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Successive changes in community structure of an ethylbenzene-degrading sulfate-reducing consortium.

The microbial community structure and successive changes in a mesophilic ethylbenzene-degrading sulfate-reducing consortium were for the first time clarified by the denaturing gradient gel electrophoresis (DGGE) analysis of the PCR amplified 16S rRNA gene fragments. At least ten bands on the DGGE gel were detected in the stationary phase. Phylogenetic analysis of the DGGE bands revealed that the consortium consisted of different eubacterial phyla including the delta subgroup of Proteobacteria, the order Sphingobacteriales, the order Spirochaetales, and the unknown bacterium. The most abundant band C was closely related to strain mXyS1, an m-xylene-degrading sulfate-reducing bacterium (SRB), and occurred as a sole band on DGGE gels in the logarithmic growth phase that 40% ethylbenzene was consumed accompanied by sulfide production. During further prolonged incubation, the dominancy of band C did not change. These results suggest that SRB corresponds to the most abundant band C and contributes mainly to the degradation of ethylbenzene coupled with sulfate reduction.

Benzene Derivatives↗

Diversity and community structure within anoxic sediment from marine salinity meromictic lakes and a coastal meromictic marine basin, Vestfold Hilds, Eastern Antarctica.

16S rDNA clone library analysis was used to examine the biodiversity and community structure within anoxic sediments of several marine-type salinity meromictic lakes and a coastal marine basin located in the Vestfolds Hills area of Eastern Antarctica. From 69 to 130 (555 total) 16S rDNA clones were analysed from each sediment sample, and restriction fragment length polymorphism (RFLP) and sequence analysis grouped the clones into 202 distinct phylotypes (a clone group with sequence similarity of >0.98). A number of phylotypes and phylotype groups predominated in all libraries, with a group of 10 phylotypes (31% of clones) forming a novel deep branch within the low G+C Gram-positive division. Other abundant phylotypes detected in several different clone libraries grouped with Prochlorococcus cyanobacteria, diatom chloroplasts, delta proteobacteria (Desulfosarcina group, Syntrophus and Geobacterl Pelobacter/Desulphuromonas group), order Chlamydiales (Parachlamydiaceae) and Spirochaetales (wall-less Antarctic spirochaetes). Most archaeal clones detected (3.1% of clones) belonged to a highly diverged group of Euryarchaeota clustering with clones previously detected in rice soil, aquifer sediments and hydrothermal vent material. Little similarity existed between the phylotypes detected in this study and other clone libraries based on marine sediment, suggesting that an enormous prokaryotic diversity occurs within marine and marine-derived sediments.

Antarctic Regions↗

Cloning of genes required for amino acid biosynthesis from Leptospira interrogans serovar icterohaemorrhagiae.

Leptospira interrogans belongs to a large family of important pathogens, which is part of the order Spirochaetales, a distinct group of eubacteria. In order to obtain a better understanding of the genetic organization of this species, we have constructed a DNA library of the serovar icterohaemorrhagiae, using the Escherichia coli vector pUC13. We have isolated Leptospira DNA fragments containing the genetic information required to complement strains of E. coli with defects in proline and leucine biosynthesis. While a 3.9 kb fragment which complemented proA also complemented proB, a 15 kb fragment complementing leuB could not complement other leu mutations. The L. interrogans origin of the cloned DNA fragments was confirmed by DNA-DNA hybridization. The hydridization was specific to the pathogenic species and was not seen with the saprophytic species L. biflexa.

Amino Acids↗

Chemical compositions of cell walls and polysaccharide fractions of spirochetes.

Cellular polysaccharide fractions of various representative members of genera of the family Spirochaetaceae were obtained by the ammonium hydroxide extraction method. The sugar composition of the polysaccharide preparations was complex and many kinds of sugars such as rhamnose, fucose, ribose, xylose, mannose, galactose, and glucose were detected in all of the spirochetes tested. Of particular interest was the presence of 4-O-methylmannose as a constituent polysaccharide in members of the genus Leptospira. This sugar was not detected in the polysaccharides of Spirochaeta, Borrelia, and Treponema. The chemical compositions of cell wall fractions were also examined. 4-O-Methylmannose was detected in the cell wall polysaccharides of the genus Leptospira but not in cell walls prepared from the Spirochaeta, Borrelia, and Treponema. The diaminopimelic acid present in cell wall peptidoglycans of the genus Leptospira was meso-diaminopimelic acid (A2pm). The molar ratios of alanine, glutamic acid, A2pm, glycine, muramic acid, and glucosamine in leptospiral cell walls were found to be approximately 2:1:1:1:1:1. In contrast to the Leptospira, the peptidoglycans of genera Spirochaeta, Borrelia, and Treponema contained ornithine (Orn) but not A2pm. Since 4-O-methylmannose and A2pm were found in the cell wall fractions of genus Leptospira but not in Spirochaeta, Borrelia, or Treponema, it was suggested that the chemical compositions of the cell wall might become an important criterion for the chemotaxonomy of Spirochaetales.

Carbohydrates↗

Molecular analysis of microbial communities in mobile deltaic muds of Southeastern Papua New Guinea.

A culture-independent examination of microbial diversity in mobile deltaic sediments from the Gulf of Papua, Papua New Guinea, was conducted by sequence analysis of 16S rDNA clone library. Universal small subunit primers were used to amplify DNA extracted from the sediment. Of 91 clones randomly selected from the library, 33 contained unique non-chimeric sequences. Analysis of these unique sequences showed that the majority of them belonged to bacteria (94.5%), with proteobacteria being the dominant division (74.8%). One sequence belonging to Crenarchaeota and another to Euryarchaeota were found as well. Bacterial sequences belonging to the following major divisions were identified: Cytophaga-Flexibacter-Bacteroides (5.5%), low GC Gram-positives (1.1%), Nitrospira group (3.3%), OP8 candidate division (2.2%), OS-K candidate division (2.2%), Planctomyces (1.1%), alpha, delta, gamma and epsilon-subdivisions of Proteobacteria (4.4, 20.9, 19.8 and 23.1%, respectively) and Spirochaetales (4.4%). The composition of 16S rDNA library of the Gulf of Papua sediments is similar to those for deepwater sediments from around the Japanese Islands, indicating a possible cosmopolitan nature of microbial communities in suboxic and anoxic sediments of the Pacific. It appears that many Papua/New Guinea sequences are loosely affiliated with bacteria involved in sulfur cycling, thus supporting the hypothesis that the sulfur cycle may be important in early diagenesis of suboxic sediments.

Journal Article↗

Nucleotide sequence analysis of a gene cloned from Leptospira biflexa serovar patoc which complements an argE defect in Escherichia coli.

The genus Leptospira, as a member of the order Spirochaetales, forms one of the most ancient evolutionary branches of the eubacteria. These spirochetes are morphologically and physiologically different from most eubacteria, and little is known about Leptospira genetics. In this communication, we report the first nucleotide sequence of a Leptospira gene. A gene which complements an argE mutation in Escherichia coli was isolated from a plasmid-based genomic library composed of Leptospira biflexa serovar patoc DNA. The functional region for the complementing activity was localized by transposon mutagenesis and restriction enzyme mapping and by subcloning. Nucleotide sequence analysis indicated a single open reading frame within the region containing argE complementing activity. The size of the predicted protein, 31,071 daltons, was in excellent agreement with data obtained from coupled transcription-translation reactions primed with cloned L. biflexa DNA. One surprising result was that the predicted amino acid sequence of this protein closely resembles portions of the beta' subunits of RNA polymerases from bacteria and chloroplasts.

Amino Acid Sequence↗

Leptospirosis in Portugal: epidemiology from 1991 to 1997.

The term leptospirosis describes a group of diseases caused by members of the order Spirochaetales, mainly the species Leptospira interrogans. Infection can be asymptomatic but disease of differing severity and clinical features (often suggestive of menin

Journal Article↗

Nucleotide sequence of the Treponema pallidum eno gene.

We determined the nucleotide sequence of the enolase (eno) gene of Treponema pallidum, the noncultivable agent of syphilis. The deduced amino acid sequence of T. pallidum enolase (Eno) is 432 amino acids long with a predicted molecular mass of 46.7 kDa. The Eno amino acid sequence has a high degree of homology to the amino acid sequences of prokaryotic and eukaryotic Eno. This is the first eno sequence reported for a bacterium in the order Spirochaetales.

Amino Acid Sequence↗

Reduction in inflammation following blockade of CD18 or CD29 adhesive pathways during the acute phase of a spirochetal-induced colitis in mice.

Colitis develops in mice infected with Brachyspira (Serpulina) hyodysenteriae. Numerous granulocytes (PMNs) are evident in cecal tissue sections 24-48 h post-infection. The role of PMNs was assessed by utilizing monoclonal antibodies specific for CD18 or CD29 to block PMN recruitment. Macroscopic lesions were less severe in mice treated with either monoclonal antibody compared to lesions observed in isotype control-treated mice. While these monoclonal antibodies may inhibit extravasation of other leukocytes, the central role of PMNs was further demonstrated in that colitis was reduced following neutrophil depletion. There was less edema and epithelial erosions in ceca of mice receiving anti-Ly6G, -CD18 or -CD29 monoclonal antibody compared to mice receiving the control. Moreover, there was a significant reduction in PMN infiltration in tissues of mice treated with anti-CD18. The reduction in infiltrating PMNs did not result from downregulation of neutrophil chemoattractant MIP-2 expression in anti-CD18-treated mice. In contrast, PMN recruitment into the cecum was apparently CD29-independent. It is noteworthy that the number of PMNs observed in anti-CD18-treated mice was significantly higher than observed in non-infected mice. The data provide evidence for a threshold number of PMNs necessary for lesion development and indicate that CD18, but not CD29, adhesive pathways are crucial for PMN recruitment in bacterial colitis.

Acute Disease↗

Serological diagnosis of erythema migrans disease and related disorders.

An indirect immunofluorescence technique for the determination of antibodies against ixodid tick spirochetes is described. Differences in the reactivity between Ixodes ricinus spirochete and Ixodes dammini spirochete antigens were not observed. Cross-reacting antibodies against Treponema pallidum and Treponema phagedenis can be eliminated by quantitative absorption with T. phagedenis. Cross-reactions with leptospira were not observed by immunofluorescence. In the IgM test, false negative reactions caused by high-titered specific IgG antibodies or false positive reactions caused by rheumatoid factor occur. This can be avoided by testing the IgM fraction (19S-IgM-test) or using sera previously treated with anti-IgG serum. Significantly elevated antibody titers against ixodid tick spirochetes were observed in 45% of 44 cases with erythema migrans disease, in 72% of 29 cases of lymphocytic meningoradiculitis, in all of nine patients with acrodermatitis chronica atrophicans and in all of four investigated patients with lymphocytoma (lymphadenosis benigna cutis).

Acrodermatitis↗

Unusual in vitro formation of cyst-like structures associated with human intestinal spirochaetosis.

Spirochaetes were isolated from rectal biopsies of three patients and successfully cultured. Enzymatic reactions and electron microscopy revealed spirochaetes resembling Brachyspira aalborgi. Examination of ultrathin sections of centrifugates of cultured spirochaetes yielded unusual cyst-like structures with an outer double membrane containing spirochaetes in different developmental stages. The protoplasmic cylinders seemed to originate from a large electron-dense focus in the cyst-like structures. The axial fibrils occurred as loosely distributed solitary structures in the cyst-like structures. Cysts were not found in the biopsy tissue. The encystment of the spirochaetes could be related to their protection, multiplication, spread and transmission.

Colitis↗

The Brachyspira hyodysenteriae ftnA gene: DNA vaccination and real-time PCR quantification of bacteria in a mouse model of disease.

The nucleotide sequence of the Brachyspira hyodysenteriae ftnA gene, encoding a putative ferritin protein (FtnA), was determined. Analysis of the sequence predicted that this gene encoded a protein of 180 amino acids. RT-PCR and Western blot showed that the ftnA gene was expressed in B. hyodysenteriae, and evidence suggests that FtnA stores iron rather than haem. ftnA was delivered as DNA and recombinant protein vaccines in a mouse model of B. hyodysenteriae infection. Vaccine efficacy was monitored by caecal pathology and quantification of B. hyodysenteriae numbers in the caeca of infected mice by real-time PCR.

Amino Acid Sequence↗

Clinical significance of human intestinal spirochetosis--a morphologic approach.

Intestinal spirochetosis (IS) is a condition defined morphologically by the presence of spirochetal microorganisms attached to the apical cell membrane of the colonic and rectal epithelium. Intestinal spirochetes comprise a heterogeneous group of bacteria. In humans Brachyspira aalborgi and Brachyspira pilosicoli predominate. Prevalence rates of IS are low where living standards are high, in contrast to poorly developed areas where IS is common. Homosexuals and HIV-infected individuals are at high risk of being colonized. Clinical significance in individual cases has remained unclear up to now. A review of the literature reveals that invasion of spirochetes beyond the surface epithelium is associated with gastrointestinal symptoms which respond to antibiotic treatment (metronidazole), whereas patients lacking this feature are mostly asymptomatic. Homosexual and HIV-positive men are more likely to be symptomatic irrespective of invasion. Rare cases of spirochetemia and multiple organ failure have been reported in critically ill patients.

Bacteremia↗

Spirochetes isolated from dairy cattle with papillomatous digital dermatitis and interdigital dermatitis.

Two groups of spirochetes were isolated from papillomatous digital dermatitis (PDD) lesions in dairy cattle. The two groups could be readily differentiated on the basis of morphologic and immunologic characteristics and enzymatic activity. A spirochete isolated from an interdigital dermatitis (IDD) lesion appeared morphologically and antigenically similar to spirochetes in one of the PDD groups and exhibited an identical enzyme activity pattern. The two groups of PDD spirochetes had characteristics most consistent with the genus Treponema. The PDD and IDD isolates differed morphologically from previously described bovine Treponema spp. Although spirochetes have been observed to be one of the predominant bacterial morphotypes in PDD and IDD and are found invading the stratum spinosum and dermal papillae in PDD lesions, the significance of these spirochetes in the etiopathogenesis of PDD and IDD is presently unknown.

Animals↗

Comparison of methods for antimicrobial susceptibility testing and MIC values for pleuromutilin drugs for Brachyspira hyodysenteriae isolated in Germany.

In Germany treatment of swine dysentery is hampered by Brachyspira hyodysenteriae strains showing elevated MIC values to the few antibiotics licensed. Therefore, susceptibility testing of clinical isolates is an important service to the swine practitioner. This study compares the established agar dilution procedure for antimicrobial susceptibility testing of this fastidious anaerobe to the broth microdilution test newly developed [Anim. Health Res. 2 (2001) 59; Vet. Microbiol. 84 (2002) 123; J. Clin. Microbiol. 41 (2003) 2596]. A total of 221 isolates were examined twice with either test procedure using tiamulin and valnemulin as antibiotics. Both methods gave reproducible results, and the MIC values for the reference strains B. hyodysenteriae B204 and Staphylococcus aureus ATCC 29213 corresponded to previously published data. However, the results for individual strains differed significantly for both tests (P < 0.001) with MIC values being on average one dilution step lower in the broth dilution method. The 221 strains used for comparing test procedures were isolated between 1989 and 2001. An additional 102 strains isolated in 2002 were tested only with the broth dilution procedure. A significant rise in the average MIC value for both pleuromutilins could be demonstrated when comparing earlier isolates to those from 2000 to 2001 (P < 0.05), while in 2002 the average MIC significantly decreased when compared to the value in 2000 (P < 0.05). However, strains with MIC values for tiamulin as high as 8 microg/ml (broth dilution) could still be isolated.

Animals↗

Protection of pigs from swine dysentery by vaccination with recombinant BmpB, a 29.7 kDa outer-membrane lipoprotein of Brachyspira hyodysenteriae.

Swine dysentery (SD) is an important endemic infection in many piggeries, and control can be problematic. In this study the efficacy of BmpB, a 29.7 kDa outer-membrane lipoprotein of Brachyspira hyodysenteriae, was evaluated as an SD vaccine. Non-lipidated BmpB was expressed in Escherichia coli as a histidine-tagged protein (His6-BmpB), or as an 8 kDa carboxy-terminal portion fused to maltose-binding protein (MBP-BmpB-F604). The purified proteins were emulsified with oil-based adjuvants for intramuscular (im) administrations. In experiment 1, 20 weaner pigs were vaccinated im with 1 mg of His6-BmpB. After 3 weeks, 10 received 1 mg of the protein orally (im/oral), and 10 received 1 mg im (im/im). Ten acted as unvaccinated controls. In experiment 2, 12 pigs were vaccinated im with 1 mg of His6-BmpB, and 12 with 1 mg of MBP-BmpB-F604. Three weeks later, each was given 1 mg of the same protein orally. Twelve pigs acted as unvaccinated controls. All pigs were challenged orally with B. hyodysenteriae 2 weeks after their second vaccination. In both experiments, all pigs vaccinated with His6-BmpB developed serum antibodies to BmpB, and oral administration provided boosting of im-induced serum antibody titres. In experiment 1, seven non-vaccinated control pigs developed dysentery and severe colitis. Three pigs vaccinated im/oral developed diarrhoea; two had severe colitis and one had mild lesions. Four pigs vaccinated im/im developed diarrhoea; one had severe colitis and the others had mild lesions. In experiment 2, six control pigs developed SD with severe colitis. Two His6-BmpB vaccinated pigs developed SD with mild colitis. Nine pigs vaccinated with MBP-BmpB-F604 developed SD and severe colitis. Overall, 50-70% of controls and 17-40% of His6-BmpB vaccinated pigs developed disease. Vaccination with MBP-BmpB-F604 did not induce serum titres against BmpB, nor confer protection. The incidence of disease for the three His6-BmpB vaccinated groups was significantly less (P = 0.047) than for the control groups, with a approximately 50% reduction. BmpB appears to have potential as an SD vaccine component.

Animals↗