미생물 오염원 추적을 위한 지표미생물로서의 somatic 대장균파지의 분자적 분석

Abstract

Viral pathogens, which are excreted with human and animal feces, can cause an infection to human through water sources. The current method by indicator bacteria has limitation to evaluate microbial water quality by viral pathogens. Somatic coliphages have been focused as an interesting alternative indicator, however, studies of somatic coliphages with nucleotide sequence analysis have not been applied for microbial source tracking. To assess the possibility of microviridae family of somatic coliphages as tool for source tracking, we investigated distinction of somatic coliphages in feces between human and animals. Fecal samples were collected from one pig excreta and each of cow (n=10), chicken (n=10), pig (n=4) and human (n=11) feces from 2010 to 2011. In these fecal samples, quantification of somatic coliphages was determined by plaque assay. Viral DNAs were extracted from the plaques, and presence of coliphages was determined by PCR, followed by sequence analysis of the amplicons. The sequence was compared with those available in the GenBank databases using the NCBI/BLAST program. Somatic coliphages from cow and human were closely related to microviridae G4-like strains, chicken and pig excreta belonged to microviridae α3-like strains and microviridae phiX 174-like strains, respectively. These data supported that somatic coliphages can be used for discriminating fecal origin sources between human and animals with microviridae family. To evaluate the utilization of somatic coliphages as origin source tracking in aquatic environments, we applied the method for feces to water samples. Water samples were collected four times in ditch and river water affected by livestock wastewater and sewage. Somatic coliphages were detected with high concentration in sites, after passing through cattle shed and pigpen in ditch water and emerging ditch water into river water. In sequence analysis, somatic coliphages in water samples were shown that the phages well fitted into three major clades of microviridae family as shown in feces. These results indicated that somatic coliphages well reflected a contamination route of fecal pollutants, which the water bodies we studied were contaminated by animal feces. Furthermore, this result confirmed that somatic coliphages were potential fecal indicator for microbial source tracking in aquatic environments. In summary, fecal pollutants could be transported to aquatic environments, which originated from untreated or maltreated wastewater of livestock and human. This indicated the necessity of understanding origin source of fecal contamination to control the quality of water and, further, to reduce public health risk. This study suggests that somatic coliphages are promising viral indicator of fecal pollution for microbial source tracking, which can discriminate origin sources of feces between human and animals.