Evolution of multidrug resistance in Salmonella enterica serovar Typhimurium as a result of biocide exposure.

该提案旨在更多地了解细菌如何对抗生素产生耐药性，以及哪些因素影响这种情况的发生率。在世界范围内，抗生素耐药性是一个主要问题，近年来越来越多的细菌对三种或更多种抗生素具有多重耐药性（MDR）。细菌可以通过多种方式对抗生素产生耐药性，其中一种机制被称为外排。外排泵能够将抗生素泵出细菌细胞;它们也泵出杀菌剂（消毒剂）。近年来，随着抗生素作为动物生长促进剂的使用减少，农业中杀生物剂的使用增加。人们担心杀生物剂通过选择产生更多外排泵的突变细菌而导致抗生素耐药性。人们感染致病菌的一种途径是通过食物，沙门氏菌是英国胃肠炎的第二大常见原因，大多数感染是通过受污染的食物。在最近的工作中，申请人已经表明，沙门氏菌暴露于农场中使用的各种杀生物剂（消毒剂）可以导致选择MDR突变体，但不能像它们的亲本一样生长。然而，这些实验使用了多次暴露于杀生物剂，目前尚不清楚需要多少次暴露才能选择MDR，以及适应性（生长和感染人类细胞的能力）的丧失是否与MDR的发展有关。该提案旨在确定需要多少杀菌剂暴露来选择MDR，以及通过进行逐步暴露实验并在每次暴露后保持突变体来确定适应性成本是否相关。然后将检查这些突变体，并测量抗生素抗性和适应性。这些实验将允许跟踪MDR随时间的发展。很可能，重复的杀生物剂暴露将选择在其基因组内具有多个突变的突变体。新的测序技术的最新发展已经允许以相对低的成本快速地对整个基因组进行测序。该提案旨在对杀生物剂选择的突变体的基因组进行测序，以鉴定涉及杀生物剂-抗生素交叉抗性的突变。将测量这些突变对所有saltinase基因表达的影响，以及突变体在超过2000种化合物存在下生长的能力。这些结果将有助于为已识别的突变分配功能，并了解它们如何影响抗生素耐药性和适应性。基因组、转录组（所有基因的表达谱）和表型（在一系列条件下生长的能力）的组装是理解抗性机制的有力工具。本研究中确定的突变的重要性将通过两种方式进行评估。首先，将在正常沙门氏菌菌株中重建特定突变，并评估对抗生素耐药性和适应性的影响。其次，将研究一组目前从动物中分离的MDR沙门氏菌是否存在这些突变，以确定它们是否存在于“真实世界”的分离株中。预期成果是：了解杀虫剂选择抗生素耐药性的机制，并评估这是否对人类健康构成威胁。更好地了解沙门氏菌的基本生物学。开发新型抗生素的新靶基因的鉴定。

This proposal aims to understand more about how bacteria become resistant to antibiotics and what factors influence the rate at which this can occur. Around the world antibiotic resistance is a major problem, in recent years increasing numbers of bacteria which are Multiply Drug Resistant (MDR) - resistant to three or more classes of antibiotics are being isolated. Bacteria can become resistant to antibiotics in a number of ways, one mechanism they use is known as efflux. Efflux pumps are able to pump antibiotics out of the bacterial cell; they also pump biocides (disinfectants). The use of biocides in farming has increased in recent years as the use of antibiotics as growth promoters in animals has been reduced. Concern has been raised about the potential for biocides to cause antibiotic resistance by selecting for mutant bacteria which produce a greater number of efflux pumps. One route by which people can become infected with pathogenic bacteria is via food, Salmonella are the second most common cause of gastroenteritis in the U.K and the majority of infections are via contaminated food. In recent work the applicants have shown that exposure of Salmonella to various biocides (disinfectants) used on farms can result in selection of mutants which are MDR but are not able to grow as well as their parent. However these experiments used multiple exposures to biocides and it is not clear how many exposures are needed to select MDR, and whether the loss of fitness (ability to grow and infect human cells) is linked to development of MDR. This proposal aims to identify how many biocide exposures are needed to select MDR and whether the fitness cost is linked by performing stepwise exposure experiments and keeping mutants after each exposure. These mutants will then be examined and the antibiotic resistance and fitness measured. These experiments will allow the development of MDR over time to be followed. It is likely that the repeated biocide exposures will select for mutants with multiple mutations within their genomes. Recent development of new sequencing technologies have allowed whole genomes to be sequenced rapidly for relatively little cost. This proposal aims to sequence the genomes of biocide selected mutants to identify mutations involved in biocide-antibiotic cross resistance. The result of these mutations on expression of all the genes of salmonella will be measured as will the ability of mutants to grow in the presence of over 2000 compounds. These results will help assign function to the mutations identified and understand how they affect antibiotic resistance and fitness. The assembly of genome, transcriptome (expression profile of all genes) and phenotype (ability to grow in a range of conditions) is a powerful tool to understand the mechanisms of resistance. The importance of mutations identified in this study will be assessed in two ways. Firstly specific mutations will be re-created in a normal Salmonella strain and the effect on antibiotic resistance and fitness evaluated. Secondly a group of current isolates of MDR Salmonella from animals will be studied for the presence of these mutations in order to identify whether they are found in 'real-world' isolates. The expected outcomes are: An understanding of the mechanisms by which biocides can select antibiotic resistance and an evaluation of whether this is a threat to human health. A better understanding of the fundamental biology of Salmonella. Identification of new target genes for development of novel antibiotics.