Identification of CTX-M-15 and CTX-M-27 in Antibiotic-Resistant Gram-Negative Bacteria Isolated from Three Rivers Running in Central Italy

The main goal of this study was to identify Gram-negative bacteria resistant to antibiotics, in particular β-lactams, in stream waters and effluents from urban wastewater treatment plants draining into Fino, Tavo, and Saline rivers of the Abruzzo region, Italy. Eight sampling sites were selected because they were the most contaminated by coliforms during previous sampling campaign. One sample for each site was collected for the detection of total and fecal coliforms, Escherichia coli and Enterococcus species by Colilert-18 and Enterolert-E Quanti-Tray/2000. Antibiotic-resistant bacteria, selected on ampicillin and cefotaxime-supplemented agar plates, were identified by EnteroPluri test systems and then confirmed by MALDI-TOF. The resistant determinants were identified and characterized by PCR and sequencing. The microbiological analysis allowed to detect E. coli, total coliforms, fecal coliforms, and enterococci with a coefficient of variation of 215.7%, 212.8%, 242.5%, and 188.5%, respectively. Several Gram-negative bacteria were identified: Serratia liquefaciens, E. coli, Enterobacter cloacae, Citrobacter freundii, Raoultella ornithinolytica, Acinetobacter johnsonii, Aeromonas veronii, Aeromonas hydrophila, and Pseudomonas koreensis. All strains possessed class 1 integrons, insertion sequences, and genes encoding for serin- and metallo-β-lactamases. Extended-spectrum β-lactamases, such as CTX-M-15 and CTX-M-27, were found in Enterobacteriaceae, whereas CphA metallo-β-lactamase was found in A. veronii and A. hydrophila. The main resistance's mechanism to β-lactams observed among the analyzed strains is represented by the production of serin β-lactamases (CTX-M-15, CTX-M-27, and SHV-1) and metallo β-lactamase (CphA).