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Evaluating extraction methods to study canine urine microbiota.

ABSTRACT: The urinary microbiota is the collection of microbes present in urine that may play a role in host health. Studies of urine microbiota have traditionally relied upon culturing methods aimed at identifying pathogens. However, recent culture-free sequencing studies of the urine microbiota have determined that a diverse array of microbes is present in health and disease. To study these microbes and their potential role in diseases like bladder cancer or interstitial cystitis, consistent extraction and detection of bacterial DNA from urine is critical. However, urine is a low biomass substrate, requiring sensitive methods to capture DNA and making the risk of contamination high. To address this challenge, we collected urine samples from ten healthy dogs and extracted DNA from each sample using five different commercially available extraction methods. Extraction methods were compared based on total and bacterial DNA concentrations and bacterial community composition and diversity assessed through 16S rRNA gene sequencing. Significant differences in the urinary microbiota were observed by dog and sex but not extraction method. The Bacteremia Kit yielded the highest total DNA concentrations (Kruskal-Wallis, p = 0.165, not significant) and the highest bacterial DNA concentrations (Kruskal-Wallis, p = 0.044). Bacteremia also extracted bacterial DNA from the greatest number of samples. Taken together, these results suggest that the Bacteremia kit is an effective option for studying the urine microbiota. This work lays the foundation to study the urine microbiome in a wide range of urogenital diseases in dogs and other species.

SUBMITTER: Mrofchak R

PROVIDER: S-EPMC8270191 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe urinary tract harbors unique microbial communities that play important roles in urogenital health and disease. Dogs naturally suffer from several of the same urological disorders as humans (e.g., urinary tract infections, neoplasia, urolithiasis) and represent a valuable translational model for studying the role of urinary microbiota in various disease states. Urine collection technique represents a critical component of urinary microbiota research study design. However, the impact of collection method on the characterization of the canine urinary microbiota remains unknown. Therefore, the objective of this study was to determine whether urine collection technique alters the microbial populations detected in canine urine samples. Urine was collected from asymptomatic dogs by both cystocentesis and midstream voiding. Microbial DNA was isolated from each sample and submitted for amplicon sequencing of the V4 region of the bacterial 16 S rRNA gene, followed by analyses to compare microbial diversity and composition between urine collection techniques.ResultsSamples collected via midstream voiding exhibited significantly higher sequence read counts (P = .036) and observed richness (P = .0024) than cystocentesis urine. Bray Curtis and Unweighted UniFrac measures of beta diversity showed distinct differences in microbial composition by collection method (P = .0050, R2 = 0.06 and P = .010, R2 = 0.07, respectively). Seven taxa were identified as differentially abundant between groups. Pasteurellaceae, Haemophilus, Friedmanniella, two variants of Streptococcus, and Fusobacterium were over-represented in voided urine, while a greater abundance of Burkholderia-Caballeronia-Paraburkholderia characterized cystocentesis samples. Analyses were performed at five thresholds for minimum sequence depth and using three data normalization strategies to validate results; patterns of alpha and beta diversity remained consistent regardless of minimum read count requirements or normalization method.ConclusionMicrobial composition differs in canine urine samples collected via cystocentesis as compared to those collected via midstream voiding. Future researchers should select a single urine collection method based on the biological question of interest when designing canine urinary microbiota studies. Additionally, the authors suggest caution when interpreting results across studies that did not utilize identical urine collection methods.

Project description:ObjectiveTo evaluate the efficacy of a novel decellularized porcine bone xenograft, produced by supercritical carbon dioxide extraction technology, on alveolar socket healing after tooth extraction compared to a commercially available deproteinized bovine bone (Bio-Oss®).Materials and methodsNine dogs (about 18 months old and weighing between 20 kg and 30 kg) underwent extractions of lower second to fourth premolars, bilaterally. The dogs were randomly selected and allocated to the following groups: Group 1: control unfilled socket; Group 2: socket filled with decellularized porcine bone xenograft (ABCcolla®) and covered by a commercially available porcine collagen membrane (Bio-Gide®); Group 3: socket filled with Bio-Oss® and covered by Bio-Gide® membrane. One dogs from each group was sacrificed at 4-, 12-, and 24-week to evaluate the socket healing after tooth extraction. The mandible bone blocks were processed without decalcification and specimens were embedded in methyl methacrylate and subjected to histopathology analyses to evaluate the bone regeneration in the extraction sockets.ResultsAt 24-week after socket healing, ABCcolla® treated defects demonstrated significantly higher histopathology score in new bone formation and bone bridging, but significantly lower score in fluorescent labeling than those of the Bio-Oss®. In the microphotographic examination, decellularized porcine bone xenograft showed similar characteristics of new bone formation to that of Bio-Oss®. However, there was significantly less remnant implant materials in the decellularized porcine bone xenograft compared to the Bio-Oss® group at 24-week. Thus, the decellularized porcine bone graft seems to have promising bone regeneration properties similar to that of Bio-Oss® with less remnant grafted material in a canine tooth extraction socket model.ConclusionsWithin the limits of the study, we concluded that ABCcolla® treated defects demonstrated significantly more new bone formation and better bone bridging, but less amount of fluorescent labeling than those of the Bio-Oss® group. However, clinical studies in humans are recommended to confirm these findings.

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Evaluating extraction methods to study canine urine microbiota.

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