ABSTRACT
The gut microbiome is a community of microorganisms that inhabits an animal host's gastrointestinal tract, with important effects on animal health that are shaped by multiple environmental, dietary, and host-associated factors. Clinical and dietary trials in companion animals are increasingly including assessment of the microbiome, but interpretation of these results is often hampered by suboptimal choices in study design. Here, we review best practices for conducting feeding trials or clinical trials that intend to study the effects of an intervention on the microbiota. Choices for experimental design, including a review of basic designs, controls, and comparison groups, are discussed in the context of special considerations necessary for microbiome studies. Diet is one of the strongest influences on the composition of gut microbiota, so applications specific to nutritional interventions are discussed in detail. Lastly, we provide specific advice for successful recruitment of colony animals and household pets into an intervention study. This review is intended to serve as a resource to academic and industry researchers, clinicians, and veterinarians alike, for studies that test many different types of interventions.
ABSTRACT
Koalas (Phascolarctos cinereus) are highly specialized herbivorous marsupials that feed almost exclusively on Eucalyptus leaves, which are known to contain varying concentrations of many different toxic chemical compounds. The literature suggests that Lonepinella koalarum, a bacterium in the Pasteurellaceae family, can break down some of these toxic chemical compounds. Furthermore, in a previous study, we identified L. koalarum as the most predictive taxon of koala survival during antibiotic treatment. Therefore, we believe that this bacterium may be important for koala health. Here, we isolated a strain of L. koalarum from a healthy koala female and sequenced its genome using a combination of short-read and long-read sequencing. We placed the genome assembly into a phylogenetic tree based on 120 genome markers using the Genome Taxonomy Database (GTDB), which currently does not include any L. koalarum assemblies. Our genome assembly fell in the middle of a group of Haemophilus, Pasteurella and Basfia species. According to average nucleotide identity and a 16S rRNA gene tree, the closest relative of our isolate is L. koalarum strain Y17189. Then, we annotated the gene sequences and compared them to 55 closely related, publicly available genomes. Several genes that are known to be involved in carbohydrate metabolism could exclusively be found in L. koalarum relative to the other taxa in the pangenome, including glycoside hydrolase families GH2, GH31, GH32, GH43 and GH77. Among the predicted genes of L. koalarum were 79 candidates putatively involved in the degradation of plant secondary metabolites. Additionally, several genes coding for amino acid variants were found that had been shown to confer antibiotic resistance in other bacterial species against pulvomycin, beta-lactam antibiotics and the antibiotic efflux pump KpnH. In summary, this genetic characterization allows us to build hypotheses to explore the potentially beneficial role that L. koalarum might play in the koala intestinal microbiome. Characterizing and understanding beneficial symbionts at the whole genome level is important for the development of anti- and probiotic treatments for koalas, a highly threatened species due to habitat loss, wildfires, and high prevalence of Chlamydia infections.
ABSTRACT
Koalas (Phascolarctos cinereus) are arboreal marsupials native to Australia that eat a specialized diet of almost exclusively eucalyptus leaves. Microbes in koala intestines are known to break down otherwise toxic compounds, such as tannins, in eucalyptus leaves. Infections by Chlamydia, obligate intracellular bacterial pathogens, are highly prevalent in koala populations. If animals with Chlamydia infections are received by wildlife hospitals, a range of antibiotics can be used to treat them. However, previous studies suggested that koalas can suffer adverse side effects during antibiotic treatment. This study aimed to use 16S rRNA gene sequences derived from koala feces to characterize the intestinal microbiome of koalas throughout antibiotic treatment and identify specific taxa associated with koala health after treatment. Although differences in the alpha diversity were observed in the intestinal flora between treated and untreated koalas and between koalas treated with different antibiotics, these differences were not statistically significant. The alpha diversity of microbial communities from koalas that lived through antibiotic treatment versus those who did not was significantly greater, however. Beta diversity analysis largely confirmed the latter observation, revealing that the overall communities were different between koalas on antibiotics that died versus those that survived or never received antibiotics. Using both machine learning and OTU (operational taxonomic unit) co-occurrence network analyses, we found that OTUs that are very closely related to Lonepinella koalarum, a known tannin degrader found by culture-based methods to be present in koala intestines, was correlated with a koala's health status. This is the first study to characterize the time course of effects of antibiotics on koala intestinal microbiomes. Our results suggest it may be useful to pursue alternative treatments for Chlamydia infections without the use of antibiotics or the development of Chlamydia-specific antimicrobial compounds that do not broadly affect microbial communities.
