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.

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.

Listeria monocytogenes Strains Underrepresented during Selective Enrichment with an ISO Method Might Dominate during Passage through Simulated Gastric Fluid and In Vitro Infection of Caco-2 Cells.

Various Listeria monocytogenes strains may contaminate a single food product, potentially resulting in simultaneous exposure of consumers to multiple strains. However, due to bias in strain recovery, L. monocytogenes strains isolated from foods by selective enrichment (SE) might not always represent those that can better survive the immune system of a patient. We investigated the effect of cocultivation in tryptic soy broth with 0.6% yeast extract (TSB-Y) at 10°C for 8 days on (i) the detection of L. monocytogenes strains during SE with the ISO 11290-1:1996/Amd 1:2004 protocol and (ii) the in vitro virulence of strains toward the Caco-2 human colon epithelial cancer cell line following exposure to simulated gastric fluid (SGF; pH 2.0)-HCl (37°C). We determined whether the strains which were favored by SE would be effective competitors under the conditions of challenges related to gastrointestinal passage of the pathogen. Interstrain competition of L. monocytogenes in TSB-Y determined the relative population of each strain at the beginning of SE. This in turn impacted the outcome of SE (i.e., favoring survival of competitors with better fitness) and the levels exposed subsequently to SGF. However, strong growth competitors could be outcompeted after SGF exposure and infection of Caco-2 cells by strains outgrown in TSB-Y and underdetected (or even missed) during enrichment. Our data demonstrate a preferential selection of certain L. monocytogenes strains during enrichments, often not reflecting a selective advantage of strains during infection. These findings highlight a noteworthy scenario associated with the difficulty of matching the source of infection (food) with the L. monocytogenes isolate appearing to be the causative agent during listeriosis outbreak investigations.IMPORTANCE This report is relevant to understanding the processes involved in selection and prevalence of certain L. monocytogenes strains in different environments (i.e., foods or sites of humans exposed to the pathogen). It highlights the occurrence of multiple strains in the same food as an important aspect contributing to mismatches between clinical isolates and infection sources during listeriosis outbreak investigations.

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.

Impact of preculture temperature on peracetic acid-induced inactivation and sublethal injury of L. monocytogenes and subsequent growth potential of single cells.

The disinfectant peracetic acid (PAA) that is used in the food industry can cause sublethal injury in L. monocytogenes. The effect of preculture temperature on the inactivation and sublethal injury of L. monocytogenes cells due to PAA was evaluated by plating on non-selective and selective agar medium supplemented with 5 % (w/v) NaCl. L. monocytogenes cells were precultured at 30 °C, 20 °C or 4 °C, and the former was used as reference temperature. Preculture of cells at 20 °C or 4 °C and subsequent exposure to PAA at the respective growth temperatures caused higher injury compared to cells grown at 30 °C and exposed to PAA 20 °C and PAA 4 °C, respectively. Survival was also affected by the preculture temperature; 20 °C-grown cultures resulted in lower survival at PAA 20 °C. Nevertheless, preculture at 4 °C resulted in a similar number of surviving cells when exposed to PAA 4 °C compared to cells precultured at 30 °C and exposed to PAA at 4 °C. Flow cytometry was subsequently used to quantify outgrowth capacity of stressed and sublethal damaged populations following sorting of single cells in nutrient rich medium (Tryptone soy broth supplemented with yeast extract [TSBY]). PAA treatment affected the outgrowth of L. monocytogenes at single-cell level resulting in increased outgrowth-times reflecting higher single cell heterogeneity. To conclude, the response of L. monocytogenes when exposed to PAA depended on the preculture conditions, and the highly heterogeneous outgrowth potential of PAA-injured cells may affect their detection accuracy and pose a food safety risk.

Understanding Wine through Yeast Interactions.

Wine is a product of microbial activities and microbe-microbe interactions. Yeasts are the principal microorganisms responsible for the evolution and fulfillment of alcoholic fermentation. Several species and strains coexist and interact with their environment and with each other during the fermentation course. Yeast-yeast interactions occur even from the early stages of fermentation, determining yeast community structure and dynamics during the process. Different types of microbial interactions (e.g., mutualism and commensalism or competition and amensalism) may exert positive or negative effects, respectively, on yeast populations. Interactions are intimately linked to yeast metabolic activities that influence the wine analytical profile and shape the wine character. In this context, much attention has been given during the last years to the interactions between Saccharomyces cerevisiae (SC) and non-Saccharomyces (NS) yeast species with respect to their metabolic contribution to wine quality. Yet, there is still a significant lack of knowledge on the interaction mechanisms modulating yeast behavior during mixed culture fermentation, while much less is known about the interactions between the various NS species or between SC and Saccharomyces non-cerevisiae (SNC) yeasts. There is still much to learn about their metabolic footprints and the genetic mechanisms that alter yeast community equilibrium in favor of one species or another. Gaining deeper insights on yeast interactions in the grape-wine ecosystem sets the grounds for understanding the rules underlying the function of the wine microbial system and provides means to better control and improve oenological practices.

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.

Evaluation of antimicrobial activities of plant aqueous extracts against Salmonella Typhimurium and their application to improve safety of pork meat.

Nine odorless laboratory-collected hydro-distilled aqueous extracts (basil, calendula, centrifuged oregano, corn silk, laurel, oregano, rosemary, spearmint, thyme) and one industrial steam-distilled oregano hydrolate acquired as by-products of essential oils purification were screened for their in vitro antimicrobial activity against three Salmonella Typhimurium strains (4/74, FS8, FS115) at 4 and 37 °C. Susceptibility to the extracts was mainly plant- and temperature-dependent, though strain dependent effects were also observed. Industrial oregano hydrolate eliminated strains immediately after inoculation, exhibiting the highest antimicrobial potential. Hydro-distilled extracts eliminated/reduced Salmonella levels during incubation at 4 °C. At 37 °C, oregano, centrifuged oregano, thyme, calendula and basil were bactericidal while spearmint, rosemary and corn silk bacteriostatic. A strain-dependent effect was observed for laurel. The individual or combined effect of marinades and edible coatings prepared of industrial hydrolate and hydro-distilled oregano extracts with or without oregano essential oil (OEO) was tested in pork meat at 4 °C inoculated with FS8 strain. Lower in situ activity was observed compared to in vitro assays. Marinades and edible coatings prepared of industrial oregano hydrolate + OEO were the most efficient in inhibiting pathogen. Marination in oregano extract and subsequent coating with either 50% oregano extract + OEO or water + OEO enhanced the performance of oregano extract. In conclusion, by-products of oregano essential oil purification may be promising alternative antimicrobials to pork meat stored under refrigeration when applied in the context of multiple hurdle approach.

Growth, detection and virulence of Listeria monocytogenes in the presence of other microorganisms: microbial interactions from species to strain level.

Like with all food microorganisms, many basic aspects of L. monocytogenes life are likely to be influenced by its interactions with bacteria living in close proximity. This pathogenic bacterium is a major concern both for the food industry and health organizations since it is ubiquitous and able to withstand harsh environmental conditions. Due to the ubiquity of Listeria monocytogenes, various strains may contaminate foods at different stages of the supply chain. Consequently, simultaneous exposure of consumers to multiple strains is also possible. In this context even strain-to-strain interactions of L. monocytogenes play a significant role in fundamental processes for the life of the pathogen, such as growth or virulence, and subsequently compromise food safety, affect the evolution of a potential infection, or even introduce bias in the detection by classical enrichment techniques. This article summarizes the impact of microbial interactions on the growth and detection of L. monocytogenes primarily in foods and food-associated environments. Furthermore it provides an overview of L. monocytogenes virulence in the presence of other microorganisms.

Growth differences and competition between Listeria monocytogenes strains determine their predominance on ham slices and lead to bias during selective enrichment with the ISO protocol.

Listeria monocytogenes strains are widespread in the environment where they live well mixed, often resulting in multiple strains contaminating a single food sample. The occurrence of different strains in the same food might trigger strain competition, contributing to uneven growth of strains in food and to bias during selective procedures. We tested the growth of seven L. monocytogenes strains (C5, 6179, ScottA, PL24, PL25, PL26, PL27) on ham slices and on nutrient-rich agar at 10°C, singly and in combinations. Strains were made resistant to different antibiotics for their selective enumeration. In addition, growth of single strains (axenic culture) and competition between strains in xenic cultures of two strains was evaluated in enrichment broth and on selective agar. According to ISO 11290-1:1996/Amd 1:2004 standard protocol for detection of L. monocytogenes, two enrichment steps both followed by streaking on ALOA were performed. Strain cultures were directly added in the enrichment broth or used to inoculate minced beef and sliced hams which were then mixed with enrichment broth. 180-360 colonies were used to determine the relative percentage of each strain recovered on plates per enrichment step. The data showed a significant impact of co-cultivation on the growth of six out of seven strains on ham and a bias towards certain strains during selective enrichment. Competition was manifested by: (i) cessation of growth for the outcompeted strain when the dominant strain reached stationary phase, (ii) reduction of growth rates or (iii) total suppression of growth (both on ham and in enrichment broth or ALOA). Outgrowth of strains by their competitors on ALOA resulted in limited to no recovery, with the outcompeting strain accounting for up to 100% of the total recovered colonies. The observed bias was associated with the enrichment conditions (i.e. food type added to the enrichment broth) and the strain-combination. The outcome of growth competition on food or nonselective agar surface did not necessarily coincide with the results of competition during enrichment. The results show that certain strains present in foods may be missed during classical detection due to strain competition and such likelihood should be taken into consideration when resolving a listeriosis outbreak.

Highly Invasive Listeria monocytogenes Strains Have Growth and Invasion Advantages in Strain Competition.

Multiple Listeria monocytogenes strains can be present in the same food sample; moreover, infection with more than one L. monocytogenes strain can also occur. In this study we investigated the impact of strain competition on the growth and in vitro virulence potential of L. monocytogenes. We identified two strong competitor strains, whose growth was not (or only slightly) influenced by the presence of other strains and two weak competitor strains, which were outcompeted by other strains. Cell contact was essential for growth inhibition. In vitro virulence assays using human intestinal epithelial Caco2 cells showed a correlation between the invasion efficiency and growth inhibition: the strong growth competitor strains showed high invasiveness. Moreover, invasion efficiency of the highly invasive strain was further increased in certain combinations by the presence of a low invasive strain. In all tested combinations, the less invasive strain was outcompeted by the higher invasive strain. Studying the effect of cell contact on in vitro virulence competition revealed a complex pattern in which the observed effects depended only partially on cell-contact suggesting that competition occurs at two different levels: i) during co-cultivation prior to infection, which might influence the expression of virulence factors, and ii) during infection, when bacterial cells compete for the host cell. In conclusion, we show that growth of L. monocytogenes can be inhibited by strains of the same species leading potentially to biased recovery during enrichment procedures. Furthermore, the presence of more than one L. monocytogenes strain in food can lead to increased infection rates due to synergistic effects on the virulence potential.

A 3-year hygiene and safety monitoring of a meat processing plant which uses raw materials of global origin.

A systematic approach in monitoring the hygiene of a meat processing plant using classical microbiological analyses combined with molecular characterization tools may assist in the safety of the final products. This study aimed: (i) to evaluate the total hygiene level and, (ii) to monitor and characterize the occurrence and spread of Salmonella spp. and Listeria monocytogenes in the environment and the final products of a meat industry that processes meat of global origin. In total, 2541 samples from the processing environment, the raw materials, and the final products were collected from a Greek meat industry in the period 2011-2013. All samples were subjected to enumeration of total viable counts (TVC), Escherichia coli (EC) and total coliforms (TCC) and the detection of Salmonella spp., while 709 of these samples were also analyzed for the presence L. monocytogenes. Pathogen isolates were serotyped and further characterized for their antibiotic resistance and subtyped by PFGE. Raw materials were identified as the primary source of contamination, while improper handling might have also favored the proliferation of the initial microbial load. The occurrence of Salmonella spp. and L. monocytogenes reached 5.5% and 26.9%, respectively. Various (apparent) cross-contamination or persistence trends were deduced based on PFGE analysis results. Salmonella isolates showed wide variation in their innate antibiotic resistance, contrary to L. monocytogenes ones, which were found susceptible to all antibiotics except for cefotaxime. The results emphasize the biodiversity of foodborne pathogens in a meat industry and may be used by meat processors to understand the spread of pathogens in the processing environment, as well as to assist the Food Business Operator (FBO) in establishing effective criteria for selection of raw materials and in improving meat safety and quality. This approach can limit the increase of microbial contamination during the processing steps observed in our study as well as the cross contamination of meat products.