Adult zebrafish infected by clinically isolated Klebsiella pneumoniae with different virulence showed increased intestinal inflammation and disturbed intestinal microbial biodiversity

**Background:** Klebsiella pneumoniae is a gram-negative bacterium that causes serious clinical infections, often complicated by high virulence and antibiotic resistance. While it is known to colonize the intestine and cause inflammation, the impact of different virulence/resistance profiles on intestinal pathology and microbial diversity is not well understood. Zebrafish are a validated model for studying K. pneumoniae virulence.

**Methods:** Three clinically isolated K. pneumoniae strains were used: KP1053 (high virulence, antibiotic-sensitive), KP1196 (high virulence, carbapenem-resistant), and KP1195 (low virulence, carbapenem-resistant). Adult female zebrafish (5 months old) were injected intraperitoneally with the bacteria. At 48 hours post-infection, intestinal tissues were collected for histopathological analysis (hematoxylin and eosin staining), ELISA for inflammatory cytokines (Il-1α, Il-1β, TNF-α), and 16S rRNA gene sequencing (V3-V4 region) to assess gut microbial composition and diversity. Survival was monitored up to 72 hours.

**Key Results:** - Survival: At 72 hours, KP1053 caused nearly 100% mortality, while KP1196 and KP1195 also significantly reduced survival (p<0.0001). - Intestinal colonization: All three strains colonized the zebrafish intestine; the order of colonization burden was KP1053 > KP1195 > KP1196. - Histopathology: KP1053 caused severe intestinal epithelial damage, reduced intestinal ridges, and a significant decrease in goblet cells. KP1195 caused epithelial dissolution and exfoliation. KP1196 showed less severe damage. - Inflammatory cytokines: All three strains significantly increased Il-1α, Il-1β, and TNF-α levels. KP1053 increased cytokine levels by up to 2-fold; KP1196 and KP1195 increased levels by 1.4–1.8 times (p<0.05 to p<0.0001). - Gut microbial diversity: The Shannon diversity index significantly increased in all infection groups, indicating altered microbial diversity. At the phylum level, Bacteroidetes increased in the KP1196 group, while Firmicutes decreased in KP1053 and KP1195 groups. At the species level, KP1053 increased Rhodococcus erythropolis, KP1196 increased Vibrio cholerae, and KP1195 increased Pseudomonas. The number of OTUs was 3564 (control), 3450 (KP1053), 3540 (KP1196), and 3612 (KP1195).

**Clinical Implications:** This study demonstrates that K. pneumoniae infection, regardless of virulence or resistance profile, induces intestinal inflammation, structural damage, and dysbiosis in a zebrafish model. The high-virulence non-resistant strain (KP1053) caused the most severe pathology, suggesting that virulence factors, not just antibiotic resistance, drive intestinal damage. The increase in harmful bacteria (e.g., Vibrio, Pseudomonas) and decrease in beneficial taxa (e.g., Firmicutes) highlight the potential for K. pneumoniae to disrupt gut homeostasis. These findings support the use of zebrafish for high-throughput screening of therapeutic agents targeting K. pneumoniae intestinal infection. However, the study is limited by the use of a non-mammalian model and the inability to control bacterial load precisely; further research in mammals is needed to confirm clinical relevance.