Sampling

Water samples were collected during the summer of 2023 (from June to September) from four locations of bathing ponds in Prague and Central Bohemia, two of which were sampled repeatedly. The repeatedly sampled sites experienced high visitor numbers on hot summer days with maximum capacity ranging from 600 to 1000 people at a time, typically reaching full occupancy. Grab samples were collected, always 1–2 samples per site, depending on the area. To capture additional strains for studying ATB resistance and the exoA gene, samples were also collected from three urban water fountains. A total of 23 water samples were collected and analyzed. The control strains used included a collection strain of P. aeruginosa CCM 1960, a strain of P. aeruginosa isolated in 2018 from a hotel swimming pool/whirpool in the Pardubice region, where it caused an outbreak of ear infections and collection strain P. otitidis CNCTC 8175.

Laboratory determination of E. coli and intestinal enterococci

E. coli was determined by β-D-glucuronidase enzyme activity by the Colilert18 Quanti/TRAY method (IDEXX). Intestinal enterococci were determined by membrane filtration and culture on agar according to Slanetz and Bartley (48 h at 36 °C) and confirmation on Bile aesculine azide agar (2 h at 44 °C). A volume of 100-mL sample was processed.

Laboratory determination of P. aeruginosa

To detect opportunistic pathogenic pseudomonades, the standard method according to EN ISO 16266 (n.d.) was used which was modified to eliminate the background microflora. Water samples (100 mL) were filtered through a 0.45-µm porosity membrane filter and transferred to Pseudomonas (CN) agar. Cultivation was carried out for 4 h at 36 °C followed by cultivation for 48 h at 44 °C. At the end of this cultivation, colonies showing light green fluorescence were counted and labeled, and the plated membrane filters were placed in 36 °C for an additional 24 h (it can appear the formation of the light green pigment pyocyanine, which is not formed at 44 °C). Acetamide and King’s medium were used as further confirmatory tests and the strains were subsequently identified by MALDI-TOF MS (Brueker Diagnostics). The confirmation at the score levels above 2000 were accepted.

Antibiotic resistance tests

To determine antibiotic resistance (sensitive, resistant, and i-strains, i.e., sensitive strains at increased exposure) isolated and identified strains were tested by the cultivation method (plate diffusion method) according to the European (2023) Committee on antimicrobial susceptibility testing; clinical breakpoints were taken from Table 13.1, valid from 29. 6. 2023. Used discs (Oxoid) with antibiotics: piperacillin/tazobactam (30/6 µg), ceftazidime (10 µg), meropenem (10 µg), imipenem (10 µg), ciprofloxacin (5 µg), and amikacin (30 µg).

PCR amplification of part of the exoA gene

The exoA gene was determined by PCR amplification of a 397 bp exoA gene portion. The amplification conditions were based on the publication by Khan and Cerniglia (1994) using Taq Purple DNA polymerase (TopBio). The PCR reaction was performed on a qTowerG instrument (Analytikjena). 10 µL of the PCR mixture was subsequently analyzed on a 1.5% agarose gel (5 V/cm) containing GelRed and visualized under UV light. Selected PCR products were verified by Sanger sequencing performed by SeqMe.