Interplay between inflammatory bowel disease therapeutics and the gut microbiome reveals opportunities for novel treatment approaches.

Inflammatory bowel disease (IBD) is a complex heterogeneous disorder defined by recurring chronic inflammation of the gastrointestinal tract, attributed to a combination of factors including genetic susceptibility, altered immune response, a shift in microbial composition/microbial insults (infection/exposure), and environmental influences. Therapeutics generally used to treat IBD mainly focus on the immune response and include non-specific anti-inflammatory and immunosuppressive therapeutics and targeted therapeutics aimed at specific components of the immune system. Other therapies include exclusive enteral nutrition and emerging stem cell therapies. However, in recent years, scientists have begun to examine the interplay between these therapeutics and the gut microbiome, and we present this information here. Many of these therapeutics are associated with alterations to gut microbiome composition and functionality, often driving it toward a "healthier profile" and preclinical studies have revealed that such alterations can play an important role in therapeutic efficacy. The gut microbiome can also improve or hinder IBD therapeutic efficacy or generate undesirable metabolites. For certain IBD therapeutics, the microbiome composition, particularly before treatment, may serve as a biomarker of therapeutic efficacy. Utilising this information and manipulating the interactions between the gut microbiome and IBD therapeutics may enhance treatment outcomes in the future and bring about new opportunities for personalised, precision medicine.

Novel, non-colonizing, single-strain live biotherapeutic product ADS024 protects against Clostridioides difficile infection challenge in vivo.

BACKGROUND: The Centers for Disease Control and Prevention estimate that Clostridioides difficile (C. difficile) causes half a million infections (CDI) annually and is a major cause of total infectious disease death in the United States, causing inflammation of the colon and potentially deadly diarrhea. We recently reported the isolation of ADS024, a Bacillus velezensis (B. velezensis) strain, which demonstrated direct in vitro bactericidal activity against C. difficile, with minimal collateral impact on other members of the gut microbiota. In this study, we hypothesized that in vitro activities of ADS024 will translate in vivo to protect against CDI challenge in mouse models. AIM: To investigate the in vivo efficacy of B. velezensis ADS024 in protecting against CDI challenge in mouse models. METHODS: To mimic disruption of the gut microbiota, the mice were exposed to vancomycin prior to dosing with ADS024. For the mouse single-dose study, the recovery of ADS024 was assessed via microbiological analysis of intestinal and fecal samples at 4 h, 8 h, and 24 h after a single oral dose of 5 × 108 colony-forming units (CFU)/mouse of freshly grown ADS024. The single-dose study in miniature swine included groups that had been pre-dosed with vancomycin and that had been exposed to a dose range of ADS024, and a group that was not pre-dosed with vancomycin and received a single dose of ADS024. The ADS024 colonies [assessed by quantitative polymerase chain reaction (qPCR) using ADS024-specific primers] were counted on agar plates. For the 28-d miniature swine study, qPCR was used to measure ADS024 levels from fecal samples after oral administration of ADS024 capsules containing 5 × 109 CFU for 28 consecutive days, followed by MiSeq compositional sequencing and bioinformatic analyses to measure the impact of ADS024 on microbiota. Two studies were performed to determine the efficacy of ADS024 in a mouse model of CDI: Study 1 to determine the effects of fresh ADS024 culture and ADS024 spore preparations on the clinical manifestations of CDI in mice, and Study 2 to compare the efficacy of single daily doses vs dosing 3 times per day with fresh ADS024. C. difficile challenge was performed 24 h after the start of ADS024 exposure. To model the human distal colon, an anerobic fecal fermentation system was used. MiSeq compositional sequencing and bioinformatic analyses were performed to measure microbiota diversity changes following ADS024 treatment. To assess the potential of ADS024 to be a source of antibiotic resistance, its susceptibility to 18 different antibiotics was tested. RESULTS: In a mouse model of CDI challenge, single daily doses of ADS024 were as efficacious as multiple daily doses in protecting against subsequent challenge by C. difficile pathogen-induced disease. ADS024 showed no evidence of colonization based on the observation that the ADS024 colonies were not recovered 24 h after single doses in mice or 72 h after single doses in miniature swine. In a 28-d repeat-dose study in miniature swine, ADS024 was not detected in fecal samples using plating and qPCR methods. Phylogenetic analysis performed in the human distal colon model showed that ADS024 had a selective impact on the healthy human colonic microbiota, similarly to the in vivo studies performed in miniature swine. Safety assessments indicated that ADS024 was susceptible to all the antibiotics tested, while in silico testing revealed a low potential for off-target activity or virulence and antibiotic-resistance mechanisms. CONCLUSION: Our findings, demonstrating in vivo efficacy of ADS024 in protecting against CDI challenge in mouse models, support the use of ADS024 in preventing recurrent CDI following standard antibiotic treatment.

Modulation of the gut microbiome with nisin.

Nisin is a broad spectrum bacteriocin used extensively as a food preservative that was identified in Lactococcus lactis nearly a century ago. We show that orally-ingested nisin survives transit through the porcine gastrointestinal tract intact (as evidenced by activity and molecular weight determination) where it impacts both the composition and functioning of the microbiota. Specifically, nisin treatment caused a reversible decrease in Gram positive bacteria, resulting in a reshaping of the Firmicutes and a corresponding relative increase in Gram negative Proteobacteria. These changes were mirrored by the modification in relative abundance of pathways involved in acetate, butyrate (decreased) and propionate (increased) synthesis which correlated with overall reductions in short chain fatty acid levels in stool. These reversible changes that occur as a result of nisin ingestion demonstrate the potential of bacteriocins like nisin to shape mammalian microbiomes and impact on the functionality of the community.

Identification of ADS024, a newly characterized strain of Bacillus velezensis with direct Clostridiodes difficile killing and toxin degradation bio-activities.

Clostridioides difficile infection (CDI) remains a significant health threat worldwide. C. difficile is an opportunistic, toxigenic pathogen that takes advantage of a disrupted gut microbiome to grow and produce signs and symptoms ranging from diarrhea to pseudomembranous colitis. Antibiotics used to treat C. difficile infection are usually broad spectrum and can further disrupt the commensal gut microbiota, leaving patients susceptible to recurrent C. difficile infection. There is a growing need for therapeutic options that can continue to inhibit the outgrowth of C. difficile after antibiotic treatment is completed. Treatments that degrade C. difficile toxins while having minimal collateral impact on gut bacteria are also needed to prevent recurrence. Therapeutic bacteria capable of producing a range of antimicrobial compounds, proteases, and other bioactive metabolites represent a potentially powerful tool for preventing CDI recurrence following resolution of symptoms. Here, we describe the identification and initial characterization of ADS024 (formerly ART24), a novel therapeutic bacterium that can kill C. difficile in vitro with limited impact on other commensal bacteria. In addition to directly killing C. difficile, ADS024 also produces proteases capable of degrading C. difficile toxins, the drivers of symptoms associated with most cases of CDI. ADS024 is in clinical development for the prevention of CDI recurrence as a single-strain live biotherapeutic product, and this initial data set supports further studies aimed at evaluating ADS024 in future human clinical trials.

Impact of nisin on Clostridioides difficile and microbiota composition in a faecal fermentation model of the human colon.

AIMS: Nisin is a bacteriocin with a broad spectrum of activity against Gram-positive bacteria. The aims were to assess nisin activity against Clostridioides difficile in a complex microbial environment and determine the minimum inhibitory concentration at which C. difficile growth is suppressed whilst having minimal impact on the faecal microbiota. METHODS AND RESULTS: Faecal slurries were prepared from fresh faecal samples and spiked with C. difficile (106  CFU per ml). Nisin was added to each fermentation at a range of concentrations from 0 to 500 µM. Following 24 h, 16S rRNA gene sequencing was performed, and the presence of viable C. difficile was assessed. There was no viable C. difficile detected in the presence of 50-500 µM nisin. There was a decrease in the diversity of the microbiota in a nisin dose-dependent manner. Nisin predominantly depleted the relative abundance of the Gram-positive bacteria whilst the relative abundance of Gram-negative bacteria such as Escherichia Shigella and Bacteroides increased. CONCLUSIONS: Using an ex vivo model of the colon, this study demonstrates the ability of purified nisin to selectively deplete C. difficile in a faecal microbial environment and establishes the minimum concentration at which this occurs whilst having a minimal impact on the composition of the microbiota. SIGNIFICANCE AND IMPACT OF THE STUDY: This study opens up the potential to use nisin as a therapeutic for clostridial gut infections.

The microbiome of deep-sea fish reveals new microbial species and a sparsity of antibiotic resistance genes.

Adaptation to life in the deep-sea can be dramatic, with fish displaying behaviors and appearances unlike those seen in any other aquatic habitat. However, the extent of which adaptations may have developed at a microbial scale is not as clear. Shotgun metagenomic sequencing of the intestinal microbiome of 32 species of deep-sea fish from across the Atlantic Ocean revealed that many of the associated microbes differ extensively from those previously identified in reference databases. 111 individual metagenome-assembled genomes (MAGs) were constructed representing individual microbial species from the microbiomes of these fish, many of which are potentially novel bacterial taxa and provide a window into the microbial diversity in this underexplored environment. These MAGs also demonstrate how these microbes have adapted to deep-sea life by encoding a greater capacity for several cellular processes such as protein folding and DNA replication that can be inhibited by high pressure. Another intriguing feature was the almost complete lack of genes responsible for acquired resistance to known antibiotics in many of the samples. This highlights that deep-sea fish microbiomes may represent one of few animal-associated microbiomes with little influence from human activity. The ability of the microbes in these samples to bioluminesce is lower than expected given predictions that this trait has an important role in their life cycle at these depths. The study highlights the uniqueness, complexity and adaptation of microbial communities living in one of the largest and harshest environments on Earth.

A multicentre analysis of Clostridium difficile in persons with Cystic Fibrosis demonstrates that carriage may be transient and highly variable with respect to strain and level.

PURPOSE: Clostridium difficile has been reported to occur in the gastrointestinal tract of 50% of Cystic Fibrosis (CF) subjects, however, clinical C. difficile infection (CDI) is a rare occurrence in this cohort despite the presence of toxigenic and hypervirulent ribotypes. Here, we present the first longitudinal, multicentre analysis of C. difficile prevalence among adult CF subjects. METHODOLOGY: Faecal samples were collected from adults with CF (selected based on confirmed Pseudomonas aeruginosa pulmonary colonisation) from Ireland, UK and Belgium as part of the CFMATTERS clinical research trial (grant No. 603038) and from non-CF controls. Faecal samples were collected on enrolment, at three monthly intervals, during pulmonary exacerbation and three months post exacerbation. C. difficile was isolated from faecal samples by ethanol shocking followed by culturing on cycloserine cefoxitin egg yolk agar. Isolates were characterised in terms of ribotype, toxin type and antibiotic susceptibility to antibiotics routinely used in the treatment of CDI (metronidazole and vancomycin) and those implicated in induction of CDI (ciprofloxacin and moxifloxacin). RESULTS: Prevalence of C. difficile among CF subjects in the three sites was similar ranging from 47% to 50% at baseline, while the healthy control cohort had a carriage rate of 7.1%. Including subjects who were positive for C. difficile at any time point there was a higher carriage rate of 71.4%, 66.7% and 63.2% in Ireland, UK, and Belgium, respectively. Ribotyping of 80 isolates from 45 CF persons, over multiple time points revealed 23 distinct ribotypes with two ribotypes (046 and 078) shared by all centres. The proportion of toxigenic isolates varied across the sites, ranging from 66.7% in Ireland to 52.9% in Belgium and 100% in the UK. Antibiotic susceptibility rates to vancomycin, metronidazole, ciprofloxacin and moxifloxacin was 100%, 97.5%, 1.3% and 63.8%, respectively. CONCLUSIONS: This study demonstrates the highest carriage rate of C. difficile to date in a CF cohort. Longitudinal data show that C. difficile can be a transient inhabitant of the CF gut, changing both in terms of strain and excretion rates.

Protecting the outside: biological tools to manipulate the skin microbiota.

Interest surrounding the role that skin microbes play in various aspects of human health has recently experienced a timely surge, particularly among researchers, clinicians and consumer-focused industries. The world is now approaching a post-antibiotic era where conventional antibacterial therapeutics have shown a loss in effectiveness due to overuse, leading to the looming antibiotic resistance crisis. The increasing threat posed by antibiotic resistance is compounded by an inadequate discovery rate of new antibiotics and has, in turn, resulted in global interest for alternative solutions. Recent studies have demonstrated that imbalances in skin microbiota are associated with assorted skin diseases and infections. Specifically, restoration of this ecosystem imbalance results in an alleviation of symptoms, achieved simply by applying bacteria normally found in abundance on healthy skin to the skin of those deficient in beneficial bacteria. The aim of this review is to discuss the currently available literature on biological tools that have the potential to manipulate the skin microbiota, with particular focus on bacteriocins, phage therapy, antibiotics, probiotics and targets of the gut-skin axis. This review will also address how the skin microbiota protects humans from invading pathogens in the external environment while discussing novel strategies to manipulate the skin microbiota to avoid and/or treat various disease states.

Encapsulated cyclosporine does not change the composition of the human microbiota when assessed ex vivo and in vivo.

Introduction. Management of steroid-refractory ulcerative colitis has predominantly involved treatment with systemic cyclosporine A (CyA) and infliximab.Aim. The purpose of this study was to assess the effect of using a colon-targeted delivery system CyA formulation on the composition and functionality of the gut microbiota.Methodology. Ex vivo faecal fermentations from six healthy control subjects were treated with coated minispheres (SmPill) with (+) or without (-) CyA and compared with a non-treated control in a model colon system. In addition, the in vivo effect of the SmPill+CyA formulation was investigated by analysing the gut microbiota in faecal samples collected before the administration of SmPill+CyA and after 7 consecutive days of administration from eight healthy subjects who participated in a pilot study.Results. Analysis of faecal samples by 16S rRNA gene sequencing indicated little variation in the diversity or relative abundance of the microbiota composition before or after treatment with SmPill minispheres with or without CyA ex vivo or with CyA in vivo. Short-chain fatty acid profiles were evaluated using gas chromatography, showing an increase in the concentration of n-butyrate (P=0.02) and acetate (P=0.32) in the faecal fermented samples incubated in the presence of SmPill minispheres with or without CyA. This indicated that increased acetate and butyrate production was attributed to a component of the coated minispheres rather than an effect of CyA on the microbiota. Butyrate and acetate levels also increased significantly (P=0.05 for both) in the faecal samples of healthy individuals following 7 days' treatment with SmPill+CyA in the pilot study.Conclusion. SmPill minispheres with or without CyA at the clinically relevant doses tested here have negligible direct effects on the gut microbiota composition. Butyrate and acetate production increased, however, in the presence of the beads in an ex vivo model system as well as in vivo in healthy subjects. Importantly, this study also demonstrates the relevance and value of using ex vivo colon models to predict the in vivo impact of colon-targeted drugs directly on the gut microbiota.

Effects of a polysaccharide-rich extract derived from Irish-sourced Laminaria digitata on the composition and metabolic activity of the human gut microbiota using an in vitro colonic model.

BACKGROUND: Brown seaweeds are known to be a rich source of fiber with the presence of several non-digestible polysaccharides including laminarin, fucoidan and alginate. These individual polysaccharides have previously been shown to favorably alter the gut microbiota composition and activity albeit the effect of the collective brown seaweed fiber component on the microbiota remains to be determined. METHODS: This study investigated the effect of a crude polysaccharide-rich extract obtained from Laminaria digitata (CE) and a depolymerized CE extract (DE) on the gut microbiota composition and metabolism using an in vitro fecal batch culture model though metagenomic compositional analysis using 16S rRNA FLX amplicon pyrosequencing and short-chain fatty acid (SCFA) analysis using GC-FID. RESULTS: Selective culture analysis showed no significant changes in cultured lactobacilli or bifidobacteria between the CE or DE and the cellulose-negative control at any time point measured (0, 5, 10, 24, 36, 48 h). Following metagenomic analysis, the CE and DE significantly altered the relative abundance of several families including Lachnospiraceae and genera including Streptococcus, Ruminococcus and Parabacteroides of human fecal bacterial populations in comparison to cellulose after 24 h. The concentrations of acetic acid, propionic acid, butyric acid and total SCFA were significantly higher for both the CE and DE compared to cellulose after 10, 24, 36 and 48 h fermentation (p < 0.05). Furthermore, the acetate:propionate ratio was significantly reduced (p < 0.05) for both CD and DE following 24, 36 and 48 h fermentation. CONCLUSION: The microbiota-associated metabolic and compositional changes noted provide initial indication of putative beneficial health benefits of L. digitata in vitro; however, research is needed to clarify if L. digitata-derived fiber can favorably alter the gut microbiota and confer health benefits in vivo.

Nisin J, a Novel Natural Nisin Variant, Is Produced by Staphylococcus capitis Sourced from the Human Skin Microbiota.

The skin microbiota is thought to play a key role in host protection from infection. Nisin J is a novel nisin variant produced by Staphylococcus capitis APC 2923, a strain isolated from the toe web space area in a screening study performed on the human skin microbiota. Whole-genome sequencing and mass spectrometry of the purified peptide confirmed that S. capitis APC 2923 produces a 3,458-Da bacteriocin, designated nisin J, which exhibited antimicrobial activity against a range of Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and Cutibacterium acnes The gene order in the nisin J gene cluster (nsjFEGBTCJP) differs from that of other nisin variants in that it is lacking the nisin regulatory genes, nisRK, as well as the nisin immunity gene nisI Nisin J has 9 amino acid changes compared to prototypical nisin A, with 8 amino acid substitutions, 6 of which are not present in other nisin variants (Ile4Lys, Met17Gln, Gly18Thr, Asn20Phe, Met21Ala, Ile30Gly, Val33His, and Lys34Thr), and an extra amino acid close to the C terminus, rendering nisin J the only nisin variant to contain 35 amino acids. This is the first report of a nisin variant produced by a Staphylococcus species and the first nisin producer isolated from human skin.IMPORTANCE This study describes the characterization of nisin J, the first example of a natural nisin variant, produced by a human skin isolate of staphylococcal origin. Nisin J displays inhibitory activity against a wide range of bacterial targets, including MRSA. This work demonstrates the potential of human commensals as a source for novel antimicrobials that could form part of the solution to antibiotic resistance across a broad range of bacterial pathogens.

A Live Bio-Therapeutic for Mastitis, Containing Lactococcus lactis DPC3147 With Comparable Efficacy to Antibiotic Treatment.

Bovine mastitis is an ongoing significant concern in the dairy and agricultural industry resulting in substantial losses in milk production and revenue. Among the predominant etiological agents of bovine mastitis are Staphylococcus aureus, Streptococcus uberis, Streptococcus dysgalactiae, and Escherichia coli. Currently, the treatment of choice for bovine mastitis involves the use of commercial therapeutic antibiotic formulations such as TerrexineTM, containing both kanamycin and cephalexin. Such antibiotics are regularly administered in more than one dose resulting in the withholding of milk for processing for a number of days. Here, we describe the optimization of a formulation of Lactococcus lactis DPC3147, that produces the two-component bacteriocin lacticin 3147, in a liquid paraffin-based emulsion (formulation hereafter designated 'live bio-therapeutic') for the first time and compare it to the commercial antibiotic formulation TerrexineTM, with a view to treating cows with clinical/sub-clinical mastitis. Critically, in a field trial described here, this 'ready-to-use' emulsion containing live L. lactis DPC3147 cells exhibited comparable efficacy to TerrexineTM when used to treat mastitic cows. Furthermore, we found that the L. lactis cells within this novel emulsion-based formulation remained viable for up to 5 weeks, when stored at 4, 22, or 37°C. The relative ease and cost-effective nature of producing this 'live bio-therapeutic' formulation, in addition to its enhanced shelf life compared to previous aqueous-based formulations, indicate that this product could be a viable alternative therapeutic option for bovine mastitis. Moreover, the single-dose administration of this 'live bio-therapeutic' formulation is a further advantage, as it can expedite the return of the milk to the milk pool, in comparison to some commercial antibiotics. Overall, in this field trial, we show that the live bio-therapeutic formulation displayed a 47% cure rate compared to a 50% cure rate for a commercial antibiotic control, with respect to curing cows with clinical/sub-clinical mastitis. The study suggests that a larger field trial to further demonstrate efficacy is warranted.

Draft Genome Sequence of Bacillus thuringiensis DPC6431, Producer of the Bacteriocin Thuricin CD.

We report the draft genome sequence of Bacillus thuringiensis DPC6431, a producer of the anticlostridial bacteriocin thuricin CD and isolated from a human fecal sample. The assembly comprises 96 contigs for a total of 5,581,839 bp, with 32.5% G+C content.

Lactobacillus gasseri APC 678 Reduces Shedding of the Pathogen Clostridium difficile in a Murine Model.

Clostridium difficile is a common cause of health-care acquired diarrhea, resulting in a spectrum of disease from mild diarrhea to life-threatening illness. Sixty Lactobacillus strains were screened for anti-C. difficile activity using a co-culture method. Based on their ability to inhibit C. difficile, L. gasseri APC 678 and L. rhamnosus DPC 6111 were selected for study in a murine model of C. difficile infection. L. gasseri ATCC 33323, was included as a control. It was established that, relative to control mice not fed Lactobacillus, feeding with L. gasseri APC 678 resulted in a significant reduction by day 7 (8-fold, p = 0.017) of viable C. difficile VPI 10463 in the feces of mice. In contrast, neither L. rhamnosus DPC 6111 nor L. gasseri ATCC 33323 significantly reduced fecal C. difficile shedding. Sequencing of the cecal microbiota showed that in mice fed L. gasseri APC 678 there was a significant increase in bacterial diversity across a number of indices when compared to the control or other Lactobacillus-fed groups. There was no significant change in the relative abundance of Firmicutes or Bacteroidetes in the group fed L. gasseri APC 678 relative to the control, while the groups fed L. rhamnosus DPC 6111 or L. gasseri ATCC 33323 showed a significant decrease in the relative abundance of Firmicutes (p = 0.002 and p = 0.019, respectively) and a significant increase in Bacteroidetes (p = 0.002 and p = 0.023, respectively). These results highlight the potential of L. gasseri APC 678 as a live therapeutic agent to target C. difficile infection.

Genus-Wide Assessment of Antibiotic Resistance in Lactobacillus spp.

Lactobacillus species are widely used as probiotics and starter cultures for a variety of foods, supported by a long history of safe usage. Although more than 35 species meet the European Food Safety Authority (EFSA) criteria for qualified presumption of safety status, the safety of Lactobacillus species and their carriage of antibiotic resistance (AR) genes is under continuing ad hoc review. To comprehensively update the identification of AR in the genus Lactobacillus, we determined the antibiotic susceptibility patterns of 182 Lactobacillus type strains and compared these phenotypes to their genotypes based on genome-wide annotations of AR genes. Resistances to trimethoprim, vancomycin, and kanamycin were the most common phenotypes. A combination of homology-based screening and manual annotation identified genes encoding resistance to aminoglycosides (20 sequences), tetracycline (18), erythromycin (6), clindamycin (60), and chloramphenicol (42). In particular, the genes aac(3) and lsa, involved in resistance to aminoglycosides and clindamycin, respectively, were found in Lactobacillus spp. Acquired determinants predicted to code for tetracycline and erythromycin resistance were detected in Lactobacillus ingluviei, Lactobacillus amylophilus, and Lactobacillus amylotrophicus, flanked in the genome by mobile genetic elements with potential for horizontal transfer.IMPORTANCELactobacillus species are generally considered to be nonpathogenic and are used in a wide variety of foods and products for humans and animals. However, many of the species examined in this study have antibiotic resistance levels which exceed those recommended by the EFSA, suggesting that these cutoff values should be reexamined in light of the genetic basis for resistance discussed here. Our data provide evidence for rationally revising the regulatory guidelines for safety assessment of lactobacilli entering the food chain as starter cultures, food preservatives, or probiotics and will facilitate comprehensive genotype-based assessment of strains for safety screening.

Protecting bactofencin A to enable its antimicrobial activity using mesoporous matrices.

There is huge global concern surrounding the emergence of antimicrobial resistant bacteria and this is resulting in an inability to treat infectious diseases. This is due to a lack of new antimicrobials coming to the market and irresponsible use of traditional antibiotics. Bactofencin A, a novel antimicrobial peptide which shows potential as an antibiotic, is susceptible to enzyme degradation. To improve its solution stability and inherent activity, bactofencin A was loaded onto a traditional silica mesoporous matrix, SBA-15, and a periodic mesoporous organosilane, MSE. The loading of bactofencin A was considerably higher onto SBA-15 than MSE due to the hydrophilic nature of SBA-15. While there was no detectable peptide released from SBA-15 into phosphate buffered saline and only 20% of the peptide loaded onto MSE was released, the loaded matrices showed enhanced activity compared to the free peptide during in vitro antimicrobial assays. In addition, the mesoporous matrices were found to protect bactofencin A against enzymatic degradation where results showed that the SBA-15 and MSE with loaded bactofencin A exposed to trypsin inhibited the growth of S. aureus while a large decrease in activity was observed for free bactofencin upon exposure to trypsin. Thus, the activity and stability of bactofencin A can be enhanced using mesoporous matrices and these matrices may enable its potential development as a novel antibiotic. This work also shows that in silico studies looking at surface functional group and size complementarity between the peptide and the protective matrix could enable the systemic selection of a mesoporous matrix for individual bacteriocins with potential antimicrobial therapeutic properties.

Human skin microbiota is a rich source of bacteriocin-producing staphylococci that kill human pathogens.

The demand for novel antimicrobial therapies due to the threat posed by antimicrobial resistance has resulted in a growing interest in the protective role of our skin bacteria and the importance of competition among bacteria on the skin. A survey of the cultivable bacteria on human skin was undertaken to identify the capacity of the skin microbiota to produce bacteriocins with activity against skin pathogens. Twenty-one bacteriocins produced by bacteria isolated from seven sites on the human body of each subject exhibited inhibition spectra ranging from broad to narrow range, inhibiting many Gram-positive bacteria, including opportunistic skin pathogens such as Propionibacterium acnes (recently renamed Cutibacterium acnes), Staphylococcus epidermidis and methicillin-resistant Staphylococcus aureus (MRSA). Sequencing indicated that the antimicrobial-producing isolates were predominately species/strains of the Staphylococcus genus. Colony mass spectrometry revealed peptide masses that do not correspond to known bacteriocins. In an era where antibiotic resistance is of major concern, the inhibitory effect of novel bacteriocins from the bacteria of skin origin demonstrates the antimicrobial potential that could be harnessed from within the human skin microbiota.

Delivery of a hydrophobic drug into the lower gastrointestinal system via an endogenous enzyme-mediated carrier mechanism: An in vitro study.

Clofazimine (CFZ) is a hydrophobic antibiotic agent which exhibits poor solubility. This poor solubility was overcome herein by the formulation of CFZ with the digestive enzyme pepsin. It is shown that pepsin can actively bind 11 CFZ molecules in the protein's native gastric environment, forming a CFZ-pepsin complex. A dynamic dissolution system, representing both the gastric and intestinal system, was used to analyze this CFZ-pepsin complex, revealing that only CFZ which binds to pepsin in the gastric environment remains in solution in the intestinal environment. The CFZ-pepsin complex displays adequate solution stability for the delivery of CFZ into the lower intestinal system. In vitro bioactivity assays against Clostridium difficile demonstrated the effectiveness of this CFZ-pepsin complex for the treatment of infectious diseases in the lower intestinal system.

Heterologous Expression of Biopreservative Bacteriocins With a View to Low Cost Production.

Bacteriocins, a heterogenous group of antibacterial ribosomally synthesized peptides, have potential as bio-preservatives in in a wide range of foods and as future therapeutics for the inhibition of antibiotic-resistant bacteria. While many bacteriocins have been characterized, several factors limit their production in large quantities, a requirement to make them commercially viable for food or pharma applications. The identification of new bacteriocins by database mining has been promising, but their potential is difficult to evaluate in the absence of suitable expression systems. E. coli has been used as a heterologous host to produce recombinant proteins for decades and has an extensive set of expression vectors and strains available. Here, we review the different expression systems for bacteriocin production using this host and identify the most important features to guarantee successful production of a range of bacteriocins.

The Use of a Mini-Bioreactor Fermentation System as a Reproducible, High-Throughput ex vivo Batch Model of the Distal Colon.

Ex vivo colon fermentation systems are highly versatile as models for analyzing gastrointestinal tract microbiota composition and functionality. Ex vivo colon models range in size and functionality from bench-top micro fermenters to large units housed in individualized cabinets. The length of set-up time (including stabilization periods) for each fermentation system can range from hours to weeks to months. The aim of this study was to investigate a single-use cassette mini-fermentation system as a reproducible batch model of the colon. The online data log from the cassettes (triplicate wells across four different cassettes, n = 12) was sensitive enough to identify real-time changes in pH, temperature, dissolved oxygen or liquid addition (sodium hydroxide) during the runs which could be addressed if an alarm set-point was triggered. The alpha diversity indices also showed little variation between cassettes with the samples clustering around the mean. The weighted beta diversity PCoA analysis illustrated that 95% of the variance between the samples was accounted for by the time-point and not the fermentation run/cassette used. The variation in taxonomic diversity between cassettes was limited to less than 20 out of 115 genera. This study provides evidence that micro-bioreactors provide some very attractive advantages as batch models for the human colon. We show for the first time the use of the micro-Matrix a 24-well sophisticated parallel controlled cassette-based bioreactors as a batch colon model. We demonstrated a high level of reproducibility across fermentation cassettes when used in conjunction with a standardized fecal microbiota. The machine can operate 24 individual fermentations simultaneously and are relatively cost effective. Based on next generation sequencing analysis, the micro-bioreactors offer a high degree of reproducibility together with high-throughput capacity. This makes it a potential system for large screening projects that can then be scaled up to large fermenters or human/animal in vivo experiments.