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The objective of this study was to investigate prebiotic potential, chemical composition, and antioxidant capacity of spice extracts. Seven culinary spices including black pepper, cayenne pepper, cinnamon, ginger, Mediterranean oregano, rosemary, and turmeric were extracted with boiling water. Major chemical constituents were characterized by RP-HPLC-DAD method and antioxidant capacity was determined by measuring colorimetrically the extent to scavenge ABTS radical cations. Effects of spice extracts on the viability of 88 anaerobic and facultative isolates from intestinal microbiota were determined by using Brucella agar plates containing serial dilutions of extracts. A total of 14 phenolic compounds, a piperine, cinnamic acid, and cinnamaldehyde were identified and quantitated. Spice extracts exhibited high antioxidant capacity that correlated with the total amount of major chemicals. All spice extracts, with the exception of turmeric, enhanced the growth of Bifidobacterium spp. and Lactobacillus spp. All spices exhibited inhibitory activity against selected Ruminococcus species. Cinnamon, oregano, and rosemary were active against selected Fusobacterium strains and cinnamon, rosemary, and turmeric were active against selected Clostridium spp. Some spices displayed prebiotic-like activity by promoting the growth of beneficial bacteria and suppressing the growth of pathogenic bacteria, suggesting their potential role in the regulation of intestinal microbiota and the enhancement of gastrointestinal health. The identification and quantification of spice-specific phytochemicals provided insight into the potential influence of these chemicals on the gut microbial communities and activities. Future research on the connections between spice-induced changes in gut microbiota and host metabolism and disease preventive effect in animal models and humans is needed., (© 2017 The Authors. Journal of Food Science published by Wiley Periodicals, Inc. on behalf of Institute of Food Technologists.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Results from our previous human pomegranate extract (POM extract) intervention study demonstrated that about seventy percent of participants were able to form urolithin A from ellagitannins in the intestine (urolithin A producers). Urolithin A formation was associated with a high proportion of Akkermansia muciniphila in fecal bacterial samples as determined by 16S rRNA sequencing. Here we investigated whether A. muciniphila counts increased in stool samples collected after the POM extract intervention compared to baseline stool samples using real-time PCR. In addition, we performed in vitro culture studies to determine the effect of POM extract and ellagic acid on the growth of A. muciniphila and to analyze ellagic acid metabolites formed in the culture broth by high-performance liquid chromatography. Supplementation of culture broth with 10 μM of ellagic acid did not change A. muciniphila growth while the addition of 0.18 mg/ml and 0.28 mg/ml of POM extract to the culture broth inhibited the growth of A. muciniphila significantly. Incubation of A. muciniphila with POM extract resulted in formation of ellagic acid and incubation of A. muciniphila with ellagic acid demonstrated hydrolysis of ellagic acid to metabolites different from urolithin A. The in vitro culture studies with A. muciniphila partially explain our in vivo findings that the presence of A. muciniphila was associated with breakdown of ellagic acid for further metabolism by other members of the microbiota. This is the first report of the role of A. muciniphila in ellagitannin hydrolysis. However, we conclude that enzymes from other bacteria must be involved in the formation of urolithin A in the human intestine., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The recent proposal by Lawson and Rainey (2015) to restrict the genus Clostridium to Clostridium butyricum and related species has ramifications for the members of the genera that fall outside this clade that should not be considered as Clostridium sensu stricto. One such organism of profound medical importance is Clostridioides difficile that is a major cause of hospital-acquired diarrhea and mortality in individuals. Based on 16S rRNA gene sequence analysis, the closest relative of Clostridium difficile is Clostridium mangenotii with a 94.7% similarity value and both are located within the family Peptostreptococcaceae that is phylogenetically far removed from C. butyricum and other members of Clostridium sensu stricto. Clostridium difficile is Clostridium mangenotii each produce abundant H2 gas when grown in PYG broth and also produce a range of straight and branched chain saturated and unsaturated fatty acids with C16:0 as a major product. The cell wall peptidoglycan contains meso-DAP as the diagnostic diamino acid. Based on phenotypic, chemotaxonomic and phylogenetic analyses, novel genus Clostridioides gen. nov. is proposed for Clostridium difficile as Clostridioides difficile gen. nov. comb. nov. and that Clostridium mangenotii be transferred to this genus as Clostridioides mangenotii comb. nov. The type species of Clostridioides is Clostridioides difficile., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: The cause of cellulitis is unclear. Streptococcus pyogenes, and to a lesser extent, Staphylococcus aureus, are presumed pathogens., Methods: We conducted a study of adults with acute cellulitis without drainage presenting to a US emergency department research network. Skin biopsy specimens were taken from the infected site and a comparable uninfected site on the opposite side of the body. Microbiology was evaluated using quantitative polymerase chain reaction (PCR), pyrosequencing, and standard culture techniques. To determine the cause, the prevalence and quantity of bacterial species at the infected and uninfected sites were compared., Results: Among 50 subjects with biopsy specimens from infected and uninfected sites, culture rarely identified a bacterium. Among 49 subjects with paired specimens from infected and uninfected sites tested with PCR, methicillin-susceptible S. aureus was identified in 20 (41%) and 17 (34%), respectively. Pyrosequencing identified abundant atypical bacteria in addition to streptococci and staphylococci. Among 49 subjects with paired specimens tested by pyrosequencing, S. aureus was identified from 11 (22%) and 15 (31%) and streptococci from 15 (31%) and 20 (41%) of the specimens, respectively. Methicillin-resistant S. aureus was not found by culture or PCR, and S. pyogenes was not identified by any technique., Conclusions: The bacterial cause of cellulitis cannot be determined by comparing the prevalence and quantity of pathogens from infected and uninfected skin biopsy specimens using current molecular techniques. Methicillin-susceptible S. aureus was detected but not methicillin-resistant S. aureus or S. pyogenes from cellulitis tissue specimens. For now, optimal treatment will need to be guided by clinical trials. Noninfectious causes should also be explored., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: It has been suggested that gut microbiota is altered in Type 2 Diabetes Mellitus (T2DM) patients., Objective: This study was to evaluate the effect of the prebiotic xylooligosaccharide (XOS) on the gut microbiota in both healthy and prediabetic (Pre-DM) subjects, as well as impaired glucose tolerance (IGT) in Pre-DM., Subjects/methods: Pre-DM (n = 13) or healthy (n = 16) subjects were randomized to receive 2 g/day XOS or placebo for 8-weeks. In Pre-DM subjects, body composition and oral glucose tolerance test (OGTT) was done at baseline and week 8. Stool from Pre-DM and healthy subjects at baseline and week 8 was analyzed for gut microbiota characterization using Illumina MiSeq sequencing., Results: We identified 40 Pre-DM associated bacterial taxa. Among them, the abundance of the genera Enterorhabdus, Howardella, and Slackia was higher in Pre-DM. XOS significantly decreased or reversed the increase in abundance of Howardella, Enterorhabdus, and Slackia observed in healthy or Pre-DM subjects. Abundance of the species Blautia hydrogenotrophica was lower in pre-DM subjects, while XOS increased its abundance. In Pre-DM, XOS showed a tendency to reduce OGTT 2-h insulin levels (P = 0.13), but had no effect on body composition, HOMA-IR, serum glucose, triglyceride, satiety hormones, and TNFα., Conclusion: This is the first clinical observation of modifications of the gut microbiota by XOS in both healthy and Pre-DM subjects in a pilot study. Prebiotic XOS may be beneficial in reversing changes in the gut microbiota during the development of diabetes., Clinical Trial Registration: NCT01944904 (https://clinicaltrials.gov/ct2/show/NCT01944904).

The present study investigated the effect of pomegranate extract (POMx) and pomegranate juice (POM juice) on the growth of major groups of intestinal bacteria: Enterobacteriaceae, Bacteroides fragilis group, clostridia, bifidobacteria, and lactobacilli, and the utilization of pomegranate polyphenols by Bifidobacterium and Lactobacillus. The total phenolic content of the pomegranate extract and juice was determined using the Folin-Ciocalteau colorimetric method and reported as gallic acid equivalent (GAE). The polyphenol composition was determined by HPLC. Stool specimens were incubated with 400, 100, and 25 μg/ml GAE POMx and POM juice and subjected to selective culture. Bifidobacterium and Lactobacillus strains were incubated with 400 μg/ml GAE POMx and POM juice and metabolites were analyzed. POMx and POM juice increased the mean counts of Bifidobacterium and Lactobacillus and significantly inhibited the growth of B. fragilis group, clostridia, and Enterobacteriaceae in a dose-response manner. Bifidobacterium and Lactobacillus utilized ellagic acid and glycosyl ellagic acid but little or no punicalin was utilized. Neither POMx nor POM juice was converted to urolithins by the test bacteria or the in vitro stool cultures. The effect of pomegranate on the gut bacteria considered to be beneficial (Bifidobacterium and Lactobacillus) suggests that pomegranate may potentially work as a prebiotic. The concept that polyphenols such as those in pomegranate impact gut microbiota populations may establish a new role for polyphenols in human health., (Published by Elsevier Ltd.)

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.

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.

During our previous studies we reclassified Clostridium coccoides and a number of misclassified ruminococci into a novel genus Blautia within the family Lachnospiraceae. However, the Rules of the Bacteriological Code currently require that the types of all species and subspecies with new names (including new combinations) be deposited in two different collections in two different countries. The type strain of Ruminococcus obeum was, at that period in time, only deposited in the American Type Culture Collection (ATCC) and a second independent deposit, as required by the Code, was not available. Consequently, the transfer of this species to the genus Blautia could not be made, because the resulting species name would not conform to the Rules governing the valid publication of species names and deposit of type material (Rules 27 and 30) and consequently would not be considered to be validly published. This resulted in a nomenclatural and taxonomic anomaly with R. obeum being phylogenetically placed among members of the genus Blautia with 16S rRNA gene sequence similarities of between 91.8 and 96.6 %. In order to rectify this unsatisfactory situation, through our discussions with the ATCC, the deposit of strain R. obeum ATCC 29174(T) to the DSMZ as strain number DSM 25238(T) was completed. Hence, the transfer of R. obeum to the genus Blautia as Blautia obeum comb. nov. is now proposed. The type strain is ATCC 29174(T) ( = DSM 25238(T) = KCTC 15206(T))., (© 2015 IUMS.)

We recently demonstrated that XOS increased the counts of Bifidobacterium in vivo without increasing Lactobacillus in healthy adults. In the current study, we evaluated the effect of XOS on the growth of 35 Bifidobacterium and 29 Lactobacillus strains in in vitro conditions. Bacteria were identified by 16S rRNA sequence analysis. The growth stimulation was determined by agar dilution technique on plates containing two-fold serial dilutions of XOS (100-0.1 mg/ml). The growth of 86% of Bifidobacterium strains was stimulated at 1.56 mg/ml XOS and 100% at 6.25 mg/ml XOS. The growth of 38% of Lactobacillus strains was stimulated at 1.56 mg/ml XOS and 62% at 6.25 mg/ml XOS; 31% of Lactobacillus were not stimulated by XOS. Our results further suggest that XOS may be beneficial in stimulating intestinal Bifidobacterium without having much effect on Lactobacillus. The potential role for XOS in managing obesity should be investigated further.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

The presence of gene 16S rRNA and genes encoding toxin A (tcdA), toxin B (tcdB), and binary toxin (cdtA/cdtB) of Clostridium difficile in stool samples from children with (110) and without (150) diarrhea was determined by using a TaqMan system. Fifty-seven (21.9%) out of 260 stool samples harbored the 16S rRNA gene. The genetic profile of tcdA+/tcdB- and cdtA+/cdtB+ was verified in one C. difficile-positive diarrhea sample and of tcdA+/tcdB+ in three C. difficile-positive nondiarrhea samples. The presence of tcdA+/tcdB+ in stools obtained from children without diarrhea, suggests that they were asymptomatic carriers of toxigenic strains.

The partial 16S rRNA, rpoB, and cpn60 genes congruently allow this study to identify all the eight isolates as the species Campylobacter showae. To our knowledge, this is the first report to reveal the interspecies and intraspecies sequence variations present in the three genes of the C. showae isolates., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Two previously uncharacterized strains of Gram-reaction-positive, anaerobic, coccus-shaped bacteria, designated strains WAL 18896(T) and WAL 18898(T), were recovered from human wound specimens and characterized using phenotypic, chemotaxonomic and molecular taxonomic methods. Comparative 16S rRNA gene sequence analysis and chemotaxonomic and biochemical characteristics demonstrated that these organisms are genotypically and phenotypically distinct and represent previously unidentified sublines within the order Clostridiales in the phylum Firmicutes. Pairwise sequence analysis demonstrated that the novel organisms had 91.9% sequence similarity to each other and were most closely related to members of the genus Peptoniphilus. The major long-chain fatty acids of both strains were C(16:0,) C(18:0), C(18:1)ω9c and C(18:2)ω6,9c. Based on the phenotypic and phylogenetic findings, strains WAL 18896(T) ( = CCUG 56065(T)  = ATCC BAA-1640(T)) and WAL 18898(T) ( = CCUG 56067(T)  = ATCC BAA-1638(T)  = DSM 22616(T)) represent two novel species, for which the names Peptoniphilus duerdenii sp. nov. and Peptoniphilus koenoeneniae sp. nov. are proposed, respectively.

The occurrence of Porphyromonas gulae, Porphyromonas macacae, Fusobacterium nucleatum and Fusobacterium canifelinum in subgingival plaque from dogs with and without periodontitis as well as their antimicrobial susceptibility were evaluated. From 50 dogs with periodontitis were identified 38 P. gulae, 8 P. macacae, 26 F. nucleatum and 15 F. canifelinum, and from 50 dogs without periodontitis were identified 15 P. gulae, 12 F. nucleatum and 11 F. canifelinum. All strains were susceptible to most of the antibiotics tested, however, different resistance rates to clarithromycin, erythromycin and metronidazole among strains were observed. The role of P. gulae, P. macacae, F. nucleatum and F. canifelinum in periodontal disease of household pets needs to be defined to a better prevention and treatment of the canine periodontitis., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (Published by Elsevier Ltd.)

Corynebacterium pyruviciproducens (C. pyruviciproducens), a newly discovered Corynebacterium, is gram-positive, non-flagellate, non-spore-forming lipophilic rod. No known pathogenic components of Corynebacteria have been found in this new bacterium, such as diphtheria toxin and tuberculostearic acid. In the present study, referring to Propionibacterium acnes (P. acnes), a well-known bacterial adjuvant, the stimulation of dendritic cells by C. pyruviciproducens was analyzed through detecting the levels of cytokine-secretion, ability of cell-proliferation and expression of membrane molecules. In addition, the effect of C. pyruviciproducens in promoting antibody production in vivo was detected. Compared with P. acnes, C. pyruviciproducens more strongly enhanced cytokine secretion including inflammatory factor IL-6 and Th1-associated molecule IL-12, and more effectively induced proliferation, activation or maturation of D2SC/1 (a murine dendritic cell line) and bone marrow-derived dendritic cells (BMDC). Vaccination studies in mice using ovalbumin (OVA) as a model antigen showed that C. pyruviciproducens effectively promoted antigen-specific humoral immune response by increasing OVA-specific antibody, Th2-biased response in spleen and high IL-4/IFN-γ ratio in serum., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Autism of the regressive variety is selected as an example of the importance of intestinal bacterial microflora in disease other than classical infection. Our studies have indicated that intestinal bacteria play a role in this disease since it responds to oral vancomycin, a drug that is not absorbed from the GI tract. Pyrosequencing studies document an abnormal gut microflora in regressive autism subjects as compared to controls. Finally, we present preliminary evidence suggesting that Desulfovibrio may play a key role in this disease., (Copyright © 2011. Published by Elsevier Ltd.)

A rapid PCR approach was developed to detect Porphyromonas gulae strains from subgingival samples of dogs with and with periodontitis. The presence of P. gulae was observed in 92% and 56%, respectively, in dogs with and without periodontitis. The new primer pair was specific to detect this microorganism, and this technique could be used to evaluate a correlation between periodontitis and P. gulae in companion animals., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Autism is a complex disorder with no specific diagnostic test so the disease is defined by its characteristics including cognitive defects, social, communication and behavioral problems, repetitive behaviors, unusual sensitivity to stimuli such as noise, restricted interests, and self stimulation. The incidence of this disease has increased remarkably in recent years and was 110/10,000 children (∼1%) in multiple areas of the US in 2007. The financial burden on families and communities is enormous. In terms of predisposing factors, heredity plays a role in some subjects, but it is clear that environmental factors are also important. Environmental toxins can affect the immune system adversely. Intestinal bacteria are recognized by a few investigators as potentially important and we have proposed that certain antimicrobial drugs may be a key factor in modifying the intestinal bacterial flora adversely, selecting out potentially harmful bacteria that are normally suppressed by an intact normal intestinal flora. We had felt that clostridia in the gut might be involved in autism because they are virulent organisms and spore-formers; spores would resist antibacterial agents so that when antibiotics were discontinued the spores would germinate and by toxin production or another mechanism lead to autism. However, a recent study of ours employing the powerful pyrosequencing technique on stools of subjects with regressive autism showed that Desulfovibrio was more common in autistic subjects than in controls. We subsequently confirmed this with pilot cultural and real-time PCR studies and found siblings of autistic children had counts of Desulfovibrio that were intermediate, suggesting possible spread of the organism in the family environment. Desulfovibrio is an anaerobic bacillus that does not produce spores but is nevertheless resistant to aerobic and other adverse conditions by other mechanisms and is commonly resistant to certain antimicrobial agents (such as cephalosporins) often used to treat ear and other infections that are relatively common in childhood. This bacterium also produces important virulence factors and its physiology and metabolism position it uniquely to account for much of the pathophysiology seen in autism. If these results on Desulfovibrio are confirmed and extended in other studies, including treatment trials with appropriate agents and careful clinical and laboratory studies, this could lead to more reliable classification of autism, a diagnostic test and therapy for regressive autism, development of a vaccine for prevention and treatment of regressive autism, tailored probiotics/prebiotics, and important epidemiologic information., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Our goal was to establish a quantitative real-time PCR (QRT-PCR) method to detect Bacteroides fragilis group and related organisms from clinical specimens. Compared to conventional anaerobic culture, QRT-PCR can provide accurate and more rapid detection and identification of B. fragilis group and similar species. B. fragilis group and related organisms are the most frequently isolated anaerobic pathogens from clinical samples. However, culture and phenotypic identification is quite time-consuming. We designed specific primers and probes based on the 16S rRNA gene sequences of Bacteroides caccae, Bacteroides eggerthii, B. fragilis, Bacteroides ovatus, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Odoribacter splanchnicus (Bacteroides splanchnicus), Parabacteroides distasonis (Bacteroides distasonis) and Parabacteroides merdae (Bacteroides merdae), and detected these species by means of QRT-PCR in 400 human surgical wound infection samples or closed abscesses. The target bacteria were detected from 31 samples (8%) by culture, but from 132 samples (33%) by QRT-PCR (p-value < 0.001). B. uniformis was the most common species (44 positive samples) according to QRT-PCR while culture showed it to be B. fragilis (16 positive samples). Additionally, for each species QRT-PCR detected higher counts than culture did; this may reflect detecting DNA of dead organisms by QRT-PCR. QRT-PCR is a rapid and sensitive method which has great potential for detection of B. fragilis group and related organisms in wound samples., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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.

A rapid real-time PCR (RT-PCR) approach was developed to detect the bft gene subtypes in Bacteroides fragilis isolated from fecal samples. DNA obtained from diarrhea (110) and nondiarrhea (150) samples was evaluated. Subtype 1 was observed in 9 (8.2%) diarrhea and 7 (4.7%) nondiarrhea samples. Subtype 2 was not detected in any DNA samples, and subtype 3 was observed in only 1 diarrhea sample. The presence of the bft-1 gene did not show any statistically significant differences between the groups of children. This technique could be used to evaluate a possible correlation between disease and the presence of B. fragilis enterotoxin.

There is evidence of genetic predisposition to autism, but the percent of autistic subjects with this background is unknown. It is clear that other factors, such as environmental influences, may play a role in this disease. In the present study, we have examined the fecal microbial flora of 33 subjects with various severities of autism with gastrointestinal symptoms, 7 siblings not showing autistic symptoms (sibling controls) and eight non-sibling control subjects, using the bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP) procedure. The results provide us with information on the microflora of stools of young children and a compelling picture of unique fecal microflora of children with autism with gastrointestinal symptomatology. Differences based upon maximum observed and maximum predicted operational taxonomic units were statistically significant when comparing autistic and control subjects with p-values ranging from <0.001 to 0.009 using both parametric and non-parametric estimators. At the phylum level, Bacteroidetes and Firmicutes showed the most difference between groups of varying severities of autism. Bacteroidetes was found at high levels in the severely autistic group, while Firmicutes were more predominant in the control group. Smaller, but significant, differences also occurred in the Actinobacterium and Proteobacterium phyla. Desulfovibrio species and Bacteroides vulgatus are present in significantly higher numbers in stools of severely autistic children than in controls. If the unique microbial flora is found to be a causative or consequent factor in this type of autism, it may have implications with regard to a specific diagnostic test, its epidemiology, and for treatment and prevention., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

A coryneform strain, 06-1773O(T) (=WAL 19168(T)), derived from a groin abscess sample was characterized using phenotypic and molecular taxonomic methods. Comparative analyses revealed more than 3 % divergence of the 16S rRNA gene sequence and about 10 % divergence of the partial rpoB gene sequence from the type strain of Corynebacterium glucuronolyticum. The strain could also be differentiated from C. glucuronolyticum by a set of phenotypic properties. A DNA-DNA relatedness study between strain WAL 19168(T) and C. glucuronolyticum CCUG 35055(T) showed a relatedness value of 13.3 % (13.7 % on repeat analysis). The genotypic and phenotypic data show that the strain merits classification within a novel species of Corynebacterium. We propose the name Corynebacterium pyruviciproducens sp. nov. for the novel species. The type strain is 06-1773O(T) (=WAL 19168(T) =CCUG 57046(T) =ATCC BAA-1742(T)).

Three strains of an unidentified Gram-stain-variable, fastidious, catalase-negative, capnophilic, non-spore-forming, coccus-shaped bacterium from human wound specimens were characterized by phenotypic and molecular taxonomic methods. Initially, these strains were anaerobic; with repeated culture, they became aerotolerant. Comparative 16S rRNA gene sequencing studies demonstrated that the unknown strains were genealogically homogeneous and constituted a novel subline within the genus Gemella. The unknown bacterium was readily distinguished from other Gemella species by biochemical tests. On the basis of both phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium from clinical specimens be classified as Gemella asaccharolytica sp. nov. The type strain is WAL 1945J(T) (=ATCC BAA-1630(T) =CCUG 57045(T)).

Two strains of previously unknown Gram-stain-positive, anaerobic, coccus-shaped bacteria from human wound specimens were characterized using phenotypic and molecular taxonomic methods. Comparative 16S rRNA gene sequencing studies and distinguishable biochemical characteristics demonstrated that these two unknown strains, WAL 1855C(T) and WAL 2038E, are genotypically homogeneous and constitute a novel lineage within Clostridium cluster XIII. There was 13-14 % 16S rRNA gene sequence divergence between the novel strains and the most closely related species, Parvimonas micra, Finegoldia magna and species of Helcococcus. Based on the phenotypic and phylogenetic findings, a novel genus and species, Murdochiella asaccharolytica gen. nov., sp. nov., are proposed. Strain WAL 1855C(T) (=ATCC BAA-1631(T) =CCUG 55976(T)) is the type strain of Murdochiella asaccharolytica.

Eleven clinical strains isolated from infected wound specimens were subjected to polyphasic taxonomic analysis. Sequence analysis of the 16S rRNA gene showed that all 11 strains were phylogenetically related to Slackia exigua. Additionally, conventional and biochemical tests of 6 of the 11 strains were performed as supplementary methods to obtain phenotypic identification by comparison with the phenotypes of the relevant type strains. S. exigua has been considered an oral bacterial species in the family Coriobacteriaceae. This organism is fastidious and grows poorly, so it may easily be overlooked. The 16S rRNA gene sequences and the biochemical characteristics of four of the S. exigua strains isolated for this study from various infections indicative of an intestinal source were almost identical to those of the validated S. exigua type strain from an oral source and two of the S. exigua strains from oral sources evaluated in this study. Thus, we show for the first time that S. exigua species can be isolated from extraoral infections as well as from oral infections. The profiles of susceptibility to selected antimicrobials of this species were also investigated for the first time.

Clostridium sordellii, an anaerobic pathogen, has recently been associated with rapidly fatal infections following medically induced abortions and injecting drug use. Patients with C. sordellii infection display few signs of inflammation such as fever, or redness and pain at the site of infection. We hypothesized that this could be due to reduced recognition of the organism by Toll-like receptors (TLRs) of the innate immune system. An ELAM-NF-kappaB luciferase reporter system in TLR-transfected HEK cells was used to measure TLR-dependent recognition of washed, heat-killed C. sordellii and other pathogenic clostridial species. Results demonstrated that all clostridia were well recognized by TLR2 alone and that responses were greatest when TLR2 was co-expressed with TLR6. Further, isolated human monocytes produced the pro-inflammatory cytokine TNFalpha and the immunoregulator IL-10 in response to C. sordellii. In addition, C. sordellii-stimulated monocytes produced 30% less TNFalpha following treatment with an anti-TLR2 blocking antibody. These data demonstrate that innate immune recognition of, and response to, cell-associated components of C. sordellii and other clostridial pathogens are mediated by TLR2 in combination with TLR6. We conclude that the characteristic absence of inflammatory signs and symptoms in C. sordellii infection is not related to inadequate immune detection of the organism, but rather is attributable to a species-specific immune system dysfunction that remains to be elucidated., (Published by Elsevier Ltd.)

Though seldom reported, Solobacterium moorei, which was first described in 2000, has been identified in specimens from patients with root canals, periradicular lesions, periodontal disease, dentoalveolar abscesses, bacteremia, septic thrombophlebitis, and halitosis. In the present study, we describe 9 cases of mixed wound infection, from a pool of 400 surgical wound infections that we have studied, in which S. moorei was isolated or found in a clone library. All isolates of S. moorei were identified by 16S rRNA gene sequence analysis, and then six were examined for their physiological and biochemical characteristics and for antimicrobial susceptibility. The results of the present study indicate that Solobacterium moorei may be a significant component in some mixed surgical wound infections and that surgical management and antimicrobial therapy may be indicated when these bacteria are identified in significant situations.

Background: The rationale and lessons learned through the evolution of the National Survey for the Susceptibility of Bacteroides fragilis Group from its initiation in 1981 through 2007 are reviewed here. The survey was conceived in 1980 to track emerging antimicrobial resistance in Bacteroides species., Methods: Data from the last 11 years of the survey (1997-2007), including 6574 isolates from 13 medical centers, were analyzed for in vitro antimicrobial resistance to both frequently used and newly developed anti-anaerobic agents. The minimum inhibitory concentrations of the antibiotics were determined using agar dilution in accordance with Clinical and Laboratory Standards Institute recommendations., Results: The analyses revealed that the carbapenems (imipenem, meropenem, ertapenem, and doripenem) and piperacillin-tazobactam were the most active agents against these pathogens, with resistance rates of 0.9%-2.3%. In the most recent 3 years of the survey (2005-2007), resistance to some agents was shown to depend on the species, such as ampicillin-sulbactam against Bacteroides distasonis (20.6%) and tigecycline against Bacteroides uniformis and Bacteroides eggerthii ( approximately 7%). Very high resistance rates (>50%) were noted for moxifloxacin and trovafloxacin, particularly against Bacteroides vulgatus. During that period of study, non-B. fragilis Bacteroides species had >40% resistance to clindamycin. Metronidazole-resistant Bacteroides strains were also first reported during that period., Conclusions: In summary, resistance to antibiotics was greater among non-B. fragilis Bacteroides species than among B. fragilis and was especially greater among species with a low frequency of isolation, such as Bacteroides caccae and B. uniformis. The emergence of resistance among the non-B. fragilis Bacteroides species underscores the need for speciation of B. fragilis group isolates and for clinicians to be aware of associations between species and drug resistance.

Fusobacterium nucleatum is a Gram-negative anaerobic rod found in dental plaque biofilms, and is an opportunistic pathogen implicated in periodontitis as well as a wide range of systemic abscesses and infections. Genomic analyses of F. nucleatum indicate considerable genetic diversity and a prominent role for horizontal gene transfer in the evolution of the species. Several plasmids isolated from F. nucleatum, including pFN1, harbor relaxase gene homologs that may function in plasmid mobilization. In this investigation we examined the RP4-mediated mobilization properties of pFN1 and the prevalence of pFN1-related sequences in a panel of F. nucleatum clinical isolates. The fusobacterial plasmid pFN1 was mobilized by RP4 at a high frequency. Deletion analyses were used to delineate the core mobilon of pFN1, which consisted of the relaxase gene (rlx), an upstream open reading frame ORF4 and a region of DNA upstream of ORF4 with potential nic sites. To examine the prevalence of pFN1 in a panel of clinical isolates, total DNA isolated from the strains was hybridized with pFN1 replication (repA) and rlx gene probes. DNA from strains harboring plasmids known to be homologous to pFN1 hybridized with both the repA and rlx probes. Five additional strains were rlx-positive but repA-negative, indicating a greater prevalence of rlx-related genes in comparison with repA-related genes. Plasmid or plasmid-related sequences were identified in 11.5% of the strains examined. These findings demonstrate mobilization properties of a fusobacterial plasmid that may be important in horizontal gene transfer., (Copyright 2009 Elsevier Inc. All rights reserved.)

The present article gives an overview of recent taxonomic changes among the Gram-negative, anaerobic rods, briefly highlighting areas where the biology and ecology have a bearing on recent nomenclatorial changes. The focus is among the genera Bacteroides, Prevotella, Porphyromonas, Leptotrichia, Dysgonomonas, Fusobacterium and the Synergistes group and additionally demonstrates the value of conserved indels and group-specific proteins for identifying and circumscribing many of these taxa and the Bacteroidetes-Chlorobi species in general.

The terminology and classification of the Anginosus group streptococci has been inconsistent. We tested the utility of 16S rRNA gene and tuf gene sequencing and conventional biochemical tests for the reliable differentiation of the Anginosus group streptococci. Biochemical testing included Rapid ID 32 Strep, API Strep, Fluo-Card Milleri, Wee-tabs, and Lancefield antigen typing. Altogether, 61 Anginosus group isolates from skin and soft tissue infections and four reference strains were included. Our results showed a good agreement between 16S rRNA gene and tuf gene sequencing. Using the full sequence was less discriminatory than using the first part of the 16S rRNA gene. The three species could not be separated with the API 20 Strep test. Streptococcus intermedius could be differentiated from the other two species by beta-galactosidase (ONPG) and beta-N-acetyl-glucosaminidase reactions. Rapid ID 32 Strep beta-glucosidase reaction was useful in separating S. anginosus strains from S. constellatus. In conclusion, both 16S rRNA gene and tuf gene sequencing can be used for the reliable identification of the Anginosus group streptococci. S. intermedius can be readily differentiated from the other two species by phenotypic tests; however, 16S rRNA gene or tuf gene sequencing may be needed for separating some strains of S. constellatus from S. anginosus.

The antimicrobial activities of telavancin and six comparators were evaluated against 460 isolates of anaerobic bacteria. Telavancin demonstrated excellent activity against gram-positive anaerobes (MIC90, 2 microg/ml) and was the most potent agent tested against Clostridium difficile (MIC90, 0.25 microg/ml). As expected, gram-negative isolates were not inhibited by telavancin.

During our investigation of the bacteriology of human wound infections and abscesses, a novel anaerobic, non-spore-forming, Gram-negative bacillus was frequently isolated. On the basis of morphological and biochemical criteria, the strains were tentatively identified as belonging to the family Bacteroidaceae, but they did not appear to correspond to any recognized species of this family. Comparative 16S rRNA gene sequencing showed that the 14 novel strains were genotypically homogeneous and confirmed their placement in the genus Porphyromonas. Sequence divergence values >10 % with respect to reference Porphyromonas species demonstrated that the strains isolated represent a novel species. On the basis of biochemical criteria and phylogenetic considerations, it is proposed that these strains isolated from human sources should be assigned to a novel species of the genus Porphyromonas, named Porphyromonas bennonis sp. nov., with WAL 1926C(T) (=ATCC BAA-1629(T) =CCUG 55979(T)) as the type strain.

Rifaximin, ampicillin-sulbactam, neomycin, nitazoxanide, teicoplanin, and vancomycin were tested against 536 strains of anaerobic bacteria. The overall MIC of rifaximin at which 50% of strains were inhibited was 0.25 microg/ml. Ninety percent of the strains tested were inhibited by 256 microg/ml of rifaximin or less, an activity equivalent to those of teicoplanin and vancomycin but less than those of nitazoxanide and ampicillin-sulbactam.

Phenotypic and phylogenetic studies were performed on 15 isolates of an unidentified Gram-positive, anaerobic, non-sporulating coccobacillus-shaped bacterium isolated from human faeces. The novel organisms were catalase-negative, indole-negative and produced acetate and succinate as end products of metabolism. Comparative 16S rRNA gene sequencing demonstrated that the 15 isolates were highly related to each other and formed a hitherto unknown subline within the clostridial rRNA cluster XIVa. The novel isolates formed a robust phylogenetic group with a number of organisms which included Clostridium coccoides, Ruminococcus luti, Ruminococcus obeum and a number of other misclassified ruminococci. On the basis of these studies, a novel genus, Blautia gen. nov., is proposed. It is suggested that Clostridium coccoides, Ruminococcus hansenii, Ruminococcus hydrogenotrophicus, Ruminococcus luti, Ruminococcus productus, and Ruminococcus schinkii are transferred to this genus as Blautia coccoides gen. nov., comb. nov., Blautia hansenii comb. nov., Blautia hydrogenotrophica comb. nov., Blautia luti comb. nov., Blautia producta comb. nov. and Blautia schinkii comb. nov. One of the new isolates, the hitherto unknown coccus-shaped bacterial strain WAL 14507T (=ATCC BAA-1564T=DSM 19850T) is proposed as representing the type strain of a novel species, Blautia wexlerae sp. nov.

This manuscript reviews evidence indicating that intestinal bacteria, specifically clostridia, may play a role in certain cases of autism and hypothesizes that the clostridial spores (which are notably resistant to antimicrobial agents and commonly used germicides) are involved in: (1) relapse in the autistic subject after a response to an agent such as oral vancomycin, after the drug is discontinued, (2) the unexplained increased incidence of autism in recent years, and (3) the unexplained increase in numbers of multiple cases in the same family. Hypothesis (1), if established as valid, would spur research to find well-tolerated and safe agents that could be given together with vancomycin (or other appropriate antimicrobial agent) to eliminate spores; this would revolutionize the therapeutic approach. Hypotheses (2) and (3) relate to widespread use of antimicrobial agents, poor hygiene in young autistic children, and difficulty in removing spores from the home environment. These latter two hypotheses have major implications with regard to the epidemiology of this important and distressing disease and would encourage research into methods to eliminate clostridial spores from the home and other environments.

Clostridium tertium has been increasingly reported as a human pathogen. This organism is an aerotolerant Gram-positive rod that is often mistaken for other organisms, such as Lactobacillus or Bacillus species. We describe a case of a patient with a history of intravenous drug use presenting to UCLA-Olive View Medical Center with gas gangrene of both upper extremities. The organism was initially misidentified as a Lactobacillus species on aerobic culture plates. However, terminal spore formation was detected in this isolate on a sub-cultured anaerobic culture plate and this isolate was confirmed as C. tertium biochemically and genetically by 16S rDNA sequencing. Additional DNA cloning libraries made from the formalin-fixed specimen revealed Peptoniphilus species and an uncultured Clostridium clone, but not C. tertium. C. tertium might be a causative organism of gas-producing myonecrosis but such an association has never been described. Clinicians should be aware of the phenomenon of aerotolerance of some anaerobes and need to clarify the identification of organisms if the clinical picture does not fit the isolated organism.

Three groups of previously unknown gram-positive, anaerobic, coccus-shaped bacteria were characterized using phenotypic and molecular taxonomic methods. Phenotypic and genotypic data demonstrate that these organisms are distinct, and each group represents a previously unknown subline within Clostridium cluster XIII. Two groups are most closely related to Peptoniphilus harei in the genus Peptoniphilus, and the other group is most closely related to Anaerococcus lactolyticus in the genus Anaerococcus. Based on the findings, three novel species, Peptoniphilus gorbachii sp. nov., Peptoniphilus olsenii sp. nov., and Anaerococcus murdochii sp. nov., are proposed. The type strains of Peptoniphilus gorbachii sp. nov., Peptoniphilus olsenii sp. nov., and Anaerococcus murdochii sp. nov. are WAL 10418(T) (= CCUG 53341(T) = ATCC BAA-1383(T)), WAL 12922(T) (= CCUG 53342(T) = ATCC BAA-1384(T)), and WAL 17230(T) (= CCUG 53340(T) = ATCC BAA-1385(T)), respectively.