Screening and characterization of Escherichia coli-specific bacteriophage isolated from sewage water

The objectives of this study were to isolate and to characterize bacteriophage specific to Escherichia coli from different sources of waste waters. Based on spot test and plaque assay, the result showed that bacteriophages could be isolated from waste water treatment plant Kudprakhow (bacteriophage named JC01), Sappasitthiprasong hospital (bacteriophage named JC02), and Khongjiam hospital (bacteriophage named JC03). Host range determination of bacteriophage revealed that all bacteriophage types had high specific host range only for E.coli. Inhibition of clinical isolates E.coli with multidrug resistant property showed that bacteriophage JC01, JC02 and JC03 inhibited the growth of E.coli at 51.7% (138/267) 52.4% (140/267), and 28.5% (76/267), respectively. Bacteriophage stability in different solutions and heat at different time points demonstrated that all bacteriophages could tolerate 0.85% normal saline and distilled water for more than 40 minutes but could not tolerate 10% ethanol and 1% hydrogen peroxide at every time point. Heat stability showed that bacteriophage JC01 had resisted at 60๐C after 60 minutes of incubation. Bacteriophage JC02 and JC03 showed the ability to resist the temperature of 60๐C after 45 minutes of incubation. The result of bacteriophage classification by genome analysis demonstrated that the extracted DNA of JC01 and JC02 could be digested with HindIII and DNase but not for RNase. For bacteriophage JC03, the extracted genome could be digested with NcoI, HindIII and DNase, but not for RNase. This result indicated that the bacteriophage JC01, JC02 and JC03 genome was a DNA virus and their genome was a double-stranded DNA (dsDNA). In addition to viral genome analysis, determination of viral particle morphology by transmission electron microscope can also be used to classify bacteriophage group. The result found that all bacteriophages had the viral particle which composed of a head with a hexagonal shape and long tails with contractile. The size from head to tail was approximately 200 nm. Based on Intraclass correlation coefficient (ICC) classification of prokaryotic (bacterial and archaeal), bacteriophage JC01, JC02 and JC03 could be classified in Family Myoviridae, Order Caudovirales. Therefore, the bacteriophages derived from this study could be used to study their potential use in further advanced steps.