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    Bacterial flora varies throughout the saltwater crocodile (Crocodylus porosus) gastrointestinal tract
    (Amer Veterinary Medical Assoc, 2023) Siddiqui, Ruqaiyyah; Maciver, Sutherland K.; Anuar, Tengku Shahrul; Khan, Naveed Ahmed
    OBJECTIVEThe objective of this study was to determine bacterial flora throughout the gastrointestinal tract of a saltwater crocodile (Crocodylus porosus) using 16S rRNA gene analysis.ANIMALSA convention on international trade in endangered species (CITES) of wild fauna and flora registered crocodile farm, provided a healthy male saltwater crocodile, Crocodylus porosus for this study.PROCEDURESThree samples were taken from the oral cavity, 3 samples from the proximal region of the small intestine (jejunum), and 3 samples from the distal part of the large intestine of the gastrointestinal tract of C. porosus were obtained using sterile cotton swabs. Next, swabs were placed in 15 mL sterile centrifuge tubes, individually, and kept on ice for immediate transportation to the laboratory. This was followed by 16S rRNA gene analysis using specific prim- ers (341F-CCTAYGGGRBGCASCAG, and 806R-GGACTACNNGGGTATCTAAT). Amplicons were sequenced on Illumina paired-end platform, and bacterial gastrointestinal communities, the relative abundance of taxa, and principal component and coordinate analysis were performed.RESULTSThe findings revealed that bacterial community structures from differing regions exhibited several differences. The number of observed bacterial operational taxonomic units (OTUs) was 153 in the oral cavity, 239 in the small intestine, and 119 in the large intestine of C. porosus. The small intestine reflects the highest richness. In contrast, the large intes- tine exhibited the least richness of microbial communities. Relative abundance of taxa showed that Proteobacteria, Bacteroidetes, and Firmicutes were dominant in all 3 sample sites. Pseudomonas differed in the oral cavity and the large intestine, with the latter exhibiting less distribution of Pseudomonas. Stenotrophomonas and Castellaniella were higher in the oral cavity, while the relative abundance of Comamonas and Salmonella was higher in the small intestine. Conversely, the relative abundance of Salmonella and Pannonibacter was augmented in the large intestine.CLINICAL RELEVANCEFor the first time, this study demonstrates the bacterial diversity along the segments of the gastrointestinal tract of C. porosus. Bacterial flora varies throughout the gastrointestinal tract. Although further studies using large cohorts are warranted; however, our findings suggest that microbiome composition may have the potential as a biomarker in determining the overall health and well-being of C. porosus.
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    Gut microbiome of Crocodylus porosus and cellular stress: inhibition of nitric oxide, interleukin 1-beta, tumor necrosis factor-alpha, and prostaglandin E2 in cerebrovascular endothelial cells
    (Springer, 2023) Siddiqui, Ruqaiyyah; Akbar, Noor; Maciver, Sutherland K.; Alharbi, Ahmad M.; Alfahemi, Hasan; Khan, Naveed Ahmed
    Crocodiles are renowned for their resilience and capacity to withstand environmental stressors, likely influenced by their unique gut microbiome. In this study, we determined whether selected gut bacteria of Crocodylus porosus exhibit anti-inflammatory effects in response to stress, by measuring nitric oxide release, interleukin 1-beta, tumor necrosis factor-alpha, and prostaglandin E2 in cerebrovascular endothelial cells. Using the Griess assay, the findings revealed that among several C. porosus gut bacterial isolates, the conditioned media containing the metabolites of two bacterial strains (CP27 and CP36) inhibited nitric oxide production significantly, in response to the positive control, i.e., taxol-treatment. Notably, CP27 and CP36 were more potent at reducing nitric oxide production than senloytic compounds (fisetin, quercetin). Using enzyme linked immunosorbent assays, the production of pro-inflammatory cytokines (IL-1 beta, TNF-alpha, PGE2), was markedly reduced by treatment with CP27 and CP36, in response to stress. Both CP27 and CP36 contain a plethora of metabolites to exact their effects [(3,4-dihydroxyphenylglycol, 5-methoxytryptophan, nifedipine, 4-chlorotestosterone-17-acetate, 3-phenoxypropionic acid, lactic acid, f-Honaucin A, l,l-Cyclo(leucylprolyl), 3-hydroxy-decanoic acid etc.], indicative of their potential in providing protection against cellular stress. Further high-throughput bioassay-guided testing of gut microbial metabolites from crocodiles, individually as well as in combination, together with the underlying molecular mechanisms, in vitro and in vivo will elucidate their value in the rational development of innovative therapies against cellular stress/gut dysbiosis.
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    Mass spectrometric analysis of bioactive conditioned media of bacteria isolated from reptilian gut
    (Future Sci Ltd, 2023) Siddiqui, Ruqaiyyah; Akbar, Noor; Soares, Nelson Cruz; Al-Hroub, Hamza Mohammad; Semreen, Mohammad Harb; Maciver, Sutherland K.; Khan, Naveed Ahmed
    Plain language summaryCrocodiles thrive in unsanitary conditions, feed on rotten meat, and endure conditions that are detrimental to human health. In addition to their immune system, we speculate that their microbial gut flora produce substances contributing to their hardiness and longevity. Herein, we showed that selected bacteria isolated from crocodile gut produced potent antibacterial properties against multiple drug-resistant pathogenic Gram-negative and Gram-positive bacteria. LC-MS/MS revealed the identity of gut microbial metabolites. These findings suggest that analyses of crocodile gut bacteria may reveal potential drug leads that can be utilized as probiotics/pre/post/antibiotics for the benefit of human health, however intensive future research is needed to realize these expectations. Aim: To determine whether selected gut bacteria of crocodile exhibit antibacterial properties. Materials & methods: Two bacteria isolated from Crocodylus porosus gut were used, namely: Pseudomonas aeruginosa and Aeromonas dhakensis. Conditioned media were tested against pathogenic bacteria and metabolites were analyzed using liquid chromatography-mass spectrometry. Results & conclusion: Antibacterial assays revealed that conditioned media showed potent effects against pathogenic Gram-positive and Gram-negative bacteria. LC-MS revealed identity of 210 metabolites. The abundant metabolites were, N-Acetyl-L-tyrosine, Acetaminophen, Trans-Ferulic acid, N, N-Dimethylformamide, Pyrocatechol, Cyclohexanone, Diphenhydramine, Melatonin, Gamma-terpinene, Cysteamine, 3-phenoxypropionic acid, Indole-3-carbinol, Benzaldehyde, Benzocaine, 2-Aminobenzoic acid, 3-Methylindole. These findings suggest that crocodile gut bacteria are potential source of novel bioactive molecules that can be utilized as pre/post/antibiotics for the benefit of human health. Tweetable abstractGut microbiome of hardy crocodile is a potential pharmacy that should be exploited for human benefit.