcriptional response.IMPORTANCEListeria monocytogenes is the etiological agent of the severe foodborne disease listeriosis. Important insight regarding the physiology and the infection biology of this microorganism has been acquired in the past 20 years. However, despite the fact that L. monocytogenes coexists with various microorganisms throughout its life cycle and during transmission from the environment to foods and then to the host, there is still limited knowledge related to the impact of surrounding microorganisms on L. monocytogenes' biological functions. In this study, we showed that L. monocytogenes modulates specific biological activities (i.e., growth and virulence potential) as a response to coexisting microorganisms and differentially alters the expression of virulence-associated genes when confronted with different bacterial genera and species. Our work suggests that the interaction with different bacteria plays a key role in the survival strategies of L. monocytogenes and supports the need to incorporate biotic factors into the research conducted to identify mechanisms deployed by this organism for establishment in different environments.

Virulence Gene Sequencing Highlights Similarities and Differences in Sequences in Listeria monocytogenes Serotype 1/2a and 4b Strains of Clinical and Food Origin From 3 Different Geographic Locations.

The prfA-virulence gene cluster (pVGC) is the main pathogenicity island in Listeria monocytogenes, comprising the prfA, plcA, hly, mpl, actA, and plcB genes. In this study, the pVGC of 36 L. monocytogenes isolates with respect to different serotypes (1/2a or 4b), geographical origin (Australia, Greece or Ireland) and isolation source (food-associated or clinical) was characterized. The most conserved genes were prfA and hly, with the lowest nucleotide diversity (π) among all genes (P < 0.05), and the lowest number of alleles, substitutions and non-synonymous substitutions for prfA. Conversely, the most diverse gene was actA, which presented the highest number of alleles (n = 20) and showed the highest nucleotide diversity. Grouping by serotype had a significantly lower π value (P < 0.0001) compared to isolation source or geographical origin, suggesting a distinct and well-defined unit compared to other groupings. Among all tested genes, only hly and mpl were those with lower nucleotide diversity in 1/2a serotype than 4b serotype, reflecting a high within-1/2a serotype divergence compared to 4b serotype. Geographical divergence was noted with respect to the hly gene, where serotype 4b Irish strains were distinct from Greek and Australian strains. Australian strains showed less diversity in plcB and mpl relative to Irish or Greek strains. Notable differences regarding sequence mutations were identified between food-associated and clinical isolates in prfA, actA, and plcB sequences. Overall, these results indicate that virulence genes follow different evolutionary pathways, which are affected by a strain's origin and serotype and may influence virulence and/or epidemiological dominance of certain subgroups.

Adaptive Response of Listeria monocytogenes to Heat, Salinity and Low pH, after Habituation on Cherry Tomatoes and Lettuce Leaves.

Pathogens found on fresh produce may encounter low temperatures, high acidity and limited nutrient availability. The aim of this study was to evaluate the effect of habituation of Listeria monocytogenes on cherry tomatoes or lettuce leaves on its subsequent response to inhibitory levels of acid, osmotic and heat stress. Habituation was performed by inoculating lettuce coupons, whole cherry tomatoes or tryptic soy broth (TSB) with a three-strains composite of L. monocytogenes, which were further incubated at 5°C for 24 hours or 5 days. Additionally, cells grown overnight in TSB supplemented with 0.6% yeast extract (TSBYE) at 30°C were used as control cells. Following habituation, L. monocytogenes cells were harvested and exposed to: (i) pH 3.5 adjusted with lactic acid, acetic acid or hydrochloric acid (HCl), and pH 1.5 (HCl) for 6 h; (ii) 20% NaCl and (iii) 60°C for 150 s. Results showed that tomato-habituated L. monocytogenes cells were more tolerant (P < 0.05) to acid or osmotic stress than those habituated on lettuce, and habituation on both foods resulted in more stress resistant cells than prior growth in TSB. On the contrary, the highest resistance to heat stress (P < 0.05) was exhibited by the lettuce-habituated L. monocytogenes cells followed by TSB-grown cells at 5°C for 24 h, whereas tomato-habituated cells were highly sensitized. Prolonged starvation on fresh produce (5 days vs. 24 h) increased resistance to osmotic and acid stress, but reduced thermotolerance, regardless of the pre-exposure environment (i.e., tomatoes, lettuce or TSB). These results indicate that L. monocytogenes cells habituated on fresh produce at low temperatures might acquire resistance to subsequent antimicrobial treatments raising important food safety implications.

Variability of Listeria monocytogenes strains in biofilm formation on stainless steel and polystyrene materials and resistance to peracetic acid and quaternary ammonium compounds.

Listeria monocytogenes is a foodborne pathogen able to tolerate adverse conditions by forming biofilms or by deploying stress resistant mechanisms, and thus manages to survive for long periods in food processing plants. This study sought to investigate the correlation between biofilm forming ability, tolerance to disinfectants and cell surface characteristics of twelve L. monocytogenes strains. The following attributes were evaluated: (i) biofilm formation by crystal violet staining method on polystyrene, and by standard cell enumeration on stainless steel and polystyrene; (ii) hydrophobicity assay using solvents; (iii) minimum inhibitory concentration (MIC) and biofilm eradication concentration (BEC) of peracetic acid (PAA) and quaternary ammonium compounds (QACs), and (iv) resistance to sanitizers (PAA 2000ppm; QACs 500ppm) of biofilms on polystyrene and stainless steel. After 72h of incubation, higher biofilm levels were formed in TSB at 20°C, followed by TSB at 37°C (P=0.087) and diluted TSB 1/10 at both 20 (P=0.005) and 37°C (P=0.004). Cells grown at 30°C to the stationary phase had significant electron donating nature and a low hydrophobicity, while no significant correlation of cell surface properties to biofilm formation was observed. Strains differed in MICPAA and BECPAA by 24- and 15-fold, respectively, while a positive correlation between MICPAA and BECPAA was observed (P=0.02). The MICQACs was positively correlated with the biofilm-forming ability on stainless steel (P=0.03). Regarding the impact of surface type, higher biofilm populations were enumerated on polystyrene than on stainless steel, which were also more tolerant to disinfectants. Among all strains, the greatest biofilm producer was a persistent strain with significant tolerance to QACs. These results may contribute to better understanding of L. monocytogenes behavior and survival on food processing surfaces.

Effect of single or combined chemical and natural antimicrobial interventions on Escherichia coli O157:H7, total microbiota and color of packaged spinach and lettuce.

Aqueous extract of Origanum vulgare (oregano), sodium hypochlorite (60 and 300 ppm of free chlorine), Citrox® (containing citric acid and phenolic compounds [bioflavonoids] as active ingredients), vinegar, lactic acid, and double combinations of Citrox, lactic acid and oregano were evaluated against Escherichia coli O157:H7 and total mesophilic microbiota on fresh-cut spinach and lettuce and for their impact on color of treated vegetables. Spinach and lettuce leaves were inoculated with E. coli O157:H7 to a level of 5-6 log CFU/g and immersed in washing solutions for 2 or 5 min at 20 °C, followed by rinsing with ice water (30s). Bacterial populations on vegetables were enumerated immediately after washing and after storage of the samples at 5 °C for 7 days under 20% CO2: 80% N2. No significant post-washing microbial reductions were achieved by chlorinated water, whereas after storage total microbiota was increased by 2.4 log CFU/g on lettuce. Vinegar wash was the most effective treatment causing E. coli O157:H7 reductions of 1.8-4.3 log CFU/g. During storage, pathogen was further decreased to below the detection limit level (<2 log CFU/g) and total microbiota exhibited the highest reductions compared to other treatments. Lactic acid reduced pathogen by 1.6-3.7 log CFU/g after washing; however levels of total microbiota increased by up to 2 log CFU/g on packaged lettuce during storage. Washing lettuce samples with oregano for 2 min resulted in 2.1 log CFU/g reduction of E. coli O157:H7. When Citrox was combined with oregano, 3.7-4.0 log CFU/g reduction was achieved on spinach and lettuce samples, with no significant effect on color parameters. Additionally, rinsing with ice water after decontamination treatments contributed to maintenance of color of the treated vegetables. In conclusion, the results indicated that vinegar, lactic acid or oregano aqueous extract alone or in combination, as alternative washing solutions to chlorine, may be effectively used to control E. coli O157:H7 and sustain acceptable appearance of fresh cut spinach and lettuce.

Modeling transfer of Escherichia coli O157:H7 and Listeria monocytogenes during preparation of fresh-cut salads: impact of cutting and shredding practices.

Cutting and shredding of leafy vegetables increases the risk of cross contamination in household settings. The distribution of Escherichia coli O157:H7 and Listeria monocytogenes transfer rates (Tr) between cutting knives and lettuce leaves was investigated and a semi-mechanistic model describing the bacterial transfer during consecutive cuts of leafy vegetables was developed. For both pathogens the distribution of log10Trs from lettuce to knife was towards low values. Conversely log10Trs from knife to lettuce ranged from -2.1 to -0.1 for E. coli O157:H7 and -2.0 to 0 for L. monocytogenes, and indicated a more variable phenomenon. Regarding consecutive cuts, a rapid initial transfer was followed by an asymptotic tail at low populations moving to lettuce or residing on knife. E. coli O157:H7 was transferred at slower rates than L. monocytogenes. These trends were sufficiently described by the transfer-model, with RMSE values of 0.426-0.613 and 0.531-0.908 for L. monocytogenes and E. coli O157:H7, respectively. The model showed good performance in validation trials but underestimated bacterial transfer during extrapolation experiments. The results of the study can provide information regarding cross contamination events in a common household. The constructed model could be a useful tool for the risk-assessment during preparation of leafy-green salads.

Listeria monocytogenes attachment to and detachment from stainless steel surfaces in a simulated dairy processing environment.

The presence of pathogens in dairy products is often associated with contamination via bacteria attached to food-processing equipment, especially from areas where cleaning/sanitation is difficult. In this study, the attachment of Listeria monocytogenes on stainless steel (SS), followed by detachment and growth in foods, was evaluated under conditions simulating a dairy processing environment. Initially, SS coupons were immersed in milk, vanilla custard, and yogurt inoculated with the pathogen (10(7) CFU/ml or CFU/g) and incubated at two temperatures (5 and 20 degrees C) for 7 days. By the end of incubation, cells were mechanically detached from coupons and used to inoculate freshly pasteurized milk which was subsequently stored at 5 degrees C for 20 days. The suspended cells in all three products in which SS coupons were immersed were also used to inoculate freshly pasteurized milk (5 degrees C for 20 days). When SS coupons were immersed in milk, shorter lag phases were obtained for detached than for planktonically grown cells, regardless of the preincubation temperature (5 or 20 degrees C). The opposite was observed when custard incubated at 20 degrees C was used to prepare the two types of inocula. However, in this case, a significant increase in growth rate was also evident when the inoculum was derived from detached cells. In another parallel study, while L. monocytogenes was not detectable on SS coupons after 7 days of incubation (at 5 degrees C) in inoculated yogurt, marked detachment and growth were observed when these coupons were subsequently transferred and incubated at 5 degrees C in fresh milk or/and custard. Overall, the results obtained extend our knowledge on the risk related to contamination of dairy products with detached L. monocytogenes cells.