Diagnostic moléculaire de l’origine des contaminations fécales dans l’environnement littoral : développement de marqueurs Bacteroidales spécifiques de l’höte
Year: 2010
Type: dissertation
Abstract: Human and animal faecal pollution affects environmental water in inland and coastal areas, with negative implications for recreational uses, public safety and shellfish sanitary status due to the presence of enteric pathogens. Starting from 2011, the revised Bathing Water European Directive (2006/7/CE) requires the establishment of bathing water profiles with an inventory and study of the pollution sources likely to affect water quality. The faecal microbiological indicators used in these regulations, Escherichia coli and enteroccoci, cannot distinguish between human and animal faecal contamination. Thus, alternative methods, that focus on target microorganisms such as bacteria belonging to the Bacteroidales order were developed or are being developed to discriminate between human and animal faecal contamination. The first markers were developed for conventional PCR; however, they were detected in very few environmental waters samples and there were never applied to shellfish. The main objective of this study was to develop and to validate a molecular approach based on quantitative real-time PCR with the Bacteroidales target, in order to identify the origin of faecal contamination in water and shellfish. Phylogenetic analysis of the partial Bacteroidales 16S rRNA gene sequences from human and animal (bovine, pig and wild birds) faeces and effluents allowed the development of 4 host-specific Bacteroidales markers to identify human (Hum-1-Bac), porcine (Pig-1-Bac and Pig-2-Bac) and ruminant (Rum-2-Bac) faecal contaminations. Applied on target and nontarget faeces and effluent samples, these markers were highly sensitive (92.3 - 100%) and specific (93.4 - 100%). The study of the Pig-1-Bac and Pig-2-Bac markers persistence in river water microcosms showed that under unfavourable conditions, aerobic and temperature of 20°C, markers persisted for at least 16 days. These results suggested that the Bacteroidales markers could still be identified after an extended period of time in river waters. This hypothesis was confirmed with their detection and quantification in river waters from the Daoulas catchment estuary and the Elorn estuary (Finistère, Brittany) with concentrations ranging from 2.7 to 6.5 U. Log10 copies/100 ml. Compared with bacterial, viral and chemical markers from runoff and environmental waters, Bacteroidales markers were better to identify the origin of the contaminations.Host-specific Bacteroidales markers were also applied for the first time in shellfish. Selection and optimisation of bacterial DNA extraction protocol from intervalvular liquid of oysters artificially contaminated, allowed detection and quantification of the markers in naturally contaminated oysters. Discrimination of the origin of faecal contamination from samples of water and intervalvular liquid of oysters from 2 different river and estuarine sites, underlined the importance of the human and ruminant faecal contamination more than the porcine contamination. Thus, this study confirms the interest of the host-specific Bacteroidales markers developed from the Bacteroidales target to identify faecal contamination in bathing waters and shellfish harvesting areas.
Author: Sophie Mieszkin
Language: French
Subfield: Water Science and Technology
Field: Environmental Science
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W1498641015