Biology subjects

Holman, D. B.

Publications and source records attributed to Holman, D. B..

8 recordsLinked to original sources

bioRxiv2023

Sequencing and Culture-based Characterization of the Vaginal and Uterine Microbiota in Beef Cattle that Became Pregnant or Non-pregnant via Artificial Insemination

In this study, we evaluated the vaginal and uterine microbiota between beef cattle that became pregnant via artificial insemination (AI) and those that did not to identify microbial signature associated with pregnancy. We also characterized the culturable fraction of these microbiota using extensive culturing and screened some vaginal and uterine bacterial isolates for their antimicrobial resistance. For this, vaginal and uterine swabs from two cohorts of Angus-crossbred cattle: mature cows (vaginal and uterine; 27 open and 31 pregnant) and heifers (vaginal; 26 open and 33 pregnant) that were collected before AI were processed for microbiota assessment using 16S rRNA gene sequencing and culturing. Twenty-nine vaginal and uterine bacterial isolates were screened for resistance against 41 antibiotics. Sequencing results revealed 11 taxa that were more abundant in the vaginal samples from non-pregnant heifers compared to pregnant heifers. No differentially abundant taxa were detected in the vaginal samples from pregnant versus non-pregnant cows. Pregnant cows had a distinct uterine microbiota community structure (P = 0.008) and interaction network structure compared to non-pregnant cows. Twenty-eight differentially abundant uterine taxa were observed between the two groups. Community structure and diversity were different between the cow vagina and uterus. A total of 733 bacterial isolates were recovered from vaginal (512) and uterine (221) swabs under aerobic (83 different species) and anaerobic (69 species) culturing. Among these isolates were pathogenic species and those mostly susceptible to tested antibiotics. Overall, our results indicate that pregnancy-associated taxonomic signatures are present in the bovine uterine and vaginal microbiota. ImportanceEmerging evidence suggests that microbiome-targeted approaches may provide a novel opportunity to reduce the incidence of reproductive failures in cattle. To develop such microbiome-based strategies, one of the first logical steps is to identify reproductive microbiome features related to fertility, and isolate the pregnancy associated microbial species for developing a future bacterial consortium that could be administered before breeding to enhance pregnancy outcomes. Here, we characterized the vaginal and uterine microbiota in beef cattle that became pregnant or not via AI and identified some microbiota features associated with pregnancy. We compared similarities between vaginal and uterine microbiota, and between heifers and cows. Using extensive culturing, we provided new insights on the culturable fraction of the vaginal and uterine microbiota, and their antimicrobial resistance. Overall, our findings will serve as an important basis for future research aimed at harnessing the vaginal and uterine microbiome for improved cattle fertility.

microbiology↗

bioRxiv2023

Whole-body Microbiota of Newborn Calves and Their Response to Prenatal Vitamin and Mineral Supplementation

Here, we investigated whether initial microbial colonization at seven different anatomical locations in newborn calves and their blood cytokines are influenced by prenatal vitamin and mineral (VTM) supplementation. Samples were collected from the hoof, liver, lung, nasal cavity, eye, rumen (tissue and fluid), and vagina of beef calves that were born from dams that received diets with or without VTM supplementation throughout gestation (n=7/group). Calves were separated from their dams immediately after birth and fed colostrum and milk replacer until euthanasia at 30 h post-initial colostrum feeding. The microbiota of all samples was assessed using 16S rRNA gene sequencing and qPCR. 15 cytokines and chemokines were quantified in their serum. The hoof, ocular, liver, respiratory, and reproductive sites of newborn calves were colonized by site-specific microbiota that differed from that of the rumen (0.64 [≥] R2 [≥] 0.12, P [≤] 0.003). Only the ruminal fluid microbiota was differed by on prenatal VTM supplementation (P<0.01 Differences (P<0.05) were detected in microbial richness (vagina), diversity (ruminal tissue and fluid, eye), composition at the phylum and genus level (ruminal tissue and fluid, and vagina), and total bacterial abundance (ocular and vagina) between VTM and control calves. The cytokine IP-10 was higher (P=0.02) in VTM calves. Overall, our results suggest that despite immediate separation from the dam upon birth, whole-body of 32-h old calves are colonized by relatively rich, diverse and site-specific bacterial communities, and that initial microbial colonization of the rumen, vagina and oculus seem to be influenced by the prenatal VTM supplementation. IMPORTANCEIncreased appreciation of maternal nutrition and microbiomes involvement in developmental programming and evidence supporting in utero microbial colonization highlight that maternal nutrition factors could impact offspring microbial colonization. Here, we investigated whether initial microbial colonization in any of 7 different anatomical sites of newborn calves was influenced by maternal vitamin and mineral (VTM) supplementation. We identified changes in ruminal, vaginal, and ocular microbiota in newborn calves in response to prenatal VTM supplementation. We provided a "holistic" view on the whole-body calf microbiota. Our data was obtained from calves of the same sex and age, and who were immediately separated from dams, and hence provides novel insights on taxonomic composition of initial bacterial microbiota colonization in those anatomical sites examined. Combined, this study provides direction for future work targeting the manipulation of early life microbiome via alteration of maternal nutrition and harnessing early life microbiota for improved cattle health and production.

microbiology↗

bioRxiv2023

The gut microbiome and resistome of conventionally- vs. pasture-raised pigs

Conventional swine production typically houses pigs indoors and in large groups, whereas pasture-raised pigs are reared outdoors in lower stocking densities. Pigs in both production systems are usually fed a grain-based diet but pasture-raised pigs may also consume plants and soil. Antimicrobial use also differs with conventionally-raised pigs often being exposed to antimicrobials directly or indirectly to control and prevent infectious disease. However, antimicrobial use can be associated with the development and persistence of antimicrobial resistance. In this study, we used shotgun metagenomic sequencing to compare the gut microbiomes and resistomes of pigs raised indoors on a conventional farm with those raised outdoors on pasture. The microbial compositions as well as the resistomes of both groups of pigs were significantly different from each other. Bacterial species such as Intestinibaculum porci, Pseudoscardovia radai, and Sharpea azabuensis were relatively more abundant in the gut microbiomes of pasture-raised pigs and Hallella faecis and Limosilactobacillus reuteri in the conventionally-raised swine. The abundance of antimicrobial resistance genes (ARGs) was significantly higher in the conventionally-raised pigs for nearly all antimicrobial classes, including aminoglycosides, beta-lactams, macrolides-lincosamides-streptogramin B, and tetracyclines. Functionally, the gut microbiomes of the two group of pigs also differed significantly based on their CAZyme profiles, with certain CAZyme families associated with host mucin degradation enriched in the conventional pig microbiomes. We also recovered 1,043 dereplicated strain-level metagenome-assembled genomes ([≥] 90% completeness and <5% contamination) to provide taxonomic context for specific ARGs and metabolic functions. Overall, the study provides insights into the differences between the gut microbiomes and resistomes of pigs raised under two very different production systems.

microbiology↗

bioRxiv2022

Novel insights into the pig gut microbiome using metagenome-assembled genomes

Pigs are among the most numerous and intensively farmed food-producing animals in the world. The gut microbiome plays an important role in the health and performance of swine and changes rapidly after weaning. Here, fecal samples were collected from pigs at 7 different times points from 7 to 140 days of age. These swine fecal metagenomes were used to assemble 1,150 dereplicated metagenome-assembled genomes (MAGs) that were at least 90% complete and had less than 5% contamination. These MAGs represented 472 archaeal and bacterial species, and the most widely distributed MAGs were the uncultured species Collinsella sp002391315, Sodaliphilus sp004557565, and Prevotella sp000434975. Weaning was associated with a decrease in the relative abundance of 69 MAGs (e.g. Escherichia coli) and an increase in the relative abundance of 140 MAGs (e.g. Clostridium sp000435835, Oliverpabstia intestinalis). Genes encoding for the production of the short-chain fatty acids acetate, butyrate, and propionate were identified in 68.5%, 18.8%, and 8.3% of the MAGs, respectively. Carbohydrate-active enzymes associated with the degradation of arabinose oligosaccharides and mixed-linkage glucans were predicted to be most prevalent among the MAGs. Antimicrobial resistance genes were detected in 327 MAGs, including 59 MAGs with tetracycline resistance genes commonly associated with pigs such as tet(44), tet(Q), and tet(W). Overall, 82% of the MAGs were assigned to species that lack cultured representatives indicating that a large portion of the swine gut microbiome is still poorly characterized. The results here also demonstrate the value of MAGs in adding genomic context to gut microbiomes. ImportanceMany of the bacterial strains found in the mammalian gut are difficult to culture and isolate due to their various growth and nutrient requirements that are frequently unknown. Here, we assembled strain-level genomes from short metagenomic sequences, so-called metagenome-assembled genomes (MAGs), that were derived from fecal samples collected from pigs at multiple time points. The majority of these MAGs represented bacterial species that have yet to be cultured or described thus underlining the need for cultivation studies that isolate and describe novel bacterial species. The genomic context of a number of antimicrobial resistance genes commonly detected in swine was also determined. In addition, our study connected taxonomy with potential metabolic functions such as carbohydrate degradation and short-chain fatty acid production.

microbiology↗

bioRxiv2021

Characterization of the microbiota associated with 12-week-old bovine fetuses exposed to divergent in utero nutrition

A recent study reported the existence of a diverse microbiota in 5-to-7-month-old calf fetuses, suggesting that colonization of the bovine gut with so-called "pioneer" microbiota may begin during mid-gestation. In the present study, we investigated 1) the presence of microbiota in bovine fetuses at early gestation (12 weeks), and 2) whether the fetal microbiota is influenced by the maternal rate of gain or dietary supplementation with vitamins and minerals (VTM) during early gestation. Amniotic and allantoic fluids, and intestinal and placental (cotyledon) tissue samples obtained from fetuses (n = 33) on day 83 of gestation were processed for the assessment of fetal microbiota using 16S rRNA gene sequencing. The sequencing results revealed that a diverse and complex microbial community was present in each of these fetal compartments evaluated. Allantoic and amniotic fluids, and fetal intestinal and placenta microbiota each had distinctly different (0.047 [≥] R2 [≥] 0.019, P [≤] 0.031) microbial community structures. Allantoic fluid had a greater (P < 0.05) microbial richness (number of OTUs) (Mean 122) compared to amniotic fluid (84), intestine (63) and placenta (66). Microbial diversity (Shannon index) was similar for the intestinal and placental samples, and both were less diverse compared with fetal fluid microbiota (P < 0.05). Thirty-nine different archaeal and bacterial phyla were detected across all fetal samples, with Proteobacteria (55%), Firmicutes (16.2%), Acidobacteriota (13.6%) and Bacteroidota (5%) predominating. Among the 20 most relatively abundant bacterial genera, Acidovorax, Acinetobacter, Brucella, Corynebacterium, Enterococcus, Exiguobacterium and Stenotrophomonas differed by fetal sample type (P < 0.05). A total of 55 taxa were shared among the four different microbial communities. qPCR of bacteria in the intestine and placenta samples as well as scanning electron microscopy imaging of fetal fluids provided additional evidence for the presence of a microbiota in these samples. Minor effects of maternal rate of gain and VTM supplementation, and their interactions on microbial richness and composition were detected. Overall, the results of this study indicate that colonization with pioneer microbiota may occur during early gestation in bovine fetuses, and that that the maternal nutritional regime during gestation may influence the early fetal microbiota.

microbiology↗

bioRxiv2021

The nasopharyngeal, ruminal, and vaginal microbiota and the core taxa shared across these microbiomes in virgin yearling heifers exposed to divergent in utero nutrition during their first trimester of gestation and in pregnant beef heifers in response to mineral supplementation

Emerging evidence has indicated that microbial transmission from the bovine dam to her fetus may take place before birth, and that the maternal microbiota during pregnancy modulates programming of fetal metabolic and nervous system development, highlighting the potential and extended role of the maternal microbiome in calf health and development. In the present study, we characterized the nasopharyngeal, ruminal and vaginal microbiota from two cohorts of beef heifers managed at the same location: 1) virgin yearling heifers (9 months old) born from dams received gestational diets which resulted in low (LG, n = 22) or medium (MG, n = 23) weight gain during the first 84 days of gestation; and 2) pregnant replacement heifers that received a vitamin and mineral supplement (VTM, n = 17) or not (Control, n = 15) during the first 6 months of gestation. Nasopharyngeal and vaginal swabs as well as ruminal fluid were collected from both cohorts and the microbiota of each sample was assessed using 16S rRNA gene sequencing. In addition to the comparison between treatment groups within each cohort, the similarity of the microbiota of the three sample types were evaluated, and shared taxa amongst these communities were identified. The bacterial genera present in the rumen and vagina that can influence methanogenic archaeal genera were predicted using a stepwise-selected generalized linear mixed model. No significant difference was observed in the alpha and beta diversity in any of the nasopharyngeal, ruminal and vaginal microbiota between LG and MG offspring virgin heifers, or between the control and VTM pregnant heifers (p > 0.05). Subtle compositional changes in the vaginal microbiota in yearling heifers, and in the nasopharyngeal and ruminal microbiota of pregnant heifers were detected in response to treatments. Forty-one archaeal and bacterial OTUs were shared by over 60% of all samples from both virgin and pregnant heifers. Two taxa within the Methanobrevibacter genus were identified as core taxa and this genus was more relatively abundant in pregnant heifers compared to virgin heifers. Among the 25 top genera, Prevotella and Prevotella UCG-003 (negative) and Christensenellaceae R-7 group (positive) were predicted to have a significant effect on ruminal Methanobrevibacter spp. The results of this study indicate that there is little impact of divergent gestational nutrition during the first trimester on the calf microbiome at 9 months postnatal, and that VTM supplementation during pregnancy may not alter the maternal microbiome. This study provides evidence that there are several microbial taxa, including methanogenic archaea, that are shared across the respiratory, gastrointestinal, and reproductive tracts, suggesting the need for a holistic evaluation of the bovine microbiota when considering potential maternal sources for seeding calves with pioneer microbiota.

microbiology↗

bioRxiv2021

A Longitudinal Study of Antimicrobial Resistance in Enterococcus spp. Isolated from a Beef Processing Plant and Retail Ground Beef

Antimicrobial use in food-producing animals has come under increasing scrutiny due to its potential association with antimicrobial resistance (AMR). Monitoring of AMR in indicator microorganisms such as Enterococcus spp. in meat production facilities and retail meat products can provide important information on the dynamics and prevalence of AMR in these environments. In this study, swabs or samples were obtained from various locations in a commercial beef packing operation (n = 600 total) and from retail ground beef (n = 60) over a 19-month period. All samples/swabs were enriched for Enterococcus spp. and suspected enterococci isolates were identified using species-specific PCR primers. Enterococcus faecalis was the most frequently isolated species followed by Enterococcus hirae, which was found mostly on hides and ground beef. Enterococcus faecium (n = 9) and E. faecalis (n = 120) isolates were further characterized for antimicrobial resistance and resistant genes due to the clinical significance of these species. Twenty-one unique AMR profiles were identified, with 90% of isolates resistant to at least two antimicrobials, and two that were resistant to nine antimicrobials. Tetracycline resistance was observed most often in E. faecalis (28.8%) and was likely mediated by tet(M). Genomic analysis of selected E. faecalis and E. faecium isolates revealed that many of the isolates in this study clustered with other publicly available genomes from ground beef, suggesting that these strains are well adapted to the beef packaging environment. IMPORTANCEAntimicrobial resistance (AMR) is a serious challenge facing the agricultural industry. Understanding the flow of antimicrobial resistant-bacteria through the beef fabrication process and into ground beef is an important step in identifying intervention points for reducing AMR. In this study we used enterococci as indicator bacteria for monitoring AMR in a commercial beef packaging facility and in retail ground beef over a 19-month period. Although washing of carcasses post-hide removal reduced the isolation frequency of Enterococcus spp., a number of antimicrobial resistant-Enterococcus faecalis isolates were recovered from ground beef produced in the packaging plant. Genome analysis showed that several E. faecalis isolates were genetically similar to publicly available isolates recovered from retail ground beef in the United States.

microbiology↗

bioRxiv2021

Weaning age and its effect on the development of the swine gut microbiome and resistome

Piglets are often weaned between 19 and 22 d of age in North America although in some swine operations this may occur at 14 d or less. Piglets are abruptly separated from their sow at weaning and are quickly transitioned from sows milk to a plant-based diet. The effect of weaning age on the long-term development of the pig gut microbiome is largely unknown. Here, pigs were weaned at either 14, 21, or 28 d of age and fecal samples collected 20 times from d 4 (neonatal) through to marketing at d 140. The fecal microbiome was characterized using 16S rRNA gene and shotgun metagenomic sequencing. The fecal microbiome of all piglets shifted significantly three to seven days post-weaning with an increase in microbial diversity. Several Prevotella spp. increased in relative abundance immediately after weaning as did butyrate-producing species such as Butyricicoccus porcorum, Faecalibacterium prausnitzii, and Megasphaera elsdenii. Within 7 days of weaning, the gut microbiome of pigs weaned at 21 and 28 days of age resembled that of pigs weaned at 14 d. Resistance genes to most antimicrobial classes decreased in relative abundance post-weaning with the exception of those conferring resistance to tetracyclines and macrolides-lincosamides-streptogramin B. The relative abundance of microbial carbohydrate-active enzymes (CAZymes) changed significantly in the post-weaning period with an enrichment of CAZymes involved in degradation of plant-derived polysaccharides. These results demonstrate that the pig gut microbiome tends change in a predictable manner post-weaning and that weaning age has only a temporary effect on this microbiome. ImportancePiglets are abruptly separated from their sow at weaning and are quickly transitioned from sows milk to a plant-based diet. This is the most important period in commercial swine production yet the effect of weaning age on the long-term development of the pig gut microbiome is largely unknown. Metagenomic sequencing allows for a higher resolution assessment of the pig gut microbiome and enables characterization of the resistome. Here we used metagenomic sequencing to identify bacterial species that were enriched post-weaning and therefore may provide targets for future manipulation studies. In addition, functional profiling of the microbiome indicated that many carbohydrate and metabolic enzymes decrease in relative abundance of after weaning. This study also highlights the challenges faced in reducing antimicrobial resistance in pigs as genes conferring tetracycline and macrolide resistance remained relatively stable from 7 days of age through to market weight at 140 d despite no exposure to antimicrobials.

microbiology↗