Bacteriophages, viruses that infect bacteria, were first noted independently by Frederick William Twort1 and Felix Hubert d’Hérelle2 between 1915 and 1917. Coliphages are a heterogeneous group of bacteriophages that infect and replicate in Gram-negative bacteria including coliforms and Escherichia coli. Coliphages are part of the gut microbiome and are excreted in the feces of humans and other warm-blooded animals. Present in large numbers in sewage, coliphages also have been isolated from sewage-contaminated sources including beaches, and other waters.
Many water quality standards use the presence of bacteria such as E. coli or enterococci as indicators of fecal contamination. However, the presence of these bacterial indicator organisms does not always have a strong correlation with the presence of fecal-borne viral pathogens. Several studies have shown that the presence of coliphages was associated with the detection of causative agents of viral illness such as noroviruses, adenoviruses, hepatitis A, rotaviruses and enteroviruses in ambient waters.3 Given that coliphages are present in the feces of humans and other warm-blooded animals and, therefore, may co-occur with other fecal-borne viral pathogens, coliphages could represent the fate and transport of other enteric viruses.3-6 In some waters, coliphages may be a more valuable indicator of human pathogenic viruses than fecal indicator bacteria. In recent years, there has been interest in using coliphages as viral indicators of fecal contamination.3,7
Similar to other viruses, coliphages are classified according to their nucleic acid content. Coliphages are further defined as either male-specific or somatic. Male-specific coliphages only infect bacterial cells that possess a plasmid encoding an F pilus, required for bacterial conjugation. In contrast, somatic coliphages can infect bacteria that do not possess the F pilus; they infect the cells through the outer membrane.
Male-specific coliphages are a broad group of coliphages, containing either DNA (referred to as FDNA, F+DNA or F-specific DNA) or RNA (referred to as FRNA, F+RNA or F-specific RNA.8 Several host bacterial strains are used to enumerate male-specific coliphage strains in water, although in the procedures described here the E. coli Famp strain is described for use as well as Salmonella enterica subspecies enterica serotype Typhimurium WG49. Escherichia coli HS (pFamp)R, or more simply referred to as E. coli Famp, (ATCC 700891), contain Famp plasmids, conferring resistance to the antibiotic ampicillin and coding for pilus production. Salmonella ser. Typhimurium are resistant to nalidixic acid and kanamycin and contain E. coli plasmids encoding the F pilus, conferring susceptibility to male-specific coliphages. MS2 phages are commonly used to represent male-specific coliphages in the laboratory.
Somatic coliphages are a large group comprising representatives from 4 bacteriophage families. Somatic coliphages are DNA viruses, but can contain either circular or linear DNA, depending on their family. The Host bacterial strain used to propagate the somatic coliphages for the procedures described here is E. coli CN13, with coliphage strain phiX174 (from the Microviridae family) used as a model for this group.
There are advantages and disadvantages to using coliphages as indicators of fecal contamination. For example, one advantage to using male-specific coliphages is that some of the ssRNA coliphages are morphologically and structurally similar to many ssRNA enteric viruses, such as the picornaviruses (poliovirus, coxsackievirus, hepatitis A), calicivirus families (norovirus), and hepatitis E virus suggesting they are similarly affected by environmental conditions.6 However, one characteristic of using the male-specific coliphage is that expression of the bacterial F pilus, necessary for infection, requires warmer temperatures, with optimal expression between 37 and 42 °C, thus limiting the coliphage replication in the environment at temperatures below 30 °C. A variety of methods are available to detect coliphages in water, including both culture-based and molecular methods. The culture-based methods described here are largely based on those developed by the US EPA (Methods 1601 and 1602).
In recent years, molecular methods for the detection of coliphage in water have been developed. However, most methods have focused on detecting the male-specific FRNA coliphage.8,9-13
The procedures described are detailed protocols for the detection of somatic and male-specific coliphages, using bacterial host cells, single-agar layer, double-agar layer after enrichment, membrane filter, and the commercial method, Fast Phage. Escherichia coli Famp is the host for the detection of male-specific coliphages (FRNA or FDNA), whereas for the detection of somatic coliphages the host is E. coli CN13. Methods are also described using the alternate host bacteria, Salmonella ser. Typhimurium WG49 expressing the F pilus to culture male-specific coliphages.14,15