Antimicrobial-resistant bacteria and microbiota identified among flies and their sympatric animals

Abstract

Antimicrobial resistance is rising globally at an alarming rate as one of the most serious public health challenges. There is increasing evidence that the environment plays a major role in the development and transmission of antimicrobial resistance genes. However, studies on the environmental connection to the spread of these important genes are scarce. Filth flies have well adapted to live in close association with humans and animals. They are attracted to microbe-rich organic materials for reproductive purposes. As a result, they can acquire bacteria including antimicrobial-resistant bacteria from animal wastes and act as a potential vector of bacterial infections. The research conducted for this dissertation aims to investigate the role of flies as a sentinel candidate for surveillance of antimicrobial resistance by isolating antimicrobial-resistant bacteria from flies and analyzing the relationship between flies, their environment, and the antimicrobial resistance shared between them. Flies were collected from six locations in Auburn, Alabama and split into three sets for analysis. Additionally, fecal samples were collected from sympatric animals in four animals’ husbandries at the same time flies were trapped. Escherichia coli and Klebsiella pneumoniae were isolated from 38.0% and 16.7%, respectively, of flies. These bacteria included multidrug-resistant and extended-spectrum beta-lactamase (ESBL)-producing strains. Notably, two flies at a dog kennel were found to have ESBL-producing E. coli isolates that belong to the same sequence type (ST68) and serotype (O25:H6). Moreover, those isolates were found to harbor the same virulence and antimicrobial resistance genes identified by whole genome sequencing, indicating that the isolates are closely related, and flies might have acquired them from the same source. Microbiota and antimicrobial resistance genes as well as mobile genetic elements (ARG/MGE) detected in flies from the second set were compared to those present in fecal samples of sympatric animals. Microbiota of the flies were found to be more similar to the microbiota of the feces of their sympatric animals than to microbiota of feces of other animals. Similarly, resistome analysis of ARG/MGE showed that 91.7% of ARG/MGE detected in flies were also found in feces. Flies in the third set were used for isolation of a total of 485 colistin-resistant bacteria, the majority of which were naturally resistant bacteria. A colistin-resistant K. pneumoniae strain was analyzed by whole genome sequencing. The genome does not harbor any known genes or mutations conferring resistance to colistin. However, the genome contains various virulence determinants involved in adhesion, iron acquisition and, most importantly, the regulator gene magA, which contributes to K. pneumoniae virulence by hypermucoviscosity phenotype. In total, the analysis of these flies and feces of sympatric animals shows that flies might mirror bacterial communities from the environment, including those with clinical importance and antimicrobial resistance phenotype. However, further investigation with long-term studies on more geospatially distinct populations is needed to determine the suitability of flies as sentinels for antimicrobial resistance surveillance programs.