2.4. Microbiological Analysis

The water samples were analyzed for each of the following microbial indicators: total mesophilic microorganisms, fecal enterococci, fecal coliforms, Escherichia coli, and Vibrio spp.

Enumeration of mesophiles was performed at 22 °C and 37 °C for 72 h, according to ISO standards [31]. Samples (1 mL) were incorporated into Plate Count Agar medium (Biokar Diagnostics, Beauvais, France) and incubated for 72 h at 22 and 37 ± 1 °C. The results were expressed in log CFU/mL.

The enumeration of fecal enterococci, fecal coliforms, and E. coli was performed after filtration of 100 mL of water through a membrane with a 0.22 µm pore size (Cellulose Nitrate Filter, Sartorius, Germany). The results were expressed in log CFU/100 mL. Typical colonies of fecal enterococci (red, maroon, or pink) were counted on Slanetz and Bartley Agar medium (Biokar Diagnostics) after 24 h of incubation at 42 ± 2 °C, according to ISO standards [31]. The typical colonies of fecal coliforms (dark blue/violet) and E. coli (pink to red) were counted on Chromogenic Coliform Agar (CCA) medium (Biokar Diagnostics) after 24 h of incubation at 44 ± 1 °C, according to ISO standards [32].

Enumeration of Vibrio spp. was conducted by filtration of 1 L water with a 0.22 µm filtration membrane (Cellulose Nitrate Filter) and incubation on Vibrio Chromogenic Agar (Candalab, Madrid, Spain) for 24 h at 36 ± 1 °C. This medium is recommended for the selective isolation and differentiation of Vibrio spp. based on colony color: V. cholerae (pink-rose), V. alginolyticus (colorless), V. parahemolyticus (green-blue), and V. vulnificus (pink-rose).

Biochemical tests were carried out to further characterize the colonies of presumptive E. coli and Vibrio spp. from the respective chromogenic media, namely, Gram staining, catalase and oxidase tests, and growth with NaCl (6% m/v) in the medium (Vibrio spp. only).

Subsequently, molecular identification was performed, targeting the 16S rRNA gene (401 bp) with the AB035924 forward primer (5′ CCC CCT GGA CGA AGA CTG A-3′) and AB035924 reverse primer (5′-ACC GCT GGC AAC AAA GGA T-3′) [33] or with the Bac27F forward primer (5-AGAGTTTGGATCMTGGCTCAG-3) and Univ1492R universal reverse primer (5-CGGTTACCTTGTTACGACTT-3) [34]. The amplified products were sequenced by STAB VIDA (Caparica, Portugal), and the resulting sequences were compared in the BLASTN gene bank [35]. The accepted percentage of identity was greater than 90% similarity [36,37].