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Asian seabass (Lates calcarifer) is a major aquaculture food fish species in Singapore. Farming of this species is increasingly threatened by frequent outbreaks of infectious diseases, resulting in mortality exceeding 50–70%. In this study, we investigated the comparative gut bacterial microbiota using 16S rRNA metasequencing between asymptomatic and diseased juvenile fish collected during a disease outbreak soon after stocking. Mild to severe chronic granulomatous enteritis was observed histopathologically in both asymptomatic and diseased fish. Kidneys of diseased fish tested PCR positive for the 'big belly' novel Vibrio spp., Streptococcus iniae and Vibrio harveyi. These bacteria were also readily detected by PCR in water samples corresponding to tanks fish were sampled from. Potentially beneficial microbes that promote gut health such as Firmicutes , Bacteroidota and Actinobacteriota were the dominant phyla in the intestinal microbiota of asymptomatic fish. Moreover, the bacteria with probiotic potential such as Lactobacillus only presented in asymptomatic fish, and Weissella was unique and prevalent (47.59%) in asymptomatic fish during the recovery phase of the disease outbreak, making them candidate biomarkers for monitoring health status of L. calcarifer. Conversely, diseased fish showed reduced diversity of their gut microbiome, with high abundance of members of the phylum Proteobacteria. Vibrio was the most dominant genus (87.3%) and Streptococcus iniae was only detected in diseased fish. These findings provide a baseline study for understanding changes in intestinal microbiota in newly stocked fish with mixed bacterial infection, biomarker assisted health monitoring, and future host-derived probiotics screening in L. calcarifer. • This study provides a baseline for understanding changes in gut microbiome in newly stocked L. calcarifer with mixed bacterial infections. • 'Big belly' novel Vibrio spp., Streptococcus iniae and Vibrio harveyi , were detected in only one asymptomatic L. calcarifer , but all diseased fish. • Gut microbiota in diseased fish was dominated by Vibrio. • Unique presence of Weissella and Lactobacillus in asymptomatic fish has potential as biomarkers assisted health monitoring in L. calcarifer. [ABSTRACT FROM AUTHOR]