Rapid Quantification of Bacteriophages of Mycobacterium spp. and Related Genera in Aquatic Systems

Viruses shape microbial communities but remain underexplored in aquatic systems. Most studies target specific viruses as contamination indicators, while the potential of phages to reflect microbial dynamics is often overlooked. We designed and optimised two qPCR assays to quantify specific phages from the order Caudoviricetes-some of the most abundant in aquatic environments-infecting Mycobacterium, Rhodococcus and Gordonia spp. Major capsid protein (MCP) sequences were retrieved from the NCBI Viral Database and degenerate and non-degenerate primers, along with a TaqMan probe, were developed targeting the MCP gene. SYBR Green and TaqMan assays were optimised, validated using an MCP sequence and applied to samples from a drinking water treatment plant (DWTP), urban lakes and activated sludge. Both assays showed strong linearity with efficiencies of 95% (TaqMan) and 94% (SYBR Green). Viral gene copies decreased across the DWTP, indicating up to four-log phage removal during treatment. Phage counts in source water paralleled bacterial counts, especially in the cold season, suggesting a potential correlation between phages and bacterial hosts. Lake samples had higher phage concentrations (up to 10⁶ copies/L), while only two wastewater treatment plants showed phage presence. These assays offer sensitive tools for monitoring phage abundance and microbial dynamics in freshwater environments.