Yeast Biodiversity of Karst Waters: Interest of Four Culture Media and an Improved MALDI-TOF MS Database.

Karst aquifers are a significant source of drinking water and highly vulnerable to pollution and microbial contamination. Microbiological regulations for the quality of drinking water mostly focus on bacterial levels and lack guidance concerning fungal contamination. Moreover, there is no standardised microbial analysis methodology for identifying fungi in water. Our main objective was to establish the most effective culture and identification methodology to examine yeast diversity in karst waters. We assessed the comparative efficacy of four culture media (CHROMagar Candida, dichloran glycerol 18% [DG18], dichloran rose Bengal chloramphenicol [DRBC], and SYMPHONY agar) for yeast isolation from karst water samples. Furthermore, we investigated the comprehensiveness of databases used in MALDI-TOF mass spectrometry (MALDI-TOF MS) for identifying environmental yeast species. In total, we analysed 162 water samples, allowing the identification of 2479 yeast isolates. We demonstrate that a combination of four culture media, each with distinct specifications, more efficiently covers a wide range of yeast species in karst water than a combination of only two or three. Supplementation of a MALDI-TOF MS database is also critical for analysing environmental microbial samples and improved the identification of yeast biodiversity. This study is an initial step towards standardising the analysis of fungal biodiversity in karst waters, enabling a better understanding of the significance of this environmental reservoir in relation to public health.

Effect of a karst system (France) on extended spectrum beta-lactamase (ESBL)-producing Escherichia coli.

Karst aquifers are an important water resource worldwide particularly exposed to anthropogenic pollution, including antibiotic-resistance. The release of antibiotic-resistant bacterial pathogens in the environment is a major public health challenge worldwide. In this One Health study, we aimed to determine the effect of karst on antibiotic-resistant bacteria. For this purpose, we determined the concentrations of extended-spectrum ß-lactamases-producing Escherichia coli (ESBL-Ec) for 92 weeks in a rural karst hydrosystem providing drinking water. ESBL-Ec isolates (n = 130) were sequenced by whole genome sequencing. We analysed the isolates at different levels of granularity, i.e., phylogroup, sequence type, presence of antibiotic-resistance genes, mutations conferring antibiotic-resistance, and virulence genes. The ESBL-Ec concentrations were spatially and temporally heterogeneous in the studied karst hydrosystem. ESBL-Ec isolates survived in the karst and their concentrations were mostly explained by the hydrodynamic of the hydrosystem. We demonstrate that the studied karst has no filtration effect on ESBL-Ec, either quantitatively (i.e., in the ESBL-Ec concentrations) or qualitatively (i.e., in the genetic characteristics of ESBL-Ec isolates).