Mechanisms of antibiotic resistance to enrofloxacin in uropathogenic Escherichia coli in dog

Escherichia coli (E. coli) urinary tract infections (UTIs) are becoming a serious problem both for pets and humans (zoonosis) due to the close contact and to the increasing resistance to antibiotics. Great progress has been made in the discovery of novel antibiotics to counteract E. coli pathogens. However, this huge progress has been undercut by the evolution of bacteria in the way of drug resistance. Enrofloxacin is one of the most efficient antibiotics against E. coli pathogens and there is considerable evidence that documents how this microorganism is becoming more resistant to this antibiotic and is developing multidrug resistance. This study has been performed in order to unravel the mechanism of induced enrofloxacin resistance in canine E. coli isolates that represent a good tool to study this pathology. For a comprehensive investigation about antibiotic resistance mechanisms, an E. coli isolate from urinary tract infection (UTI) was induced to grow under a concentration of 10 µg/ml of enrofloxacin. Proteins represent the main contributors to the mechanisms involved in antibiotic resistance in bacteria. Their abundance profile provides reliable information about the mechanisms involved in this process. Three biological replicates of control and resistant isolate have been analyzed both through 2D DIGE and shotgun proteomics. In our case, prior to perform the proteomics analysis, the screening with MS profiling of control and resistant isolates was performed highlighting the differences among strains and successfully clustering experimental strains according to PC analysis (PCA). The results clearly demonstrate differential profiles in peptide expression among clusters and between isolate and reference strain. According to these preliminary results, the protein profile of the resistant group was compared with the one of its sensitive counterpart by a proteomic analysis based on 2D-DIGE to separate proteins combined to MALDI MS analysis and database search to identify differentially expressed proteins. This analysis showed the modulation of 19 proteins between the two conditions. At the same time, another comparative proteomics investigation was also carried out by free-label nLC-MSE. This method has allowed the qualitative and quantitative analysis of total protein extracts of the two groups for the simultaneous screening of a larger number of proteins. A total of 57 differentially expressed proteins were identified and a comprehensive study on the proteins associated with enrofloxacin resistance was carried out. We classified these modulated proteins by their molecular functions and pathways where they are principally involved. This approach allowed to highlight some pathways that can be involved in an increased antibiotic resistance. Two major mechanisms were discovered. The first one was related to the stabilization of DNA structure through the up-regulation of Dsp protein. The second one was the downregulation of outer membrane protein W in order to reduce the membrane permeability to enrofloxacin. Discovered differentilally expressed proteins are principally involved in antibiotic resistance and linked to the oxidative stress response, to DNA protection and in membrane permeability. Moreover, since enrofloxacin is an inhibitor of DNA gyrase, the overexpression of DNA starvation/stationary phase protection protein (Dsp) could be a central point to discover the mechanism of this clone to counteract the effects of enrofloxacin. In parallel, the dramatic decrease of the synthesis of the outer membrane protein W, which represents one of the main gates for enrofloxacin entrance, could explain additional mechanism of E. coli defense against this antibiotic. The proteins differentially expressed could represent putative targets for the development of new strategies to counteract drug and multidrug resistance.

大肠埃希菌（Escherichia coli，E. coli）尿路感染（urinary tract infection，UTI）作为一种人畜共患病（zoonosis），由于人与宠物间的密切接触以及细菌对抗生素的耐药性持续攀升，正逐渐成为威胁宠物与人类健康的严峻问题。学界在研发可对抗埃希菌病原体的新型抗生素方面已取得重大进展，然而，细菌耐药性的演化极大地抵消了这一研究进展。恩诺沙星（enrofloxacin）是对抗埃希菌病原体最有效的抗生素之一，现有大量研究证据表明，该菌对该抗生素的耐药性不断增强，且正逐步发展出多重耐药性。本研究旨在阐明犬源埃希菌分离株中诱导型恩诺沙星耐药性的产生机制，这类分离株是研究该病理过程的理想模型。为全面探究抗生素耐药性的相关机制，研究人员将一株源自尿路感染的埃希菌分离株置于浓度为10 µg/ml的恩诺沙星环境中，诱导其产生耐药性。蛋白质是细菌抗生素耐药性相关机制的核心介导因子，其表达丰度谱可为该过程的相关机制提供可靠的研究依据。研究采用双向差异凝胶电泳（two-dimensional difference gel electrophoresis，2D DIGE）与鸟枪法蛋白质组学（shotgun proteomics）两种技术，对对照组与耐药株分离株各3次生物学重复样本进行了分析。在开展蛋白质组学分析前，研究人员先通过质谱分析（mass spectrometry profiling，MS profiling）对对照组与耐药株分离株进行了筛选，明确了菌株间的差异，并通过主成分分析（principal component analysis，PCA）成功实现了实验菌株的聚类。分析结果清晰显示，不同聚类组间以及分离株与参考菌株间的肽段表达谱存在显著差异。基于上述初步结果，研究人员采用基于双向差异凝胶电泳的蛋白质组学分析方法分离蛋白质，结合基质辅助激光解吸电离质谱（matrix-assisted laser desorption/ionization mass spectrometry，MALDI MS）分析与数据库检索，对耐药组与敏感对照组的蛋白质谱进行了对比，以鉴定差异表达蛋白质。该分析共鉴定出19种在两组间存在表达调控差异的蛋白质。与此同时，研究人员还采用无标记液相色谱-质谱联用法（label-free nLC-MSE）开展了另一项对比蛋白质组学研究。该方法可对两组的总蛋白质提取物进行定性与定量分析，实现对大量蛋白质的同步筛选。该研究共鉴定出57种差异表达蛋白质，并对与恩诺沙星耐药性相关的蛋白质开展了全面分析。研究人员根据这些差异调控蛋白质的分子功能及其参与的主要通路对其进行了分类。通过该分类方法，研究人员明确了若干可能与抗生素耐药性增强相关的通路。研究发现了两种主要的耐药机制：其一为通过上调Dsp蛋白质（Dsp protein）稳定DNA结构；其二为下调外膜蛋白W（outer membrane protein W）的表达，以降低细菌细胞膜对恩诺沙星的通透性。所鉴定出的差异表达蛋白质主要参与抗生素耐药性过程，并与氧化应激应答、DNA保护以及细胞膜通透性调控相关。此外，由于恩诺沙星是DNA旋转酶（DNA gyrase）的抑制剂，DNA饥饿/静止期保护蛋白（DNA starvation/stationary phase protection protein，Dsp）的过表达可能是该菌株对抗恩诺沙星作用机制的核心环节。与此同时，作为恩诺沙星进入细菌的主要通道之一的外膜蛋白W的合成量大幅下降，这可解释埃希菌对抗该抗生素的另一重防御机制。这些差异表达蛋白质有望成为研发新型策略以对抗单药及多重耐药性的潜在靶点。