Deposition and hydrolysis of serine dipeptide lipids of Bacteroidetes bacteria in human arteries: relationship to atherosclerosis.

Multiple reaction monitoring-MS analysis of lipid extracts from human carotid endarterectomy and carotid artery samples from young individuals consistently demonstrated the presence of bacterial serine dipeptide lipid classes, including Lipid 654, an agonist for human and mouse Toll-like receptor (TLR)2, and Lipid 430, the deacylated product of Lipid 654. The relative levels of Lipid 654 and Lipid 430 were also determined in common oral and intestinal bacteria from the phylum Bacteroidetes and human serum and brain samples from healthy adults. The median Lipid 430/Lipid 654 ratio observed in carotid endarterectomy samples was significantly higher than the median ratio in lipid extracts of common oral and intestinal Bacteroidetes bacteria, and serum and brain samples from healthy subjects. More importantly, the median Lipid 430/Lipid 654 ratio was significantly elevated in carotid endarterectomies when compared with control artery samples. Our results indicate that deacylation of Lipid 654 to Lipid 430 likely occurs in diseased artery walls due to phospholipase A2 enzyme activity. These results suggest that commensal Bacteriodetes bacteria of the gut and the oral cavity may contribute to the pathogenesis of TLR2-dependent atherosclerosis through serine dipeptide lipid deposition and metabolism in artery walls.

Prebiotic Potential and Chemical Composition of Seven Culinary Spice Extracts.

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.

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.

Pomegranate ellagitannins stimulate the growth of Akkermansia muciniphila in vivo.

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.

Reclassification of Clostridium difficile as Clostridioides difficile (Hall and O'Toole 1935) Prévot 1938.

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.

Xylooligosaccharide supplementation alters gut bacteria in both healthy and prediabetic adults: a pilot study.

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).

Inability of polymerase chain reaction, pyrosequencing, and culture of infected and uninfected site skin biopsy specimens to identify the cause of cellulitis.

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.

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.

In vitro study of the prebiotic xylooligosaccharide (XOS) on the growth of Bifidobacterium spp and Lactobacillus spp.

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.

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.

Pomegranate ellagitannins stimulate growth of gut bacteria in vitro: Implications for prebiotic and metabolic effects.

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.

Reclassification of Ruminococcus obeum as Blautia obeum comb. nov.

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)).

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.

Genes Encoding Toxin of Clostridium difficile in Children with and without Diarrhea.

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.

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.

Serine lipids of Porphyromonas gingivalis are human and mouse Toll-like receptor 2 ligands.

The total cellular lipids of Porphyromas gingivalis, a known periodontal pathogen, were previously shown to promote dendritic cell activation and inhibition of osteoblasts through engagement of Toll-like receptor 2 (TLR2). The purpose of the present investigation was to fractionate all lipids of P. gingivalis and define which lipid classes account for the TLR2 engagement, based on both in vitro human cell assays and in vivo studies in mice. Specific serine-containing lipids of P. gingivalis, called lipid 654 and lipid 430, were identified in specific high-performance liquid chromatography fractions as the TLR2-activating lipids. The structures of these lipids were defined using tandem mass spectrometry and nuclear magnetic resonance methods. In vitro, both lipid 654 and lipid 430 activated TLR2-expressing HEK cells, and this activation was inhibited by anti-TLR2 antibody. In contrast, TLR4-expressing HEK cells failed to be activated by either lipid 654 or lipid 430. Wild-type (WT) or TLR2-deficient (TLR2(-/-)) mice were injected with either lipid 654 or lipid 430, and the effects on serum levels of the chemokine CCL2 were measured 4 h later. Administration of either lipid 654 or lipid 430 to WT mice resulted in a significant increase in serum CCL2 levels; in contrast, the administration of lipid 654 or lipid 430 to TLR2(-/-) mice resulted in no increase in serum CCL2. These results thus identify a new class of TLR2 ligands that are produced by P. gingivalis that likely play a significant role in mediating inflammatory responses both at periodontal sites and, potentially, in other tissues where these lipids might accumulate.

Single nucleotide polymorphisms are randomly dispersed and mostly synonymous in partial rpoB and cpn60 genes of Campylobacter showae human isolates.

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.

Occurrence and antimicrobial susceptibility of Porphyromonas spp. and Fusobacterium spp. in dogs with and without periodontitis.

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.

Peptoniphilus duerdenii sp. nov. and Peptoniphilus koenoeneniae sp. nov., isolated from human clinical specimens.

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.