Listeria Monocytogenes Biofilm Research Articles

Variability of RNA Quality Extracted from Biofilms of Foodborne Pathogens Using Different Kits Impacts mRNA Quantification by qPCR

The biofilm formation by foodborne pathogens is known to increase the problem related with surface disinfection procedure in the food processing environment and consequent transmission of these pathogens into the population. Messenger RNA has been increasingly used to understand the action and the consequences of disinfectants in the virulence on such biofilms. RNA quality is an important requirement for any RNA-based analysis since the quality can impair the mRNA quantification. Therefore, we evaluated five different RNA extraction kits using biofilms of the foodborne pathogens Listeria monocytogenes, Escherichia coli, and Salmonella enterica. The five kits yielded RNA with different quantities and qualities. While for E. coli the variability of RNA quality did not affect the quantification of mRNA, the same was not true for L. monocytogenes or S. enterica. Therefore, our results indicate that not all kits are suitable for RNA extraction from bacterial biofilms, and thus, the selection of RNA extraction kit is crucial to obtain accurate and meaningful mRNA quantification.

Inactivation of Listeria monocytogenes on ready-to-eat food processing equipment by chlorine dioxide gas

The effectiveness of chlorine dioxide (ClO2) gas to control Listeria contamination on food contact and environmental surfaces was investigated in order to comply with the zero tolerance policy. Different ClO2 gas concentrations (0.3, 0.5, 1 and 2 mg/l) were evaluated in order to inactivate Listeria monocytogenes (planktonic cells and biofilms) on stainless steel coupons. An ideal condition was then selected, and its applicability was studied on a commercial meat slicer and an industrial hot-dog peeler (using a surrogate organism). L. monocytogenes biofilm cells initially showed more sensitivity to ClO2 treatments, as compared to planktonic cells, but after 10 min, ∼4 log CFU/cm2 reduction was observed for all the concentrations used. The treatment at 2 mg/l for 30 min was selected for the further validation study. Complete pathogen inactivation, >5 log CFU/cm2, was obtained on both the meat slicer and peeler, demonstrating the potential applicability of ClO2 gas as a sanitizing agent for RTE meat processing equipment.

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.

Identification of genes involved in Listeria monocytogenes biofilm formation by mariner-based transposon mutagenesis

Listeria monocytogenes is a ubiquitous food-borne pathogen, whose distribution and survival in food-processing environments are associated with the ability to form biofilms. The process of biofilm formation is complex and its molecular mechanism is relatively poorly understood in L. monocytogenes. To better understand the genetics of this process, a mariner-based transposon mutagenesis strategy was used to identify genes involved in biofilm formation of L. monocytogenes. A library of 6,500 mutant colonies was screened for reduced biofilm formation using a microtiter plate biofilm assay. Forty biofilm-deficient mutants of L. monocytogenes were identified based on DNA sequences of the transposon-flanking regions and Southern hybridization with a transposon-based probe. The insertions harbored by these mutants led to the identification of 24 distinct loci, 18 of which, to our knowledge, have not been previously reported to function in the biofilm formation in L. monocytogenes. Genetic complementation confirmed the importance of lmo1386, a gene encoding a putative DNA translocase, for biofilm formation. Molecular analyses of mutants indicated that the majority of the 24 identified genes are related to flagella motility, gene regulation, and cell surface structures.

Activity of South African medicinal plants against Listeria monocytogenes biofilms, and isolation of active compounds from Acacia karroo

In South Africa, the antimicrobial activity of many indigenous plants has been investigated. In general, studies have focused on planktonic bacteria, with less attention given to bacterial biofilms. Many organisms, however, including the opportunistic pathogen Listeria monocytogenes occur more frequently as biofilms. The aim of this study was to identify and select plants that exhibit the best antilisterial activity, isolate the bioactive compounds and determine their effect on the architecture of listerial biofilms. The ethyl acetate and chloroform extracts of thirteen plants were investigated for antilisterial activity. The ethyl acetate extract of Acacia karroo and Plectranthus ecklonii showed the best antilisterial activity, exhibiting a minimum inhibitory concentration (MIC) of 3.1 mg/ml and 0.5 mg/ml, respectively. These were further selected for the identification of bioactive compounds. Column chromatographic purification of the ethyl acetate extracts of the leaves of A. karroo led to the isolation of three known pure compounds, namely epicatechin ( 1), β-sitosterol ( 2) and epigallocatechin ( 3). Confocal scanning laser microscopy (CSLM) showed that the biomass of the listerial biofilm was reduced when the isolated compounds were added. The aggregation of cells that were exposed to β-sitosterol and epigallocatechin was reduced from 25 μm as observed in untreated cells to < 10 μm in length.

Effect of neutral electrolyzed water and nisin on Listeria monocytogenes biofilms, and on listeriolysin O activity

All food processing surfaces are potential sites for biofilm formation of foodborne pathogens, which may result in increased virulence and better adaptation to survival in foods. This study was aimed to evaluate the effect of two antimicrobials, neutral electrolyzed water (NEW) and nisin, and their combination, on Listeria monocytogenes Scott A biofilms formed on glass and stainless steel surfaces. We also examined the effects of sub-lethal doses of NEW on listeriolysin O (LLO) activity from free and biofilm listerial cells. Coupons inoculated with L. monocytogenes cells were used to produce biofilms by incubation for four days at 37°C. An orthogonal experimental design with two replicates was used to test the effect of four factors on biofilm population. The factors were antimicrobial agents: NEW (65ppm), nisin (6976IU/per coupon), and their combination; temperature: 20°C and 37°C; contact time: 5, 10, 20 and 45min; and type of material: glass or stainless steel. Antimicrobial compounds and exposure time significantly affected L. monocytogenes populations in biofilms from both surfaces. A bactericidal effect was shown by NEW on free listerial cells at 30ppm for 0.5min of exposure, regardless treatment temperature. Same effect was observed on listerial biofilms at 65ppm or higher concentrations, after 10min contact time. A sub-lethal concentration of NEW acting on listerial biofilms resulted in an increased LLO activity, while non-treated biofilms exhibited a reduced activity, but higher than that found for free cells. The use of NEW as a sanitizer may be effective in reducing bacterial contamination. In addition because of its safety, which would benefit the food industry and its environmental friendliness, NEW may be of significant use in the food industry.

The effect of polyesterurethane belt surface roughness on Listeria monocytogenes biofilm formation and its cleaning efficiency

Contamination of surfaces in direct contact with food in the processing line is the major cause of finished product cross-contamination, especially conveyor belt surfaces that transfer food products. Polyesterurethane (PSU) is one of the materials widely used in conveyor belt systems; therefore, the objective of this study was to examine the effect of PSU conveyor belt surface roughness on the ability of biofilm formation by two strains of Listeria monocytogenes, i.e. ATCC19114 and ATCC51782. Additionally, the efficiency of a cleaning and sanitizing procedure adopted by a chicken meat plant in Thailand in eliminating biofilm formation on belt surfaces was assessed. The roughness values (Ra) were 0.05 ± 0.00 μm and 1.44 ± 0.01 μm for new (unused) and old conveyor belts (in use for 5 years), respectively. It was found that at 30 °C, both strains of L. monocytogenes formed robust biofilms regardless of differences in surface roughness. Conversely, at 15 °C, the ability of L. monocytogenes to form biofilms decreased with the lower Ra value. Under a simulated processing environment, cleaning and sanitizing reduced L. monocytogenes biofilms from both old and new conveyor belt surfaces to certain levels; however, a total reduction could not be achieved. In addition, more highly concentrated cleaning and sanitizing agents resulted in greater reduction. Results from this study clearly illustrate that surface roughness of material is also an important factor which may hinder effective cleaning and sanitizing. Therefore, the roughness value may be used as an indicator for evaluating the life of conveyor belts being used in the food industry.

Mixed species biofilms of Listeria monocytogenes and Lactobacillus plantarum show enhanced resistance to benzalkonium chloride and peracetic acid

We investigated the formation of single and mixed species biofilms of Listeria monocytogenes strains EGD-e and LR-991, with Lactobacillus plantarum WCFS1 as secondary species, and their resistance to the disinfectants benzalkonium chloride and peracetic acid. Modulation of growth, biofilm formation, and biofilm composition was achieved by addition of manganese sulfate and/or glucose to the BHI medium. Composition analyses of the mixed species biofilms using plate counts and fluorescence microscopy with dual fluorophores showed that mixed species biofilms were formed in BHI (total count, 8-9 log10 cfu/well) and that they contained 1-2 log10 cfu/well more L. monocytogenes than L. plantarum cells. Addition of manganese sulfate resulted in equal numbers of both species (total count, 8 log10 cfu/well) in the mixed species biofilm, while manganese sulfate in combination with glucose, resulted in 1-2 log10 more L. plantarum than L. monocytogenes cells (total count, 9 log10 cfu/well). Corresponding single species biofilms of L. monocytogenes and L. plantarum contained up to 9 log10 cfu/well. Subsequent disinfection treatments showed mixed species biofilms to be more resistant to treatments with the selected disinfectants. In BHI with additional manganese sulfate, both L. monocytogenes strains and L. plantarum grown in the mixed species biofilm showed less than 2 log10 cfu/well inactivation after exposure for 15min to 100μg/ml benzalkonium chloride, while single species biofilms of both L. monocytogenes strains showed 4.5 log10 cfu/well inactivation and single species biofilms of L. plantarum showed 3.3 log10 cfu/well inactivation. Our results indicate that L. monocytogenes and L. plantarum mixed species biofilms can be more resistant to disinfection treatments than single species biofilms.

Bioactivity of selected essential oils and some components on Listeria monocytogenes biofilms

Listeria monocytogenes is a Gram-positive bacterium, able to survive and grow in water, soil, agricultural products, various foods and the food-processing environment. The ubiquitous nature of the organism, coupled with its ability to colonise food-processing surfaces by forming biofilms, causes it to be of a major concern to the industry. Increased foodborne pathogen resistance and negative consumer perceptions regarding the use of synthetic bacteriocides, has resulted in natural antimicrobials being sourced from the plant kingdom. The listerial antibiofilm activities of Syzygium aromaticum (clove), Mentha spicata (spearmint), Lippia rehmannii and Cymbopogon citratus (lemongrass) essential oils and their major components were evaluated using the crystal violet assay and confocal scanning laser microscopy. Listerial biofilms treated with S. aromaticum or M. spicata essential oils, or the pure compounds nerol and citral, exhibited a similar biofilm biomass to the positive control. However, the essential oils of lemongrass, L. rehmannii, eugenol and R-carvone caused biofilm enhancement, rather than inhibition. L. rehmannii and lemongrass essential oils did not display any antibiofilm properties. Results obtained were confirmed by microscopic observations indicating either a reduction (inhibition) or an increase (enhancement) in biofilm biomass when exposed to the essential oils or pure compounds. The present study revealed that M. spicata and S. aromaticum essential oils as well as pure citral and nerol are good candidates for further development of ecofriendly disinfectants.

INTERACTION OF LISTERIA MONOCYTOGENES AND STAPHYLOCOCCUS EPIDERMIDIS IN DUAL SPECIES BIOFILMS

ABSTRACT The development of mixed biofilms of Listeria monocytogenes and Staphylococcus epidermidis was studied on two model surfaces. Growth tests at 30C for mono and mixed cultures of the two bacteria in defined Hsiang‐Ning Tsai Medium, in tryptic soy broth (TSB)+1% glucose, TSB + 0.6% yeast extract (TSBYE) and TSBYE + 1% glucose was performed. The optimal growth was seen in TSBYE + 1% glucose, and hence, biofilm formation in this medium was studied using a polystyrene microtiter plate assay. Mono and mixed cultures with different ratios of the two bacteria were assayed for (1) biofilm strength, expressed as the amount of cell‐bound crystal violet and (2) amount of matrix polysaccharide measured by ruthenium red staining. The mixture with a 40:60 ratio of L. monocytogenes to S. epidermidis yielded the highest values in both assays. The biofilm strength measured for this ratio indicated a synergistic interaction between the two bacterial species. Scanning electron microscopy of biofilms formed on cellulose membranes also revealed such an interaction.PRACTICAL APPLICATIONSWithin food processing environments, bacterial biofilms are of concern, in particular if they contain foodborne pathogens such as Listeria monocytogenes which may (re‐)contaminate food products. Our study shows that the intrinsically weak biofilm former L. monocytogenes strain EGD‐e, can form more robust biofilms in association with bacteria like Staphylococcus epidermidis, which as such are harmless in food, but form strong biofilms. This suggests that also such synergistically acting bacteria should be monitored and controlled.

Antibiofilm Effect of Trans-Cinnamaldehyde on Uropathogenic Escherichia coli

Urinary tract infections are the most common hospital acquired infections in humans, caused primarily by uropathogenic Escherichia coli. Indwelling urinary catheters for bladder drainage in humans become encrusted with uropathogenic E. coli biofilms that are resistant to common antibiotics, resulting in chronic infections. We studied the efficacy of the cinnamon ingredient trans-cinnamaldehyde (Sigma) for preventing uropathogenic E. coli biofilm. We also determined the efficacy of trans-cinnamaldehyde as an ingredient in catheter lock solution to inactivate preformed uropathogenic E. coli biofilm. Polystyrene plates and urinary catheters inoculated with uropathogenic E. coli (5 to 6.0 log cfu) were treated with trans-cinnamaldehyde (0%, 0.1%, 0.25% or 0.5%) at 37C. Catheters with uropathogenic E. coli biofilm were also treated with lock solution containing trans-cinnamaldehyde (0%, 1%, 1.25% or 1.5%). Uropathogenic E. coli biofilm on control and trans-cinnamaldehyde treated plates and catheters was determined on incubation days 0, 1, 3 and 5. Trans-cinnamaldehyde potential cytotoxity, if any, was determined in HTB-4 bladder epithelial cells (ATCC). At all concentrations trans-cinnamaldehyde effectively prevented uropathogenic E. coli biofilm on plates and catheters. As a constituent in catheter lock solution, it inactivated uropathogenic E. coli biofilm on catheters. Trans-cinnamaldehyde produced no cytotoxic effects on human bladder epithelial cells at the tested concentrations. Results suggest that trans-cinnamaldehyde may be applied as a catheter surface coating or as an ingredient in catheter lock solution to prevent urinary tract infection in humans.

Effect of Batch and Fed-Batch Growth Modes on Biofilm Formation by Listeria monocytogenes at Different Temperatures

The influence of Listeria monocytogenes (L. monocytogenes) biofilm formation feeding conditions (batch and fed-batch) at different temperatures on biofilm biomass and activity was determined. Biofilm biomass and cellular metabolic activity were assessed by Crystal Violet (CV) staining and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) colorimetric method, respectively. Live/Dead staining was also performed in order to get microscopic visualization of the different biofilms. Results revealed that at refrigeration temperature (4 degrees C) a higher amount of biofilm was produced when batch conditions were applied, while at higher temperatures the fed-batch feeding condition was the most effective on biofilm formation. Moreover, independently of the temperature used, biofilms formed under fed-batch conditions were metabolically more active than those formed in batch mode. In conclusion, this work shows that different growth modes significantly influence L. monocytogenes biofilm formation on abiotic surfaces as well as the metabolic activity of cells within biofilms.

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 μm) and electropolished stainless steel (Ra = 0.16 ± 0.06, 0.40 ± 0.003, and 0.67 ±.02 μm) 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°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 electro-polished 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 10μm 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.

The effect of five common essential oil components on Listeria monocytogenes biofilms

The effect of five essential oil components (EOC’s) on biofilms were investigated on two pathogenic Listeria monocytogenes isolates. Growth of the biofilm was assessed using the crystal violet (CV) staining assay and the XTT reduction assay. Scanning electron microscopy (SEM) was used to assess the architectural changes of the biofilm. Treatment of a 6 h preformed biofilm with each of the EOC’s at a concentration of 1 mg/ml enhanced growth of the biofilm which was confirmed by SEM. Conversely, the presence of the essential oil components caused a reduction in the metabolic activity of the biofilm as shown by the XTT reduction assay.

Effect of Biofilm Dryness on the Transfer of Listeria monocytogenes Biofilms Grown on Stainless Steel to Bologna and Hard Salami

Listeria monocytogenes continues to be a major cause of class I food recalls in the United States. Very little is known about its transfer and cross-contamination in processing scenarios. The objective of this study was to evaluate the effect of hydration level on L. monocytogenes biofilms grown on stainless steel and its effect on the biofilm transfer to foods. Biofilms were grown on stainless steel in diluted tryptic soy broth 1:20 for 48 h at 32°C. After this, biofilms were equilibrated over saturated salt solutions at 20°C for 24 h (94, 75, 58, and 33% relative humidity; % RH) prior to transferring. Transfer experiments were conducted from inoculated stainless steel to bologna and hard salami at a constant pressure (45 kPa) and time (30 s) with a universal testing machine. The experiment was designed with a factorial design 4 × 2 (biofilms equilibrated at 4% RH and two foods) and duplicated every day, and the whole experiment was repeated nine times. The results were analyzed with an analysis of variance by SAS Statistical Analysis Software. Our results showed that more bacteria were transferred to bologna (mean efficiency of transfer [EOT] = 3.0) than to hard salami (mean EOT = 0.35, P < 0.01). As biofilms became drier, the transfer of Listeria from stainless steel to both foods increased (P < 0.05). The EOT increased from 2 to 3.8 and from 0.2 to 0.51 upon transfer when drying the biofilm for bologna and hard salami, respectively. This study may be an indication that as biofilms were dried, the cell-cell and cell-surface interactions became weaker, and bacterial transfer increased. This phenomenon was enhanced in foods containing higher water activity levels. We hypothesize that this increased in transfer was due to the presence of capillary forces in the food.

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

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

Effect of Lactococcus lactis UQ2 and its bacteriocin on Listeria monocytogenes biofilms

Listeria monocytogenes is a foodborne pathogen of major concern in food processing due to its wide environmental distribution and ability to form biofilms. Lactococcus lactis UQ2 is a bacteriocinogenic strain isolated from Mexican-style cheese, genetically identified as nisin A. It was grown on supplemented whey medium, representing an alternative, and probably more economical way to obtain this bacteriocin. A spray-dried crude bacteriocin fermentate (CBF) of L. lactis UQ2, or L. lactis UQ2 cells were used to test their antimicrobial activity against planktonic and sessile cells of L. monocytogenes Scott A on stainless steel chips. L. lactis UQ2 reduced >5 log cycles of this pathogen biofilms per chip, in competitive-exclusion tests at 37 °C. A new approach was developed to evaluate the antagonic effect of L. lactis UQ2 which outnumbered Listeria cells, by using fluorescent in situ hybridization utilizing labeled specific probes. An in vitro study showed that the combination of the two main antimicrobials produced by L. lactis UQ2 (lactic acid and nisin A) can be more efficient to suppress L. monocytogenes growth than each one on its own. This study may provide information to develop strategies to suppress or control L. monocytogenes biofilms on stainless steel surfaces, one of the most common materials used for food processing equipment.

Chlorine Resistance of Listeria monocytogenes Biofilms and Relationship to Subtype, Cell Density, and Planktonic Cell Chlorine Resistance

Strains of Listeria monocytogenes vary in their ability to produce biofilms. This research determined if cell density, planktonic chlorine resistance, or subtype are associated with the resistance of L. monocytogenes biofilms to chlorine. Thirteen strains of L. monocytogenes were selected for this research based on biofilm accumulation on stainless steel and rep-PCR subtyping. These strains were challenged with chlorine to determine the resistance of individual strains of L. monocytogenes. Planktonic cells were exposed to 20 to 80 ppm sodium hypochlorite in 20 ppm increments for 5 min in triplicate per replication, and the experiment was replicated three times. The number of tubes with surviving L. monocytogenes was recorded for each isolate at each level of chlorine. Biofilms of each strain were grown on stainless steel coupons. The biofilms were exposed 60 ppm of sodium hypochlorite. When in planktonic culture, four strains were able to survive exposure to 40 ppm of chlorine, whereas four strains were able to survive 80 ppm of chlorine in at least one of three tubes. The remaining five strains survived exposure to 60 ppm of chlorine. Biofilms of 11 strains survived exposure to 60 ppm of chlorine. No association of biofilm chlorine resistance and planktonic chlorine resistance was observed; however, biofilm chorine resistance was similar for strains of the same subtype. Biofilm cell density was not associated with chlorine resistance. In addition, biofilms that survived chlorine treatment exhibited different biofilm morphologies. These data suggest that chlorine resistance mechanisms of planktonic cells and biofilms differ, with planktonic chlorine resistance being more affected by inducible traits, and biofilm chlorine resistance being more affected by traits not determined in this study.

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°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.

Generation of antifouling layers on stainless steel surfaces by plasma‐enhanced crosslinking of polyethylene glycol

AbstractPolyethylene glycol (PEG) structures were deposited onto stainless steel (SS) surfaces by spin coating and argon radio frequency (RF)‐plasma mediated crosslinking. Electron spectroscopy for chemical analysis (ESCA) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR‐FTIR) indicated the presence of CH2CH2O structure and CCC linkage, as a result of the plasma crosslinking, on PEG‐modified SS surfaces. Scanning electron microscopy (SEM) indicated complete deposition, and water contact angle analysis revealed higher hydrophilicity on PEG‐modified surfaces compared to unmodified SS surfaces. Surface morphology and roughness analysis by atomic force microscopy (AFM) revealed smoother SS surfaces after PEG modification. The evaluation of antifouling ability of the PEG‐modified SS surfaces was carried out. Compared to the unmodified SS, PEG‐modified surfaces showed about 81–96% decrease in Listeria monocytogenes attachment and biofilm formation (p &lt; 0.05). This cold plasma mediated PEG crosslinking provided a promising technique to reduce bacterial contamination on surfaces encountered in food‐processing environments. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 485–497, 2005