Bacterial biofilms in food processing environments: a review of recent developments in chemical and biological control

Biofilms are immobile communities of micro‐organisms attached to any surface, such as stainless steel or a food matrix surface or on packaging material. They may be composed of a single species, but more generally, in the natural environment, they consist of mixed species together with an extracellular matrix. Biofilms provide a common mechanism of persistence for a number of bacterial species especially in food processing environments, and therefore, prevention of biofilm formation and the removal of preformed biofilms are an important issue for the food industry. This article reviews the current understanding on the formation of biofilms and recent developments in biological and chemical methods for prevention and removal.     

丁香酚对解淀粉芽孢杆菌气液界面生物膜的抑制作用

为研究丁香酚对解淀粉芽孢杆菌DY1a生物膜的抑制作用及成膜早期界面黏附聚集能力的影响,分析了不同质量浓度的丁香酚对生物膜形成、生物膜表面微观结构、膜内活菌数及生物膜基质多糖和蛋白质含量的影响,并评价其对成熟生物膜的清除能力,通过细菌运动能力实验、细胞表面疏水性、细胞表面Zeta电位及细胞自聚集能力,综合分析丁香酚对生物膜形成早期界面黏附聚集能力的影响。结果表明:丁香酚对解淀粉芽孢杆菌的最低生物膜抑制质量浓度(MBIC)为1.500mg/mL,MBIC的丁香酚对成熟生物膜清除率为28.85%。添加1/2 MBIC、1/4 MBIC的丁香酚培养基气液界面形成的生物膜较为单薄,表面较为光滑平整,生物膜内活菌数显著降低。丁香酚对腐败菌泳动能力抑制率为22.16%~100.00%,对丛集能力的抑制率为43.86%~97.50%,使细胞表面疏水性、细胞表面负Zeta电位和自聚集率均降低。此外,丁香酚显著抑制了腐败菌胞外多糖和蛋白质合成。丁香酚对芽孢杆菌生物膜的抑制作用主要是通过抑制细菌运动能力,降低细胞表面的疏水性、自聚集性,从而干扰早期菌体在成膜界面上的黏附能力,并通过抑制胞外聚合物组分的合成分泌延迟生物膜的形成和成熟。丁香酚可作为抑制腐败解淀粉芽孢杆菌气液界面生物膜形成的潜在抗生物膜剂。     

Physiology and behavior of Pseudomonas fluorescens single and dual strain biofilms under diverse hydrodynamics stresses

Three selected Pseudomonas fluorescens strains (the type strain and two strains originally isolated from a dairy processing plant - D3-348 and D3-350) were used to form turbulent and laminar flow-generated biofilms under laboratorial conditions using flow cell reactors with stainless steel substrata. The D3-348 and D3-350 strains were also used to form dual biofilms. Biofilm phenotypic characteristics, such as respiratory activity, total and culturable cells, biomass, total and matrix proteins and polysaccharides were compared. Biofilm mechanical stability, as a major feature involved in biofilm persistence, was also assessed using a rotating device system. The results indicate that hydrodynamic conditions have a remarkable impact on biofilm phenotype. Turbulent biofilms were more active, had more mass per adhesion surface area, a higher number of total and culturable cells, a higher amount of total proteins per gram of biofilm, similar matrix proteins and identical (D3-348 and D3-350 single and dual biofilms) or smaller (type strain) total and matrix polysaccharides content than their laminar counterparts. Biofilms formed by the type strain revealed a considerable higher amount of total and culturable cells and a higher amount of total proteins (turbulent biofilms) and total and matrix polysaccharides per gram of biofilm than single and dual biofilms formed by the other strains. Mechanical stability assays disclosed that biofilms formed by both type and D3-348 strains had the highest resistance to removal when exposed to mechanical stress. Dual strain biofilms population analysis revealed an apparent co-existence, evidencing neutral interactions. The overall results provided useful information regarding a broad spectrum of P. fluorescens biofilm phenotypic parameters, which can contribute to control and model biofilm processes in food industry.     

Biofilm Formation Characteristics of Pseudomonas lundensis Isolated from Meat

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

Microscopic observation of multispecies biofilm of various structures on whey concentration membranes

The objective of this study was to evaluate biofilm formation on polyamide reverse osmosis (RO) whey concentration membranes. Biofilms were observed with scanning electron and fluorescence microscopy. For scanning electron microscopy, pieces of 6-, 12-, and 14-mo-old membranes were allowed to air dry at room temperature (22°C) for 24 h followed by sputter coating with a 5-nm layer of gold and microscopic observations. Scanning electron microscopy images revealed that the hydrophilic layer, used to prevent membrane plugging, was not evenly distributed on the surface. Although this hydrophilic layer seemed to prevent the attachment of proteins, it supported biofilm formation. Three different structures of multispecies biofilm were observed on the retentate side of the membrane: 1) a mono layer, 2) a 3-dimensional structure of a dense matrix of extracellular polymeric substances where different types of bacterial cells were embedded, and 3) cell aggregates. In some of the biofilms, a smooth layer (shell) covered cell aggregates. In the 6-mo-old membranes, part of the shell layer was broken off. Biofilms as observed on the RO membrane were described as having a hill-and-valley type of structure, with hills showing a mushroom-like appearance and valleys comprising dense matrices of extracellular polymers with embedded bacterial cells. Fluorescence microscopy showed live cells on the surface of the biofilm. It is concluded that both cells in the deep layers of biofilm and surface cells may resist cleaning and sanitation. The extent of biofilm formation and the presence of live cells on RO membranes after regular clean in place cycles indicate the need for a more effective cleaning regimen customized for dairy separation systems.     

A review of current and emergent biofilm control strategies

Microbial adhesion to surfaces and the consequent biofilm formation has been documented in many different environments. Biofilms constitute a protected mode of growth that allows microorganisms to survival in hostile environments, being their physiology and behavior significantly different from their planktonic counterparts. In dairy industry, biofilms may be a source of recalcitrant contaminations, causing food spoilage and are possible sources of public health problems such as outbreaks of foodborne pathogens. Biofilms are difficult to eradicate due to their resistant phenotype. However, conventional cleaning and disinfection regimens may also contribute to inefficient biofilm control and to the dissemination of resistance. Consequently, new control strategies are constantly emerging with main incidence in the use of biosolutions (enzymes, phages, interspecies interactions and antimicrobial molecules from microbial origin).The present review will focus on describing the mechanisms involved in biofilm formation and behavior, deleterious effects associated with their presence, and some of the current and emergent control strategies, providing new insight of concern for food industry.     

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.     

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

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

Biofilm formation and contamination of cheese by nonstarter lactic acid bacteria in the dairy environment

Defects in cheese, such as undesirable flavors, gas formation, or white surface haze from calcium lactate crystals, can result from growth of nonstarter lactic acid bacteria (NSLAB). The potential for biofilm formation by NSLAB during cheese manufacturing, the effect of cleaning and sanitizing on the biofilm, and bacterial growth and formation of defects during ripening of the contaminated cheese were studied. Stirred-curd Cheddar cheese was made in the presence of stainless steel chips containing biofilms of either of two strains of erythromycin-resistant NSLAB (Lactobacillus curvatus strain JBL2126 or Lactobacillus fermentum strain AWL4001). During ripening, the cheese was assayed for total lactic acid bacteria, numbers of NSLAB, and percentage of lactic acid isomers. Biofilms of L. curvatus formed during cheese making survived the cleaning process and persisted in a subsequent batch of cheese. The starter culture also survived the cleaning process. Additionally, L. curvatus biofilms present in the vat dislodged, grew to high numbers, and caused a calcium lactate white haze defect in cheese during ripening. On the other hand, biofilms of L. fermentum sloughed off during cheese making but could not compete with other NSLAB present in cheese during ripening. Pulsed-field gel electrophoresis results verified the presence of the two biofilm strains during cheese making and in the ripening cheese. Probable contamination sites in the plant for other NSLAB isolated in the cheese were identified, thus supporting the hypothesis that resident NSLAB biofilms are a viable source of contamination in the dairy environment.     

Promising strategies to control persistent enemies: Some new technologies to combat biofilm in the food industry—A review

Biofilm is an advanced form of protection that allows bacterial cells to withstand adverse environmental conditions. The complex structure of biofilm results from genetic-related mechanisms besides other factors such as bacterial morphology or substratum properties. Inhibition of biofilm formation of harmful bacteria (spoilage and pathogenic bacteria) is a critical task in the food industry because of the enhanced resistance of biofilm bacteria to stress, such as cleaning and disinfection methods traditionally used in food processing plants, and the increased food safety risks threatening consumer health caused by recurrent contamination and rapid deterioration of food by biofilm cells. Therefore, it is urgent to find methods and strategies for effectively combating bacterial biofilm formation and eradicating mature biofilms. Innovative and promising approaches to control bacteria and their biofilms are emerging. These new approaches range from methods based on natural ingredients to the use of nanoparticles. This literature review aims to describe the efficacy of these strategies and provide an overview of recent promising biofilm control technologies in the food processing sector.     

Multipopulation model of membrane-aerated biofilms

Biofilms cultivated on oxygen-filled gas-permeable membranes grow differently than conventional biofilms, as the chemical species required for growth diffuse from different sides of the biofilm. Oxygen is delivered directly to the base of the biofilm by the membrane, while organic substrates and other soluble nutrients are provided to the upper surface of the biofilm via the water in which the membranes are immersed. This counterdiffusion of nutrients results in a growth environment very different from that of conventional biofilms that receive both oxygen and other nutrients from the water. In recent years, membrane-supported biofilms have been shown to simultaneously remove chemical oxygen demand (COD) and inorganic nitrogen from wastewater in laboratory studies. Several investigators have developed computer models of these biofilms, but they have all focused on a single population of aerobic bacteria. While these models are useful in characterizing the behavior of these biofilms in pure cultures, they are not useful in modeling the behavior of the biofilms in mixed cultures such as those found in wastewater treatment. In this study, a multipopulation biofilm model was developed that includes aerobic heterotrophs, nitrifiers, denitrifiers, and acetoclastic methanogens. The model was constructed with Aquasim software and can predict the COD and inorganic nitrogen removal behavior observed previously in experimental studies. In this paper we present examples of predicted biofilm behavior and compare the results of this multiple-population model with the single-population models published previously. In addition, the behavior of the biofilm is discussed in terms of application to wastewater treatment.     

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.     

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

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

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.     

Survival of Campylobacter jejuni in biofilms isolated from chicken houses

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

Biofilm hydrous manganese oxyhydroxides and metal dynamics in acid rock drainage

Biofilms in shallow, tailings-associated acid rock drainage (ARD) accumulated metals from May to September, indicating scavenging is stable within these biological solids over seasonal time frames. Results indicate a doubling (Mn, Cr) to over a 6-fold increase (Ni, Co) in biofilm metal concentrations. Biofilm oxygen and pH gradients measured over diel time scales with microelectrodes were observed to be both spatially and temporally variable, indicating that biofilms are highly dynamic geochemical environments. Biofilm metal retention and affinities were element specific indicating different processes control their sequestration. Metals were specifically scavenged by the organic constituents of the biofilm itself (Ni, Co) and associated biominerals of amorphous Mn oxyhydroxides (HMO; Ni, Co, and Cr). Results are consistent with sorption and coprecipitation processes controlling Ni and Co biofilm association, while Cr dynamics appear linked to those of Mn through redox processes. Biofilm HMO concentrations increased seasonally but showed significant diel fluctuations, indicating that both formation and dissolution processes occurred over rapid time scales in these biofilms. Biofilm HMO concentrations increased nocturnally but decreased during daylight hours to late afternoon minima. Under the geochemical conditions of the streams, observed HMO formation rates can only be explained by microbial catalysis. These results are the first to quantitatively examine microbial biofilm metal dynamics using microscale, geochemical techniques at both diel and seasonal time scales. They provide strong evidence for the significant role that microbial activity can play in metal geochemistry in natural environments.     

Proteolysis produced within biofilms of bacterial isolates from raw milk tankers

In this study, six bacterial isolates that produced thermo-resistant enzymes isolated from the internal surfaces of raw milk tankers were evaluated for their ability to produce proteolysis within either single culture biofilms or co-culture biofilms. Biofilms were formed in an in vitro model system that simulated the upper internal surface of a raw milk tanker during a typical summer's day of milk collection in New Zealand. The bacterial isolates were further evaluated for their ability to form biofilms at 25, 30 and 37°C. Mutual and competitive effects were observed in some of the co-culture biofilms, with all isolates being able to form biofilms in either single culture or co-culture at the three temperatures. The proteolysis was also evaluated in both biofilms and corresponding planktonic cultures. The proteolysis per cell decreased as the temperature of incubation (20-37°C) increased. Furthermore, mutualistic interactions in terms of proteolysis were observed when cultures were grown as co-culture biofilms. This is the first study to show that proteolytic enzymes can be produced in biofilms on the internal surfaces of raw milk tankers. This has important implications for the cleaning and the temperature control of raw milk transport tankers.     

Naturally occurring biofilms on alfalfa and other types of sprouts

Scanning electron microscopy was used to examine the cotyledons, hypocotyls, and roots of alfalfa, broccoli, clover, and sunflower sprouts purchased from retail outlets as well as alfalfa sprouts grown in the laboratory using a tray system equipped with automatic irrigation. Biofilms were observed on all plant parts of the four types of commercially grown sprouts. Biofilms were also commonly observed on alfalfa sprouts grown in the laboratory by 2 days of growth. Rod-shaped bacteria of various sizes were predominant on all sprouts examined both as free-living cells and as components of biofilms. Occasionally, cocci-shaped bacteria as well as yeast cells were also present in biofilms. The microbes contained in the biofilms appeared to be attached to each other and to the plant surface by a matrix, most likely composed of bacterial exopolysaccharides. Biofilms were most abundant and of the largest dimensions on cotyledons, sometimes covering close to the entire cotyledon surface (approximately 2 mm in length). Naturally occurring biofilms on sprouts may afford protected colonization sites for human pathogens such as Salmonella and Escherichia coli O157:H7, making their eradication with antimicrobial compounds difficult.     

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.     

The adherence of Salmonella Enteritidis PT4 to stainless steel: the importance of the air-liquid interface and nutrient availability

Biofilm formation on stainless steel by Salmonella enterica serovar Enteritidis PT4 during growth in three different nutritious conditions was studied. The ability of micro-organisms to generate biofilms on the stainless steel surfaces was studied for a total period of 18 days at 20 degrees C, under three different experimental treatments: (i) growth medium (tryptone soy broth) was not refreshed (no further nutrients were provided) during the incubation period, (ii) growth medium was renewed every 2 days and (iii) growth medium was renewed every 2 days and at the same time the planktonic cells from the old medium were transferred to the new fresh medium. It was found that biofilms developed better and a higher number of adherent cells (ca. 10(7) cfu/cm(2)) were recovered when the organism was grown in periodically renewed nutrient medium than when the growth medium was not refreshed. Regardless of the availability of nutrients, biofilm development was better (range 2-3 logs greater) when coupons were not totally covered by the growth medium and part of the surface was exposed to the air-liquid interface, than when coupons were submerged in the medium. The results suggest that existence of air-liquid interface and adequate nutrient conditions provide the best environment for Salmonella Enteritidis PT4 biofilm formation on stainless steel. The possible role of stationary phase planktonic cells in biofilm development by sessile/attached microbial cells is also discussed.