Effect of Chitosan Microparticles on the Uterine Microbiome of Dairy Cows with Metritis.

The objective of this study was to evaluate the effect of chitosan microparticles on the uterine microbiome of cows with metritis. Dairy cows with metritis (n = 89) were assigned to 1 of 3 treatments: chitosan microparticles (n = 21), in which the cows received an intrauterine infusion of chitosan microparticles at metritis diagnosis (day 0), day 2, and day 4; ceftiofur (n = 25), in which the cows received a subcutaneous injection of ceftiofur on day 0 and day 3; and no intrauterine or subcutaneous treatment (n = 23). Nonmetritic cows (n = 20) were healthy cows matched with cows with metritis by the number of days postpartum at metritis diagnosis. Uterine swab samples collected on days 0, 3, 6, 9, and 12 were used for 16S rRNA gene sequencing and 16S RNA gene copy number quantification by quantitative PCR. Principal-coordinate analysis showed that the microbiome of the ceftiofur-treated and metritic untreated groups progressed toward that of the nonmetritic group by day 3, whereas that of the chitosan microparticle-treated group remained unchanged. The differences on day 3 were mainly due to a greater relative abundance of Fusobacteria, particularly Fusobacterium, in the chitosan microparticle-treated group than in the ceftiofur-treated and metritic untreated groups. Furthermore, the microbiome of the ceftiofur-treated group became similar to that of the nonmetritic group by day 9, whereas the microbiome of the chitosan microparticle-treated and metritic untreated groups became similar to that of the nonmetritic group only by day 12. The total bacterial 16S rRNA gene counts in the chitosan microparticle-treated group were greater than those in the metritic untreated controls on days 6 and 9, whereas the ceftiofur treatment group was the only group in which the total bacterial 16S rRNA gene count became similar to that in the nonmetritic group by day 12. In summary, chitosan microparticles slowed the progression of the uterine microbiome toward a healthy state, whereas ceftiofur hastened the progression toward a healthy state.IMPORTANCE Third-generation cephalosporins, such as ceftiofur, are commonly used to treat metritis in dairy cows. Chitosan microparticles has been shown to have a broad spectrum of activity in vitro and to be effective against uterine pathogens in vivo; therefore, they have been hailed as a possible alternative to traditional antibiotics. Nonetheless, in the present study, we saw that chitosan microparticle treatment slowed the progression of the uterine microbiome of cows with metritis toward a healthy state, whereas ceftiofur treatment hastened the progression toward a healthy state. Given the lack of an effective alternative to traditional antibiotics and an increased concern about antimicrobial resistance, a greater effort should be devoted to the prevention of metritis in dairy cows.

Prevalence of extended-spectrum ß-lactamases in the local farm environment and livestock: challenges to mitigate antimicrobial resistance.

The effectiveness of antibiotics has been challenged by the increasing frequency of antimicrobial resistance (AR), which has emerged as a major threat to global health. Despite the negative impact of AR on health, there are few effective strategies for reducing AR in food-producing animals. Of the antimicrobial resistant microorganisms (ARMs), extended-spectrum ß-lactamases (ESBLs)-producing Enterobacteriaceae are an emerging global threat due to their increasing prevalence in livestock, even in animals raised without antibiotics. Many reviews are available for the positive selection of AR associated with antibiotic use in livestock, but less attention has been given to how other factors including soil, water, manure, wildlife, and farm workers, are associated with the emergence of ESBL-producing bacteria. Understanding of antibiotic resistance genes and bacteria transfer at the interfaces of livestock and other potential reservoirs will provide insights for the development of mitigation strategies for AR.

Uterine infection alters the transcriptome of the bovine reproductive tract three months later.

Infection of the postpartum uterus with pathogenic bacteria is associated with infertility months later in dairy cattle. However, it is unclear whether these bacterial infections lead to long-term changes in the reproductive tract that might help explain this infertility. Here we tested the hypothesis that infusion of pathogenic bacteria into the uterus leads to changes in the transcriptome of the reproductive tract 3 months later. We used virgin Holstein heifers to avoid potential confounding effects of periparturient problems, lactation, and negative energy balance. Animals were infused intrauterine with endometrial pathogenic bacteria Escherichia coli and Trueperella pyogenes (n = 4) and compared with control animals (n = 6). Three months after infusion, caruncular and intercaruncular endometrium, isthmus and ampulla of the oviduct, and granulosa cells from ovarian follicles >8 mm diameter were profiled by RNA sequencing. Bacterial infusion altered the transcriptome of all the tissues when compared with control. Most differentially expressed genes were tissue specific, with 109 differentially expressed genes unique to caruncular endometrium, 57 in intercaruncular endometrium, 65 in isthmus, 298 in ampulla, and 83 in granulosa cells. Surprisingly, despite infusing bacteria into the uterus, granulosa cells had more predicted upstream regulators of differentially expressed genes than all the other tissues combined. In conclusion, there were changes in the transcriptome of the endometrium, oviduct and even granulosa cells, 3 months after intrauterine infusion of pathogenic bacteria. These findings imply that long-term changes throughout the reproductive tract could contribute to infertility after bacterial infections of the uterus.

Polar delivery of Legionella type IV secretion system substrates is essential for virulence.

A recurrent emerging theme is the targeting of proteins to subcellular microdomains within bacterial cells, particularly to the poles. In most cases, it has been assumed that this localization is critical to the protein's function. Legionella pneumophila uses a type IVB secretion system (T4BSS) to export a large number of protein substrates into the cytoplasm of host cells. Here we show that the Legionella export apparatus is localized to the bacterial poles, as is consistent with many T4SS substrates being retained on the phagosomal membrane adjacent to the poles of the bacterium. More significantly, we were able to demonstrate that polar secretion of substrates is critically required for Legionella's alteration of the host endocytic pathway, an activity required for this pathogen's virulence.

Genomic Comparison Reveals Natural Occurrence of Clinically Relevant Multidrug-Resistant Extended-Spectrum-ß-Lactamase-Producing Escherichia coli Strains.

The effectiveness of antibiotics has been challenged by the increasing frequency of antimicrobial resistance (AMR), which has emerged as a major threat to global health. Despite its negative impact on the development of AMR, there are few effective strategies for reducing AMR in food-producing animals. Using whole-genome sequencing and comparative genomics of 36 multidrug-resistant (MDR) Escherichia coli strains isolated from beef cattle with no previous exposure to antibiotics, we obtained results suggesting that the occurrence of MDR E. coli also arises in animals with no antibiotic selective pressure. Extended-spectrum-ß-lactamase-producing E. coli strains with enhanced virulence capacities for toxin production and adherence have evolved, which implies important ramifications for animal and human health. Gene exchanges by conjugative plasmids and insertion elements have driven widespread antibiotic resistance in clinically relevant pathogens. Phylogenetic relatedness of E. coli strains from various geographic locations and hosts, such as animals, environmental sources, and humans, suggests that transmission of MDR E. coli strains occurs intercontinentally without host barriers.IMPORTANCE Multidrug-resistant (MDR) Escherichia coli isolates pose global threats to public health due to the decreasing availability of treatment options. To better understand the characteristics of MDR E. coli isolated from food-producing animals with no antibiotic exposure, we employed genomic comparison, high-resolution phylogenetics, and functional characterization. Our findings highlight the potential capacity of MDR E. coli to cause severe disease and suggest that these strains are widespread intercontinentally. This study underlines the occurrence of MDR E. coli in food-producing animals raised without antibiotic use, which has alarming, critical ramifications within animal and human medical practice.

In situ structure of the Legionella Dot/Icm type IV secretion system by electron cryotomography.

Type IV secretion systems (T4SSs) are large macromolecular machines that translocate protein and DNA and are involved in the pathogenesis of multiple human diseases. Here, using electron cryotomography (ECT), we report the in situ structure of the Dot/Icm type IVB secretion system (T4BSS) utilized by the human pathogen Legionella pneumophila This is the first structure of a type IVB secretion system, and also the first structure of any T4SS in situ While the Dot/Icm system shares almost no sequence similarity with type IVA secretion systems (T4ASSs), its overall structure is seen here to be remarkably similar to previously reported T4ASS structures (those encoded by the R388 plasmid in Escherichia coli and the cag pathogenicity island in Helicobacter pylori). This structural similarity suggests shared aspects of mechanism. However, compared to the negative-stain reconstruction of the purified T4ASS from the R388 plasmid, the L. pneumophila Dot/Icm system is approximately twice as long and wide and exhibits several additional large densities, reflecting type-specific elaborations and potentially better structural preservation in situ.

A model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes.

Bacterial infection of the uterus causes clinical endometritis in 15 to 20% of postpartum dairy cows and reduces fertility, even after the resolution of disease. However, it is difficult to disentangle the mechanisms linking reduced fertility with endometritis because cows have multiple confounding postpartum conditions. The aim of the present experiment was to develop an in vivo model of clinical endometritis in Holstein heifers using pathogenic Escherichia coli and Trueperella pyogenes. Estrous cycles of heifers were synchronized using a 5-d Co-Synch protocol, and subsequently received exogenous progesterone to elevate circulating progesterone at the time of uterine infusion. Endometrial scarification was performed before uterine infusion of live pathogenic Escherichia coli and Trueperella pyogenes, or sterile vehicle. Effects of infusion were evaluated by measuring rectal temperature, plasma haptoglobin, hematology, grading pus in the vaginal mucus, quantifying 16S rRNA in vaginal mucus, and transrectal ultrasonography. Bacterial infusion increased the median vaginal mucus to grade 2 by d 3 postinfusion, and to grade 3 from d 4 to 6 postinfusion. Control heifers maintained a median vaginal mucus grade ≤1 from d 1 to 6. Transrectal ultrasound revealed the accumulation of echogenic fluid in the uterus of heifers following bacterial infusion, which was absent in control heifers. Total 16S rRNA in vaginal mucus was elevated in bacteria-infused heifers compared with control heifers at d 5. Rectal temperature was increased in bacteria-infused heifers. Plasma haptoglobin, general health, and appetite did not differ between groups. As indicated by increased vaginal mucus grade after bacterial infusion and absence of systemic signs of illness, this model successfully induced symptoms resembling clinical endometritis in virgin Holstein heifers. The model allows the isolation of effects of uterine disease on fertility from confounding factors that can occur during the postpartum period in dairy cows.

A study at the wildlife-livestock interface unveils the potential of feral swine as a reservoir for extended-spectrum ß-lactamase-producing Escherichia coli.

Wildlife is known to serve as carriers and sources of antimicrobial resistance (AMR). Due to their unrestricted movements and behaviors, they can spread antimicrobial resistant bacteria among livestock, humans, and the environment, thereby accelerating the dissemination of AMR. Extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is one of major concerns threatening human and animal health, yet transmission mechanisms at the wildlife-livestock interface are not well understood. Here, we investigated the mechanisms of ESBL-producing bacteria spreading across various hosts, including cattle, feral swine, and coyotes in the same habitat range, as well as from environmental samples over a two-year period. We report a notable prevalence and clonal dissemination of ESBL-producing E. coli in feral swine and coyotes, suggesting their persistence and adaptation within wildlife hosts. In addition, in silico studies showed that horizontal gene transfer, mediated by conjugative plasmids and insertion sequences elements, may play a key role in spreading the ESBL genes among these bacteria. Furthermore, the shared gut resistome of cattle and feral swine suggests the dissemination of antibiotic resistance genes at the wildlife-livestock interface. Taken together, our results suggest that feral swine may serve as a reservoir of ESBL-producing E. coli.

Evaluating the potential of lignosulfonates and chitosans as alfalfa hay preservatives using in vitro techniques.

Our objectives were to compare the antifungal activity of 5 lignosulfonates, and 2 chitosans against fungi isolated from spoiled hay, and assess the effects of an optimized lignosulfonate, chitosan, and propionic acid (PRP) on high-moisture alfalfa hay. In experiment 1, we determined the minimum inhibitory concentration and minimum fungicidal concentration of 4 sodium lignosulfonates, 1 magnesium lignosulfonate, 2 chitosans, and PRP (positive control) against Aspergillus amoenus, Mucor circinelloides, Penicillium solitum, and Debaromyces hansenii at pH 4 and 6. Among sodium lignosulfonates, the one from Sappi Ltd. (NaSP) was the most antifungal at pH 4. However, chitosans had the strongest fungicidal activity with the exception of M. circinelloides at both pH 4 and 6. PRP had more antifungal effects than NaSP and was only better than chitosans for M. circinelloides. In experiment 2, we evaluated the effects of 3 additives (ADV): optimized NaSP (NaSP-O, UMaine), naïve chitosan (ChNv, Sigma-Aldrich), and PRP on high-moisture alfalfa hay. The experimental design was a randomized complete block design replicated 5 times. Treatment design was the factorial combination of 3 ADV× 5 doses (0, 0.25, 0.5, 1, and 2% w/w fresh basis). Additives were added to 35 g of sterile alfalfa hay (71.5 ± 0.23% DM), inoculated with a mixture of previously isolated spoilage fungi (5.8 log cfu/fresh g), and aerobically incubated in vitro for 23 d (25°C). After incubation, DM losses were reduced by doses as low as 0.25% for both NaSP-O and PRP (x¯=1.61) vs. untreated hay (24.0%), partially due to the decrease of mold and yeast counts as their doses increased. Also, hay NH3-N was lower in NaSP-O and PRP, with doses as low as 0.25%, relative to untreated hay (x¯=1.13 vs. 7.80% of N, respectively). Both NaSP-O and PRP increased digestible DM recovery (x¯=69.7) and total volatile fatty acids (x¯=94.3), with doses as low as 0.25%, compared with untreated hay (52.7% and 83.8 mM, respectively). However, ChNv did not decrease mold nor yeast counts (x¯=6.59 and x¯=6.16 log cfu/fresh g, respectively) and did not prevent DM losses relative to untreated hay. Overall, when using an alfalfa hay substrate in vitro, NaSP-O was able to prevent fungal spoilage to a similar extent to PRP. Thus, further studies are warranted to develop NaSP-O as a hay preservative under field conditions.

In our first experiment, we assessed the antifungal activity of two major types of byproducts, one known as lignosulfonates (5 types), which are generated by paper mills, and another known as chitosans (2 types), which are generated from shellfish. These were tested against four fungi isolated from spoiled hay. We observed that acidic conditions are not necessary for chitosans but are crucial to activate the antifungal properties of lignosulfonates. Also, we found that sodium lignosulfonate from Sappi Ltd. was the most antifungal relative to other sodium lignosulfonates from other manufacturers. Chitosans had stronger fungicidal activity than propionic acid or lignosulfonates against all but one mold tested. In our second experiment, we compared the best treatments from experiment 1 against propionic acid using alfalfa hay as a substrate to grow the same fungi tested in experiment 1. None of the doses of chitosan prevented spoilage on high moisture hay, showing results similar to untreated hay. In contrast, an optimized sodium lignosulfonate and propionic acid prevented fungal spoilage of alfalfa hay with doses as low as 0.25%.

A novel combination therapy for multidrug resistant pathogens using chitosan nanoparticles loaded with ß-lactam antibiotics and ß-lactamase inhibitors.

Antimicrobial resistance is one of the greatest global threats. Particularly, multidrug resistant extended-spectrum ß-lactamase (ESBL)-producing pathogens confer resistance to many commonly used medically important antibiotics, especially beta-lactam antibiotics. Here, we developed an innovative combination approach to therapy for multidrug resistant pathogens by encapsulating cephalosporin antibiotics and ß-lactamase inhibitors with chitosan nanoparticles (CNAIs). The four combinations of CNAIs including two cephalosporin antibiotics (cefotaxime and ceftiofur) with two ß-lactamase inhibitors (tazobactam and clavulanate) were engineered as water-oil-water emulsions. Four combinations of CNAIs showed efficient antimicrobial activity against multidrug resistant ESBL-producing Enterobacteriaceae. The CNAIs showed enhanced antimicrobial activity compared to naïve chitosan nanoparticles and to the combination of cephalosporin antibiotics and ß-lactamase inhibitors. Furthermore, CNAIs attached on the bacterial surface changed the permeability to the outer membrane, resulting in cell damage that leads to cell death. Taken together, CNAIs have provided promising potential for treatment of diseases caused by critically important ESBL-producing multidrug resistant pathogens.

Draft Genome Sequence of NDM-Encoding Klebsiella pneumoniae Isolated from Feral Swine.

New Delhi metallo-ß-lactamase (NDM)-producing Enterobacteriaceae pose a great threat to public health globally. Most known NDM-producing Enterobacteriaceae are associated with human hospital or community infections. Here, we report the draft genome sequence of an NDM-1-encoding Klebsiella pneumoniae strain isolated from feral swine (Sus scrofa) captured in Florida, USA.

Draft Genome Sequence of a Burkholderia cepacia Complex Strain Isolated from a Human Intra-abdominal Abscess.

Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens with high transmissibility and mortality. Here, we report the draft genome sequence of a Bcc strain isolated from a deep abscess culture in an immunocompetent patient with no relevant prior medical history.

The Gut Microbiota of Newborn Calves and Influence of Potential Probiotics on Reducing Diarrheic Disease by Inhibition of Pathogen Colonization.

Calf diarrhea is one of the most concerning challenges facing both the dairy and beef cattle industry. Maintaining healthy gut microbiota is essential for preventing gastrointestinal disorders. Here, we observed significantly less bacterial richness in the abnormal feces with watery or hemorrhagic morphology compared to the normal solid feces. The normal solid feces showed high relative abundances of Osllospiraceae, Christensenellaceae, Barnesiella, and Lactobacillus, while the abnormal feces contained more bacterial taxa of Negativicutes, Tyzzerella, Parasutterella, Veillonella, Fusobacterium, and Campylobacter. Healthy calves had extensive bacterial-bacterial correlations, with negative correlation between Lactobacillus and potential diarrheagenic Escherichia coli-Shigella, but not in the abnormal feces. We isolated Lactobacillus species (L. reuteri, L. johnsonii, L. amylovorus, and L. animalis), with L. reuteri being the most abundant, from the healthy gut microbiota. Isolated Lactobacillus strains inhibited pathogenic strains including E. coli K88 and Salmonella Typhimurium. These findings indicate the importance of a diverse gut microbiota in newborn calf's health and provide multiple potential probiotics that suppress pathogen colonization in the gastrointestinal tract to prevent calf diarrhea.

Host genetics exerts lifelong effects upon hindgut microbiota and its association with bovine growth and immunity.

The gut microbiota is a complex ecological community that plays multiple critical roles within a host. Known intrinsic and extrinsic factors affect gut microbiota structure, but the influence of host genetics is understudied. To investigate the role of host genetics upon the gut microbiota structure, we performed a longitudinal study in which we evaluated the hindgut microbiota and its association with animal growth and immunity across life. We evaluated three different growth stages in an Angus-Brahman multibreed population with a graduated spectrum of genetic variation, raised under variable environmental conditions and diets. We found the gut microbiota structure was changed significantly during growth when preweaning, and fattening calves experienced large variations in diet and environmental changes. However, regardless of the growth stage, we found gut microbiota is significantly influenced by breed composition throughout life. Host genetics explained the relative abundances of 52.2%, 40.0%, and 37.3% of core bacterial taxa at the genus level in preweaning, postweaning, and fattening calves, respectively. Sutterella, Oscillospira, and Roseburia were consistently associated with breed composition at these three growth stages. Especially, butyrate-producing bacteria, Roseburia and Oscillospira, were associated with nine single-nucleotide polymorphisms (SNPs) located in genes involved in the regulation of host immunity and metabolism in the hindgut. Furthermore, minor allele frequency analysis found breed-associated SNPs in the short-chain fatty acids (SCFAs) receptor genes that promote anti-inflammation and enhance intestinal epithelial barrier functions. Our findings provide evidence of dynamic and lifelong host genetic effects upon gut microbiota, regardless of growth stages. We propose that diet, environmental changes, and genetic components may explain observed variation in critical hindgut microbiota throughout life.

Prevalence of Field-Collected House Flies and Stable Flies With Bacteria Displaying Cefotaxime and Multidrug Resistance.

Antibiotic use in livestock accounts for 80% of total antibiotic use in the United States and has been described as the driver for resistance evolution and spread. As clinical infections with multidrug-resistant pathogens are rapidly rising, there remains a missing link between agricultural antibiotic use and its impact on human health. In this study, two species of filth flies from a livestock operation were collected over the course of 11 mo: house flies Musca domestica (L.) (Diptera: Muscidae), representing a generalist feeder, and stable flies Stomoxys calcitrans (L.) (Diptera: Muscidae), representing a specialist (blood) feeder. The prevalence of flies carrying cefotaxime-resistant (CTX-R) bacteria in whole bodies and dissected guts were assayed by culturing on antibiotic-selective media, with distinct colonies identified by Sanger sequencing. Of the 149 flies processed, including 81 house flies and 68 stable flies, 18 isolates of 12 unique bacterial species resistant to high-level cefotaxime were recovered. These isolates also showed resistance to multiple classes of antibiotics. The CTX-R isolates were predominantly recovered from female flies, which bore at least two resistant bacterial species. The majority of resistant bacteria were isolated from the guts encompassing both enteric pathogens and commensals, sharing no overlap between the two fly species. Together, we conclude that house flies and stable flies in the field could harbor multidrug-resistant bacteria. The fly gut may serve as a reservoir for the acquisition and dissemination of resistance genes.

Dissemination mechanisms of NDM genes in hospitalized patients.

BACKGROUND: NDM-producing Enterobacteriaceae are a major clinical concern worldwide. We characterized NDM-positive pathogens isolated from patients and assessed the dissemination patterns of the bla NDM genes in a hospital setting. METHODS: Eleven NDM-positive Enterobacteriaceae (three Enterobacter hormaechei, six Klebsiella pneumoniae and two Escherichia coli) were isolated from nine patients over a 1 year period. Antimicrobial susceptibility was assessed by MICs. A combination of short- and long-read WGS was used for genome analysis. Clinical treatment history of patients was linked with genetic features of individual isolates to investigate the dissemination patterns of the bla NDM genes and NDM-positive strains. RESULTS: bla NDM in clonal K. pneumoniae were transmitted between two patients. In other instances, an identical IncC plasmid encoding NDM-1 was transmitted between E. coli and K. pneumoniae isolated from the same patient, and an IncX3 plasmid, carrying bla NDM-1 or bla NDM-5, was harboured in non-clonal E. hormaechei. Varying patterns of IS elements were identified as a critical transmission mechanism in association with bla NDM genes. CONCLUSIONS: Multiple transmission patterns were identified in hospitalized patients, including dissemination of clonal bacterial strains carrying resistance genes and horizontal transfer of resistance genes among divergent bacterial strains. Controlling spread of NDM is complex: while attention to standard infection control practices is critically important, this needs to be matched by aggressive efforts to limit unnecessary antimicrobial use, to minimize the selection for and risk of transfer of 'high mobility' resistance genes among Enterobacteriaceae.

Survival of Escherichia coli O157 in autoclaved and natural sandy soil mesocosms.

While the soil microbiome may influence pathogen survival, determining the major contributors that reduce pathogen survival is inconclusive. This research was performed to determine the survival of E. coli O157 in autoclaved and natural (unautoclaved) sandy soils. Soils were inoculated with three different E. coli O157 strains (stx1+/stx2+, stx1-/stx2-, and stx1-/stx2+), and enumerated until extinction at 30°C. There was a significant difference in the survival of E. coli O157 based on soil treatment (autoclaved versus natural) at 30°C on days 1 (P = 0.00022), 3, (P = 2.53e-14), 7 (P = 5.59e-16), 14 (P = 1.072e-12), 30 (P = 7.18e-9), and 56 (P = 0.00029), with greater survival in autoclaved soils. The time to extinction (two consecutive negative enrichments) for all three strains was 169 and 84 days for autoclaved and natural soils, respectively. A separate E. coli O157 trial supplemented with 16S rRNA gene sequencing of the soil microbiome was performed at 15°C and 30°C on days 0, 7, 14, and 28 for each soil treatment. Greater species richness (Chao1, P = 2.2e-16) and diversity (Shannon, P = 2.2e-16) was observed in natural soils in comparison with autoclaved soils. Weighted UniFrac (beta-diversity) showed a clear distinction between soil treatments (P = 0.001). The greatest reduction of E. coli O157 was observed in natural soils at 30°C, and several bacterial taxa positively correlated (relative abundance) with time (day 0 to 28) in these soils (P < 0.05), suggesting that the presence of those bacteria might cause the reduction of E. coli O157. Taken together, a clear distinction in E. coli O157 survival, was observed between autoclaved and natural soils along with corresponding differences in microbial diversity in soil treatments. This research provides further insights into the bacterial taxa that may influence E. coli O157 in soils.

Host genetic effects upon the early gut microbiota in a bovine model with graduated spectrum of genetic variation.

Multiple synergistic factors affect the development and composition of mammalian gut microbiota, but effects of host genetics remain unclear. To illuminate the role of host genetics on gut microbiota, we employed animals with a graduated spectrum of genetic variation with minimal environmental influences. We bred 228 calves with linearly varying breed composition from 100% Angus (Bos taurus) to 100% Brahman (Bos indicus), as a proxy for genetic variation, and then raised the offspring in the same environment with identical diets. We hypothesized each breed would harbor distinct gut microbiota due to genetic influence. We found that the gut microbiota of preweaning calves at 3 months old is significantly affected by host genetics, profoundly by paternal genome. We also demonstrate that single nucleotide polymorphisms in host mucin-encoding genes, critical for gut mucosal health, are significantly correlated with both breed composition and mucin-degrading gut bacteria. We further demonstrate host genetics indirectly changes gut microbiota composition via microbe-microbe interactions. These findings indicate a strong contribution by host genetics in shaping the gut microbiota during early life stages, shedding light on impact of animal breeding on gut microbiota, which is associated with animal growth and health.

Uterine infusion of bacteria alters the transcriptome of bovine oocytes.

Postpartum uterine infection reduces fertility in dairy cattle; however, the mechanisms of uterine infection-mediated infertility are unknown. Paradoxically, infection-induced infertility persists after the resolution of disease. Oocytes are a finite resource, which are present at various stages of development during uterine infection. It is likely that oocyte development is influenced by uterine infection-induced changes to the follicular microenvironment. To better understand the impact of infection on oocyte quality we employed global transcriptomics of oocytes collected from heifers after receiving intrauterine infusion of pathogenic Escherichia coli and Trueperella pyogenes. We hypothesized that the oocyte transcriptome would be altered in response to intrauterine infection. A total of 452 differentially expressed genes were identified in oocytes collected from heifers 4 days after bacteria infusion compared to vehicle infusion, while 539 differentially expressed genes were identified in oocytes collected from heifers 60 days after bacteria infusion. Only 42 genes were differentially expressed in bacteria-infused heifers at both Day 4 and Day 60. Interferon, HMGB1, ILK, IL-6, and TGF-beta signaling pathways were downregulated in oocytes collected at Day 4 from bacteria-infused heifers, while interferon, ILK, and IL-6 signaling were upregulated in oocytes collected at Day 60 from bacteria-infused heifers. These data suggest that bacterial infusion alters the oocyte transcriptome differently at Day 4 and Day 60, suggesting different follicle stages are susceptible to damage. Characterizing the long-term impacts of uterine infection on the oocyte transcriptome aids in our understanding of how infection causes infertility in dairy cattle.

Molecular architecture, polar targeting and biogenesis of the Legionella Dot/Icm T4SS.

Legionella pneumophila survives and replicates inside host cells by secreting ~300 effectors through the defective in organelle trafficking (Dot)/intracellular multiplication (Icm) type IVB secretion system (T4BSS). Here, we used complementary electron cryotomography and immunofluorescence microscopy to investigate the molecular architecture and biogenesis of the Dot/Icm secretion apparatus. Electron cryotomography mapped the location of the core and accessory components of the Legionella core transmembrane subcomplex, revealing a well-ordered central channel that opens into a large, windowed secretion chamber with an unusual 13-fold symmetry. Immunofluorescence microscopy deciphered an early-stage assembly process that begins with the targeting of Dot/Icm components to the bacterial poles. Polar targeting of this T4BSS is mediated by two Dot/Icm proteins, DotU and IcmF, that, interestingly, are homologues of the T6SS membrane complex components TssL and TssM, suggesting that the Dot/Icm T4BSS is a hybrid system. Together, these results revealed that the Dot/Icm complex assembles in an 'axial-to-peripheral' pattern.