Elucidating the mechanisms and factors influencing biofilm formation in foods

该申请要求继续支持生物膜抗菌素耐药性研究。细菌生物膜在表面形成，在那里它们对抗菌剂和其他压力源的抵抗力比在液体悬浮液中生长的对应细菌更强。因此，生物膜往往在食品加工厂的抗菌素处理和卫生制度下存活下来，在那里它们迅速再生，并导致下游食品质量损失、产品保质期缩短或食源性疾病。抗菌药物治疗后生物膜存活的机制尚不完全清楚；因此，有效的生物膜控制策略仍然是一个具有社会和经济重要性的目标。我的研究项目的核心集中在阐明生物膜的结构和代谢反应途径，这些生物膜有助于增强对抗菌药物（如苯扎氯铵、氯己定和三氯生）的耐药性，使用野生型和突变型肠沙门氏菌、大肠杆菌O157菌株和定义的共培养系统。据推测，先前的胁迫事件（低温生长）和共培养生物膜中保护性微生境的形成都诱导了胁迫反应途径，从而增强了抗性。将使用共聚焦激光显微镜（CLM）和全细胞蛋白表达模式对预处理（适应）和未适应的生物膜进行比较研究，以记录效果。荧光探针和CLM将在处理和未处理的生物膜中提供生物膜细胞状况的原位物理和代谢信息，而蛋白质分析将揭示对抗菌剂的适应以及生物膜内的位置如何影响生物膜代谢过程。通过将生物膜和重悬的生物膜细菌置于一组施加的压力（例如，抗菌剂和热量）并确定它们各自的存活率，将评估“先前生物膜压力”的总体影响。本研究还将采用相关显微镜方法（同步软x射线透射显微镜，原子力显微镜，共聚焦显微镜）来检查表面细菌-抗菌相互作用，以及食品级纳米材料对细菌的影响。

This submission requests continued support for the study of biofilm antimicrobial resistance. Bacterial biofilms develop on surfaces where they become more resistant to antimicrobial agents and other stressors than their counterpart bacteria grown in liquid suspension. Biofilms thus tend to survive antimicrobial treatment and sanitation regimes in food processing plants, where they rapidly regrow and contribute to downstream losses in food quality, reduced product shelf life, or food borne disease. The mechanisms responsible for biofilm survival following antimicrobial treatment are not entirely clear; consequently, effective biofilm control strategies remain a goal of social and economic importance. The core of my research program focuses on elucidating structural and metabolic response pathways of biofilms that contribute to enhanced resistance to antimicrobial agents such as benzalkonium chloride, chlorhexidine and triclosan, using wild-type and mutant strains of Salmonella enterica, Escherichia coli O157, and defined co-culture systems. It is hypothesized that prior stress events (growth at low temperature) and formation of protective microniches in co-culture biofilms, both induce stress response pathways that lead to enhanced resistance. Comparative studies of pre-stressed (adapted) and unadapted biofilms will be performed using confocal laser microscopy (CLM) and whole cell protein expression patterns to document effects. Fluorescent probes and CLM will provide in situ physical and metabolic information on biofilm cell condition in treated and untreated biofilms, whereas protein analyses will reveal how adaptation to antimicrobial agents, as well as location within the biofilm, affect biofilm metabolic processes. The overall effect of 'prior biofilm stress' will be evaluated by subjecting biofilms and resuspended biofilm bacteria to a panel of imposed stresses (e.g., antimicrobial agents and heat) and determining their respective survival. This research will also employ correlative microscopy approaches (synchrotron soft x-ray transmission microscopy, atomic force microscopy, confocal microscopy) to examine bacterial-antimicrobial interactions on surfaces, as well as the effects of food-grade nanomaterials on bacteria.