Bacteriophages as indicators

Bacteriophages of gut bacteria have characteristics and behavior outside the gut that make them suitable for being used as indicators of different aspects related with fecal pollution. Coliphages have been proposed as indicators of fecal/viral pollution (IAWPRC, 1991; Ashbolt et al., 2001; Jofre et al., 2016; McMinn et al., 2017) and phages infecting Bacteroides have been proposed as markers of the source of fecal pollution (Jofre et al., 2014).

3.1 Coliphages

A coliphage is defined as a bacteriophage able to infect Escherichia coli. This bacterium is included among the coliform bacteria group, which is a group of genotypically related bacteria. This kind of bacteria are the most abundant aerobic prokaryotes in the gut for both human and animals, and hence easy to culture in the laboratory. Consequently, coliphages are also the most abundant bacteriophages present in the gut that are easy to cultivate.

Just like any other bacteriophages, coliphages have a narrow range of possible hosts. Some coliphages can infect coliform bacteria other than E. coli (for example Shigella or Klebsiella). The specificity is affected by the way of entering the cell. As we explained when defining bacteriophages, we differentiate between somatic (entering the cell through the cell wall) and F-specific bacteriophages (entering the cell using the sexual pilli), and this can be applied as well to coliphages. As for the morphology of coliphages, every group previously described (Myoviridae, Syphoviridae, Podoviridae, Microviridae, Inoviridae and Leviviridae) is represented among them.

Two groups of bacteriophages infecting E. coli, somatic and F-specific coliphages, have been investigated in academia for many years as both fecal and viral indicators.

The high concentrations of coliphages found in raw wastewater and in many other matrixes contaminated with fecal remains; the easy, fast and cost effective detection and enumeration methods; and the persistence in the water environment and the resistance to treatments that resemble those of viruses, make indicator bacteriophages good surrogate indicator candidates for various set-ups.

For practical purposes, coliphages are organized in operational groups rather than in taxons, mostly because of the current methods for their detection and quantification. Somatic, F-specific and the subset F-specific RNA coliphages have been extensively studied for this purpose. Humans and animals excrete both groups.

Coliphages have the properties required for an indicator of fecal and/or viral pollution: they are present in high numbers in sources of fecal pollution; they are more resistant to natural and anthropogenic stressors than bacterial indicators and as resistant as viruses; they are present in water environments whenever fecal pollution exists; they are specific for fecal or sewage pollution; their chances of multiplying in water environments is, if any, extremely low; they are not pathogenic and detectable by simple, rapid and inexpensive methods.

Additionally, they present some attractive features for working with them:

Somatic coliphages are those infecting E. coli through the cell wall. Among them we find bacteriophages from the families Myoviridae, Syphoviridae, Podoviridae, Microviridae. There is consensus about using the host strain (CN13, WG5) to detect phages by standardized methods; other host strains might detect different numbers. Their strength relies both on the concentration found in sources of fecal pollution and method simplicity.

F-specific coliphages, also named sexual coliphages, are those infecting E. coli through the sexual pili and comprise phages of the families Inoviridae and Leviviridae. E. coli (HS) and Salmonella (WG49) strains have been designed to detect mostly F-specific bacteriophages. Their strength is due to their morphology, similar to that of many animal viruses. (Kuprovic et al, 2016)

Historically, the F-specific phages strategy was developed since it was the common belief that somatic coliphages were replicating in the environment, and since pili are not synthesized below 32ºC, F-specific were less prone to replicate outside the gut. However, nowadays there are many arguments to believe that the replication of somatic coliphages or F-specific coliphages outside the gut is very unlikely.

F-specific RNA phages can be further differentiated by counting plaques on detection media with and without RNAse. Since the process of infection of Leviviridae is sensitive to RNAse in the detection medium, the difference between the counts gives the numbers of F-specific RNA bacteriophages (link ISO 10705-1)

The sum of somatic and F-specific coliphages constitutes the group total coliphages. Host strains (for example CB390) counting total coliphages have been developed (Guzman et al., 2008).

3.2 Coliphages in guidelines and regulations

Furthermore, regulatory authorities in different areas of the world are beginning to consider coliphages as indicators for water quality criteria. Examples of ruling guidelines that include bacteriophages are displayed in table 3.1

Year Country: Regulation sector Description
1989 USA Integrity membranes and UV USEPA. 1989. Drinking Water; national Primary Drinking Water Regulations; Filtration; Disinfection; Turbidity, Giardia lamblia; Viruses; Legionella; and Heterotrophic Bacteria; Final Rule. 40CFR Parts 141 and 142. Federal register 54: 27486-27541. Washington D.C.
1999 United Kingdom Integrity membranes and UV DWI. 1999. The Water Supply (Water Quality) (Amendment) Regulations 1999: Cryptosporidiumem> in Water Supplies. Department for Environment, Food and Rural Affairs. Statutory Instruments No. 1524. United Kingdom Legislation (available at http://united-kingdom-legislation.vlex.co.uk/vid/water-supply-quality-amendment-28393731)
2001 Canada (Quebec) Drinking Water Loi sur la qualité de l’environnement : règlement sur la qualité de l'eau potable c. Q.-2, r. 18.1.1. Gazette Officielle du Québec 24, 3561. Government of Quebec, Montreal, Quebec, Canada
2005 Australia (Queensland) Reclaimed water Queensland Government. 2005. Queensland Water Recycling Guidelines. Queensland Environmental Protection Agency. Brisbane. Australia.
2006 Australia (Northern Territory) Reclaimed water DRAFT Northern Territory Interim Guidelines for Management of Recycled Water Schemes 2006
2006 Australia (Northern Territory) Reclaimed water Australian Guidelines for Water Recycling: Managing Health and Environmental Risks (Phase 1), 2006
2006 Australia (Western Australia) Reclaimed water Australian Guidelines for Water Recycling: Managing Health and Environmental Risks (Phase 1) 2006
2006 USA Groundwater 40 CFR Parts 9, 141, and 142 National Primary Drinking Water Regulations: Ground Water Rule; Final Rule.
2001 USA Integrity membranes and UV USEPA. 2001. Low-pressure membrane filtration for pathogen removal: application, implementation and regulatory issues. EPA 815-C-01-001. Environmental Protection Agency. Washington D.C.
2007 Australia (New South Wales) Recycled Water Interim NSW Guidelines for Management of Private Recycled Water Schemes, 2007
2008 Australia Recreational Water Guidelines for Managing Risks in Recreational Water (Emerging interest)
2008 Canada Recreational Water Guidelines for Canadian Recreational Water Quality (Third edition 2012)
2009 Australia (Western Australia) Recycled Water Draft Government of Western Australia Department of Health. Draft Guidelines for the Use of Recycled Water in Western Australia. Initial External Consultation
2009 USA Molluscan shellfish Guide for the control of Molluscan Shellfish. National shellfish Sanitation program
2011 Australia Drinking Water National Water Quality Management Strategy. Australian Drinking Water Guidelines 6 (version 3.2 updated February 2016)
2011 Canada Drinking Water Guidelines for Canadian Drinking Water Quality: Guideline Technical Document - Enteric Viruses
2011 USA (North Caroline) Reclaimed water North Carolina Environmental Quality. North Carolina Adm. Code 15A NCAC 2U Reclaimed Water; North Carolina Department of Environment and Natural Resources. Regulatory Review of North Caroline.
2012 Australia (Western Australia) Biosolids Western Australian guidelines for biosolids management. Department of Environment and Conservation
2014 Colombia Biosolids Decreto Número 1287 por el cual se establecen criterios para el uso de los biosólidos generados en plantas de tratamiento de aguas residuales municipales,
2014 França Reclaimed water Arrêté du 25 juin 2014 modifiant l’arrêté du 2 août 2010 relatif à l’utilisation d’eaux issues du traitement d’épuration des eaux résiduaires urbaines pour l’irrigation de cultures ou d’espaces verts.
2016 Europe Drinking Water DRAFT: Revision of Annex I of the Council Directive on the Quality of Water intended for Human consumption (drinking water Directive) Background paper on microbiologically safe water and microbiological parameters
2018 USA Recreational water Review of coliphages as possible indicators of fecal contamination for ambient water quality. EPA 820-R-15-098
2017 Europe Reclaimed Water DRAFT: Development of minimum quality requirements for water reuse in agricultural irrigation and aquifer recharge

3.3 Phages infecting Bacteroides as markers of the source of fecal pollution

Bacteroides is one of the most abundant bacterial genera in the human gut. It is a strict anaerobe. This limits even more its possibilities to reproduce outside the gut as it passes to aerobic environments. The most abundant phages infecting bacteroides are tailed (at least the ones described so far), and most of them are classified among the Siphoviridae. They are somatic bacteriophages and their host range is quite narrow. They are pretty abundant in fecally-contaminated water. Different strains of bacteroides detect bacteriophages present in the gut of different animals, which is useful for detecting whether the source of contamination is human or animal. The main drawback of this system is that there are doubts about their geographical continuity, so possible hosts have to be isolated in different geographical areas. Bacteriophages infecting bacteroides are useful for microbial source tracking (Jofre et al., 2014).

References

Ashbolt, N.J., Grabow, O.K. and Snozzi, M. (2001). Indicators of microbial water quality. In Water Quality: Guidelines, Standards and Health, ed. Fewtrell, L. and Bartram, J. pp. 289–315. London, UK: IWA Publishing.

Guzmán, C, Mocé-Llivina, L., Lucena, F. and J. Jofre (2008). Evaluation of the Escherichia coli host strain (CB390) for simultaneous detection of somatic and F-specific coliphages. Applied and Environmental Microbiology 74: 531-534    

IAWPRC, Study Group on Health Related Water Microbiology (1991). Bacteriophages as model viruses in water quality control. Water Res 25, 529–545.

Jofre, J., A.R. Blanch, F. Lucena and M. Muniesa. (2014). Bacteriophages infecting Bacteroides as markers for microbial source tracking. Water Research 55: 1-11.

Jofre, J., F. Lucena, A. R. Blanch and M. Muniesa. (2016). Review. Coliphages as Model Organisms in the Characterization and Management of Water Resources. Water 8, 199; doi:10.3390/w8050199.

Krupovic, M., Dutilh, B.E., Adriaenssens, E.M. et al. Taxonomy of prokaryotic viruses: update from the ICTV bacterial and archaeal viruses subcommittee. Arch Virol (2016) 161: 1095. https://doi.org/10.1007/s00705-015-2728-0.

McMinn, B.R., Ashbolt, N.J. and Korajkic, A. (2017). Bacteriophages as indicators of faecal pollution and enteric virus removal. Lett ApplMicrobiol 65, 11-26.

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