Ribotypes of clinical Vibrio cholerae non-O1 non-O139 strains in relation to O-serotypes.
The emergence of Vibrio cholerae O139 in 1992 and reports of an increasing number of other non-O1 serogroups being associated with diarrhoea, stimulated us to characterize V. cholerae non-O1 non-O139 strains received at the National Institute of Infectious Diseases, Japan for serotyping. Ribotyping with the restriction enzyme BglI of 103 epidemiological unrelated mainly clinical strains representing 10 O-serotypes yielded 67 different typing patterns. Ribotype similarity within each serotype was compared by using the Dice coefficient (Sd) and different levels of homogeneity were observed (serotypes O5, O41 and O17, Sd between 82 and 90%: serotypes O13 and O141 Sd of 72; and O2, O6, O7, O11, O24 Sd of 62-66%). By cluster analysis, the strains were divided into several clusters of low similarity suggesting a high level of genetic diversity. A low degree of similarity between serotypes and ribotypes was found as strains within a specific serotypes often did not cluster but clustered with strains from other serotypes. However, epidemiological unrelated O5 strains showed identical or closely related ribotypes suggesting that these strains have undergone few genetic changes and may correspond to a clonal line. Surprisingly, 10 of 16 O141 strains studied contained a cholera toxin (CT) gene, including 7 strains recovered from stool and water samples in the United States. This is to our knowledge the first report of CT-positive clinical O141 strains. The closely related ribotypes shown by eight CT-positive strains is disturbing and suggest that these strains may be of a clonal origin and have the potential to cause cholera-like disease. Despite the low degree of correlation found between ribotypes and serotypes, both methods appears to be valuable techniques in studying the epidemiology of emerging serotypes of V. cholerae. (+info)
Molecular differentiation of Renibacterium salmoninarum isolates from worldwide locations.
Renibacterium salmoninarum is a genospecies that is an obligate pathogen of salmonid fish and is capable of intracellular survival. Conventional typing systems have failed to differentiate isolates of R. salmoninarum. We used two methods to assess the extent of molecular variation which was present in isolates from different geographic locations. In one analysis we investigated possible polymorphisms in a specific region of the genome, the intergenic spacer (ITS) region between the 16S and 23S rRNA genes. In the other analysis we analyzed differences throughout the genome by using randomly amplified polymorphic DNA (RAPD). We amplified the spacer region of 74 isolates by using PCR and performed a DNA sequence analysis with 14 geographically distinct samples. The results showed that the 16S-23S ribosomal DNA spacer region of R. salmoninarum is highly conserved and suggested that only a single copy of the rRNA operon is present in this slowly growing pathogen. DNA sequencing of the spacer region showed that it was the same length in all 14 isolates examined, and the same nucleotide sequence, sequevar 1, was obtained for 11 of these isolates. Two other sequevars were found. No tRNA genes were found. We found that RAPD analysis allows reproducible differentiation between isolates of R. salmoninarum obtained from different hosts and different geographic regions. By using RAPD analysis it was possible to differentiate between isolates with identical ITS sequences. (+info)
Effect of phenylurea herbicides on soil microbial communities estimated by analysis of 16S rRNA gene fingerprints and community-level physiological profiles.
The effect of three phenyl urea herbicides (diuron, linuron, and chlorotoluron) on soil microbial communities was studied by using soil samples with a 10-year history of treatment. Denaturing gradient gel electrophoresis (DGGE) was used for the analysis of 16S rRNA genes (16S rDNA). The degree of similarity between the 16S rDNA profiles of the communities was quantified by numerically analysing the DGGE band patterns. Similarity dendrograms showed that the microbial community structures of the herbicide-treated and nontreated soils were significantly different. Moreover, the bacterial diversity seemed to decrease in soils treated with urea herbicides, and sequence determination of several DGGE fragments showed that the most affected species in the soils treated with diuron and linuron belonged to an uncultivated bacterial group. As well as the 16S rDNA fingerprints, the substrate utilization patterns of the microbial communities were compared. Principal-component analysis performed on BIOLOG data showed that the functional abilities of the soil microbial communities were altered by the application of the herbicides. In addition, enrichment cultures of the different soils in medium with the urea herbicides as the sole carbon and nitrogen source showed that there was no difference between treated and nontreated soil in the rate of transformation of diuron and chlorotoluron but that there was a strong difference in the case of linuron. In the enrichment cultures with linuron-treated soil, linuron disappeared completely after 1 week whereas no significant transformation was observed in cultures inoculated with nontreated soil even after 4 weeks. In conclusion, this study showed that both the structure and metabolic potential of soil microbial communities were clearly affected by a long-term application of urea herbicides. (+info)
Anaerobic oxidation of o-xylene, m-xylene, and homologous alkylbenzenes by new types of sulfate-reducing bacteria.
Various alkylbenzenes were depleted during growth of an anaerobic, sulfate-reducing enrichment culture with crude oil as the only source of organic substrates. From this culture, two new types of mesophilic, rod-shaped sulfate-reducing bacteria, strains oXyS1 and mXyS1, were isolated with o-xylene and m-xylene, respectively, as organic substrates. Sequence analyses of 16S rRNA genes revealed that the isolates affiliated with known completely oxidizing sulfate-reducing bacteria of the delta subclass of the class Proteobacteria. Strain oXyS1 showed the highest similarities to Desulfobacterium cetonicum and Desulfosarcina variabilis (similarity values, 98.4 and 98.7%, respectively). Strain mXyS1 was less closely related to known species, the closest relative being Desulfococcus multivorans (similarity value, 86.9%). Complete mineralization of o-xylene and m-xylene was demonstrated in quantitative growth experiments. Strain oXyS1 was able to utilize toluene, o-ethyltoluene, benzoate, and o-methylbenzoate in addition to o-xylene. Strain mXyS1 oxidized toluene, m-ethyltoluene, m-isoproyltoluene, benzoate, and m-methylbenzoate in addition to m-xylene. Strain oXyS1 did not utilize m-alkyltoluenes, whereas strain mXyS1 did not utilize o-alkyltoluenes. Like the enrichment culture, both isolates grew anaerobically on crude oil with concomitant reduction of sulfate to sulfide. (+info)
High-affinity methane oxidation by a soil enrichment culture containing a type II methanotroph.
Methanotrophic bacteria in an organic soil were enriched on gaseous mixing ratios of <275 parts per million of volume (ppmv) of methane (CH4). After 4 years of growth and periodic dilution (>10(20) times the initial soil inoculum), a mixed culture was obtained which displayed an apparent half-saturation constant [Km(app)] for CH4 of 56 to 186 nM (40 to 132 ppmv). This value was the same as that measured in the soil itself and about 1 order of magnitude lower than reported values for pure cultures of methane oxidizers. However, the Km(app) increased when the culture was transferred to higher mixing ratios of CH4 (1,000 ppmv, or 1%). Denaturing gradient gel electrophoresis of the enrichment grown on <275 ppmv of CH4 revealed a single gene product of pmoA, which codes for a subunit of particulate methane monooxygenase. This suggested that only one methanotroph species was present. This organism was isolated from a sample of the enrichment culture grown on 1% CH4 and phylogenetically positioned based on its 16S rRNA, pmoA, and mxaF gene sequences as a type II strain of the Methylocystis/Methylosinus group. A coculture of this strain with a Variovorax sp., when grown on <275 ppmv of CH4, had a Km(app) (129 to 188 nM) similar to that of the initial enrichment culture. The data suggest that the affinity of methanotrophic bacteria for CH4 varies with growth conditions and that the oxidation of atmospheric CH4 observed in this soil is carried out by type II methanotrophic bacteria which are similar to characterized species. (+info)
Polynucleotide probes that target a hypervariable region of 16S rRNA genes to identify bacterial isolates corresponding to bands of community fingerprints.
Temperature gradient gel electrophoresis (TGGE) is well suited for fingerprinting bacterial communities by separating PCR-amplified fragments of 16S rRNA genes (16S ribosomal DNA [rDNA]). A strategy was developed and was generally applicable for linking 16S rDNA from community fingerprints to pure culture isolates from the same habitat. For this, digoxigenin-labeled polynucleotide probes were generated by PCR, using bands excised from TGGE community fingerprints as a template, and applied in hybridizations with dot blotted 16S rDNA amplified from bacterial isolates. Within 16S rDNA, the hypervariable V6 region, corresponding to positions 984 to 1047 (Escherichia coli 16S rDNA sequence), which is a subset of the region used for TGGE (positions 968 to 1401), best met the criteria of high phylogenetic variability, required for sufficient probe specificity, and closely flanking conserved priming sites for amplification. Removal of flanking conserved bases was necessary to enable the differentiation of closely related species. This was achieved by 5' exonuclease digestion, terminated by phosphorothioate bonds which were synthesized into the primers. The remaining complementary strand was removed by single-strand-specific digestion. Standard hybridization with truncated probes allowed differentiation of bacteria which differed by only two bases within the probe target site and 1.2% within the complete 16S rDNA. However, a truncated probe, derived from an excised TGGE band of a rhizosphere community, hybridized with three phylogenetically related isolates with identical V6 sequences. Only one of the isolates comigrated with the excised band in TGGE, which was shown to be due to identical sequences, demonstrating the utility of a combined TGGE and V6 probe approach. (+info)
Immunochemical detection and isolation of DNA from metabolically active bacteria.
Most techniques used to assay the growth of microbes in natural communities provide no information on the relationship between microbial productivity and community structure. To identify actively growing bacteria, we adapted a technique from immunocytochemistry to detect and selectively isolate DNA from bacteria incorporating bromodeoxyuridine (BrdU), a thymidine analog. In addition, we developed an immunocytochemical protocol to visualize BrdU-labeled microbial cells. Cultured bacteria and natural populations of aquatic bacterioplankton were pulse-labeled with exogenously supplied BrdU. Incorporation of BrdU into microbial DNA was demonstrated in DNA dot blots probed with anti-BrdU monoclonal antibodies and either peroxidase- or Texas red-conjugated secondary antibodies. BrdU-containing DNA was physically separated from unlabeled DNA by using antibody-coated paramagnetic beads, and the identities of bacteria contributing to both purified, BrdU-containing fractions and unfractionated, starting-material DNAs were determined by length heterogeneity PCR (LH-PCR) analysis. BrdU-containing DNA purified from a mixture of DNAs from labeled and unlabeled cultures showed >90-fold enrichment for the labeled bacterial taxon. The LH-PCR profile for BrdU-containing DNA from a labeled, natural microbial community differed from the profile for the community as a whole, demonstrating that BrdU was incorporated by a taxonomic subset of the community. Immunocytochemical detection of cells with BrdU-labeled DNA was accomplished by in situ probing with anti-BrdU monoclonal antibodies and Texas red-labeled secondary antibodies. Using this suite of techniques, microbial cells incorporating BrdU into their newly synthesized DNA can be quantified and the identities of these actively growing cells can be compared to the composition of the microbial community as a whole. Since not all strains tested could incorporate BrdU, these methods may be most useful when used to gain an understanding of the activities of specific species in the context of their microbial community. (+info)
Dissimilatory reduction of Fe(III) and other electron acceptors by a Thermus isolate.
A thermophilic bacterium that can use O2, NO3-, Fe(III), and S0 as terminal electron acceptors for growth was isolated from groundwater sampled at a 3.2-km depth in a South African gold mine. This organism, designated SA-01, clustered most closely with members of the genus Thermus, as determined by 16S rRNA gene (rDNA) sequence analysis. The 16S rDNA sequence of SA-01 was >98% similar to that of Thermus strain NMX2 A.1, which was previously isolated by other investigators from a thermal spring in New Mexico. Strain NMX2 A.1 was also able to reduce Fe(III) and other electron acceptors. Neither SA-01 nor NMX2 A.1 grew fermentatively, i.e., addition of an external electron acceptor was required for anaerobic growth. Thermus strain SA-01 reduced soluble Fe(III) complexed with citrate or nitrilotriacetic acid (NTA); however, it could reduce only relatively small quantities (0.5 mM) of hydrous ferric oxide except when the humic acid analog 2,6-anthraquinone disulfonate was added as an electron shuttle, in which case 10 mM Fe(III) was reduced. Fe(III)-NTA was reduced quantitatively to Fe(II); reduction of Fe(III)-NTA was coupled to the oxidation of lactate and supported growth through three consecutive transfers. Suspensions of Thermus strain SA-01 cells also reduced Mn(IV), Co(III)-EDTA, Cr(VI), and U(VI). Mn(IV)-oxide was reduced in the presence of either lactate or H2. Both strains were also able to mineralize NTA to CO2 and to couple its oxidation to Fe(III) reduction and growth. The optimum temperature for growth and Fe(III) reduction by Thermus strains SA-01 and NMX2 A.1 is approximately 65 degrees C; their optimum pH is 6.5 to 7.0. This is the first report of a Thermus sp. being able to couple the oxidation of organic compounds to the reduction of Fe, Mn, or S. (+info)