Role of sigma B factor in the alkaline tolerance response of Listeria monocytogenes 10403S and cross-protection against subsequent ethanol and osmotic stress

Many of the considerable abilities of Listeria monocytogenes to persist and grow in a wide range of adverse environmental conditions are thought to be at least partly under the control of the alternative sigma factor (sigmaB), encoded by the sigB gene. However, little is known about the role of this master regulon in the impressive ability of Listeria to persist and grow under conditions of alkaline pH. In this study, Northern blot analysis of parent Listeria mRNA revealed that alkali adaptation (pH 9.5 for 1 h) significantly increased the expression of sigB-derived mRNA. The study included a comparison of the relative survival of mid-exponential populations of adapted and nonadapted parent type (sigmaB expressing) and mutant (not sigmaB expressing, deltasigB) Listeria strains during subsequent alkaline (pH 12.0), osmotic (25% NaCl, wt/vol), or ethanol (16.5%) stress. Alkali-adapted parent strains were more resistant to pH 12.0 than were adapted deltasigB type strains, but both alkali-adapted parent and deltasigB strains were more resistant to pH 12.0 than were nonadapted strains. Alkali-adapted parent strains were more resistant to osmotic stress than were adapted deltasigB type strains. No significant differences in viability were observed between alkali-adapted parent and deltasigB strains after ethanol stress, suggesting that cross-protection against osmotic stress is mediated by sigmaB whereas cross-protection against ethanol is sigmaB independent. Overall, alkali-induced cross-protection against osmotic and ethanol challenges may have serious implications for food safety and human health because such stress conditions are routinely used as part of food preservation and surface cleaning processes.     

环境胁迫下沙门氏菌应激反应及抗性机制研究进展

细菌为了生存能够适应多种不利于生长的环境条件，因此许多食源性致病菌能够在某些食品保鲜处理中存活下来。沙门氏菌（Salmonella）是世界范围内常见的食源性致病细菌，在食品保鲜中可承受极端pH、温度、渗透压、寡营养和高压等胁迫。在与宿主相互作用过程中，沙门氏菌应对不同环境胁迫，可产生应激蛋白、进行系统自我调节和表达相关基因等，进而影响其致病能力，产生交叉抗性从而更好的生存和繁殖。因此，沙门氏菌能在遗传和表型水平上适应不同的极端环境。本文综述了沙门氏菌耐受环境胁迫，暴露于这些环境胁迫时发生的适应性变化，对细菌致病性的影响，以及产生的交叉保护机制。这些方面将有助于了解沙门氏菌的发病机制，对于制定抗击沙门氏菌感染的新策略，保障食品安全具有重要意义。     

大肠杆菌在食品加工贮藏中胁迫响应机制的研究进展

大肠杆菌能够感受环境信号并对环境的变化迅速做出反应。因此,在食品加工贮藏中,大肠杆菌在面对物理、化学因子胁迫时会产生应激反应,使其仍然能够生存和保持毒力,给食品安全带来极大的威胁。本文总结了在常见的食品加工贮藏胁迫因子下,包括热激、冷激、干燥、高渗透压、抗菌肽和酸,大肠杆菌的分子及生理响应机制及其在食品工业中的应用,并对大肠杆菌胁迫响应的未来研究做出展望。     

The physiology of Campylobacter species and its relevance to their role as foodborne pathogens

Campylobacter jejuni and C. coli are recognised as the leading causes of bacterial foodborne diarrhoeal disease throughout the development world. While most foodborne bacterial pathogens are considered to be relatively robust organisms, as a consequence of the necessity to survive the inimical conditions imposed by food processing and preservation, Campylobacter species have uniquely fastidious growth requirements and an unusual sensitivity to environmental stress. Campylobacters also lack many of the well characterised adaptive responses that can be collated with resistance to stress in other bacteria. The aim of this review is to outline the unusual physiology of campylobacters (C. jejuni and C. coli) and to describe how this influences their role as foodborne pathogens.     

环境胁迫调控植物乳杆菌细菌素合成的研究进展

植物乳杆菌作为具有重要经济价值的乳酸菌被广泛应用于食品发酵与保鲜领域,由于其代谢过程中会产生具有广谱抑菌特性、对热稳定且易被蛋白酶水解的细菌素,因此有作为天然食品生物防腐剂的较大应用潜力。研究表明,在发酵过程中菌体的生长和细菌素的合成受多种环境因素如盐胁迫、酸胁迫、氧胁迫及低高温胁迫的影响,但目前环境因素调节信号分子产生以及调控相关基因合成细菌素的具体机制仍然有待研究,另一方面,通用的调控通路还未被发现。因此,本文介绍了植物乳杆菌抵御胁迫的反应机制并详细阐述了环境胁迫下与细菌素合成密切相关的调控基因和重要调控蛋白,为食品发酵加工过程中合理控制发酵条件,促进细菌素合成从而延长食品货架期提供理论依据。     

Membrane fluidity-related adaptive response mechanisms of foodborne bacterial pathogens under environmental stresses

The maintenance of bacterial membrane fluidity plays an important role in a variety of cell physiological functions such as nutrient transport, protection from external adverse environments, and cell morphology. The fluidity of membranes is modified in response to several environmental cues, enabling bacterial survival in otherwise unfavorable conditions. Many foodborne bacterial pathogens are able to survive a variety of food preservation treatments used to prevent microbial contamination. These pathogens are able to successfully exploit membrane fluidity-related adaptation strategies under unfavorable conditions, resulting in food hygiene failures. Factors involved in food preservation include pH, temperature, osmotic stress, antimicrobial agents, and high pressure. The fluidity of bacterial membrane lipid bilayer is altered mainly via the adjustment of membrane fatty acid composition. Under undesirable conditions, Gram-negative bacteria alter their membrane fluidity primarily by regulating the ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs) and, to a lesser extent, the levels of cyclopropane fatty acids (CFAs), or by cis/trans isomerization. Gram-positive bacteria typically alter their membrane fluidity with changes in fatty acyl chain length or by forming branched-chain fatty acids (BCFAs), besides changes to the ratio of UFA to SFA. This review encompasses various modulators of membrane fluidity, particularly with respect to foodborne pathogens, which often survive even the hostile environments associated with food processing.     

Heat acclimation and the role of RpoS in prolonged heat shock of Escherichia coli O157

Escherichia coli, a commensal mesophile that primarily inhabits the gastro-intestinal tract, responds to temperature up-shifts with transient expression of stress-response proteins. The goal of this study was to identify adaptive proteins of E. coli O157 crucial for growth resumption of this human pathogen after heat shock, with specific focus on the role of the RpoS sigma factor. Using the comparative proteomic analysis of hyper-thermally acclimatized wild-type strain B-1 and rpoS-mutant strain SV521, we identified 39 proteins that underwent significantly-different induction upon temperature shock at 45°C or rpoS mutation. All identified proteins of the heat post-acclimation stimulon fell into two large sub-groups: (i) stress proteins, including molecular chaperons, proteases, DNA/RNA stabilizing enzymes, and anti-oxidant proteins, and (ii) housekeeping proteins. It was found that in the heat stress stimulon RpoS has significantly (P=0.012) limited control over the key stress proteins involved in translation, translational elongation, protein folding and refolding. However, RpoS showed a significant (P=0.035) control over the cellular metabolic processes that included NADPH regeneration, pentose-phosphate shunt, nicotinamide nucleotide and NADP metabolic processes, reflecting its specific importance in promoting resource utilization (energy, protein synthesis etc.) during proliferation of hyperthermally-adapted cells. Pathogenic strains, like E. coli O157, have the ability to survive a variety of harsh stress conditions, leading to their entry into the food chain, and subsequent pathogenesis. This research offers insights into the physiological response of this pathogen during the critical period following adaptation to thermal stress and subsequent resumption of growth.     

双组分调控系统及其对细菌诱导性耐酸响应调控机理的研究进展

双组分调控系统（two-component regulatory system,TCS）是维持细菌在压力环境中存活的重要结构。食品加工过程极易产生高渗、弱酸等压力环境,双组分系统能够帮助细菌感受外部环境的胁迫,及时动员体内对抗机制,这一过程容易产生耐酸、耐渗透压、耐高温甚至是高毒性的菌株,威胁食品安全。本文主要对TCS的结构组成、识别信号及调控作用等进行概述,并与细菌诱导耐酸响应（acid tolerance response,ATR）中的酸休克蛋白、细胞膜系统和氨基酸代谢等产生机制进行联系,综述了细菌在酸性条件下通过TCS响应信号分子激发ATR的具体过程。     

An overview of molecular stress response mechanisms in Escherichia coli contributing to survival of Shiga toxin-producing Escherichia coli during raw milk cheese production

The ability of foodborne pathogens to survive in certain foods mainly depends on stress response mechanisms. Insight into molecular properties enabling pathogenic bacteria to survive in food is valuable for improvement of the control of pathogens during food processing. Raw milk cheeses are a potential source for human infections with Shiga toxin-producing Escherichia coli (STEC). In this review, we focused on the stress response mechanisms important for allowing STEC to survive raw milk cheese production processes. The major components and regulation pathways for general, acid, osmotic, and heat shock stress responses in E. coli and the implications of these responses for the survival of STEC in raw milk cheeses are discussed.     

Evaluation of currently employed food preservation conditions to tackle biofilm forming food pathogens

Controlling biofilm forming, pathogenic bacteria is an emerging challenge in the food industry, and the present study aims to test the efficacy of existing food preservation methods against these bacteria. We isolated 10 different biofilm forming pathogenic bacteria viz. Klebsiella quasipneumoniae, Bacillus cereus, Bacillus amyloliquefaciens, Bacillus anthracis, and Myroides odoratimimus from spoiled milk, biscuits, and cakes. K. quasipneumonia and B. amyloliquifaciens formed strong biofilm (OD₅₅₀ > 0.85), whereas B. cereus, B. anthracis, and M. odoratimimus made moderate biofilm (OD₅₅₀₅ > 0.6) in 72 hr. The isolates, B. amyloliquifaciencs, B. anthracis, and M. odoratimimus were found positive for protease activity. The isolates showed significant tolerance to preservation conditions: pH (4–10), salt (up to 5%), temperature (up to 50°C) and sodium benzoate (up to 0.1%). The isolates also exhibited high antibiotic resistance, maximum by M. odoratimimus (MIC ≤ 256 µg/ml) against chloramphenicol which can have serious implications especially in tackling food borne illness caused by the biofilm forming food pathogens.

Practical applications

The present study discloses the presence of biofilm forming pathogenic bacterial strains in common food stuff and their tolerance of preservation conditions and resistance to antibiotics. The occurrence of pathogens tolerant to common preservation conditions and their resistance to antibiotic should be addressed very cautiously. The significant findings of this study warrant the need for more stringent preservation methods to control food pathogens, especially the biofilm forming type. Moreover, findings of the present study will help to design novel preservation techniques such as incorporating antibiofilm coatings in food processing units and packaging materials, which will ensure food safety and public health.     

单核细胞增生李斯特菌对食品加工中的胁迫响应及其机制研究进展

单核细胞增生李斯特菌（简称单增李斯特菌）在食品加工过程中经常会遇到环境胁迫,如酸胁迫、热胁迫、冷胁迫、干燥和高渗透压胁迫以及交叉胁迫等,并产生应激反应,而经过环境胁迫的单增李斯特菌可显著增强其抵抗致死性环境胁迫的能力,给食品安全带来极大危害。单增李斯特菌的酸胁迫响应机制主要包括F0F1-ATPase系统、谷氨酸脱羧酶系统以及精氨酸脱亚胺酶系统。热休克蛋白、冷休克蛋白、相容性溶质在单增李斯特菌的热胁迫、冷胁迫和干燥及高渗透压胁迫反应中起重要作用。本文从以上几方面就单增李斯特菌对环境胁迫响应及其机制的研究现状进行了综述,并提出了今后可能的研究方向,为单增李斯特菌在食品加工过程中的胁迫响应研究及防控提供指导。     

Food preservation by high pressure

Novel non-thermal food processing technologies aim to provide safe, high quality foods with desirable nutritional, physico-chemical and sensorical properties. More recently with the use of minimal processing treatment concepts have been added to the already existing food processing requirements. Some of them might be beneficial for the improvement of hygiene and the extension of shelf life. This presentation will focus on the current practice, the knowledge and future developments of high pressure processing (HPP). Hydrostatic high pressure technology is relatively new to food industry and is more and more considered as an alternative to traditional preservation methods like heat processing. Inactivation of bacteria, spores, virus has been demonstrated. Relevant aspects of the European legislation on novel foods will be discussed. International trends and recent developments in machinery will be reviewed.     

The Acetic Acid Tolerance Response induces cross-protection to salt stress in Salmonella typhimurium

Salmonella typhimurium induces an Acid Tolerance Response (ATR) upon exposure to mildly acidic conditions in order to protect itself against severe acid shock. This response can also induce cross-protection to other stresses such as heat and salt. We investigated whether both the acetic acid induced and lactic acid induced ATR in S. typhimurium provided cross-protection to a salt stress at 20 degrees C. Acid-adapted cells were challenged with both a sodium chloride (NaCl) and potassium chloride (KCl) shock and their ability to survive ascertained. Acetic acid adaptation provided cells with protection against both NaCl and KCl stress. However, lactic acid adaptation did not protect against either osmotic stressor and rendered cells hypersensitive to NaCl. These results have implications for the food industry where hurdle technology means multiple sub-lethal stresses such as mild pH and low salt are commonly used in the preservation of products.     

Effects of environmental stresses on the activities of the uspA, grpE and rpoS promoters of Escherichia coli O157:H7

Heat shock proteins and RNA polymerase sigma factor play an important role in protecting cells against environmental stresses, including starvation, osmotic and oxidative stresses, and cold shock. In this study, the effect of environmental stresses on activity of the auto-fluorescent Escherichia coli O157:H7 generated by the fusion of gfp(uv) to E. coli uspA, grpE and rpoS promoters were examined. Osmotic shock caused about a 4-fold increase in green fluorescence of E. coli O157:H7 harboring uspA::gfp(uv) or rpoS::gfp(uv) at 37 degrees C and room temperature whereas osmotic shock at 5 degrees C did not induce green fluorescence. When starved, E. coli O157:H7 possessing grpE::gfp(uv) was more sensitive for evaluating stress at low temperature while uspA::gfp(uv) was better suited for detecting the stress response at higher temperature. The uspA, grpE and rpoS promoters were up-regulated to varying degrees by stresses commonly encountered during food processing.     

Heat stress adaptation induces cross-protection against lethal acid stress conditions in Arcobacter butzleri but not in Campylobacter jejuni

The ability of many bacteria to adapt to stressful conditions may later protect them against the same type of stress (specific adaptive response) or different types of stresses (multiple adaptive response, also termed cross-protection). Arcobacter butzleri and Campylobacter jejuni are close phylogenetic relatives that occur in many foods of animal origin and have been linked with human illness (mainly diarrhoea). In the present study, sublethal stress adaptation temperatures (48 °C and 10 °C) and mild and lethal acid conditions (pH 5.0 and pH 4.0) were determined for A. butzleri and C. jejuni. In addition, it was evaluated whether these sublethal stress adaptations cause specific adaptive responses or cross-protection against subsequent mild or lethal acid stresses in these bacteria. The studies were conducted in broth adjusted to the different conditions and the results were determined by the dilution series plating method. It was shown that heat stress adapted A. butzleri (incubated for 2 h at 48 °C) were significantly more resistant to subsequent lethal acid stress (pH 4.0) than non-adapted cells at the 1 h time-point (p < 0.01 in Wilcoxon rank sum test). No specific adaptive responses against the stresses in A. butzleri or C. jejuni and no cross-protection in C. jejuni were found. The ability of heat stressed A. butzleri to tolerate later lethal acid conditions should be taken into account when designing new food decontamination and processing strategies.     

Development of Wide-Spectrum Hybrid Bacteriocins for Food Biopreservation

The food industry demands new procedures and methods to produce minimally processed, ready to eat food with intact nutritional, taste, and flavor properties. The biopreservation and the use of both bacteriocins produced by lactic acid bacteria (LAB) and bacteriocinogenic strains as an alternative to substitute chemical antimicrobial for food preservation became increasingly important in the last two decades. When the new proposed natural preservatives techniques are applied, probiotics food can be obtained and, simultaneously, foodborne pathogens and spoilage contaminants can diminish. However, bacteriocins produced by LAB have a narrow antibacterial spectrum and are inactive against Gram-negative bacteria like Salmonella and the emergent enterohemorrhagic Escherichia coli. Knowing the mechanism of action and the structural features of microcins synthesized by Gram-negative bacteria and with potent antimicrobial activity against the mentioned microorganism, the proposal is to obtain hybrid peptides (microcin–bacteriocin) with broad antimicrobial spectrum. This review explains how the inability of bacteriocins to cross the outer membrane of Gram-negative bacteria unable them to act on the bacteria. It will also be discussed how a hybrid bacteriocin can be obtained.