Genetic Characterization of Antimicrobial-Resistant Escherichia coli Isolated from a Mixed-Use Watershed in Northeast Georgia, USA

Abstract

In order to determine the role of surface water in the development and spread of antibiotic-resistant (AR) bacteria, water samples were collected quarterly from 2015 to 2016 from a mixed-use watershed in Georgia. In our previous study, 496 Escherichia coli were isolated from surface water, out of which, 34 isolates were resistant to antimicrobials. For the current study, these 34 AR E. coli were characterized using pulsed-field gel electrophoresis, AR gene detection, plasmid replicon typing, class I integron detection, and multi-locus sequence typing. Genes were identified as conferring resistance to azithromycin (mph(A)); β-lactams (bla_CMY, bla_CTX, bla_TEM); chloramphenicol (floR); streptomycin (strA, strB); sulfisoxazole (sul1, sul2); tetracycline (tetA, tetB, tetC); and trimethoprim/sulfamethoxazole (dhfr5, dhfr12). Five ciprofloxacin- and/or nalidixic-resistant isolates contained point mutations in gyrA and/or parC. Most of the isolates (n = 28) carried plasmids and three were positive for class I integrons. Twenty-nine sequence types (ST) were detected, including three epidemic urinary-tract-infection-associated ST131 isolates. One of the ST131 E. coli isolates exhibited an extended-spectrum β-lactamase (ESBL) phenotype and carried bla_CTX-M-15 and bla_TEM-1. To our knowledge, this is the first study on the emergence of an ESBL-producing E. coli ST131 from environmental water in the USA, which poses a potential risk to human health through the recreational, agricultural, or municipal use of this natural resource. This study identified E. coli with AR mechanisms to commonly used antimicrobials and carrying mobile genetic elements, which could transfer AR genes to other bacteria in the aquatic environment.

Keywords: E. coli; ST131; antimicrobial resistance; extended spectrum β-lactamase (ESBL); surface water.

Figure 1

Map of water sampling sites in the Upper Oconee Watershed near Athens, GA. Sampling sites where antimicrobial resistant E. coli were isolated are labeled and are symbolized as red circles. Points of interest (circles—cities, triangles—water reclamation facilities, and pentagons—hospitals) are also labeled. The National Hydrography Dataset (NHD) streams are shown for reference. Inset map shows the Upper Oconee watershed in grey and Athens, GA, as a black triangle.

Figure 2

Dendrogram of 34 antimicrobial-resistant E. coli isolates recovered from Upper Oconee Watershed near Athens, GA. Their pulsed-field gel electrophoresis (PFGE) analysis, antimicrobial resistance patterns, replicon types, multilocus sequence typing (MLST), and phylogenetic groups are shown. Black boxes represent resistance to antimicrobials or the presence of plasmids, and beige boxes represent susceptibility to antimicrobials or absence of plasmids. Antimicrobials: azithromycin (AZI), chloramphenicol (CHL), gentamicin (GEN), nalidixic acid (NAL), streptomycin (STR), tetracycline (TET), ceftiofur (TIO), ceftriaxone (AXO), ampicillin (AMP), amoxicillin/clavulanic acid (AMO), cefoxitin (FOX), ciprofloxacin (CIP), sulfisoxazole (SUL), and trimethoprim/sulfamethoxazole (TRI).

Figure 3

Map of water sampling sites in the Brooklyn Creek watershed in Athens, GA. Sampling site where extended-spectrum β-lactamase (ESBL)-producing E. coli was isolated (MIDO 826) is colored red. Sampling sites where Non-ESBL-producing E. coli were isolated are in blue. Hospitals are symbolized as black triangles.