Reduction of Escherichia coli O157:H7 in Biofilms Using Bacteriophage BPECO 19

Biofilm formation is a growing concern in the food industry. Escherichia coli O157:H7 is one of the most important foodborne pathogens that can persists in food and food‐related environments and subsequently produce biofilms. The efficacy of bacteriophage BPECO 19 was evaluated against three E. coli O157:H7 strains in biofilms. Biofilms of the three E. coli O157:H7 strains were grown on abiotic (stainless steel, rubber, and minimum biofilm eradication concentration [MBEC^TM] device) and biotic (lettuce) surfaces at different temperatures. The effectiveness of bacteriophage BPECO 19 in reducing preformed biofilms on these surfaces was further evaluated by treating the surfaces with a phage suspension (10⁸ PFU/mL) for 2 h. The results indicated that the phage treatment significantly reduced (P  < 0.05) the number of adhered cells in all the surfaces. Following phage treatment, the viability of adhered cells was reduced by ≥3 log CFU/cm², 2.4 log CFU/cm², and 3.1 log CFU/peg in biofilms grown on stainless steel, rubber, and the MBEC^TM device, respectively. Likewise, the phage treatment reduced cell viability by ≥2 log CFU/cm² in biofilms grown on lettuce. Overall, these results suggested that bacteriophages such as BPECO 19 could be effective in reducing the viability of biofilm‐adhered cells.     

Bactericidal Effect of Calcium Oxide (Scallop‐Shell Powder) Against Pseudomonas aeruginosa Biofilm on Quail Egg Shell,Stainless Steel,Plastic, and Rubber

The aim of this study was to evaluate the bactericidal effect of calcium oxide (CaO) against Pseudomonas aeruginosa biofilms on quail eggshells and major egg contacting surfaces (stainless steel, plastic, and rubber). The samples were subjected to CaO treatments (0%, 0.01%, 0.05%, 0.10%, 0.15%, 0.20%, 0.25%, and 0.30%) for 1 min. All the CaO treatments significantly reduced P. aeruginosa biofilms on all tested surfaces as compared to controls. In comparison of biofilm stability, the strongest and most resistant biofilm was formed on eggshell against the CaO treatment, followed by rubber, stainless steel, and plastic. In evaluation of bactericidal effect, the largest reduction (3.16 log CFU) was observed in plastic even at the lowest concentration of CaO (0.01%), whereas the least reduction was found in eggshells, regardless of CaO concentration. In addition, stainless steel showed a significant reduction in biofilm formation at all concentrations except 0.10% to 0.15% CaO. At 0.30% CaO, the reduction of P. aeruginosa in biofilms on stainless steel, plastic, rubber, and eggshell were 5.48, 6.37, 4.87, and 3.14 log CFU/cm² (CFU/egg), respectively. Biofilm reduction after CaO treatment was also observed by field emission scanning electron microscopy (FE‐SEM). Based on the FE‐SEM images, we observed that P. aeruginosa biofilms formed compact aggregations on eggshell surfaces with CaO treatments up to 0.30%. More specifically, a 0.20% CaO treatment resulted in the reductions of 3 to 6 log CFU in all materials.     

Combination of ultraviolet light-C and clove essential oil to inactivate Salmonella Typhimurium biofilms on stainless steel

Salmonella typhimurium is able to form biofilms as a resistance mechanism against antimicrobials; therefore, it represents a problem for assuring food safety and highlights the importance of research on anti-biofilm technologies. In this study, S. typhimurium biofilms were inactivated with the combination of clove essential oil (CEO) and ultraviolet light (UV-C). The volatile composition of the CEO determined by gas chromatography showed eugenol as the major constituent (82%). A combination of CEO with UV-C achieved a complete bacterial reduction (6.8 log/cm²) on biofilms with doses of 1.2 mg/ml and 76.41 mJ/cm², respectively. Individually, the CEO at 1.2 mg/ml caused a reduction of 1.8 log CFU/cm² of attached bacteria cells on stainless steel, while UV-C individually used at 620.4 mJ/cm² caused a 2.9 log CFU/cm² reduction compared to control biofilms. In conclusion, this study demonstrated a synergistic effect of combining CEO and UV-C irradiation to inactivate biofilms of S. typhimurium.     

Feed substrates influence biofilm formation on reverse osmosis membranes and their cleaning efficiency

The dairy industry is increasingly using reverse osmosis (RO) membranes for concentration of various fluid feed materials such as whey and ultrafiltration (UF) permeate. This study compared the effect of UF permeate and whey on membrane biofilm formation. A Bacillus sp., previously isolated in our laboratory from a cleaning-resistant membrane biofilm, was used to develop 48-h-old static biofilms on RO membrane pieces, using the different feed substrates (UF permeate, whey, and an alternating whey/UF feed). Biofilms were analyzed for viable counts by the swab technique, and we used scanning electron and atomic force microscopy for microstructure imaging. The membrane cleaning process included 6 sequential steps. We observed differences in the resistance pattern of the 3 types of biofilms to the typical cleaning process. The mean pretreatment counts of the 48-h UF permeate biofilms were 5.39 log cfu/cm², much higher than the whey biofilm counts of 3.44 log, and alternating whey/UF biofilm counts of 4.54 log. After a 6-step cleaning cycle, we found 2.54 log survivors of the Bacillus isolate on UF biofilms, whereas only 1.82 log survivors were found in whey biofilm, and 2.14 log survivors on whey/UF permeate biofilms. In conclusion, the UF permeate biofilms was more resistant to the biofilm cleaning process compared with the whey or whey/UF permeate biofilms. Scanning electron micrographs showed different microstructures of biofilms based on the type of feed. For UF permeate and whey/UF permeate biofilms, bacilli were present in multilayers of cells in aggregates or irregular clusters with foulant layers. In contrast, those in whey biofilms were in monolayers, with a smoother, flatter appearance. Atomic force microscopy analysis indicated that UF permeate biofilms had the greatest surface roughness among the biofilms, reflecting intensified bacterial colonization. The biofilm micro- and nanostructure variations for the 2 feed substrates and their combination may have resulted in differences in their resistance to the cleaning process.     

The probiotic,Leuconostoc mesenteroides,inhibits Listeria monocytogenes biofilm formation

Listeria monocytogenes biofilm formation renders these cells highly resistant to current sanitation methods, and probiotics may be a promising approach to the efficient inhibition of Listeria biofilms. In the present study, three Leuconostoc mesenteroides strains of lactic acid bacteria isolated from kimchi were shown to be effective probiotics for inhibiting Listeria biofilm formation. Biofilms of two L. monocytogenes serotypes, 1/2a (ATCC15313) and 4b (ATCC19115), in dual-species culture with each probiotic strain were decreased by more than 40-fold as compared with single-species Listeria biofilms; for instance, a reduction from 5.4 × 10⁶ colony forming units (CFU)/cm² L. monocytogenes ATCC19115 in single-species biofilms to 1.1 × 10⁵ CFU/cm² in dual-species biofilms. Most likely, one of the Leuconostoc strains, L. mesenteroides W51, led to the highest Listeria biofilm inhibition without affecting the growth of L. monocytogenes. The cell-free supernatant from the L. mesenteroides W51 culture containing large protein molecules (>30 kDa) also inhibited Listeria biofilms. These data indicate that Leuconostoc probiotics can be used to repress L. monocytogenes biofilm contamination on surfaces at food processing facilities.     

Synergistic effect between Helichrysum italicum essential oil and cold nitrogen plasma against Staphylococcus aureus biofilms on different food‐contact surfaces

Staphylococcus aureus is the most common pathogen in human, and the most diseases produced by S. aureus are associated with biofilms. Helichrysum italicum essential oil (EO), as a natural and safe spice, was employed to disperse S. aureus biofilm by cold nitrogen plasma (CNP) assist. After S. aureus biofilm formation on food container surfaces, they were exposed to CNP and then treated with Helichrysum italicumEO for biofilm dispersion. The population of S. aureus biofilm was approximately reduced by 2 logs after individual treatment of 0.5 mg mL^?1 Helichrysum italicumEO or 400 W CNP. Interestingly, the combined treatment of Helichrysum italicumEO (0.5 mg mL^?1, 40 min) and CNP (400 W, 1 min) reduced the S. aureus viable count in biofilm below 2 logs CFU per cm² after 1‐day storage. Therefore, the synergetic treatment holds great promise to improve the current treatment systems of bacterial contamination on different food‐contact surfaces.     

Optimisation of Bacillus cereus biofilm removal in the dairy industry using an in vitro model of cleaning‐in‐place incorporating serine protease

This article addresses a major hygienic issue in the dairy industry, namely biofilm removal. Response surface methodology was deployed to optimise Bacillus cereus biofilm removal conditions using serine protease. The currently practiced alkali cleaning‐in‐place (CIP) method was also optimised, and while the optimised protease CIP resulted in complete removal of biofilm cells, the reference CIP (with alkali) resulted in a reduction of 4.08 log biofilm cells/cm² and the optimised alkali CIP resulted in a reduction of ～4.92 log biofilm cells/cm². Furthermore, the amount of the biofilm matrix removed in the optimised protease CIP was significantly higher than that of alkali CIP. It was concluded that the optimised protease CIP has better applicability.     

Removal of Microbial Biofilms from Dispense Equipment: The Effect of Enzymatic Pre‐digestion and Detergent Treatment

Microbial biofilms can form in dispense outlets as a result of poor or inadequate cleaning and can be difficult to remove using conventional practices. Enzymatic cleaners might help to remove biofilms by degrading the exopolysaccharide layers in which the microbes are embedded. A multispecies biofilm comprising wild type dispense isolates of Flavimonas oryzihabitans, Lactobacillus brevis, Leuconostoc mesenteroides and Saccharomyces cerevisiae was generated on a section of tubing and fitted into a pilot dispense system, which was left uncleaned for 12 weeks. After cleaning approximately 10⁴ viable aerobes and 10³ viable anaerobes were still present. Stainless steel coupons and pieces of dispense line contaminated with biofilm were incubated in the laboratory with an enzyme mix containing varying proportions of α‐amylase, β‐glucuronidase, glucose oxidase, dextranase, protease and pectinase. Cultures grown on stainless steel had significantly (F pr. > 0.05) less viable cells than non‐enzyme treated biofilms, but this was dependent on the microbial species. Typically Lactobacillus brevis was most susceptible to the enzyme treatment. Cultures grown on dispense line were much more resistant to enzymatic digestion. Pre‐digestion with protease was most effective for removal of Lactobacillus brevis and Leuconostoc mesenteroides but not for Saccharomyces cerevisiae and Flavimonas oryzihabitans. In the simulated bar, pre‐digestion with protease reduced the viable cell count by 0.64 log units for the aerobes and 1.9 log units for the anaerobes. This study demonstrates that pre‐digestion with enzyme solutions before line cleaning is useful for treating heavily contaminated lines in trade.     

Evaluation of a novel antimicrobial (lauric arginate ester) substance against biofilm of Escherichia coli O157:H7, Listeria monocytogenes,and Salmonella spp.

The purpose of this study was to evaluate the activity of a novel antimicrobial substance lauric arginate ester (LAE) against selected foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes and Salmonella spp.) in biofilm. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined and showed that LAE exhibits a strong antimicrobial activity. Biofilms were grown on abiotic stainless steel, rubber, MBEC biofilm device) and biotic (lettuce) surfaces. The efficacy of LAE (50, 100 and 200 ppm) at reducing the biofilm cells on these surfaces was examined by applying LAE for 2 h. Results revealed that LAE exhibited the reduction in biofilm bacteria up to 7 log CFU cm^?2, 3.5 log CFU cm^?2, 4.0 log CFU peg^?1 and 1.5 log CFU cm^?2 on stainless steel, rubber, MBEC and lettuce surfaces, respectively. Overall, these results suggest that LAE has been shown to be a potential alternative to control bacteria in biofilm mode in food industry.     

Reduction effect of the selected chemical and physical treatments to reduce L. monocytogenes biofilms formed on lettuce and cabbage

As people shift their attention away from unhealthy foods, healthy fresh produce has become popular. However, fresh produce has contributed to many outbreaks of Listeria monocytogenes, which can form a mature biofilm within 24 h. Recent control strategies have proved ineffective in ensuring safe food production. This study focuses on L. monocytogenes biofilms formed on lettuces and cabbages using a viable plate count method and field emission electron microscopy. We investigated the reduction efficacy of treatment with 200 parts per million (ppm) chlorine, 2% each of citric, lactic, and malic acids, 32 Hz ultrasonication (US), 390 mJ/cm² ultraviolet-C (UV-C), or 750 mJ/cm² cold oxygen plasma (COP) on L. monocytogenes biofilms. Following treatment, the quality of the vegetables was analyzed with standard procedures. UV-C and COP showed the best CFU reduction, regardless of the nature of the vegetable surface, while US failed to produce any significant reduction (P > 0.05). Furthermore, chemical treatments reduced count by < 1 log colony forming unit (CFU)/cm² on lettuces, whereas a > 2 log reduction was observed on cabbages. The effect of chemical treatment largely depended on the particular vegetable, while UV-C and COP achieved high reduction regardless of the vegetable, and had no effect on quality. We, therefore, speculate that UV-C and COP show promise in overcoming L. monocytogenes biofilms on food produce.     

Potential of Escherichia coli O157:H7 to persist and form viable but non-culturable cells on a food-contact surface subjected to cycles of soiling and chemical treatment

Our aim was to assess the potential of Escherichia coli O157:H7 to persist in a processing environment. We studied E. coli behaviour under conditions modelling those of meat plants to establish one initial bacterial load that allows persistence and another that does not. Polyurethane coupons (3.5 cm²) were contaminated once with E. coli in meat exudate before being subjected daily to a cleaning product and a disinfectant, both at half the recommended in-use concentrations, and a further soiling with the exudate. This procedure aimed to model what occurs in harbourage sites. Because previous experiments showed that persistence could not be achieved at 15 °C (temperature of slaughter halls), we incubated the coupons at 20 °C. Viable cells were determined by ethidium monoazide-qPCR (EMA-qPCR). When the first chemical treatment (CT) was applied to 24-hour biofilms with 5.4 log CFU/cm², cells were no longer detectable after the first week. However, on 66-hour biofilms with 6.7 log CFU/cm², after initially decreasing, E. coli numbers reached 6.6 log CFU/cm² and 8.3 log viable cells/cm² on the 11th day. When E. coli was cultured with a Comamonas testosteroni previously shown to increase E. coli biofilm formation, and subjected to CT on alternate days, E. coli stabilized at 4.6 log CFU/cm² before the CT, from the 5th day of the experiment. The killing and detachment effects of the CT decreased over time and PCR quantification detected a resumption of growth after 2 days (CT on alternate days) or 3 days (daily CT). Intracellular pH (pHi) of individual cells was determined during an experiment in which the CT was applied on alternate days. The proportion of cells with no proton gradient towards the environment (pHi ≤ 5.4) increased after the CT as expected. But during the first week of the experiment only, a further increase in this proportion occurred 24 h after the CT, suggesting that some of the surviving viable but non-culturable cells finally died.This study shows that conditions leading to E. coli O157:H7 persistence are not likely to arise when good refrigeration and hygiene practices are applied, and highlights the usefulness of EMA or PMA-qPCR as a complement to CFU determination in studying bacterial survival after cleaning and disinfection.     

Inactivation and removal of Enterobacter aerogenes biofilm in a model piping system using plasma-activated water (PAW)

Plasma-activated water (PAW) was explored as a clean-in-place agent for inactivating biofilms of Enterobacter aerogenes on the inner surface of a model piping system. Abilities of PAW or chlorine solution or distilled water to inactivate the biofilm (~1 × 10⁸ CFU/cm²) attached to the pipe inner surface were compared. Distilled water flowing at 0.11 m/s through the piping system reduced the Enterobacter aerogenes counts on the surface by 0.35, 0.2, and 0.2 log CFU/cm² on the tees, elbows, and tubing, respectively. However, PAW reduced the E. aerogenes counts on tees, elbows, and tubing by 3.0, 3.2, and 3.8 log CFU/cm², respectively, which were similar to 100-ppm chlorine water. Increasing the flow velocity to 0.22 m/s made only small difference in terms of biofilm removal. The antimicrobial effect of PAW for Enterobacter aerogenes biofilm on the inner surface of a piping system was comparable to 100-ppm of chlorine solution.     

Effect of carvacrol and thymol on Salmonella spp. biofilms on polypropylene

The present study evaluated the effects of carvacrol and thymol against Salmonella spp. biofilm on polypropylene. The efficacy of the compounds was assessed by quantifying Salmonella spp. cells during and after biofilm formation on polypropylene and performing scanning electron microscopy. During biofilm formation, carvacrol and thymol, at subinhibitory concentrations, reduced bacterial counts about 1–2 log, while established Salmonella spp. biofilms were reduced about 1–5 log by carvacrol and thymol, at MIC or 2× MIC. The greatest reduction in carvacrol‐treated biofilms, about 5 log, was observed with 156 and 312 μg mL^?1 (MIC and 2× MIC) in established Salmonella Typhimurium ATCC 14028 biofilms. Thymol showed the greatest reduction, about 4 log, at 624 μg mL^?1 (2× MIC) against mature Salmonella Enteritidis biofilm. Carvacrol and thymol reduced the number of Salmonella spp. cells on polypropylene, suggesting their potential for the control of Salmonella spp. biofilms.     

Salmonella enterica Enteritidis biofilm formation and viability on regular and triclosan-impregnated bench cover materials

Contamination of food contact surfaces by microbes such as Salmonella is directly associated with substantial industry costs and severe foodborne disease outbreaks. Several approaches have been developed to control microbial attachment; one approach is the development of food contact materials incorporating antimicrobial compounds. In the present study, Salmonella enterica Enteritidis adhesion and biofilm formation on regular and triclosan-impregnated kitchen bench stones (silestones) were assessed, as was cellular viability within biofilms. Enumeration of adhered cells on granite, marble, stainless steel, and silestones revealed that all materials were prone to bacterial colonization (4 to 5 log CFU/cm(2)), and no significant effect of triclosan was found. Conversely, results concerning biofilm formation highlighted a possible bacteriostatic activity of triclosan; smaller amounts of Salmonella Enteritidis biofilms were formed on impregnated silestones, and significantly lower numbers of viable cells (1 × 10(5) to 1 × 10(6) CFU/cm(2)) were found in these biofilms than in those on the other materials (1 × 10(7) CFU/cm(2)). All surfaces tested failed to promote food safety, and careful utilization with appropriate sanitation of these surfaces is critical in food processing environments. Nevertheless, because of its bacteriostatic activity, triclosan incorporated into silestones confers some advantage for controlling microbial contamination.     

Inactivation of biofilm cells of foodborne pathogen by aerosolized sanitizers

The objective of this study was to determine the effect of aerosolized sanitizers on the inactivation of Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes biofilms. Biofilms were formed on a stainless steel and polyvinyl chloride (PVC) coupon by using a mixture of three strains each of three foodborne pathogens. Six day old biofilms on stainless steel and PVC coupons were treated with aerosolized sodium hypochlorite (SHC; 100 ppm) and peracetic acid (100, 200, and 400 ppm) in a model cabinet for 5, 10, 30, and 50 min. Treatment with 100 ppm PAA was more effective than the same concentration of SHC with increasing treatment time. Exposure to 100 ppm SHC and PAA for 50 min significantly (p<0.05) reduced biofilm cells of three foodborne pathogens (0.50 to 3.63 log CFU/coupon and 2.83 to more than 5.78 log CFU/coupon, respectively) compared to the control treatment. Exposure to 200 and 400 ppm PAA was more effective in reducing biofilm cells. Biofilm cells were reduced to below the detection limit (1.48 log CFU/coupon) between 10 and 30 min of exposure. The results of this study suggest that aerosolized sanitizers have a potential as a biofilm control method in the food industry.     

Influence of plasma characteristics on the efficacy of Cold Atmospheric Plasma (CAP) for inactivation of Listeria monocytogenes and Salmonella Typhimurium biofilms

The biofilm mode of growth protects bacterial cells from applied disinfection methods for abiotic (food) contact surfaces. Therefore, new inactivation technologies such as Cold Atmospheric Plasma (CAP) should be considered. However, the influence of different plasma characteristics on the CAP efficacy for biofilm inactivation requires further study. In this research, the influence of (i) the applied plasma configuration (Dielectric Barrier Discharge (DBD) and Surface Barrier Discharge (SBD)), (ii) the oxygen level of the gas flow (He + 0.0/0.5/1.0 (v/v) % O₂), and (iii) the plasma intensity (13.88, 17.88, and 21.88 V input voltage) on the CAP efficacy for inactivation of L. monocytogenes and S. Typhimurium biofilms was investigated. Depending on the applied plasma characteristics, log₁₀-reductions up to approximately 3.5 log(CFU/cm²) were obtained. Nevertheless, it could be concluded that the highest log-reductions were in general obtained while using the DBD electrode, 0.0 (v/v) % O₂, and an input voltage of 21.88 V.Industrial relevanceThis study demonstrated the potential application of CAP for inactivation of pathogenic biofilms developed on abiotic (food) contact surfaces. The effect of different plasma characteristics on the CAP inactivation efficacy was investigated and determined optimal conditions resulted in promising reductions of the biofilm-associated cells. By incorporating this novel technology in a complete cleaning and disinfection process, the risk of (cross) contamination of food products might extensively be reduced.     

Evaluation of sodium dichloroisocyanurate treatment on recovered concentrations of Salmonella enterica,Escherichia coli O157:H7, and Listeria monocytogenes from cattle hide surfaces and culture medium

A major concern of the cattle industry is cross-contamination of meat with pathogens. Cattle are exposed to fecal material, mud, and other contaminants which harbor pathogens that can be shed onto meat and meat processing equipment. Due to increased chances of meat contamination during processing, new antimicrobial formulations for carcass washing before hide removal needs to be identified and tested. Sodium dichloroisocyanurate (SDIC) has biocidal properties and belongs to the N-halamine group of compounds. Disk diffusion assays revealed, 1,000 ppm SDIC effectively reduced pathogen concentrations. SDIC was evaluated for its effects on pathogens in Tryptic Soy Broth and results revealed that 1,000 ppm SDIC had a strong correlation with time and treatment with no bacterial growth in log CFU ml⁻¹ observed at the lowest detection level. Treatment of inoculated hides with 1,000 ppm SDIC for 5 min resulted in reduction of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes at 1.97, 2.02, and 2.84 log CFU cm⁻², respectively.     

Biofilm Formation and Control in Food Processing Facilities

Microorganisms on wet surfaces have the ability to aggregate, grow into microcolonies, and produce biofilm. Growth of biofilms in food processing environments leads to increased opportunity for microbial contamination of the processed product. These biofilms may contain spoilage and pathogenic microorganisms. Microorganisms within biofilms are protected from sanitizers increasing the likelihood of survival and subsequent contamination of food. This increases the risk of reduced shelf life and disease transmission. Extracellular polymeric substances associated with biofilm that are not removed by cleaning provide attachment sites for microorganisms newly arrived to the cleaned system. Biofilm formation can also cause the impairment of heat transfer and corrosion to metal surfaces. Some of the methods used to control biofilm formation include mechanical and manual cleaning, chemical cleaning and sanitation, and application of hot water.     

Biofilm Formation of O157 and Non‐O157 Shiga Toxin‐Producing Escherichia coli and Multidrug‐Resistant and Susceptible Salmonella Typhimurium and Newport and Their Inactivation by Sanitizers

This study compared biofilm formation by 7 serogroups of pathogenic Escherichia coli and 2 or 3 phenotypes of Salmonella (susceptible, multidrug‐resistant [MDR], and/or multidrug resistant with ampC gene [MDR‐AmpC]). One‐week mature biofilms were also exposed to water, quaternary ammonium compound‐based (QAC), and acid‐based (AB) sanitizers. Seven groups (strain mixture) of above‐mentioned pathogens were separately spot‐inoculated onto stainless steel coupons surfaces for target inoculation of 2 log CFU/cm², then stored statically, partially submerged in 10% nonsterilized meat homogenate at 4, 15, and 25 °C. Biofilm cells were enumerated on days 0, 1, 4, and 7 following submersion in 30 mL for 1 min in water, QAC, and AB. Counts on inoculation day ranged from 1.6 ± 0.4 to 2.4 ± 0.6 log CFU/cm² and changed to 1.2 ± 0.8 to 1.9 ± 0.8 on day 7 at 4 °C with no appreciable difference among the 7 pathogen groups. After treatment with QAC and AB on day 7, counts were reduced (P < 0.05) to less than 0.7 ± 0.6 and 1.2 ± 0.5, respectively, with similar trends among pathogens. Biofilm formation at higher temperatures was more enhanced; E. coli O157:H7, as an example, increased (P < 0.05) from 1.4 ± 0.6 and 2.0 ± 0.3 on day 0 to 4.8 ± 0.6 and 6.5 ± 0.2 on day 7 at 15 and 25 °C, respectively. As compared to 4 °C, after sanitation, more survivors were observed for 15 and 25 °C treatments with no appreciable differences among pathogens. Overall, we observed similar patterns of growth and susceptibility to QAC and AB sanitizers of the 7 tested pathogen groups with enhanced biofilm formation capability and higher numbers of treatment survivors at higher temperatures.     

Cell viability of Listeria monocytogenes biofilms

Several authors have reported biofilm formation by Listeria monocytogenes, and it is suspected that biofilms form a unique niche for extended survival of this foodborne pathogen in food-processing environments. We have evaluated growth of two L. monocytogenes strains (Murray and 7148) in biofilms and analysed the relationship between culturable and viable-but-non-culturable (VBNC) cells. Biofilms were grown on glass slides in static conditions at 37°C for up to 10 days. Culturable cells for L. monocytogenes Murray grew to 10⁵cfu cm⁻²within 2 days, while L. monocytogenes 7148 required 4 days to reach these cell numbers. After 2 days, cell counts of L. monocytogenes Murray decreased, followed by another increase with cell numbers reaching almost 10⁶cfu cm⁻²on day 10. In contrast, cell counts of L. monocytogenes 7148 stayed close to 10⁵cfu cm⁻²until day 10. VBNC cells of L. monocytogenes Murray increased with biofilm age while this was not seen for strain 7148. Also, swabbing removed biofilms of strain Murray more easily than strain 7148. Comparisons of viable counts obtained for swabbed and in situ biofilms indicated that these strain differences are due either to variable composition of extracellular polymeric substances in the two biofilms or to different cell physiology of the two strains.