The widespread dissemination of integrons throughout bacterial communities in a riverine system

Abstract

Anthropogenic inputs increase levels of antimicrobial resistance (AMR) in the environment, however, it is unknown how these inputs create this observed increase, and if anthropogenic sources impact AMR in environmental bacteria. The aim of this study was to characterise the role of waste water treatment plants (WWTPs) in the dissemination of class 1 integrons (CL1s) in the riverine environment. Using sample sites from upstream and downstream of a WWTP, we demonstrate through isolation and culture-independent analysis that WWTP effluent significantly increases both CL1 abundance and antibiotic resistance in the riverine environment. Characterisation of CL1-bearing isolates revealed that CL1s were distributed across a diverse range of bacteria, with identical complex genetic resistance determinants isolated from both human-associated and common environmental bacteria across connected sites. Over half of sequenced CL1s lacked the 3'-conserved sequence ('atypical' CL1s); surprisingly, bacteria carrying atypical CL1s were on average resistant to more antibiotics than bacteria carrying 3'-CS CL1s. Quaternary ammonium compound (QAC) resistance genes were observed across 75% of sequenced CL1 gene cassette arrays. Chemical data analysis indicated high levels of boron (a detergent marker) downstream of the WWTP. Subsequent phenotypic screening of CL1-bearing isolates demonstrated that ~90% were resistant to QAC detergents, with in vitro experiments demonstrating that QACs could solely select for the transfer of clinical antibiotic resistance genes to a naive Escherichia coli recipient. In conclusion, this study highlights the significant impact of WWTPs on environmental AMR, and demonstrates the widespread carriage of clinically important resistance determinants by environmentally associated bacteria.

Fig. 1

Prevalence of CL1s, 3’-CS region of CL1s, and QAC resistance genes qacE and qacH, as determined by qPCR for three sites downstream of WWTP Finham (DS1, DS2 and DS3) and three sites upstream of WWTP Finham (US1, US2 and US3). Error bars are two standard error units based on three biological replicates

Fig. 2

Resistant quotients for bacteria isolated from a river downstream of an effluent discharge in comparison to upstream. a Escherichia coli, b presumptive coliforms except for E. coli, c Gram-negative Proteobacteria (non-coliforms). * denotes significant differences

Fig. 3

Species and sequence type composition of CL1-containing isolates broken into three bacterial groups: Gram-negative non-coliforms, presumptive coliforms excluding E. coli (PCEs) and E. coli. DS non-coliforms n = 44, US non-coliforms n = 26 DS PCEs n = 13, US PCEs n = 6, DS E. coli STs n = 45, US E. coli STs = 17

Fig. 4

Schematic representation of the different types of gene cassette arrays found throughout CL1-bearing bacteria. CL1s with 100 % sequence nucleotide identity between identified features were grouped, with N = the number of isolates recovered carrying the specified gene cassette array. C1–C8 refer to clinical CL1s. A1–A10 refer to atypical CL1s. Recombination sites (attC and attI) are denoted by grey boxes. All sequences of CL1s have been submitted to Genbank (Accession MF686098-MF686115).

Fig. 5

Comparison of the number of isolates resistant to a panel of 12 tested antibiotics for (a) DS Atypical CL1s, vs. DS 3′-CS CL1s (b) DS Enterobacteriaceae vs. US Enterobacteriaceae. * denotes significant differences