Protocol for inoculating bottled mineral water with bacteria to receive artificial contaminated water samples.

Here, we present a protocol for inoculating drinking water samples with a variety of pathogens or facultative pathogen bacteria. We describe steps for preparing bacterial solutions, inoculating mineral water bottles and other drinking water samples, filtration and incubation of the agar plates, and counting colony-forming unit per mL. We also detail procedures for determining selected chemical properties, such as anions and cations, which can also affect the bacterial growth. For complete details on the use and execution of this protocol, please refer to Schalli et al.1.

The behaviour of Escherichia coli and Pseudomonas aeruginosa in bottled mineral water.

Microbial contamination of bottled water during the filling and capping procedure is a problem which should be avoided. The examination of the influence of carbon dioxide (CO2) on bacterial growth of Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa) in bottled mineral water was the aim of this study. Commercially available glass bottles with plastic screw caps filled with natural mineral water (without additional CO2 "still" (StMW) and with CO2 "sparkling" (SpMW) were obtained from a manufacturer in the province of Styria, Austria. The artificial contamination was performed in the lab by opening the bottle with subsequent addition of a bacterial solution with a defined number of bacteria. For each bacterial strain, 12 bottles were prepared. Samples (100 mL) were taken after a specific number of days, filtrated and placed on Endo Agar for cultivation. After incubation for 24 h bacterial colonies were counted. In this study CO2 addition to bottled water reduced colony forming units of the two investigated bacterial strains over time.

Dissolved Carbon Dioxide: The Lifespan of Staphylococcus aureus and Enterococcus faecalis in Bottled Carbonated Mineral Water.

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.

Cefsulodin and Vancomycin: A Supplement for Chromogenic Coliform Agar for Detection of Escherichia coli and Coliform Bacteria from Different Water Sources.

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.

Comparison of Updated Methods for Legionella Detection in Environmental 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.

Effect of solar radiation on survival of indicator bacteria in bathing waters.

Sunlight exposure is considered to be the most important cause of "natural disinfection" in surface water environments. The UV-B portion of the solar spectrum is the most bactericidal, causing direct (photo-biological) DNA damage. In the present experimental study, the effect of solar radiation on the elimination of bacteria in water, especially in surface water, was studied. The influence of depth and UV-B transmittance of water was determined. Comparing Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa and Staphylococcus aureus, Enterococcus faecalis proved to be the most resistant organism. Pseudomonas aeruginosa was shown to be the most sensitive indicator bacterium among the tested microorganisms. Results show a significant correlation between radiation intensity and reduction rates. Best elimination of microorganisms occurs on the water surface; with increasing water depth, there is less UV radiation to inactivate bacteria. High turbidity substantially reduces UV-B transmittance in water causing decreased elimination efficiency. The results of the present study show that sunlight, given an appropriate intensity and good water transparency is suitable to inactivate fecal indicator bacteria within a few hours in surface waters and therefore also in bathing waters.