Direct and culture-enriched 16S rRNA sequencing of cecal content of healthy horses and horses with typhlocolitis

Abstract

Next generation sequencing has demonstrated that alpha diversity of the fecal microbiota is significantly altered in horses with typhlocolitis. The objective of this study was to evaluate the bacterial composition of the cecum content of horses with and without typhlocolitis through direct and culture-enriched 16S gene sequencing of six healthy horses and six horses with acute typhlocolitis; a case-control study design. Cecal content was collected after euthanasia. An aliquot was used for direct 16S gene sequencing. Another was serially diluted with brain heart infusion (BHI) and plated onto five different agar media. All culture medias, except for MacConkey, were incubated anaerobically. Bacterial colonies were harvested in bulk and used for DNA extraction, 16S PCR amplification, and sequenced using the Illumina MiSeq platform. Predominant phyla in healthy and diseased horses were Firmicutes, followed by Bacteroidetes in all cultured medias, except for MacConkey agar, in which Proteobacteria was the dominant phylum. Greater bacterial richness was identified in sequenced cecal contents as compared to cultured plates (P < 0.05). Culture-enriched molecular profiling combined with 16S rRNA gene sequencing offer an alternative method for the study of the gut microbiota of horses. For direct cecum content 16S gene amplification, the alpha diversity indices were lower in diarrheic horses compared to healthy horses (P < 0.05). A higher relative abundance of Fusobacteriota was found in 2/6 samples from diarrheic horses. The role of Fusobacteriota in equine colitis deserves investigation.

Fig 1. Culture-enriched 16S rRNA gene sequencing workflow.

Cecal samples were collected from horses. One aliquot was submitted for direct 16S rRNA sequencing. Other aliquots were plated onto 6 different media types and incubated anaerobically and anaerobically. Colonies were harvested and submitted for culture-enriched 16S rRNA sequencing. Abbreviations: MacConkey agar (Mac), fastidious agar with horse’s blood added (FAA), Columbia agar with horse’s blood added (CBA+HB), Columbia agar with rumen fluid added (CBA+RF), BHI3 with carbohydrate added (BHI), and AS (samples plated on CBA+HB after alcohol shock).

Fig 2. Observed and Shannon diversity of different media types divided by the groups of healthy horses and horses with diarrhea.

Abbreviations: MacConkey agar (Mac), fastidious agar with horse’s blood added (FAA), Columbia agar with horse’s blood added (CBA+HB), Columbia agar with rumen fluid added (CBA+RF), BHI3 with carbohydrate added (BHI), AS (samples plated on CBA+HB after alcohol shock), and Cecum (samples sequenced directly, without culture).

Fig 3. Principal coordinate analysis (PCoA) Plot generated on weighted UniFrac distances of cecal samples from healthy horses and horses with acute colitis either sequenced directly (cecum) or after selective media culture (Mac, FAA, CBA+HB, CBA+RF, BHI and AS).

Abbreviations: MacConkey agar (Mac), fastidious agar with horse’s blood added (FAA), Columbia agar with horse’s blood added (CBA+HB), Columbia agar with rumen fluid added (CBA+RF), BHI3 with carbohydrate added (BHI), AS (samples plated on CBA+HB after alcohol shock), and Cecum (samples sequenced directly, without culture).

Fig 4. Distribution and relative abundance of bacterial phyla across sample types.

Microbiota for each animal ID are partitioned by seven sample types. Bacterial phyla under 2% relative abundance in each sample were grouped. Abbreviations: MacConkey agar (Mac), fastidious agar with horse’s blood added (FAA), Columbia agar with horse’s blood added (CBA+HB), Columbia agar with rumen fluid added (CBA+RF), BHI3 with carbohydrate added (BHI), AS (samples plated on CBA+HB after alcohol shock), and Cecum (samples sequenced directly, without culture). Healthy horses: S1-S6. Acute typhlocolitis horses: S7-S12.

Fig 5. Distribution of culture-derived equine microbiota across different culture media.

Venn diagrams display the number of bacterial taxa in common between different culture medias. Microbiota of CBA+HB and CBA+RF (CBA) were grouped together, as well as BHI and FAA (BF), for easier visualization of Venn diagrams. Bacterial taxa were grouped to different taxonomy levels in each individual Venn diagram (from top to bottom: phylum, family, genus, species, individual ASV). Venn diagram sections are shaded based on bacterial taxa count. Abbreviations: MacConkey agar (Mac), fastidious agar with horse’s blood added (FAA), Columbia agar with horse’s blood added (CBA+HB), Columbia agar with rumen fluid added (CBA+RF), BHI3 with carbohydrate added (BHI), AS (samples plated on CBA+HB after alcohol shock), and Cecum (samples sequenced directly, without culture).

Fig 6. Relative abundance of ASVs in culture-derived samples.

Only ASVs that were present in at least one culture-derived sample but absent in all cecum samples are included. Relative abundances were partitioned by culture media and individual animal ID. ASVs were grouped at family taxonomy. Families with relative abundances less than 0.5% were grouped together. Abbreviations: MacConkey agar (Mac), fastidious agar with horse’s blood added (FAA), Columbia agar with horse’s blood added (CBA+HB), Columbia agar with rumen fluid added (CBA+RF), BHI3 with carbohydrate added (BHI), and AS (samples plated on CBA+HB after alcohol shock). Healthy horses: S1-S6. Acute typhlocolitis horses: S7-S12.

Fig 7. Relative abundance of genus in culture-derived and cecum samples.

Abbreviations: MacConkey agar (Mac), fastidious agar with horse’s blood added (FAA), Columbia agar with horse’s blood added (CBA+HB), Columbia agar with rumen fluid added (CBA+RF), BHI3 with carbohydrate added (BHI), AS (samples plated on CBA+HB after alcohol shock), and Cecum (samples sequenced directly, without culture). Healthy horses: S1-S6. Acute typhlocolitis horses: S7-S12.

Fig 8. Relative abundance of the top 20 genus in cecal samples of healthy horses and horses with diarrhea.

Fig 9

Differential abundance analysis to the cecum genus level bacteria of diarrheic horses showing the difference in abundance of each genus compared to healthy horses (A). The prevalence of significant genera in cecum and cultured samples (B). Abbreviations: MacConkey agar (Mac), fastidious agar with horse’s blood added (FAA), Columbia agar with horse’s blood added (CBA+HB), Columbia agar with rumen fluid added (CBA+RF), BHI3 with carbohydrate added (BHI), AS (samples plated on CBA+HB after alcohol shock), and Cecum (samples sequenced directly, without culture).

Fig 10. Number of bacterial taxa present in each sample type at multiple taxonomic ranks.

The number of unique bacterial taxa are displayed for each sample type at A) phylum, B) family, C) genus, D) species, and E) ASV level. E) The y-axis is square-rooted for easier visualization of major differences between ASV counts between sample type. Unaltered number of ASVs are present within the stacked bars. Abbreviations: MacConkey agar (Mac), fastidious agar with horse’s blood added (FAA), Columbia agar with horse’s blood added (CBA+HB), Columbia agar with rumen fluid added (CBA+RF), BHI3 with carbohydrate added (BHI), AS (samples plated on CBA+HB after alcohol shock), and Cecum (samples sequenced directly, without culture). Healthy horses: S1-S6. Acute typhlocolitis horses: S7-S12.

Fig 11. Phylogenetic tree of ASV distribution across each sample type.

Maximum likelihood tree tips are coloured by family-level taxonomic classification. Tips were labeled for ASVs that were present in multiple samples, and if species-level taxonomy was available. Colored tiles represent ASVs being present in at least one sample of each sample type. Stacked bar plots on outer ring demonstrate the relative abundance of each ASV and are colored by sample type. Abbreviations: MacConkey agar (Mac), fastidious agar with horse’s blood added (FAA), Columbia agar with horse’s blood added (CBA+HB), Columbia agar with rumen fluid added (CBA+RF), BHI3 with carbohydrate added (BHI), AS (samples plated on CBA+HB after alcohol shock), and Cecum (samples sequenced directly, without culture).