Vibrio cholerae Infection Alters Zebrafish Intestinal Microbiome and Metabolite Profiles

Title: Vibrio cholerae Infection Induces Strain-Specific Modulation of the Zebrafish Intestinal Microbiome

Journal: Infection and Immunity

Published: 2021

Background

The study investigates the impact of various Vibrio cholerae strains on the intestinal microbiota of zebrafish, focusing on colonization patterns and associated changes in microbial diversity and metabolites. Zebrafish serve as a model organism due to their genetic tractability and physiological relevance to human intestinal microbiota studies. The study aims to elucidate how V. cholerae infections alter the zebrafish gut microbiome, potentially influencing host health and disease.

Materials & Methods

Bacterial Strains and Culture Conditions

Five V. cholerae strains (254-93, AM-19226, V52, E7946, N16961) were cultured in LB broth with streptomycin at 37°C. Culture densities were adjusted prior to zebrafish exposure.

Zebrafish Husbandry and Experimental Design

Wild-type AB zebrafish were housed under controlled conditions and fasted before experimentation. Fish were exposed to V. cholerae via immersion in sterile infection water, with control groups exposed to 1× PBS.

Intestinal Colonization Assessment

After exposure, zebrafish intestines were homogenized, and bacterial colonies were quantified on selective media. Tank water was also sampled and processed similarly.

DNA Extraction and Sequencing

DNA was extracted from zebrafish intestines and water samples for 16S rRNA gene sequencing using Illumina MiSeq. Data were processed using DADA2 to identify amplicon sequence variants (ASVs).

Diversity Analysis

Alpha (Chao1, Shannon-Wiener, inverse Simpson indices) and beta (Jaccard, Bray-Curtis dissimilarity) diversity indices were calculated. PCoA and PERMANOVA were used to assess microbiome variation among samples.

Metabolomics and Functional Analysis

Untargeted metabolomics of zebrafish intestinal homogenates was conducted using UPLC-TOF-MS. Sample preparation involved extraction with 80% methanol. The analysis focused on identifying metabolites such as succinic acids and their relative abundances in zebrafish intestinal samples.

Statistical Analysis

Data were analyzed using various statistical tests, including ANOVA and Tukey's test, to assess significance between experimental groups.

Results

Intestinal Colonization by V. cholerae

Intestinal colonization by V. cholerae strains was assessed through bacterial enumeration in zebrafish intestines. Significant differences were observed in colonization levels among the different strains (V. cholerae 254-93, AM-19226, V52, E7946, N16961). Strain E7946 exhibited the highest colonization levels, followed by strains AM-19226 and V52, whereas strains 254-93 and N16961 showed lower colonization efficiency.

Microbiome Diversity Analysis

Alpha Diversity

Alpha diversity indices (Chao1, Shannon-Wiener, inverse Simpson) were calculated to assess species richness and evenness in zebrafish intestines following V. cholerae infection. Compared to controls, infected groups exhibited altered alpha diversity metrics, with significant differences observed in richness and evenness indices.

Beta Diversity

Beta diversity analysis using PCoA based on Bray-Curtis dissimilarity matrices revealed distinct clustering patterns among V. cholerae-infected and control group. PERMANOVA analysis confirmed significant differences in microbiome composition between infection groups and controls.

β-Diversity of the zebrafish intestinal microbiome following V. cholerae 254-93 infection in a zebrafish host.

β-Diversity analysis of the zebrafish intestinal microbiome after V. cholerae V52 infection, illustrating microbial community differences in the host. β-Diversity of the zebrafish intestinal microbiome following V. cholerae V52 infection in a zebrafish host.

Metabolomics Analysis

Untargeted metabolomics identified differential metabolic profiles in zebrafish intestines following V. cholerae infection. Metabolites associated with immune response and gut health were significantly altered in infected groups compared to controls.

PCoA plots comparing zebrafish intestinal microbiome β-diversities after a 24-hour V. cholerae infection using Jaccard and Bray-Curtis dissimilarity indices, with accompanying P values showing statistical differences. Comparison of zebrafish intestinal microbiome β-diversities following V. cholerae infections. (A) β-Diversities of the zebrafish intestinal microbiome following a 24-hour V. cholerae infection expressed as PCoA plots based on Jaccard and Bray-Curtis dissimilarity indices. (B) P values indicating statistical differences of the PCoA plots shown in A.

Secreted Mucin Quantification

The Microtiter PAS assay quantified mucin levels in infection water, a biomarker for diarrhea. Significant increases in mucin secretion were observed in V. cholerae-infected groups compared to controls, correlating with infection severity.

Reference

Breen, Paul, et al. "Vibrio cholerae infection induces strain-specific modulation of the zebrafish intestinal microbiome." Infection and Immunity 89.9 (2021). https://doi.org/10.1128/iai.00157-21

* For Research Use Only. Not for use in diagnostic procedures.

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