Simultaneous detection of protozoa, bacteria, and viruses from environmental water through membrane-adsorption followed by direct nucleic acid extraction

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 m) achieved the highest concentration efficiency for Cryptosporidium parvum, Legionella pneumophila, and murine norovirus (MNV), outperforming other methods utilizing the membrane with alternative pore sizes or membrane with another material (i.e., nylon). Furthermore, 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, 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 further revealed 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 without introducing measurement bias. Overall, the combination of membrane adsorption and bead-beating-based direct nucleic acid extraction offers a sensitive, all-in-one 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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/669200v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@d0e9f4org.highwire.dtl.DTLVardef@1afc9fcorg.highwire.dtl.DTLVardef@1f5c355org.highwire.dtl.DTLVardef@1eb14eb_HPS_FORMAT_FIGEXP M_FIG C_FIG