Tichowtungia aerotolerans gen. nov., sp. nov., a novel representative of the phylum Kiritimatiellaeota and proposal of Tichowtungiaceae fam. nov., Tichowtungiales ord. nov. and Tichowtungiia class. nov.

Kiritimatiellaeota is widespread and ecologically important in various anoxic environments. However, the portion of culturable bacteria within this phylum is quite low and, in fact, there is only one currently described species. In this study, a novel anaerobic, non-motile, coccoid, Gram-stain-negative bacterial strain, designated S-5007T, was isolated from surface marine sediment. The 16S rRNA gene sequence was found to have very low 16S rRNA gene sequence similarity to the nearest known type strain, Kiritimatiella glycovorans L21-Fru-ABT (84.9 %). The taxonomic position of the novel isolate was investigated using a polyphasic approach and comparative genomic analysis. Phylogenetic analyses based on 16S rRNA genes and genomes indicated that strain S-5007T branched within the radiation of the phylum Kiritimatiellaeota. Different from the type strain, strain S-5007T can grow under microaerobic conditions, and the genomes of strain S-5007T and the other strains in its branch have many more antioxidant-related genes. Meanwhile, other different metabolic features deduced from genome analysis supported the separate evolution of the proposed class (strain S-5007T branch) and K. glycovorans L21-Fru-ABT. Based on phylogenetic and phenotypic characterization studies, Tichowtungia aerotolerans gen. nov., sp. nov. is proposed with S-5007T (=MCCC 1H00402T=KCTC 15876T) as the type strain, as the first representative of novel taxa, Tichowtungiales ord. nov., Tichowtungiaceae fam. nov. in Tichowtungiia class. nov.

Analysis of the intraimplant microflora of two-piece dental implants.

OBJECTIVE: Information about the spectrum of microorganisms in the intraimplant cavities of two-piece dental implants is scarce. The purpose of this study was to assess the intraimplant microflora of two-piece dental implants by conventional biochemical testing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and 16 s rDNA gene sequencing. MATERIALS AND METHODS: Ten patients (six men and four women; average age = 66.7 years; age range = 58-78 years) received 35 two-piece titanium implants carrying ball attachments. Biofilm sampling was performed with sterile microbrushes, and nonadherent microbial samples were obtained by injection and reuptake of predefined volumes of NaCl solution. The samples were cultured and analyzed by conventional biochemical testing, MALDI-TOF MS, and 16 s rDNA gene sequencing. RESULTS: Of the 103 species detected, 27 and 33 were identified only in the biofilm and nonadherent microbial samples, respectively. Forty-three species were identified in both types of samples. CONCLUSIONS: Two-piece dental implants harbored a broad spectrum of gram-positive and gram-negative aerobes and anaerobes, especially rods and cocci. CLINICAL RELEVANCE: These findings confirm bacterial translocation from the oral cavity to intraimplant cavities. Microbiological methods as used in this study are necessary to reveal the complete vital microflora of intraimplant cavities.

Phycisphaera mikurensis gen. nov., sp. nov., isolated from a marine alga, and proposal of Phycisphaeraceae fam. nov., Phycisphaerales ord. nov. and Phycisphaerae classis nov. in the phylum Planctomycetes.

Three strains, FYK2301M01(T), FYK2301M18 and FYK2301M52, all being Gram-negative, spherical, motile and facultatively anaerobic, were isolated from a marine alga (Porphyra sp.) collected on Mikura Island, Japan. Colonies of the strains were circular and pink-pigmented on Marine Agar 2216 (Difco) at 25 degrees C. Cells of the strains reproduced by binary fission. The G+C content of the DNA was 73 mol%. The major isoprenoid quinone was MK-6. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strains are the members of the WPS-1 group (Nogales et al., 2001) comprising no validly described taxa within the phylum Planctomycetes. The highest similarity value of the 16S rRNA gene sequences of the strains to those in the established bacterial taxa was only 78.7% to Planctomyces brasiliensis DSM 5305(T). From the taxonomic data obtained in this study, it is proposed that the new marine isolates be placed in a novel genus and species named Phycisphaera mikurensis gen. nov., sp. nov. within a new family, order and class Phycisphaeraceae fam. nov., Phycisphaerales ord. nov. and Phycisphaerae classis nov. in the phylum Planctomycetes. The type strain of Phycisphaera mikurensis is FYK2301M01(T) (= NBRC 102666(T) = KCTC 22515(T)).

European surveillance study on antimicrobial susceptibility of Gram-positive anaerobic cocci.

Gram-positive anaerobic cocci (GPAC) are a heterogeneous group of microorganisms frequently isolated from local and systemic infections. In this study, the antimicrobial susceptibilities of clinical strains isolated in 10 European countries were investigated. After identification of 299 GPAC to species level, the minimum inhibitory concentrations of penicillin, imipenem, clindamycin, metronidazole, vancomycin and linezolid were determined by the agar dilution method according to the Clinical and Laboratory Standards Institute. The majority of isolates were identified as Finegoldia magna and Parvimonas micra (formerly Peptostreptococcus micros), isolated from skin and soft tissue infections. All isolates were susceptible to imipenem, metronidazole, vancomycin and linezolid. Twenty-one isolates (7%) were resistant to penicillin (n=13) and/or to clindamycin (n=12). Four isolates were resistant to both agents. The majority of resistant isolates were identified as F. magna and originated from blood, abscesses and soft tissue infections.

Digestive tract microbiota in healthy volunteers.

PURPOSE: The aim of this study was to standardize the methods of sample collection of mucus from the digestive tract and to determine the microbiota in healthy volunteers from Brazil, collecting samples from the mouth, esophagus, stomach, duodenum, jejunum, ileum, colon, and rectum. METHODS: Microbiota of selected healthy volunteers from the oral cavity (n=10), the esophagus (n=10), the upper digestive tract (n=20), and the lower digestive tract (n=24) were evaluated through distinct collection methods. Collection methods took into account the different sites, using basic scraping and swabbing techniques, stimulated saliva from the oral cavity, irrigation-aspiration with sterile catheters especially designed for the esophagus, a probe especially designed for upper digestive tract, and a special catheter for the lower digestive tract. RESULTS: (i) Mixed microbiota were identified in the oral cavity, predominantly Gram-positive aerobic and anaerobic cocci; (ii) transitional flora mainly in the esophagus; (iii) Veillonella sp, Lactobacillus sp, and Clostridium sp in the stomach and duodenum; (iv) in the jejunum and upper ileum, we observed Bacteroides sp, Proteus sp, and Staphylococcus sp, in addition to Veillonella sp; (v) in the colon, the presence of "nonpathogenic" anaerobic bacteria Veillonella sp (average 10(5) UFC) indicates the existence of a low oxidation-reduction potential environment, which suggests the possibility of adoption of these bacteria as biological markers of total digestive tract health. CONCLUSIONS: The collection methods were efficient in obtaining adequate samples from each segment of the total digestive tract to reveal the normal microbiota. These procedures are safe and easily reproducible for microbiological studies.

[Infectious spondylodiskitis and epidural abscess after spinal puncture for pilonidal sinus excision]. / Espondilodiscitis infecciosa y absceso epidural después de una punción subaracnoidea para escisión de sinus pilonidal.

Vertebral infections after spinal puncture are rare and often inadequately documented. Their incidence does not exceed that of spontaneous epidural abscesses and we should therefore be cautious about assuming a causal relation between puncture and an abscess. After analyzing 10 published cases we saw that only half of them reported on aseptic conditions and only 2 patients seem to have had a prior infection. In 3 cases, the abscesses appeared after technically simple punctures whereas half the reports did not even mention the type of puncture. This complication should be considered whenever a patient develops back pain and fever, even if there are no neurological deficits and even after a simple spinal puncture. Given that early diagnosis and treatment have proven effective in improving the survival rate and reducing the rate of neurological sequelae, magnetic resonance images should be ordered urgently so that early treatment can be established.

Victivallis vadensis gen. nov., sp. nov., a sugar-fermenting anaerobe from human faeces.

A novel strictly anaerobic, cellobiose-degrading bacterium, strain CelloT, was isolated from a human faecal sample by combining enrichments in liquid and soft-agar basal media. A noteworthy characteristic was its inability to grow on normal agar plates and in roll tubes. The cells were coccus shaped and non-motile, with an extracellular slime layer. Growth of strain CelloT occurred between 20 and 40degrees C, with optimal growth at 37 degrees C. The pH range for growth was 5-7.5 with an optimum at 6.5. In pure culture, strain CelloT could only grow on a variety of sugars. Glucose was converted to acetate, ethanol and H2. The doubling time on glucose was 0.5 h. In a syntrophic co-culture with Methanospirillum hungatei strain JF-1T, strain CelloT converted glucose to acetate and H2. The G+C content was 59.2 mol%. 16S rDNA analysis revealed that the closest relatives of strain CelloT were two uncultured bacteria from anaerobic digesters, both with 94% 16S rDNA sequence similarity. The closest cultured representatives belong to genera of the bacterial division 'Verrucomicrobia'. The name Victivallis vadensis gen. nov., sp. nov. is proposed for strain CelloT (=DSM 14823T =ATCC BAA-548T).

[Metabolism of rumen microbial populations formed on biosubstrates with different assimilation under the effect of pentachlorophenol]. / Metabolizm mikrobnykh populiatsii rubtsia, sformovanykh na biosubstratakh riznoï dostupnosti, za diï pentakhlorfenolu.

It has been established that metabolism of mixed microbial population formed on easy assimilated sources of energy and nitrogen (concentrate diet) progressed on higher level. There is increase of amilolytic activity, formation of lactate, ammonia, low molecular carbonic acids with predomination of propionate molar fraction. The increased resistance to effect of pentachlorophenol (PCP) is characteristic nature of the latter. The role of the most resistant synthrophic bacteria to PCP increases. The pure strains of Streptococcus bovis and Megasphaera elsdenii do not stop metabolism at 100 microM of PCP. Mixed population of microorganisms formed on hard accessible biosubstrates (cellulose) and Butyrivibrio fibrisolvens have the increased cellulosolytic activity and while the high sensibility even to low doses of PCP (10-40 microM) is observed. It has been supposed that mechanism of PCP effect is ambiguous for various species of microbial complex of rumen. It's effect strength on all main chains of metabolism (membrane transport, energetic exchange, protein biosynthesis, etc.) significantly depends on capacity of pool of metabolic intermediates formed as a result of definite program of biotechnology of nurture, but significantly decreases the harmful effect of biocides.

Serogroups of the beer spoilage bacterium Megasphaera cerevisiae correlate with the molecular weight of the major EDTA-extractable surface protein.

Megasphaera cerevisiae is a Gram-negative obligate anaerobe that causes turbidity and off-flavour and aroma in beer. Seven isolates of M. cerevisiae were obtained worldwide, and their extractable surface antigens were focused upon to determine if there is more than one serogroup of this bacterium. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of ethylenediaminetetraacetic acid (EDTA) bacterial extracts revealed a predominant protein with apparent molecular weights of 46,000, 45,000, and 43,000 for three, two, and two isolates, respectively. When mouse anti-serum generated against any of the EDTA extracts was reacted with denatured bacterial proteins in immunoblots, all bacterial isolates exhibited extensive cross-reactivity involving three antigens, one being the major EDTA-extractable protein. In contrast, when the sera were tested for surface reactivity with intact bacteria, three cross-reactivity groups were observed, with the groups individually comprised of bacteria having the same size major EDTA-extractable surface protein. When BALB/c mice immunized with a bacterium from each of the three serogroups were used for monoclonal antibody (Mab) hybridoma production, bacterial surface-reactive Mabs were obtained whose reactivities parallel the three polyclonal antibody-defined serogroups. Through combining these surface-reactive Mabs, it will be possible to rapidly detect and identify beer contamination by M. cerevisiae belonging to any serogroup.

Removal of phenolic compounds from a petrochemical effluent with a methanogenic consortium.

A methanogenic consortium was used to degrade phenol and ortho- (o-) cresol from a specific effluent of a petrochemical refinery. This effluent did not meet the local environmental regulations for phenolic compounds (178 mg/L), oils and greases (61 mg/L), ammoniacal nitrogen (75 mg/L) or sulfides (3.2 mg/L). The consortium, which degrades phenol via its carboxylation to benzoic acid, was progressively adapted to the effluent. Despite the very high effluent toxicity (EC50 of 2% with Microtox), the adapted consortium degraded 97% of 156 mg/L phenol in the supplemented effluent after 13 days in batch cultures (serum bottle). The addition of proteose peptone to the effluent is essential for phenol degradation. o-cresol was also transformed but not meta- or para-cresols. A continuous flow fixed-film anaerobic bioreactor was developed with the consortium. Treating the effluent with the bioreactor reduced phenol and phenolic compounds concentrations by 97 and 83%, respectively, for a hydraulic residence time of 6 h. This treatment also reduced by about half the effluent toxicity. Oils and greases and ammoniacal nitrogen were not affected. Similar microbiological forms were observed in serum bottles and in the bioreactors with or without the petrochemical effluent. These results indicate that this methanogenic consortium can treat efficiently the phenolic compounds in this specific petrochemical effluent.

Isolation of Lautropia mirabilis from oral cavities of human immunodeficiency virus-infected children.

Lautropia mirabilis, a pleomorphic, motile, gram-negative coccus, has been isolated from the oral cavities of 32 of 60 (53.3%) children infected with human immunodeficiency virus (HIV) and 3 of 25 (12.0%) HIV-uninfected controls; the association of L. mirabilis isolation with HIV infection is significant (P < 0.001). All children in the study, both HIV-infected children and controls, were born to HIV-infected mothers. The presence of this bacterium was not associated with clinical disease in these children. The HIV-infected children with L. mirabilis did not differ from the HIV-infected children without L. mirabilis in immunological status, clinical status, or systemic medications. The role of HIV infection itself or concomitant factors in the establishment of L. mirabilis in the oral cavity remains to be elucidated.

Isolation of Lautropia mirabilis from sputa of a cystic fibrosis patient.

An aggregate-forming coccus, isolated twice as the predominant microorganism in sputa from a cystic fibrosis patient on consecutive days, was shown to belong to the species Lautropia mirabilis on the bases of similarities of 16S rRNA gene sequences and phenotype. These isolates of L. mirabilis appear to be the first reported from a patient with cystic fibrosis and outside of Denmark.

Glutaconate CoA-transferase from Acidaminococcus fermentans: the crystal structure reveals homology with other CoA-transferases.

BACKGROUND: Coenzyme A-transferases are a family of enzymes with a diverse substrate specificity and subunit composition. Members of this group of enzymes are found in anaerobic fermenting bacteria, aerobic bacteria and in the mitochondria of humans and other mammals, but so far none have been crystallized. A defect in the human gene encoding succinyl-CoA: 3-oxoacid CoA-transferase causes a metabolic disease which leads to severe ketoacidosis, thus reflecting the importance of this family of enzymes. All CoA-transferases share a common mechanism in which the CoA moiety is transferred from a donor (e.g. acetyl CoA) to an acceptor, (R)-2-hydroxyglutarate, whereby acetate is formed. The transfer has been described by a ping-pong mechanism in which CoA is bound to the active-site residue of the enzyme as a covalent thiol ester intermediate. We describe here the crystal structure of glutaconate CoA-transferase (GCT) from the strictly anaerobic bacterium Acidaminococcus fermentans. This enzyme activates (R)-2-hydroxyglutarate to (R)-2-hydroxyglutaryl-CoA in the pathway of glutamate fermentation. We initiated this project to gain further insight into the function of this enzyme and the structural basis for the characteristics of CoA-transferases. RESULTS: The crystal structure of GCT was solved by multiple isomorphous replacement to 2.55 A resolution. The enzyme is a heterooctamer and its overall arrangement of subunits can be regarded as an (AB)4tetramer obeying 222 symmetry. Both subunits A and B belong to the open alpha/beta-protein class and can be described as a four-layered alpha/alpha/beta/alpha type with a novel composition and connectivity of the secondary structure elements. The core of subunit A consists of seven alpha/beta repeats resulting in an all parallel central beta sheet, against which helices pack from both sides. In contrast, the centre of subunit B is formed by a ninefold mixed beta sheet. In both subunits the helical C terminus is folded back onto the N-terminal domain to form the third layer of helices. CONCLUSIONS: The active site of GCT is located at the interface of subunits A and B and is formed by loops of both subunits. The funnel-shaped opening to the active site has a depth and diameter of about 20 A with the catalytic residue, Glu54 of subunit B, at the bottom. The active-site glutamate residue is stabilized by hydrogen bonds. Despite very low amino acid sequence similarity, subunits A and B reveal a similar overall fold. Large parts of their structures can be spatially superimposed, suggesting that both subunits have evolved from a common ancestor.

Conversion of glutaconate CoA-transferase from Acidaminococcus fermentans into an acyl-CoA hydrolase by site-directed mutagenesis.

The heterooctameric (alphabeta)4 glutaconate CoA-transferase (EC 2.8.3.12) from the anaerobic bacterium Acidaminococcus fermentans catalyses the transfer of CoASH from acetyl-CoA to the 1-carboxylate of glutaconate. During this reaction the glutamate residue 54 of the beta-subunit (betaE54) forms a CoA-ester. The single amino acid replacement betaE54D resulted in a drastic change of enzymatic function. The CoA-transferase activity decreased from 140 to less than 0.01 s(-1), whereas the acyl-CoA hydrolase activity increased from less than 0.01 to 16 s(-1). The new enzyme was able to catalyse the hydrolysis of glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA. Since the mutants betaE54A and betaE54N showed neither acyl-CoA hydrolase nor CoA-transferase activity, it was concluded that the aspartate carboxylate of the mutant betaE54D acted as a general base which facilitated the attack of water at the thiolester carbonyl. Surprisingly, Km for glutaryl-CoA hydrolysis by the mutant (0.7 microM) as compared to CoA-transfer by the wild-type (28 microM) was 40 times lower. A 65 kDa protein, obtained by fusing the genes, gctA-gctB, coding for glutaconate CoA-transferase, retained 30% of the wild-type activity. Comparison of the amino acid sequences of 13 related enzymes demonstrated that Nature already has applied gene fusion in the case of pig heart CoA-transferase and has been using the E --> D mutation for catalysis by a yeast acetyl-CoA hydrolase.

Anaerobic cocci of clinical importance.

The current knowledge is reviewed concerning the anaerobic cocci, in particular those of clinical relevance. The anaerobic cocci are defined and their current taxonomic positions discussed. It is clear that new genera and species await to be characterised fully. The overwhelming majority found in clinical material belong to the genus Peptostreptococcus, with the remainder belonging to the veillonellae and, possibly, ruminococci. Human infections with other anaerobic cocci are extremely rare. Their morphology, metabolism and culture, and role in clinical infections are assessed. The methods for isolation and identification, which for some species are difficult, are presented, together with brief summaries of the clinically important species. The review concludes with the current status of antibiotic susceptibilities and the methods used to test susceptibility in vitro. There is no current consensus as to which susceptibility test method is the method of choice.

Sequence of the gene encoding the 16S rRNA of the beer spoilage organism Megasphaera cerevisiae.

The 16S ribosomal RNA gene from the beer-spoilage organism, Megasphaera cerevisiae was polymerase chain reaction (PCR)-amplified and sequenced. Analysis confirmed the phylogenetic position of M. cerevisiae as a sister taxon of Megasphaera elsdenii, within the obligately anaerobic, Gram-negative cocci. The sequence obtained should facilitate the development of DNA probes for early detection of this spoilage organism.

Interaction of the rumen fungus Orpinomyces joyonii with Megasphaera elsdenii and Eubacterium limosum.

The degradation and fermentation of microcrystalline cellulose were studied in monoculture of the polycentric anaerobic fungus Orpinomyces joyonii and in co-cultures with the rumen bacteria Megasphaera elsdenii and Eubacterium limosum. More than 25% of cellulose hydrolysis products (glucose and cellodextrins) were released by the fungus into the medium after 8 d of cultivation. These products were metabolized by bacteria in mixed cultures. In co-culture with the fungus M. elsdenii and E. limosum increased the extent of microcrystalline cellulose degradation by 10.12% and 7.96%, respectively. Biomass yield in co-cultures was increased by 89.9% and 59.4% for M. elsdenii and E. limosum. Y cellulose for fungus alone was 52.29 g dry matter mol-1 glucose. These values were 64.93 and 55.92 g mol-1 glucose unit in co-culture with M. elsdenii and E. limosum, respectively.

The apical border plaque in severe periodontitis. An ultrastructural study.

This study concerns the apical border (AB) plaque in relation to severe forms of periodontitis (SP), including juvenile, post-juvenile, and rapidly progressing periodontitis. Twenty-four (24) teeth from 16 patients with SP were examined by transmission electron microscopy (TEM). The AB was not discrete, with islands of bacteria in the so-called plaque-free zone (PFZ). Coronal to the AB the established plaque consisted of a layer of Gram-positive cocci and ghost cells and a superficial layer mainly of Gram-negative morphotypes, including cocci, rods, filaments, fusiforms, and spirochetes. The most apical apparently intact organisms in the PFZ were in bacterial islands or in isolation and were predominantly Gram-negative cocci and rods, with ghost cells in abundance. Ruthenium red, alcian blue-lanthanum nitrate, and safranin O were used to label matrix polyanionic macromolecules, and periodic acid (thiosemicarbazide) silver proteinate for intracellular polysaccharide (IPS). The matrix components were mainly fibrillar. Many intact bacteria exhibited extracellular polysaccharides or glycocalyces associated with their cell wall, and cytoplasmic IPS granules. The latter varied in distribution and were evident even in the most apically advanced intact microorganisms. The results indicate that IPS and some matrix features of the apical border plaque in severe periodontitis in certain aspects resemble those of sub-contact area plaque on children's teeth, in health or associated with early chronic gingivitis, and with those in chronic adult periodontitis. They also suggest the establishment of acidic regions in the microniche at the bottom of the periodontal pocket in the various forms of periodontitis differing in rate of progression. It was concluded that there was a limited range of intact bacterial morphotypes in the apical border plaque in severe periodontitis, similar to those in chronic adult periodontitis.

Lautropia mirabilis gen. nov., sp. nov., a gram-negative motile coccus with unusual morphology isolated from the human mouth.

An organism that seems to be identical to Orskov's 'Sarcina mirabilis' [Orskov, J. (1930) Acta Pathol Microbiol Scand Suppl III, 519-541] has been rediscovered in specimens from the upper respiratory tract of humans. Six strains were studied, and the results, which conformed to Orskov's description of S. mirabilis, were as follows. Rough to smooth colonies grow on many plated media and show extremely polymorphic cell morphology with round cells with diameters from 1 to > 10 microns. The smallest cells were often motile with circular movements. Strains were Gram-negative, facultatively anaerobic, oxidase and urease positive, and weakly catalase positive. Nitrate and nitrite were reduced, and glucose, fructose, sucrose and mannitol were fermented. Polysaccharide was produced on sucrose agar. Electron microscopy showed coccoid cells with a bundle of three to nine flagella, a Gram-negative cell-wall morphology, and aggregates of irregular cells held together by a common surface layer. The mean mol% (G+C) of the organisms was 65.0. 16S-ribosomal RNA sequencing revealed that the organism belongs to the beta subgroup of Proteobacteria, separate from all other described genera, but most closely related to Burkholderia. The name Lautropia mirabilis is proposed for this organism.