ABSTRACT: Crocodiles thrive in unsanitary conditions, feed on rotten meat, are exposed to heavy metals and are among the very few species to endure the catastrophic Cretaceous-Tertiary extinction event, and yet they can live up to 100 years. We hypothesized that crocodiles have developed mechanisms to achieve such longevity while surviving under stressful conditions. We speculate that their microbial gut flora may produce substances contributing to their “hardiness” and “longevity”. Previously we characterized selected microbial gut bacteria colonizing the gastrointestinal tract of Crocodylus porosus (CP) using 16S rDNA sequencing. Next, bacterial conditioned media containing gut microbial metabolites were prepared. Bioassay-guided testing of selected bacterial conditioned media using LC-TIMS-QTOF MS, revealed the identity of gut microbial metabolites. Among two bacterial conditioned media, i.e., CP27 and 36, the analyses resulted in 141 highly confidently (MS/MS) identified metabolites in both samples. The pairwise comparison of the two samples indicated that 109 metabolites change significantly between them (p <0.05). Among abundant metabolites more prevalent in CP36 there were 2-Methyl-4-nitroimidazole, N-Acetyl-L-tyrosine, Acetaminophen, Trans-Ferulic acid, N, N-Dimethylformamide, Pyrocatechol, Cyclohexanone, 3, 4-Dihydrozphenylglycol, Diphenhydramine, Melatonin, Gamma –terpinene. Whereas in CP27 samples the most abundant metabolites were Carbamazepin, deoxyninosine, Cysteamine, Benzylnicotinate, 3-phenoxypropionic acid, Indole-3-carbinol, Benzaldehyde, Benzocaine, 2-Aminobenzoic acid, 3-Methylindole. Functional enrichment analysis of all identified metabolites with metabolite sets based on drug pathways showed that they were enriched for drug action of top ten pathways associating with enalapril metabolism pathway, diphenhydramine H1-Antihistamine action, enalarpil action pathway, benzocaine action pathway, mepivacaine action pathway, oxybuprocaine action pathways, nifedipine action pathway, propranolol action pathway, acetaminophen metabolism pathway, carbamazepine metabolism pathway. These findings suggest that analyses of crocodile gut bacteria may reveal potential drug leads, intensive future research is needed to realize these expectations.
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