Elevated systemic antibodies towards commensal gut microbiota in autoinflammatory condition

Background

Familial Mediterranean fever (FMF) is an autoinflammatory condition, which is characterized by acute, self-limiting episodes of fever and serositis and chronic subclinical inflammation in remission. Here we investigated the consequence of this condition on the level of systemic antibodies directed towards common intestinal bacteria.

Methodology/Principal Findings

The level of systemic antibodies towards the antigens of Bacteroides, Parabacteroides, Escherichia, Enteroccocus and Lactobaccilus was measured by ELISA in FMF patients at various stages of the disease and in healthy controls. The difference between remission and attack was not significant. IgG antibodies against the antigens of Bacteroides, Parabacteroides, Escherichia and Enteroccocus were significantly increased in FMF compared to control while IgA levels were not significantly affected. Western blot analyses demonstrated the IgG reactivity against multiple antigens of commensal bacteria in FMF. Serological expression cloning was performed to identify these antigens. No single dominant antigen was identified; the response was generalized and directed against a variety of proteins from Bacteroides, Parabacteroides, Escherichia, and other gut commensals.

Conclusions/Significance

This autoinflammatory syndrome is characterized by the increased systemic reactivity against commensal gut microbiota. This is probably the consequence of hypersensitivity of the inflammasome in FMF that triggers the inflammation and contributes to the excessive translocation of bacteria and bacterial antigens through the gut barrier.     

A flow cytometric assay to monitor antimicrobial activity of defensins and cationic tissue extracts

To determine the antibacterial activity of defensins and other antimicrobial peptides in biopsy extracts, we evaluated a flow cytometric method with the membrane potential sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. This assay enables us to discriminate intact non-fluorescent and depolarized fluorescent bacteria after exposure to antimicrobial peptides by measurement at the direct target, the cytoplasmic membrane and the membrane potential. The feasibility of the flow cytometric assay was evaluated with recombinant human beta-defensin 3 (HBD-3) against 25 bacterial strains representing 12 species. HBD-3 showed a broad-spectrum dose dependent activity and the minimal dose to cause depolarization ranged from 1.25 to >15 microg/ml HBD-3, depending on the species tested. The antibacterial effect was diminished with sodium chloride or dithiothreitol and could be abrogated with a HBD-3 antibody. Additionally, isolated cationic extracts from human intestinal biopsies showed a strong bactericidal effect against Escherichia coli K12, E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923, which was diminished towards E. coli at 150 mM NaCl, whereas the activity towards S. aureus ATCC 25923 remained unaffected at physiological salt concentrations. DTT blocked the bactericidal effect of biopsy extracts completely.     

Ferritin-like family proteins in the anaerobe Bacteroides fragilis: when an oxygen storm is coming, take your iron to the shelter

Bacteroides are gram-negative anaerobes and one of the most abundant members the lower GI tract microflora where they play an important role in normal intestinal physiology. Disruption of this commensal relationship has a great impact on human health and disease. Bacteroides spp. are significant opportunistic pathogens causing infections when the mucosal barrier integrity is disrupted following predisposing conditions such as GI surgery, perforated or gangrenous appendicitis, perforated ulcer, diverticulitis, trauma and inflammatory bowel diseases. B. fragilis accounts for 60–90 % of all anaerobic infections despite being a minor component of the genus (<1 % of the flora). Clinical strains of B. fragilis are among the most aerotolerant anaerobes. When shifted from anaerobic to aerobic conditions B. fragilis responds to oxidative stress by inducing the expression of an extensive set of genes involved in protection against oxygen derived radicals and iron homeostasis. In Bacteroides, little is known about the metal/oxidative stress interactions and the mobilization of intra-cellular non-heme iron during the oxidative stress response has been largely overlooked. Here we present an overview of the work carried out to demonstrate that both oxygen-detoxifying enzymes and iron-storage proteins are essential for B. fragilis to survive an adverse oxygen-rich environment. Some species of Bacteroides have acquired multiple homologues of the iron storage and detoxifying ferritin-like proteins but some species contain none. The proteins found in Bacteroides are classical mammalian H-type non-heme ferritin (FtnA), non-specific DNA binding and starvation protein (Dps) and the newly characterized bacterial Dps-Like miniferritin protein. The full contribution of ferritin-like proteins to pathophysiology of commensal and opportunistic pathogen Bacteroides spp. still remains to be elucidated.     

Expression of 4kD scorpion defensin and its in vitro synergistic activity with conventional antibiotics

The 4kD scorpion defensin (SD) is a potent disulfide-linked peptide. In this study, we expressed it in methylotrophic yeast Pichia pastoris and purified it using Ni–NTA His Bind Resin. We investigated its in vitro antibacterial activity and effect in combination with several conventional antibiotics. We first examined its antibacterial activity towards several Gram-positive and Gram-negative bacteria. Then we used the broth microdilution method to test drugs alone and in combination and used the fractional inhibitory concentration (FIC index) to classify the drug interactions. Our study showed the expressed SD peptide has antibacterial activity against Salmonella typhimurium, E. coli and S. aureus etc. Synergy or additive interaction was observed between SD and Norfloxacin, Polymyxin B and Ampicillin. Cell growth tests showed that combination of SD and Norfloxacin can improve their activity against bacteria. This result maybe permit lower using of the conventional antibiotic agents more effectively and safely.     

A novel insect defensin mediates the inducible antibacterial activity in larvae of the dragonfly Aeschna cyanea (Paleoptera, Odonata).

The injection of low doses of bacteria into the aquatic larvae of dragonflies (Aeschna cyanea, Odonata, Paleoptera) induces the appearance in their hemolymph of a potent antibacterial activity. We have isolated a 38-residue peptide from this hemolymph which is strongly active against Gram-positive bacteria and also shows activity against one of the Gram-negative bacteria which was tested. The peptide is a novel member of the insect defensin family of inducible antibacterial peptides, which had so far only been reported from the higher insect orders believed to have evolved 100 million years after the Paleoptera. Aeschna defensin is more potent than defensin from the dipteran Phormia, from which its structure differs in several interesting aspects, which are discussed in the paper.     

Bacterial Population Adherent to the Epithelium on the Roo of the Dorsal Rumen of Sheep

By anaerobic procedures, the total number of adherent bacteria was determined on tissue samples obtained from the roof of the dorsal rumen of three sheep. After four washings, 1.91 × 10⁷, 0.34 × 10⁷, and 1.23 × 10⁷ bacteria per cm² were still attached to the rumen epithelium in sheep 1, 2, and 3, respectively. A total of 95 strains of bacteria were isolated from these three samples. Based on morphology, Gram stain, anaerobiosis, motility, and fermentation end products, they were presumptively identified as follows: Butyrivibrio fibrisolvens, 30 strains; atypical Butyrivibrio, 5 strains; Bacteroides ruminicola, 22 strains; Lactobacillus, 1 strain; and unknown Bacteroides species, 37 strains. For sheep 3, washing the rumen epithelium a total of 10 times reduced the adherent bacterial population by 93% (8.4 × 10⁵ bacteria per cm²). Of 30 strains isolated from this sample, 22 were presumptively identified as Butyrivibrio and Bacteroides types. These results suggest that the epithelium on the roof of the dorsal rumen is primarily colonized by two genera of bacteria, Butyrivibrio and Bacteroides. Most Butyrivibrio and Bacteroides ruminicola strains appeared to be similar to previously isolated rumen strains. However, the unknown Bacteroides species differed considerably from the three species of this genus which are commonly isolated from rumen contents.     

Characteristics of Bacterial Isolates from the Gut of Freshwater Fish, Labeo rohita that May be Useful as Potential Probiotic Bacteria

In this study, we aimed to evaluate the in vitro probiotic characteristics of three bacteria, Lactobacillus plantarum VSG3, Pseudomonas aeruginosa VSG2, and Bacillus subtilis VSG1, isolated from the gut of Labeo rohita. The bacterial isolates tolerated low pH and high bile concentrations in the fish well. The bacterial adhesion capacity to fish intestinal mucosa revealed that the three potential probiotic isolates had a significantly higher adhesion capacity compared to the pathogenic strains tested. L. plantarum VSG3 exhibited the best adhesion capacity (19.1?%) to the intestinal mucosa. Among the isolates, L. plantarum VSG3 and P. aeruginosa VSG2 showed strong antibacterial activities against fish pathogens as measured in spent culture liquids. Moreover, all the isolates were susceptible to each tested antibiotic, which ensured their inability to exhibit antibiotic-resistance properties. Considering these promising results, selected strains should be further studied to determine their probiotic effects in vivo in fish.     

Induction of human defensins by intestinal Caco-2 cells after interactions with opportunistic Candida species

In this study we investigated the effects of Candida albicans, Candida krusei, Candida tropicalis and Candida parapsilosis on human beta-defensin 2 (HBD-2) production in Caco-2 intestinal cell line, and the production of alpha-defensins (human neutrophil peptides, HNP 1–3) in peripheral blood. Opportunistic pathogen yeasts can modulate the host immune function by inducing defensins, the natural antimicrobial peptides. Here we show that Candida spp. stimulated HBD-2 expression in and release from Caco-2 cells, with C. albicans inducing the highest levels of HBD-2. Similarly, HNP 1–3 secretion was significantly increased in whole blood after exposure to Candida yeast cells, with C. albicans producing the greatest effect. Our investigations underscore the important role of beta and alpha defensins produced by intestinal epithelial cells locally and neutrophils systemically in the antifungal defense against Candida.     

Alterations of the Ileal and Colonic Mucosal Microbiota in Canine Chronic Enteropathies

Background

The intestinal microbiota is increasingly linked to the pathogenesis of chronic enteropathies (CE) in dogs. While imbalances in duodenal and fecal microbial communities have been associated with mucosal inflammation, relatively little is known about alterations in mucosal bacteria seen with CE involving the ileum and colon.

Aim

To investigate the composition and spatial organization of mucosal microbiota in dogs with CE and controls.

Methods

Tissue sections from endoscopic biopsies of the ileum and colon from 19 dogs with inflammatory bowel disease (IBD), 6 dogs with granulomatous colitis (GC), 12 dogs with intestinal neoplasia, and 15 controls were studied by fluorescence in situ hybridization (FISH) on a quantifiable basis.

Results

The ileal and colonic mucosa of healthy dogs and dogs with CE is predominantly colonized by bacteria localized to free and adherent mucus compartments. CE dogs harbored more (P < 0.05) mucosal bacteria belonging to the Clostridium-coccoides/Eubacterium rectale group, Bacteroides, Enterobacteriaceae, and Escherichia coli versus controls. Within the CE group, IBD dogs had increased (P < 0.05) Enterobacteriaceae and E. coli bacteria attached onto surface epithelia or invading within the intestinal mucosa. Bacterial invasion with E. coli was observed in the ileal and colonic mucosa of dogs with GC (P < 0.05). Dogs with intestinal neoplasia had increased (P < 0.05) adherent (total bacteria, Enterobacteriaceae, E. coli) and invasive (Enterobacteriaceae, E. coli, and Bacteroides) bacteria in biopsy specimens. Increased numbers of total bacteria adherent to the colonic mucosa were associated with clinical disease severity in IBD dogs (P < 0.05).

Conclusion

Pathogenic events in canine CE are associated with different populations of the ileal and colonic mucosal microbiota.     

Intestinal inflammation alters mucosal carbohydrate foraging and monosaccharide incorporation into microbial glycans

Endogenous carbohydrates released from the intestinal mucus represent a constant source of nutrients to the intestinal microbiota. Mucus‐derived carbohydrates can also be used as building blocks in the biosynthesis of bacterial cell wall components, thereby influencing host mucosal immunity. To assess the uptake of endogenous carbohydrates by gut microbes in healthy mice and during intestinal inflammation, we applied azido‐monosaccharides that can be tracked on bacterial cell walls after conjugation with fluorophores. In interleukin‐10 deficient mice, changes in the gut microbiota were accompanied by decreased carbohydrate hydrolase activities and increased lumenal concentrations of host glycan‐derived monosaccharides. Tracking of the monosaccharide N‐azidoacetylglucosamine (GlcNAz) in caecum bacteria revealed a preferential incorporation of this carbohydrate by Xanthomonadaceae in healthy mice and by Bacteroidaceae in interleukin‐10 deficient mice. These GlcNAz‐positive Bacteroidaceae fractions mainly belonged to the species B. acidifaciens and B. vulgatus. Growth of Bacteroides species in the presence of specific monosaccharides changed their stimulatory activity toward CD11c⁺ dendritic cells. Expression of activation markers and cytokine production was highest after stimulation of dendritic cells with B. vulgatus. The variable incorporation of monosaccharides by related Bacteroides species underline the necessity to investigate intestinal bacteria down to the species level when addressing microbiota‐host interactions.     

Novel strategies of essential oils,chitosan, and nano- chitosan for inhibition of multi-drug resistant: E. coli O157:H7 and Listeria monocytogenes

Despite the wide range of available antibiotics, food borne bacteria demonstrate a huge spectrum of resistance. The current study aims to use natural components such as essential oils (EOs), chitosan, and nano-chitosan that have very influential antibacterial properties with novel technologies like chitosan solution/film loaded with EOs against multi-drug resistant bacteria. Two strains of Escherichia coli O157:H7 and three strains of Listeria monocytogenes were used to estimate antibiotics resistance. Ten EOs and their mixture, chitosan, nano-chitosan, chitosan plus EO solutions, and biodegradable chitosan film enriched with EOs were tested as antibacterial agents against pathogenic bacterial strains. Results showed that E. coli O157:H7 51,659 and L. monocytogenes 19,116 relatively exhibited considerable resistance to more than one single antibiotic. Turmeric, cumin, pepper black, and marjoram did not show any inhibition zone against L. monocytogenes; Whereas, clove, thyme, cinnamon, and garlic EOs exhibited high antibacterial activity against L. monocytogenes with minimum inhibitory concentration (MIC) of 250–400 μl 100^?1 ml and against E. coli O157:H7 with an MIC of 350–500 μl 100^?1 ml, respectively. Among combinations, clove, and thyme EOs showed the highest antibacterial activity against E. coli O157:H7 with MIC of 170 μl 100^?1 ml, and the combination of cinnamon and clove EOs showed the strongest antibacterial activity against L. monocytogenes with an MIC of 120 μl 100^?1 ml. Both chitosan and nano-chitosan showed a promising potential as an antibacterial agent against pathogenic bacteria as their MICs were relatively lower against L. monocytogenes than for E. coli O157:H7. Chitosan combined with each of cinnamon, clove, and thyme oil have a more effective antibacterial activity against L. monocytogenes and E. coli O157:H7 than the mixture of oils alone. Furthermore, the use of either chitosan solution or biodegradable chitosan film loaded with a combination of clove and thyme EOs had the strongest antibacterial activity against L. monocytogenes and E. coli O157:H7. However, chitosan film without EOs did not exhibit an inhibition zone against the tested bacterial strains.     

Fish oil enhances recovery of intestinal microbiota and epithelial integrity in chronic rejection of intestinal transplant

Background

The intestinal chronic rejection (CR) is the major limitation to long-term survival of transplanted organs. This study aimed to investigate the interaction between intestinal microbiota and epithelial integrity in chronic rejection of intestinal transplantation, and to find out whether fish oil enhances recovery of intestinal microbiota and epithelial integrity.

Methods/Principal Findings

The luminal and mucosal microbiota composition of CR rats were characterized by DGGE analysis at 190 days after intestinal transplant. The specific bacterial species were determined by sequence analysis. Furthermore, changes in the localization of intestinal TJ proteins were examined by immunofluorescent staining. PCR-DGGE analysis revealed that gut microbiota in CR rats had a shift towards Escherichia coli, Bacteroides spp and Clostridium spp and a decrease in the abundance of Lactobacillales bacteria in the intestines. Fish oil supplementation could enhance the recovery of gut microbiota, showing a significant decrease of gut bacterial proportions of E. coli and Bacteroides spp and an increase of Lactobacillales spp. In addition, CR rats showed pronounced alteration of tight junction, depicted by marked changes in epithelial cell ultrastructure and redistribution of occuldin and claudins as well as disruption in TJ barrier function. Fish oil administration ameliorated disruption of epithelial integrity in CR, which was associated with an improvement of the mucosal structure leading to improved tight junctions.

Conclusions/Significance

Our study have presented novel evidence that fish oil is involved in the maintenance of epithelial TJ integrity and recovery of gut microbiota, which may have therapeutic potential against CR in intestinal transplantation.     

Antimicrobial activity of human α‐defensin 6 analogs: insights into the physico‐chemical reasons behind weak bactericidal activity of HD6 in vitro

Human α‐defensin 6 (HD6), unlike other mammalian defensins, does not exhibit bactericidal activity, particularly against aerobic bacteria. Monomeric HD6 has a tertiary structure similar to other α‐defensins in the crystalline state. However, the physico‐chemical reasons behind the lack of antibacterial activity of HD6 are yet to be established unequivocally. In this study, we have investigated the antimicrobial activity of HD6 analogs. A linear analog of HD6, in which the distribution of arginine residues was similar to active α‐defensins, shows broad‐spectrum antimicrobial activity, indicating that atypical distribution of arginine residues contributes to the inactivity of HD6. Peptides spanning the N‐terminal cationic segment were active against a wide range of organisms. Antimicrobial potency of these shorter analogs was further enhanced when myristic acid was conjugated at the N‐terminus. Cytoplasmic localization of the analogs without fatty acylation was observed to be necessary for bacterial killing, while they exhibited fungicidal activity by permeabilizing Candida albicans membranes. Myristoylated analogs and the linear full‐length arginine analog exhibited activity by permeabilizing bacterial and fungal membranes. Our study provides insights into the lack of bactericidal activity of HD6 against aerobic bacteria. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Decrease in anaerobe-related bacteraemias and increase in Bacteroides species isolation rate from 1998 to 2007: A retrospective study

Conflicting data have accumulated in recent years regarding the incidence of anaerobic bacteraemias. The aim of this study was to determine the prevalence of bacteraemias due to anaerobic bacteria and evaluate the importance of anaerobic blood cultures in a university hospital in Israel. A retrospective survey which focused on anaerobic blood culture bottles was performed on blood cultures received in our laboratory during the decade from January 1998 to December 2007. Anaerobic-related bacteraemias decreased during that period, whereas a significant increase was observed in Bacteroides species isolated from the blood cultures (from 18% during 1998–2002 to 43% during 2003–2007). Comparison of the medical records of 54 patients with Bacteroides-related bacteraemia during the two end periods (1998–1999 and 2006–2007) revealed a marked increase in complex underlying diseases. Hypertension and diabetes mellitus type II were found in 29% of the patients in 1998–1999 and increased to 43–45% of the patients in 2006–2007. Ischemic heart disease also increased from 14% of the patients in 1998–1999 to 43% in 2006–2007. We conclude that although positive anaerobic blood cultures account for a small percentage of positive blood samples, the growing involvement of Bacteroides species-related bacteraemias together with an increase in complex underlying diseases in these patients emphasize the importance of anaerobic blood cultures, particularly in patients with co-morbidities.     

Antibiotic-resistant bacteria in the guts of insects feeding on plants: prospects for discovering plant-derived antibiotics

Background

Although plants produce many secondary metabolites, currently none of these are commercial antibiotics. Insects feeding on specific plants can harbour bacterial strains resistant to known antibiotics suggesting that compounds in the plant have stimulated resistance development. We sought to determine whether the occurrence of antibiotic-resistant bacteria in insect guts was a widespread phenomenon, and whether this could be used as a part of a strategy to identify antibacterial compounds from plants.

Results

Six insect/plant pairs were selected and the insect gut bacteria were identified and assessed for antibiotic susceptibilities compared with type strains from culture collections. We found that the gut strains could be more or less susceptible to antibiotics than the type strains, or show no differences. Evidence of antibacterial activity was found in the plant extracts from five of the six plants, and, in one case Catharanthus roseus (Madagascar Periwinkle), compounds with antibacterial activity were identified.

Conclusion

Bacterial strains isolated from insect guts show a range of susceptibilities to antibiotics suggesting a complex interplay between species in the insect gut microbiome. Extracts from selected plants can show antibacterial activity but it is not easy to isolate and identify the active components. We found that vindoline, present in Madagascar Periwinkle extracts, possessed moderate antibacterial activity. We suggest that plant-derived antibiotics are a realistic possibility given the advances in genomic and metabolomic methodologies.

     

Discovery of new human beta-defensins using a genomics-based approach

Epithelial beta-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a <1 Mb region of chromosome 8p22-p23. To identify new defensin genes, the DNA sequence from a contig of large-insert genomic clones from the region containing human beta-defensin-2 (HBD-2) was analyzed for the presence of defensin genes. This sequence survey identified a novel beta-defensin, termed HBD-3. The HBD-3 gene contains two exons, is located 13 kb upstream from the HBD-2 gene, and it is transcribed in the same direction. A partial HBD-3 cDNA clone was amplified from cDNA derived from IL-1beta induced fetal lung tissue. The cDNA sequence encodes for a 67 amino acid peptide that is approximately 43% identical to HBD-2 and shares the beta-defensin six cysteine motif. By PCR analysis of two commercial cDNA panels, HBD-3 expression was detected in adult heart, skeletal muscle, placenta and in fetal thymus. From RT-PCR experiments, HBD-3 expression was observed in skin, esophagus, gingival keratinocytes, placenta and trachea. Furthermore, in fetal lung explants and gingival keratinocytes, HBD-3 mRNA expression was induced by IL-1beta. Additional sequence analysis identified the HE2 (human epididymis secretory protein) gene 17 kb upstream from the HBD-3 gene. One splice variant of this gene (HE2beta1) encodes a beta-defensin consensus cysteine motif, suggesting it represents a defensin gene product. HE2beta1 mRNA expression was detected in gingival keratinocytes and bronchial epithelia using RT-PCR analysis. The discovery of these novel beta-defensin genes may allow further understanding of the role of defensins in host immunity at mucosal surfaces.     

A Defensin from the Model Beetle Tribolium castaneum Acts Synergistically with Telavancin and Daptomycin against Multidrug Resistant Staphylococcus aureus

The red flour beetle Tribolium castaneum is a common insect pest and has been established as a model beetle to study insect development and immunity. This study demonstrates that defensin 1 from T. castaneum displays in vitro and in vivo antimicrobial activity against drug resistant Staphylococcus aureus strains. The minimum inhibitory concentration (MIC) of defensin 1 against 11 reference and clinical staphylococcal isolates was between 16–64 μg/ml. The putative mode of action of the defensin peptide is disruption of the bacterial cell membrane. The antibacterial activity of defensin 1 was attenuated by salt concentrations of 1.56 mM and 25 mM for NaCl and CaCl₂ respectively. Treatment of defensin 1 with the reducing agent dithiothreitol (DTT) at concentrations 1.56 to 3.13 mM abolished the antimicrobial activity of the peptide. In the presence of subinhibitory concentrations of antibiotics that also target the bacterial cell envelope such as telavancin and daptomycin, the MIC of the peptide was as low as 1 μg/ml. Moreover, when tested against an S. aureus strain that was defective in D-alanylation of the cell wall, the MIC of the peptide was 0.5 μg/ml. Defensin 1 exhibited no toxicity against human erythrocytes even at 400 μg/ml. The in vivo activity of the peptide was validated in a Caenorhabditis elegans-MRSA liquid infection assay. These results suggest that defensin 1 behaves similarly to other cationic AMPs in its mode of action against S. aureus and that the activity of the peptide can be enhanced in combination with other antibiotics with similar modes of action or with compounds that have the ability to decrease D-alanylation of the bacterial cell wall.