Validation of gavage sampling as tool for longitudinal sampling of microbiota of the mouse gastric lumen.

BACKGROUND: Fecal samples are commonly used for longitudinal studies of the gut lumen microbiome to track the course of response to infection or drug treatment, but no comparable method has been evaluated for longitudinal analysis of the gastric lumen microbiome in mice. Herein, a buffer flush of the stomach with a flexible gavage needle was used to collect gastric contents at one or several time points without harming the mouse. These samples were compared to samples collected by sacrifice and dissection of the mouse stomach. Microbiota from these samples were sequenced and evaluated in two ways: the composition of samples as measured by beta diversity and the richness of samples as measured by alpha diversity. Additionally, the effect of multiple sampling every two days on these metrics were studied. DNA was extracted from each of these samples and Illumina 16S rRNA gene sequencing was performed. RESULTS: First, taxonomic richness of gavage and dissection samples was compared. A greater number of taxa was detected in gavage samples than in dissection samples. Second, taxonomic richness was analyzed over time. No significant difference in taxonomic richness was observed with repeated gavage flushes. Third, a comparison was made of the taxonomic composition of samples collected by gavage versus dissection followed by a comparison of samples collected over multiple samplings. Nonmetric multidimensional scaling analysis revealed no clear differences between collection by gavage flushing or dissection. Using weighted Unifrac and Aitchison taxonomic distances between gavage and dissection samples were not significantly different from distances between gavage samples themselves, and no significant difference was found in the taxonomic composition of mice which were sampled repeatedly. Finally, relative abundances of specific identified taxa were compared, and eleven taxa were found to differ in frequency between collection methods. Using the more stringent Analysis of Composition of Microbiomes (ANCOM), seven was found to differ. Similarly, no significant differences were uncovered using these analyses over multiple samples by gastric flush. CONCLUSION: In summary, the consistency of the microbiota collected by gastric flushing recommends its use for microbiome analysis of gastric fluid similar to the use of fecal sampling to study the gut lumen microbiome.

Control of Helicobacter pylori with engineered probiotics secreting selective guided antimicrobial peptides.

Helicobacter pylori is the primary cause of 78% of gastric cancer cases, providing an opportunity to prevent cancer by controlling a single bacterial pathogen within the complex gastric microbiota. We developed highly selective antimicrobial agents against H. pylori by fusing an H. pylori-binding guide peptide (MM1) to broad-spectrum antimicrobial peptides. The common dairy probiotic Lactococcus lactis was then engineered to secrete these guided antimicrobial peptides (gAMPs). When co-cultured in vitro with H. pylori, the gAMP probiotics lost no toxicity compared to unguided AMP probiotics against the target, H. pylori, while losing >90% of their toxicity against two tested off-target bacteria. To test binding to H. pylori, the MM1 guide was fused to green fluorescent protein (GFP), resulting in enhanced binding compared to unguided GFP as measured by flow cytometry. In contrast, MM1-GFP showed no increased binding over GFP against five different off-target bacteria. These highly selective gAMP probiotics were then tested by oral gavage in mice infected with H. pylori. As a therapy, the probiotics outperformed antibiotic treatment, effectively eliminating H. pylori in just 5 days, and also protected mice from challenge infection as a prophylactic. As expected, the gAMP probiotics were as toxic against H. pylori as the unguided AMP probiotics. However, a strong rebound in gastric species diversity was found with both the selective gAMP probiotics and the non-selective AMP probiotics. Eliminating the extreme microbial dysbiosis caused by H. pylori appeared to be the major factor in diversity recovery. IMPORTANCE Alternatives to antibiotics in the control of Helicobacter pylori and the prevention of gastric cancer are needed. The high prevalence of H. pylori in the human population, the induction of microbial dysbiosis by antibiotics, and increasing antibiotic resistance call for a more sustainable approach. By selectively eliminating the pathogen and retaining the commensal community, H. pylori control may be achieved without adverse health outcomes. Antibiotics are typically used as a therapeutic post-infection, but a more targeted, less disruptive approach could be used as a long-term prophylactic against H. pylori or, by extension, against other gastrointestinal pathogens. Furthermore, the modular nature of the guided antimicrobial peptide (gAMP) technology allows for the substitution of different guides for different pathogens and the use of a cocktail of gAMPs to avoid the development of pathogen resistance.

Spiking immunotherapy with a bacterial cocktail brings T cells back to the fight.

A recent study by Montalban-Arques et al.1 in Cell Host & Microbe shows that augmenting the function of the gut microbiota reduces tumor burden. Four Clostridiales species blocked tumor growth as efficient as chemotherapy or immunotherapy in colorectal cancer and melanoma.

Exploration of the Vaginal and Gut Microbiome in African American Women by Body Mass Index, Class of Obesity, and Gestational Weight Gain: A Pilot Study.

OBJECTIVE: This study determines the differences in the distal gut and vaginal microbiome in African American (AA) women by prepregnancy body mass index and gestational weight gain (GWG) comparing women with and without obesity and by obesity class. STUDY DESIGN: We prospectively sampled the vaginal and distal gut microbiome in pregnant AA women at two time points during pregnancy. Samples were analyzed using high-throughput sequencing of the V4 region of the 16S ribosomal ribonucleic acid gene. RESULTS: Distinct differences in vaginal and distal gut α-diversity were observed at time point 1 between women with and without obesity by total GWG. Significant differences in distal gut ß-diversity were also found at time point 1 in obese women by GWG. Within the Bacteroides genus, a significant association was observed by total GWG among obese women which was absent in nonobese women. Women with class III obesity who experienced low GWG had the lowest abundance of distal gut Bacteroides and appreciably higher relative abundance of a consortia of vaginal taxa including Atopobium, Gardnerella, Prevotella, and Sneathia. CONCLUSION: These results contribute new evidence showing that GWG in combination with obesity and obesity class is associated with an altered distal gut and vaginal composition early in pregnancy among AA women.

Repurposing a drug targeting peptide for targeting antimicrobial peptides against Staphylococcus.

OBJECTIVES: Targeted therapies seek to selectively eliminate a pathogen without disrupting the resident microbial community. However, with selectivity comes the potential for developing bacterial resistance. Thus, a diverse range of targeting peptides must be made available. RESULTS: Two commonly used antimicrobial peptides (AMPs), plectasin and eurocin, were genetically fused to the targeting peptide A12C, which selectively binds to Staphylococcus species. The targeting peptide did not decrease activity against the targeted Staphylococcus aureus and Staphylococcus epidermidis, but drastically decreased activity against the nontargeted species, Enterococcus faecalis, Bacillus subtilis, Lactococcus lactis and Lactobacillus rhamnosus. This effect was equally evident across two different AMPs, two different species of Staphylococcus, four different negative control bacteria, and against both biofilm and planktonic forms of the bacteria. CONCLUSIONS: A12C, originally designed for targeted drug delivery, was repurposed to target antimicrobial peptides. This illustrates the wealth of ligands, both natural and synthetic, which can be adapted to develop a diverse array of targeting antimicrobial peptides.

Variation of Pharmacokinetic Profiles of Some Antidiabetic Drugs from Nanostructured Formulations Administered Through Pulmonary Route.

BACKGROUND: Diabetes is a chronic disease that occurs when the pancreas does not produce enough insulin, or when the body cannot effectively use the insulin it produces. WHO projects that diabetes death will be doubled between 2005 and 2030, where 347 million people worldwide had diabetes as per the report of 2013. The increase in the prevalence of diabetes is due to three influences - lifestyle, ethnicity, and age. METHODS: The present review summarizes the pharmacokinetic parameters and challenges in the field of nanoparticles and nanoliposomes of insulin and other antidiabetic drugs given through pulmonary route to treat diabetes effectively. RESULTS: Current challenges in diabetes management include optimizing the use of the already available therapies to ensure adequate glycemic condition, blood pressure, lipid control and to reduce complications. At present, several pieces of research have been focusing on new management options for diabetes. Among these options, the use of nanomedicine is becoming an eye catching and most promising one. Currently, nanoparticles and nanoliposomes are thrust areas of research to treat any deadly disease like diabetes. These drug delivery systems ultimately result in longer circulation half-lives, improved drug pharmacokinetics, and reduced side effects of therapeutically active substances that may be insulin and non-insulin. CONCLUSIONS: Thus, the pulmonary route is the most promising alternative route of drug delivery since it is non-invasive and lungs have a large surface area, richly supplied by the capillary network, for absorption of drugs.

Size dependent variations of phospholipid based vesicular drug carriers in systemic drug activity.

Lipid based vesicular drug delivery system, one of the emerging technologies designed for addressing the delivery challenges of conventional drug delivery methods, has widespread applications in chemotherapeutics, immunotherapeutics, recombinant DNA technology, membrane biology and also as a diagnostic tool in different biological field. The enclosed phospholipid bilayer spherical structure, typically known as liposome, is a versatile vesicular delivery system to carry hydrophilic/hydrophobic drug generally efficiently to the site of action leading to reduced non-specific toxicity and improved bioavailability of the therapeutic moiety. Efficacy of drug encapsulated in liposome depends mainly on the circulation amount of liposome and its residence time, in vivo drug release, drug accumulation in the target site and uptake of the formulation in the reticuloendothelial system. Liposomal formulation factors that dictate those actions are liposomal size (hydrodynamic diameter), surface charge, lipid composition and steric stabilization. Variation in liposomal size shows around 100 fold alterations in pharmacokinetic parameters and systemic activity while the other factors such as surface charge, lipid composition and steric stabilization bring only about 10 fold changes in those properties. The findings indicate the critical role of vesicular size in liposomal efficacy. In the present review the effect of size-variation of liposome on systemic activity of drug as well as its pharmacokinetic profile will be discussed to understand the rational designing of liposomal preparation to maximize therapeutic activity of a drug at desired magnitude and to provide a wide range of product applications such as immunological vaccines, chemotherapy, antimicrobial therapy etc.