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Complete mineralisation of dimethylformamide by Ochrobactrum sp. DGVK1 isolated from the soil samples collected from the coalmine leftovers.

A bacterial strain DGVK1 capable of using N,N-dimethylformamide (DMF) as sole source of carbon and nitrogen was isolated from the soil samples collected from the coalmine leftovers. The molecular phylogram generated using the complete sequence of 16S rDNA of the strain DGVK1 showed close links to the bacteria grouped under Brucellaceae family that belongs to alphaproteobacteria class. Specifically, the 16S rDNA sequence of strain DGVK1 has shown 97% similarity to Ochrobactrum anthropi LMG 3331 (D12794). This bacterium has also shown impressive growth on dimethylamine, methylamine, formaldehyde and formate that are considered to be the prominent catabolic intermediates of DMF. DMF degradation has led to the accumulation of ammonia and dimethylamine contributing to the increase of pH of the medium. The DMF-grown resting cells of Ochrobactrum sp. DGVK1 have also contributed for the release of ammonia when resting cell suspension was added to phosphate buffer containing DMF. Similar experiments done with the glucose-grown cultures have not produced ammonia and thus indicating the inducible nature of DMF-degrading enzymes in Ochrobactrum sp. DGVK1. Further, dimethylformamidase, dimethylamine dehydrogenase and methylamine dehydrogenase, the key enzymes involved in the degradation of DMF, were assayed, and the activities of these enzymes were found only in DMF-grown cultures further confirming the inducible nature of the DMF degradation. Based on these results, DMF degradation pathway found in Ochrobactrum sp. DGVK1 has been proposed.

Biodegradation, Environmental↗

Non-specific biodegradation of the organophosphorus pesticides, cadusafos and ethoprophos, by two bacterial isolates.

An enrichment culture technique was used for the isolation of microorganisms responsible for the enhanced biodegradation of the nematicide cadusafos in soils from a potato monoculture area in Northern Greece. Mineral salts medium supplemented with nitrogen (MSMN), where cadusafos (10 mg l(-1)) was the sole carbon source, and soil extract medium (SEM) were used for the isolation of cadusafos-degrading bacteria. Two pure bacterial cultures, named CadI and CadII, were isolated and subsequently characterized by sequencing of 16S rRNA genes. Isolate CadI showed 97.4% similarity to the 16S rRNA gene of a Flavobacterium strain, unlike CadII which showed 99.7% similarity to the 16S rRNA gene of a Sphingomonas paucimobilis. Both isolates rapidly metabolized cadusafos in MSMN and SEM within 48 h with concurrent population growth. This is the first report for the isolation and characterization of soil bacteria with the ability to degrade rapidly cadusafos and use it as a carbon source. Degradation of cadusafos by both isolates was accelerated when MSMN was supplemented with glucose. In contrast, addition of succinate in MSMN marginally reduced the degradation of cadusafos. Both isolates were also able to degrade completely ethoprophos, a nematicide chemical analog of cadusafos, but did not degrade the other organophosphorus nematicides tested such as isazofos and isofenphos. Inoculation of a soil freshly treated with cadusafos or ethoprophos (10 mg l(-1)) with high inoculum densities (4.3 x 10(8) cells g(-1)) of Sphingomonas paucimobilis resulted in the rapid degradation of both nematicides. These results indicate the potential of this bacterium to be used in the clean-up of contaminated pesticide waste in the environment.

Antinematodal Agents↗

Impact of chromium-contaminated wastewaters on the microbial community of a river.

The influence of chromium on the microbial community structure was analyzed in a river system subjected to long-term chromium contamination, by plating and by sequencing 16S rRNA genes cloned from DNA extracted from the river sediments. We also analyzed the influence of chromium on the ability of the microbial community to resist and reduce Cr(VI) and on its resistance to antibiotics. Shifts in the microbial community structure were analyzed by amplified ribosomal DNA restriction analysis fingerprinting. The isolates obtained were phylogenetically related to Actinobacteria, Firmicutes, Bacteroidetes and Proteobacteria, whereas Acidobacteria and Deltaproteobacteria were only revealed by clone analyses. Cr(VI)-resistant and Cr(VI)-reducing strains were isolated in all sites examined. However, each sample site had a microbial community with a different antibiotic resistance pattern. Our study seems to indicate that in this river ecosystem chromium influenced the microbial communities, altering some of their functional characteristics, such as the percentage of the microbial community able to resist or to reduce Cr(VI) and the phylogenetic groups isolated, but it did not affect the structural diversity. Furthermore, the concentration of Cr(VI) in the sediments could not be correlated with a lower number of bacteria or lower index of generic diversity, neither with the ability of the microbial community to resist or to reduce higher Cr(VI) concentrations.

Anti-Bacterial Agents↗

T-RFLP analysis of bacterial communities in cyclodextrin-amended bioreactors developed for biodegradation of polychlorinated biphenyls.

In this study, T-RFLP analysis was used to determine the structure and spatial distribution of the indigenous bacterial community of an actual-site PCB-contaminated soil treated in aerobic packed-bed loop reactors (PBLRs) in the absence or in the presence of a mixture of randomly methylated beta-cyclodextrins (RAMEB) at 0.5 or 1% w/w. RAMEB was found to significantly enhance the aerobic bioremediation of soil with effects that increased proportionally with the concentration at which it was applied. At the end of treatment (180 days), T-RFLP analysis of the soil samples collected from the top and bottom regions of the PBLRs showed a series of 50 single T-RFs. Remarkably, the number of T-RFs was significantly lower (13-22) in samples collected from different sections of the RAMEB-amended bioreactors with respect to equivalent samples collected from the RAMEB-free reactor. Cluster analysis based on the presence or the absence of T-RFs peaks revealed high similarity, inside each reactor, between the top and bottom parts of its soil bed. Soil samples collected at the top and bottom regions of the two bioreactors amended with RAMEB, clustered together while the equivalent samples of the bioreactor without RAMEB formed a separate cluster which was distantly related to the soil samples obtained from the parallel amended bioreactor. Notably, T-RFLP analyses combined with extensive sequencing of 16S rDNA allowed us to tentatively allocate a series of bacterial species corresponding to specific peaks of the T-RFLP profiles and to determine their phylogenetic affiliation.

Biodegradation, Environmental↗

A RAPD-based comparison of thermophilic bacilli from milk powders.

The similarity of strains of thermophilic Geobacillus stearothermophilus (formerly Bacillus stearothermophilus), Anoxybacillus flavithermus (formerly Bacillus flavothermus), Bacillus licheniformis and Bacillus subtilis isolated from separate milk powder production runs from multiple factories was examined using a random amplified polymorphic DNA (RAPD) protocol. As a result of the analysis of the RAPD fingerprints and data relating to general growth and biochemical tests, over 98% of the 1470 isolates examined (grown at 55 degrees C) were assigned to the species G. stearothermophilus, A. flavithermus, B. licheniformis and B. subtilis. The G. stearothermophilus isolates were identified as being nearly identical to G. stearothermophilus (DSMZ 22; equivalent to ATCC 12980), or G. stearothermophilus var. calidolactis (DSMZ 1550). Three groups of isolates were found to be related to A. flavithermus (DSMZ 2641) by partial small ribosomal subunit (16S) sequence comparisons and shown to be interrelated by RAPD analyses with multiple primer sets. The thermophilic isolates of B. licheniformis were positively identified by comparison with type strains of B. licheniformis DSMZ 13 and DSMZ 8785. All of the B. subtilis strains shared bands in their RAPD profiles and were similar to a common B. subtilis type strain (DSMZ 10 and DSMZ 347). Overall, the most common and prevalent group of strains (group A) was demonstrated to be closely related to G. stearothermophilus (DSMZ 22).

Animals↗

Genotypic diversity among Bacillus licheniformis strains from various sources.

Bacillus licheniformis is exploited industrially for the production of enzymes and has been shown to exhibit pathogenic properties. Because of these divergent characteristics, questions arise concerning intraspecies diversity. A comparative study by means of combined repetitive polymerase chain reaction, rpoB and gyrA sequencing, 16S rDNA targeted probe analysis, DNA-DNA hybridizations, gelatinase tests and antibiotic susceptibility tests was performed on a set of strains from diverse sources, including strains with pathogenic potential. B. licheniformis was found to consist of two lineages that are distinguished genotypically.

Bacillus↗

Nucleotide sequences of accessible regions of 23S RNA in 50S ribosomal subunits.

Nucleotide sequences around kethoxal-reactive guanine residues of 23S RNA in 50S ribosomal subunits have been determined. By use of the diagonal paper electrophoresis method )Noller, H.F. (1974), Biochemistry 13, 4694-4703), 41 ribonuclease T1 oligonucleotides, originating from about 25 sites, were identified and sequenced. These sites are single stranded and accessible in free 50S subunits, and are thus potential sites for interaction with functional ligands during protein synthesis. Examination of these sequences for potential intermolecular base-pairing reveals the following: (1) There are 19 possible complementary combinations between exposed sequences in 16S and 23S RNA containing more than 4 base pairs: 15 containing 5 base pairs and 4 containing 6 base pairs. Nine of these complementary combinations contain 16S RNA sequences which we have previously shown to be protected from kethoxall by 50S subunits (Chapman, N.M., and Noller, H.F. (1977), J. Mol. Biol. 109, 131-149). (2) One of the exposed sites in 23S RNA has a sequence which is complementary to the invariant GT psi CR sequence in tRNA.

Aldehydes↗

Plastid transformation in Lesquerella fendleri, an oilseed Brassicacea.

A plastid transformation protocol was developed for Lesquerella fendleri, a species with a high capacity for plant regeneration in tissue culture. Transformation vector pZS391B carried an aadA16gfp marker gene conferring streptomycin-spectinomycin resistance and green fluorescence under UV light. Biolistic transformation of 51 Lesquerella leaf samples, followed by spectinomycin selection, yielded two transplastomic clones. The AAD-GFP fusion protein, the marker gene product, was localized to chloroplasts by confocal laser microscopy. Fertile plants and seed progeny were obtained in line Lf-pZS391B-1. In the 51 samples a large number (108) of spontaneous mutants were identified. In five of the lines spectinomycin resistance was localized to a conserved stem structure by sequencing 16S rRNA genes. Success in L. fendleri, a wild oilseed species, extends plastid transformation beyond Arabidopsis thaliana in the Brassicaceae family.

Anti-Bacterial Agents↗

Isolated pituitary granuloma by atypical Mycobacterium in a nonimmunosuppressed woman.

A 32-year-old woman presented with a 10-day history of fever (38.0 degrees C), headaches, nausea, vomiting and a 6-month history of diabetes insipidus and amenorrhoea. Two months previously she had undergone a surgical drilling of the right mastoid area because of mastoiditis. Endocrine investigation showed elevated serum prolactin levels, secondary adrenal and gonadal failure and a normal thyroid function. Cranial MRI scan revealed a contrast enhancing intrasellar mass (approximately 2 cm) of heterogeneous appearance with suprasellar extension and thickening of the pituitary stalk. Lumbar puncture was suggestive of aseptic meningitis. The Ziehl-Neelsen stain of cerebrospinal fluid (CSF) and the tuberculin skin test were both negative. The pituitary mass was removed with a transsphenoidal approach. Histological examination demonstrated destruction of the adenohypophysis by epithelioid granulomas with partial caseous necrosis and microabscess formation, suggestive of a mycobacterial infection. A polymerase chain reaction analysis performed on paraffin-embedded tissue was positive for mycobacterial DNA. According to the individual 16S sequence, it was identified as Mycobacterium malmoense, an atypical nontuberculous mycobacterium (NTM). In conclusion, this is the first case of an isolated pituitary granuloma caused by an NTM infection in a nonimmunosuppressed patient.

Adult↗

The localization of multiple sites on 16S RNA which are cross-linked to proteins S7 and S8 in Escherichia coli 30S ribosomal subunits by treatment with 2-iminothiolane.

RNA-protein cross-links were introduced into E. coli 30S ribosomal subunits by reaction with 2-iminothiolane followed by a mild ultraviolet irradiation treatment. After removal of non-reacted protein and partial nuclease digestion of the cross-linked 16S RNA-protein moiety, a number of individual cross-linked complexes could be isolated and the sites of attachment of the proteins to the RNA determined. Protein S8 was cross-linked to the RNA at three different positions, within oligo-nucleotides encompassing positions 629-633, 651-654, and (tentatively) 593-597 in the 16S sequence. Protein S7 was cross-linked within two oligonucleotides encompassing positions 1238-1240, and 1377-1378. In addition, a site at position 723-724 was observed, cross-linked to protein S19, S20 or S21.

Cross-Linking Reagents↗

Class-level relationships in the phylum Cnidaria: molecular and morphological evidence.

The evolutionary history of cnidarian life cycles has been debated since the 1880s, with different hypotheses favored even by current textbooks. Contributing to the disagreement is the fact that the systematic relationships of the four cnidarian classes have received relatively little examination using modern systematic methods. Here we present analyses of class-level relationships based on 18S ribosomal DNA (rDNA) sequence, mitochondrial 16S rDNA sequence, mitochondrial genome structure, and morphological characters. DNA sequences were aligned using a repeatable parsimony-based approach incorporating a range of alignment parameters. Analyses of individual data sets and of all data combined are unanimous in grouping the classes possessing a medusa stage, leaving the holobenthic Anthozoa basal within the phylum.

Animals↗

Isolation and identification of methylobacterium species from the tap water in hospitals in Japan and their antibiotic susceptibility.

Contamination of tap water by Methylobacterium species has become a serious concern in hospitals. This study was planned to examine the distribution of Methylobacterium species inhabiting tap water used in Japanese hospitals and antibiotic sensitivity of the isolates in 2004. Species identification of 58 isolates was performed based on the homology of a partial sequence of 16S rDNA. The dominant Methylobacterium species in hospital water were M. aquaticum and M. fujisawaense. To examine the biochemical properties of these isolates, a carbon source utilization was tested using an API50CH kit. The phenotypic character varied widely, and was not necessarily consistent with the results of phylogenic analysis based on the partial 16S rDNA sequence, suggesting that the biochemical properties are not suitable for identification of Methylobacterium species. The isolates were also subjected to antibiotic sensitivity tests. They were resistant to 8 antibiotics, but highly sensitive to imipenem (MIC90 = 1 microg/ml) and tetracycline (MIC90 = 8 microg/ml). These findings concerning the isolates revealed the presence of Methylobacterium species with resistance to multiple antibiotics in hospital tap water.

Anti-Bacterial Agents↗

Molecular phylogeny and typing of blackflies (Diptera: Simuliidae) that serve as vectors of human or bovine onchocerciasis.

A subregion of the mitochondrial large subunit (16s) rRNA gene was amplified by polymerase chain reaction (PCR) from nine species of blackflies (Diptera: Simuliidae) which serve as natural or experimental vectors of human or bovine Onchocerca parasites. PCR products from each species of blackfly were tested by directed heteroduplex analysis (DHDA), and their genotypes established according to diagnostic banding patterns of the heteroduplex products. Three alleles of mitochondrial 16s rRNA were found to exist in members of the Simulium (Ewardsellum) damnosum sensu lato complex from West Africa, and two alleles were found in the Neotropical Simulium (Psilopelmia) ochraceum Walker complex and the Simulium (Simulium) metallicum Bellardi complex. Different single alleles were detected in Austrosimulium bancrofti, in English S.(S)noelleri and in two North American laboratory vectors: Simulium (Psilozia) vittatum Zetterstedt and S.(S.)decorum Walker. Phylogenetic analysis of 16s sequences indicated that blackflies from West Africa and the Americas formed distinct clades. Neotropical onchocerciasis vectors were found to be more closely related to Nearctic and Palaearctic non-vector Simulium species than to the African vectors of onchocerciasis.

Animals↗

Marine diatom species harbour distinct bacterial communities.

We examined bacterial dynamics in batch cultures of two axenic marine diatoms (Thalassiosira rotula and Skeletonema costatum). The axenic diatoms were inoculated with natural bacterial assemblages and monitored by 4,6-diamidino-2-phenolindole (DAPI) counts, denaturing gradient gel electrophoresis (DGGE) with subsequent analysis of excised, sequenced 16S rRNA gene fragments, and fluorescence in situ hybridization (FISH) with group-specific 16S rRNA oligonucleotide probes. Our results show that algal growth exhibited pronounced differences in axenic treatments and when bacteria were present. Bacterial abundance and community structure greatly depended on species, growth and physiological status of even closely related algae. Free-living and phytoplankton-associated bacteria were very different from each other and were dominated by distinct phylogenetic groups. The diatom-associated bacteria mainly belonged to the Flavobacteria-Sphingobacteria group of the Bacteroidetes phylum whereas free-living bacteria, which were rather similar in both cultures, comprised mainly of members of the Roseobacter group of alpha-Proteobacteria. Presence and disappearance of specific bacteria during algal growth indicated pronounced differences in environmental conditions over time and selection of bacteria highly adapted to the changing conditions. Tight interactions between marine bacteria and diatoms appear to be important for the decomposition of organic matter and nutrient cycling in the sea.

Alphaproteobacteria↗

Comparison of three different PCR methods for detection of Brucella spp in human blood samples.

In the diagnosis of human brucellosis, PCR could be a more sensitive technique than blood cultures and more specific than conventional serological tests. We compared three different PCR methods for the detection of Brucella spp. and we studied whether human genomic DNA affect the sensitivity of three primer pairs for the detection of Brucella DNA in a peripheral-blood PCR assay. These three pairs of primers amplified three different fragments included in: (i). a gene encoding a 31-kDa Brucella abortus antigen (primers B4/B5), (ii). a sequence 16S rRNA of B. abortus (primers F4/R2), and (iii). a gene encoding an outer membrane protein (omp-2) (primers JPF/JPR). The three primers assayed showed a difference in sensitivity for detecting purified Brucella DNA, ranging between 8 fg and 20 pg. However, the sensitivity of the primers F4/R2 and B4/B5 was affected by the presence of human DNA while the primers JPF/JPR were not. Therefore, although the sensitivity of PCR using primers F4/R2 is affected by human DNA, they are still the most sensitive and they could provide a useful tool for the diagnosis of human brucellosis.

Brucella↗

Detection and isolation of novel rhizopine-catabolizing bacteria from the environment

Microbial rhizopine-catabolizing (Moc) activity was detected in serial dilutions of soil and rhizosphere washes. The activity observed generally ranged between 10(6) and 10(7) catabolic units per g, and the numbers of nonspecific culture-forming units were found to be approximately 10 times higher. A diverse set of 37 isolates was obtained by enrichment on scyllo-inosamine-containing media. However, none of the bacteria that were isolated were found to contain DNA sequences homologous to the known mocA, mocB, and mocC genes of Sinorhizobium meliloti L5-30. Twenty-one of the isolates could utilize an SI preparation as the sole carbon and nitrogen source for growth. Partial sequencing of 16S ribosomal DNAs (rDNAs) amplified from these strains indicated that five distinct bacterial genera (Arthrobacter, Sinorhizobium, Pseudomonas, Aeromonas, and Alcaligenes) were represented in this set. Only 6 of these 21 isolates could catabolize 3-O-methyl-scyllo-inosamine under standard assay conditions. Two of these, strains D1 and R3, were found to have 16S rDNA sequences very similar to those of Sinorhizobium meliloti. However, these strains are not symbiotically effective on Medicago sativa, and DNA sequences homologous to the nodB and nodC genes were not detected in strains D1 and R3 by Southern hybridization analysis.

Journal Article↗

First report of bacteremia by Asaia bogorensis, in a patient with a history of intravenous-drug abuse.

We report the first documented case of bacteremia caused by Asaia bogorensis in a young patient with a history of intravenous-drug abuse. A. bogorensis was identified by sequencing the 16S rRNA gene. The isolate was exceptionally resistant to almost all antibiotics that are routinely tested for gram-negative rods but was susceptible to netilmicin, gentamicin, and doxycycline.

Acetobacteraceae↗

Comparison of major antigenic proteins of six strains of the human granulocytic ehrlichiosis agent by Western immunoblot analysis.

The etiologic agent of human granulocytic ehrlichiosis (HGE) is an obligate intracellular bacterium. In 1996, blood specimens from 53 patients suspected of having HGE were examined by indirect fluorescent antibody (IFA) testing with the HGE agent no. 13 isolate as the antigen, by nested PCR, and by culture. All patients resided in Westchester County, N.Y. Twelve patient specimens were positive for IFA (titer > or = 1:40). Seven of these were also positive by PCR. Of the seven specimens positive by both IFA testing and PCR, the HGE agent was isolated from four (no. 2, 3, 6, and 11) and continuously cultured in HL-60 cells. These were confirmed as the HGE agent by sequencing of 16S rDNA. Both purified whole-cell organisms and the outer membrane fractions of the new isolates were compared with no. 13 isolate and a tick (USG) isolate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot analysis. No. 11 and 13 isolates had identical SDS-PAGE patterns with respect to 49- and 47-kDa proteins. No. 3 and USG isolates lacked the 47-kDa protein, and no. 6 isolate lacked the 49-kDa protein. Both 49- and 47-kDa bands were absent in no. 2 isolate. Western blot results with seven different sera, including five convalescent-phase sera from these patients, one dog anti-USG isolate, and one horse anti-BDS isolate, showed that all major antigens in six isolates were recognized by all sera. However, the molecular sizes and the numbers of major antigens recognized varied among the six isolates. Overall, HGE agent no. 3, 6, 11, and 13, and USG isolates had similar patterns, with 1 or 2 major antigens with molecular masses of around 49 and 47 kDa. No. 2 isolate was quite distinct in having a major antigen of 43 kDa. This indicates that although these antigenic epitopes are all cross-reactive among strains, the HGE agent has a strain pleomorphism in its major antigenic proteins. The major antigen profiles of the outer membrane protein fractions and of whole organisms of six HGE agent isolates were similar, suggesting that 49- and 47-kDa major antigens are the outer membrane proteins of the HGE agent.

Animals↗