Gut bacterial communities in healthy and compromised green turtles (Chelonia mydas) and an alternative treatment for gastrointestinal disorders

Green turtles (Chelonia mydas) are endangered marine herbivores that digest food, primarily sea grasses through microbial fermentation. The gut microbiota and its role in health and disease are largely unexplored although microbial dysbiosis is believed to be associated with the pathogenesis of several intestinal and extra-intestinal disorders. Gut microbial dysbiosis primarily refers to the microbial alteration and or imbalance within the gastrointestinal tract. Debilitated sea turtles are often treated and nursed to health in turtle hospitals and antibiotic therapy may be one of the contributing factors of microbial dysbiosis together with other collateral damages. The objective of this project was to understand gastrointestinal (GI) diseases of green turtles in rehabilitation by identifying normal and potentially pathogenic microbes, their antimicrobial resistance, and establishing the use of bacteriophages as an alternative to antibiotics. Additionally, the impact of the broad-spectrum antibiotic, enrofloxacin and bacteriophage therapy on the gut bacterial flora of green turtles were investigated. In this study, both culture dependent and independent techniques were employed to identify the gut bacteria in green turtles. Cloacal swabs were taken from a total of 73 green turtles between 2015 and 2016 for culture dependent identification of Enterobacterales. A total of 16 different bacterial species that represented nine different genera were identified. The predominant isolates were Citrobacter, Edwardsiella, Escherichia and Klebsiella. Antimicrobial resistance against 12 different antibiotics from six different classes was evaluated. The bacterial isolates showed highest resistance to β-lactam antibiotics followed by quinolone and tetracycline classes. Approximately one-third of the isolates identified exhibited multidrug-resistance. The high-throughput sequencing targeting the V1-V3 regions of the bacterial 16S rRNA gene identified a total of 19 bacterial phyla from a total of 12 samples. The faecal bacterial community of green turtles was largely dominated by Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria. Firmicutes predominated among wild-captured green turtles while Proteobacteria prevailed in stranded turtles. The predominance of the genus Bacteroides in all groups indicates the importance of these bacteria in turtle gut health. Wildcaptured green turtles showed the highest microbial diversity and richness compared to stranded green turtles. This study also investigated and compared the faecal bacterial communities between prehospitalisation (PH) and post-rehabilitation (PR) stranded green turtles. Bacteria within the phylum Proteobacteria dominated in both PH and PR samples without any significant difference. The significant abundance of Campylobacter fetus, Escherichia coli, Clostridium botulinum and Vibrio parahaemolyticus in PH samples indicates pathogenic associations of zoonotic potential within stranded turtles. In this study, all post-rehabilitation green turtles exhibited similar bacterial communities irrespective of their microbial compositions at prehospitalisation. The marked differences in the gut microbiota of PH and PR turtles indicate the outcome of dietary, management and environmental shift during rehabilitation. The mucosa-associated bacterial communities across the GI tract of green turtles were investigated. Bacterial diversity and richness decreased longitudinally along the GI tract from oesophagus to the small intestine while the large intestine showed a higher bacterial diversity and richness compared to the small intestine. The GI tract mucosa-associated microbial community of green turtles was largely dominated by Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes and Fusobacteria. However, the detailed composition of these phyla was notably distinct for different GI regions. This study provides a proof-of-concept for the application of bacteriophage (phages) to eliminate targeted bacteria as an alternative to antibiotics. Bacteria-specific phage cocktails were found to significantly reduce the targeted Acinetobacter in phage-treated turtles during the therapy. Compared to control turtles, no significant difference was observed in the gut bacterial diversity and compositions in the phage-treated turtles. In contrast, bacterial diversity was significantly reduced in antibiotic-treated turtles during the therapy at day 15 and throughout the trial. The alteration in the bacterial gut communities of antibiotic-treated turtles was largely due to an increase in abundance of Gram-positive Firmicutes and a concurrent decrease in abundance of Gram-negative Bacteroidetes, Proteobacteria and Verrucomicrobia. Additionally, the relative abundance of several bacteria at lower taxonomic levels was much less affected by phages than by antibiotic enrofloxacin. In conclusion, this is the first detailed characterisation of gut bacterial communities of green turtles in the context of their different health and environmental conditions. The findings offer a helpful reference for further investigations of sea turtle gut microbiome and their metabolic functions to improve their health and nutrition during rehabilitation. The phage treatment described here provides a targeted alternative to antibiotics, with the possibility to manipulate transient as well as indigenous bacterial flora with a broad application in many gut-related dysbiosis of turtles