Lethality of chlorine, chlorine dioxide, and a commercial produce sanitizer to Bacillus cereus and Pseudomonas in a liquid detergent, on stainless steel, and in biofilm

Many factors that are not fully understood may influence the effectiveness of sanitizer treatments for eliminating pathogens and spoilage microorganisms in food or detergent residues or in biofilms on food contact surfaces. This study was done to determine the sensitivities of Pseudomonas cells and Bacillus cereus cells and spores suspended in a liquid dishwashing detergent and inoculated onto the surface of stainless steel to treatment with chlorine, chlorine dioxide, and a commercial produce sanitizer (Fit). Cells and spores were incubated in a liquid dishwashing detergent for 16 to 18 h before treatment with sanitizers. At 50 microg/ml, chlorine dioxide killed a significantly higher number of Pseudomonas cells (3.82 log CFU/ml) than did chlorine (a reduction of 1.34 log CFU/ml). Stainless steel coupons were spot inoculated with Pseudomonas cells and B. cereus cells and spores, with water and 5% horse serum as carriers. Chlorine was more effective than chlorine dioxide in killing cells and spores of B. cereus suspended in horse serum. B. cereus biofilm on stainless steel coupons that were treated with chlorine dioxide or chlorine at 200 microg/ml had total population reductions (vegetative cells plus spores) of > or = 4.42 log CFU per coupon; the number of spores was reduced by > or = 3.80 log CFU per coupon. Fit (0.5%) was ineffective for killing spot-inoculated B. cereus and B. cereus in biofilm, but treatment with mixtures of Fit and chlorine dioxide caused greater reductions than did treatment with chlorine dioxide alone. In contrast, when chlorine was combined with Fit, the lethality of chlorine was completely lost. This study provides information on the survival and sanitizer sensitivity of Pseudomonas and B. cereus in a liquid dishwashing detergent, on the surface of stainless steel, and in a biofilm. This information will be useful for developing more effective strategies for cleaning and sanitizing contact surfaces in food preparation and processing environments.     

Survival of Campylobacter jejuni in biofilms isolated from chicken houses

Campylobacter jejuni is a thermophilic and microaerophilic enteric pathogen associated with poultry. Biofilms may be a source of C. jejuni in poultry house water systems since they can protect constituent microorganisms from environmental stress. In this study, the viability of C. jejuni in biofilms of gram-positive chicken house isolates (P1, Y1, and W1) and a Pseudomonas sp. was determined using a cultural method (modified brucella agar) and direct viable count (DVC). Two-day biofilms grown on polyvinyl chloride (PVC) coupons in R2A broth at 12 and 23 degrees C were incubated with C. jejuni for a 6-h attachment period. Media were then refreshed every 24 h for 7 days to allow biofilm growth. Two-day biofilms of P1, Y1, and Pseudomonas spp. enhanced attachment (P < 0.01) of C. jejuni (4.74, 4.62, and 4.78 log cells/cm2, respectively) compared to W1 and controls without preexisting biofilm (4.31 and 4.22 log cells/cm2, respectively). On day 7, isolates P1 and Y1 and Pseudomonas biofilms covered 5.4, 7.0, and 21.5% of the surface, respectively, compared to 4.9% by W1. Viable C. jejuni on the surface decreased (P < 0.05) with time, with the greatest reduction occurring on surfaces without a preexisting biofilm. The number of viable C. jejuni determined by DVC was greater than that determined by the cultural method, indicating that C. jejuni may form a viable but nonculturable state within the biofilm. Both DVC and the cultural method indicate that biofilms enhance (P < 0.01) the survival of C. jejuni during incubation at 12 and 23 degrees C over a 7-day period.     

Inactivation of Listeria monocytogenes/Pseudomonas biofilms by peracid sanitizers.

The ability of peracetic acid and peroctanoic acid sanitizers to inactivate mixed-culture biofilms of a Pseudomonas sp. and Listeria monocytogenes on stainless steel was investigated. Types of biofilms tested included a 4-h attachment of the mixed-cell suspension and a 48-h biofilm of mixed culture formed in skim milk or tryptic soy broth. Biofilm-containing coupons were immersed in solutions of hypochlorite, peracetic acid, and peroctanoic acid either with or without organic challenge. Organic challenge consisted of either coating the biofilms with milk that were then allowed to dry, or adding milk to the sanitizing solution to achieve a 5% concentration. Surviving cells were enumerated by pouring differential agar directly on the treated surfaces. The peracid sanitizers were more effective than chlorine for inactivating biofilm in the presence of organic challenge. The 48-h mixed-culture biofilm grown in milk was reduced to less than 3 CFU/cm2 by 160 ppm of peracid sanitizer after 1 min of exposure. Peroctanoic acid was more effective than peracetic acid against biofilm cells under conditions of organic challenge. Pseudomonas and L. monocytogenes were inactivated to similar levels by the sanitizer treatments, even though Pseudomonas predominated in the initial biofilm population.     

Effectiveness of chemical sanitizers against Campylobacter jejuni-containing biofilms

Survival of Campylobacter jejuni in mixed-culture biofilms was determined after treatment with chemical sanitizers including chlorine, quaternary ammonia, peracetic acid (PAA), and a PAA/peroctanoic acid mixture (PAA/POA). Biofilm-producing bacteria (gram-positive rods, Y1 and W1) were isolated from chicken house nipple drinkers. A meat plant isolate (Pseudomonas sp.) was also included as a biofilm producer. Two-day-old biofilms grown on polyvinyl chloride (PVC) plastic coupons in R2A broth at 12 degrees C were incubated with 10(6) CFU/ml C jejuni for 6 h to allow attachment. The coupons were then rinsed and incubated in fresh media for an additional 24 h. C. jejuni-containing biofilms were detached by vortexing with glass beads in modified brucella broth, which was then enumerated for C. jejuni on selective/differential media. The presence of biofilm enhanced (P < 0.01) the attachment and survival of C. jejuni After the 24-h incubation, only 20 CFU/cm2 of C. jejuni were recovered from the control without biofilms compared to 2,500 to 5,000 CFU/cm2 in samples with preexisting biofilms. The presence of biofilm microflora decreased (P < 0.01) the effectiveness of sanitizers against C. jejuni. Chlorine was the most effective sanitizer since it completely inactivated C. jejuni in the biofilms after treatment at 50 ppm for 45 s. C. jejuni in biofilms was susceptible to all sanitizers tested but was not completely inactivated by treatment with quaternary ammonia, PAA, or PAA/POA mixture at 50 and 200 ppm for 45 s.     

Enhancing the bactericidal effect of electrolyzed water on Listeria monocytogenes biofilms formed on stainless steel

Biofilms are potential sources of contamination to food in processing plants, because they frequently survive sanitizer treatments during cleaning. The objective of this research was to investigate the combined use of alkaline and acidic electrolyzed (EO) water in the inactivation of Listeria monocytogenes biofilms on stainless steel surfaces. Biofilms were grown on rectangular stainless steel (type 304, no. 4 finish) coupons (2 by 5 cm) in a 1:10 dilution of tryptic soy broth that contained a five-strain mixture of L. monocytogenes for 48 h at 25 degrees C. The coupons with biofilms were then treated with acidic EO water or alkaline EO water or with alkaline EO water followed by acidic EO water produced at 14 and 20 A for 30, 60, and 120 s. Alkaline EO water alone did not produce significant reductions in L. monocytogenes biofilms when compared with the control. Treatment with acidic EO water only for 30 to 120 s, on the other hand, reduced the viable bacterial populations in the biofilms by 4.3 to 5.2 log CFU per coupon, whereas the combined treatment of alkaline EO water followed by acidic EO water produced an additional 0.3- to 1.2-log CFU per coupon reduction. The population of L. monocytogenes reduced by treatments with acidic EO water increased significantly with increasing time of exposure. However, no significant differences occurred between treatments with EO water produced at 14 and 20 A. Results suggest that alkaline and acidic EO water can be used together to achieve a better inactivation of biofilms than when applied individually.     

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.     

Sodium hypochlorite-mediated inactivation of Cronobacter spp. biofilms on conveyor belt chips

Chlorine bactericidal effects on Cronobacter vegetative cells and biofilms on conveyor belt chips were evaluated. Cronobacter vegetative cell isolates were inactivated after 10 min chlorine exposure (10 ppm). No efficacy difference was observed between pH-adjusted and pHunadjusted sodium hypochlorite solutions. The pH-adjusted solution was more effective at reducing the bacterial population in a biofilm. The number of cells in the biofilm on a Buna-N chip decreased by 3 log after exposure to 100 ppm of pH-adjusted chlorine and for PVC by 2.21 log after exposure to 400 ppm of pH-adjusted solution. The rate of biofilm reduction is dependent on the composition of the conveyor belt, concentration of chlorine, and pH of the chlorine solution.     

Removal of Pseudomonas putida biofilm and associated extracellular polymeric substances from stainless steel by alkali cleaning

Alkali (NaOH)-based compounds are commonly used in the food industry to clean food contact surfaces. However, little information is available on the ability of alkali and alkali-based cleaning compounds to remove extracellular polymeric substances (EPS) produced by biofilm bacteria. The objectives of this study were to determine the temperature and NaOH concentration necessary to remove biofilm EPS from stainless steel under turbulent flow conditions (clean-in-place simulation) and to determine the ability of a commercial alkaline cleaner to remove biofilm EPS from stainless steel when applied under static conditions without heat. Biofilms were produced by growing Pseudomonas putida on stainless steel for 72 h at 25 degrees C in a 1:10 dilution of Trypticase soy broth. The biofilms were treated using NaOH at concentrations of 1.28 to 6.0% and temperatures ranging from 66 to 70 degrees C. Other biofilms were treated with commercial alkaline cleaner at 25 or 4 degrees C for 1 to 30 min. Removal of EPS was determined by direct microscopic observation of samples stained with fluorescent-labeled peanut agglutinin lectin. Treatment with 1.2% NaOH at 66 degrees C for 3 min was insufficient to remove biofilm EPS. A minimum of 2.5% NaOH at 66 degrees C and 2.0% NaOH at 68 degrees C for 3 min were both effective for EPS removal. Commercial alkaline cleaner removed over 99% of biofilm EPS within 1 min at 4 and 25 degrees C under static conditions. Selection of appropriated cleaning agent formulation and use at recommended concentrations and temperatures is critical for removal of biofilm EPS from stainless steel.     

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.     

Biofilm Formation Characteristics of Pseudomonas lundensis Isolated from Meat

Biofilms formations of spoilage and pathogenic bacteria on food or food contact surfaces have attracted increasing attention. These events may lead to a higher risk of food spoilage and foodborne disease transmission. While Pseudomonas lundensis is one of the most important bacteria that cause spoilage in chilled meat, its capability for biofilm formation has been seldom reported. Here, we investigated biofilm formation characteristics of P. lundensis mainly by using crystal violet staining, and confocal laser scanning microscopy (CLSM). The swarming and swimming motility, biofilm formation in different temperatures (30, 10, and 4 °C) and the protease activity of the target strain were also assessed. The results showed that P. lundensis showed a typical surface‐associated motility and was quite capable of forming biofilms in different temperatures (30, 10, and 4 °C). The strain began to adhere to the contact surfaces and form biofilms early in the 4 to 6 h. The biofilms began to be formed in massive amounts after 12 h at 30 °C, and the extracellular polysaccharides increased as the biofilm structure developed. Compared with at 30 °C, more biofilms were formed at 4 and 10 °C even by a low bacterial density. The protease activity in the biofilm was significantly correlated with the biofilm formation. Moreover, the protease activity in biofilm was significantly higher than that of the corresponding planktonic cultures after cultured 12 h at 30 °C.     

Elimination of Listeria monocytogenes biofilms by ozone, chlorine, and hydrogen peroxide

This study evaluated the efficacy of ozone, chlorine, and hydrogen peroxide to destroy Listeria monocytogenes planktonic cells and biofilms of two test strains, Scott A and 10403S. L. monocytogenes was sensitive to ozone (O3), chlorine, and hydrogen peroxide (H2O2). Planktonic cells of strain Scott A were completely destroyed by exposure to 0.25 ppm O3 (8.29-log reduction, CFU per milliliter). Ozone's destruction of Scott A increased when the concentration was increased, with complete elimination at 4.00 ppm O3 (8.07-log reduction, CFU per chip). A 16-fold increase in sanitizer concentration was required to destroy biofilm cells of L. monocytogenes versus planktonic cells of strain Scott A. Strain 10403S required an ozone concentration of 1.00 ppm to eliminate planktonic cells (8.16-log reduction, CFU per milliliter). Attached cells of the same strain were eliminated at a concentration of 4.00 ppm O3 (7.47-log reduction, CFU per chip). At 100 ppm chlorine at 20 degrees C, the number of planktonic cells of L. monocytogenes 10403S was reduced by 5.77 log CFU/ml after 5 min of exposure and by 6.49 log CFU/ml after 10 min of exposure. Biofilm cells were reduced by 5.79 log CFU per chip following exposure to 100 ppm chlorine at 20 degrees C for 5 min, with complete elimination (6.27 log CFU per chip) after exposure to 150 ppm at 20 degrees C for 1 min. A 3% H2O2 solution reduced the initial concentration of L. monocytogenes Scott A planktonic cells by 6.0 log CFU/ml after 10 min of exposure at 20 degrees C, and a 3.5% H2O2 solution reduced the planktonic population by 5.4 and 8.7 log CFU/ml (complete elimination) after 5 and 10 min of exposure at 20 degrees C, respectively. Exposure of cells grown as biofilms to 5% H2O2 resulted in a 4.14-log CFU per chip reduction after 10 min of exposure at 20 degrees C and in a 5.58-log CFU per chip reduction (complete elimination) after 15 min of exposure.     

Resistance of pathogenic bacteria on the surface of stainless steel depending on attachment form and efficacy of chemical sanitizers

Various bacteria including food spoilage bacteria and pathogens can form biofilms on different food processing surfaces, leading to potential food contamination or spoilage. Therefore, the survival of foodborne pathogens (Escherichia coli O157:H7, Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, Cronobacter sakazakii) in different forms (adhered cells, biofilm producing in TSB, biofilm producing at RH 100%) on the surface of stainless steel and stored at various relative humidities (RH 23%, 43%, 68%, 85%, and 100%) at room temperature for 5 days was investigated in this study. Additionally, the efficacy of chemical sanitizers (chlorine-based and alcohol-based commercial sanitizers) on inhibiting various types of biofilms of E. coli O157:H7 and S. aureus on the surface of stainless steel was investigated. The number of pathogens on the surface of stainless steel in TSB stored at 25 °C for 7 days or RH 100% at 25 °C for 7 days was significantly increased and resulted in the increase of 3 log₁₀ CFU/coupon after 1 day, and these levels were maintained for 7 days. When stainless steel coupons were stored at 25 °C for 5 days, the number of pathogens on the surface of stainless steel was significantly reduced after storage at RH 23%, 43%, 68%, and 85%, but not at 100%. When the bacteria formed biofilms on the surface of stainless steel in TSB after 6 days, the results were similar to those of the attached form. However, levels of S. aureus and C. sakazakii biofilms were more slowly reduced after storage at RH 23%, 43%, 68%, and 85% for 5 days than were those of the other pathogens. Formation of biofilms stored at RH 100% for 5 days displayed the highest levels of resistance to inactivation. Treatment with the alcohol sanitizer was very effective at inactivating attached pathogens or biofilms on the surface of stainless steel. Reduction levels of alcohol sanitizer treatment ranged from 1.91 to 4.77 log and from 4.35 to 5.35 log CFU/coupon in E. coli O157:H7 and S. aureus, respectively. From these results, the survival of pathogens contaminating the surfaces of food processing substrates such as stainless steel varied depending on RH and attachment form. Also, alcohol-based sanitizers can be used as a potential method to remove microbial contamination on the surfaces of utensils, cooking equipment, and other related substrates regardless of the microbial attached form.     

Influence of extended acid stressing in fresh beef decontamination runoff fluids on sanitizer resistance of acid-adapted Escherichia coli O157:H7 in biofilms

This study evaluated resistance to sanitizing solutions of Escherichia coli O157:H7 cells forming biofilms on stainless steel coupons exposed to inoculated meat decontamination runoff fluids (washings). A previously acid-adapted culture of a rifampicin-resistant derivative of E. coli O157:H7 strain ATCC 43895 was inoculated in unsterilized or sterilized combined hot-water (85 degrees C) and cold-water (10 degrees C) (50/50 [vol/vol]) composite water (W) washings (pH 6.29 to 6.47) and in W washings mixed with 2% acetic acid (pH 4.60 to 4.71) or in 2% lactic acid W washings (pH 4.33 to 4.48) at a ratio of 1/99 (vol/vol). Stainless steel coupons (2 by 5 by 0.08 cm) were submerged in the inoculated washings and stored for up to 14 days at 15 degrees C. Survival of E. coli O157:H7 was determined after exposure (0 to 60 s for cells in suspension and 0 to 300 s for attached cells) to two commercial sanitizers (150 ppm peroxyacetic acid and 200 ppm quaternary ammonium compound) at 2, 7, and 14 days. E. coli O157:H7 attached more rapidly to coupons submerged in washings containing the natural flora than to those without. The attached cells were more resistant to the effects of the sanitizers than were the cells in suspension, and survival was highest in the presence of the natural flora. Attached cells in the presence of dilute acid washings were more sensitive to subsequent sanitizer treatments than were cells generated in the presence of W washings. Under the conditions of this study, cells of E. coli O157:H7 in W washings were more sensitive to acidic (peroxyacetic acid) than to alkaline (quaternary ammonium) sanitizers during storage. These results suggest that meat processing plants that apply no decontamination or that use only water washings of meat should consider using acidic sanitizers to enhance biofilm removal. Plants that apply both water and acidic washings may create a sublethal acid-stressing environment in the runoff fluids, sensitizing biofilm cells to subsequent sanitizing treatments.     

Fate of Enterobacter sakazakii attached to or in biofilms on stainless steel upon exposure to various temperatures or relative humidities

Survival of Enterobacter sakazakii dried on the surface of stainless steel and exposed to 43% relative humidity, as affected by temperature, was studied. Populations of E. sakazakii (7.4 to 8.6 log CFU per coupon) on coupons dried for 2 h at 22 degrees C decreased significantly (P < or = 0.05) at 4, 25, and 37 degrees C within 10, 3, and 1 day(s), respectively, but the pathogen remained viable for up to 60 days. At a given storage temperature and time, reductions were significantly greater when cells had been suspended in water rather than in infant formula before drying. Formation of biofilm by E. sakazakii on stainless steel immersed in M9 medium, which contains minimal concentrations of nutrients, and infant formula at 25 degrees C and subsequent survival of cells at 25 degrees C as affected by exposure to 23, 43, 68, 85, and 100% relative humidity were investigated. Some of the cells in these biofilms survived under all test relative humidities for up to 42 days. The overall order of survival as affected by relative humidity was 100 > 23 = 43 = 68 > 85% relative humidity, regardless of the medium in which the biofilm was formed. Reduction in viability of cells was significantly greater in biofilm that had formed in M9 medium than in biofilm formed in infant formula. Results indicate that infant formula provides protection for attached cells, as well as cells in biofilm, against lethality on exposure to desiccation. These results are useful when predicting the survival characteristics of E. sakazakii on stainless steel surfaces in processing and preparation kitchen environments.     

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.     

A predictive model for heat inactivation of Listeria monocytogenes biofilm on stainless steel

Heat treatment of potential biofilm-forming sites is sometimes used for control of Listeria monocytogenes in food processing plants. However, little information is available on the heat treatment required to kill L. monocytogenes present in biofilms. The purpose of this study was to develop a predictive model for the heat inactivation of L. monocytogenes in monoculture biofilms (strains Scott A and 3990) and in biofilms with competing bacteria (Pseudomonas sp. and Pantoea agglomerans) formed on stainless steel in the presence of food-derived soil. Biofilms were produced on stainless steel coupons with diluted tryptic soy broth incubated for 48 h at 25 degrees C. Duplicate biofilm samples were heat treated for 1, 3, 5, and 15 min at 70, 72, 75, 77, and 80 degrees C and tested for survivors using enrichment culture. The experiment was repeated six times. A predictive model was developed using logistic regression analysis of the fraction negative data. Plots showing the probability of L. monocytogenes inactivation in biofilms after heat treatment were generated from the predictive equation. The predictive model revealed that hot water sanitation of stainless steel can be effective for inactivating L. monocytogenes in a biofilm on stainless steel if time and temperature are controlled. For example, to obtain a 75% probability of total inactivation of L. monocytogenes 3990 biofilm, a heat treatment of 80 degrees C for 11.7 min is required. The model provides processors with a risk management tool that provides predicted probabilities of L. monocytogenes inactivation and allows a choice of three heat resistance assumptions. The predictive model was validated using a five-strain cocktail of L. monocytogenes in the presence of food soil.     

Efficiency of different sanitation methods on Listeria monocytogenes biofilms formed under various environmental conditions

The resistance of Listeria monocytogenes biofilms formed under food processing conditions, against various sanitizing agents and disinfection procedures was evaluated in the present study. The first sanitation procedure included biofilm formation on stainless steel coupons (SS) placed in tryptic soy broth supplemented with 0.6% yeast extract (TSBYE) of various concentrations of NaCl (0.5, 7.5 and 9.5%) at different temperatures (5 and 20 °C). The biofilms formed were exposed to warm (60 °C) water for 20 min, or to peroxyacetic acid (2% PAA) for 1, 2, 3 and 6 min. Treatment with warm water caused no significant (P ≥ 0.05) reductions in the attached populations. Conversely, surviving bacteria on SS coupons decreased as the exposure time to 2% PAA increased and could not be detected by culture after 6 min of exposure. Biofilms formed at 20°C were more resistant to PAA than biofilms formed at 5 °C. Salt concentration in the growth medium had no marked impact on the resistance to PAA. The second sanitation procedure included biofilm formation of nonadapted (NA) and acid-adapted (AA) cells in TSBYE of pH 5.0 and 7.0 (i.e., NA-5.0, NA-7.0 and AA-5.0, AA-7.0) at 4 °C. Coupons bearing attached cells of L. monocytogenes were periodically exposed to chlorine (0.465% Cl(-)), quaternary ammonium compound (1% QAC) and 2% PAA. The resistance of attached cells to QAC, PAA and Cl(-) followed the order: AA-5.0>NA-7.0 ≥ AA-7.0>NA-5.0. The most effective sanitizer was QAC followed by PAA and Cl(-). The results can lead to the development of efficient sanitation strategies in order to eliminate L. monocytogenes from the processing environment. Furthermore, such results may explain the presence of L. monocytogenes after sanitation as a result of cell attachment history.     

Biofilm formation and sporulation by Bacillus cereus on a stainless steel surface and subsequent resistance of vegetative cells and spores to chlorine, chlorine dioxide, and a peroxyacetic acid-based sanitizer

Biofilm formation by Bacillus cereus 038-2 on stainless steel coupons, sporulation in the biofilm as affected by nutrient availability, temperature, and relative humidity, and the resistance of vegetative cells and spores in biofilm to sanitizers were investigated. Total counts in biofilm formed on coupons immersed in tryptic soy broth (TSB) at 12 and 22 degrees C consisted of 99.94% of vegetative cells and 0.06% of spores. Coupons on which biofilm had formed were immersed in TSB or exposed to air with 100, 97, 93, or 85% relative humidity. Biofilm on coupons immersed in TSB at 12 degrees C for an additional 6 days or 22 degrees C for an additional 4 days contained 0.30 and 0.02% of spores, respectively, whereas biofilm exposed to air with 100 or 97% relative humidity at 22 degrees C for 4 days contained 10 and 2.5% of spores, respectively. Sporulation did not occur in biofilm exposed to 93 or 85% relative humidity at 22 degrees C. Treatment of biofilm on coupons that had been immersed in TSB at 22 degrees C with chlorine (50 microg/ml), chlorine dioxide (50 microg/ml), and a peroxyacetic acid-based sanitizer (Tsunami 200, 40 microg/ml) for 5 min reduced total cell counts (vegetative cells plus spores) by 4.7, 3.0, and 3.8 log CFU per coupon, respectively; total cell counts in biofilm exposed to air with 100% relative humidity were reduced by 1.5, 2.4, and 1.1 log CFU per coupon, respectively, reflecting the presence of lower numbers of vegetative cells. Spores that survived treatment with chlorine dioxide had reduced resistance to heat. It is concluded that exposure of biofilm formed by B. cereus exposed to air at high relative humidity (> or =97%) promotes the production of spores. Spores and, to a lesser extent, vegetative cells embedded in biofilm are protected against inactivation by sanitizers. Results provide new insights to developing strategies to achieve more effective sanitation programs to minimize risks associated with B. cereus in biofilm formed on food contact surfaces and on foods.     

Effect of stress induced by suboptimal growth factors on survival of Escherichia coli O157:H7.

This study investigated the growth and survival of E. coli O157:H7 exposed to a combination of suboptimal factors (22 degrees C, 7 degrees C, -18 degrees C/0.5% NaCl, 5.0% NaCl/pH 7.0, pH 5.4, pH 4.5/addition of lactic acid) in a simulation medium for red meat (beef gravy). Prolonged survival was noted as the imposed stress was more severe, and as multiple growth factors became suboptimal. At a defined temperature (7 degrees C or -18 degrees C), survival was prolonged at the more acid, more suboptimal pH (pH 4.5 > pH 5.4 > pH 7.0) while at a defined pH (pH 4.5), better survival was observed at 7 degrees C than at 22 degrees C. This suggests that application of the hurdle concept for preservation of food may inhibit outgrowth but induce prolonged survival of E. coli O157:H7 in minimal processed foods. At both 22 degrees C and 7 degrees C, the addition of lactic acid instead of HCl to reduce pH (to pH 4.5) resulted in a more rapid decrease of E. coli O157:H7. High survival was observed in beef gravy, pH 5.4 at -18 degrees C (simulation of frozen meat)-reduction of log 3.0 to log 1.9 after 43 days--and in beef gravy, pH 4.5 and 5% NaCl at 7 degrees C (simulation of a fermented dried meat product kept in refrigeration)--less than 1 log reduction in 43 days. In these circumstances, however, a high degree of sublethal damage of the bacterial cells was noted. The degree of sublethal damage can be estimated from the difference in recovery of the pathogen on the non-selective TSA medium and the selective SMAC medium.     

A predictive model for heat inactivation of Listeria monocytogenes biofilm on buna-N rubber

The purpose of this study was to develop a predictive model for the heat inactivation of Listeria monocytogenes in monoculture (strains Scott A and 3990) and with competing bacteria (Pseudomonas sp. and Pantoea agglomerans) formed on buna-N rubber with and without the presence of food-derived soil. Biofilms were produced on rubber disks in dilute Tryptic Soy broth (dTSB) with incubation for 48 h at 25 °C. Duplicate biofilm samples were heat treated for 1, 3, 5, and 15 min at 70, 72, 75, 77 and 80 °C and tested for survivors using enrichment media. The experiment was repeated six times. A predictive model was developed and plots were generated showing the percent probability of L. monocytogenes inactivation in biofilms after heat treatment. For example, to achieve a 95% probability level of complete inactivation required heat treatment of 76 °C for 6 min. The predicted model was validated using a five-strain cocktail of L. monocytogenes. The validated prediction model indicates that with proper maintenance of the time/temperature controls L. monocytogenes in biofilms on rubber surfaces will be inactivated. This model can be used as a tool in the selection of hot water sanitation processes for rubber surfaces.