Sabine Platzer

During the process of mineral water production, many possible contamination settings can influence the quality of bottled water. Microbial contamination can originate from different sources, for example, the ambient air, the bottles, the caps, and from the bottling machine itself. The aim of this study was to investigate the influence of three different carbon dioxide (CO₂) concentrations (3.0 g/L, 5.5 g/L, and 7.0 g/L; 20 bottles each) in bottled mineral water on the bacterial growth of Staphylococcus aureus (S. aureus) and Enterococcus faecalis (Ent. faecalis). The examined mineral water was artificially contaminated before capping the bottles inside the factory. After a specific number of days, water samples were taken from freshly opened bottles and after filtration (100 mL), filters were placed on Columbia Agar with 5% Sheep blood to cultivate S. aureus and Slanetz and Bartley Agar to cultivate Ent. faecalis. The respective colony-forming units (CFU) were counted after incubation times ranging from 24 to 120 h. Colony-forming units of S. aureus were not detectable after the 16th and 27th day, whereas Ent. faecalis was not cultivable after the 5th and 13th day when stored inside the bottles. The investigation of the bottles that were stored open for a certain amount of time with CO₂ bubbling out showed only single colonies for S. aureus after the 5th day and no CFUs for Ent. faecalis after the 17th day. A reduction in the two investigated bacterial strains during storage in carbonated mineral water bottles means that a proper standardized disinfection and cleaning procedure, according to valid hygiene standards of industrial bottling machines, cannot be replaced by carbonation.

Background microorganism growth on Chromogenic Coliform Agar (CCA) can be challenging. For this reason, a new alternative method with a Cefsulodin/Vancomycin (CV)-supplemented CCA should be developed in this study. CCA supplemented with CV was validated according to ÖNORM EN ISO 16140-4:2021 using water from natural sources in Styria, Austria. Results show that the alternative method using the supplemented CCA has similar values in relation to sensitivity (82.2%), specificity (98.6%) and higher selectivity (59%) compared to the reference method. Repeatability and reproducibility were acceptable for the alternative method and showed similar results with the reference method. The alternative method shows a very low false positive rate and a low false negative rate paired with good performance regarding the inclusion study. The exclusion study shows the advantage of our method by suppressing background microorganisms and facilitating the process of enumeration of Escherichia coli and other coliform bacteria on CCA plates. Aeromonas hydrophila and Pseudomonas aeruginosa growth was inhibited using the supplement. To conclude, the coliform CV selective supplement combined with CCA is an appropriate tool for coliform bacteria detection in water samples.

The difficulty of cultivation of Legionella spp. from water samples remains a strenuous task even for experienced laboratories. The long incubation periods for Legionellae make isolation difficult. In addition, the water samples themselves are often contaminated with accompanying microbial flora, and therefore require complex cultivation methods from diagnostic laboratories. In addition to the recent update of the standard culture method ISO 11731:2017, new strategies such as quantitative PCR (qPCR) are often discussed as alternatives or additions to conventional Legionella culture approaches. In this study, we compared ISO 11731:2017 with qPCR assays targeting Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1. In samples with a high burden of accompanying microbial flora, qPCR shows an excellent negative predictive value for Legionella pneumophila, thus making qPCR an excellent tool for pre-selection of negative samples prior to work-intensive culture methods. This and its low limit of detection make qPCR a diagnostic asset in Legionellosis outbreak investigations, where quick-risk assessments are essential, and are a useful method for monitoring risk sites.

Waterborne diseases are a serious threat because of their ability to infect a high number of individuals in a short time span, such as during outbreaks of Legionellosis. This significantly highlights the need for the rapid detection and quantification of bacteria in environmental water samples. The aim of this study was to investigate the feasibility of quantitative Polymerase Chain Reaction (qPCR) for the detection of Legionellapneumophila (L. pneumophila) in environmental water samples and comparison of standard culture methods for Legionella detection with qPCR. Our study reached a negative predictive value (NPV) for L. pneumophila of 80.7% and for L. pneumophila serogroup 1 (sg1) the calculated NPV was 87.0%. The positive predictive value (PPV) for L. pneumophila was 53.9% and for L. pneumophila sg1 PPV was 21.4%. Results showed a correlation between qPCR and culture with an R² value of 0.8973 for L. pneumophila, whereas no correlation was observed for the detection of L. pneumophila sg1. In our study, qPCR proved useful for the identification of L. pneumophila negative samples. However, despite the obvious benefits (sample handling, rapid generation of results), qPCR needs to be improved regarding the PPV before it can replace culture in water quality assessment.