Molecular epidemiology and antimicrobial resistance spread of environmental Escherichia coli isolates: A one health approach

Abstract

Antibiotic resistant bacteria are present in wastewaters as their elimination during treatment in wastewater treatment plants is often impossible. Water plays an important role in the spread of these microorganisms among humans, animals and the environment. Unfortunately, in Greece knowledge on prevalence and diversity of antibiotic resistance bacteria in environmental habitats is very limited. Therefore, this doctoral dissertation was designed to study antimicrobial resistance under the One Health approach and aimed to a) assess the antibiotic resistance patterns and detect the antibiotic resistance genes related to resistant phenotypes, b) identify molecular genotypes, c) compare resistance patterns and genotypes between clinical and environmental E. coli isolates and d) identify molecular mechanism contributing to antimicrobial resistance spread both in clinical settings and in environment (aquatic and wastewater). For this reason, during the thesis, a total of 139 clinical and 502 environmental E. coli isolates were collected. Environmental isolates were obtained from semi-treated hospital wastewater, treated wastewater, and river water samples. All these isolates (clinical and environmental) are spatially and temporally related. In order to examine the circulated phylogenies in the clinical settings and in different environmental habitats all isolates were subjected to the molecular typing technique of phylogrouping. This method shown that the phylogenetic group B2 was predominant in clinical settings (60%; 84/139) and the second most frequent among wastewaters, whereas group A was dominant in all environmental isolates (48%, 242/502). To determine the prevalent resistance patterns, all isolates (both clinical and environmental) were evaluated for their susceptibility to 18 commonly used antibiotics. Based on the results, the vast majority of both environmental and clinical isolates were resistant, particularly to penicillins. In addition, 84 isolates (73 environmental and 11 clinical) exhibiting resistant or multidrug-resistant profiles associated with β-lactamases were identified and analyzed for β-lactamase genes. The blaCTX-M-group 1 gene was found in 52 isolates (62%; 52/84), making it the most frequently encountered β-lactamase gene among both clinical and environmental isolates. Other β-lactamase genes detected included blaCTX-M-group 9 (8.4%; 7/84), blaTEM (14.3%; 12/84), blaSHV (20.2%; 17/84), blaOXA-244 (1.2%; 1/84), blaCMY-2 (2.4%; 2/84), blaDHA-1 (1.2%; 1/84), and blaFOX-17 (1.2%; 1/84). Finally, plasmid analysis, conjugation assay and plasmid sequencing were implemented in certain β- lactamase producing isolates to investigate the molecular environment of resistance genes and others molecular mechanisms which probable contributing to resistance dissemination. Out of the 33 isolates initially selected for the conjugation assay, only thirteen (39.4%; 13/33) appeared to contain conjugative plasmids and consequently the ability to transmit resistance το β-lactamases. Sequencing analysis was applied in three plasmids which were isolated from one clinical and two environmental E. coli and carried β-lactamase genes. Specifically, the three plasmids were ptrc203cli, ptrc618, and ptrc297, which respectively carried the β-lactamase genes blaDHA-1, blaCTX-M-14, and blaSHV-12. The first two plasmids belong to the compatibility group IncFII, while the last one belongs to the IncX3 group. Additionally, these conjugative plasmids not only carried the aforementioned β-lactamase genes but also additional resistance genes related to resistance to other categories of antibiotics. Specifically, ptrc203cli also co- carried resistance genes for sulfonamides (sul1), trimethoprim (drfA17), and fluoroquinolones (qnrB4); the plasmid ptrc618 harbored resistance genes for aminoglycosides (aac6’-Ib3), macrolides (mphA), and chloramphenicol (cmlA1); and ptrc297 carried a resistance gene for quinolones (qnrS1). The results also showed that all of the resistance genes were embedded within mobile elements (IS elements and integrons), which contribute to the further spread of multidrug resistance. In conclusion, this doctoral thesis reports confirming data that river water and wastewater serve as reservoirs of antibiotic resistant bacteria and as vehicles for the transmission of resistance genes to various bacterial species.