Phages in water: alternative indicators viral bacterial indicators. Qualitative and quantitative methods (EPA 1601 and 1602, for Somatic phages and F +).

(EPA Method 1601: 2001. Male-specific (F +) and Somatic Coliphage in Water by Two-step Enrichment Procedure) (EPA Method 1602: 2001. Male-specific (F +) and Somatic Coliphage in Water by Single Agar Layer - Sal Procedure)

Information 16/04/07.

Inland freshwater and marine waters can become contaminated with human and animal excreta. Human excreta may contain enteric pathogens such as viruses Enterovirus, Adenovirus, and that can cause outbreaks Norovirus or isolated cases of acute gastroenteritis, infectious hepatitis (hepatitis A) and other diseases. Human enteric viruses can survive after treatment better than other indicators of fecal contamination and pathogenic bacteria wastewater, and often the treatment of wastewater is inadequate to prevent pollution of marine waters and bioaccumulation of molluscs bivalves ingest.

Sewage can contain bacteria and viruses that cause diseases, as well as safe as are some enteric bacteria and bacteriophages fecal indicators. Bacteriophages can be used as indicators for determining fecal water quality, based on that controlling these as an indicator, the possible presence of other less prevalent pathogens is controlled.

Common methods for detecting faecal contamination indicator bacteria (e.g. coliforms, Enterococcus -.. Streptococcus fecales-) not reliably predict the presence of viruses in water or shellfish.

The most common fecal indicator testing waters are coliforms and Escherichia coli. However, other bacteria such as Enterococcus spp. and a bacterial viruses as coliphages (Escherichia coli bacteriophage) have been recognized as markers of faecal contamination equivalent to Escherichia coli. Coliphages are bacterial viruses and some of them are structurally similar to enteric pathogenic viruses (with a similar capsid of enteric virus protein coat), with a DNA genome or RNA single stranded (ss) or double stranded (ds) . Some phages are called phages "male" or F +, by infesting the bacteria through sexual pili possessing the "male" bacteria (F +). These phages are morphologically similar to enteroviruses and have similar chemical treatments water resistance. Among such phages are the Leviviridae (ssRNA, 24 nm in diameter, phage MS2) and Inoviridae (ssDNA, 810 x 6 nm, fd phage). Other phages are called somatic coliphages or F-, because they require the presence of sex pili to infect bacteria, and the infected through contact with the bacterial cell wall. These include the Myoviridae (dsDNA, 95 x 65 nm, phage T2), the Styloviridae (dsDNA, 54 nm, ? phage), the Podoviridae (dsDNA, 47 nm, phage T7), and Microviridae (ssDNA, 30 nm , phage ?X174).

Coliphages removed with faecal excretions and not replicated in water unless coliforms are present, so are a useful indicator of fecal contamination. Therefore, they are considered a good indicator to determine the viral risk in a water supply. It has been shown that the monitoring of phages can be a useful tool for monitoring and control of the low levels of contamination, especially in chlorinated water as coliphages have a resistance higher than the Enterovirus chlorine, as it is the case of type 1 poliovirus.

Detection of phages is an alternative to bacterial indicators of water contamination as they are indicators of fecal contamination, while the possible presence of enterically transmitted virus such as infectious hepatitis -hepatitis of A-, Norovirus, or other enteroviruses, so currently detecting coliphages, is considered an indicator of water quality. The most common biological contamination comes from wastewater contaminated with human or animal excreta.

Detection methods Coliphages

EPA 1601 method is a qualitative method (presence or absence) enrichment in two stages, using two types of host bacteria for the presence of both groups of phages (F + and somatic), previously cited. After an incubation period of water in the presence of indicator bacteria, samples should be deposited on a layer of specific bacterial growth for each type of phage which are incubated and examined for the presence of circular areas of lysis. This method is considered the most sensitive to exclude the presence of phages in a sample of water. EPA 1602 method uses an assay in one step and quantifies the amount of phages present.

It has been recommended various methods (water treatment with chloroform, water filtration membrane, treating the water with detergents, ...), to try to eliminate possible bacterial flora present in the water passenger, which can interfere with the development of Escherichia coli reporter strains of, or prevent visualization of the areas of lysis caused by replication coliphages. However, all recommended methods reduce to some extent the detection of phages, so should not be used if it is to detect small amounts of phages.

Also, there are proposed various methods to concentrate the phages in water samples, adsorption to hydroxylapatite and elution with sodium phosphate, filtration ion exchange resins and adsorption filter with micropores, adsorption filters micropores and elution with meat extract , etc.). However these methods are not suitable for concentrating phages for their sensitivity to pH levels that are used in some methods for adsorption. These methods are based on that low pH viruses possess positive charges and adhere to negatively charged membranes; while for circumvention of the membranes a high pH is required. These changes can damage extreme pH and make coliphages not be detected with the methods based on replication.

In general, to make a quantitative determination is necessary to prepare serial dilutions of the water sample and inoculate duplicate dilutions on plates with the indicator bacteria. However, since it is considered that the presence of phages should not be detected, the EPA 1601 method recommended not dilute the sample and perform exposure of 100 mL of water to each of the two strains of indicator Escherichia coli and the medium culture, to thereby prepare a series of agar plates which will determine the absence of phages of either groups.

The limit is 0 phages in 100 mL of drinking water.

Tests in IVAMI

  • Qualitative test for detection of phages F + (males) and somatic (F-), according to EPA standard 1601: 2001.
  • Quantitative test for detection of phages F + (males) and somatic (F-) according to standard EPA 1602: 2001.

Delivery of results

  • The minimum necessary for carrying out the cultures (48 to 72 hours).

Form with product features and variables for testing

If applying for conducting tests with the product you must send the corresponding completed form.

The minimum volume of water required is February 00 mL (100 mL for each of the types of phages).

If the water is chlorinated, it should be treated with 1 to 3 mg / liter of sodium thiosulfate to remove chlorine.

Preservation and shipment of sample:

  • Refrigerated (preferred) for a maximum time of 24 hours.  

Cost of the test:

  • EPA Method 1601 (isolates)
  • EPA Method 1602 (isolates)
  • Several samples received simultaneously
  • Consult