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Carbapenemase-producing Enterobacterales (CPE) are a growing concern, representing a major public health threat to humans, especially in healthcare settings. In the present study, we evaluated the persistent contamination by carbapenem-resistant Enterobacterales in water from Douro River, Portugal. KPC-producing Enterobacterales were detected in five water samples separated chronologically by 15 days each. Susceptibility testing was performed by disk-diffusion-method according to Clinical and Laboratory Standards Institute (CLSI), phenotypic carbapenemase activity was evaluated by carbapenem inactivation method, presumptive identification of the isolates was performed by CHROMagar orientation and confirmed by API-20E. Carbapenemase genes were screened by PCR and the clonality of all isolates was assessed by XbaI-Pulsed Field Gel Electrophoresis (PFGE). Fifteen KPC-producing Enterobacterales isolates were selected, identified as multidrug-resistant and showed a resistance profile to non-beta-lactam antibiotics: sulfamethoxazole + trimethoprim (7/15), ciprofloxacin (3/15), fosfomycin (3/15) and chloramphenicol (2/15). Isolates were identified as (6) Escherichia coli and (9) Klebsiella pneumoniae. Our results suggest a punctual contamination with KPC-producing Enterobacterales continued through the time. The absence of clonality between the isolates suggests a circulation of mobile genetic element harbouring KPC gene in the origin of contamination. This work provides a better understanding on the impacts of water pollution resulting from human activities on aquatic environments.

Resistances to extended-spectrum cephalosporins (ESC) and colistin are One Health issues since genes encoding these resistances can be transmitted between all sectors of the One Health concept, i.e., human, animal, and the environment. Among food-producing animals, sheep farming has long been overlooked. To fill in this knowledge gap, we looked for ESC- and colistin resistance in 21 faecal samples collected from sheep in one farm in the south of Portugal. ESC-resistant isolates were selected on MacConkey agar plates supplemented with cefotaxime. Susceptibility testing was performed by the disk-diffusion method according to CLSI, while colistin MIC was determined by broth microdilution. ESC- and colistin-resistance genes were identified by PCR, and the clonality of all isolates was assessed by XbaI-PFGE. The replicon content was determined by PCR according to the PCR-based replicon typing (PBRT) scheme. Sixty-two non-duplicate ESC-resistant E. coli isolates were identified, which all presented an extended-spectrum beta-lactamase (ESBL) phenotype, mostly due to the presence of CTX-M genes. One CTX-M-1-producing E. coli was concomitantly colistin-resistant and presented the plasmid-mediated mcr-1 gene. Nearly all isolates showed associated resistances to non-beta-lactam antibiotics, which could act as co-selectors, even in the absence of beta-lactam use. The results showed a high proportion of ESBL-producing E. coli in sheep faeces. Their dissemination was very dynamic, with the spread of successful clones between animals, but also a large diversity of clones and plasmids, sometimes residing in the same animal. This study highlights the need for global surveillance in all food-producing sectors, in order to avoid the dissemination of genes conferring resistance to last-resort antibiotics in human medicine.

Food producing animal is a global challenge in terms of antimicrobial resistance spread. Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae are relevant opportunistic pathogens that may spread in many ecological niches of the One Health approach as human, animal and environment due to intestinal selection of antimicrobial resistant commensals in food production animals. Cattle production is a relevant ecological niche for selection of commensal bacteria with antimicrobial resistance from microbiota. Enterobacteriaceae show importance in terms of circulation of resistant-bacteria and antimicrobial resistance genes via food chain creating a resistance reservoir, setting up a threat for colonization of humans and consequent health risk. ESBL-producing Enterobacteriaceae are a threat in terms of human health responsible for life threatening outbreaks and silent enteric colonization of community populations namely the elder population. Food associated colonization is a risk difficult to handle and control. In a time of globalization of food trading, population intestinal colonization is a mirror of food production and in that sense this work aims to make a picture of ESBL-producing Enterobacteriaceae in animal production for food over the world in order to make some light in this reality of selection of resistant threats in food producing animal.