A large sampling study on the occurrence and characteristics of Pseudomonas aeruginosa and heterotrophic bacteria in mineral water over seasons and in different containers

Abstract

This study investigated the presence of Pseudomonas aeruginosa and heterotrophic bacteria in 1150 samples of bottled mineral water. P. aeruginosa was initially isolated using membrane filtration on selective agar and subsequently confirmed by PCR. Further characterization included pulsed-field gel electrophoresis (PFGE), detection of virulence genes, antimicrobial resistance profiling, Caco-2 cell invasion, and biofilm formation on different packaging materials. P. aeruginosa was detected in 11.5 % of samples, with the highest prevalence in reusable 20 L plastic jugs. PFGE revealed 41 distinct genetic profiles, indicating high diversity. The most frequent virulence genes detected were phzM (89.5 %), ExoS (88.8 %), toxA (86.8 %), and lasB (79.8 %). The more clinically relevant gene ExoU was found in 10.5 % of isolates. Antibiotic susceptibility tests showed that 14 % of isolates were multidrug-resistant, with resistance to piperacillin-tazobactam (26 %), gentamicin (18 %), and fluoroquinolones (12 %). Caco-2 cell assays showed that 73 % of strains exhibited high invasion potential (37.9–62.3 %), comparable to clinical isolates. Biofilm assays demonstrated strong adherence to materials commonly used in bottled water packaging, with the highest biofilm density observed on polypropylene. These findings suggest that reusable containers may be more prone to persistent contamination. Although the overall occurrence of P. aeruginosa was low, the presence of multidrug-resistant and virulent strains raises concerns, especially for immunocompromised individuals. These results emphasize the need for strict hygienic practices, particularly in reusable packaging systems, and routine microbial monitoring to ensure the microbiological safety of bottled mineral water.