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Paneer is a fresh, soft ready-to-eat cheese that is susceptible to Listeria monocytogenes contamination, exemplified by product recalls in Australia, Canada, and the USA. Previous research demonstrates that L. monocytogenes grows in paneer, however there are no paneer-specific predictive models that quantify the effect of environmental conditions on L. monocytogenes viability. This study measured the viability of a five-strain cocktail of L. monocytogenes in freshly prepared paneer incubated at 4-40 °C. Growth rates were fitted with the extended Ratkowsky square root model, with growth rates ranging from 0.014 to 0.352 log 10  CFU/h. In comparison with published models, only the ComBase L. monocytogenes broth model acceptably predicted growth (B f = 1.01, A f = 1.12) versus the developed model. The influence of paneer pH (5.0-6.0) and storage temperature (41-45 °C) on L. monocytogenes growth at the upper temperature growth boundary was described using a logistic model. These models provide quantitative tools to improve the safety of paneer processing conditions, shelf-life estimation, food safety management plans, and risk assessment., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The ability of Listeria monocytogenes isolates to survive within the food production environment (FPE), as well as virulence, varies greatly between strains. There are specific genetic determinants that have been identified which can strongly influence a strains ability to survive in the FPE and/or within human hosts. In this study, we assessed the FPE fitness and virulence potential, including efficacy of selected hygiene or treatment intervention, against 52 L. monocytogenes strains isolated from various food and food environment sources. Phenotypic tests were performed to determine the minimum inhibitory concentration of cadmium chloride and benzalkonium chloride and the sensitivities to five clinically relevant antibiotics. A genomic analysis was also performed to identify resistance genes correlating to the observed phenotypic resistance profiles, along with genetic determinants of interest which may elude to the FPE fitness and virulence potential. A transposon element containing a novel cadmium resistance gene, cadA7, a Tn916 variant insert in the hypervariable Listeria genomic island 1 region and an LGI2 variant were identified. Resistance to cadmium and disinfectants was prevalent among isolates in this study, although no resistance to clinically important antimicrobials was observed. Potential hypervirulent strains containing full length inlA, LIPI-1 and LIPI-3 were also identified in this study. Cumulatively, the results of this study show a vast array of FPE survival and pathogenicity potential among food production-associated isolates, which may be of concern for food processing operators and clinicians regarding L. monocytogenes strains colonising and persisting within the FPE, and subsequently contaminating food products then causing disease in at risk population groups., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Listeria monocytogenes is a ubiquitous bacterium capable of colonising and persisting within food production environments (FPEs) for many years, even decades. This ability to colonise, survive and persist within the FPEs can result in food product cross-contamination, including vulnerable products such as ready to eat food items. Various environmental and genetic elements are purported to be involved, with the ability to form biofilms being an important factor. In this study we examined various mechanisms which can influence colonisation in FPEs. The ability of isolates (n = 52) to attach and grow in biofilm was assessed, distinguishing slower biofilm formers from isolates forming biofilm more rapidly. These isolates were further assessed to determine if growth rate, exopolymeric substance production and/or the agr signalling propeptide influenced these dynamics and could promote persistence in conditions reflective of FPE. Despite no strong association with the above factors to a rapid colonisation phenotype, the global transcriptome suggested transport, energy production and metabolism genes were widely upregulated during the initial colonisation stages under nutrient limited conditions. However, the upregulation of the metabolism systems varied between isolates supporting the idea that L. monocytogenes ability to colonise the FPEs is strain-specific.

Listeria monocytogenes is a foodborne pathogen capable of colonizing and persisting in the food production environment (FPE). While there are a variety of factors involved in L. monocytogenes' ability to persist in FPE, the ability to form biofilms has the potential to increase their chance of survival and long-term colonization. Understanding the mechanisms involved in L. monocytogenes ability to form biofilms may potentially help food safety managers optimize control strategies targeting it in the FPE. In this chapter, a high-throughput method to determine L. monocytogenes ability to attach and form biofilms utilizing FPE-grade stainless steel is described. This method provides fast and efficient results, facilitating scaling up to large numbers of isolates to measure their ability to form biofilms, where lower-throughput approaches can then be utilized to further characterize isolates of interest.

Contamination of industrial and domestic food usage environments by the attachement of bacterial food-borne pathogen Listeria monocytogenes has public health and economic implications. Comprehensive proteomics experiments using label-free liquid chromatography/tandem mass spectrometry were used to compare the proteomes of two different L. monocytogenes strains (Siliken_1/2c and F2365_4b), which show very different capacities to attach to surfaces. Growth temperature and strain type were highly influential on the proteomes in both attached and planktonic cells. On the basis of the proteomic data, it is highly unlikely that specific surface proteins play a direct role in adherence to inanimate surfaces. Instead, strain-dependent responses related to cell envelope polymer biosynthesis and stress response regulation likely contribute to a different ability to attach and also to survive external stressors. Collectively, the divergent proteome-level responses observed define strain- and growth-temperature-dependent differences relevant to attachment efficacy, highlight relevant proteins involved in stress protection in attached cells, and suggest that strain differences and growth conditions are important in relation to environmental persistence.

The ability of Listeria monocytogenes to adapt to various food and food- processing environments has been attributed to its robustness, persistence and prevalence in the food supply chain. To improve the present understanding of molecular mechanisms involved in hyperosmotic and low-temperature stress adaptation of L. monocytogenes, we undertook transcriptomics analysis on three strains adapted to sub-lethal levels of these stress stimuli and assessed functional gene response. Adaptation to hyperosmotic and cold-temperature stress has revealed many parallels in terms of gene expression profiles in strains possessing different levels of stress tolerance. Gene sets associated with ribosomes and translation, transcription, cell division as well as fatty acid biosynthesis and peptide transport showed activation in cells adapted to either cold or hyperosmotic stress. Repression of genes associated with carbohydrate metabolism and transport as well as flagella was evident in stressed cells, likely linked to activation of CodY regulon and consequential cellular energy conservation.

Listeria monocytogenes is a foodborne human pathogen capable of causing life-threatening disease in susceptible populations. Previous proteomic analysis we performed demonstrated that different strains of L. monocytogenes initiate a stringent response when subjected to alkaline growth conditions. Here, using multidimensional protein identification technology (MudPIT), we show that in L. monocytogenes EGD-e this response involves an energy shift to anaerobic pathways in response to the extracellular pH environment. Importantly we show that this supports a reduction in relative lag time following an abrupt transition to low oxygen tension culture conditions. This has important implications for the packaging of fresh and ready-to-eat foods under reduced oxygen conditions in environments where potential exists for alkaline adaptation.

Alkaline solutions are used to clean food production environments but the role of alkaline resistance in persistent food factory contamination by Listeria monocytogenes is unknown. We used shotgun proteomics to characterise alkaline adapted L. monocytogenes recovered as persistent and transient food factory contaminants. Three unrelated strains were studied including two persistent and a transient food factory contaminant determined using multilocus sequence typing (MLST). The strains were adapted to growth at pH 8.5 and harvested in exponential phase. Protein extracts were analysed using multidimensional protein identification technology (MudPIT) and protein abundance compared by spectra counting. The strains elicited core responses to alkaline growth including modulation of intracellular pH, stabilisation of cellular processes and reduced cell-division, independent to lineage, MLST or whether the strains were transient or persistent contaminants. Alkaline adaptation by all strains corresponded to that expected in stringent-response induced cells, with protein expression supporting metabolic shifts concordant with elevated alarmone production and indicating that the alkaline-stringent response results from energy rather than nutrient limitation. We believe this is the first report describing induction of a stringent response in different L. monocytogenes strains by alkaline pH under non-limiting growth conditions. The work emphasises the need for early intervention to avoid persistent food factory contamination by L. monocytogenes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The global proteomic responses of the foodborne pathogen Listeria monocytogenes strain Scott A, during active growth and transition to the stationary growth phase under progressively more acidic conditions, created by addition of lactic acid and HCl, were investigated using label-free liquid chromatography/tandem mass spectrometry. Approximately 56% of the Scott A proteome was quantitatively assessable, and the data provides insight into its acquired acid tolerance response (ATR) as well as the relation of the ATR to the growth phase transition. Alterations in protein abundance due to acid stress were focused in proteins belonging to the L. monocytogenes common genome, with few strain-dependent proteins involved. However, one of the two complete prophage genomes appeared to enter lysogeny. During progressive acidification, the growth rate and yield were reduced 55% and 98%, respectively, in comparison to nonacidified control cultures. The maintenance of the growth rate was determined to be connected to activation of cytoplasmic pH homeostatic mechanisms while cellular reproductive-related and cell component turnover proteins were markedly more abundant in acid stressed cultures. Cell biomass accumulation was impeded predominantly due to repression of phosphodonor-linked enzymes involved with sugar phosphotransfer, glycolysis, and cell wall polymer biosynthesis. Acidification caused a shift from heterofermentation to an oxidatively stressed state in which ATP appears to be generated mainly through the pyruvate dehydrogenase/pyruvate oxidase/phosphotransacetylase/acetate kinase and branched chain acid dehydrogenase pathways. Analysis of regulons indicated energy conservation occurs due to repression by the GTP/isoleucine sensor CodY and also the RelA mediated stringent response. Whole proteome analysis proved to be an effective way to highlight proteins involved with the acquisition of the ATR.

This study aimed to identify factors that influence the development of biofilm by Listeria monocytogenes strains and to determine the extent to which biofilm production protects against quaternary ammonium compound (QAC) disinfectant challenge. A total of 95 L. monocytogenes strains were studied and biofilm production was assessed as a function of incubation temperature, media pH, strain origin, serotype, and environmental persistence status. Attachment and biofilm development (inferred by the level of attached biomass) were measured in vitro using a colourimetric 96-well microtitre plate method in nutritive media (Brain-Heart Infusion). Increased biofilm production correlated with increasing temperature and the most acidic, or most alkaline, growth conditions tested. Clinical and environmental (food factory) strains were observed to increase biofilm production at higher and lower incubation temperatures respectively, independent of their rate of planktonic growth. Serotype 1/2a strains produced significantly more biofilm. Biofilm maturity, rather than strain, was correlated with resistance to QAC. Carbohydrate containing exopolymeric material could not be detected in the biofilm of representative strains, and no correlation between strains recovered as persistent food factory contaminants and biofilm production was identified. Although limited to in vitro inference based on the assay system used, our results suggest that environmental conditions determine the level of biofilm production by L. monocytogenes strains, independent of the rate of planktonic growth, and that this may manifest from selection pressures to which a given strain grows optimally., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Application of simultaneous low pH (pH 3.5) and low water activity (a(w)=0.9; 2.5M NaCl) conditions to Listeria monocytogenes strains ScottA and FW03/0035, and growth permissive temperatures from 25 degrees C up to 45 degrees C result in increasingly accelerated inactivation rates. This phenomenon was related to i) increased cell permeability as suggested by ethidium homodimer-1 uptake and ii) de-energization as indicated by rapidly reduced ATP basal levels. Enrichment-based recovery experiments indicated that the stress conditions eventually lead to complete loss of reproductive capacity, possibly corresponding to an irreversible collapse of pH homeostasis. Transcriptomic analyses were used to obtain further insights into the physiology of the inactivation process occurring at 25 degrees C where inactivation times were more prolonged. QPCR, mRNA decay and microarray experiments revealed transcripts of tufA and other genes become substantially more stable during inactivation resulting from exposure to combined low pH/a(w) and from non-growth permissive temperature exposure. Genes that appear to be important for initial survival of combined low pH/a(w) were delineated by K-means clustering of expression data and included an overrepresentation of SigB-activated genes, the overall response of which fades with increasing time of inactivation exposure., (2010 Elsevier B.V. All rights reserved.)

In an experiment delineating aciduric strains, food and clinical Listeria monocytogenes isolates tended to produce the most biomass whereas ovine and avian strains produced comparatively less biomass when exposed to high levels of sodium diacetate (SD) and potassium sorbate. Compared to reference strains that exhibited greater acid sensitivity, representative food isolates with comparatively good growth capacities in the presence of 21 mM SD at pH 5.0 accumulated reduced levels of acetate anion and K(+) ion. The aciduric nature of SD-resistant strains was also reflected by comparatively high tolerance to pH 2.4 (HCl) acid challenges, a property boosted by the presence of SD. Exposure to elevated levels of SD (21 mM SD at pH 5.0) was found to have broad effects on gene expression, as differentiated from effects caused by mildly acidic conditions (pH 5.0). SD-resistant strain FW04/0025 was more responsive to elevated SD, increasing the expression of 222 genes (>2-fold change [P < 0.05]), compared to the more sensitive EGD reference strain, which exhibited increases in expression of 112 genes. Key differences between the strains in relation to SD-enhanced transcripts were notably associated with the cell envelope, oxidative stress management, and intermediary metabolism. SD thus appears to differentially influence growth efficiency and survival of strains, under conditions relevant to acidic foods, that could be due to altered cell wall and metabolic phenotypes.

High hydrostatic pressure processing (HPP) is currently being used as a treatment for certain foods to control the presence of food-borne pathogens, such as Listeria monocytogenes. Genomic microarray analysis was performed to determine the effects of HPP on L. monocytogenes in order to understand how it responds to mechanical stress injury. Reverse transcriptase PCR analysis of tufA and rpoC indicated that the reduction of mRNA expression in HPP-treated cells was dependent on intensity and time of the treatment. Treatments of 400 and 600 MPa for 5 min on cells in the exponential growth phase, though leading to partial or complete cellular inactivation, still resulted in measurable relative differential gene expression. Gene set enrichment analysis indicated that HPP induced increased expression of genes associated with DNA repair mechanisms, transcription and translation protein complexes, the septal ring, the general protein translocase system, flagella assemblage and chemotaxis, and lipid and peptidoglycan biosynthetic pathways. On the other hand, HPP appears to suppress a wide range of energy production and conversion, carbohydrate metabolism and virulence-associated genes accompanied by strong suppression of the SigB and PrfA regulons. HPP also affected genes controlled by the pleotrophic regulator CodY. HPP-induced cellular damage appears to lead to increased expression of genes linked to sections of the cell previously shown in bacteria to be damaged or altered during HPP exposure and suppression of gene expression associated with cellular growth processes and virulence.