Bacterial Quorum Sensing and Food Industry

Abstract: Food spoilage and biofilm formation by food‐related bacteria are significant problems in the food industry. Even with the application of modern‐day food preservative techniques, excessive amounts of food are lost due to microbial spoilage. A number of studies have indicated that quorum sensing plays a major role in food spoilage, biofilm formation, and food‐related pathogenesis. Understanding bacterial quorum‐sensing signaling systems can help in controlling the growth of undesirable food‐related bacteria. This review focusses on the various signaling molecules produced by Gram‐negative and Gram‐positive bacteria and the mechanism of their quorum‐sensing systems, types of signaling molecules that have been detected in different food systems using biosensors, the role of signaling molecules in biofilm formation, and significance of biofilms in the food industry. As quorum‐sensing signaling molecules are implicated in food spoilage, based on these molecules potential, quorum‐sensing inhibitors/antagonists can be developed to be used as novel food preservatives for maintaining food integrity and enhancing food safety. Practical Application: Bacteria use signaling molecules for inter‐ and intracellular communication. This phenomenon of bacterial cell‐to‐cell communication is known as quorum sensing. Quorum‐sensing signals are implicated in bacterial pathogenicity and food spoilage. Therefore, blocking the quorum‐sensing signaling molecules in food‐related bacteria may possibly prevent quorum‐sensing‐regulated phenotypes responsible for food spoilage. Quorum‐sensing inhibitors/antagonists could be used as food preservatives to enhance the shelf life and also increase food safety.     

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.     

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.     

Biofilms in the Spotlight: Detection,Quantification, and Removal Methods

Microorganisms can colonize and subsequently form biofilms on surfaces, which protect them from adverse conditions and make them more resistant than their planktonic free‐living counterparts. This is a major concern in the food industry because the presence of biofilms has significant implications for microbial food contamination and, therefore, for the transmission of foodborne diseases. Adequate hygienic conditions and various preventive and control strategies have consequently been developed to ensure the provision of safe, good‐quality food with an acceptable shelf‐life. This review focuses on the significance of biofilms in the food industry by describing the factors that favor their formation. The interconnected process among bacteria known as “quorum sensing,” which plays a significant role in biofilm development, is also described. Furthermore, we discuss recent strategic methods to detect, quantify, and remove biofilms formed by pathogenic bacteria associated with food processing environments, focusing on the complexity of these microbial communities.     

Biofilm Formation and Control in Food Processing Facilities

Microorganisms on wet surfaces have the ability to aggregate, grow into microcolonies, and produce biofilm. Growth of biofilms in food processing environments leads to increased opportunity for microbial contamination of the processed product. These biofilms may contain spoilage and pathogenic microorganisms. Microorganisms within biofilms are protected from sanitizers increasing the likelihood of survival and subsequent contamination of food. This increases the risk of reduced shelf life and disease transmission. Extracellular polymeric substances associated with biofilm that are not removed by cleaning provide attachment sites for microorganisms newly arrived to the cleaned system. Biofilm formation can also cause the impairment of heat transfer and corrosion to metal surfaces. Some of the methods used to control biofilm formation include mechanical and manual cleaning, chemical cleaning and sanitation, and application of hot water.     

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.     

Biofilm formation characteristics of bacterial isolates retrieved from a reverse osmosis membrane

High-quality water purification systems using reverse osmosis (RO) membrane separation have faced a major challenge related to biofilm formation on the membrane surface, or biofouling. To understand this issue, the biofilm formation characteristics of four bacterial isolates previously retrieved from an RO membrane treating potable water were investigated. Biofilm formation of all four isolates occurred to different extents in microtiter plates and could be related to one or more cell properties (hydrophobicity, surface charge, and motility). For Dermacoccus sp. strain RO12 and Microbacterium sp. strain RO18, bacterial adhesion was facilitated by cell surface hydrophobicity, and for Rhodopseudomonas sp. strain RO3, adhesion was assisted by its low surface charge. Sphingomonas sp. strain RO2 possessed both twitching and swarming motilities, which could be important in mediating surface colonization. Further, strains RO2, RO3, and RO12 did not exhibit swimming motility, suggesting that they could be transported to RO membrane surfaces by other mechanisms such as convective permeate flow. The biofilm formation of RO2 was further tested on different RO membranes made of cellulose acetate, polyamide, and thin film composite in continuous flow cell systems. The resultant RO2 biofilms were independent of membrane surface properties and this was probably related to the ex-opolysaccharides secreted bythe biofilm cells. These results suggested that RO2 could colonize RO membranes effectively and could be a potential fouling organism in RO membranes for freshwater purification.     

群体感应抑制剂调控食源性微生物生物膜形成的研究进展

食源性微生物的生物膜是附着在固体表面上形成的具有空间组织的群落,因其能够附着在食品工业环境中的生物或非生物表面,且对消毒剂和抗菌剂能产生抗药性,通常难以控制,被认为是造成设备损坏、能源成本增加、食物变质和引起食源性疾病的原因之一,给食品工业带来了巨大的挑战。研究发现群体感应在生物膜的形成中起至关重要的作用,可以通过阻断群体感应系统来控制生物膜的形成。因此,群体感应抑制剂可以作为控制生物膜形成的新策略,在食品工业中对控制生物膜的形成具有巨大潜力。本文综述了生物膜的形成、群体感应系统及其对生物膜的调控作用,介绍了群体感应抑制剂的调控机制及其分类,为通过群体感应抑制剂调控生物被膜的形成提供研究思路。     

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.     

Removal of Salmonella enterica serovar Typhimurium and Cronobacter sakazakii biofilms from food contact surfaces through enzymatic catalysis

Bacterial biofilms are highly difficult to control, hence significant economic resources have been allocated to develop strategies to eradicate them. This study evaluated the effect of an enzymatic treatment to be used as a cleaning product to control the presence of biofilms. Two different materials used in the food industry, polystyrene and stainless steel, were tested using Salmonella Typhimuirum and Cronobacter sakazakii. Biofilm formation was carried out by inoculating the surfaces with a standardized concentration of 4 log (CFU cm⁻²) and incubated for 48 hr with renewal of nutrients. The biofilm formation and subsequent enzymatic treatment were quantified using fluorescence microscopy and the conventional culture method. The enzymatic treatment showed significant reductions of 2–3 log (CFU cm⁻²) in biofilm cells, which was attributed to the degradation of the extracellular matrix and the further detachment of both microorganisms. The maximum biofilm detachment obtained with the preventive formula was 46.67%; however, this percentage could be increased by applying an aggressive treatment or by adding a subsequent disinfection step that would eliminate adhered microbial cells. Further, the enzymatic cleaning treatment could be exploited as a potent technology to control bacterial adherence and biofilm formation in the food industry.     

食源性混合菌生物被膜的形成和相互作用机制

在食品加工中,食源性病原菌生物被膜的存在会引发大量食品污染等安全问题。目前,国内外学者深入研究了单一菌生物被膜,然而对混合菌生物被膜的研究较少。本文在现有的研究基础上,归纳总结了食源性混合菌生物被膜的形成规律及混合菌生物膜中菌种间的相互作用,初步探讨了群体感应在混合菌生物膜中的作用,以期为食品领域应对和破解食源性混合菌生物被膜污染问题提供思路。     

Biofilms in the food industry: problems and potential solutions

Contamination of food by spoilage and pathogenic micro‐organisms costs the food industry millions of dollars annually. Much of this contamination may be attributed to the presence of biofilms in the processing plant. In this review, we examine the properties of micro‐organisms and the surfaces of processing equipment that influence the formation of biofilms. Of particular concern is the increased resistance of biofilms to cleaning and disinfection processes. Alternative means of controlling biofilm development are described and some aspects where more information is required are identified.     

Aeromonas biofilm on stainless steel: efficiency of commonly used disinfectants

Aeromonas spp. are ubiquitous bacteria widely distributed among aquatic environments that have the ability to form biofilms. This aptitude allows them to persist in water distribution systems, contaminating drinking water, food processing surfaces and ultimately food. For this study, the biofilm‐forming ability of aeromonads was evaluated after 48‐h incubation on stainless steel discs at both 4 and 20 °C. Subsequently, disinfectants based on amphoteric surfactants and chlorine compounds were evaluated regarding the capacity to eradicate preformed biofilm and inhibit biofilm formation. Results obtained demonstrated that all strains under analysis were able to form biofilm at both room and refrigeration temperatures. The chlorine‐based disinfectant demonstrated to be efficient in removing preformed biofilm, but both were unsuccessful in preventing biofilm formation, highlighting the importance of adequate cleaning and disinfection procedures, with emphasis on food processing surfaces.     

Biofilm Formation of Staphylococcus aureus on Various Surfaces and Their Resistance to Chlorine Sanitizer

This study investigated the effect of material types (polystyrene, polypropylene, glass, and stainless steel) and glucose addition on Staphylococcus aureus biofilm formation, and the relationship between biofilm formation measured by crystal violet (CV) staining and the number of biofilm cells determined by cell counts was studied. We also evaluated the efficacy of chlorine sanitizer on inhibiting various different types of S. aureus biofilms on the surface of stainless steel. Levels of biofilm formation of S. aureus were higher on hydrophilic surfaces (glass and stainless steel) than on hydrophobic surfaces (polypropylene and polystyrene). With the exception of biofilm formed on glass, the addition of glucose in broth significantly increased the biofilm formation of S. aureus on all surfaces and for all tested strains (P ≤ 0.05). The number of biofilm cells was not correlated with the biomass of the biofilms determined using the CV staining method. The efficacy of chlorine sanitizer against biofilm of S. aureus was not significantly different depending on types of biofilm (P > 0.05). Therefore, further studies are needed in order to determine an accurate method quantifying levels of bacterial biofilm and to evaluate the resistance of bacterial biofilm on the material surface.     

食品接触表面生物被膜形成机制及防控方法研究进展

食源性致病菌的生物被膜是固着在食品接触表面上形成的具有一定空间组织的多细胞群体结构。因生物被膜具有极强的黏附性和抗逆性,常规消杀手段难以对其进行有效防控,造成食品安全隐患并严重威胁消费者身体健康。本文归纳了近年来国内外食源性致病菌生物被膜在形成机制及防控方法方面的相关研究。以生物被膜黏附性提高、菌体状态改变和抗逆性增强的3个主要特点为核心,总结讨论了细菌的长链附属结构、群体感应系统及胞外聚合物在生物被膜的形成过程中的作用,并将生物被膜的防控策略分为物理、化学和生物法3类,分别分析了各类方法的作用原理及优缺点,旨在为食品领域生物被膜的高效防控方法的开发提供理论指导,以期更好地实现食品微生物的安全有效防控。     

食源性金黄色葡萄球菌生物膜形成调控与生物防控策略研究进展

生物膜是很多食源性致病菌应对各种极端环境、杀菌理化因子以及维持菌体内环境稳定的重要基质屏障。数据显示,有超过80%的细菌感染由生物膜引起,尤以金黄色葡萄球菌感染多见。食源性金黄色葡萄球菌是引发细菌性食物中毒和食品安全事故的重要风险源, 特别是多重耐药菌株。金黄色葡萄球菌的耐药性、致病性和免疫逃逸与生物膜复杂的三维结构有重大关系。由于生物膜结构基因和调控因子结构相对保守, 现已成为金黄色葡萄球菌生物防控新的重要的效应靶点。本文从多糖细胞间黏附素、胞外DNA和生物膜形成相关蛋白等角度阐明了生物膜形成机制,从群体感应系统（如Agr系统和LuxS/AI-2群体感应系统）、全局性调控因子（如附属调节因子Sar和转录因子SigB）以及双组分信号转导系统（如SrrAB系统、SaeRS系统、ArlSR系统、LytRS系统和WalKR系统）等角度系统阐述了生物膜形成调控机制。最后,从抗菌肽（蛋白）、植物源化合物、生物酶、抗菌药物等角度提出新的防控策略, 以期为食源性金黄色葡萄球菌的生物防控提供指导。     

Compositions of maple sap microflora and collection system biofilms evaluated by scanning electron microscopy and denaturing gradient gel electrophoresis

The bacterial microflora of maple sap and biofilms in collection system tubing were studied through the use of bacterial counts, scanning electron microscopy (SEM) of surfaces and the analysis of 16S rRNA gene by denaturing gradient gel electrophoresis (DGGE). Samples were taken at five times during the 2002 and 2003 seasons in order to follow the changes in the microflora of this complex ecosystem. Bacterial counts showed the growth of bacterial populations as the season advanced. These populations were mainly composed of psychrotrophic bacteria and Pseudomonas spp. SEM results confirmed the suspected presence of biofilms on the inner surfaces of tubing samples. Bacterial colonization and biofilm formation progressively increased during the season for both lateral and main line surfaces, and biofilms were mainly composed of rod shape bacteria. The bacterial microflora profiles obtained for sap and corresponding biofilm by DGGE showed up to 12 major bands. The Shannon-Weaver index of diversity (H) calculated from DGGE bands were statistically higher for sap samples compared to biofilm. The diversity index was relatively stable or increasing for lateral line sap and biofilm samples during the season while the diversity index for sap and biofilm samples of the main line showed a decreasing profile as the season progressed. Sequence analysis of major DGGE bands revealed the predominance of bacteria from the genera Pseudomonas, Rahnella and another, unidentified genus. The results describe the composition of sap collection system microflora as well as the formation of biofilms and will be useful for further studies on factors affecting maple product quality.     

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.     

细菌群体感应系统调控及淬灭机制研究进展

细菌利用群体感应系统实现种内和种间信息的交流,调控菌体的生理特性,如生物膜的形成、菌体的运动、毒素的分泌、细菌素及抗生素的产生,群体感应系统贯穿细菌生长繁殖的整个过程,并对细菌群体的稳定性具有重要作用.近年来,越来越多的群体感应系统被发现和深入研究,其调控机制也逐渐清晰,这为群体感应系统更加广泛地应用于实际奠定了基础....     

Surfactant cleaning of ultrafiltration membranes fouled by whey

A polyethersulphone ultrafiltration membrane was prepared for concentration of whey. The membrane was fouled by whey and the effect of different cleaning agents on flux recovery of the fouled membrane was studied. The optimum cleaning procedure for membrane regeneration was elucidated. The results showed that a combination of surfactants (anionic, cationic and nonionic) may be employed as the optimum cleaning agent for maximum flux recovery. The fluorescence studies revealed that the cationic surfactant interact with proteins by breaking the intra‐chain hydrophobic bonding and providing electrostatic repulsion. Changing the alkyl chain from dodecyl to hexadecyl increases the interaction of surfactant–protein. Dodecyltrimethylammonium bromide (DTAB) provided a weak interaction with whey proteins than to tetradecyltrimethylammonium bromide (TTAB) and cetyltrimethylammonium bromide (CTAB). All data obtained in this study support a surfactant–protein interaction in which hydrophobic forces play a dominant role. The nonionic surfactants poly(oxyethylene) isooctyl phenyl ether (TX‐100) and anionic surfactants SDS interact with amino acids in the inner protein structure thus denaturate tertiary protein structure and reduce hydrophobic interaction of proteins by membrane surface.