Biofilms In Food Processing Environments Research Articles

Staphylococcus aureus in Dairy Industry: Enterotoxin Production, Biofilm Formation, and Use of Lactic Acid Bacteria for Its Biocontrol.

Staphylococcus aureus is a well-known pathogen capable of producing enterotoxins during bacterial growth in contaminated food, and the ingestion of such preformed toxins is one of the major causes of food poisoning around the world. Nowadays 33 staphylococcal enterotoxins (SEs) and SE-like toxins have been described, but nearly 95% of confirmed foodborne outbreaks are attributed to classical enterotoxins SEA, SEB, SEC, SED, and SEE. The natural habitat of S. aureus includes the skin and mucous membranes of both humans and animals, allowing the contamination of milk, its derivatives, and the processing facilities. S. aureus is well known for the ability to form biofilms in food processing environments, which contributes to its persistence and cross-contamination in food. The biocontrol of S. aureus in foods by lactic acid bacteria (LAB) and their bacteriocins has been studied for many years. Recently, LAB and their metabolites have also been explored for controlling S. aureus biofilms. LAB are used in fermented foods since in ancient times and nowadays characterized strains (or their purified bacteriocin) can be intentionally added to prolong food shelf-life and to control the growth of potentially pathogenic bacteria. Regarding the use of these microorganism and their metabolites (such as organic acids and bacteriocins) to prevent biofilm development or for biofilm removal, it is possible to conclude that a complex network behind the antagonistic activity remains poorly understood at the molecular level. The use of approaches that allow the characterization of these interactions is necessary to enhance our understanding of the mechanisms that govern the inhibitory activity of LAB against S. aureus biofilms in food processing environments.

Eugenol nanoemulsion reduces Listeria monocytogenes biofilm by modulating motility, quorum sensing, and biofilm architecture

Listeria monocytogenes is a major foodborne pathogen in the United States that is capable of forming sanitizer-tolerant biofilms on diverse food contact surfaces and under varying temperature conditions. A plethora of research in the last decade has explored the potential of phytochemicals as antibiofilm agents. However, the low solubility of phytochemicals is a significant challenge that needs to be addressed to develop plant-based disinfectants that can be applied in the industry for controlling L. monocytogenes biofilms and improving food safety. This study investigated the efficacy of eugenol nanoemulsion (EGNE) in inhibiting biofilm formation in two strains of L. monocytogenes (Scott A and AT19115) on stainless steel surfaces at two temperatures (25 or 10°C). In addition, the effect of EGNE on pathogen motility, extracellular polymeric substances (EPS) production, eDNA production, and quorum sensing activity during biofilm formation was studied using standard bioassays. Moreover, the efficacy of EGNE in killing mature L. monocytogenes biofilm was also investigated against both the strains and temperature combinations. All experiments had a completely randomized design with duplicate samples and were repeated at least three times. EGNE had a particle size of ~75 nm, a polydispersity index of 0.25, and a high negative surface charge. EGNE 700 mg/L inhibited L. monocytogenes biofilm formation significantly by ~1.89 log in 72 h at 25°C and ~1.25 log on day 16 at 10°C, when compared to control (p < 0.05). EGNE at 2,750 mg/L concentration completely inactivated (~7 log CFU/coupon reduction as compared to control) L. monocytogenes biofilm cells developed at 25 or 10°C as early as 1 min of treatment time (p < 0.05). In addition, EGNE was able to significantly reduce the motility, EPS, eDNA production, and quorum sensing activity which plays a major role in biofilm formation. Both L. monocytogenes Scott A and AT19115 strains exhibited similar sensitivity to EGNE treatments. The results suggest that EGNE could potentially be used as a natural sanitizer to effectively control L. monocytogenes biofilms in food processing environments.

Two-component system virS/virR regulated biofilm formation of Listeria monocytogenes 10403S

Listeria monocytogenes is a foodborne pathogen that is ubiquitous in the natural environment.Listeria monocytogenes is capable of forming persistent biofilms in food processing environments, which can lead to persistent contamination of foods. It poses a significant threat to human health and food safety. Two-component system (TCS) plays a crucial role in various aspects of bacterial biofilm formation and regulation of virulence factors. The wild-type strain L. monocytogenes 10403S was utilized to build the gene deletion strain ΔvirS/virR and the gene complementation strain CΔvirS/virR. The biofilm assay with crystal violet staining and a motility assay showed that deletion of virS/virR resulted in reduced biofilm formation and motility in L. monocytogenes 10403S. Complementation strain CΔvirS/virR restored the phenotypes to level of wild-type strain. Mutation the 52nd aspartate (Asp52) of VirR with alanine resulted in significantly reduce in both biofilm formation and motility. The GFP reporter system and qRT-PCR demonstrated that deletion virS/virR significantly downregulated the transcriptional level of the chemotactic system cheY, which may be the main reason for the reduced motility and lower biofilm formation capacity of ΔvirS/virR. These findings demonstrated that the two-component system virS/virR positively regulated motility, thereby influencing biofilm formation in L. monocytogenes 10403S. This provides clues to control the biofilm risks of L. monocytogenes in food safety.

Risk factor-based clustering of Listeria monocytogenes in food processing environments using principal component analysis

Listeria monocytogenes has a range of strategies that allow it to persist as biofilms in food processing environments (FPE), making it a pathogen of concern to the food industry. The properties of these biofilms are highly variable among strains, and this significantly affects the risk of food contamination. The present study therefore aims to conduct a proof-of-concept study to cluster strains of L. monocytogenes by risk potential using principal component analysis, a multivariate approach.A set of 22 strains, isolated from food processing environments, were typed by serogrouping and pulsed-field gel electrophoresis, showing a relatively high diversity. They were characterized in terms of several biofilm properties that might pose a potential risk of food contamination. The properties studied were tolerance to benzalkonium chloride (BAC), the structural parameters of biofilms (biomass, surface area, maximum and average thickness, surface to biovolume ratio and roughness coefficient) measured by confocal laser scanning microscopy and (3) transfer of biofilm cells to smoked salmon.The PCA correlation circle revealed that the tolerance of biofilms to BAC was positively correlated with roughness, but negatively with biomass parameters. On the contrary, cell transfers were not related to three-dimensional structural parameters, which suggests the role of other variables yet unexplored. Additionally, hierarchical clustering grouped strains into three different clusters. One of them included the strains with high tolerance to BAC and roughness. Another one consisted of strains with enhanced transfer ability, whereas the third cluster contained those that stood out for the thickness of biofilms. The present study represents a novel and effective way to classify L. monocytogenes strains according to biofilm properties that condition the potential risk of reaching the consumer through food contamination. It would thus allow the selection of strains representative of different worst-case scenarios for future studies in support of QMRA and decision-making analysis.

The HD-GYP domain protein of Shewanella putrefaciens YZ08 regulates biofilm formation and spoilage activities

Shewanella putrefaciens is an important spoilage bacteria in seafood and its ability to form biofilms in food processing environments increases the chances of food spoilage. Exploring the regulatory factors associated with biofilm formation and spoilage activity in S. putrefaciens is of great significance for extending the shelf life of seafood. In this work, the regulatory function of HD-GYP domain protein K2227_17660 in spoilage microorganism S. putrefaciens YZ08 was studied. The deletion mutant Δ17660 was developed to explore the effects of K2227_17660 in c-di-GMP content regulation, motility, biofilm formation, extracellular protease activity, and spoilage potential by phenotypic and transcriptional comparison with wild-type (WT) strain. Deletion of K2227_17660 significantly increased c-di-GMP content, biofilm biomass, the production of extracellular polysaccharide, trimethylamine (TMA), and putrescine compared with WT strains, and also affected membrane fatty acid composition. Furthermore, RT-qPCR results revealed the expression levels of genes associated with biofilm biomass, spoilage and unsaturated fatty acids (UFAs) synthesis changed in a manner consistent with the phenotypes. Our results indicated that K2227_17660 possesses phosphodiesterase (PDE) activity that controls the biofilm biomass and spoilage potential of S. putrefaciens. This study provided a basis for a correlation between c-di-GMP and food spoilage in S. putrefaciens, providing new insights into the control of food quality and safety.

Antibiofilm efficacy of Leuconostoc mesenteroides J.27-derived postbiotic and food-grade essential oils against Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Escherichia coli alone and in combination, and their application as a green preservative in the seafood industry

Foodborne pathogen-mediated biofilms in food processing environments are severe threats to human lives. In the interest of human and environmental safety, natural substances with antimicrobial properties and generally regarded as safe (GRAS) status are the futuristic disinfectants of the food industry. In this study, the efficacy of bioactive, soluble products (metabolic by-products) from lactic acid bacteria (LAB) and plant-derived essential oils (EO) were investigated as biocidal agents. The postbiotic produced by kimchi-derived Leuconostoc mesenteroides J.27 isolate was analyzed for its metabolic components to reveal its antimicrobial potential against three pathogenic microorganisms (Vibrio parahaemolyticus, Pseudomonas aeruginosa, and Escherichia coli). Additionally, the efficacy of food-grade EO (eugenol and thymol, respectively) was also assessed in our study. Determination of the minimum inhibitory concentration (MIC) of postbiotic and EO against three tested pathogens revealed that the sub-MIC (0.5 MIC) of postbiotic and EO could efficiently inhibit the biofilm formation on both seafood (squid) and seafood-processing surfaces (rubber and low-density polyethylene plastic). Moreover, the polymerase chain reaction (PCR) analysis confirmed that the LAB J.27 isolate possesses bacteriocin- and enzyme-coding genes. The residual antibacterial activity of the produced postbiotic was maintained over a diverse pH range (pH 1–6) but was entirely abolished at neutral or higher pH values. However, the activity was unaffected by exposure to high temperatures (100 and 121 °C) and storage (30 days). Notably, the leakage of intracellular metabolites, damage to DNA, and the down-regulation of biofilm-associated gene expression in the pathogens increased significantly (p > 0.05) following the combination treatment of postbiotic with thymol compared to postbiotic with eugenol. Nonetheless, all in vitro results indicated the prospective use of combining Leu. mesenteroides J.27-derived postbiotic with both EO as a “green preservative” in the seafood industry to inhibit the formation of pathogenic microbial biofilms.

Combating biofilms of foodborne pathogens with bacteriocins by lactic acid bacteria in the food industry.

Most foodborne pathogens have biofilm-forming capacity and prefer to grow in the form of biofilms. Presence of biofilms on food contact surfaces can lead to persistence of pathogens and the recurrent cross-contamination of food products, resulting in serious problems associated with food safety and economic losses. Resistance of biofilm cells to conventional sanitizers urges the development of natural alternatives to effectively inhibit biofilm formation and eradicate preformed biofilms. Lactic acid bacteria (LAB) produce bacteriocins which are ribosomally synthesized antimicrobial peptides, providing a great source of nature antimicrobials with the advantages of green and safe properties. Studies on biofilm control by newly identified bacteriocins are increasing, targeting primarily onListeria monocytogenes, Staphylococcus aureus, Salmonella, and Escherichia coli. This review systematically complies and assesses the antibiofilm property of LAB bacteriocins in controlling foodborne bacterial-biofilms on food contact surfaces. The bacteriocin-producing LAB genera/species, test method (inhibition and eradication), activity spectrum and surfaces are discussed, and the antibiofilm mechanisms are also argued. The findings indicate that bacteriocins can effectively inhibit biofilm formation in a dose-dependent manner, but are difficult to disrupt preformed biofilms. Synergistic combination with other antimicrobials, incorporation in nanoconjugates and implementation of bioengineering can help to strengthen their antibiofilm activity. This review provides an overview of the potential and application of LAB bacteriocins in combating bacterial biofilms in food processing environments, assisting in the development and widespread use of bacteriocin as a promising antibiofilm-agent in food industries.

The role of the Listeria monocytogenes surfactome in biofilm formation.

Summary Listeria monocytogenes is a highly pathogenic foodborne bacterium that is ubiquitous in the natural environment and capable of forming persistent biofilms in food processing environments. This species has a rich repertoire of surface structures that enable it to survive, adapt and persist in various environments and promote biofilm formation. We review current understanding and advances on how L. monocytogenes organizes its surface for biofilm formation on surfaces associated with food processing settings, because they may be an important target for development of novel antibiofilm compounds. A synthesis of the current knowledge on the role of Listeria surfactome, comprising peptidoglycan, teichoic acids and cell wall proteins, during biofilm formation on abiotic surfaces is provided. We consider indications gained from genome‐wide studies and discuss surfactome structures with established mechanistic aspects in biofilm formation. Additionally, we look at the analogies to the species L. innocua, which is closely related to L. monocytogenes and often used as its model (surrogate) organism.

Role of the VirSR-VirAB system in biofilm formation of Listeria monocytogenes EGD-e

The ability of Listeria monocytogenes, an important foodborne pathogen, to form biofilms in food processing environments leads to increased opportunity for contamination of food products, which is a major concern for food safety. In this study, the role of a complex system composed of the VirSR two-component signal transduction system (TCS) and the ATP-binding cassette (ABC) transporter VirAB in biofilm formation of L. monocytogenes EGD-e was investigated. Biofilm formation was measured using the microplate assay with crystal violet staining, confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM), and attachment and swarming motility were compared between strain EGD-e and its isogenic deletion mutants. Additionally, the relative expression levels of genes associated with the early steps of biofilm development in the wild-type and mutant strains were also determined by RT-qPCR. Results from microplate assay, CLSM and SEM showed that VirR is not required for biofilm formation in L. monocytogenes EGD-e. A central finding of this study is that both VirAB and VirS are essential for biofilm formation and they could function as a whole in biofilm formation of L. monocytogenes EGD-e. The results also demonstrated that both VirAB and VirS are involved in attachment, but they are not associated with swarming motility. Results from RT-qPCR showed that flaA, motA and motB were downregulated in the mutant strains ΔvirAB and ΔvirS, which could be the possible reason for reduced attachment and biofilm formation in these mutants. This study provides a better understanding of the mechanisms involved in biofilm formation of L. monocytogenes, leading to improved processes to control this biofilm-forming foodborne pathogen.

Biofilm Formation by Listeria monocytogenes 15G01, a Persistent Isolate from a Seafood-Processing Plant, Is Influenced by Inactivation of Multiple Genes Belonging to Different Functional Groups.

Listeria monocytogenes is a ubiquitous foodborne pathogen that results in a high rate of mortality in sensitive and immunocompromised people. Contamination of food with L. monocytogenes is thought to occur during food processing, most often as a result of the pathogen producing a biofilm that persists in the environment and acting as the source for subsequent dispersal of cells onto food. A survey of seafood-processing plants in New Zealand identified the persistent strain 15G01, which has a high capacity to form biofilms. In this study, a transposon library of L. monocytogenes 15G01 was screened for mutants with altered biofilm formation, assessed by a crystal violet assay, to identify genes involved in biofilm formation. This screen identified 36 transposants that showed a significant change in biofilm formation compared to the wild type. The insertion sites were in 27 genes, 20 of which led to decreased biofilm formation and seven to an increase. Two insertions were in intergenic regions. Annotation of the genes suggested that they are involved in diverse cellular processes, including stress response, autolysis, transporter systems, and cell wall/membrane synthesis. Analysis of the biofilms produced by the transposants using scanning electron microscopy and fluorescence microscopy showed notable differences in the structure of the biofilms compared to the wild type. In particular, inactivation of uvrB and mltD produced coccoid-shaped cells and elongated cells in long chains, respectively, and the mgtB mutant produced a unique biofilm with a sandwich structure which was reversed to the wild-type level upon magnesium addition. The mltD transposant was successfully complemented with the wild-type gene, whereas the phenotypes were not or only partially restored for the remaining mutants.IMPORTANCE The major source of contamination of food with Listeria monocytogenes is thought to be due to biofilm formation and/or persistence in food-processing plants. By establishing as a biofilm, L. monocytogenes cells become harder to eradicate due to their increased resistance to environmental threats. Understanding the genes involved in biofilm formation and their influence on biofilm structure will help identify new ways to eliminate harmful biofilms in food processing environments. To date, multiple genes have been identified as being involved in biofilm formation by L. monocytogenes; however, the exact mechanism remains unclear. This study identified four genes associated with biofilm formation by a persistent strain. Extensive microscopic analysis illustrated the effect of the disruption of mgtB, clsA, uvrB, and mltD and the influence of magnesium on the biofilm structure. The results strongly suggest an involvement in biofilm formation for the four genes and provide a basis for further studies to analyze gene regulation to assess the specific role of these biofilm-associated genes.

Antimicrobial and antifouling polymeric coating mitigates persistence of Pseudomonas aeruginosa biofilm

Food wasted due to food spoilage remains a global challenge to the environmental sustainability and security of food supply. In food manufacturing, post-processing contamination of food can occur due to persistent bacterial biofilms, which can be resistant to conventional cleaning and sanitization. The objective was to characterize the efficacy of a polymeric coating in reducing Pseudomonas aeruginosa biofilm establishment and facilitating its removal. Viable cell density of a 48 h biofilm was reduced by 2.10 log cfu cm−2 on the coated surface, compared to native polypropylene. Confocal laser scanning and electron microscopy indicated reductions in mature biofilm viability and thickness on the coated material. The antifouling coating improved cleanability, with ∼2.5 log cfu cm−2 of viable cells remaining after 105 min cleaning by water at 65 °C, compared to 4.5 log cfu cm−2 remaining on native polypropylene. Such coatings may reduce the persistence of biofilms in food processing environments, in support of reducing food spoilage and waste

Biofilms in Food Processing Environments: Challenges and Opportunities.

This review examines the impact of microbial communities colonizing food processing environments in the form of biofilms on food safety and food quality. The focus is both on biofilms formed by pathogenic and spoilage microorganisms and on those formed by harmless or beneficial microbes, which are of particular relevance in the processing of fermented foods. Information is presented on intraspecies variability in biofilm formation, interspecies relationships of cooperativism or competition within biofilms, the factors influencing biofilm ecology and architecture, and how these factors may influence removal. The effect on the biofilm formation ability of particular food components and different environmental conditions that commonly prevail during food processing is discussed. Available tools for the in situ monitoring and characterization of wild microbial biofilms in food processing facilities are explored. Finally, research on novel agents or strategies for the control of biofilm formation or removal is summarized.

Inhibition and Inactivation of Escherichia coli O157:H7 Biofilms by Selenium

The present study investigated the efficacy of selenium (Se) in reduction of enterohemorrhagic Escherichia coli (EHEC) exopolysaccharide (EPS) synthesis, inhibition of biofilm formation at 25 and 4°C on polystyrene surface, and inactivation of mature EHEC biofilms in combination with hot water. Sterile 96-well polystyrene plates inoculated with EHEC (∼6.0 log CFU per well) were treated with a subinhibitory concentration (SIC) of Se, and biofilms were allowed to mature at 4 and 25°C for 96 h. Biofilm-associated bacterial population was determined by scraping and plating, whereas the extent of EPS production was determined using ruthenium red staining assay. Solid surface assay was used to study the effect of Se on early attachment of EHEC cells to polystyrene. The efficacy of Se in rapid inactivation of preformed, mature EHEC biofilm was investigated by treating biofilms on polystyrene plates with the MBC of Se in combination with hot water at 80°C with a contact time of 0 min, 30 s, 2 min, and 5 min. Furthermore, the effect of Se on EHEC biofilm architecture was visualized using confocal microscopy, whereas the effect of Se on EHEC biofilm genes was determined using real-time quantitative PCR (RT-qPCR). Finally, the potential feasibility of coating stainless steel surfaces with Se nanoparticles to inhibit EHEC biofilm formation was studied. Se reduced early attachment of planktonic cells, biofilm formation, and EPS synthesis in EHEC ( P < 0.05). Se in combination with hot water reduced biofilm-associated bacterial counts by 3 to 4 log CFU/mL at 5 min of exposure compared with the control ( P < 0.05). However, hot water treatment alone decreased biofilm-associated bacterial counts by only 1.0 log CFU/mL. RT-qPCR results revealed that Se down-regulated the transcription of critical genes associated with biofilm synthesis in EHEC ( P < 0.05). The results collectively suggest that Se could potentially be used to control EHEC biofilms in food processing environments, but appropriate applications need to be validated.

Single and binary applications of essential oils effectively control Listeria monocytogenes biofilms

The high environmental impact and the increasing resistance of microorganisms to conventional disinfectants applied in the food industry have led to an increasing interest in the antimicrobial properties of plant essential oils (EOs). The present study aimed to develop highly effective EO-based treatments against Listeria monocytogenes biofilms formed on stainless steel and polystyrene surfaces under food-related conditions. EOs with an efficacy comparatively higher or similar to peracetic acid and sodium hypochlorite against L. monocytogenes planktonic cells were selected, including for the first time Cordia verbenacea and Pimenta pseudochariophyllus oils. Selected EOs were subsequently characterized chemically by GC/MS analysis and applied in single treatments, binary combinations and combinations with peracetic acid to evaluate their efficacy against 24-h-old L. monocytogenes biofilms. Lippia sidoides, Thymus vulgaris and Pimenta pseudochariophyllus oils were highly effective against planktonic cells and biofilms of L. monocytogenes. Thymol was the main compound of Lippia sidoides and Thymus vulgaris oils, whereas Pimenta pseudochariophyllus contained high concentrations of chavibetol. However, only Lippia sidoides oil was able to completely eradicate L. monocytogenes biofilms at relatively high doses (2.75% v/v). The application of selected EOs in binary combinations decreased considerably doses required to kill 99.99% of biofilm cells. Moreover, the application of peracetic acid combined with these EOs enhanced its efficacy against L. monocytogenes biofilms. Therefore, EO-based treatments proposed in this study, particularly those combining L. sidoides with T. vulgaris or peracetic acid, could represent an effective and environmentally-friendly strategy to control L. monocytogenes biofilms in food-processing environments.

Role of cell surface composition and lysis in static biofilm formation by Lactobacillus plantarum WCFS1

Next to applications in fermentations, Lactobacillus plantarum is recognized as a food spoilage organism, and its dispersal from biofilms in food processing environments might be implicated in contamination or recontamination of food products. This study provides new insights into biofilm development by L. plantarum WCFS1 through comparative analysis of wild type and mutants affected in cell surface composition, including mutants deficient in the production of Sortase A involved in the covalent attachment of 27 predicted surface proteins to the cell wall peptidoglycan (ΔsrtA) and mutants deficient in the production of capsular polysaccharides (CPS1–4, Δcps1–4). Surface adhesion and biofilm formation studies revealed none of the imposed cell surface modifications to affect the initial attachment of cells to polystyrene while biofilm formation based on Crystal Violet (CV) staining was severely reduced in the ΔsrtA mutant and significantly increased in mutants lacking the cps1 cluster, compared to the wild-type strain. Fluorescence microscopy analysis of biofilm samples pointed to a higher presence of extracellular DNA (eDNA) in cps1 mutants and this corresponded with increased autolysis activity. Subsequent studies using Δacm2 and ΔlytA derivatives affected in lytic behaviour revealed reduced biofilm formation measured by CV staining, confirming the relevance of lysis for the build-up of the biofilm matrix with eDNA.

Bacterial biofilms in food processing environments: a review of recent developments in chemical and biological control

SummaryBiofilms are immobile communities of micro‐organisms attached to any surface, such as stainless steel or a food matrix surface or on packaging material. They may be composed of a single species, but more generally, in the natural environment, they consist of mixed species together with an extracellular matrix. Biofilms provide a common mechanism of persistence for a number of bacterial species especially in food processing environments, and therefore, prevention of biofilm formation and the removal of preformed biofilms are an important issue for the food industry. This article reviews the current understanding on the formation of biofilms and recent developments in biological and chemical methods for prevention and removal.

Synergistic bactericidal action of phytic acid and sodium chloride against Escherichia coli O157:H7 cells protected by a biofilm

The food industry must prevent the build-up of strong Escherichia coli O157:H7 biofilms in food processing environments. The present study examined the bactericidal action of phytic acid (PA), a natural extract from rice bran and the hulls/peels of legumes, against E. coli O157:H7 biofilms. The synergistic bactericidal effects of PA plus sodium chloride (NaCl) were also examined. E. coli O157:H7 biofilms were allowed for form on stainless steel coupons by culture in both rich (tryptic soy broth, TSB) and minimal (M9) medium at 22°C for 6days. Bacterial cells within biofilms grown in M9 medium were significantly more resistant to PA than those grown in TSB (p<0.05); thus M9 medium was selected for further experiments. The anti-biofilm effect of PA was significantly increased by addition of NaCl (2–4%) (p<0.05); indeed, the combination of 0.4% PA plus 3–4% NaCl completely inactivated E. coli O157:H7 biofilms without recovery (a>6.5logCFU/cm2 reduction). Neither PA nor NaCl alone were this effective (PA, 1.6–2.7logCFU/cm2 reduction; NaCl, <0.5logCFU/cm2 reduction). Confocal laser scanning microscopy images of propidium iodide-treated cells showed that PA (0.4%) plus NaCl (2–4%) had marked membrane permeabilizing effects. These results suggest that a sanitizer that combines these two naturally occurring antimicrobial agents may be useful to food safety managers who encounter thick biofilm formation in food processing environments.

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.

Antibiofilm effect of plant derived antimicrobials on Listeria monocytogenes

The present study investigated the efficacy of sub-inhibitory concentrations (SICs, concentrations not inhibiting bacterial growth) and bactericidal concentrations (MBCs) of four, generally recognized as safe (GRAS), plant-derived antimicrobials (PDAs) in inhibiting Listeria monocytogenes (LM) biofilm formation and inactivating mature LM biofilms, at 37, 25 and 4 °C on polystyrene plates and stainless-steel coupons. In addition, the effect of SICs of PDAs on the expression of LM genes critical for biofilm synthesis was determined by real-time quantitative PCR. The PDAs and their SICs used for inhibition of biofilm were trans-cinnamaldehyde (TC 0.50, 0.75 mM), carvacrol (CR 0.50, 0.65 mM), thymol (TY 0.33, 0.50 mM), and eugenol (EG 1.8, 2.5 mM), whereas the PDA concentrations used for inactivating mature biofilms were 5.0 and 10.0 mM (TC, CR), 3.3 and 5.0 mM (TY), 18.5 and 25.0 mM (EG). All PDAs inhibited biofilm synthesis and inactivated fully formed LM biofilms on both matrices at three temperatures tested (P < 0.05). Real-time quantitative PCR data revealed that all PDAs down-regulated critical LM biofilm-associated genes (P < 0.05). Results suggest that TC, CR, TY, and EG could potentially be used to control LM biofilms in food processing environments, although further studies under commercial settings are necessary.

Phenotypic, Proteomic, and Genomic Characterization of a Putative ABC-Transporter Permease Involved in Listeria monocytogenes Biofilm Formation

The foodborne pathogen Listeria monocytogenes is able to form biofilms in food processing environments. Previously, we have reported that an lm.G_1771 gene (encoding a putative ABC-transporter permease) was involved in negative regulation of L. monocytogenes biofilm formation using LM-49, a biofilm-enhanced mutant isolated on Tn917 mutagenesis (AEM 2008 p.7675-7683). Here, the possible action of this ABC-transporter permease in L. monocytogenes biofilm formation was characterized by phenotypic, proteomic, and genomic analyses using an lm.G_1771 gene deletant (Δ1771). The Δ1771 mutant exhibited the same enhanced ability for biofilm formation as the LM-49 strain using a crystal violet staining assay. DNA microarrays and two-dimensional gel electrophoresis revealed 49 and 11 differentially expressed (twofold or more) genes or proteins in Δ1771, respectively. The transcriptomics study indicated that lm.G_1771 could play a vital role in regulating candidate genes involved in biofilm formation such as genes encoding cell surface proteins (Dlt), cell surface anchor proteins (SrtA), and transcriptional regulators (GntR) contributing to negative regulation of biofilm formation by L. monocytogenes. The mutant Δ1771 was more sensitive to Triton X-100 and less resistant to cationic antibiotics, which might be explained by the down-regulation of dlt operon in this deletant and the fact that dlt involves the incorporation of D-alanine residues into lipoteichoic acids, resulting in a positive net charge on the teichoic acids. Therefore, lm.G_1771 is considered to be involved in negative regulation of biofilm formation, and the results from this work provide a possible molecular mechanism of biofilm formation regulated by lm.G_1771 in L. monocytogenes.