Frontiers in Microbiology · 10 authors, 2 centres
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This study compared the gut and oral microbiomes of healthy bullfrogs and those with meningitis-like infectious disease (MID) using 16S rRNA sequencing. Diseased frogs showed significantly increased microbial diversity and richness in both sites, with a marked rise in the abundance of the pathogenic genus Elizabethkingia. These findings suggest that Elizabethkingia is a key pathogen in MID and that disease disrupts the normal microbial balance, potentially worsening outcomes.
**Background:** Bullfrog (Lithobates catesbeiana) farming is economically important but faces disease outbreaks, including meningitis-like infectious disease (MID), which causes high mortality. MID is characterized by neurological symptoms, corneal opacity, and systemic lesions. Previous work identified Elizabethkingia as a potential pathogen, but the impact on the host microbiome was unknown. This study aimed to characterize gut and oral microbial community changes in MID-affected bullfrogs compared to healthy controls.
**Methods:** Ten bullfrogs (5 healthy, 5 with MID) were collected from a single farm in Anhui Province, China, in December 2021. Gut and oral swabs were collected under sterile conditions. DNA was extracted using the CTAB method, and the 16S rRNA gene V3–V4 region was amplified and sequenced on the Illumina MiSeq platform. Sequences were clustered into operational taxonomic units (OTUs) at 97% identity. Alpha diversity (Observed species, Chao1, Shannon, Simpson) was compared between groups using t-tests. Beta diversity was assessed via non-metric multidimensional scaling (NMDS) based on Bray–Curtis dissimilarity. Linear discriminant analysis effect size (LEfSe) identified taxa with significant abundance differences (LDA score >2.0, p<0.05).
**Key Results:** Sequencing yielded 135 OTUs in gut samples (78 shared, 45 unique to MID, 12 unique to HC) and 299 OTUs in oral samples (191 shared, 66 unique to MID, 42 unique to HC). In the oral cavity, all α-diversity indices were significantly higher in MID vs. HC: Observed species (p=6.014×10⁻⁵), Chao1 (p=1.237×10⁻⁵), Shannon (p=0.001), Simpson (p=0.0099). In the gut, differences were also significant: Observed species (p=1.079×10⁻⁸), Chao1 (p=2.439×10⁻⁶), Shannon (p=0.00013), Simpson (p=0.0004). NMDS showed clear separation between MID and HC groups for both gut (stress=0.0012) and oral (stress=0.0131) microbiota. At the phylum level, Firmicutes abundance decreased and Bacteroidota increased in MID vs. HC in both sites. At the genus level, Lactococcus (a beneficial probiotic) was nearly absent in MID, while Flavobacterium, Acinetobacter, and Elizabethkingia increased markedly. LEfSe identified 16 significantly different clades in the gut and 14 in the oral cavity; in both sites, Firmicutes and Bacilli were enriched in HC, while Bacteroidota had the highest LDA score in MID.
**Clinical Implications:** This study demonstrates that MID in bullfrogs is associated with profound dysbiosis in both gut and oral microbiomes, characterized by increased diversity and a shift from beneficial (Lactococcus) to potentially pathogenic bacteria (Elizabethkingia, Flavobacterium, Acinetobacter). The significant rise in Elizabethkingia abundance supports its role as a primary pathogen in MID. The loss of Lactococcus may weaken host defenses, while overgrowth of opportunistic pathogens could exacerbate disease. These findings provide a microbial basis for understanding MID pathogenesis and suggest that monitoring Elizabethkingia levels and restoring beneficial microbiota could aid in disease prevention and control in bullfrog aquaculture. Future studies should include physiological data (e.g., weight, blood parameters) to correlate microbiome changes with host health.