Integrating culture-based and molecular methods provides an improved assessment of microbial quality in a coastal lagoon.

Faecal pollution in aquatic environments is a worldwide public health concern, yet the reliability and comprehensiveness of the methods used to assess faecal contamination are still debated. We compared three approaches, namely a culture-based method to enumerate Faecal Indicator Bacteria (FIB), a FIB-targeting qPCR assay, and High-Throughput Sequencing (HTS) to detect faeces- and sewage-associated taxa in water and sediment samples of an impacted model lagoon and its adjacent sea across one year. Despite at different levels, all approaches agreed in showing a higher contamination in the lagoon than in the sea, and higher in sediments than water. FIB significantly correlated when considering separately sediment and water, and when using both cultivation and qPCR. Similarly, FIB correlated between cultivation and qPCR, but qPCR provided consistently higher estimates of FIB. Faeces-associated bacteria positively correlated with cultivated FIB in both compartments, whereas sewage-associated bacteria did only in water. Considering their benefits and limitations, we conclude that, in our study site, improved quali-quantitative information on contamination is provided when at least two approaches are combined (e.g., cultivation and qPCR or HTS data). Our results provide insights to move beyond the use of FIB to improve faecal pollution management in aquatic environments and to incorporate HTS analysis into routine monitoring.

DNA extraction procedure: a critical issue for bacterial diversity assessment in marine sediments.

In order to evaluate whether different DNA extraction procedures can affect estimates of benthic bacterial diversity, based on 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) fingerprinting technique, we compared two in situ lysis procedures (a SDS-based protocol and a commercial kit for DNA recovery) and one cell-extraction protocol on a variety of marine sediments. Despite the two in situ lysis procedures resulted in significantly different DNA yields (highest with the SDS in situ lysis), estimates of bacterial diversity provided a not significantly different ribotype richness, as well as similar values of the Shannon-Wiener (H') and Margalef (d) indices of biodiversity and of evenness (Pielou index, J). Conversely, the cell-extraction procedure for DNA extraction resulted always in a significantly lower ribotype richness and diversity. The analysis of similarities (anosim) among the T-RFLP electropherograms allowed concluding that ribotypes composition did not change significantly using different protocols. However, the analysis of beta-diversity (turnover diversity) revealed that a large number of ribotypes was observed exclusively with one of the three protocols utilized. When unshared ribotypes from in situ lysis and cell extraction were pooled together, total ribotype richness resulted much higher (up to 80%). Our results indicate that estimates of ribotype diversity based on a single protocol of DNA extraction can significantly underestimate the total number of bacterial ribotypes present in the benthic domain. We recommend that future studies will not only integrate different DNA extraction procedures, but also will explore the possibility of integrating two or more different genetic markers in order to increase our ability to detect the actual bacterial diversity in environmental samples.