Development of a multiplex droplet digital PCR assay for simultaneous detection and quantification of <i>Escherichia coli, E. marmotae, </i>and <i>E. ruysiae</i> in water samples

<p dir="ltr"><a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/escherichia" target="_blank"><i>Escherichia</i></a><i> </i><i>coli</i> are widely used by water quality managers as Fecal Indicator Bacteria, but current quantification methods do not differentiate them from benign, environmental <i>Escherichia</i> species such as <i>E.</i><i> </i><a href="https://www.sciencedirect.com/topics/immunology-and-microbiology/marmota" target="_blank">marmotae</a> (formerly named cryptic clade V) or <i>E. ruysiae</i> (cryptic clades III and IV). Reliable and specific techniques for their identification are required to avoid confounding microbial water quality assessments. To address this, a multiplex <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/droplet-digital-polymerase-chain-reaction" target="_blank">droplet digital PCR</a> (ddPCR) assay targeting <i>lipB</i> (<i>E. coli</i> and <i>E. ruysiae</i>) and <i>bglC</i> (<i>E.</i><i> </i><a href="https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/marmota" target="_blank"><i>marmotae</i></a>) was designed. The ddPCR performance was assessed using <i>in silico</i> analysis; <a href="https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/genomic-dna" target="_blank">genomic DNA</a> from 40 local, international, and reference strains of target and non-target <a href="https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/coliform-bacterium" target="_blank">coliforms</a>; and spiked water samples in a range relevant to water quality managers (1 to 1000 cells/100 mL). Results were compared to an analogous <a href="https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/real-time-polymerase-chain-reaction" target="_blank">quantitative PCR</a> (qPCR) and the Colilert method. Both PCR assays showed excellent sensitivity with a limit of detection of 0.05 pg/μL and 0.005 pg/μl for ddPCR and qPCR respectively, and of quantification of 0.5 pg/μL of genomic DNA. The ddPCR allowed differentiation and quantification of three <i>Escherichia</i> species per run by amplitude multiplexing and showed a high concordance with concentrations measured by Colilert once proportional bias was accounted for. <i>In silico</i> specificity testing underlined the possibility to further detect and distinguish <i>Escherichia</i> cryptic clade VI. Finally, the applicability of the ddPCR was successfully tested on environmental water samples where <i>E. marmotae</i> and <i>E. ruysiae</i> potentially confound <i>E. coli</i> counts based on the Most Probable Number method, highlighting the utility of this novel ddPCR as an efficient and rapid discriminatory test to improve water quality assessments.</p>