Identification of genes involved in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> biofilm formation by <Emphasis Type="Italic">mariner</Emphasis>-based transposon mutagenesis

Listeria monocytogenes is a ubiquitous food-borne pathogen, whose distribution and survival in food-processing environments are associated with the ability to form biofilms. The process of biofilm formation is complex and its molecular mechanism is relatively poorly understood in L. monocytogenes. To better understand the genetics of this process, a mariner-based transposon mutagenesis strategy was used to identify genes involved in biofilm formation of L. monocytogenes. A library of 6,500 mutant colonies was screened for reduced biofilm formation using a microtiter plate biofilm assay. Forty biofilm-deficient mutants of L. monocytogenes were identified based on DNA sequences of the transposon-flanking regions and Southern hybridization with a transposon-based probe. The insertions harbored by these mutants led to the identification of 24 distinct loci, 18 of which, to our knowledge, have not been previously reported to function in the biofilm formation in L. monocytogenes. Genetic complementation confirmed the importance of lmo1386, a gene encoding a putative DNA translocase, for biofilm formation. Molecular analyses of mutants indicated that the majority of the 24 identified genes are related to flagella motility, gene regulation, and cell surface structures.     

Interference of sanitizers,NaCl and curing salts on Listeria monocytogenes adhesion and subsequent biofilm formation

Listeria monocytogenes, a well-known foodborne pathogen and the causative agent of listeriosis, has the ability to persist in food processing environments due to its high adhesion ability in different surfaces, playing an important role in the food industry. The aim of this study was to assess how the main stressing conditions, usually observed in meat processing facilities (sanitizers, NaCl, curing salts), interfere in L. monocytogenes adhesion and biofilm formation. The isolates, representatives of different L. monocytogenes lineages (n = 6) were subjected to four different sanitizers (S1: quaternary ammonium; S2: peracetic acid, hydrogen peroxide and glacial acetic acid, S3: biguanide polyhexamethylene hydrochloride, S4: hydrogen peroxide) to verify adhesion ability and susceptibility based on minimum inhibitory concentration (MIC). In addition, the isolates adhesion and biofilm were assessed up to 72 h under different conditions: sanitizers (MIC values), curing salts and NaCl (both at 5, 7·5, 10%), at different temperatures (4, 12 and 37°C). Despite the effectiveness of sanitizers, isolates presented higher biofilm development when compared to controls in the presence of quaternary ammonium (S1, 1: 1,024) at 4°C, over the tested time (P < 0·05). Furthermore, different responses were observed for the different L. monocytogenes strains tested, providing a better understanding of the persistence of this pathogen in the food processing facilities.     

Biofouling of stainless steel surfaces by four common pathogens: the effects of glucose concentration,temperature and surface roughness

There is a wide range of factors affecting bacterial adhesion and biofilm formation. However, in both food processing and medical settings, it is very hard to obtain suitably controlled conditions so that the factors that reduce surface colonisation and biofouling can be studied. The aim of this study was to evaluate the effect of glucose concentration, temperature and stainless steel (SS) surface roughness on biofouling by four common pathogens (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and L. monocytogenes). Among the tested variables, the untreated SS surface (3C) was shown to be fouled more than 3D polished, brushed or electropolished SS surfaces. Although an array of parameters influenced biofouling, the most promising control measure was the influence of low temperature (4?°C) that reduced biofouling even in the case of the psychrophilic Listeria monocytogenes. The study findings could significantly contribute to the prevention of SS surface contamination and consequential biofouling by food and healthcare associated pathogens.     

Variation in Biofilm Formation among Strains of Listeria monocytogenes

Contamination of food by Listeria monocytogenes is thought to occur most frequently in food-processing environments where cells persist due to their ability to attach to stainless steel and other surfaces. Once attached these cells may produce multicellular biofilms that are resistant to disinfection and from which cells can become detached and contaminate food products. Because there is a correlation between virulence and serotype (and thus phylogenetic division) of L. monocytogenes, it is important to determine if there is a link between biofilm formation and disease incidence for L. monocytogenes. Eighty L. monocytogenes isolates were screened for biofilm formation to determine if there is a robust relationship between biofilm formation, phylogenic division, and persistence in the environment. Statistically significant differences were detected between phylogenetic divisions. Increased biofilm formation was observed in Division II strains (serotypes 1/2a and 1/2c), which are not normally associated with food-borne outbreaks. Differences in biofilm formation were also detected between persistent and nonpersistent strains isolated from bulk milk samples, with persistent strains showing increased biofilm formation relative to nonpersistent strains. There were no significant differences detected among serotypes. Exopolysaccharide production correlated with cell adherence for high-biofilm-producing strains. Scanning electron microscopy showed that a high-biofilm-forming strain produced a dense, three-dimensional structure, whereas a low-biofilm-forming strain produced a thin, patchy biofilm. These data are consistent with data on persistent strains forming biofilms but do not support a consistent relationship between enhanced biofilm formation and disease incidence.     

Identification of Listeria monocytogenes Determinants Required for Biofilm Formation

Listeria monocytogenes is a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA), as this bacterium can easily contaminate ready-to-eat (RTE) foods and cause an invasive, life-threatening disease (listeriosis). Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC) microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes.     

Kinetics of biofilm formation and desiccation survival of Listeria monocytogenes in single and dual species biofilms with Pseudomonas fluorescens,Serratia proteamaculans or Shewanella baltica on food-grade stainless steel surfaces

This study investigated the dynamics of static biofilm formation (100% RH, 15 °C, 48–72 h) and desiccation survival (43% RH, 15 °C, 21 days) of Listeria monocytogenes, in dual species biofilms with the common spoilage bacteria, Pseudomonas fluorescens, Serratia proteamaculans and Shewanella baltica, on the surface of food grade stainless steel. The Gram-negative bacteria reduced the maximum biofilm population of L. monocytogenes in dual species biofilms and increased its inactivation during desiccation. However, due to the higher desiccation resistance of Listeria relative to P. fluorescens and S. baltica, the pathogen survived in greater final numbers. In contrast, S. proteamaculans outcompeted the pathogen during the biofilm formation and exhibited similar desiccation survival, causing the N_{21 days} of Serratia to be ca 3 Log₁₀(CFU cm^?2) greater than that of Listeria in the dual species biofilm. Microscopy revealed biofilm morphologies with variable amounts of exopolymeric substance and the presence of separate microcolonies. Under these simulated food plant conditions, the fate of L. monocytogenes during formation of mixed biofilms and desiccation depended on the implicit characteristics of the co-cultured bacterium.     

Listeria monocytogenes Biofilm-Associated Protein (BapL) May Contribute to Surface Attachment of L. monocytogenes but Is Absent from Many Field Isolates

Listeria monocytogenes is a food-borne pathogen capable of adhering to a range of surfaces utilized within the food industry, including stainless steel. The factors required for the attachment of this ubiquitous organism to abiotic surfaces are still relatively unknown. In silico analysis of the L. monocytogenes EGD genome identified a putative cell wall-anchored protein (Lmo0435 [BapL]), which had similarity to proteins involved in biofilm formation by staphylococci. An insertion mutation was constructed in L. monocytogenes to determine the influence of this protein on attachment to abiotic surfaces. The results show that the protein may contribute to the surface adherence of strains that possess BapL, but it is not an essential requirement for all L. monocytogenes strains. Several BapL-negative field isolates demonstrated an ability to adhere to abiotic surfaces equivalent to that of BapL-positive strains. BapL is not required for the virulence of L. monocytogenes in mice.     

Listeria monocytogenes is common in wild birds in Helsinki region and genotypes are frequently similar with those found along the food chain

Aims: To evaluate the prevalence and genetic diversity of Listeria monocytogenes in wild birds and to compare the genotypes with isolates previously collected from foods and food processing environments. Methods and Results: Samples of wild birds’ faeces (n = 212) were collected from a municipal landfill site and from urban areas in the Helsinki region and analysed by two‐step enrichment and plating onto L. monocytogenes‐selective agar. The overall prevalence of L. monocytogenes in bird faeces was 36% (95% CI 30–43%), and prevalence on the landfill site was significantly higher. All isolates were analysed with pulsed‐field gel electrophoresis and compared with the L. monocytogenes profiles in an existing collection. Similar pulsotypes were found in birds and in isolates collected along the food chain. Conclusions: Birds commonly carry L. monocytogenes, and strains are frequently similar with those detected in foods and food processing environments. Thus, birds may disseminate L. monocytogenes in nature and may also contaminate foods when entering the food processing environments and outdoor market places. Significance and Impact of the Study: Populations of L. monocytogenes in wild birds and along the food processing chain overlap. Our findings add to the epidemiological data on this significant foodborne pathogen.     

Colorimetric assay for biofilms in wet processing conditions

Controlling bacterial biofilms is necessary for food safety and industrial processing in clean room environments. Our goal was to develop a method to quantitatively measure biofilm produced by pathogens under wet poultry production and processing conditions. Stainless steel and glass coupons were incubated in aqueous media containing reduced nutrients and exposed to Listeria monocytogenes under static temperature and humidity conditions. Samples were measured separately by biofilm assay and viable cell density, and then confirmed by spectrophotometry and microscopy. The biofilm assay resulted in different t groupings from the cell density. The mean from the biofilm assay was 0.50, and the error% was 0.595. The mean of the log₁₀ density (cfu/cm²) was 5.90, and the standard deviation ranged from 0.127 to 0.438 on 24 coupons. The typical sequence of biofilm development, followed by microscopy of biofilm grown on glass coupons, exhibited a change from dispersed single cells to an all-over pattern of clumps with few dispersed cells. L. monocytogenes formed biofilms on all of the substrata tested. Bacterial counts from planktonic cultures at 24, 48, 72, and 144 h confirmed that L. monocytogenes remained viable throughout the experiment and reached equilibrium between 6 and 24 h. The cell density log₁₀/ml was 8.01, 8.03, 7.69, and 6.66, respectively; and the standard deviation ranged from 0.156 to 0.394. The data will be used to grow stable biofilms of Listeria spp. collected from the food processing environment for further study. This is the first use of the crystal violet assay for measurement of bacterial biofilms on stainless steel under these conditions. The methods tested are applicable to other bacteria and substrata.     

A study on the effects of some laboratory-derived genetic mutations on biofilm formation by Listeria monocytogenes

Biofilms formed by the human pathogen Listeria monocytogenes in food-processing environments can be a potential source of contamination. In this study, we investigated the ability of L. monocytogenes wild type and its laboratory-derived isogenic mutants in cwhA, prfA, agrA, flaA, degU, ami and sigB to adhere to and form biofilms on abiotic surfaces. The results suggest that inactivation of the two component regulatory system degU completely abolished biofilm formation, while inactivation of the flagellar gene flaA, two component response regulator agrA and the autolysin-adhesin gene ami lead to severe impairment of initial attachment and the subsequent development of a mature biofilm by L. monocytogenes. Mutants in the global regulator of virulence prfA and the alternative sigma factor sigB were unaffected and formed biofilms similar to wild type L. monocytogenes.     

Antibacterial isoeugenol coating on stainless steel and polyethylene surfaces prevents biofilm growth

Aims

Pathogenic bacteria can spread between individuals or between food items via the surfaces they share. Limiting the survival of pathogens on surfaces, therefore, presents an opportunity to limit at least one route of how pathogens spread. In this study, we propose that a simple coating with the essential oil isoeugenol can be used to circumvent the problem of bacterial transfer via surfaces.

Methods and Results

Two commonly used materials, stainless steel and polyethylene, were coated by physical adsorption, and the coatings were characterized by Raman spectroscopy, atomic force microscopy and water contact angle measurements. We quantified and visualized the colonization of coated and uncoated surfaces by three bacteria: Staphylococcus aureus, Listeria monocytogenes and Pseudomonas fluorescens. No viable cells were detected on surfaces coated with isoeugenol.

Conclusions

The isoeugenol coating prepared with simple adsorption proved effective in preventing biofilm formation on stainless steel and polyethylene surfaces. The result was caused by the antibacterial effect of isoeugenol, as the coating did not diminish the adhesive properties of the surface.

Significance and Impact of the Study

Our study demonstrates that a simple isoeugenol coating can prevent biofilm formation of S. aureus, L. monocytogenes and P. fluorescens on two commonly used surfaces.     

Sources and survival of Listeria monocytogenes on fresh,leafy produce

Listeria monocytogenes is an intracellular human pathogen which enters the body through contaminated food stuffs and is known to contaminate fresh leafy produce such as spinach, lettuce and rocket. Routinely, fresh leafy produce is grown and processed on a large scale before reaching the consumer through various products such as sandwiches and prepared salads. From farm to fork, the fresh leafy produce supply chain (FLPSC) is complex and contains a diverse range of environments where L. monocytogenes is sporadically detected during routine sampling of produce and processing areas. This review describes sources of the bacteria in the FLPSC and outlines the physiological and molecular mechanisms behind its survival in the different environments associated with growing and processing fresh produce. Finally, current methods of source tracking the bacteria in the context of the food supply chain are discussed with emphasis on how these methods can provide additional, valuable information on the risk that L. monocytogenes isolates pose to the consumer.     

Does Campylobacter jejuni Form Biofilms in Food-Related Environments?

Campylobacter jejuni is one of the most frequent causes of bacterial gastrointestinal food-borne infection worldwide. This species is part of the normal flora of the gastrointestinal tracts of animals used for food production, including poultry, which is regarded as the primary source of human Campylobacter infections. The survival and persistence of C. jejuni in food processing environments, especially in poultry processing plants, represent significant risk factors that contribute to the spread of this pathogen through the food chain. Compared to other food-borne pathogens, C. jejuni is more fastidious in its growth requirements and is very susceptible to various environmental stressors. Biofilm formation is suggested to play a significant role in the survival of C. jejuni in the food production and processing environment. The aims of this minireview were (i) to examine the evidence that C. jejuni forms biofilms and (ii) to establish the extent to which reported and largely laboratory-based studies of C. jejuni biofilms provide evidence for biofilm formation by this pathogen in food processing environments. Overall existing studies do not provide strong evidence for biofilm formation (as usually defined) by most C. jejuni strains in food-related environments under the combined conditions of atmosphere, temperature, and shear that they are likely to encounter. Simple attachment to and survival on surfaces and in existing biofilms of other species are far more likely to contribute to C. jejuni survival in food-related environments based on our current understanding of this species.     

Multivariate analysis reveals differences in biofilm formation capacity among Listeria monocytogenes lineages

Biofilm formation capacity evaluated under identical conditions differs among Listeria monocytogenes lineages. The approach of using one set of factors or one variable at a time fails to explain why some lineages are more prevalent than others in certain environments. This study proposes the use of multivariate analysis to compare biofilm formation by various strains and describes the ecological niches of L. monocytogenes lineages. Nutrient availability, temperature, pH and water activity (a_w) at three different levels were used to determine biofilm formation by 41 strains. Despite the high degree of similarity (≤ 80%), distinct lineage-associated biofilm formation patterns were identified. A linear regression model for each strain and a principal component analysis of regression coefficients indicated that Lineages I and III have different, but overlapping, ecological niches. This study is the first to report the use of multivariate analyses to compare biofilm formation by various isolates of L. monocytogenes.     

Cellular hydrophobicity of Listeria monocytogenes involves initial attachment and biofilm formation on the surface of polyvinyl chloride

Aims: To clarify the cellular properties of Listeria monocytogenes involved in adhesion to and biofilm formation on polyvinyl chloride, a widely used material in the food manufacturing process. Methods and Results: A significant correlation between the ability of initial adherence to and biofilm formation on PVC was observed for 24 L. monocytogenes strains (Spearman rank‐correlation coefficient, r_s = 0·89). The swimming motility assay revealed no relationship between initial adherence and motility of L. monocytogenes. The microbial adhesion to solvent assay revealed an interaction of L. monocytogenes cells with nonpolar solvents, and a significant correlation was also observed between the degree of interaction with nonpolar solvents and initial adherence to PVC (r_s = 0·87 and r_s = 0·84, between initial adherence and affinities to decane and hexadecane, respectively). Conclusions: Results indicate that cellular hydrophobicity of L. monocytogenes is an important property involved in the initial adherence to and biofilm formation on PVC. Significance and Impact of Study: This study clarified the factors involved in the adherence to and biofilm formation ability of L. monocytogenes strains with PVC.     

Development of a biofilm model for Listeria monocytogenes EGD-e

The ability to form persistent biofilms makes the pathogenic bacterium Listeria monocytogenes a hazardous contaminant in food processing environments. Growth and biofilm formation of L. monocytogenes EGD-e were studied in defined medium (HTM) and in tryptic soy broth (TSB) with different supplements. TSB + 1% glucose gave optimal results. Using this medium, biofilm development on the model surface polystyrene (microtiter plate) was monitored by the standard crystal violet staining for adherent cells after bacterial cultivation for 24 and 48 h at five different temperatures (4, 18, 25, 30 and 37°C). In parallel, the matrix exopolysaccharide formed after 48 h of incubation was quantified by staining with ruthenium red. In both assays incubation at 30°C yielded the highest values. The formation of larger scale biofilms on dialysis membranes, placed on TSB agar with 1% glucose for 48 h, was studied by scanning electron microscopy. Contiguous and multilayered biofilms were observed at 18, 25, 30 and 37°C incubation temperature. The methodology is suitable for quantitative and microscopic studies and, in addition, yields sufficient cell mass for subsequent biochemical and molecular biological analyses.     

Listeria monocytogenes Behaviour in Presence of Non-UV-Irradiated Titanium Dioxide Nanoparticles

Listeria monocytogenes is the agent of listeriosis, a food-borne disease. It represents a serious problem for the food industry because of its environmental persistence mainly due to its ability to form biofilm on a variety of surfaces. Microrganisms attached on the surfaces are a potential source of contamination for environment and animals and humans. Titanium dioxide nanoparticles (TiO₂ NPs) are used in food industry in a variety of products and it was reported that daily exposure to these nanomaterials is very high. Anti-listerial activity of TiO₂ NPs was investigated only with UV-irradiated nanomaterials, based on generation of reactive oxigen species (ROS) with antibacterial effect after UV exposure. Since both Listeria monocytogenes and TiO₂ NPs are veicolated with foods, this study explores the interaction between Listeria monocytogenes and non UV-irradiated TiO₂ NPs, with special focus on biofilm formation and intestinal cell interaction. Scanning electron microscopy and quantitative measurements of biofilm mass indicate that NPs influence both production and structural architecture of listerial biofilm. Moreover, TiO₂ NPs show to interfere with bacterial interaction to intestinal cells. Increased biofilm production due to TiO₂ NPs exposure may favour bacterial survival in environment and its transmission to animal and human hosts.     

Effect of Batch and Fed-Batch Growth Modes on Biofilm Formation by <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> at Different Temperatures

The influence of Listeria monocytogenes (L. monocytogenes) biofilm formation feeding conditions (batch and fed-batch) at different temperatures on biofilm biomass and activity was determined. Biofilm biomass and cellular metabolic activity were assessed by Crystal Violet (CV) staining and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) colorimetric method, respectively. Live/Dead staining was also performed in order to get microscopic visualization of the different biofilms. Results revealed that at refrigeration temperature (4°C) a higher amount of biofilm was produced when batch conditions were applied, while at higher temperatures the fed-batch feeding condition was the most effective on biofilm formation. Moreover, independently of the temperature used, biofilms formed under fed-batch conditions were metabolically more active than those formed in batch mode. In conclusion, this work shows that different growth modes significantly influence L. monocytogenes biofilm formation on abiotic surfaces as well as the metabolic activity of cells within biofilms.