Detection of Clostridium perfringens toxin genes in the gut microbiota of autistic children.

We studied stool specimens from 33 autistic children aged 2-9 years with gastrointestinal (GI) abnormalities and 13 control children without autism and without GI symptoms. We performed quantitative comparison of all Clostridium species and Clostridium perfringens strains from the fecal microbiota by conventional, selective anaerobic culture methods. We isolated C. perfringens strains and performed PCR analysis for the main C. perfringens toxin genes, alpha, beta, beta2, epsilon, iota and C. perfringens enterotoxin gene. Our results indicate that autistic subjects with gastrointestinal disease harbor statistically significantly (p = 0.031) higher counts of C. perfringens in their gut compared to control children. Autistic subjects also harbor statistically significantly (p = 0.015) higher counts of beta2-toxin gene-producing C. perfringens in their gut compared to control children, and the incidence of beta2-toxin gene-producing C. perfringens is significantly higher in autistic subjects compared to control children (p = 0.014). Alpha toxin gene was detected in all C. perfringens strains studied. C. perfringens enterotoxin gene was detected from three autistic and one control subject. Beta, epsilon, and iota toxin genes were not detected from autistic or control subjects.

Antimicrobial Activity of Pomegranate and Green Tea Extract on Propionibacterium Acnes, Propionibacterium Granulosum, Staphylococcus Aureus and Staphylococcus Epidermidis.

We used pomegranate extract (POMx), pomegranate juice (POM juice) and green tea extract (GT) to establish in vitro activities against bacteria implicated in the pathogenesis of acne. Minimum inhibitory concentrations (MIC) of 94 Propionibacterium acnes, Propionibacterium granulosum, Staphylococcus aureus, and Staphylococcus epidermidis strains were determined by Clinical and Laboratory Standards Institute-approved agar dilution technique. Total phenolics content of the phytochemicals was determined using the Folin-Ciocalteu method and the polyphenol composition by HPLC. Bacteria were identified by 16S rRNA sequence analysis. GT MIC of 400 µg/ml or less was obtained for 98% of the strains tested. 64% of P. acnes strains had POMx MICs at 50 µg/ml whereas 36% had MIC >400 µg/ml. POMx, POM juice, and GT showed inhibitory activity against all the P. granulosum strains at ≤100 µg/ml. POMx and GT inhibited all the S. aureus strains at 400 µg/ml or below, and POM juice had an MIC of 200 µg/ml against 17 S. aureus strains. POMx inhibited S. epidermidis strains at 25 µg/ml, whereas POM juice MICs were ≥200 µg/ml. The antibacterial properties of POMx and GT on the most common bacteria associated with the development and progression of acne suggest that these extracts may offer a better preventative/therapeutic regimen with fewer side effects than those currently available.

Endogenous cholinergic tone modulates spontaneous network level neuronal activity in primary cortical cultures grown on multi-electrode arrays.

BACKGROUND: Cortical cultures grown long-term on multi-electrode arrays (MEAs) are frequently and extensively used as models of cortical networks in studies of neuronal firing activity, neuropharmacology, toxicology and mechanisms underlying synaptic plasticity. However, in contrast to the predominantly asynchronous neuronal firing activity exhibited by intact cortex, electrophysiological activity of mature cortical cultures is dominated by spontaneous epileptiform-like global burst events which hinders their effective use in network-level studies, particularly for neurally-controlled animat ('artificial animal') applications. Thus, the identification of culture features that can be exploited to produce neuronal activity more representative of that seen in vivo could increase the utility and relevance of studies that employ these preparations. Acetylcholine has a recognised neuromodulatory role affecting excitability, rhythmicity, plasticity and information flow in vivo although its endogenous production by cortical cultures and subsequent functional influence upon neuronal excitability remains unknown. RESULTS: Consequently, using MEA electrophysiological recording supported by immunohistochemical and RT-qPCR methods, we demonstrate for the first time, the presence of intrinsic cholinergic neurons and significant, endogenous cholinergic tone in cortical cultures with a characterisation of the muscarinic and nicotinic components that underlie modulation of spontaneous neuronal activity. We found that tonic muscarinic ACh receptor (mAChR) activation affects global excitability and burst event regularity in a culture age-dependent manner whilst, in contrast, tonic nicotinic ACh receptor (nAChR) activation can modulate burst duration and the proportion of spikes occurring within bursts in a spatio-temporal fashion. CONCLUSIONS: We suggest that the presence of significant endogenous cholinergic tone in cortical cultures and the comparability of its modulatory effects to those seen in intact brain tissues support emerging, exploitable commonalities between in vivo and in vitro preparations. We conclude that experimental manipulation of endogenous cholinergic tone could offer a novel opportunity to improve the use of cortical cultures for studies of network-level mechanisms in a manner that remains largely consistent with its functional role.

Emergence of a small-world functional network in cultured neurons.

The functional networks of cultured neurons exhibit complex network properties similar to those found in vivo. Starting from random seeding, cultures undergo significant reorganization during the initial period in vitro, yet despite providing an ideal platform for observing developmental changes in neuronal connectivity, little is known about how a complex functional network evolves from isolated neurons. In the present study, evolution of functional connectivity was estimated from correlations of spontaneous activity. Network properties were quantified using complex measures from graph theory and used to compare cultures at different stages of development during the first 5 weeks in vitro. Networks obtained from young cultures (14 days in vitro) exhibited a random topology, which evolved to a small-world topology during maturation. The topology change was accompanied by an increased presence of highly connected areas (hubs) and network efficiency increased with age. The small-world topology balances integration of network areas with segregation of specialized processing units. The emergence of such network structure in cultured neurons, despite a lack of external input, points to complex intrinsic biological mechanisms. Moreover, the functional network of cultures at mature ages is efficient and highly suited to complex processing tasks.

Fretibacterium fastidiosum gen. nov., sp. nov., isolated from the human oral cavity.

SGP1(T), a strain belonging to a lineage of the phylum Synergistetes with no previously cultivated representatives was subjected to a comprehensive range of phenotypic and genotypic tests. For good growth the strain was dependent on co-culture with, or extracts from, selected other oral bacteria. Cells of strain SGP1(T) were asaccharolytic and major amounts of acetic acid and moderate amounts of propionic acid were produced as end products of metabolism in peptone-yeast extract-glucose broth supplemented with a filtered cell sonicate of Fusobacterium nucleatum subsp. nucleatum ATCC 25586(T) (25 %, v/v). Hydrogen sulphide was produced and gelatin was weakly hydrolysed. The major cellular fatty acids were C(14 : 0), C(18 : 0) and C(16 : 0). The DNA G+C content of strain SGP1(T) was 63 mol%. Phylogenetic analysis of the full-length 16S rRNA gene showed that strain SGP1(T) represented a novel group within the phylum Synergistetes. A novel species in a new genus, Fretibacterium fastidiosum gen. nov., sp. nov., is proposed. The type strain of Fretibacterium fastidiosum is SGP1(T) ( = DSM 25557(T) = JCM 16858(T)).

Description of Alloprevotella rava gen. nov., sp. nov., isolated from the human oral cavity, and reclassification of Prevotella tannerae Moore et al. 1994 as Alloprevotella tannerae gen. nov., comb. nov.

Five strains of anaerobic, gram-negative bacilli isolated from the human oral cavity were subjected to a comprehensive range of phenotypic and genotypic tests and were found to comprise a homogeneous group. Phylogenetic analysis of full-length 16S rRNA gene sequences showed that these strains represented a novel group within the family Prevotellaceae, and the most closely related species was Prevotella tannerae. P. tannerae and the novel taxon are deeply branched from the genus Prevotella, with sequence identities to the type strain of the type species of Prevotella, Prevotella melaninogenica, of 82.2 and 85.6 %, respectively. The novel genus Alloprevotella gen. nov. is proposed to accommodate the novel species Alloprevotella rava gen. nov., sp. nov. and the previously named Prevotella tannerae Moore et al. 1994 as Alloprevotella tannerae gen. nov., comb. nov. The type species is Alloprevotella tannerae. The type strain of Alloprevotella rava is 81/4-12(T) ( = DSM 22548(T)  = CCUG 58091(T)) and the type strain of Alloprevotella tannerae is ATCC 51259(T)  = CCUG 34292(T)  = CIP 104476(T)  = NCTC 13073(T). Alloprevotella rava is weakly to moderately saccharolytic and produces moderate amounts of acetic acid and major amounts of succinic acid as end products of fermentation. Strains are sensitive to 20 % bile and hydrolyse gelatin. The principal cellular long-chain fatty acids are anteiso-C15 : 0, iso-C15 : 0, C16 : 0, iso-C17 : 0 and iso-C17 : 0 3-OH. The G+C content of the DNA of the type strain is 47 mol%.

Microbiology of regressive autism.

This manuscript summarizes some of our earlier work on the microbiology of autism subjects' stool specimens, as compared with stools from control subjects. Our most recent data indicating that Desulfovibrio may play an important role in regressive autism is also presented. In addition, we present information on antimicrobial susceptibility patterns of Desulfovibrio using the CLSI agar dilution susceptibility technique. In addition, we summarize data from our earlier studies showing the impact of various antimicrobial agents on the indigenous bowel flora. This shows that penicillins and cephalosporins, as well as clindamycin, have a major impact on the normal bowel flora and therefore might well predispose subjects to overgrowth of such organisms as Clostridium difficile, and of particular importance for autism, to Desulfovibrio.

Prevotella fusca sp. nov. and Prevotella scopos sp. nov., isolated from the human oral cavity.

Two strains of anaerobic, Gram-negative bacilli isolated from the human oral cavity were subjected to a comprehensive range of phenotypic and genotypic tests and were found to belong to two separate taxa. Phylogenetic analysis of full-length 16S rRNA gene sequences showed that the strains were both related to, but distinct from, the type strain of Prevotella melaninogenica. Two novel species, Prevotella fusca sp. nov. and Prevotella scopos sp. nov., are proposed to accommodate these strains. Both strains were saccharolytic and produced acetic and succinic acids, with lesser amounts of lactic and isovaleric acids, as end products of fermentation, and both were sensitive to 20 % bile. The principal cellular long-chain fatty acids of both strains were ai-C(15 : 0), 3-OH i-C(17 : 0), 3-OH C(16 : 0), i-C(15 : 0) and C(16 : 0). The DNA G+C contents of the type strains of Prevotella fusca (W1435(T)  = DSM 22504(T)  = CCUG 57946(T)) and Prevotella scopos (W2052(T)  = DSM 22613(T ) = CCUG 57945(T)) were 43 and 41 mol%, respectively. The two species could be differentiated by gelatin hydrolysis, cellobiose and ribose fermentation, and production of ß-glucosidase.

Scardovia wiggsiae sp. nov., isolated from the human oral cavity and clinical material, and emended descriptions of the genus Scardovia and Scardovia inopinata.

Six strains of anaerobic, pleomorphic Gram-positive bacilli, isolated from the human oral cavity and an infected arm wound, were subjected to a comprehensive range of phenotypic and genotypic tests and were found to comprise a homogeneous group. 16S rRNA gene sequence analysis revealed that the isolates were most closely related to Scardovia inopinata CCUG 35729(T) (94.8-94.9 % 16S rRNA gene sequence similarity). The isolates were saccharolytic and produced acetic and lactic acids as end products of fermentation. The major fatty acids were C(16 : 0) (49.8 %) and C(18 : 1)ω9c (35.8 %). Polar lipid analysis revealed a variety of glycolipids, diphosphatidylglycerol, an unidentified phospholipid and an unidentified phosphoglycolipid. No respiratory quinones were detected. The peptidoglycan was of the type A4α L-Lys-Thr-Glu, with L-lysine partially replaced by L-ornithine. The DNA G+C content of one of the strains, C1A_55(T)(,) was 55 mol%. A novel species, Scardovia wiggsiae sp. nov., is proposed to accommodate the six isolates, with the type strain C1A_55(T) (=DSM 22547(T)=CCUG 58090(T)).

Prevotella saccharolytica sp. nov., isolated from the human oral cavity.

Two strains of anaerobic, Gram-stain-negative bacilli isolated from the human oral cavity (D033B-12-2(T) and D080A-01) were subjected to a comprehensive range of phenotypic and genotypic tests and were found to be distinct from any previously described species. 16S rRNA gene sequence analysis revealed that the strains were related most closely to the type strain of Prevotella marshii (93.5 % sequence identity). The novel strains were saccharolytic and produced acetic acid and succinic acid as end products of fermentation. The principal cellular long-chain fatty acids were C16 :0), iso-C14:0, C14:0, anteiso-C15:0, iso-C16 :0 and C16:0) 3-OH. The G+C content of the DNA of strain D033B-12-2(T) was 44 mol%. Strains D033B-12-2(T) and D080A-01 are considered to represent a single novel species of the genus Prevotella, for which the name Prevotella saccharolytica sp. nov. is proposed. The type strain is D033B-12-2(T) (=DSM 22473(T) =CCUG 57944(T)).

Pomegranate extract induces ellagitannin metabolite formation and changes stool microbiota in healthy volunteers.

The health benefits of pomegranate (POM) consumption are attributed to ellagitannins and their metabolites, formed and absorbed in the intestine by the microbiota. In this study twenty healthy participants consumed 1000 mg of POM extract daily for four weeks. Based on urinary and fecal content of the POM metabolite urolithin A (UA), we observed three distinct groups: (1) individuals with no baseline UA presence but induction of UA formation by POM extract consumption (n = 9); (2) baseline UA formation which was enhanced by POM extract consumption (N = 5) and (3) no baseline UA production, which was not inducible (N = 6). Compared to baseline the phylum Actinobacteria was increased and Firmicutes decreased significantly in individuals forming UA (producers). Verrucomicrobia (Akkermansia muciniphila) was 33 and 47-fold higher in stool samples of UA producers compared to non-producers at baseline and after 4 weeks, respectively. In UA producers, the genera Butyrivibrio, Enterobacter, Escherichia, Lactobacillus, Prevotella, Serratia and Veillonella were increased and Collinsella decreased significantly at week 4 compared to baseline. The consumption of pomegranate resulted in the formation of its metabolites in some but not all participants. POM extract consumption may induce health benefits secondary to changes in the microbiota.

Characterisation of Eubacterium-like strains isolated from oral infections.

The genus Eubacterium currently includes a heterogeneous group of gram-positive, non-spore-forming anaerobic bacilli, many of which are slow growing, fastidious and generally unreactive in biochemical tests. As a consequence, cultivation and identification of isolates are difficult and the taxonomy of the group remains indifferent. In this study, 105 isolates from odontogenic infections, infections associated with dental implants or saliva from healthy subjects and provisionally assigned to the genus Eubacterium were subjected to phenotypic and genotypic analysis. Ninety-one of the isolates were identified as belonging to one of 14 previously described species: Atopobium parvulum (5 isolates), A. rimae (29), Bulleidia extructa (2), Cryptobacterium curtum (1), Dialister pneumosintes (1), Eubacterium saburreum (2), E. sulci (8), E. yurii subsp. yurii (1), Filifactor alocis (3), Lactobacillus uli (1), Mogibacterium timidum (13), M. vescum (6), Pseudoramibacter alactolyticus (6) and Slackia exigua (13). The remaining 14 isolates did not correspond to existing species. This study confirms the diversity of organisms provisionally assigned to the genus Eubacterium by conventional identification methods. This group of organisms is frequently isolated from oral infections but their role in the aetiology of these conditions has yet to be determined.

Pomegranate extract exhibits in vitro activity against Clostridium difficile.

OBJECTIVE: To determine the possible utility of pomegranate extract in the management or prevention of Clostridium difficile infections or colonization. METHOD: The activity of pomegranate was tested against 29 clinical C. difficile isolates using the Clinical and Laboratory Standards Institute-approved agar dilution technique. Total phenolics content of the pomegranate extract was determined by Folin-Ciocalteau colorimetric method and final concentrations of 6.25 to 400 µg/mL gallic acid equivalent were achieved in the agar. RESULTS: All strains had MICs at 12.5 to 25 mg/mL gallic acid equivalent range. Our results suggest antimicrobial in vitro activity for pomegranate extract against toxigenic C. difficile. CONCLUSION: Pomegranate extract may be a useful contributor to the management and prevention of C. difficile disease or colonization.

Xylooligosaccharide increases bifidobacteria but not lactobacilli in human gut microbiota.

This study was conducted to determine the tolerance and effects of the prebiotic xylooligosaccharide (XOS) on the composition of human colonic microbiota, pH and short chain fatty acids (SCFA) in order to determine whether significant changes in the microbiota would be achievable without side effects. Healthy adult subjects (n = 32) were recruited in a double-blind, randomized, placebo-controlled study. Subjects received 1.4 g XOS, 2.8 g XOS or placebo in daily doses. The study consisted of a 2 week run-in, an 8 week intervention, and a 2 week washout phase. Stool samples were collected at baseline, after 4 and 8 weeks of intervention and 2 weeks after cessation of intervention. Samples were subjected to culture, pyrosequencing of community DNA, pH and SCFA analyses. Tolerance was evaluated by daily symptom charts. XOS was tolerated without significant gastrointestinal side effects. Bifidobacterium counts increased in both XOS groups compared to the placebo subjects, the 2.8 g per day group showed significantly greater increases than the 1.4 g per day group. Total anaerobic counts and Bacteroides fragilis group counts were significantly higher in the 2.8 g per day XOS group. There were no significant differences in the counts of Lactobacillus, Enterobacteriaceae and Clostridium between the three groups. XOS intervention had no significant effect on stool pH, SCFA or lactic acid. Pyrosequencing showed no notable shifts in bacterial diversity. XOS supplementation may be beneficial to gastrointestinal microbiota and 2.8 g per day may be more effective than 1.4 g per day. The low dose required and lack of gastrointestinal side effects makes the use of XOS as a food supplement feasible.

Multiscale evolving complex network model of functional connectivity in neuronal cultures.

Cultures of cortical neurons grown on multielectrode arrays exhibit spontaneous, robust, and recurrent patterns of highly synchronous activity called bursts. These bursts play a crucial role in the development and topological self-organization of neuronal networks. Thus, understanding the evolution of synchrony within these bursts could give insight into network growth and the functional processes involved in learning and memory. Functional connectivity networks can be constructed by observing patterns of synchrony that evolve during bursts. To capture this evolution, a modeling approach is adopted using a framework of emergent evolving complex networks and, through taking advantage of the multiple time scales of the system, aims to show the importance of sequential and ordered synchronization in network function.

Phosphorylated dihydroceramides from common human bacteria are recovered in human tissues.

Novel phosphorylated dihydroceramide (PDHC) lipids produced by the periodontal pathogen Porphyromonas gingivalis include phosphoethanolamine (PE DHC) and phosphoglycerol dihydroceramides (PG DHC) lipids. These PDHC lipids mediate cellular effects through Toll-like receptor 2 (TLR2) including promotion of IL-6 secretion from dendritic cells and inhibition of osteoblast differentiation and function in vitro and in vivo. The PE DHC lipids also enhance (TLR2)-dependent murine experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. The unique non-mammalian structures of these lipids allows for their specific quantification in bacteria and human tissues using multiple reaction monitoring (MRM)-mass spectrometry (MS). Synthesis of these lipids by other common human bacteria and the presence of these lipids in human tissues have not yet been determined. We now report that synthesis of these lipids can be attributed to a small number of intestinal and oral organisms within the Bacteroides, Parabacteroides, Prevotella, Tannerella and Porphyromonas genera. Additionally, the PDHCs are not only present in gingival tissues, but are also present in human blood, vasculature tissues and brain. Finally, the distribution of these TLR2-activating lipids in human tissues varies with both the tissue site and disease status of the tissue suggesting a role for PDHCs in human disease.

Revealing ensemble state transition patterns in multi-electrode neuronal recordings using hidden Markov models.

In order to harness the computational capacity of dissociated cultured neuronal networks, it is necessary to understand neuronal dynamics and connectivity on a mesoscopic scale. To this end, this paper uncovers dynamic spatiotemporal patterns emerging from electrically stimulated neuronal cultures using hidden Markov models (HMMs) to characterize multi-channel spike trains as a progression of patterns of underlying states of neuronal activity. However, experimentation aimed at optimal choice of parameters for such models is essential and results are reported in detail. Results derived from ensemble neuronal data revealed highly repeatable patterns of state transitions in the order of milliseconds in response to probing stimuli.

Propionibacterium acidifaciens sp. nov., isolated from the human mouth.

Three strains of anaerobic, pleomorphic, Gram-positive-staining bacilli, which were isolated from human carious dentine, were subjected to a comprehensive range of phenotypic and genotypic tests and were found to comprise a homogeneous group. The strains were saccharolytic and produced acetic and propionic acids in large amounts, and succinic acid in moderate amounts, as the end products of fermentation. 16S rRNA gene and RpoB protein sequence analyses revealed that the strains constituted a novel group within the genus Propionibacterium, most closely related to Propionibacterium australiense but sharing only 8 % DNA-DNA relatedness with the type strain of that species. Therefore, a novel species, Propionibacterium acidifaciens sp. nov., is proposed to accommodate these strains. The DNA G+C content of the type strain is 70 mol%. The type strain is C3M_31(T) (=DSM 21887(T) =CCUG 57100(T)).

Prevotella micans sp. nov., isolated from the human oral cavity.

Four strains of anaerobic Gram-negative bacilli isolated from the human mouth were characterized using a variety of phenotypic and genotypic tests. The strains were found to comprise a homogeneous group and 16S rRNA gene sequence analysis revealed them to be distinct from but related to a loose cluster of Prevotella species including Prevotella buccalis, Prevotella nanceiensis and Prevotella marshii. A novel species, Prevotella micans sp. nov., is proposed to accommodate these strains. Prevotella micans is saccharolytic and produces acetic, isovaleric and succinic acids and minor amounts of isobutyric acid as end products of fermentation. The G+C content of the DNA of the type strain is 46 mol%. The type strain of Prevotella micans is E7.56(T) (=DSM 21469(T )=CCUG 56105(T)).

Pyramidobacter piscolens gen. nov., sp. nov., a member of the phylum 'Synergistetes' isolated from the human oral cavity.

Four strains of anaerobic, Gram-negative bacilli isolated from the human oral cavity were subjected to a comprehensive range of phenotypic and genotypic tests and were found to comprise a homogeneous group distinct from any species with validly published names. 16S rRNA and 23S rRNA gene sequence analyses and DNA-DNA reassociation data revealed that the strains constituted a novel group within the phylum 'Synergistetes' and were most closely related to Jonquetella anthropi. Two libraries of randomly cloned DNA were prepared from strain W5455(T) and were sequenced to provide a genome survey as a resource for metagenomic studies. A new genus and novel species, Pyramidobacter piscolens gen. nov., sp. nov., is proposed to accommodate these strains. The genus Pyramidobacter comprises strains that are anaerobic, non-motile, asaccharolytic bacilli that produce acetic and isovaleric acids and minor to trace amounts of propionic, isobutyric, succinic and phenylacetic acids as end products of metabolism. P. piscolens gen. nov., sp. nov. produced hydrogen sulphide but was otherwise largely biochemically unreactive. Growth was stimulated by the addition of glycine to broth media. The G+C content of the DNA of the type strain was 59 mol%. The type strain of Pyramidobacter piscolens sp. nov. is W5455(T) (=DSM 21147(T)=CCUG 55836(T)).