Diversity of the heterotrophic microbial populations for distinguishing natural mineral waters

In the recent years the consumption of natural mineral waters has risen all over the world, becoming a usual alternative for tap water and other beverages. Natural mineral waters are complex environments containing a high diversity of autochthonous microbiota. The identification and characterization of this indigenous microbiota may help to detect changes occurring in the different steps of the bottling process and take preventive measures before the bottled water arrives to the consumer. The aims of this study were to describe the bacterial heterotrophic populations in natural mineral waters with a cultivation-dependent method and determine whether their autochthonous microbiota were specific enough to be clearly distinguished from that of other natural mineral waters with a phenotypic-based method. For this purpose, water from three independent Spanish springs was sampled in two seasons (winter and summer) and heterotrophic aerobic bacterial strains were isolated at two temperatures (22 ± 2°C and 36 ± 2°C) on R2A agar. Isolates were phenotyped biochemically with Php-48 plates (Bactus AB, Sweden), and the indexes of diversity and similarity between populations were calculated. The 16S rRNA gene of the most representative strains of each biochemical cluster was sequenced for its identification. Finally, a ten-fold cross-validation method was assayed for the identification of the origin of a natural mineral water when phenotyping a set of isolates. High levels of diversity were found at all sites. One of the sources was found to present less diversity due to a confirmed contamination with Pseudomonas aeruginosa. The study of the similarities showed that growing temperatures and seasons caused significant differences in structures and composition at the sources. In addition, several bacterial species were isolated and identified, some of them rarely isolated in natural mineral waters, revealing the complexity and lack of knowledge of these ecosystems. Consequently, the applied phenotypic methodology was found to be feasible for differential identification of microbiota in these environments. Moreover, the experimental model assayed was strong enough to identify the origin of a natural mineral water. It may thus be possible to confirm that the evaluation of diversity of heterotrophic aerobic bacterial populations could be applied to identify bottled water sources.