Coliphage-based Microbial Source Tracking in Shellfish Growing Area of South Korea

Abstract

Waterborne infectious diseases from fecal contamination have been emerged as a major issue in South Korea. Most of the waterborne illnesses particularly by virus have been implicated in outbreaks due to shellfish, whose contaminations caused by the fecal pollution in sea water. Microbial source tracking (MST) is one of the methods used to identify the source of the contamination in water samples. Among the MST tools, genotyping of male-specific (F+, F-specific) RNA coliphages have been shown to be effective as a target MST microorganism. We applied MST method to determine fecal sources contaminating surface water and sea water in three shellfish growing areas in South Korea using F+ coliphages and somatic coliphages. In total, 144 surface water and 336 sea water samples were collected. F+ coliphages as well as somatic coliphages were prevalent and abundant in sampling areas. More than 150 F+ RNA coliphages were isolated from water samples by the single agar layer method from US EPA method 1602. We further analyzed F+ RNA coliphage isolates using RNase sensitivity assays, Reverse transcription (RT)-PCR, and nucleic acid sequencing. F+ RNA coliphages were mostly predominant in surface water (61.4 % positive) and seawater (38.6 %positive) samples. Area A was the most contaminated area among three shellfish growing areas in South Korea. Genotyping of 44 F+ RNA coliphages revealed that most of isolated F+ RNA coliphages belonged to Group I coliphages, which are likely originated from human and non-human fecal sources. Coliphages were better indicator than bacterial indicators such as total coliform and fecal coliform. Somatic coliphage have high correlation than F+ coliphage with enterovirus. These results demonstrate that appropriate analysis of F+ RNA coliphages could effectively distinguish the contamination source. Also from these results, the water in shellfish growing areas of South Korea might be contaminated by human and animal fecal sources from surroundings near the surface water. Furthermore, somatic coliphages could be effective viral indicator in water sources with F+ coliphages.