Determination of crAssphage in water samples and applicability for tracking human faecal pollution

Abstract

SummaryIn recent decades, considerable effort has been devoted to finding microbial source‐tracking (MST) markers that are suitable to assess the health risks of faecally polluted waters, with no universal marker reported so far. In this study, the abundance and prevalence of a crAssphage‐derived DNA marker in wastewaters of human and animal origins were studied by a new qPCR assay with the ultimate aim of assessing its potential as an MST marker. crAssphage showed up to 106 GC/ml in the sewage samples of human origin, in both the total DNA and the viral DNA fraction. In wastewaters containing animal faecal remains, 39% of the samples were negative for the presence of the crAssphage sequence, while those showing positive results (41% of the samples) were at least 1 log10 unit lower than the samples of human origin. Noteworthy, the log10 values of the ratio (R) crAssphage (GC/ml)/Escherichia coli (CFU/ml) varied significantly depending on the human or animal origin (R > 1.5 for human samples and R < −1.5 for animal wastewater samples. This study opens the way for further research to explore if different specific animal variants of crAssphage exist and whether other zones of the crAssphage genome are better suited to source discrimination.

Quantitative CrAssphage real‐time PCR assay derived from data of multiple geographically distant populations

“…For this purpose, Stacher et al derived the candidate set of primers from a single CrAssphage genome assembly deposited in the databases, without aiming for detection of a broader range of its variants. Also Garcia‐Aljaro et al used CrAssphage detection for microbial source tracking—they derived their primers from ten human sewage samples. These primer‐probe combinations annealed suboptimally to a substantial proportion of our multinational sample set (for data see Supplementary Material S1 where presumed matching of the primers and probes to the viral genomes is analyzed for our samples with more than 10 copies CrAssphage /μL).…”

“…Understandably, the assay might fail in any CrAssphage variants with the orf00042 deleted or too genetically distant—such virus types were however absent from our multinational metagenomic set. Our oligonucleotide design also aimed for resistance to minor genetic variation: we purposedly used relatively long primers and a classic TaqMan probe design, which is known to tolerate mismatches under its sequence much better than short minor groove binder (MGB) probes that had been used in the previous three assays …”

“…Our oligonucleotide design also aimed for resistance to minor genetic variation: we purposedly used relatively long primers and a classic TaqMan probe design, which is known to tolerate mismatches under its sequence much better than short minor groove binder (MGB) probes that had been used in the previous three assays. [5][6][7] A weakness of our study lies in the lack of a reference method to which any novel CrAssphage PCR assay might be related. The virus is unculturable and its host unknown.…”

Fecal pollution: new trends and challenges in microbial source tracking using next‐generation sequencing

“…Recently, García‐Aljaro et al . () developed a qPCR‐based assay which targets crAssphage for human fecal pollution. While these bacteriophages have been reported to be source‐specific, there have been contradicting reports of their geographical distribution, cross‐reactivity and variable detection rates (McMinn et al ., ).…”

Pathogens, faecal indicators and human-specific microbial source-tracking markers in sewage

“…Recently, a novel bacteriophage predicted by computational analysis to be highly abundant in human faeces has been used in combination with E. coli to differentiate human from animal faecal contamination in sewage samples (García‐Aljaro et al . ).…”