Influence of peroxyacetic acid and nisin and coculture with Enterococcus faecium on Listeria monocytogenes biofilm formation

Biofilm formation is a matter of concern in food industries because biofilms facilitate the survival of pathogenic bacteria such as Listeria monocytogenes, which may contaminate food-processing equipment and products. In this study, nisin and two Enterococcus faecium strains were evaluated for their effect on biofilm formation by L. monocytogenes cultured in brain heart infusion broth and on stainless steel coupons. Elimination of preformed L. monocytogenes biofilms by peroxyacetic acid also was tested. Adhesion control experiments were performed with pure cultures of L. monocytogenes after swab collection of adhered cells, which were then enumerated on PALCAM agar plates and visualized by scanning electron microscopy. Formation of a biofilm was recorded when the number of adhered cells was at least 10(3) CFU/cm2. When L. monocytogenes was cocultured with E. faecium bac-, the number of adhered L. monocytogenes cells was 2.5 log lower (P = 0.002) when initially compared with the control culture, but after 6 h of incubation a biofilm was again detected. However, in coculture on stainless steel coupons, E. faecium bac+ inhibited L. monocytogenes adherence and did not allow biofilm formation for up to 48 h (P < 0.001). In the presence of nisin or after treatment with peroxyacetic acid, bacterial growth was reduced (P < 0.001) up to 4.6 and 5.6 log CFU/cm2, respectively, when compared with L. monocytogenes cultures on untreated coupons. However, after these treatments, cells were still present, and after 24 h of incubation, a renewed biofilm was detected in L. monocytogenes cultures treated with nisin. Although all tested conditions reduced L. monocytogenes growth to some extent, only coculture with E. faecium bac+ efficiently reduced biofilm formation, suggesting a potential control strategy for this pathogen.     

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.     

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.     

Interactions in biofilms between Listeria monocytogenes and resident microorganisms from food industry premises

Twenty nine bacterial strains were grown as binary culture biofilms with Listeria monocytogenes to assess their influence on the settlement of the latter on stainless steel coupons. Most of the strains had been isolated from food processing plants after cleaning and disinfection and were tentatively identified by the APILAB Plus 3.3.3 database (bioMerieux). Sixteen of them decreased L. monocytogenes biofilm colony forming units (CFU) counts. Three strains, Bacillus sp. CCL 9 an unidentified Gram-positive strain CCL 59 and Pseudomonas fluorescens E9. 1, led to a 3-log difference in CFU counts between the pure L. monocytogenes biofilms and the mixed biofilms. Eleven strains had no effect and only four, Kocuria varians CCL 73, Staphylococcus capitis CCL 54, Stenotrophomonas maltophilia CCL 47 and Comamonas testosteroni CCL 24, had a positive effect, with a 0.5- to 1.0-log increase in the L. monocytogenes biofilm CFU counts. On its own, L. monocytogenes settled as single cells, but in binary biofilms, different spatial arrangements were observed: (i) with K. varians CCL 73, K. varians CCL 56 and S. capitis CCL 54, L. monocytogenes cells gathered around the microcolonies of the partner strain; (ii) with the two Gram-negative strains, C. testosteroni CCL 24 and CCL 25, L. monocytogenes cells formed its own microcolonies. No link could be found between the exopolysaccharide production capacity of the bacterial strains in pure-culture biofilms and their effect on the L. monocytogenes population in mixed biofilms. With one strain, C. testosteroni CCL 24, adding filter-sterilized supernatant from a pure-culture biofilm to a pure culture of L. monocytogenes increased the number of L. monocytogenes cells adhering to the stainless steel coupons and forming microcolonies. This study suggests that the "house flora" can have a strong effect on the likelihood of finding L. monocytogenes on inert surfaces.     

Evaluation of Methods To Assess the Biofilm-Forming Ability of Listeria monocytogenes

The contamination of ready-to-eat products with Listeria monocytogenes has been related to the presence of biofilms in production lines, as biofilms protect cells from chemical sanitizers. The ability of L. monocytogenes to produce biofilms is often evaluated using in vitro methodologies. This work aims to compare the most frequently used methodologies, including high-throughput screening methods based on microplates (crystal violet and the Calgary Biofilm Device) and methods based on CFU enumeration and microscopy after growth on stainless steel. Thirty isolates with diverse origins and genetic characteristics were evaluated. No (or low) correlations between methods were observed. The only significant correlation was found between the methods using stainless steel. No statistically significant correlation (P > 0.05) was detected among genetic lineage, serovar, and biofilm-forming ability. Because results indicate that biofilm formation is influenced by the surface material, the extrapolation of results from high-throughput methods using microplates to more industrially relevant surfaces should be undertaken with caution.     

Surface material, temperature, and soil effects on the survival of selected foodborne pathogens in the presence of condensate

The effects of surface type (stainless steel, acetal resin, and fiberglass reinforced plastic wall paneling [FRP]), soil, and temperature on the survival of Listeria monocytogenes, Salmonella spp., and Yersinia enterocolitica, in the presence of condensate were evaluated. Surface coupons--half soiled with sterile porcine serum--were exposed to cell suspensions made from individual five-strain cocktails composed of organisms from the same genus (10(7) CFU/ml) in Butterfield's phosphate buffer and incubated for 2 h at 25 degrees C allowing attachment of cells to coupon surfaces. Coupons were rinsed to remove unattached cells, incubated at either 4 or 10 degrees C under condensate-forming conditions, and sampled at six time intervals over a 15-day period. For enumeration, cells were removed from the coupons by vigorous shaking in 100 ml of Butterfield's phosphate buffer with 3 g of glass beads and plated on tryptic soy agar with 0.6% yeast extract. Stainless steel did not support the survival of Listeria as well as acetal resin or FRP. Acetal resin and stainless steel were less supportive of Salmonella than FRP. All surfaces supported the survival of Yersinia over the 15-day trial equally. Temperature had little effect on survival of all organisms across all surfaces with one exception. However, Yersinia displayed growth on FRP at 10 degrees C. but death at 4 degrees C. Serum had a protective effect on L. monocytogenes on all surfaces, with populations sustained at significantly (P < or = 0.05) higher numbers over time than unsoiled coupons. Serum didnot effect survival of Salmonella or Yersinia on stainless steel, acetal resin, or FRP.     

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.     

Effect of surface roughness and stainless steel finish on Listeria monocytogenes attachment and biofilm formation

The purpose of this study was to evaluate the effect of surface roughness (Ra) and finish of mechanically polished stainless steel (Ra = 0.26 +/- 0.05, 0.49 +/- 0.10, and 0.69 +/- 0.05 microm) and electropolished stainless steel (Ra = 0.16 +/- 0.06, 0.40 +/- 0.003, and 0.67 +/- 0.02 microm) on Listeria adhesion and biofilm formation. A four-strain cocktail of Listeria monocytogenes was used. Each strain (0.1%) was added to 200 ml of tryptic soy broth (TSB), and coupons were inserted to the mixture for 5 min. For biofilm formation, coupons with adhesive cells were incubated in 1:20 diluted TSB at 32 degrees C for 48 h. The experiment was performed by a randomized block design. Our results show that the level of Listeria present after 48 h of incubation (mean = 7 log CFU/cm2) was significantly higher than after 5 min (mean = 6.0 log CFU/cm2) (P < 0.01). No differences in initial adhesion were seen in mechanically finished (mean = 6.7 log CFU/cm2) when compared with electropolished stainless steel (mean = 6.7 log CFU/cm2) (P > 0.05). Listeria initial adhesion (values ranged from 5.9 to 6.1 log CFU/cm2) or biofilm formation (values ranged from 6.9 to 7.2 log CFU/cm2) was not significantly correlated with Ra values (P > 0.05). Image analysis with an atomic force microscope showed that bacteria did not colonize the complete surface after 48 h but were individual cells or grouped in microcolonies that ranged from 5 to 10 microm in diameter and one to three cell layers in thickness. Exopolymeric substances were observed to be associated with the colonies. According to our results, electropolishing stainless steel does not pose a significant advantage for food sanitation over mechanically finished stainless steel.     

Effects of epiphytic Enterobacteriaceae and pseudomonads on the growth of Listeria monocytogenes in model media

Four Enterobacteriaceae (Enterobacter agglomerans and Rhanella aquatilis) and six pseudomonads (Pseudomonas fluorescens, Pseudomonas chlororaphis, Pseudomonas putida) isolated from minimally processed green endive were coinoculated at 10 degrees C with Listeria monocytogenes in a minimal medium. Pseudomonads did not modify the growth of L. monocytogenes, whereas Enterobacteriaceae reduced its maximal population by 2 to 3 log CFU/ml. The same effect was observed in a diluted yeast extract medium supplemented with amino acids and glucose, in which L. monocytogenes grown alone reached 10(9) to 10(10) CFU/ml. In the same diluted yeast extract medium, not supplemented with glucose and amino acids, the maximal population of L. monocytogenes in the presence of both Enterobacteriaceae and pseudomonads was only slightly reduced (less than 0.5 log CFU/ml). Culture filtrates of the Enterobacteriaceae had no inhibitory activity on L. monocytogenes. The effect of the Enterobacteriaceae on L. monocytogenes growth was presumably due to a competition for glucose and/or amino acids.     

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.     

Reduction of Listeria monocytogenes in queso fresco cheese by a combination of listericidal and listeriostatic GRAS antimicrobials

Single and combined effects of three GRAS (generally recognized as safe) antimicrobials including, bacteriophage P100 (phage P100), lauric arginate (LAE), and potassium lactate-sodium diacetate mixture (PL-SD) were evaluated against Listeria monocytogenes cold growth in queso fresco cheese (QFC). The fate of phage P100 when exposed to LAE (200 ppm) or PL-SD (2.8% PL and 0.2% SD) was determined at 4°C and 30°C in a broth model. Phage P100 was found to be stable in the presence of these antimicrobial agents as plaque forming units (PFU) did not vary between control, LAE or PL-SD treatments. When 9 log CFU/ml of stationary phase cells of L. monocytogenes was exposed to these antimicrobials in tryptic soy broth, there was a 3 to 5 log CFU/ml reduction with phage P100 and a complete 9 log CFU/ml reduction with LAE but no measurable reduction with PL-SD after 24h at 4°C or 30°C. In QFC, the L. monocytogenes populations increased from the initial 3.5 log CFU/cm(2) to 7.7 log CFU/cm(2) in 28 days at 4°C. Treatment with 7.8 log PFU/cm(2) of phage P100 or 200 ppm of LAE showed strong listericidal effect initially by reducing L. monocytogenes counts by 2 to 3.5-4 log CFU/cm(2) while there was a subsequent regrowth of L. monocytogenes at 4°C. Treatment with PL-SD showed strong listeriostatic effect without decreasing L. monocytogenes counts but growth was prevented for 28 days at 4°C. Only the combined treatment of listericidal phage P100 or LAE with listeriostatic PL-SD reduced the initial L. monocytogenes counts by 2-4 log CFU/cm(2) and also kept the L. monocytogenes counts at that reduced level in QFC for 28 days at 4°C.     

Formation of biofilms by Listeria monocytogenes under various growth conditions

Eight strains of Listeria monocytogenes (7644, 19112, 15313, Scott A, LCDC, 10403S, SLCC, and 1370) produce biofilms when grown on polyvinyl chloride microtiter well plates. The growth medium (tryptic soy broth [TSB] or modified Welshimer's broth [MWB] at 32 degrees C) influenced the amount of biofilm formed; maximum biofilms were formed in MWB by six strains and in TSB by the remaining two strains. This result suggests that the growth medium is critical in development of L. monocytogenes biofilm. This organism also produced biofilms on stainless steel chips. Biofilm formation on these chips was observed following growth in TSB at 4, 20, and 37 degrees C. After 20 h of incubation at 20 or 37 degrees C, the cell density was approximately 10(6) CFU per chip, and after 4 days incubation at 4 degrees C, the cell density was 10(5) CFU per chip. L. monocytogenes strain Scott A biofilm formation on stainless steel chips was visualized using scanning electron microscopy, which revealed dense aggregates of cells held together by meshlike webbing.     

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.     

Temperature and biological soil effects on the survival of selected foodborne pathogens on a mortar surface

The survival of three foodborne pathogens (Listeria monocytogenes, Yersinia enterocolitica, and Salmonella) attached to mortar surfaces, with or without biological soil (porcine serum) and incubated at either 4 or 10 degrees C in the presence of condensate, was evaluated. Soiled and unsoiled coupons were inoculated by immersion into a five-strain cocktail (approximately 10(7) CFU/ml) of each organism type and evaluated. Coupons were incubated at 25 degrees C for 2 h to allow attachment of cells, rinsed to remove unattached cells, and incubated at either 4 or 10 degrees C at high humidity to create condensate on the surface. Sonication was used to remove the attached cells, and bacteria (CFU per coupon) was determined at 9 to 10 sampling periods over 120 h. Yersinia populations decreased more than 5 log units in the presence of serum in a 24-h period. Listeria and Salmonella had better survival on mortar in the presence of serum than Yersinia throughout the 120-h incubation period. Populations of L. monocytogenes declined more rapidly at 10 than at 4 degree C after 24 h. In general, differences in temperature did not affect the survival of Salmonella or Yersinia. Serum had a protective effect on the survival of all three organisms, sustaining populations at significantly (P < or = 0.05) higher numbers over time than on corresponding unsoiled coupons. There were no significant differences (P > 0.05) among the mean number (CFU per coupon) of L. monocytogenes, Y. enterocolitica, or Salmonella on initial attachment onto the mortar surfaces (unsoiled). The results indicate relatively rapid destruction of selected pathogenic bacteria on unsoiled mortar surfaces compared with those that contained biological soil, thus highlighting the need for effective cleaning to reduce harborage of these microbes in the food factory environment.     

Comparative evaluation of adhesion and biofilm formation of different Listeria monocytogenes strains

Thirteen Listeria monocytogenes strains were used to grow biofilms on glass surfaces in static conditions at 37 degrees C for up to 4 days. After the initial 3-h adhesion and in subsequent 1-day intervals, cell numbers were determined using standard plate count after swabbing the cells from the glass surface. The three-dimensional structure of in situ biofilms was determined by confocal scanning laser microscopy (CSLM). After 3 h incubation, bacterial cells for all 13 strains of L. monocytogenes were found attached to glass slides and all strains formed biofilms within 24 h. The strains varied significantly in their ability to adhere to the surface and significant differences for cell numbers after 24 h biofilm growth were found. Cell counts in biofilms formed by five L. monocytogenes strains were monitored over 4 days. The counts increased for the first 2 days reaching 10(5) cfu/cm2, except for L. monocytogenes 7148 (10(4) cfu/cm2). After 2 days, cell counts remained at 10(5) cfu/cm2 for four strains (tested on days 3 and 4), while L. monocytogenes 7148 continued to grow and reached 10(5) cfu/cm2 on day 4. This difference in biofilm growth was not related to variations in growth rates of planktonic cells suggesting that growth behaviour of Listeria in biofilms may be different from their planktonic growth. CSLM revealed that the biofilms grown under static conditions consisted of two distinct layers with 0.5 log10 higher cell numbers in the bottom layer as compared to the upper layer.     

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.     

Characteristics of microbial biofilm on wooden vats ('gerles') in PDO Salers cheese

The purpose of this study was to characterize microbial biofilms from 'gerles' (wooden vats for making PDO Salers cheese) and identify their role in milk inoculation and in preventing pathogen development. Gerles from ten farms producing PDO Salers cheese were subjected to microbial analysis during at least 4 periods spread over two years. They were distinguished by their levels of Lactobacillus (between 4.50 and 6.01 log CFU/cm(2)), Gram negative bacteria (between 1.45 and 4.56 log CFU/cm(2)), yeasts (between 2.91 and 5.57 log CFU/cm(2)), and moulds (between 1.72 and 4.52 log CFU/cm(2)). They were then classed into 4 groups according their microbial characteristics. These 4 groups were characterized by different milk inoculations (with either sour whey or starter culture, daily or not), and different washing procedures (with water or whey from cheese making). The farm gerles were not contaminated by Salmonella, Listeria monocytogenes or Staphylococcus aureus. Only one slight, punctual contamination was found on one gerle among the ten studied. Even when the milk was deliberately contaminated with L. monocytogenes and S. aureus in the 40 L experimental gerles, these pathogens were found neither on the gerle surfaces nor in the cheeses. Using 40 L experimental gerles it was shown that the microbial biofilms on the gerle surfaces formed in less than one week and then remained stable. They were mainly composed of a great diversity of lactic acid bacteria (Leuconostoc pseudomesenteroides, Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus hilgardii,…), Gram positive catalase positive bacteria (Curtobacterium flaccumfaciens, Curtobacterium oceanosedimentum Citrococcus spp., Brachybacterium rhamnosum, Kocuria rhizophila, Arthrobacter spp.…) and yeast (Kluyveromyces lactis, Kluyveromyces marxianus). In less than 1 min, even in a 500 L farm gerle, the gerle's microbial biofilm can inoculate pasteurized milk with micro-organisms at levels superior to those in raw milk.     

Efficacy of cetylpyridinium chloride against Listeria monocytogenes and its influence on color and texture of cooked roast beef

Sliced (cut) and exterior (intact) surfaces of restructured cooked roast beef were inoculated with Listeria monocytogenes, treated with cetylpyridinium chloride (CPC; immersion in 500 ml of 1% solution for 1 min), individually vacuum packaged, and stored for 42 days at 0 or 4 degrees C. Noninoculated samples were similarly treated, packaged, and stored to determine effects on quality (color and firmness) and on naturally occurring bacterial populations, including aerobic plate counts and lactic acid bacteria. Immediately after CPC treatment, regardless of inoculation level, L. monocytogenes populations were reduced (P = 0.05) by about 2 log CFU/cm2 on sliced surfaces and by about 4 log CFU/cm2 on exterior surfaces. Throughout 42 days of refrigerated storage (at both 0 and 4 degrees C), L. monocytogenes populations on CPC-treated samples remained lower (P = 0.05) than those of nontreated samples for both surface types. After 42 days of storage at both 0 and 4 degrees C, aerobic plate count and lactic acid bacteria populations of treated samples were 1 to 1.5 log CFU/cm2 lower (P = 0.05) than those of nontreated samples for both surface types. CPC treatment resulted in negligible effects (P > 0.05) on the color (L*, a*, and b* values) of exterior and sliced roast beef surfaces during storage. For both sliced and exterior surfaces, CPC-treated samples were generally less firm than nontreated samples. CPC treatment effectively reduced L. monocytogenes populations on roast beef surfaces and resulted in relatively minor impacts on color and texture attributes. CPC treatment, especially when applied to products prior to slicing, may serve as an effective antimicrobial intervention for ready-to-eat meat products.     

Transfer of surface-dried Listeria monocytogenes from stainless steel knife blades to roast turkey breast

Listeria contamination of food contact surfaces can lead to cross-contamination of ready-to-eat foods in delicatessens. Recognizing that variations in Listeria biofilm-forming ability exist, the goal of this study was to determine whether these differences in biofilm formation would affect the Listeria transfer rate during slicing of delicatessen turkey meat. In this study, six previously identified strong and weak biofilm-forming strains of Listeria monocytogenes were grown at 22 degrees C for 48 h on Trypticase soy agar containing 0.6% yeast extract and harvested in 0.1% peptone. Thereafter, the strains were combined to obtain two 3-strain cocktails, resuspended in turkey slurry, and inoculated onto flame-sterilized AISI grade 304 stainless steel knife blades that were subjected to 6 and 24 h of ambient storage at approximately 78% relative humidity. After mounting on an Instron Universal Testing Machine, these blades were used to obtain 16 slices of retail roast turkey breast. Based on an analysis of the slices by direct plating, Listeria populations decreased 3 to 5 log CFU per slice after 16 slices. Overall, total transfer to turkey was significantly greater for strong (4.4 log CFU total) as opposed to weak (3.5 log CFU total; P < 0.05) biofilm formers. In addition, significantly more cells were transferred at 6 (4.6 log CFU total) than at 24 h (3.3 log CFU total; P < 0.05) with Listeria quantifiable to the 16th slice, regardless of the inoculation level. Increased survival by the strong biofilm formers, as evidenced by viability staining, suggests that these strains are better adapted to survive stressful conditions than their weak biofilm-forming counterparts.