Identifying potential polymicrobial pathogens: Moving beyond differential abundance to driver species

It is now recognized that some diseases of aquatic animals are attributed to polymicrobial pathogen infections. Thus, the traditional view of "one pathogen, one disease" might mislead the identification of multiple pathogens, which in turn impedes the design of probiotics. To address this gap, we explored polymicrobial pathogens bases on the origin and timing of increased abundance over shrimp white feces syndrome (WFS) progression. OTU70848 Vibrio fluvialis, OTU35090 V.coralliilyticus and OTU28721 V.tubiashii were identified as the primary colonizers, whose abundances increased only in individuals that eventually showed disease signs but were stable in healthy subjects over the same timeframe. Notably, the random Forest model revealed that the profiles of three primary colonizers contributed an overall 91.4% of diagnosing accuracy of shrimp health status. Additionally, NetShift analysis quantified that the three primary colonizers were important "drivers" in the gut microbiotas from healthy to WFS shrimp. For these reasons, the primary colonizers were potential pathogens that contributed the exacerbation of WFs. By this logic, we further identified a few "divers" commensales in healthy individuals, such as OTU50531 Demequina sediminicola and OTU74495 Ruegeria mobilis, which directly antagonized the three primary colonizers. The predicated funtional pathways involved in energy metabolism, genetic information processing, terpenoids and polyketides metabolism, lipid and amino acid metabolism significantly decreased in diseased shrimp compared to healthy cohorts, in concordant with the knowledge that the attenuations of these functional pathways increase shrimp sensitivity to pathogen infection. Collectively, we provide an ecological framework for inferring polymicrobial pathogens and designing antagonized probiotics by quantifying their changed "divers" feature that intimately links shrimp WFS progression. This approach might generalize to the exploring disease etiology for other aquatic animals.