• Simultaneous detection of protozoa, bacteria, and viruses in water • 0.8 µm MCE membrane with MgCl 2 offers highest recovery efficiency • rRNA targeting and preamplification improve detection sensitivity • This method achieves as high sensitivity as IFAT for Giardia detection Waterborne outbreaks caused by protozoan, bacterial, and viral pathogens continue to pose serious public health threats. However, comprehensive monitoring of all three major pathogen types remains uncommon, primarily due to the technical difficulty of simultaneously concentrating and characterizing these diverse microorganisms from a single water sample. Here, we evaluated a membrane adsorption followed by bead-beating-based direct nucleic acid extraction for its sensitivity in detecting waterborne pathogens. We further applied the optimized protocol to river water samples. Our results demonstrated that the use of magnesium chloride in combination with a mixed cellulose ester membrane filter (pore size: 0.8 μm) achieved the highest concentration efficiency for Cryptosporidium parvum, Legionella pneumophila , and murine norovirus (MNV) in spiked water samples, outperforming other methods utilizing membranes with alternative pore sizes or membrane with another material (i.e., nylon). Furthermore, under controlled spiking conditions, targeting rRNA of C. parvum instead of rDNA, or incorporating a preamplification step for the L. pneumophila mip gene and MNV, significantly improved the sensitivity. Notably, under the spiking conditions, the 50% limit of detection for C. parvum using our method was comparable to that of traditional immunofluorescent antibody testing. Application of this method to river water demonstrated its environmental applicability, revealing a higher abundance of Bacteroides rRNA markers compared to their corresponding rDNA, underscoring the potential of rRNA-based targets for more sensitive bacterial detection. Importantly, when the target is genomic DNA or RNA, preamplification enables the use of a larger nucleic acids input volume, thereby enhancing sensitivity. Overall, the combination of membrane adsorption and bead-beating-based direct nucleic acid extraction offers a sensitive approach for simultaneous detection of protozoa, bacteria, and viruses in environmental water. This method serves as an effective ‘screening’ tool for the presence of the three major types of waterborne microorganisms.
Torii et al. (Sun,) studied this question.