Benjamin P. Willing

The Microbiota and Health Promoting Characteristics of the Fermented Beverage Kefir

Kefir is a complex fermented dairy product created through the symbiotic fermentation of milk by lactic acid bacteria and yeasts contained within an exopolysaccharide and protein complex called a kefir grain. As with other fermented dairy products, kefir has been associated with a range of health benefits such as cholesterol metabolism and angiotensin-converting enzyme (ACE) inhibition, antimicrobial activity, tumor suppression, increased speed of wound healing, and modulation of the immune system including the alleviation of allergy and asthma. These reports have led to increased interest in kefir as a focus of research and as a potential probiotic-containing product. Here, we review those studies with a particular emphasis on the microbial composition and the health benefits of the product, as well as discussing the further development of kefir as an important probiotic product.

Lipid Emulsion Formulation of Parenteral Nutrition Affects Intestinal Microbiota and Host Responses in Neonatal Piglets

Background: Total parenteral nutrition (TPN) is a cause of intestinal microbial dysbiosis and impaired gut barrier function. This may contribute to life-threatening parenteral nutrition–associated liver disease and sepsis in infants. We compared the effects of a lipid emulsion containing long-chain &ohgr;-3 polyunsaturated fatty acids (PUFAs; SMOFlipid) and a predominantly &ohgr;-6 PUFA emulsion (Intralipid) on microbial composition and host response at the mucosal surface. Materials and Methods: Neonatal piglets were provided isocaloric, isonitrogenous TPN for 14 days versus sow-fed (SF) controls. Equivalent lipid doses (10 g/kg/d) were given of either SMOFlipid (ML; n = 10) or Intralipid (SO; n = 9). Ileal segments and mucosal scrapings were used to characterize microbial composition by 16S rRNA gene sequencing and quantitative gene expression of tight junction proteins, mucins, antimicrobial peptides, and inflammatory cytokines. Results: The microbial composition of TPN piglets differed from SF, while ML and SO differed from each other (analysis of molecular variance; P < .05); ML piglets were more similar to SF, as indicated by UniFrac distance (P < .05). SO piglets showed a specific and dramatic increase in Parabacteroides (P < .05), while ML showed an increase in Enterobacteriaceae (P < .05). Gene expression of mucin, claudin 1, &bgr;-defensin 2, and interleukin 8 were higher in TPN; overall increases were significantly less in ML versus SO (P < .05). Conclusion: The formulation of parenteral lipid is associated with differences in the gut microbiota and host response of TPN-fed neonatal piglets. Inclusion of &ohgr;-3 long-chain PUFAs appears to improve host-microbial interactions at the mucosal surface, although mechanisms are yet to be defined.

Early life antibiotic exposure affects pancreatic islet development and metabolic regulation.

Childhood antibiotic exposure has been recently linked with increased risk of metabolic disease later in life. A better understanding of this association would potentially provide strategies to reduce the childhood chronic disease epidemic. Therefore, we explored the underlying mechanisms using a swine model that better mimics human infants than rodents, and demonstrated that early life antibiotic exposure affects glucose metabolism 5 weeks after antibiotic withdrawal, which was associated with changes in pancreatic development. Antibiotics exerted a transient impact on postnatal gut microbiota colonization and microbial metabolite production, yet changes in the expression of key genes involved in short-chain fatty acid signaling and pancreatic development were detected in later life. These findings suggest a programming effect of early life antibiotic exposure that merits further investigation.

Dietary Pea Fiber Supplementation Improves Glycemia and Induces Changes in the Composition of Gut Microbiota, Serum Short Chain Fatty Acid Profile and Expression of Mucins in Glucose Intolerant Rats

Several studies have demonstrated the beneficial impact of dried peas and their components on glucose tolerance; however, the role of gut microbiota as a potential mediator is not fully examined. In this study, we investigated the effect of dietary supplementation with raw and cooked pea seed coats (PSC) on glucose tolerance, microbial composition of the gut, select markers of intestinal barrier function, and short chain fatty acid profile in glucose intolerant rats. Male Sprague Dawley rats were fed high fat diet (HFD) for six weeks to induce glucose intolerance, followed by four weeks of feeding PSC-supplemented diets. Cooked PSC improved glucose tolerance by approximately 30% (p < 0.05), and raw and cooked PSC diets reduced insulin response by 53% and 56% respectively (p < 0.05 and p < 0.01), compared to HFD (containing cellulose as the source of dietary fiber). 16S rRNA gene sequencing on fecal samples showed a significant shift in the overall microbial composition of PSC groups when compared to HFD and low fat diet (LFD) controls. At the family level, PSC increased the abundance of Lachnospiraceae and Prevotellaceae (p < 0.001), and decreased Porphyromonadaceae (p < 0.01) compared with HFD. This was accompanied by increased mRNA expression of mucin genes Muc1, Muc2, and Muc4 in ileal epithelium (p < 0.05). Serum levels of acetate and propionate increased with raw PSC diet (p < 0.01). These results indicate that supplementation of HFD with PSC fractions can improve glycemia and may have a protective role against HFD-induced alterations in gut microbiota and mucus layer.

Host Immune Selection of Rumen Bacteria through Salivary Secretory IgA

The rumen microbiome is integral to efficient production in cattle and shows strong host specificity, yet little is known about what host factors shape rumen microbial composition. Secretory immunoglobulin A (SIgA) is produced in large amounts in the saliva, can coat both commensal and pathogenic microbes within the gut, and presents a plausible mechanism of host specificity. However, the role salivary SIgA plays in commensal bacteria selection in ruminants remains elusive. The main objectives of this study were to develop an immuno-affinity benchtop method to isolate SIgA-tagged microbiota and to determine if salivary SIgA preferentially binds selected bacteria. We hypothesized that SIgA-tagged bacteria would differ from total bacteria, thus supporting a potential host-derived mechanism in commensal bacterial selection. Whole rumen (n = 9) and oral secretion samples (n = 10) were incubated with magnetic beads conjugated with anti-secretory IgA antibodies to enrich SIgA-tagged microbiota. Microbial DNA from the oral secretion, whole rumen, SIgA-tagged oral secretion, and SIgA-tagged rumen was isolated for amplicon sequencing of V1–V3 region of 16S rDNA genes. Whole rumen and oral secretion had distinctive (P < 0.05) bacterial compositions indicated by the non-parametric multidimensional scaling plot using Euclidean distance metrics. The SIgA-tagged microbiota from rumen and oral secretion had similar abundance of Bacteroidetes, Actinobacteria, Fibrobacter, candidate phyla TM7, and Tenericutes and are clustered tightly. Composition of SIgA-tagged oral secretion microbiota was more similar to whole rumen microbiota than whole oral secretion due to enrichment of rumen bacteria (Lachnospiraceae) and depletion of oral taxa (Streptococcus, Rothia, Neisseriaceae, and Lactobacillales). In conclusion, SIgA-tagged oral secretion microbiota had an increased resemblance to whole rumen microbiota, suggesting salivary SIgA-coating may be one host-derived mechanism impacting commensal colonization. Further studies, to explore the variations in antibody affinity between different animals as a driver of microbial composition are warranted.

High Fructose Intake During Pregnancy in Rats Influences the Maternal Microbiome and Gut Development in the Offspring

Studies in pregnant women indicate the maternal microbiome changes during pregnancy so as to benefit the mother and fetus. In contrast, disruption of the maternal microbiota around birth can compromise normal bacterial colonisation of the infant’s gastrointestinal tract. This may then inhibit development of the gut so as to increase susceptibility to inflammation and reduce barrier function. The impact of modulating fructose intake on the maternal microbiome through pregnancy is unknown, therefore we examined the effect of fructose supplementation on the maternal microbiome together with the immediate and next generation effects in the offspring. Wistar rat dams were divided into control and fructose fed groups that received 10% fructose in their drinking water from 8 weeks of age and throughout pregnancy (10–13 weeks). Maternal fecal and blood samples were collected pre-mating (9 weeks) and during early (gestational day 4–7) and late pregnancy (gestational day 19–21). We show supplementation of the maternal diet with fructose appears to significantly modulate the maternal microbiome, with a significant reduction in Lactobacillus and Bacteroides. In offspring maintained on this diet up to pregnancy and term there was a reduction in gene expression of markers of gut barrier function that could adversely affect its function. An exacerbated insulin response to pregnancy, reduced birth weight, but increased fat mass was also observed in these offspring. In conclusion dietary supplementation with fructose modulates the maternal microbiome in ways that could adversely affect fetal growth and later gut development.

Establishing a model for childhood obesity in adolescent pigs: Pig model for childhood obesity

Rising worldwide prevalence of obesity and metabolic diseases in children has accentuated the importance of developing prevention and management strategies. The objective of this study was to establish a model for childhood obesity using high‐fat feeding of adolescent pigs, as pigs have a longer developmental period and are physiologically more similar to humans than rodents.

Impact of Clinical Use of Parenteral Lipid Emulsions on Bile Acid Metabolism and Composition in Neonatal Piglets

BACKGROUND Neonates with intestinal failure dependent on parenteral nutrition (PN) are at risk of intestinal failure-associated liver disease (IFALD). PN lipid composition relates to the risk of IFALD, but the mechanisms are poorly understood. We investigated the effects of soybean oil (SO), a mixed-lipid (ML) emulsion containing fish oil (FO), and a pure FO. We hypothesized FO-containing PN lipids would result in increased gene expression of canalicular bile acid transporters and a larger, more hydrophilic bile acid pool, predictive of increased bile flow. METHODS Neonatal piglets were allocated to receive 1 of SO, ML, or FO throughout 14 days of PN feeding. Relative expression of genes involved in bile acid synthesis and transport were determined through quantitative polymerase chain reaction. Bile secreted from the liver was collected and measured. Bile acid composition was determined using tandem mass spectrometry. Regression analysis was used to determine predictors of bile flow. RESULTS PN reduced bile acid secretion (P < .001). FO-containing PN lipids were associated with greater expression of bile acid and organic solute transport genes (P < .05) and greater secretion of hydrophobic bile acids (P < .001). Farnesoid X receptor (P = .01), bile salt export pump (P < .01), multidrug resistant protein 2 (P < .01), and unconjugated hyocholic acid (P < .001) independently predicted bile flow. CONCLUSIONS PN lipid modulation altered bile acid metabolism and composition. These alterations may explain the hepatoprotective effects of FO-containing PN lipids and support their use in the prevention and treatment of IFALD.

Hepatic Expression of PEMT, but Not Dietary Choline Supplementation, Reverses the Protection against Atherosclerosis in Pemt−/−/Ldlr−/− Mice

Background Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine to phosphatidylcholine. Pemt-/-/low density lipoprotein receptor (Ldlr)-/- mice have significantly reduced plasma lipids and are protected against atherosclerosis. Recent studies have shown that choline can be metabolized by the gut flora into trimethylamine-N-oxide (TMAO), which is an emerging risk factor for atherosclerosis. Objective The objective of this study was to determine whether ectopic hepatic PEMT expression or choline supplementation would promote atherosclerosis in Pemt-/-/Ldlr-/- mice. Methods Male 8- to 10-wk-old Pemt+/+/Ldlr-/- (SKO) and Pemt-/-/Ldlr-/- (DKO) mice were injected with an adeno-associated virus (AAV) expressing green fluorescent protein (GFP) or human PEMT and fed a Western diet (40% of calories from fat, 0.5% cholesterol) for 8 wk. In a separate experiment, 8- to 10-wk-old SKO and half of the DKO male mice were fed a Western diet with normal (3 g/kg) choline for 12 wk. The remaining DKO mice [choline-supplemented (CS) DKO] were fed a CS Western diet (10 g choline/kg). Plasma lipid concentrations, choline metabolites, and aortic atherosclerosis were measured. Results Plasma cholesterol, plasma TMAO, and aortic atherosclerosis were reduced by 60%, 40%, and 80%, respectively, in DKO mice compared with SKO mice. AAV-PEMT administration increased plasma cholesterol and TMAO by 30% and 40%, respectively, in DKO mice compared with AAV-GFP-treated DKO mice. Furthermore, AAV-PEMT-injected DKO mice developed atherosclerotic lesions similar to SKO mice. In the second study, there was no difference in atherosclerosis or plasma cholesterol between DKO and CS-DKO mice. However, plasma TMAO concentrations were increased 2.5-fold in CS-DKO mice compared with DKO mice. Conclusions Reintroducing hepatic PEMT reversed the atheroprotective phenotype of DKO mice. Choline supplementation did not increase atherosclerosis or plasma cholesterol in DKO mice. Our data suggest that plasma TMAO does not induce atherosclerosis when plasma cholesterol is low. Furthermore, this is the first report to our knowledge that suggests that de novo choline synthesis alters TMAO status.

Bacterial resistance to antibiotic alternatives: a wolf in sheep’s clothing?1

With the growing concern of antibiotic resistance (Aminov and Mackie, 2007; Zaman et al., 2017), there has been a strong push to reduce the use of antibiotics in animal production systems (Van Boeckel et al., 2015; Ventola, 2015). Many antibiotic alternatives have been developed, with varying degrees of success in improving health outcomes and growth performance (Gresse et al., 2017). These alternatives use very different approaches to regulate both commensal and pathogenic bacterial populations. Antibiotic alternatives such as phage and bacteriocins have very clear mechanisms of antimicrobial activity (Figure 1), whereas others, such as essential oils/phytosterols, have less defined modes of action. Irrespective of mode of action, there has been insufficient attention given to the ability of bacteria to develop resistance to these antibiotic alternatives. Considering the development of resistance will be essential in finding long-term solutions. In this review, we present what is known about the ability of bacteria to become resistant to these antibiotic alternatives, and more importantly, identify where they contribute to antibiotic resistance. Prudence is required, as avoiding further contribution to antibiotic resistance is necessary. This review is not exhaustive but is intended to give a good representation from different classes of antibiotic alternatives. In particular, we focus on phage, essential oils, direct-fed microbials and bacteriocins, metals and minerals, and organic acids. Some consideration is given to their application, effectiveness, and modes of action. Open in a separate window Figure 1. (A) Phage interact with specific receptors to inject DNA into the bacterial cell, causing viral proliferation and cell lysi (i). Essential oils (EOs) disrupt efflux/influx, membrane receptors and stability (ii). Copper disrupts bacterial lipids, proteins, and DNA through oxidization (iii). Bacteriocins cause cell wall lysis, disrupt the plasma membrane structure (pore formation), and interfere with DNA function (iv). (B) Bacterial resistance to phage is conferred through either blockage/removal of the receptor or cutting of phage DNA in the cell by CRISPR/CAS (i). Bacteria form aggregates to minimize cell surface exposure to EOs, thus preventing membrane associated disruptions (ii). Glutathione chelates Cu+, ATPase efflux system exports Cu+/Cu2+, and siderophores sequester Cu2+ to prevent it entering the cell (iii). Modifications of the cell wall and membrane affect fluidity and charge, impairing bacteriocin binding (iv).