We compared the resistomes within polluted and unpolluted rivers, focusing on extended-spectrum beta-lactamase (ESBL) genes, in particular bla _CTX-M . Twelve rivers from a Portuguese hydrographic basin were sampled. Physicochemical and microbiological parameters of water quality were determined, and the results showed that 9 rivers were classified as unpolluted (UP) and that 3 were classified as polluted (P). Of the 225 cefotaxime-resistant strains isolated, 39 were identified as ESBL-producing strains, with 18 carrying a bla _CTX-M gene (15 from P and 3 from UP rivers). Analysis of CTX-M nucleotide sequences showed that 17 isolates produced CTX-M from group 1 (CTX-M-1, -3, -15, and -32) and 1 CTX-M that belonged to group 9 (CTX-M-14). A genetic environment study revealed the presence of different genetic elements previously described for clinical strains. IS Ecp1 was found in the upstream regions of all isolates examined. Culture-independent bla _CTX-M -like libraries were comprised of 16 CTX-M gene variants, with 14 types in the P library and 4 types in UP library, varying from 68% to 99% similarity between them. Besides the much lower level of diversity among CTX-M-like genes from UP sites, the majority were similar to chromosomal ESBLs such as bla _RAHN-1 . The results demonstrate that the occurrence and diversity of bla _CTX-M genes are clearly different between polluted and unpolluted lotic ecosystems; these findings favor the hypothesis that natural environments are reservoirs of resistant bacteria and resistance genes, where anthropogenic-driven selective pressures may be contributing to the persistence and dissemination of genes usually relevant in clinical environments.

Carbapenems are last-resort antibiotics to handle serious infections caused by multiresistant bacteria. The incidence of resistance to these antibiotics has been increasing and new resistance mechanisms have emerged. The dissemination of carbapenem resistance in the environment has been overlooked. The main goal of this research was to assess the prevalence and diversity of carbapenem-resistant bacteria in riverine ecosystems. The presence of frequently reported carbapenemase-encoding genes was inspected. The proportion of imipenem-resistant bacteria was on average 2.24 CFU/ml. Imipenem-resistant strains ( n =110) were identified as Pseudomonas spp., Stenotrophomonas maltophilia , Aeromonas spp., Chromobacterium haemolyticum , Shewanella xiamenensis , and members of Enterobacteriaceae . Carbapenem-resistant bacteria were highly resistant to other beta-lactams such as quinolones, aminoglycosides, chloramphenicol, tetracyclines, and sulfamethoxazole/trimethoprim. Carbapenem resistance was mostly associated with intrinsically resistant bacteria. As intrinsic resistance mechanisms, we have identified the bla _CphA gene in 77.3% of Aeromonas spp., bla _L1 in all S. maltophilia , and bla _OXA-48-like in all S. xiamenensis . As acquired resistance mechanisms, we have detected the bla _VIM-2 gene in six Pseudomonas spp. (5.45%). Integrons with gene cassettes encoding resistance to aminoglycosides ( aacA and aacC genes), trimethoprim ( dfrB1b ), and carbapenems ( bla _VIM-2 ) were found in Pseudomonas spp. Results suggest that carbapenem resistance dissemination in riverine ecosystems is still at an early stage. Nevertheless, monitoring these aquatic compartments for the presence of resistance genes and its host organisms is essential to outline strategies to minimize resistance dissemination.