Synergistic Effects of Sodium Chloride,Glucose, and Temperature on Biofilm Formation by Listeria monocytogenes Serotype 1/2a and 4b Strains

Biofilm formation by Listeria monocytogenes is generally associated with its persistence in the food-processing environment. Serotype 1/2a strains make up more than 50% of the total isolates recovered from food and the environment, while serotype 4b strains are most often associated with major outbreaks of human listeriosis. Using a microplate assay with crystal violet staining, we examined biofilm formation by 18 strains of each serotype in tryptic soy broth with varying concentrations of glucose (from 0.25% to 10.0%, wt/vol), sodium chloride (from 0.5% to 7.0%, wt/vol) and ethanol (from 1% to 5.0%, vol/vol), and at different temperatures (22.5°C, 30°C, and 37°C). A synergistic effect on biofilm formation was observed for glucose, sodium chloride, and temperature. The serotype 1/2a strains generally formed higher-density biofilms than the 4b strains under most conditions tested. Interestingly, most serotype 4b strains had a higher growth rate than the 1/2a strains, suggesting that the growth rate may not be directly related to the capacity for biofilm formation. Crystal violet was found to stain both bacterial cells and biofilm matrix material. The enhancement in biofilm formation by environmental factors was apparently due to the production of extracellular polymeric substances instead of the accumulation of viable biofilm cells.Listeria monocytogenes, a Gram-positive bacterium, is capable of causing severe food-borne infections in both humans and animals. The organism is ubiquitous in the environment and can grow in a wide variety of foods, including those stored at refrigeration temperatures. It is particularly difficult to eliminate this bacterium from ready-to-eat foods and food-processing equipment (19). The ability to form biofilms protects the bacterium from stresses in food-processing environments (13, 25). Among the 13 different serotypes described, serotypes 1/2a, 1/2b, and 4b are involved in the majority of human cases of listeriosis. Serotype 4b strains have accounted for most human outbreaks, whereas the majority of L. monocytogenes strains isolated from foods or food-processing plants belong to serotype 1/2a (19).Comparative studies to link the phenotypic attributes of L. monocytogenes strains to serotypes have obtained variable results. Buncic et al. (4) have shown that serotype 1/2a isolates were more resistant to antilisterial bacteriocins than serotype 4b strains at 4°C. They also found that 4b isolates exhibited greater resistance to heat treatments at 60°C and were easier to recover than 1/2a strains immediately following cold storage. Bruhn et al. (3) observed that 1/2a strains (lineage II) grew faster than 4b and 1/2b (lineage I) strains in commonly used enrichment broth media (University of Vermont media I and II). However, other studies have indicated that similar differences could not be linked to a serotype (14), and sequencing results have shown a syntenic relationship between strains of the two serotypes (27).Some L. monocytogenes strains have consistently been isolated from food-processing plants over many years (1, 28). Although several studies have been carried out to identify differences in cell adherence and biofilm formation among different serotypes, conflicting results were obtained. Lineage I isolates (including serotypes 4b, 1/2b, 3c, and 3b) were found to produce higher-density biofilms than lineage II isolates (including serotypes 1/2a, 1/2c, and 3a) (8, 28). However, this conclusion was not supported by other studies (1, 7, 18). For serotype 4b strains, the capacity to form biofilms was reduced when the nutrient level in a medium decreased, while serotype 1/2a strains were not similarly affected (11).It has been suggested that the formation of a biofilm is a stress response by bacterial cells (15, 16). Biofilm research under laboratory conditions may not reflect biofilm formation in the environment. To investigate the behavior of L. monocytogenes in biofilms, a simulated food-processing (SFP) system including several stresses was designed (30). The SFP system was used to study 1/2a and 4b strains in mixed-culture biofilms (31). Bacterial cells from a 1/2a cocktail predominated over 4b strains when exposed to the SFP system for 4 weeks, but no competitive inhibition was observed. Environmental factors, including temperature, sugar, salt, pH, and nutrients that are common in foods and food-processing environments, have been demonstrated to have impacts on L. monocytogenes adhesion and biofilm formation (25). The objectives of this study were to investigate and compare biofilm formation between L. monocytogenes serotype 1/2a strains and serotype 4b strains under a variety of environmental conditions, including different temperatures and varying concentrations of salt, sugar, and ethanol, and to examine the synergistic effects of these factors on biofilm formation by both serotypes.     

Biofilm Production Potential of Salmonella Serovars Isolated from Chickens in North West Province,South Africa

Bacterial biofilms have recently gained considerable interest in the food production and medical industries due to their ability to resist destruction by disinfectants and other antimicrobials. Biofilms are extracellular polymer matrices that may enhance the survival of pathogens even when exposed to environmental stress. The effect of incubation temperatures (25°C, 37°C, and 40°C) and Salmonella serotype on biofilm-forming potentials was evaluated. Previously typed Salmonella serotypes (55) isolated from the gut of chickens were accessed for biofilms formation using a standard assay. Salmonella Typhimurium ATCC 14028^TM and Salmonella Enteritidis ATCC 13076^TM (positive controls), Escherichia coli (internal control) and un-inoculated Luria Bertani (LB) broth (negative control) were used. The isolates formed no biofilm (11.86–13.56%), weak (11.86–45.76%), moderate (18.64–20.34%), strong biofilms (23.73–54.24%) across the various temperatures investigated. Serotypes, Salmonella Heidelberg and Salmonella Weltevreden were the strongest biofilm formers at temperatures (25°C, 37°C, and 40°C, respectively). The potential of a large proportion (80%) of Salmonella serotypes to form biofilms increased with increasing incubation temperatures but decreased at 40°C. Findings indicate that average temperature favours biofilm formation by Salmonella serotypes. However, the influence of incubation temperature on biofilm formation was greater when compared to serotype. A positive correlation exists between Salmonella biofilm formed at 25°C, 37°C and 40°C (p ≥ 0.01). The ability of Salmonella species to form biofilms at 25°C and 37°C suggests that these serotypes may present severe challenges to food-processing and hospital facilities.Key words: Salmonella, biofilm, biofilm production potential, crystal violet microtitre     

Ability of the Listeria monocytogenes Strain Scott A To Cause Systemic Infection in Mice Infected by the Intragastric Route

Listeriosis is an important food-borne disease that causes high rates of morbidity and mortality. For reasons that are not clear, most large outbreaks of human listeriosis involve Listeria monocytogenes serotype 4b. Relatively little is known about the pathogenesis of listeriosis following gastrointestinal exposure to food-borne disease isolates of L. monocytogenes. In the present study, we investigated the pathogenesis of systemic infection by the food-borne isolate Scott A in an intragastric (i.g.) mouse challenge model. We found that the severity of infection with L. monocytogenes Scott A was increased in mice made neutropenic by administration of monoclonal antibody RB6-8C5. This observation was similar to a previous report on a study with the laboratory strain L. monocytogenes EGD. Prior administration of sodium bicarbonate did not enhance the virulence of L. monocytogenes strain Scott A for i.g. inoculated mice. Following i.g. inoculation of mice, two serotype 4b strains of L. monocytogenes (Scott A and 101M) achieved a greater bacterial burden in the spleen and liver and elicited more severe histopathological damage to those organs than did a serotype 1/2a strain (EGD) and a serotype 1/2b stain (CM). Of the four strains tested, only strain CM exhibited poor survival in synthetic gastric fluid in vitro. The other three strains exhibited similar patterns of survival at pHs of greater than 5 and relatively rapid (<30 min) loss of viability at pHs of less than 5.0. Growth of L. monocytogenes Scott A at temperatures of 12.5 to 37°C did not affect its ability to cause systemic infection in i.g. inoculated mice. These observations suggest that the serotype 4b L. monocytogenes strains Scott A and 101M possess one or more virulence determinants that make them better able to cause systemic infection following inoculation via the g.i. tract than do the serotype 1/2 strains EGD and CM.     

Control of Listeria monocytogenes in a Biofilm by Competitive-Exclusion Microorganisms

Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37°C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10³ CFU of L. monocytogenes/ml and 10⁵ CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37°C for 24 h, 15°C for 14 days, 8°C for 21 days, and 4°C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37°C, two at 15 and 8°C, and three at 4°C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4°C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log₁₀ CFU of L. monocytogenes/cm²). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37°C.     

Heavy-Metal and Benzalkonium Chloride Resistance of Listeria monocytogenes Isolates from the Environment of Turkey-Processing Plants

The resistance of Listeria monocytogenes to cadmium and arsenic has been used extensively for strain subtyping. However, limited information is available on the prevalence of such resistance among isolates from the environment of food-processing plants. In addition, it is not known whether the resistance of such isolates to heavy metals may correlate with resistance to quaternary ammonium compounds extensively used as disinfectants in the food-processing industry. In this study, we characterized 192 L. monocytogenes isolates (123 putative strains) from the environment of turkey-processing plants in the United States for resistance to cadmium and arsenic and to the quaternary ammonium disinfectant benzalkonium chloride (BC). Resistance to cadmium was significantly more prevalent among strains of serotypes 1/2a (or 3a) and 1/2b (or 3b) (83% and 74%, respectively) than among strains of the serotype 4b complex (19%). Resistance to BC was encountered among 60% and 51% of the serotype 1/2a (or 3a) and 1/2b (or 3b) strains, respectively, and among 7% of the strains of the serotype 4b complex. All BC-resistant strains were also resistant to cadmium, although the reverse was not always the case. In contrast, no correlation was found between BC resistance and resistance to arsenic, which overall was low (6%). Our findings suggest that the processing environment of turkey-processing plants may constitute a reservoir for L. monocytogenes harboring resistance to cadmium and to BC and raise the possibility of common genetic elements or mechanisms mediating resistance to quaternary ammonium disinfectants and to cadmium in L. monocytogenes.     

Competition of Listeria monocytogenes Serotype 1/2a and 4b Strains in Mixed-Culture Biofilms

The majority of Listeria monocytogenes isolates recovered from foods and the environment are strains of serogroup 1/2, especially serotypes 1/2a and 1/2b. However, serotype 4b strains cause the majority of human listeriosis outbreaks. Our investigation of L. monocytogenes biofilms used a simulated food-processing system that consisted of repeated cycles of growth, sanitation treatment, and starvation to determine the competitive fitness of strains of serotypes 1/2a and 4b in pure and mixed-culture biofilms. Selective enumeration of strains of a certain serotype in mixed-culture biofilms on stainless steel coupons was accomplished by using serotype-specific quantitative PCR and propidium monoazide treatment to prevent amplification of extracellular DNA or DNA from dead cells. The results showed that the serotype 1/2a strains tested were generally more efficient at forming biofilms and predominated in the mixed-culture biofilms. The growth and survival of strains of one serotype were not inhibited by strains of the other serotype in mixed-culture biofilms. However, we found that a cocktail of serotype 4b strains survived and grew significantly better in mixed-culture biofilms containing a specific strain of serotype 1/2a (strain SK1387), with final cell densities averaging 0.5 log₁₀ CFU/cm² higher than without the serotype 1/2a strain. The methodology used in this study contributed to our understanding of how environmental stresses and microbial competition influence the survival and growth of L. monocytogenes in pure and mixed-culture biofilms.A prominent food-borne pathogen, Listeria monocytogenes can cause severe infections in humans, primarily in high-risk populations, though the disease (listeriosis) is relatively rare (11, 30, 43). Outbreaks of listeriosis have resulted from the contamination of a variety of foods by L. monocytogenes, especially meat and dairy products (27). L. monocytogenes is ubiquitous in the environment, able to grow at refrigeration temperature, and tolerant of the low pHs (3 to 4) typical of acidified foods (28, 32, 44). The capacity to produce biofilms confers protection against stresses common in the food-processing environment (13, 33).Biofilms are characterized by dense clusters of bacterial cells embedded in extracellular polymeric substances which are secreted by cells to aid in adhesion to surfaces and to other cells (4, 5). Strains of L. monocytogenes have been known to persist for years in food-processing environments, presumably in biofilms. Of the 13 known serotypes of L. monocytogenes, three (1/2a, 1/2b, and 4b) account for >95% of the isolates from human illness (21). Serotype 1/2a accounts for >50% of the L. monocytogenes isolates recovered from foods and the environment, while most major outbreaks of human listeriosis have been caused by serotype 4b strains (1, 3, 14, 15, 17, 22, 29, 31, 41, 47, 49,). No correlation between L. monocytogenes strain fitness and serotype has been identified (16, 19). Some studies have reported that strains repeatedly isolated from food and environmental samples (defined as persistent strains) had a higher adherence capacity than strains that were sporadically isolated (2, 36), while this phenomenon was not observed by others (7). Serotype 4b strains exhibited a higher capacity for biofilm formation than did serotype 1/2a strains (36), whereas this was not observed by Di Bonaventura and colleagues (6). It has been suggested that serotype 1/2a strains could be more robust than serotype 4b strains in biofilm formation under a variety of environmental conditions. Furthermore, strains of these serotypes differ in terms of the medium that promotes biofilm formation. Biofilm formation by serotype 4b strains was higher in full-strength tryptic soy broth than in diluted medium, whereas the opposite was observed with serotype 1/2a strains, which produced more biofilm in diluted medium (12).There is limited information on microbial competition between strains of different serotypes in biofilms or on how the environmental stresses present in food-processing environments may affect the biofilm formation and survival of L. monocytogenes of different serotypes. In food-processing plants, the environmental stresses encountered by bacteria are more complex and variable than most laboratory systems used for microbial ecology and biofilm studies. A simulated food-processing (SFP) system has been developed to address this issue (38). The SFP system incorporates several stresses that may affect bacteria in biofilms in the food-processing environment, including exposure to sanitizing agents, dehydration, and starvation. When biofilms were subjected to the SFP regimen over a period of several weeks, the cell numbers of L. monocytogenes strains in the biofilms initially were reduced and then increased as the culture adapted (38). The development of resistance to sanitizing agents was specific to the biofilm-associated cells and was not apparent in the detached cells (38). This suggested that extracellular polymeric substances present in the biofilm matrix were responsible for the resistance to sanitizing agents. It was subsequently found that real-time PCR, in combination with propidium monoazide (PMA) treatment of samples prior to DNA isolation, was an effective method for enumerating viable cells in biofilms (37).The objective of this study was to determine if strains of serotype 1/2a or 4b have a selective advantage under stress conditions. We investigated and compared the initial attachment and biofilm formation capabilities of L. monocytogenes strains of these two serotypes and analyzed the survival and growth of bacteria of each serotype in mixed-serotype biofilms in the SFP system by using PMA with quantitative PCR.     

Rapid methods to assess sanitizing efficacy of benzalkonium chloride to Listeria monocytogenes biofilms

Different methods were used to investigate biofilm growth including crystal violet staining, ATP bioluminescence and total viable count. Seven strains of Listeria monocytogenes and 8 of their derivative strains were screened for their capacity to form biofilms. Both adaptation to benzalkonium chloride (BC) and curing of plasmids did not significantly affect biofilm-forming ability. The strains of L. monocytogenes belonging to serotype 1 formed biofilms significantly better as compared to serotype 4 (P = 0.0003). To estimate the efficacy of BC for biofilm elimination the best and the poorest biofilm-formers were used (C719 and LJH 381). It was observed that, L. monocytogenes strain C719 in biofilms is at least 1000 times more resistant to BC than in planktonic form. Cells present in biofilms were shown to recover and grow after BC treatment thus providing a source of recontamination. It was shown that ATP bioluminescence provides good correlation with bacterial counts of L. monocytogenes in biofilms. Staining with crystal violet, on the contrary, did not correlate with bacterial growth in biofilms in the presence of high concentrations of BC but provided information on the concentration of bacterial cells, both live and dead, attached to the surface. ATP bioluminescence was found to be a reliable method for rapid estimation of the efficacy of sanitizers for biofilm disinfection. Crystal violet staining, on the other hand, was shown to be a suitable method to monitor removal of biofilms. Our investigation showed that for Listeria biofilms concentrations of BC higher then 10 mg/ml should be applied for at least 30 min to kill almost all the live cells in biofilms. However, this concentration was still not enough to remove biofilms from the surface of plastic.     

Comparative Proteomic Analysis of Listeria monocytogenes Strains F2365 and EGD

Listeria monocytogenes is a gram-positive, food-borne pathogen that causes disease in both humans and animals. There are three major genetic lineages of L. monocytogenes and 13 serovars. To further our understanding of the differences that exist between different genetic lineages/serovars of L. monocytogenes, we analyzed the global protein expression of the serotype 1/2a strain EGD and the serotype 4b strain F2365 during early-stationary-phase growth at 37°C. Using multidimensional protein identification technology with electrospray ionization tandem mass spectrometry, we identified 1,754 proteins from EGD and 1,427 proteins from F2365, of which 1,077 were common to both. Analysis of proteins that had significantly altered expression between strains revealed potential biological differences between these two L. monocytogenes strains. In particular, the strains differed in expression of proteins involved in cell wall physiology and flagellar biosynthesis, as well as DNA repair proteins and stress response proteins.     

Inflammasome-Mediated Inhibition of Listeria monocytogenes-Stimulated Immunity Is Independent of Myelomonocytic Function

Activation of the Nlrc4 inflammasome results in the secretion of IL-1β and IL-18 through caspase-1 and induction of pyroptosis. L. monocytogenes engineered to activate Nlrc4 by expression of Legionella pneumophilia flagellin (L. monocytogenes L.p.FlaA) are less immunogenic for CD8⁺ T cell responses than wt L. monocytogenes. It is also known that IL-1β orchestrates recruitment of myelomonocytic cells (MMC), which have been shown to interfere with T cell-dendritic cells (DC) interactions in splenic white pulp (WP), limiting T cell priming and protective immunity. We have further analyzed the role of MMCs in the immunogenicity of L. monocytogenes L.p.FlaA. We confirmed that MMCs infiltrate the WP between 24–48 hours in response to wt L. monocytogenes infection and that depletion of MMCs enhances CD8⁺ T cell priming and protective memory. L. monocytogenes L.p.FlaA elicited accelerated recruitment of MMCs into the WP. While MMCs contribute to control of L. monocytogenes L.p.FlaA, MMC depletion did not increase immunogenicity of L.p.FlaA expressing strains. There was a significant decrease in L. monocytogenes L.p.FlaA in CD8α⁺ DCs independent of MMCs. These findings suggest that limiting inflammasome activation is important for bacterial accumulation in CD8α⁺ DCs, which are known to be critical for T cell response to L. monocytogenes.     

Potentially Pathogenic Escherichia coli Can Form a Biofilm under Conditions Relevant to the Food Production Chain

The biofilm-producing abilities of potentially human-pathogenic serotypes of Escherichia coli from the ovine reservoir were studied at different temperatures and on different surfaces. A possible influence of the hydrophobicity of the bacterial cells, as well as the presence of two virulence factors, the Shiga toxin-encoding (Stx) bacteriophage and the eae gene, was also studied. A total of 99 E. coli isolates of serotypes O26:H11, O103:H2, and O103:H25 isolated from sheep feces were included. The results show that isolates of all three E. coli serotypes investigated can produce biofilm on stainless steel, glass, and polystyrene at 12, 20, and 37°C. There was a good general correlation between the results obtained on the different surfaces. E. coli O103:H2 isolates produced much more biofilm than those of the other two serotypes at all three temperatures. In addition, isolates of serotype O26:H11 produced more biofilm than those of O103:H25 at 37°C. The hydrophobicity of the isolates varied between serotypes and was also influenced by temperature. The results strongly indicated that hydrophobicity influenced the attachment of the bacteria rather than their ability to form biofilm once attached. Isolates with the eae gene produced less biofilm at 37°C than isolates without this gene. The presence of a Stx bacteriophage did not influence biofilm production. In conclusion, our results show that potentially human-pathogenic E. coli from the ovine reservoir can form biofilm on various surfaces and at several temperatures relevant for food production and handling.     

Effects of Above-Optimum Growth Temperature and Cell Morphology on Thermotolerance of Listeria monocytogenes Cells Suspended in Bovine Milk

The thermotolerances of two different cell forms of Listeria monocytogenes (serotype 4b) grown at 37 and 42.8°C in commercially pasteurized and laboratory-tyndallized whole milk (WM) were investigated. Test strains, after growth at 37 or 42.8°C, were suspended in WM at concentrations of approximately 1.5 × 10⁸ to 3.0 × 10⁸ cells/ml and were then heated at 56, 60, and 63°C for various exposure times. Survival was determined by enumeration on tryptone-soya-yeast extract agar and Listeria selective agar, and D values (decimal reduction times) and Z values (numbers of degrees Celsius required to cause a 10-fold change in the D value) were calculated. Higher average recovery and higher D values (i.e., seen as a 2.5- to 3-fold increase in thermotolerance) were obtained when cells were grown at 42.8°C prior to heat treatment. A relationship was observed between thermotolerance and cell morphology of L. monocytogenes. Atypical Listeria cell types (consisting predominantly of long cell chains measuring up to 60 μm in length) associated with rough (R) culture variants were shown to be 1.2-fold more thermotolerant than the typical dispersed cell form associated with normal smooth (S) cultures (P ≤ 0.001). The thermal death-time (TDT) curves of R-cell forms contained a tail section in addition to the shoulder section characteristic of TDT curves of normal single to paired cells (i.e., S form). The factors shown to influence the thermoresistance of suspended Listeria cells (P ≤ 0.001) were as follows: growth and heating temperatures, type of plating medium, recovery method, and cell morphology. Regression analysis of nonlinear data can underestimate survival of L. monocytogenes; the end point recovery method was shown to be a better method for determining thermotolerance because it takes both shoulders and tails into consideration. Despite their enhanced heat resistance, atypical R-cell forms of L. monocytogenes were unable to survive the low-temperature, long-time pasteurization process when freely suspended and heated in WM.     

Changes in Listeria monocytogenes Membrane Fluidity in Response to Temperature Stress

Listeria monocytogenes is a food-borne pathogen that has been implicated in many outbreaks associated with ready-to-eat products. Listeria adjusts to various stresses by adjusting its membrane fluidity, increasing the uptake of osmoprotectants and cryoprotectants, and activating the σ^B stress factor. The present work examines the regulation of membrane fluidity through direct measurement based on fluorescent anisotropy. The membrane fluidities of L. monocytogenes Scott A, NR30, wt10403S, and cld1 cells cultured at 15 and 30°C were measured at 15 and 30°C. The membrane of the cold-sensitive mutant (cld1) was more rigid than the membranes of the other strains when grown at 30°C, but when grown at 15°C, it was able to adjust its membrane to approach the rigidity of the other strains. The difference in rigidities, as determined at 15 and 30°C, was greater in liposomes than in whole cells. The rates of fluidity adjustment and times required for whole cells to adjust to a different temperature were similar among strains but different from those of liposomes. This suggests that the cells had a mechanism for homeoviscous adaptation that was absent in liposomes.     

Attachment of and Biofilm Formation by Enterobacter sakazakii on Stainless Steel and Enteral Feeding Tubes

Enterobacter sakazakii has been reported to form biofilms, but environmental conditions affecting attachment to and biofilm formation on abiotic surfaces have not been described. We did a study to determine the effects of temperature and nutrient availability on attachment and biofilm formation by E. sakazakii on stainless steel and enteral feeding tubes. Five strains grown to stationary phase in tryptic soy broth (TSB), infant formula broth (IFB), or lettuce juice broth (LJB) at 12 and 25°C were examined for the extent to which they attach to these materials. Higher populations attached at 25°C than at 12°C. Stainless steel coupons and enteral feeding tubes were immersed for 24 h at 4°C in phosphate-buffered saline suspensions (7 log CFU/ml) to facilitate the attachment of 5.33 to 5.51 and 5.03 to 5.12 log CFU/cm², respectively, before they were immersed in TSB, IFB, or LJB, followed by incubation at 12 or 25°C for up to 10 days. Biofilms were not produced at 12°C. The number of cells of test strains increased by 1.42 to 1.67 log CFU/cm² and 1.16 to 1.31 log CFU/cm² in biofilms formed on stainless steel and feeding tubes, respectively, immersed in IFB at 25°C; biofilms were not formed on TSB and LJB at 25°C, indicating that nutrient availability plays a major role in processes leading to biofilm formation on the surfaces of these inert materials. These observations emphasize the importance of temperature control in reconstituted infant formula preparation and storage areas in preventing attachment and biofilm formation by E. sakazakii.     

Restriction Fragment Length Polymorphisms Detected with Novel DNA Probes Differentiate among Diverse Lineages of Serogroup 4 Listeria monocytogenes and Identify Four Distinct Lineages in Serotype 4b

Listeria monocytogenes of serotype 4b has been implicated in numerous outbreaks of food-borne listeriosis and in ca. 40% of sporadic cases. Strains of this serotype appear to be relatively homogeneous genetically, and molecular markers specific for distinct serotype 4b lineages have not been frequently identified. Here we show that DNA fragments derived from the putative mannitol permease locus of Listeria monocytogenes had an unexpectedly high potential to differentiate among different strains of serotype 4b when used as probes in Southern blotting of EcoRI-digested genomic DNA, yielding four distinct restriction fragment length polymorphism (RFLP) patterns. Strains of two epidemic-associated lineages, including the major epidemic clone implicated in several outbreaks in Europe and North America, had distinct RFLPs which differed from those of all other serotype 4b strains that we screened but which were encountered among strains of serotypes 1/2b and 3b. In addition, three serogroup 4 lineages were found to have unique RFLPs that were not encountered among any other L. monocytogenes strains. One was an unusual lineage of serotype 4b, and the other two were members of the serotype 4a and 4c group. The observed polymorphisms may reflect evolutionary relationships among lineages of L. monocytogenes and may facilitate detection and population genetic analysis of specific lineages.     

Expression of ActA, Ami, InlB, and Listeriolysin O in Listeria monocytogenes of Human and Food Origin

Expression of proteins involved in the adhesion of Listeria monocytogenes to mammalian cells or in the intracellular life cycle of this bacterium, including listeriolysin O (LLO), ActA, Ami, and InlB, was used to compare two populations of L. monocytogenes strains. One of the populations comprised 300 clinical strains, and the other comprised 150 food strains. All strains expressed LLO, InlB, and ActA. No polymorphism was observed for LLO and InlB. Ami was detected in 283 of 300 human strains and in 149 of 150 food strains. The strains in which Ami was not detected were serovar 4b strains. Based on the molecular weights of the proteins detected, the strains were divided into two groups with Ami (groups Ami1 [75% of the strains] and Ami2 [21%]) and into four groups with ActA (groups ActA1 [52% of the strains], ActA2 [18%], ActA3 [30%], and ActA4 [one strain isolated from food]). Logistic regression showed that food strains were more likely to belong to group ActA3 than human strains (odds ratio [OR] = 2.90; P = 1 × 10⁻⁴). Of the strains isolated from patients with non-pregnancy-related cases of listeriosis, bacteremia was predominantly associated with group Ami1 strains (OR = 1.89; P = 1 × 10⁻²) and central nervous system infections were associated with group ActA2 strains (OR = 3.04; P = 1 × 10⁻³) and group ActA3 strains (OR = 3.91; P = 1 × 10⁻³).     

Examination of Food Chain-Derived Listeria monocytogenes Strains of Different Serotypes Reveals Considerable Diversity in inlA Genotypes,Mutability, and Adaptation to Cold Temperatures

Listeria monocytogenes strains belonging to serotypes 1/2a and 4b are frequently linked to listeriosis. While inlA mutations leading to premature stop codons (PMSCs) and attenuated virulence are common in 1/2a, they are rare in serotype 4b. We observed PMSCs in 35% of L. monocytogenes isolates (n = 54) recovered from the British Columbia food supply, including serotypes 1/2a (30%), 1/2c (100%), and 3a (100%), and a 3-codon deletion (amino acid positions 738 to 740) seen in 57% of 4b isolates from fish-processing facilities. Caco-2 invasion assays showed that two isolates with the deletion were significantly more invasive than EGD-SmR (P < 0.0001) and were either as (FF19-1) or more (FE13-1) invasive than a clinical control strain (08-5578) (P = 0.006). To examine whether serotype 1/2a was more likely to acquire mutations than other serotypes, strains were plated on agar with rifampin, revealing 4b isolates to be significantly more mutable than 1/2a, 1/2c, and 3a serotypes (P = 0.0002). We also examined the ability of 33 strains to adapt to cold temperature following a downshift from 37°C to 4°C. Overall, three distinct cold-adapting groups (CAG) were observed: 46% were fast (<70 h), 39% were intermediate (70 to 200 h), and 15% were slow (>200 h) adaptors. Intermediate CAG strains (70%) more frequently possessed inlA PMSCs than did fast (20%) and slow (10%) CAGs; in contrast, 87% of fast adaptors lacked inlA PMSCs. In conclusion, we report food chain-derived 1/2a and 4b serotypes with a 3-codon deletion possessing invasive behavior and the novel association of inlA genotypes encoding a full-length InlA with fast cold-adaptation phenotypes.     

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.     

Microplate Fluorescence Assay for Measurement of the Ability of Strains of Listeria monocytogenes from Meat and Meat-Processing Plants To Adhere to Abiotic Surfaces

Listeria monocytogenes is a significant food-borne pathogen that is capable of adhering to and producing biofilms on processing equipment, making it difficult to eliminate from meat-processing environments and allowing potential contamination of ready-to-eat (RTE) products. We devised a fluorescence-based microplate method for screening isolates of L. monocytogenes for the ability to adhere to abiotic surfaces. Strains of L. monocytogenes were incubated for 2 days at 30°C in 96-well microplates, and the plates were washed in a plate washer. The retained cells were incubated for 15 min at 25°C with 5,6-carboxyfluorescein diacetate and washed again, and then the fluorescence was read with a plate reader. Several enzymatic treatments (protease, lipase, and cellulase) were effective in releasing adherent cells from the microplates, and this process was used for quantitation on microbiological media. Strongly adherent strains of L. monocytogenes were identified that had 15,000-fold-higher levels of fluorescence and 100,000-fold-higher plate counts in attachment assays than weakly adherent strains. Strongly adherent strains of L. monocytogenes adhered equally well to four different substrates (glass, plastic, rubber, and stainless steel); showed high-level attachment on microplates at 10, 20, 30, and 40°C; and showed significant differences from weakly adherent strains when examined by scanning electron microscopy. A greater incidence of strong adherence was observed for strains isolated from RTE meats than for those isolated from environmental surfaces. Analysis of surface adherence among Listeria isolates from processing environments may provide a better understanding of the molecular mechanisms involved in attachment and suggest solutions to eliminate them from food-processing environments.     

Cold Shock Induction of Thermal Sensitivity in Listeria monocytogenes

Cold shock at 0 to 15°C for 1 to 3 h increased the thermal sensitivity of Listeria monocytogenes. In a model broth system, thermal death time at 60°C was reduced by up to 45% after L. monocytogenes Scott A was cold shocked for 3 h. The duration of the cold shock affected thermal tolerance more than did the magnitude of the temperature downshift. The Z values were 8.8°C for controls and 7.7°C for cold-shocked cells. The D values of cold-shocked cells did not return to control levels after incubation for 3 h at 28°C followed by heating at 60°C. Nine L. monocytogenes strains that were cold shocked for 3 h exhibited D₆₀ values that were reduced by 13 to 37%. The D-value reduction was greatest in cold-shocked stationary-phase cells compared to cells from cultures in either the lag or exponential phases of growth. In addition, cold-shocked cells were more likely to be inactivated by a given heat treatment than nonshocked cells, which were more likely to experience sublethal injury. The D values of chloramphenicol-treated control cells and chloramphenicol-treated cold-shocked cells were no different from those of untreated cold-shocked cells, suggesting that cold shock suppresses synthesis of proteins responsible for heat protection. In related experiments, the D values of L. monocytogenes Scott A were decreased 25% on frankfurter skins and 15% in ultra-high temperature milk if the inoculated products were first cold shocked. Induction of increased thermal sensitivity in L. monocytogenes by thermal flux shows potential to become a practical and efficacious preventative control method.