Bluephage, a method for efficient detection of somatic coliphages in one hundred milliliter water samples

Abstract

Emerging water quality guidelines and regulations require the absence of somatic coliphages in 100 mL of water, yet the efficiency of standardized methods to test this volume of sample is questionable. A recently described procedure, Bluephage, using a modified E. coli host strain, overcomes some of the methodological limitations of standardized methods. In a maximum of 6.5 hours (2.5 hours for pre-growing the host strain and 4 hours for the presence/absence test), Bluephage allows the direct detection of one plaque-forming unit (PFU) in a 100 mL water sample. The test shows high levels of specificity for somatic coliphages and comparable accuracy with standardized methods.Coliphages, viruses infecting Escherichia coli, have been included or are being considered for inclusion in water quality regulations and guidelines around the world 1-6 . Some of these regulations, such as those for different types of drinking water 5,6 , prescribe or recommend the absence of somatic coliphages in 100 mL water samples.Widely accepted standardized methods for determining somatic coliphages are available 7-9 . The host strains and media endorsed in the ISO (International Organization for Standardization) standard 10705-2:2000 7 and the USEPA (U.S. Environmental Protection Agency) 1601 and 1602 methods 8,9 provide similar results 10-12 . To determine coliphage absence in 100 mL water samples, which requires analysis of the full 100 mL volume, the following standardized procedures are currently used: (1) the presence/ absence test according to ISO or USEPA; (2) the double agar layer (DAL) assay as indicated in ISO 7 , which requires the concentration of phages in 100 mL of water sample followed by phage enumeration in the full concentrate, and (3) the USEPA's single agar layer (SAL) assay 8 , which is applied to 10 replicas of 10 mL. According to ISO and USEPA standards, the presence/absence test requires 2 steps (enrichment and spot test) and needs more than one working day to obtain results. On the other hand, both the concentration 13,14 and SAL procedures are complex and suffer from loss of efficiency in coliphage recovery 10,15 . Feasible, efficient, fast and user-friendly methods to determine the presence of coliphages in 100 mL water samples are therefore required. Recent progress in this field [16][17][18] includes the development of Bluephage method 18 , whose application can facilitate the routine implementation of somatic coliphage determination in laboratories with minimal equipment and expertise requirements.To detect phage concentrations as low as one in a given volume is challenging, as viral particles have an uneven spatial distribution in watery suspensions, theoretically following the Poisson model 19 . Accordingly, to assess the accuracy and detection limit of different phage determination methods, a statistical approach was used to calculate the distribution in aliquots of suspension and consequently the expected percentage of positive samples in a set of recipients prepared to contain about one phage per 100 m...

Comparison of methods for the enumeration of coliphages in 100 mL water samples

“…In wastewater samples, the lower counts obtained by techniques using a SAL rather than DAL assay are consistent with the scant data available in the literature (Méndez et al, 2020;Mooijman et al, 2001). When describing the SAL procedure in the late 1980s, Grabow and Coubrough compared different media and host bacterial strains, but recognised the difficulty of conducting a comparative study of SAL and DAL assays in 100 mL samples due to the highly variable recoveries produced by each technique (Grabow and Coubrough, 1986).…”

Colifagi Somatyczne jako Wskaźnik w Ocenie Jakości wody do Picia

“…Testy te mogą być oparte na znormalizowanych metodach ISO i EPA. Umożliwiają one oznaczenia jakościowe oraz ilościowe colifagów somatycznych [6,27,39,49,52,53]. Ich zastosowanie może obejmować różne matryce, w tym wodę, ścieki i osady.…”

Biological Indicators for Fecal Pollution Detection and Source Tracking: A Review

“…The substrate resulting from the overexpressed β-glucuronidase is chromogenic and produces a blue color that indicates coliphage density [185]. This method features an excellent sensitivity (as low as 1 PFU/100 mL), an excellent specificity ensured by the coliphage, and a rapid procedure within 6.5 h (2.5 h for pre-growing host E. coli strains, 4 h for the assay) [186]. Thus, the Bluephage method can provide a rapid and sensitive estimation of fecal pollution in water samples.…”