Assessment of a regulatory sanitization process in Egyptian dairy plants in regard to the adherence of some food-borne pathogens and their biofilms

Food-borne pathogens may develop certain strategies that enable them to defy harsh conditions such as chemical sanitization. Biofilm formation represents a prominent one among those adopted strategies, by which food-borne pathogens protect themselves against external threats. Thus, bacterial biofilm is considered as a major hazard for safe food production. This study was designed to investigate the adherence and the biofilm formation ability of some food-borne pathogens on stainless steel and polypropylene surfaces using chip assay, and to validate regular sanitizing process (sodium hypochlorite 250mg/L) for effective elimination of those pathogens. Sixteen pathogenic bacterial strains, previously isolated from raw milk and dairy products at Zagazig city, Egypt (9 Staphylococcus aureus, 4 Cronobacter sakazakii and 3 Salmonella enterica serovar Typhimurium), were chosen for this study. Strains showed different patterns of adherence and biofilm formation on tested surfaces with minor significance between surfaces. The ability of sodium hypochlorite to completely eradicate either adhered or biofilm-embedded pathogens varied significantly depending on the strain and type of surface used. Whilst, sodium hypochlorite reduced tested pathogens counts per cm(2) of produced biofilms, but it was not able to entirely eliminate neither them nor adherent Cronobacter sakazakii to stainless steel surface. This study revealed that biofilm is considered as a sustainable source of contamination of dairy products with these pathogens, and also emphasized the need of paying more attention to the cleaning and sanitizing processes of food contact surfaces.     

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.     

An Ecological Perspective of Listeria monocytogenes Biofilms in Food Processing Facilities

Listeria monocytogenes can enter the food chain at virtually any point. However, food processing environments seem to be of particular importance. From an ecological point of view, food processing facilities are microbial habitats that are constantly disturbed by cleaning and sanitizing procedures. Although L. monocytogenes is considered ubiquitous in nature, it is important to recognize that not all L. monocytogenes strains appear to be equally distributed; the distribution of the organism seems to be related to certain habitats. Currently, no direct evidence exists that L. monocytogenes-associated biofilms have played a role in food contamination or foodborne outbreaks, likely because biofilm isolation and identification are not part of an outbreak investigation, or the definition of biofilm is unclear. Because L. monocytogenes is known to colonize surfaces, we suggest that contamination patterns may be studied in the context of how biofilm formation is influenced by the environment within food processing facilities. In this review, direct and indirect epidemiological and phenotypic evidence of lineage-related biofilm formation capacity to specific ecological niches will be discussed. A critical view on the development of the biofilm concept, focused on the practical implications, strengths, and weaknesses of the current definitions also is discussed. The idea that biofilm formation may be an alternative surrogate for microbial fitness is proposed. Furthermore, current research on the influence of environmental factors on biofilm formation is discussed.     

食品接触表面对金黄色葡萄球菌生物被膜形成的影响

在食品工业中,如果金黄色葡萄球菌粘附在食品接触表面并形成生物被膜,不但会造成设备的腐蚀,更重要的是会污染食品,影响了食品的安全性.生物被膜形成的第一步是微生物在食品接触表面的粘附.本文研究了4种不同食品接触表面对金黄色葡萄球菌生物被膜形成的影响.结果表明：与玻璃和PE塑料相比,不锈钢表面粘附形成生物被膜的活菌数较多.不同的食品接触表面影响了生物被膜的形成.     

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.     

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.     

沙门氏菌生物菌膜形成的分子调控研究进展

沙门氏菌是全球范围内危害公众健康的主要食源性致病菌，每年造成千万人食物中毒。黏附于食品加工接触面的生物菌膜是沙门氏菌交叉污染的源头，其对消毒剂具有极强的耐受抵抗作用，可引发大规模食物中毒并导致食品召回。本文在论述细菌黏附辅助体（菌毛和鞭毛）和胞外多聚物对生物菌膜的具体作用及其在mRNA转录层面调控效应的基础上，重点阐述了转录后层面上的非编码小RNA（non-coding small RNA，sRNA）及其对移动性、胞外多聚物等核心mRNA通路的调控机制，并归纳出sRNA调控生物菌膜的4 种作用模式，最后对该领域的研究前景进行了前瞻性展望，以期为拓展研究思路、完善生物菌膜的科学理论和研发针对性控制技术提供参考。     

Prevalence of microbial biofilms on selected fresh produce and household surfaces

Investigations of biofilms in domestic environments are sparsely represented in the literature. In this study, samples of various household surfaces, including food, laundry and kitchen items, were analyzed for evidence of biofilm presence. Visualization of the surfaces was carried out using cryostage scanning electron microscopy (CSEM) and light microscopy. Qualitative evidence of the presence of biofilm formation was obtained from all of the sample groups analyzed, suggesting the widespread existence of microorganisms in biofilms on domestic surfaces. This suggests that biofilms may be important in household hygiene, and highlights the need for standardized, approved biofilm methods suitable for consumer products testing.     

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。     

Salmonella biofilms: An overview on occurrence,structure, regulation and eradication

The ability of Salmonella to form complex surface-associated communities, called biofilms, contributes to its resistance and persistence in both host and non-host environments and is especially important in food processing environments. In this review, the different types of abiotic (plastic, glass, cement, rubber, and stainless steel) and biotic surfaces (plant surfaces, epithelial cells, and gallstones) on which Salmonella biofilms have been described are discussed, as well as a number of commonly used laboratory setups to study Salmonella biofilm formation (rdar morphotype, pellicle formation, and biofilms on polystyrene pegs). Furthermore, the structural components important during Salmonella biofilm formation are described (curli and other fimbriae, BapA, flagella, cellulose, colanic acid, anionic O-antigen capsule and fatty acids), with special attention to the structural variations of biofilms grown on different surfaces and under different conditions. Indeed, biofilm formation is strongly influenced by different environmental signals, via a complex regulatory network. An extensive overview is given on the current understanding of this genetic network and the interactions between its different components (CsgD, RpoS, Crl, OmpR, IHF, H-NS, CpxR, MlrA, c-di-GMP, BarA/SirA, Csr, PhoPQ, RstA, Rcs, metabolic processes and quorum sensing). To further illustrate that biofilm formation is a mechanism of Salmonella to adapt to different environments, the resistance of Salmonella biofilms against different stress factors including desiccation stress, disinfectants (e.g. hypochlorite, glutaraldehyde, cationic tensides and triclosan) and antibiotics (e.g. ciprofloxacin) is described. Finally, a number of Salmonella biofilm inhibitors, identified through bottom-up- and top-down-approaches, are discussed, such as surfactin, glucose, halogenated furanones, 4(5)-aryl 2-aminoimidazoles, furocoumarins and salicylates. Also the potential of combination therapy (e.g. combinations of triclosan and quaternary ammonium salts or halogenated furanones and antibiotics/disinfectants) and nano- and micro-emulsions to inhibit Salmonella biofilm formation is discussed. Insight into the pathogen's complex biofilm process will eventually lead to further unraveling of its intricacies and more efficient strategies to combat Salmonella biofilms.     

Cold Plasma Synthesis of Poly(ethylene glycol)-like Layers on Stainless-Steel Surfaces to Reduce Attachment and Biofilm Formation by Listeria monocytogenes

ABSTRACT Poly(ethylene glycol) (PEG)-like structures were generated on stainless steel under di(ethylene glycol) vinyl ether (DiEGVE) radio frequency-plasma environments. Electron spectroscopy for chemical analysis and attenuated total reflectance Fourier transform infrared spectroscopy indicated a PEG-like deposition, which was stable to cleaning, sanitizing, and storage for up to 2 mo. Atomic force microscopy and water contact angle analysis indicated that the modified stainless-steel surfaces were less rough and more hydrophilic than the unmodified surfaces. Listeria monocytogenes attachment and biofilm formation on modified surfaces decreased more than 90% compared with the unmodified stainless steel ( P < 0.01). DiEGVE cold plasma was demonstrated to be a promising technique to reduce bacterial contamination on surfaces encountered in food-processing environments.     

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.     

群体感应在食源性微生物生物膜形成中的作用

生物膜(Biofilm)是由众多微生物聚集黏附在物体表面形成的多细胞群体结构,多种食源性微生物均能形成相应的生物膜,且该现象的产生对食品加工与安全有着重要影响。群体感应(Quorum sensing,QS)已被证明是调控生物膜形成的重要因素。文章主要介绍了群体感应对几种食源性细菌及真菌生物膜形成的调控作用,旨在为食品加工过程中微生物生物膜的控制与利用提供参考。     

食品工业中阪崎克罗诺杆菌生物膜形成的影响因素与控制策略研究进展

阪崎克罗诺杆菌是一种食源性条件致病菌,它可以引起不同年龄段人群的感染,对于新生儿和免疫功能不全的婴幼儿的威胁尤为严重,引起的症状包括坏死性小肠结肠炎、菌血症、脑膜炎和术后骨髓炎等。在食品生产环境以及设备表面由阪崎克罗诺杆菌形成的生物膜是持久性污染食品的重要来源。因此本文主要综述了在食品工业中阪崎克罗诺杆菌流行病学、生物膜形成的机制以及生物膜控制的策略,旨在为建立安全的无生物膜食品生产环境体系,控制阪崎克罗诺杆菌所致食品的潜在污染以及临床感染提供理论指导。      

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。     

从腐败食品中分离的乳酸菌生物被膜形成的影响因素

从腐败的蔬菜和肉质食品中分离筛选乳酸菌(LAB),并以其作为研究对象,对乳酸菌生物成膜不同影响因素进行研究。生化分离鉴定乳酸菌,在不同的营养物质浓度及培养条件下,用96孔板法检测乳酸菌成膜。在无外添加物,37℃和42℃的培养温度,pH4有利于乳酸菌生物膜的形成,低温不利于生物膜的形成。低浓度的NaCl可促进LAB形成生物膜,但高于某浓度,就抑制LAB成膜。不同LAB菌株对不同葡萄糖浓度成膜效果不同,且与温度交互作用。结果表明,腐败食品中乳酸菌具有一定的生物被膜形成能力,控制乳酸菌生物膜的形成对于防治食品的腐败变质具有一定的意义。     

食品中细菌生物被膜及其形成机制的研究进展

生物被膜是自然条件下细菌在固态表面生长时采取的一种较为独特的群体生活方式,该方式较浮游状态存在显著生物学特征差异,对食品的危害更为严重,但目前国内外食品领域对其研究较少.本文在收集、研究现有文献的基础上归纳介绍了细菌生物被膜的特点及其形成过程,概述了各种影响细菌生物被膜形成的因素与机制,旨在提高人们对细菌生物被膜的认识,防范和减少细菌生物被膜对食品质量与安全的危害,同时推动该领域的研究发展.     

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.     

Inactivation of Listeria monocytogenes/Pseudomonas biofilms by peracid sanitizers.

The ability of peracetic acid and peroctanoic acid sanitizers to inactivate mixed-culture biofilms of a Pseudomonas sp. and Listeria monocytogenes on stainless steel was investigated. Types of biofilms tested included a 4-h attachment of the mixed-cell suspension and a 48-h biofilm of mixed culture formed in skim milk or tryptic soy broth. Biofilm-containing coupons were immersed in solutions of hypochlorite, peracetic acid, and peroctanoic acid either with or without organic challenge. Organic challenge consisted of either coating the biofilms with milk that were then allowed to dry, or adding milk to the sanitizing solution to achieve a 5% concentration. Surviving cells were enumerated by pouring differential agar directly on the treated surfaces. The peracid sanitizers were more effective than chlorine for inactivating biofilm in the presence of organic challenge. The 48-h mixed-culture biofilm grown in milk was reduced to less than 3 CFU/cm2 by 160 ppm of peracid sanitizer after 1 min of exposure. Peroctanoic acid was more effective than peracetic acid against biofilm cells under conditions of organic challenge. Pseudomonas and L. monocytogenes were inactivated to similar levels by the sanitizer treatments, even though Pseudomonas predominated in the initial biofilm population.     

Attachment and biofilm formation by various serotypes of Salmonella as influenced by cellulose production and thin aggregative fimbriae biosynthesis

This study was undertaken to quantify thin aggregative fimbriae and cellulose produced by Salmonella and to evaluate their roles in attachment and biofilm formation on polystyrene and glass surfaces. Thin aggregative fimbriae and cellulose produced by four wild-type and two pairs of Salmonella, representing four different colony morphotypes (rdar: red, dry, and rough; pdar: pink, dry, and rough; bdar: brown, dry, and rough; and saw: smooth and white), were quantified. The ability of the Salmonella cells to attach and form biofilms on the selected surfaces was evaluated in Luria-Bertani (LB) broth with or without salt (0.5%) or glucose (2%) at 28 degrees C during a 7-day period. The cells expressing the rdar or pdar colony morphotypes produced significantly greater amounts of thin aggregative fimbriae and cellulose on LB no salt agar, respectively. The cells expressing the rdar colony morphotype attached in higher numbers and formed more biofilm than did the cells expressing the pdar colony morphotype. The members of the pairs expressing the bdar colony morphotype attached more efficiently and formed more biofilm on the tested surfaces than did their counterparts expressing the saw colony morphotype. These results indicated that thin aggregative fimbriae impart attachment ability to Salmonella and, upon coexpression with cellulose, enhance biofilm formation on certain abiotic surfaces. The knowledge acquired in the study may help develop better cleaning strategies for food processing equipment.