Development and use of Bacteroides 16S rRNA Polymerase Chain Reaction Assay for Source Tracking Dog Faecal Pollution in Bathing Waters

Abstract

Faecal pollution on bathing beaches poses a potential threat to human health and as a result may also negatively affect the local economy. In instances where the source of such pollution is not obvious, it may be necessary to track such sources using a host-specific genetic markers technique. Bacteroides species are potential indicators for source tracking of faecal pollution in bathing waters. This study designed specific primer sets to amplify sections of the 16S rRNA gene unique to Bacteroides from domestic dogs and used quantitative PCR (qPCR) to quantify such genetic markers in environmental samples. The sensitivity and specificity of the primer sets was determined; they were specific in silico against known dog Bacteroides sequences and in vitro against Bacteroides sequences originating from human and livestock faeces. Dog faecal Bacteroides contamination was then detected in sea water during the bathing season at a local beach where dogs are banned during the summer months, in spite of the fact that these waters had met EU directive standards based on the culture-based enumeration of faecal indicator bacteria. Quantitative PCR was used to determine the limit of detection (LOD) of the dog Bacteroides genetic markers in these water samples. The copy number of dog Bacteroides genetic markers in the water was low and the LOD of those markers was 4 copies per reaction. The use of these dog primers has the potential to supply important additional information when source tracking faecal pollution at bathing beaches and maintaining water quality.

Strategies for Monitoring Microbial Life in Beach Sand for Protection of Public Health

“…Currently, library-independent methods using PCR or quantitative PCR (qPCR) to target specific genes of host-associated bacteria are preferred [91]. This latter approach is possible due to the extensive effort in developing primers sets that are specific to several biological groups, including humans [92], seagulls [93], cattle [94], and dogs [95].…”

Host-Associated Bacteroides 16S rDNA-Based Markers for Source Tracking of Fecal Pollution in Laguna Lake, Philippines

“…Several assays have been designed to target specific sequences within the Bacteroides 16S rRNA gene, and to differentiate human-derived contamination from that of other animals. Markers for the estimation of fecal pollution from pigs [ 6 ], dogs [ 9 ], humans [ 10 , 11 ], cows [ 10 , 11 ], and poultry [ 12 ] have been designed and evaluated. However, due to their variability in validity and efficiency when applied in different regions, there are no gold standards for human and nonhuman markers that are applicable to all geographical areas [ 13 , 14 ].…”

Microbial Source Tracking as a Method of Determination of Beach Sand Contamination

“…Usually, contamination events arise from combined sewer overflows or discharges of wastewater with inadequate or non-existing treatment. Animal feces can be classified as being from domestic or feral animals, with the most common ones being from dogs, cattle and other ruminants, some wild animals, and birds (such as seagulls) [19][20][21][22][23][24][25][26].…”