Development of two microbial source tracking markers for detection of wastewater-associatedEscherichia coliisolates

Abstract

ABSTRACTEscherichia colihas been used as an indicator of fecal pollution in environmental waters. However, its presence in environmental waters does not provide information on the source of water pollution. Identifying the source of water pollution is paramount to be able to effectively reduce contamination. The present study aimed to identifyE. colimicrobial source tracking (MST) markers that can be used to identify domestic wastewater contamination in environmental waters. We first analyzed wastewaterE. coligenomes sequenced by us (n = 50) and RefSeq animalE. coligenomes of fecal origin (n = 82), and identified 144 candidate wastewater-associated marker genes. The sensitivity and specificity of the candidate marker genes were then assessed by screening the genes in 335 RefSeq wastewaterE. coligenomes and 3,318 RefSeq animalE. coligenomes. We finally identified two MST markers, namely W_nqrC and W_clsA_2, which could be used for detection of wastewater-associatedE. coliisolates. These two markers showed higher performance than the previously developed human wastewater-associatedE. colimarkers H8 and H12. When used in combination, W_nqrC and W_clsA_2 showed specificity of 98.9% and sensitivity of 25.7%. PCR assays to detect W_nqrC and W_clsA_2 were also developed and validated. The developed PCR assays are potentially useful for detectingE. coliisolates of wastewater origin in environmental waters, though users should keep in mind that the sensitivity of these markers is not high. Further studies are needed to assess the applicability of the developed markers to a culture-independent approach.