Molecular Characterization of Pathogenic Strains of Bacteria Causing Motile Aeromonas Septicemia in Heteropneustes fossilis: Implications for Potential Drug Development

Abstract

Background: Dysbiosis of harmonized microbial communities of fish are crucial to identify for better health management of aquaculture species. In addition to classical methods, high-throughput sequencing techniques are now frequently used to analyze the 16S rRNA gene amplicon to assess the shifting of microbial populations as biotic stressors for fish. This study elucidated the different microbiome community by both culture dependent and culture independent methods isolated from healthy and diseased stinging catfish (Heteropneustes fossilis) and associated environment. Moreover, the isolated pathogenic Aeromonas hydrophila strains from diseased fish skin were identified and confirmed by pathogenic gene amplification and experimental infection. In addition, an attempted was made to design potential inhibitor (drug like compound) against virulent serine protease. Methods: Healthy and diseased stinging catfishes, soil and water samples were collected from fifteen locations of Bangladesh. Bacterial community from fish tissues and environmental samples were isolated and identified by conventional culture methods and then confirmed by 16S rRNA gene sequencing. Different microbiome community were identified and compared by 16S rRNA gene specific metagenomics approach. Pathogenic Aeromonas hydrophila was isolated from diseased fish skin lesions using selective Rimler Shotts medium with novobiocin supplement. Pathogenicity of isolated A. hydrophila was confirmed by PCR amplification of five pathogenic genes. After confirming virulence factors, two isolates (AGM2 and AYN7) were selected for experimental infection. The pathogens were later identified based on pathogenic gene specific markers and characterized by antibiogram profiling. One of the pathogenic genes coded for the serine protease was selected for identification of effective drug using in silico drug designing bioinformatics tools. Results: Higher bacterial community was evident in healthy fish tissue than diseased fish; and found similarities with the communities of soil and water samples by culture dependent method. Significant difference of bacterial load was observed between healthy and diseased fish tissues. Similarly, the richer and diversified microbiome communities were found in healthy fish skin than diseased fish skin by culture independent 16S rRNA metagenomics. At the genus level, Pseudomonas had the highest richness in healthy groups and was almost completely absent in the diseased groups, but Flavobacterium was quite prevalent in diseased fishes. The diseased group harbored the Aeromonas hydrophila as minor dominant which might reflect their opportunistic characteristics. Two isolated of A. hydrophila (AGM2 and AYN7) showed highest resistance in antibiogram test (9 and 6 antibiotics, respectively) against 11 antibiotics. In Mueller-Hinton (MH) agar media, the growth of these two isolates was inhibited by ceftriaxone, cefepime, gentamycin, azithromycin, and ciprofloxacin. Challenge test with AGM2 showed higher pathogenicity than AYN7 in post injection of healthy stinging catfish. Pathological signs and symptoms (lesions) and excess mucus (typical signs of MAS) were observed in injected fish with both isolates. The virulent gene ahpA encoded for serine protease was characterized and the homology modelling was performed to find potential inhibitor against the protein. An important parameter Ramachandran plot exhibited maximum amino acid residues (88.1%) in the favorable region which was also supported by the accepted G-factor value (overall score 0.38). The molecular docking of 3D structure showed the highest affinity (-7.6) with ceftriaxone from six selected compounds. Conclusion: Fish health can be impacted by the highly varied microbial communities associated with skin across populations and species. Several factors can alter the community structure to make dominant as single or mixed pathogens. The findings of this study will improve the understanding of microbiota modulation and would act as a baseline information to develop possible drug (s) like compound that would be helpful to avoid indiscriminate use of antibiotic in aquaculture sector.