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.

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.

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)).

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.

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.

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.

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.

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.

State of the art; microbiology in health and disease. Intestinal bacterial flora in autism.

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.

Application of quantitative real-time PCR for rapid identification of Bacteroides fragilis group and related organisms in human wound samples.

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.

Detection of Porphyromonas gulae from subgingival biofilms of dogs with and without periodontitis.

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.

Lessons learned from the anaerobe survey: historical perspective and review of the most recent data (2005-2007).

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.

Phenotypic and molecular characterization of Solobacterium moorei isolates from patients with wound infection.

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.

Characterization of Slackia exigua isolated from human wound infections, including abscesses of intestinal origin.

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.

Mobilization and prevalence of a Fusobacterial plasmid.

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.