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

Highlights

• Urinary tract infections and dog as reservoir and animal model

• E. coli isolates from UTI and antibiotic resistance to enrofloxacin

• Differential protein expression of enrofloxacin-induced E. coli isolate

• Resistance mechanisms involve membrane permeability and stabilization of DNA.

Abstract

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.

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.

The isolated E. coli has been induced with enrofloxacin and studied through 2D DIGE and shotgun MS.

Discovered differentially expressed proteins are principally involved in antibiotic resistance and linked to oxidative stress response, to DNA protection and to 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. All 2D DIGE and MS data have been deposited into the ProteomeXchange Consortium with identifier PXD002000 and DOI http://dx.doi.org/10.6019/PXD002000.

This article is part of a Special Issue entitled: HUPO 2014.

Introduction

Antibiotic resistance in microbes in general and particularly in Escherichia coli represents a phenomenon that is dramatically increasing in the last decade. This is due to the use of high amounts of antibiotics in all fields of zootechnical industry [1], [2], [3], [4], [5].

E. coli also represents a serious burden for human health because of its spread in the environment. It could be found in skin, in surgical infections and in several other types of epithelial tissues. Because of its high presence in the environment, its infection could be quite common and antibiotic resistance actually represents a serious problem especially in the light of last experimental evidences that document how E. coli is developing high rates of multidrug resistance [6].

One of the most frequent infections of this pathogen for humans is caused by extra-intestinal pathogenic E. coli (ExPEC). It can colonize urinary tract causing urinary tract infections (UTIs). In worst cases, septicemia and meningitis can occur [7]. It has been estimated that about 50% of all women can suffer from UTI at least one time in their life [8] and that in most cases it could be recurrent. The analysis of the genome of several E. coli isolates demonstrated the direct link between production animals, meat and human UTI giving the basis to consider UTI as a real zoonosis [9]. The mechanisms behind the consumption of food from animal production and UTI are still unknown, however, there are evidences that document how resistant E. coli can jump from food to human extraintestinal pathogenic E. coli [10]. The similarity between UTI animal infections and human was documented by Johnson and colleagues who also proposed the dog as animal model and natural reservoir [11], [12]. In this article, the role of dog as reservoir of “human” uropathogenic E. coli for acquisition by susceptible human hosts is well described [11], [13], [14]. According to the described experimental evidences it is possible to hypothesize the jump of mutated or resistant strains of E. coli from dog to humans. The antibiotic resistance of E. coli isolates from dogs has been as well already documented [15] and in particular to enrofloxacin that actually represents one of the most used antibiotics to counteract this infection [16]. Fluoroquinolones are inhibitors of bacterial DNA replication through the block of topoisomerase and DNA gyrase [17]. Thus, this results in DNA supercoiling and in related DNA damage [18]. The genetic mechanism of E. coli resistance to fluoroquinolone action has already been documented [19]. Different experiments show how the development of antibiotic resistance could be due to mutations of the genes encoding DNA gyrase and topoisomerase IV [20]. However, this adaptation is not the only one responsible for the development of E. coli fluoroquinolone resistance, that depends not directly from genome mutations but for example from the loss of membrane porins or the augmented drug extrusion through the efflux pumps [20].

Apart from these evidences, the cellular response and the adaptive mechanisms developed by E. coli to counteract the action of this drug still have to be elucidated through the analysis of the differential proteome. In the last decades, the study of proteome in antibiotic resistance provided a valid contribution to a better understanding of the mechanisms of this phenomenon. Therefore, the aim of this study was to perform a deeper investigation of possible molecular pathways involved in the antibiotic resistance.

The differential proteomic profiling between sensitive and enrofloxacin-resistant (induced) E. coli isolates from UTIs of dogs has been evaluated. The E. coli isolate was treated with growing concentration of enrofloxacin up to 10 μg/ml. Both sensitive and exposed isolates were discriminated through the biochemical and antibiotic resistance profiles and the proteomic analysis has been performed by two complementary methodologies based on 2D DIGE and shotgun MS analysis. These approaches led us to highlight some proteins differentially expressed that could play a key role in antibiotic resistance and could become possible targets to counteract multidrug resistance.