# Reference Whole Genome Sequence Analyses and Characterization of a Novel Carnobacterium maltaromaticum Distinct Sequence Type Isolated from a North American Gray Wolf (Canis lupus) Gastrointestinal Tract

## TL;DR

A new type of Carnobacterium bacteria was found in a Gray Wolf's gut, which could have antimicrobial uses and be a potential probiotic.

## Contribution

This is the first report of Carnobacterium maltaromaticum isolated from a member of the genus Canis.

## Key findings

- A novel Carnobacterium maltaromaticum sequence type was identified from a Gray Wolf's gastrointestinal tract.
- The genome contains genes for antimicrobial properties, including bacteriocin expression.
- The isolate is the first axenic C. maltaromaticum culture from the genus Canis.

## Abstract

Worldwide, the animal kingdom contains a largely untapped resource of unknown bacteria that could significantly contribute to the health and safety of human and animal well-being. Specifically, free-ranging species of animals harbor diverse, yet-to-be-discovered bacteria that may have potentially useful applications. Across species, analyzing and categorizing microbes and their genomic components will increase the discovery of potential antibacterial and anti-fungal applications. These applications include use as new probiotics, that is, microbes that contribute to an animal’s gastroenteric health. During this investigation, a member of the Carnobacteria was discovered in the gastrointestinal tract of a North American Gray Wolf. These bacteria have known anti-listeria properties and may help support the gut health of animals. Genotypically, the bacterial isolate was demonstrated to be a unique member of the Carnobacteria. This is the first report of these types of bacteria isolated from any member of the genus Canis, including dogs and their progenitor, the Gray wolf. Because only a small percentage of bacteria have been discovered, this investigation also further supports the vital need to protect the biodiversity of our planet.

We hypothesize that bacteria isolated from free-ranging animals could potentially be useful for practical applications. To meet this objective a Gram-positive bacterium was isolated from the gastrointestinal (GI) tract of a Gray Wolf (Canis lupus) using Brucella broth with hemin and vitamin K (BBHK). By small ribosomal RNA (16S) gene sequencing the bacterium was initially identified as a novel Carnobacterium maltaromaticum strain. The bacterium could be propagated both anaerobically and aerobically and was both catalase/oxidase negative and negative by the starch hydrolysis as well as negative using lipase assays. The reference whole genome sequence (WGS) was obtained using both Illumina and Nanopore sequencing. The genome assembly was 3,512,202 bp in length, encoding core bacterial genes with a GC% content of 34.48. No lysogenic bacteriophage genes were detected, although the genome harbors genes for the expression of bacteriocin and other secondary metabolites with potential antimicrobial properties. Multilocus sequence typing (MLST), WGS phylogenetics, average nucleotide identity (ANI), and single nucleotide polymorphism (SNP) analyses of the isolate’s genome indicate this bacterium is a newly identified Carnobacterium maltaromaticum sequence type (ST). Members of the Carnobacteria have anti-listeria activities, highlighting their potential functional properties. Consequently, the isolate could be a potential probiotic for canids and this is the first report on an axenic C. maltaromaticum culture from the genus Canis.