Source tracking of faecal indicator bacteria of human pathogens in bathing waters; an evaluation and development

Abstract

Bacterial water pollution is a significant problem because it is associated with reduction in the ‘quality’ of water systems with a potential impact on human health. Faecal indicator bacteria (FIB) are usually used to monitor the quality of water, and to indicate the presence of pathogens in water bodies. However, enumeration alone does not enable identification of the precise origin of these pathogens. This study aimed to monitor the quality of bathing water and associated fresh water in and out of the ‘bathing season’ in the UK, and to evaluate the use of microbial source tracking (MST) such as the host-specific based polymerase chain reaction (PCR) and quantitative PCR (qPCR) to recognize human and other animal sources of faecal pollution. The culture-dependent EU method of estimating FIB in water and sediment samples was performed on beach in the South Sands, Kingsbridge estuary, Devon, UK- a previously ‘problematic’ site. FIB were present at significant levels in the sediments, especially mud, as well as fresh water from the stream and pond flowing onto South Sands beach. However, the quality of bathing water was deemed to be ‘good’ and met with the EU bathing water directive 2006. Using MST it was possible to successfully classify the nature of the source from which the bacteria came. PCR was applied to detect the Bacteroides species 16S rRNA genetic markers from human sewage and animal faeces. All water and sediment samples displayed positive results with a general Bacteroides marker indicating the presence of Bacteroides species. Host-specific PCR showed the human Bacteroides genetic marker only in the sediment of the stream. However, limitations in the ‘types’ of probes available and in the persistence of these markers were identified. Thus, novel dog-specific Bacteroides conventional PCR and qPCR primer sets were developed to amplify a section of the 16S rRNA gene unique to the Bacteroides genetic marker from domestic dog faeces, and these were successfully used to quantify those markers in water samples at a ‘dog permitted’ and ‘dog banned’ beach (Bigbury-on-Sea, Devon, UK). Generic, human and dog Bacteroides PCR primer sets were also used to evaluate the persistence of Bacteroides genetic markers in controlled microcosms of water and sediment at differing salinities (< 0.5 and 34 psu) and temperature (10 and 17 ºC). The rates of decline were found did not differ significantly over 14 and 16 days for the water and sediment microcosms, respectively. Beach sediments which were studied in this project may act as a reservoir for adhesive FIB, and this was confirmed using fluorescence in situ hybridisation (FISH). The similarity in the persistence of these Bacteroides 16S rRNA genetic markers in environmental water and sediment suggests that viable but non-culturable (VBNC) Bacteroides spp. do not persist in the natural environment for long. Therefore, 16S rRNA genetic markers can be of value as additional faecal indicators of bathing water pollution and in source tracking. Thus, in this study MST methods were successfully used and in future applications, dog-specific primer sets can be added to the suite of host-specific Bacteroides genetic markers available to identify the source(s) of problem bacteria found on failing beaches.