CTX-M-Producing Bacteria Isolated from a Highly Polluted River System in Portugal

Abstract

Enterobacteriaceae resistant to third-generation cephalosporins are a great concern for public health, as these are first-line drugs to treat infections. The production of carbapenemases and extended spectrum beta-lactamases (ESBLs) and/or the overexpression of AmpC β-lactamases are the main mechanisms of resistance to these antibiotics. Among the ESBLs, CTX-M β-lactamases are the most prevalent worldwide. Our aims were to determine the prevalence of cefotaxime-resistant Enterobacteriaceae along a heavily polluted river and characterize bla_CTX-M carriers. River water was collected in 11 sites along the main course and tributaries, in two sampling moments. Water quality was evaluated and a collection of cefotaxime-resistant isolates was obtained. bla_CTX-M carriers were characterized regarding phylogenetic affiliation, clonality, antibiotic susceptibility, gene diversity, and context. Water presented very low quality in all sites. From 147 cefotaxime-resistant isolates, 46% carried bla_CTX-M and were affiliated with Escherichia, Klebsiella, Enterobacter, and Citrobacter. Molecular typing revealed clonal isolates in different sites and over the two years, suggesting survival of the strains in the river or continuous pollution inputs from the same sources. Eight variants of bla_CTX-M were found, with bla_CTX-M-15 being the most prevalent (52.5%). Sites with a lower water quality showed the highest resistance rates and prevalence of bla_CTX-M, suggesting that river water may embody human health risks.

Keywords: Enterobacteriaceae; antibiotic resistance; blaCTX-M; pig farms; river pollution; β-lactamases.

Figure 1

Sampling sites (P1–P15), coordinates, and other information and map showing sites (red dots) located in the Lis River main course (P1–P3, P6, P9, P12–P15) and tributaries (P4, P5, P7, P8, P10). WWTPs are indicated using yellow dots and the density of swine farms is in orange (from Teixeira et al., 2022 [39]).

Figure 2

Proportion of cefotaxime-resistant bacteria determined in all sampling sites located in the Lis River from spring (P1) to mouth (P15) (P1–P3, P6, P9, P12–P15; black) and three affluents (P4/P5, P7/P8, and P10/P11; light grey); Sites located downstream WWTP and in the vicinities of pig farms are indicated by * and #, respectively.

Figure 3

CTX-M encoding genes detected in the Lis riverine system. Black dots for Escherichia isolates, red for Klebsiella, and blue for Citrobacter or Enterobacter. Sites located downstream WWTP and in the vicinities of pig farms are indicated by * and #, respectively.

Figure 4

Genomic environments for the variants of the bla_CTX-M gene found in the isolates. Ten different environments were observed, and they are represented from (A–J).