Development of a predictive in-vitro model to study preservative resistance development in Pseudomonas aeruginosa

化学防腐剂是食品、营养和健康领域的重要工具，被添加到家庭和个人护理（HPC）产品中，以防止微生物生长，并确保数百万消费者每天使用这些产品时保持产品的安全性。消费者如何使用HPC产品的性质导致潜在的微生物接种，例如护肤霜和联合利华销售的许多HPC产品依赖于防腐剂来防止微生物腐败。因此，HC和PC液体配方的未来防腐系统是过去几年研发所面临的最大技术挑战之一。化学防腐剂正在接受消费者安全、环境影响和公共关系意见的严格审查。HPC行业正在通过去除快速作用的防腐剂（如甲醛供体和异噻唑啉酮），并用具有较慢杀灭效力的温和化学品（如有机酸）取代它们来应对;这会带来更大的防腐剂抗性风险。对防腐剂的耐药性可能是由于：低于有效浓度或微生物生物负荷压倒防腐剂。工业生产中抗菌素耐药性的主要促进因素是生物膜的形成，生物膜的斯劳会短暂地对生产批次造成不同的污染。生物膜阻碍了化学物质的扩散，导致生物膜上的浓度梯度，微生物暴露于亚致死浓度的抗菌剂，从而有机会产生耐药性。对抗抗菌剂耐药性是一项日益严峻的全球挑战;在过去3年中，由于微生物污染而召回的HPC产品有所增加。公共召回的微生物污染事件成本> 75万欧元，贸易召回> 5万欧元;对品牌的声誉损害可能会给企业带来更大的成本。在2014/15财政年度，由于微生物污染，联合利华对HPC进行了两次公开召回和一次贸易召回，费用超过150万欧元。假单胞菌是HPC产品最常见的工业微生物污染物。假单胞菌属是一种普遍存在的微生物属，通常从水中分离。因此，铜绿假单胞菌是引起产品腐败的主要微生物，也是了解防腐剂耐药性的极好模式生物。了解假单胞菌的防腐剂抗性机制和生物膜形成的重要性对于优化新的防腐策略非常重要。本项目将在假单胞菌分离株中开发对一系列防腐剂的体外抗性，阐明对新的渐进式杀灭防腐剂的抗性机制。将通过基因组和转录组学分析对体外耐药假单胞菌分离株和天然存在的耐药工业污染假单胞菌分离株进行比较，以确定体外耐药机制是否反映了联合利华工厂中产生的耐药机制。特别是，我们的目标是实现以下目标：验证对一系列防腐化学品产生抗性的选定方法，以确定利用更广泛的防腐化学品进一步产生抗性的首选方法，假单胞菌属分离株对策略性防腐化学品（包括二元和三元组合）的抗性的产生。使用体外和离体抗性假单胞菌属分离株的下一代测序分析来确定抗性的基因组和转录组学基础。比较耐药性机制，以确定体外产生的耐药性是否代表工业环境中自然产生的耐药性。评价体外耐药性生成模型作为防腐剂耐药性发展研究工具的能力。

Chemical preservatives, essential tools in the context of food, nutrition and health, are added to home and personal care (HPC) products to prevent microorganism growth and ensure products remain safe to use by the millions of consumers that utilise these products daily. The nature of how consumers use HPC products results in potential microbial inoculation e.g. skin creams and many of the HPC products Unilever sells rely on preservatives to prevent microbial spoilage. As a result, future-proofing preservative systems for HC and PC liquid formulations is one of the biggest technical challenges faced by R&D over the past couple of years.Chemical preservatives are undergoing intense scrutiny for consumer safety, environmental impact and public relation opinion. The HPC industry is responding by removing fast acting preservatives such as formaldehyde donors and isothiazolinones and replacing them with milder chemicals that have a slower kill efficacy, such as organic acids; which introduces greater risk of preservative resistance. Resistance to preservatives can occur due to: sub-effective concentrations or microorganism bioburden overwhelming the preservative. A leading facilitator of antimicrobial resistance for industrial manufacturing is the formation of biofilms that slough transiently giving variable contamination of manufactured batches. Biofilms occlude the diffusion of chemicals resulting in a concentration gradient across the biofilm and microorganisms exposed to sub-lethal concentration of antimicrobials giving an opportunity to develop resistance.Combatting antimicrobial resistance is an increasing global challenge; HPC products recalled due to microbial contamination has increased over the past 3 years. A microbiological contamination incident for a public recall costs >750K euro and a trade recall >50K euro; the reputational damage to a brand can be a significantly greater cost to the business. In the financial year 2014/15 two public and one trade recall in HPC due to microbiological contamination cost Unilever >1.5 million euros.Pseudomonas spp are the most common industrial microbial contaminants of HPC products. Pseudomonas spp are a ubiquitous genus of microorganisms routinely isolated from water. Consequently, P. aeruginosa is a leading microorganism of concern for product spoilage and an excellent model organism to develop understanding of preservative resistance. Understanding preservative resistance mechanisms and the importance of biofilm formation of Pseudomonas spp is very important to optimise new preservation strategies.This project will develop in-vitro resistance to a range of preservatives in Pseudomonas isolates, elucidating mechanisms of resistance against new, progressive kill preservation chemicals. A comparison of in-vitro resistant Pseudomonas isolates and naturally occurring resistant industrial contamination Pseudomonas isolates will be performed by genomic and transcriptomic analysis to identify if in-vitro resistance mechanisms reflect those generated in Unilever factories.In particular, we aim to achieve the following:Validation of selected methods for generating resistance to a range of preservation chemicals to determine the preferred methodology for further resistance development utilising a wider range of preservation chemicals and isolates.Generation of resistance to strategic preservation chemicals including binary and tertiary combinations in Pseudomonas isolates.Use of next generation sequencing analysis of in-vitro and ex-vivo resistant Pseudomonas isolates to determine genomic and transcriptomic basis for resistance. Comparison of resistance mechanisms to determine whether resistance generated in-vitro is representative of resistance occurring naturally within an industrial environment. Evaluation of in-vitro resistance generation model capability as an investigative tool for preservative resistance development.