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.     

Beneficial biofilm formation by industrial bacteria Bacillus subtilis and related species

Biofilms are densely packed multicellular communities of microorganisms attached to a surface or interface. Bacteria seem to initiate biofilm formation in response to specific environmental cues, such as nutrient and oxygen availability. Biofilms undergo dynamic changes during their transition from free-living organisms to sessile biofilm cells, including the specific production of secondary metabolites and a significant increase in the resistivity to biological, chemical, and physical assaults. Bacillus subtilis is an industrially important bacterium exhibiting developmental stages. It forms rough biofilms at the air-liquid interface rather than on the surface of a solid phase in a liquid, due to the aerotaxis of the cells. Biofilm formation by B. subtilis and related species permits the control of infection caused by plant pathogens, the reduction of mild steel corrosion, and the exploration of novel compounds. Although it is obviously important to control harmful biofilm formation, the exploitation of beneficial biofilms formed by such industrial bacteria may lead to a new biotechnology.     

益生菌生物被膜的研究进展

益生菌是指当以足够数量存在时可对机体健康产生有益作用的活的微生物。近年来,由于其对机体具有保健和治疗作用,因而受到越来越多的关注。生物被膜是细菌分泌的多糖,纤维蛋白和脂蛋白等物质将细菌自身包裹其中,吸附在生物材料或机体腔道等表面而形成的膜样复合物,是自然状态下许多细菌所选择的生存方式。但是关于益生菌生物被膜的研究还比较少,且尚处于起步阶段。本综述围绕益生菌生物被膜的形成、阶段特征、影响因素、优势及调控机制等展开分析,并指出益生菌生物被膜的相关研究将会是益生菌研究领域的一个重要发展方向。     

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.     

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.     

The Paradox of Mixed‐Species Biofilms in the Context of Food Safety

Formation of mixed‐species biofilms constitutes a common adaptation of foodborne pathogens and indigenous microbiota for prolonged survival in their food niche. Nevertheless, the potential role of mixed‐species biofilms in food safety remains to be elucidated. The formation of mixed‐species biofilms on food and food processing surfaces depends on various physical, chemical, and biological processes including species composition, especially of the indigenous microbiota and nutrients, food types, temperature, quorum sensing, extracellular polymeric substance (EPS) production, biofilms maturation, and dispersal steps. Compared to monospecies, mixed‐species are highly resistant to antimicrobials, possibly due to higher EPS production, internalization into food, fitness of species, denser and thicker biofilms maturation, and interspecific protection of 1 species by others, although there are much debate among studies. The fitness of mixed‐species biofilms populations is suggested to be of a cooperative, competitive, or neutral nature based on the genetic background of the involved species. Currently, various methods using microarray, confocal microscopy, proteomics, and selective media are being explored for the detection of mixed‐species biofilms to resolve the conflict issues. Here, we review recent progress in this emerging field in the context of food safety and propose that novel and alternative techniques like antiquorum sensing, antibiofilms, enzymes, hurdle techniques, and bacteriophages will significantly help to control the formation of mixed‐species biofilms for enhanced food safety. The next challenge will be to integrate the fitness and resistance patterns of mixed‐species biofilms in the laboratory with those of natural settings.     

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 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.     

食品工业中混合菌生物被膜的形成、相互作用与新型控制策略

细菌黏附在食品或食品接触表面并形成生物被膜可能导致设备损坏、食品变质甚至人类疾病。混合菌生物被膜作为细菌在食品工业中的主要存在形式,与单菌生物被膜相比,对消毒剂和抗菌素往往具有更强的抗性。然而,混合菌生物被膜的形成与种间相互作用十分复杂,其在食品工业中的潜在作用仍有待探索。本文总结了混合菌生物被膜的形成和种间相互作用以及近年来的新型控制策略,并对未来食品工业中混合菌生物被膜的污染防控进行了展望,旨在为混合菌生物被膜在食品工业中的深入研究以及制定高效的新型控制策略提供理论依据和参考,以期更好地保障食品安全与公众健康。     

A photoacoustic technique for depth-resolved in situ monitoring of biofilms

Biofilms occur in natural and engineered water systems. Biofouling in technical processes lowers the water quality and increases the frictional resistance in tubes. In wastewater treatment plants, biofilms are used for removal of organic an inorganic pollutants. For improvement of antifouling strategies and for process optimization in wastewater treatments plants, an analytical technique for online monitoring of biofilms is needed. In this article, a new setup for in situ monitoring of biofilms by photoacoustic spectroscopy is presented. To produce a biofilm, a mixture of microorganisms was grown in a nutrient solution inside a tube reactor. The content of the tube reactor was pumped through a flow channel, and biofilms were generated at the inner surfaces. Three photoacoustic sensor heads were integrated at different positions into the base plate of the flow channel. By photoacoustic spectroscopy, growth, thickness, and detachment of biofilms can be monitored on-line and nondestructively. Experiments presented in this article showed that the flow conditions influence the structure and thickness of biofilms. By changing the pH value, electrostatic interactions inside the biofilm matrix were influenced, and the subsequent detachment processes were observed online. The interaction of iron(III) oxide particles with biofilms led to particle adsorption on the outer and inner surfaces of the biofilm. Afterwards, biofilm flocs were sloughed off from the base biofilm.     

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

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

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.     

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.     

Biofilm − An unrecognised source of spoilage enzymes in dairy products?

Enzymes play an important role in food processing, where they either increase or decrease the value of food commodities. Within the dairy context, undesirable enzymes include indigenous enzymes originating from the cow and microbial enzymes produced by the natural bacteria associated with the cow and its environment. Some of the heat-stable enzymes can remain active after the heat treatments applied during processing and may eventually reduce the quality of the final product. Biofilms may play a role in promoting enzyme production in microorganisms because of the microenvironments created within the biofilm structure. Many studies have been carried out on indigenous and bacterial enzymes that occur in milk, but few studies have looked at the relationship between spoilage enzymes and biofilms. We suggest that bacterial biofilms in dairy manufacture can be a source of dairy spoilage enzymes.     

Candida krusei development on turbulent flow regimes: Biofilm formation and efficiency of cleaning and disinfection program

In food processing lines or in complex equipment such as pumps or valves, microorganisms are exposed to varying hydrodynamic conditions caused by the flow of liquid food, and biofilms are thus grown under a wide distribution of local hydrodynamic strengths. Using an industrially relevant strain of Candida krusei, we demonstrated that biofilms formed on stainless steel for 4 days at Reynolds (Re) numbers ranging from 294,000 to 1.2 × 10⁶ proceeds through three distinct developmental phases. These growth phases transform adherent blastospores to well-defined cellular communities encased in an extracellular matrix and biofilm formation increases when increasing Reynolds number and time. In all growth phases, the morphology of C. krusei biofilm revealed the influence of hydrodynamic drag. Indeed, we study the effect of cleaning and sanitation procedure in the control of turbulent flow-generated biofilm. This procedure involves alkali (NaOH 0.5%) and sodium hypochlorite (500 ppm). In terms of total biofilm mass, removal decreases with increasing biofilm age. The largest reduction post-treatment (between 57% and 62%) was observed, to all Reynolds numbers, on 24 and 48 h-old biofilms. Removal was between 39% and 46% on 72 h-old biofilms and was close to 30% for all Reynolds numbers on 96 h-old biofilm.     

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.     

Disruption of Staphylococcus aureus biofilms using rhamnolipid biosurfactants

Staphylococcus aureus is an important pathogen that has shown ability to establish biofilm communities that can represent a source of contamination and resistance in food processing. Rhamnolipids (RL) have attracted attention as candidates to replace synthetic surfactants, exhibiting high surface activity combined with its microbial origin, biodegradability, and low toxicity. In this work, an RL biosurfactant was evaluated regarding its ability to disrupt or remove S. aureus biofilms established on polystyrene plates using nutrient broth and skim milk as the growth media. Rhamnolipid treatment was performed at different surfactant concentrations and temperatures. Rhamnolipid removes up to 88.9% of milk-based biofilms, whereas for nutrient medium 35% removal was attained. The RL concentration affects the disruption of nutrient medium-based biofilms. High carbohydrate content of milk-based biofilms favors disruption by RL and the organization of RL molecules in solution showed a predominance of aggregates from 1 to 10 and 100 to 1,000 nm in all conditions studied. Biofilm disruption activity of RL is nutrient-specific and dependent on biofilm matrix composition. Staphylococcus aureus biofilms established in milk were significantly reduced using RL at low concentrations and temperatures. These findings suggest potential application of RL in milk (dairy) processing industries where low temperatures are applied.     

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

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

Combating biofilms of foodborne pathogens with bacteriocins by lactic acid bacteria in the food industry

Most foodborne pathogens have biofilm-forming capacity and prefer to grow in the form of biofilms. Presence of biofilms on food contact surfaces can lead to persistence of pathogens and the recurrent cross-contamination of food products, resulting in serious problems associated with food safety and economic losses. Resistance of biofilm cells to conventional sanitizers urges the development of natural alternatives to effectively inhibit biofilm formation and eradicate preformed biofilms. Lactic acid bacteria (LAB) produce bacteriocins which are ribosomally synthesized antimicrobial peptides, providing a great source of nature antimicrobials with the advantages of green and safe properties. Studies on biofilm control by newly identified bacteriocins are increasing, targeting primarily onListeria monocytogenes, Staphylococcus aureus, Salmonella, and Escherichia coli. This review systematically complies and assesses the antibiofilm property of LAB bacteriocins in controlling foodborne bacterial-biofilms on food contact surfaces. The bacteriocin-producing LAB genera/species, test method (inhibition and eradication), activity spectrum and surfaces are discussed, and the antibiofilm mechanisms are also argued. The findings indicate that bacteriocins can effectively inhibit biofilm formation in a dose-dependent manner, but are difficult to disrupt preformed biofilms. Synergistic combination with other antimicrobials, incorporation in nanoconjugates and implementation of bioengineering can help to strengthen their antibiofilm activity. This review provides an overview of the potential and application of LAB bacteriocins in combating bacterial biofilms in food processing environments, assisting in the development and widespread use of bacteriocin as a promising antibiofilm-agent in food industries.     

CALCOFLUOR AS A FLUORESCENT PROBE TO DETECT BIOFILMS OF FOODBORNE PATHOGENS

Biofilms enable foodborne pathogens to resist removal from surfaces, survive disinfection and elude detection. This study evaluated the use of Calcofluor, which binds to polysaccharides containing β-D-glucans, to detect biofilms produced by Salmonella enterica serovar Berta and Salmonella enterica serovar Typhimurium DT104 (St DT104), Escherichia coli, Aeromonas hydrophila, Vibrio cholerae O139 and Hyphomonas adhaerens. Biofilms produced by St DT104, S. berta and V. cholerae on five types of surfaces (glass, polypropylene, Teflon™, stainless steel and aluminum) were detected by Calcofluor. Results suggest the potential use of Calcofluor as probes of foodborne pathogens in biofilms.