Mechanisms of antibiotic resistance to enrofloxacin in uropathogenic Escherichia coli in dog☆
Graphical abstract
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.
Section snippets
Bacterial culture
E. coli strain was chosen among the collection of canine isolates from urine of dogs affected by cystitis. Briefly, 200 μl of urine sample was streaked onto blood agar plates (Oxoid, Italy) and incubated at 37 °C for 18–24 h under aerobic condition.
Bacterial identification was performed by evaluating the colony morphology, using Gram staining and biochemical tests; also API 20E (BioMériéux, France) was used to identify E. coli strain more accurately.
For induction of antibiotic resistance,
Results
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 groups have been analyzed both through 2D DIGE and shotgun proteomics. The whole dataset is described below.
Discussion
In the present study, a comparative proteomic investigation was performed to identify proteins associated with resistance to enrofloxacin in E. coli, allowing us to get deeper insights in the complex mechanism of acquired resistance to this antibiotic.
Biochemical and antibiotic resistance profiling were performed in order to give more completeness to this study. Data showed in Table 2, Table 3 of supplementary material highlighted that both control and resistant bacteria had exactly the same
Conclusion
The applied complementary approach described allowed the identification of key proteins and pathways involved in the development of antibiotic resistance due to E. coli growth in presence of enrofloxacin. Bioinformatics analysis, as showed in Fig. 5, demonstrates how the proteins involved in cell replication process are more expressed in the control isolates. These proteins are involved in cell division process as (FtsZ), replicative DNA helicase and ATP-dependent Lon protease. All these
Acknowledgments and funding
Authors are grateful to the COST action 1002 FAP for the network provided and to the project “Piano di Sviluppo - UNIMI” (to A.S.) for the support.
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This article is part of a Special Issue entitled: HUPO 2014.