Evaluation of extended-spectrum β-lactamase (ESBL) producing bacteria in feces of shelter dogs as a biomarker for altered gut microbial taxa and functional profiles

Reta Duguma Abdi ^1* Srinka Datta ² Akshaykumar Zawar ² Pratap Kafle ³

• ¹ Department of Veterinary Biomedical Sciences, College of Veterinary Medicine, Long Island University, Brookville, New York, United States
• ² GeneSpectrum Life Sciences LLP, Pune, Maharashtra, India
• ³ Department of Diagnostic Medicine and Pathobiology Shreiber School of Veterinary medicine, Rowan University, Mullica Hill, New Jersey 08082, United States

The USA is home to 83-88 million dogs, with 3-7 million living in shelters. Shelter dogs move through the supply chain from their geographical origin to adoptive homes, with possible exposure to pathogens and shift in their gut microbiota. However, research in this area is limited. This study examined the effects of intestinal colonization by ESBL bacteria on gut taxa abundance, diversity, and functions in 52 shelter dogs of various ages, sexes, and fertility statuses.We isolated fecal DNA, sequenced their 16S, processed the sequences using DADA2, identified taxa profiles in each dog by Phyloseq, and analyzed Chao1, Shannon, and Simpson alpha diversity by ggplot2 and Wilcoxon test. We analyzed beta diversity using Bray-Curtis dissimilarity matrix from the vegan package. Differential abundance of taxa, gut microbiome functions, and differential abundance of microbiome functions were analyzed using DESeq2, PICRUSt2, and ALDEx2, respectively, with Wilcoxon rank and Kruskal-Wallis tests for comparisons between dog groups.Firmicutes (69.3%), Bacteroidota (13.5%), Actinobacteriota (6.77%), Proteobacteria (5.54%), and Fusobacteriota (4.75%) were the major phyla in the gut of shelter dogs. ESBL bacteria colonized dogs had reduced gut microbiota alpha diversity than non-colonized dogs. The abundance levels of the following phyla (Proteobacteria, Deferribacterota, Bacteroidota, Fusobacteriota, and Spirochaetota), class (Gammaproteobacteria, Bacteroidia, Deferribacteres, Brachyspirae, and Fusobacteria), and families (Enterobacteriaceae, Peptostreptococcaceae, Lactobacillaceae, Lachnospiraceae, Prevotellaceae, and Peptostreptococcaceae) were significantly (p< 0.05) varied between the two dog groups. Further stratified analysis by age, sex, and spaying/neutering status influenced the abundance of taxa in ESBL bacteria colonized dogs, indicating these covariates act as effect modifiers. Most gut metabolic and biosynthetic pathways were downregulated in ESBL bacteria colonized dogs compared to non-colonized dogs. However, alpha-linolenic acid metabolism and shigellosis, fluorobenzoate degradation, allantoin degradation, toluene degradation, glycol degradation, fatty acid and beta-oxidation, and glyoxylate metabolism bypass pathways were increased in dogs colonized by ESBL bacteria.Colonization by ESBL bacteria marks altered gut microbiota. Dog`s demography and fertility status modify the alterations, indicating host factors and ESBL bacteria interplay to shape gut microbiota. ESBL bacteria or other factors reprogram gut microbiome functions through down and upregulating multiple metabolic and biosynthesis pathways to promote ESBL bacteria colonization.