Detection of KPC-producing Enterobacteria and Antimicrobial Resistance in Brazilian Sewage Treatment Plants

Background and Aims: Pathogenic microorganisms can be found in sewage and may cause several infections and deleterious health effects. In this scenario, Enterobacterales can trigger several diseases in humans and animals, since antimicrobial resistance bacteria, as well as antimicrobial resistance genes, are considered a global public health problem that tends to increase. Therefore, this study aims to identify Enterobacterales resistant to third- and fourth-generation cephalosporins and carbapenems isolated from Sewage Treatment Plants (STP) in Brazil.

Methodology: Four sampling campaigns were performed weekly from the affluent and effluent of two STPs at the Oswaldo Cruz Foundation. In each campaign, four samples were collected in different points in the affluent at the entrance to the STP and the effluent at the exit. A chromogenic medium infused with third-generation cephalosporin was employed, which Gram-negative and oxidase-negative bacteria underwent conventional screening and biochemical tests. Identification was performed with an automated method in VITEK 2® (BioMérieux). The antimicrobial susceptibility tests (AST) and phenotypic assays for carbapenemase detection were evaluated using the agar diffusion methodology in Müeller-Hinton agar, and antibiotic disks suitable for Enterobacterales. The profile of enterobacteria was categorized as Sensitive (S), Intermediate (I), and Resistant (R). The identification of genes related to bacterial resistance were based on PCR reactions, where DNA was extracted using the heat shock method.

Results: Eight species were identified in the 19 Enterobacterales isolates. Of these, 26% were Klebsiella pneumoniae, 16% Escherichia coli, 16% Citrobacter freundii, 16% Kluyvera cryocrescens, 11% Kluyvera intermedia, 5% Enterobacter asburiae, 5% Citrobacter farmeri and 5% Citrobacter amalonaticus. K. pneumoniae was isolated in all samples of raw sewage from STP B, which was already expected since STP B receives hospital waste. However, it was not found in STP A or any sample of treated sewage from either STP. Among these isolates, 75% were non-sensitive to ciprofloxacin, 71% to ceftriaxone, 57% to ceftazidime, 46% to cefoxitin, 36% to cefepime, 28% to piperacillin + tazobactam, and 11% to gentamicin, respectively. Phenotypic tests effectively screened carbapenem resistance mechanisms, displaying high concordance with molecular tests. These findings indicate the presence of the blaKPC gene, reflecting the predominance of this enterobacteria in Brazil.

Conclusion: A high profile of non-sensitivity to most antimicrobials tested, especially third-generation cephalosporins, quinolones, and carbapenems were reported. These results emphasize the dissemination of potentially pathogenic and antimicrobial-resistant microorganisms in the environment from STP’s final effluents, regardless of the type of system adopted. Therefore, the concern related to environmental contamination and the spread of bacterial resistance to antimicrobials in aquatic reservoirs must be addressed.