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.     

Efficacy of two cleaning and sanitizing combinations on Listeria monocytogenes biofilms formed at low temperature on a variety of materials in the presence of ready-to-eat meat residue

Biofilms in the food-processing industry are a serious concern due to the potential for contamination of food products, which may lead to decreased food quality and safety. The effect of two detergent and sanitizer combinations on the inactivation of Listeria monocytogenes biofilms was studied. Combination A uses a chlorinated-alkaline, low-phosphate detergent, and dual peracid sanitizer. Combination B uses a solvated-alkaline environmental sanitation product and hypochlorite sanitizer. The survival of bacterial biofilms placed at 4 and 10 degrees C and held for up to 5 days was also addressed. To simulate conditions found in a ready-to-eat meat-processing environment, biofilms were developed in low-nutrient conditions at 10 degrees C (with and without meat and fat residue) on a variety of materials found in a plant setting. Included were two types of stainless steel, three materials for conveyor use, two rubber products, a wall, and floor material. Biofilms developed on all surfaces tested; numbers at day 2 ranged from 3.2 log on silicone rubber to 4.47 log CFU/cm2 on Delrin, an acetal copolymer. Biofilm survival during storage was higher at 4 degrees C (36.3 to 1,621%) than 10 degrees C (4.5 to 83.2%). Small amounts of meat extract, frankfurters, or pork fat reduced biofilm formation initially; with time, the biofilm cell number and survival percentage increased. Cleaning efficacy was surface dependent and decreased with residue-soiled surfaces; biofilms developed on the brick and conveyor material were most resistant. Both detergents significantly (P < 0.05) removed or inactivated biofilm bacteria. The sanitizers further reduced biofilm numbers; however, the reduction was not significant in most cases for the dual peracid. Using a benchmark efficacy of >3-log reduction, combination A was only effective on 50.0% of the samples, Combination B, at 86.1%, was more effective.     

Biofilm formation and biocides sensitivity of Pseudomonas marginalis isolated from a maple sap collection system

The susceptibility of planktonic and biofilm cells of Pseudomonas marginalis toward four commonly used biocides at different temperatures (15 and 30 degrees C) and biofilm growth times (24 and 48 h) was assessed. Using the MBEC biofilm device, biofilm production in maple sap was shown to be highly reproducible for each set of conditions tested. Biofilm formation was influenced by growth temperature and time. A temperature of 15 degrees C and incubation time of 24 h yielded fewer CFU per peg and showed fewer adhered cells and typical biofilm structures, based on scanning electron microscopy observations as compared with other conditions. Minimal biofilm eradication concentration values for P. marginalis were significantly greater (P. < 0.001) than were MBCs for planktonic cells and for every biocide tested, with the exception of minimal biofilm eradication concentration values for peracetic acid at 15 degrees C and 24 h. Sodium hypochlorite and peracetic acid sanitizers were able to eliminate P. marginalis biofilms at lower concentrations as compared with hydrogen peroxide- and quaternary ammonium-based sanitizers (P < 0.001). According to the results obtained, sodium hypochlorite and peracetic acid sanitizers would be more appropriate for maple sap collection system sanitation.     

Biofilms of Listeria monocytogenes produced at 12 °C either in pure culture or in co-culture with Pseudomonas aeruginosa showed reduced susceptibility to sanitizers

The biofilm-forming ability of 21 Listeria monocytogenes isolates, previously pulsotyped and corresponding to 16 strains, from different origins was evaluated using the Calgary Biofilm Device, at 37 °C. Biofilms of 4 selected strains were also produced either on pure cultures or on co-cultures with Pseudomonas aeruginosa (PAO1), at 12 °C and at 37 °C. For these biofilms, the minimum biofilm eradication concentrations (MBECs) of 4 commercial dairy sanitizers (1 alkyl amine acetate based--T99, 2 chlorine based--T66 and DD, and 1 phosphoric acid based--BP) were determined. Listeria monocytogenes biofilms grown, either at 37 °C or 12 °C, were able to achieve similar cell densities by using different incubation periods (24 h and 7 d, respectively). In co-culture biofilms, P. aeruginosa was the dominant species, either at 37 °C or at 12 °C, representing 99% of a total biofilm population of 6 to 7 log CFU/peg. Co-culture biofilms were generally less susceptible than L. monocytogenes pure cultures. More interestingly, the biofilms produced at 12 °C were usually less susceptible to the sanitizers than when produced at 37 °C. Single or co-culture biofilms of L. monocytogenes and PAO1, particularly produced at 12 °C, retrieved MBEC values for agents T99 and BP that were, at times, above the maximum in-use recommended concentrations for these agents. The results presented here reinforce the importance of the temperature used for biofilm formation, when susceptibility to sanitizers is being assessed. PRACTICAL APPLICATION: Since most food plants have cold wet growth niches in production and storage areas, susceptibility testing should be performed on biofilms produced at refrigeration temperatures. Moreover, the efficiency of the sanitizers used in food industries should be performed on mixed culture biofilms, since in field conditions these will predominate. The results presented here highlight the importance of the temperature used for biofilm formation, when susceptibility to disinfectants is being assessed, as biofilms produced at lower temperature were less susceptible to sanitizers.     

Effect of chemical sanitizers with and without ultrasonication on Listeria monocytogenes as a biofilm within polyvinyl chloride drain pipes

As part of a biofilm in a floor drain, Listeria monocytogenes is exceedingly difficult to eradicate with standard sanitizing protocols. The objective of these studies was to test the use of ultrasonication to break up biofilm architecture and allow chemical sanitizers to contact cells directly. L. monocytogenes biofilms were created in model polyvinyl chloride drain pipes. Chemical sanitizers (quaternary ammonium, peroxide, or chlorine) were applied to the drain pipes with and without a 30-s ultrasonication treatment. Controls using sterile water were included for comparison. L. monocytogenes cells were enumerated from the liquid in the drain and the inside wall surface of the pipe. All chemicals lowered numbers of planktonic cells from 6.6 log CFU/ml in the water control to < 100 CFU/ml. Attached cells were also affected by the chemical sanitizers. Approximately 6.0 log CFU/cm2 of the inner wall surface was detected in water control pipes, and ultrasonication did not lower these numbers. With or without ultrasonication, the peroxide-based sanitizer was effective for reducing the numbers of attached L. monocytogenes cells, resulting in approximately 2.0 log CFU/cm2. Both the chlorine- and quaternary ammonium-based sanitizers reduced the number of attached L. monocytogenes cells to a lesser degree, resulting in 4.2 to 4.4 log CFU/cm2. However, addition of ultrasonication improved the performance of both these sanitizers, causing a further reduction to 3.1 and 2.9 CFU/ cm2 for quaternary ammonium- and chlorine-based chemicals, respectively. These results indicate that a peroxide-based sanitizer alone can be very effective against biofilm L. monocytogenes in drain pipes, and the addition of ultrasonication can improve the effectiveness of chlorine or quaternary ammonium sanitizers.     

Evaluation of dairy and food plant sanitizers against Salmonella typhimurium and Listeria monocytogenes

Six commonly used dairy and food plant sanitizers were evaluated against Salmonella typhimurium and Listeria monocytogenes. Of these six, two were acid anionic sanitizers, one contained a quaternary ammonium compound, one was based on active iodine, and two contained active chlorine. Of the last two, one contained hypochlorite and the other contained active chlorine in organic form. The chlorine-based sanitizers were effective at 100 ppm of available chlorine against both these organisms. The sanitizer containing iodine was effective at 12.5 and 25 ppm titratable iodine against L. monocytogenes and S. typhimurium, respectively. The acid anionic sanitizers were effective at 200 ppm of active agent against both the bacteria, and the quaternary ammonium-based sanitizer was effective at 100 and 200 ppm of active compound against L. monocytogenes and S. typhimurium, respectively. The sanitizer containing iodine at 12.5 and 25 ppm of titratable iodine showed activity equivalent to 50 and 200 ppm of available chlorine, respectively, against L. monocytogenes and 100 and 200 ppm of available chlorine, respectively, against S. typhimurium.     

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.     

Different responses of planktonic and attached Bacillus subtilis and Pseudomonas fluorescens to sanitizer treatment.

Three commercial sanitizers containing iodophor (I), peracetic acid/ hydrogen peroxide (PAH), or chlorhexidine gluconate (CG) were evaluated in vitro against planktonic and sessile Bacillus subtilis or Pseudomonas fluorescens cells grown in Standard One Nutrient Broth. Sessile cells were attached to stainless steel or polyurethane test surfaces. Planktonic and attached cells of both bacteria were enumerated by plate counts after sanitizer treatment for 1, 3, or 5 min. Sessile cells were dislodged from test surfaces by shaking them with beads. Cell morphologies were monitored by scanning electron microscopy (SEM). Attached B. subtilis and P. fluorescens cells on both surface types were less susceptible to all three sanitizers than their planktonic counterparts. PAH, I, and CG were equally effective against planktonic P. fluorescens cells, which were reduced by 99.999% after 1, 3, and 5 min exposure. PAH was the only sanitizer effective against attached P. fluorescens cells on both surface types; it reduced counts by < or = 99.9% after 1, 3, and 5 min exposure. PAH was also the most effective sanitizer against planktonic B. subtilis cells, reducing counts by 99.9% after 1, 3, and 5 min. Sessile B. subtilis cells on both surface types were the least susceptible to all sanitizers; counts were reduced by only 99.5% or less after exposure to PAH for 5 min. SEM revealed that planktonic and attached cells of both bacteria exhibited symptoms of surface roughness, indentations, and shape distortions after treatment with any of the sanitizers.     

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.     

Biofilm Formation by Shiga Toxin-Producing Escherichia coli O157:H7 and Non-O157 Strains and Their Tolerance to Sanitizers Commonly Used in the Food Processing Environment

Shiga toxin-producing Escherichia coli (STEC) strains are important foodborne pathogens. Among these, E. coli O157:H7 is the most frequently isolated STEC serotype responsible for foodborne diseases. However, the non-O157 serotypes have been associated with serious outbreaks and sporadic diseases as well. It has been shown that various STEC serotypes are capable of forming biofilms on different food or food contact surfaces that, when detached, may lead to cross-contamination. Bacterial cells at biofilm stage also are more tolerant to sanitizers compared with their planktonic counterparts, which makes STEC biofilms a serious food safety concern. In the present study, we evaluated the potency of biofilm formation by a variety of STEC strains from serotypes O157:H7, O26:H11, and O111:H8; we also compared biofilm tolerance with two types of common sanitizers, a quaternary ammonium chloride-based sanitizer and chlorine. Our results demonstrated that biofilm formation by various STEC serotypes on a polystyrene surface was highly strain-dependent, whereas the two non-O157 serotypes showed a higher potency of pellicle formation at air-liquid interfaces on a glass surface compared with serotype O157:H7. Significant reductions of viable biofilm cells were achieved with sanitizer treatments. STEC biofilm tolerance to sanitization was strain-dependent regardless of the serotypes. Curli expression appeared to play a critical role in STEC biofilm formation and tolerance to sanitizers. Our data indicated that multiple factors, including bacterial serotype and strain, surface materials, and other environmental conditions, could significantly affect STEC biofilm formation. The high potential for biofilm formation by various STEC serotypes, especially the strong potency of pellicle formation by the curli-positive non-O157 strains with high sanitization tolerance, might contribute to bacterial colonization on food contact surfaces, which may result in downstream product contamination.     

Biofilm formation by acid-adapted and nonadapted Listeria monocytogenes in fresh beef decontamination washings and its subsequent inactivation with sanitizers

The antimicrobial effects of sodium hypochlorite (SH, 200 ppm, at an adjusted pH of 6.80 +/- 0.20 and at an unadjusted pH of 10.35 +/- 0.25), quaternary ammonium compound (pH 10.20 +/- 0.12, 200 ppm), and peroxyacetic acid (PAA, pH 3.45 +/- 0.20, 150 ppm) on previously acid-adapted or nonadapted Listeria monocytogenes inoculated (10(5) CFU/ml) into beef decontamination water washings were evaluated. The effects of the sanitizers on suspended cells (planktonic or deattached) and on cells attached to stainless steel coupons obtained from inoculated washings stored at 15 degrees C for up to 14 days were studied. Cells were exposed to sanitizers on days 2, 7, and 14. The pathogen had formed a biofilm of 5.3 log CFU/cm2 by day 2 of storage (which was reduced to 4.6 log CFU/cm2 by day 14), while the total microbial populations showed more extensive attachment (6.1 to 6.6 log CFU/cm2). The sanitizers were more effective in reducing populations of cells in suspension than in reducing populations of attached cells. Overall, there were no differences between previously acid-adapted and nonadapted L monocytogenes with regard to sensitivity to sanitizers. The total microbial biofilms were the most sensitive to all of the sanitizers on day 2, but their resistance increased during storage, and they were at their most resistant on day 14. Listeria monocytogenes displayed stronger resistance to the effects of the sanitizers on day 7 than on day 2 but had become sensitized to all sanitizers by day 14. SH at the adjusted pH (6.80) (ASH) was generally more effective in reducing bacterial populations than was SH at the unadjusted pH. PAA generally killed attached cells faster at 30 to 300 s of exposure than did the other sanitizers, except for ASH on day 2. PAA was more effective in killing attached cells than in killing cells treated in suspension, in contrast to the other sanitizers.     

Germicidal efficacy of sanitizers on food-borne bacteria and effect of sanitizes in CIP and SIP simulation

The germicidal efficacy of six sanitizers against food-borne bacteria and spores (Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis spores) and the effect of these sanitizers in simulation of cleaning and sterilizing in place were evaluated. The most effective sanitizer solution was peracetic acid (PAA), which was capable of reducing E. coli populations by more than 5 log CFU/g at 60 mg/L. The rest of the sanitizers resulted in a population reduction of less than 5 log CFU/g at 150 mg/L in phenol coefficient test. The effect of acid-anionic sanitizer (ABF) and PAA against E. coli and S. aureus was affected by both pH and temperature. The synergism of sanitizers on germicidal efficacy has also been examined in this study. It was observed that the combination of PAA and ABF presented high efficacy against spores of B. subtilis. 0.2 % PAA in combination with 0.1 % ABF reduced 7.6 log spores. The simulation of cleaning-in-place (CIP) and sterilization-in-place (SIP) revealed that most of organisms were eliminated during CIP. B. subtilis spores holding strong attachment and heat resistance could be eliminated with the combination of 0.2 % PAA and 0.1 % ABF. These findings showed that germicidal efficacy against bacteria and spores was affected by the type of sanitizer, concentration, and environmental conditions, which provides the guidelines in lowering the concentration of sanitizers through synergistic activity and further may be valuable references for sanitation to compete the cycle of good hygiene practices in a proper CIP/SIP process.     

Behavior of Listeria monocytogenes in a Pseudomonas putida biofilm on a condensate-forming surface

Listeria monocytogenes has been isolated from condensate-forming surfaces in food processing plants. The objective of this research was to observe the behavior of L. monocytogenes on condensate-covered stainless steel with a Pseudomonas putida biofilm. L. monocytogenes-containing biofilms, either with or without added chicken protein, were incubated in a high humidity chamber at 12 degrees C to allow formation of condensate. Samples were analyzed for attached and unattached L. monocytogenes and total plate count periodically for 35 days. Samples were also taken for microscopic observation of Listeria and bacterial extracellular polymeric substances (EPS). L. monocytogenes attached in significantly greater numbers (> 3-log difference) to surfaces with preexisting P. putida biofilms than to Pseudomonas-free surfaces. L. monocytogenes survived in the presence or absence of P. putida with no added nutrients for 35 days, with numbers of survivors in the range of 3 to 4 log CFU/cm2 in the presence of P. putida and less than 2.9 log CFU/cm2 in pure culture. Attached and unattached L. monocytogenes were at similar levels throughout the incubation under all conditions studied. The addition of protein to the biofilms allowed growth of L. monocytogenes in pure culture during the first 7 days of incubation. Numbers of L. monocytogenes were not affected by the presence of P. putida when protein was present. Unattached L. monocytogenes were at levels of 3.6 to 6.7 log CFU/cm2 on the protein-containing surfaces. Microscopic observation of the condensate-covered biofilms indicated that L. monocytogenes formed microcolonies embedded within an EPS matrix over a 28-day period. This research demonstrates that L. monocytogenes can survive on condensate-forming stainless steel in low and high nutrient conditions, with or without the presence of Pseudomonas biofilm. The Listeria can detach and, therefore, have the potential to contaminate product.     

Effect of enterocin AS-48 in combination with biocides on planktonic and sessile Listeria monocytogenes

Enterocin AS-48 was tested on a cocktail of Listeria monocytogenes strains in planktonic and sessile states, singly or in combination with biocides benzalkonium chloride, cetrimide, hexadecylpyridinium chloride, didecyldimethylammonium bromide, triclosan, poly-(hexamethylen guanidinium) hydrochloride, chlorhexidine, hexachlorophene, and the commercial sanitizers P3 oxonia and P3 topax 66. Combinations of sub-inhibitory bacteriocin concentrations and biocide concentrations 4 to 10-fold lower than their minimum inhibitory concentrations (MIC) completely inhibited growth of the planktonic listeriae. Inactivation of Listeria in biofilms formed on polystyrene microtiter plates required concentrations of enterocin AS-48 greater than 50 μg/ml, and biocide concentrations ten to 100-fold higher. In combination with enterocin AS-48 (25 or 50 μg/ml), microbial inactivation increased remarkably for all biocides except P3 oxonia and P3 topax 66 solutions. Polystyrene microtiter plates conditioned with enterocin solutions (0.5-25 μg/ml) decreased the adherence and biofilm formation of the L. monocytogenes cell cocktail, avoiding biofilm formation for at least 24 h at a bacteriocin concentration of 25 μg/ml.     

Biofilm Formation in Milk Production and Processing Environments; Influence on Milk Quality and Safety

Abstract: Bacteria in milk have the ability to adhere and aggregate on stainless steel surfaces, resulting in biofilm formation in milk storage tanks and milk process lines. Growth of biofilms in milk processing environments leads to increased opportunity for microbial contamination of the processed dairy products. These biofilms may contain spoilage and pathogenic microorganisms. Bacteria within biofilms are protected from sanitizers due to multispecies cooperation and the presence of extracellular polymeric substances, by which their survival and subsequent contamination of processed milk products is promoted. This paper reviews the most critical factors in biofilm formation, with special attention to pseudomonads, the predominant spoilage bacteria originating from raw milk. Biofilm interactions between pseudomonads and milk pathogens are also addressed, as emerging risks and future research perspectives, specifically related to the milk processing environment.     

Resistance to benzalkonium chloride, peracetic acid and nisin during formation of mature biofilms by Listeria monocytogenes

Increase of resistance to the application of benzalkonium chloride (BAC), peracetic acid (PA) and nisin during biofilm formation at 25 °C by three strains of Listeria monocytogenes (CECT 911, CECT 4032, CECT 5873 and BAC-adapted CECT 5873) in different scenarios was compared. For this purpose, resistance after 4 and 11-days of biofilm formation was quantified in terms of lethal dose 90% values (LD₉₀), determined according with a dose-response logistic mathematical model. Microscopic analyses after 4 and 11-days of L. monocytogenes biofilm formation were also carried out. Results demonstrated a relation between the microscopic structure and the resistance to the assayed biocides in matured biofilms. The worst cases being biofilms formed by the strain 4032 (in both stainless steel and polypropylene), which showed a complex “cloud-type” structure that correlates with the highest resistance of this strain against the three biocides during biofilm maturation. However, that increase in resistance and complexity appeared not to be dependent on initial bacterial adherence, thus indicating mature biofilms rather than planctonic cells or early-stage biofilms must be considered when disinfection protocols have to be optimized. PA seemed to be the most effective of the three disinfectants used for biofilms. We hypothesized both its high oxidizing capacity and low molecular size could suppose an advantage for its penetration inside the biofilm. We also demonstrated that organic material counteract with the biocides, thus indicating the importance of improving cleaning protocols. Finally, by comparing strains 5873 and 5873 adapted to BAC, several adaptative cross-responses between BAC and nisin or peracetic acid were identified.     

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.     

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.     

Current and Recent Advanced Strategies for Combating Biofilms

Biofilms are matrix‐enclosed microbial aggregates that adhere to a biological or nonbiological surface. Biofilm formation is a significant problem in the medical, food, and marine industries and can lead to substantial economic and health problems. The complex microbial community of a biofilm is highly resistant to antibiotics and sanitizers and confers persistent survival that is a challenge to overcome. There are several conventional approaches to combating biofilms, physical and/or mechanical removal, chemical removal, and the use of antimicrobials, sanitizers, or disinfectants to kill biofilm organisms. However, biofilms are highly resistant to these approaches as opposed to planktonic cells. Thus, novel approaches other than the conventional methods are urgently needed. In this review, we discuss current and new advanced antibiofilm strategies that are superior to the conventional method in terms of addressing the biofilm problem for the improvement of healthcare, food safety, and in industrial processes.     

Assessing biofilm formation by Listeria monocytogenes strains

When a microtitre plate assay was used to quantify biofilm production by Listeria monocytogenes strains following growth in Tryptone Soy Broth (TSB) for 48 h at 20 degrees C, 127 of 138 strains (92.0%) were classified as weak, 9 of 138 strains (6.5%) as moderate and only 2 of 138 strains (1.5%) as strong biofilm formers. The strains included environmental, animal, food (persistent and sporadic strains) and clinical isolates previously typed using esterase electrophoresis (ESE) and multi-locus enzyme electrophoresis (MEE). Strains from different sources produced similar quantities of biofilm, whereas biofilm production by ESE type II strains, irrespective of source, was greater than that observed for other ESE types. No correlation between MEE type and biofilm production was observed. A Petri dish assay which allowed parallel quantification and microscopic examination of biofilms was used to examine biofilm formation by selected L. monocytogenes strains during growth in TSB for 14 days at 20 degrees C. Results from these assays showed that following prolonged incubation, some L. monocytogenes strains categorized as weak biofilm formers by the 48 h microtitre assay, were able to form biofilms similar in terms of quantity and structure to those produced by strains classified as strong or medium biofilm formers. Results from 14-day Petri dish assays confirmed 48 h microtitre assays regarding greater biofilm production by ESE type II strains compared to other ESE types of L. monocytogenes. Biofilm production was similar for ESE type II persistent and sporadic food isolates but reduced for ESE type II clinical strains.