Milk microbiome in dairy cattle and the challenges of low microbial biomass and exogenous contamination.

BACKGROUND: The blanket usage of antimicrobials at the end of lactation (or "drying off") in dairy cattle is under increasing scrutiny due to concerns about antimicrobial resistance. To lower antimicrobial usage in dairy farming, farmers are now encouraged to use "selective dry cow therapy" whereby only cows viewed as at high risk of mastitis are administered antimicrobial agents. It is important to gain a better understanding of how this practice affects the udder-associated microbiota and the potential knock-on effects on antimicrobial-resistant bacterial populations circulating on the farm. However, there are challenges associated with studying low biomass environments such as milk, due to known contamination effects on microbiome datasets. Here, we obtained milk samples from cattle at drying off and at calving to measure potential shifts in bacterial load and microbiota composition, with a critical assessment of contamination effects. RESULTS: Several samples had no detectable 16S rRNA gene copies and crucially, exogenous contamination was detected in the initial microbiome dataset. The affected samples were removed from the final microbiome analysis, which compromised the experimental design and statistical analysis. There was no significant difference in bacterial load between treatments (P > 0.05), but load was lower at calving than at drying off (P = 0.039). Escherichia coli counts by both sequence and culture data increased significantly in the presence of reduced bacterial load and a decreasing trend of microbiome richness and diversity. The milk samples revealed diverse microbiomes not reflecting a typical infection profile and were largely comprised of gut- and skin-associated taxa, with the former decreasing somewhat after prolonged sealing of the teats. CONCLUSIONS: The drying off period had a key influence on microbiota composition and bacterial load, which appeared to be independent of antimicrobial usage. The interactions between drying off treatment protocol and milk microbiome dynamics are clearly complex, and our evaluations of these interactions were restricted by low biomass samples and contamination effects. Therefore, our analysis will inform the design of future studies to establish whether different selection protocols could be implemented to further minimise antimicrobial usage.

Temporal and nutritional effects on the weaner pig ileal microbiota.

BACKGROUND: The porcine gastrointestinal microbiota has been linked to both host health and performance. Most pig gut microbiota studies target faecal material, which is not representative of microbiota dynamics in other discrete gut sections. The weaning transition period in pigs is a key development stage, with gastrointestinal problems being prominent after often sudden introduction to a solid diet. A better understanding of both temporal and nutritional effects on the small intestinal microbiota is required. Here, the development of the porcine ileal microbiota under differing levels of dietary protein was observed over the immediate post-weaning period. RESULTS: Ileal digesta samples were obtained at post-mortem prior to weaning day (day - 1) for baseline measurements. The remaining pigs were introduced to either an 18% (low) or 23% (high) protein diet on weaning day (day 0) and further ileal digesta sampling was carried out at days 5, 9 and 13 post-weaning. We identified significant changes in microbiome structure (P = 0.01), a reduction in microbiome richness (P = 0.02) and changes in the abundance of specific bacterial taxa from baseline until 13 days post-weaning. The ileal microbiota became less stable after the introduction to a solid diet at weaning (P = 0.036), was highly variable between pigs and no relationship was observed between average daily weight gain and microbiota composition. The ileal microbiota was less stable in pigs fed the high protein diet (P = 0.05), with several pathogenic bacterial genera being significantly higher in abundance in this group. Samples from the low protein and high protein groups did not cluster separately by their CAZyme (carbohydrate-active enzyme) composition, but GH33 exosialidases were found to be significantly more abundant in the HP group (P = 0.006). CONCLUSIONS: The weaner pig ileal microbiota changed rapidly and was initially destabilised by the sudden introduction to feed. Nutritional composition influenced ileal microbiota development, with the high protein diet being associated with an increased abundance of significant porcine pathogens and the upregulation of GH33 exosialidases-which can influence host-microbe interactions and pathogenicity. These findings contribute to our understanding of a lesser studied gut compartment that is not only a key site of digestion, but also a target for the development of nutritional interventions to improve gut health and host growth performance during the critical weaning transition period.

Prostaglandin E2 promotes intestinal inflammation via inhibiting microbiota-dependent regulatory T cells.

The gut microbiota fundamentally regulates intestinal homeostasis and disease partially through mechanisms that involve modulation of regulatory T cells (Tregs), yet how the microbiota-Treg cross-talk is physiologically controlled is incompletely defined. Here, we report that prostaglandin E2 (PGE2), a well-known mediator of inflammation, inhibits mucosal Tregs in a manner depending on the gut microbiota. PGE2 through its receptor EP4 diminishes Treg-favorable commensal microbiota. Transfer of the gut microbiota that was modified by PGE2-EP4 signaling modulates mucosal Treg responses and exacerbates intestinal inflammation. Mechanistically, PGE2-modified microbiota regulates intestinal mononuclear phagocytes and type I interferon signaling. Depletion of mononuclear phagocytes or deficiency of type I interferon receptor diminishes PGE2-dependent Treg inhibition. Together, our findings provide emergent evidence that PGE2-mediated disruption of microbiota-Treg communication fosters intestinal inflammation.

Microbial Stimulation Reverses the Age-Related Decline in M Cells in Aged Mice.

Aging has a profound effect on the immune system, termed immunosenescence, resulting in increased incidence and severity of infections and decreased efficacy of vaccinations. We previously showed that immunosurveillance in the intestine, achieved primarily through antigen sampling M cells in the follicle associated epithelium (FAE) of Peyer's patches, was compromised during aging due to a decline in M cell functional maturation. The intestinal microbiota also changes significantly with age, but whether this affects M cell maturation was not known. We show that housing of aged mice on used bedding from young mice, or treatment with bacterial flagellin, were each sufficient to enhance the functional maturation of M cells in Peyer's patches. An understanding of the mechanisms underlying the influence of the intestinal microbiota on M cells has the potential to lead to new methods to enhance the efficacy of oral vaccination in aged individuals.

Resistance to change: AMR gene dynamics on a commercial pig farm with high antimicrobial usage.

Group antimicrobial administration is used to control disease in livestock, but we have little insight into how this impacts antimicrobial resistance (AMR) gene dynamics. Here, a longitudinal study was carried out during a single production cycle on a commercial pig unit with high historic and current antimicrobial usage. Quantitative PCR, 16S rRNA gene metabarcoding and shotgun metagenomic sequencing were used to track faecal AMR gene abundance and diversity and microbiome alpha diversity. Shotgun metagenomic sequencing identified 144 AMR genes in total, with higher AMR gene diversity present in young pigs compared to dry sows. Irrespective of in-feed antibiotic treatment or changes in microbiome diversity, mean AMR gene copy number was consistently high, with some AMR genes present at copy numbers comparable to the bacterial 16S rRNA gene. In conclusion, AMR gene prevalence and abundance were not influenced by antibiotic use, either during the production cycle or following whole-herd medication. The high levels of certain genes indicate they are widely disseminated throughout the microbial population, potentially aiding stability. Despite the high and relatively stable levels of resistance genes against the main antimicrobials used, these compounds continue to control production limiting diseases on this unit.

Changes in the Ileal, but Not Fecal, Microbiome in Response to Increased Dietary Protein Level and Enterotoxigenic Escherichia coli Exposure in Pigs.

The relationship between porcine gut microbiota composition and health is an important area of research, especially due to the need to find alternatives to antimicrobial use to manage disease in livestock production systems. Previous work has indicated that lower crude dietary protein levels can reduce the impacts of postweaning colibacillosis, which is a porcine diarrheal disease caused by enterotoxigenic Escherichia coli (ETEC). Here, to explore the complex interactions between the gut microbiota, protein nutrition, and ETEC exposure, the microbial compositions of both ileal digesta and feces were analyzed with or without ETEC exposure from pigs fed a low- or high-protein diet. Since ETEC colonization is mostly localized to the ileum, changes in the small intestinal microbiota were expected in response to ETEC exposure. This was supported by the study findings, which identified significant microbiota changes in ileal samples but not in fecal samples. Both increased dietary protein and ETEC exposure impacted on ileal microbiota alpha diversity (richness and diversity indices) and beta diversity (structure, stability, and relative taxon abundances) at certain sampling points, although the combination of a high-protein diet and ETEC exposure had the most profound impact on ileal microbiota composition. An understanding of how infection and nutrition lead to microbiota changes is likely to be required if dietary strategies are to be developed for the management of enteric diseases.IMPORTANCE Gut bacterial communities have been shown to play a key role in pig health and development and are strongly influenced by host diet, but studies highlighting the complex interactions between nutrition, gut infections and the microbiome tend to focus on bacterial populations in the feces and not other important gut locations. We found that alteration of dietary protein level and exposure to a pathogenic microorganism, enterotoxigenic Escherichia coli (ETEC), changed bacterial populations in the distal small intestine (i.e., the ileum). We found that the most profound changes occurred in pigs fed a high-protein diet in combination with exposure to ETEC, showing a clear interaction between dietary composition and exposure to a key pathogen. These changes were not observed in the fecal samples, revealing the importance of studying biologically pertinent sites in the gut, and so the data will help to inform the development of alternative management strategies for enteric disorders.

Analysis of temporal fecal microbiota dynamics in weaner pigs with and without exposure to enterotoxigenic Escherichia coli1,2.

The primary aim of this work was to study potential effects of subclinical enterotoxigenic Escherichia coli (ETEC) exposure on porcine fecal microbiota composition, with a secondary aim of profiling temporal shifts in bacterial communities over the weaning transition period. 16S rRNA gene metabarcoding and quantitative PCR (qPCR) were used to profile the fecal microbiota and quantify ETEC excretion in the feces, respectively. Temporal shifts in fecal microbiota structure and stability were observed across the immediate postweaning period (P < 0.05), including significant shifts in the relative levels of specific bacterial phylotypes (P < 0.05). ETEC exposure did not change the fecal microbiota structure (P > 0.05), but significant variations in fecal community structure and stability were linked to variations in ETEC excretion level at particular time points (P < 0.05). In this study, marked temporal changes in microbiota structure and stability were evident over the short weaning transition period, with a relationship between ETEC excretion level and fecal microbiota composition being observed. This study has provided a detailed analysis of fecal microbiota dynamics in the pig, which should help to inform the development of novel management strategies for enteric disorders based on an improved understanding of microbial populations during the challenging postweaning period.

The Madness of Microbiome: Attempting To Find Consensus "Best Practice" for 16S Microbiome Studies.

The development and continuous improvement of high-throughput sequencing platforms have stimulated interest in the study of complex microbial communities. Currently, the most popular sequencing approach to study microbial community composition and dynamics is targeted 16S rRNA gene metabarcoding. To prepare samples for sequencing, there are a variety of processing steps, each with the potential to introduce bias at the data analysis stage. In this short review, key information from the literature pertaining to each processing step is described, and consequently, general recommendations for future 16S rRNA gene metabarcoding experiments are made.

Variability of the Sheep Lung Microbiota.

UNLABELLED: Sequencing technologies have recently facilitated the characterization of bacterial communities present in lungs during health and disease. However, there is currently a dearth of information concerning the variability of such data in health both between and within subjects. This study seeks to examine such variability using healthy adult sheep as our model system. Protected specimen brush samples were collected from three spatially disparate segmental bronchi of six adult sheep (age, 20 months) on three occasions (day 0, 1 month, and 3 months). To further explore the spatial variability of the microbiotas, more-extensive brushing samples (n = 16) and a throat swab were taken from a separate sheep. The V2 and V3 hypervariable regions of the bacterial 16S rRNA genes were amplified and sequenced via Illumina MiSeq. DNA sequences were analyzed using the mothur software package. Quantitative PCR was performed to quantify total bacterial DNA. Some sheep lungs contained dramatically different bacterial communities at different sampling sites, whereas in others, airway microbiotas appeared similar across the lung. In our spatial variability study, we observed clustering related to the depth within the lung from which samples were taken. Lung depth refers to increasing distance from the glottis, progressing in a caudal direction. We conclude that both host influence and local factors have impacts on the composition of the sheep lung microbiota. IMPORTANCE: Until recently, it was assumed that the lungs were a sterile environment which was colonized by microbes only during disease. However, recent studies using sequencing technologies have found that there is a small population of bacteria which exists in the lung during health, referred to as the "lung microbiota." In this study, we characterize the variability of the lung microbiotas of healthy sheep. Sheep not only are economically important animals but also are often used as large animal models of human respiratory disease. We conclude that, while host influence does play a role in dictating the types of microbes which colonize the airways, it is clear that local factors also play an important role in this regard. Understanding the nature and influence of these factors will be key to understanding the variability in, and functional relevance of, the lung microbiota.

Human Streptococcus agalactiae strains in aquatic mammals and fish.

BACKGROUND: In humans, Streptococcus agalactiae or group B streptococcus (GBS) is a frequent coloniser of the rectovaginal tract, a major cause of neonatal infectious disease and an emerging cause of disease in non-pregnant adults. In addition, Streptococcus agalactiae causes invasive disease in fish, compromising food security and posing a zoonotic hazard. We studied the molecular epidemiology of S. agalactiae in fish and other aquatic species to assess potential for pathogen transmission between aquatic species and humans. METHODS: Isolates from fish (n = 26), seals (n = 6), a dolphin and a frog were characterized by pulsed-field gel electrophoresis, multilocus sequence typing and standardized 3-set genotyping, i.e. molecular serotyping and profiling of surface protein genes and mobile genetic elements. RESULTS: Four subpopulations of S. agalactiae were identified among aquatic isolates. Sequence type (ST) 283 serotype III-4 and its novel single locus variant ST491 were detected in fish from Southeast Asia and shared a 3-set genotype identical to that of an emerging ST283 clone associated with invasive disease of adult humans in Asia. The human pathogenic strain ST7 serotype Ia was also detected in fish from Asia. ST23 serotype Ia, a subpopulation that is normally associated with human carriage, was found in all grey seals, suggesting that human effluent may contribute to microbial pollution of surface water and exposure of sea mammals to human pathogens. The final subpopulation consisted of non-haemolytic ST260 and ST261 serotype Ib isolates, which belong to a fish-associated clonal complex that has never been reported from humans. CONCLUSIONS: The apparent association of the four subpopulations of S. agalactiae with specific groups of host species suggests that some strains of aquatic S. agalactiae may present a zoonotic or anthroponotic hazard. Furthermore, it provides a rational framework for exploration of pathogenesis and host-associated genome content of S. agalactiae strains.