Improved method for purification of bacterial DNA from bovine milk for detection of Brucella spp. by PCR.
(49/6674)
Different methods of extraction of bacterial DNA from bovine milk to improve the direct detection of Brucella by PCR were evaluated. We found that the use of a lysis buffer with high concentrations of Tris, EDTA, and NaCl, high concentrations of sodium dodecyl sulfate and proteinase K, and high temperatures of incubation was necessary for the efficient extraction of Brucella DNA. The limit of detection by PCR was 5 to 50 Brucella CFU/ml of milk. (+info)
Comparison and recovery of Escherichia coli and thermotolerant coliforms in water with a chromogenic medium incubated at 41 and 44.5 degrees C.
(50/6674)
This study compared the performance of a commercial chromogenic medium, CHROMagarECC (CECC), and CECC supplemented with sodium pyruvate (CECCP) with the membrane filtration lauryl sulfate-based medium (mLSA) for enumeration of Escherichia coli and non-E. coli thermotolerant coliforms (KEC). To establish that we could recover the maximum KEC and E. coli population, we compared two incubation temperature regimens, 41 and 44.5 degrees C. Statistical analysis by the Fisher test of data did not demonstrate any statistically significant differences (P = 0.05) in the enumeration of E. coli for the different media (CECC and CECCP) and incubation temperatures. Variance analysis of data performed on KEC counts showed significant differences (P = 0.01) between KEC counts at 41 and 44.5 degrees C on both CECC and CECCP. Analysis of variance demonstrated statistically significant differences (P = 0.05) in the enumeration of total thermotolerant coliforms (TTCs) on CECC and CECCP compared with mLSA. Target colonies were confirmed to be E. coli at a rate of 91.5% and KEC of likely fecal origin at a rate of 77.4% when using CECCP incubated at 41 degrees C. The results of this study showed that CECCP agar incubated at 41 degrees C is efficient for the simultaneous enumeration of E. coli and KEC from river and marine waters. (+info)
Establishing the independent culture of a strictly symbiotic bacterium Symbiobacterium thermophilum from its supporting Bacillus strain.
(51/6674)
Symbiobacterium thermophilum is a strictly symbiotic thermophile, the growth of which is dependent on the coexistence of an associating thermophilic Bacillus sp., strain S. S. thermophilum grows only in mixed culture with the Bacillus strain in liquid media, and does not form visible colonies on solid media. To measure the growth of this symbiotic bacterium and to analyze its growth requirements, we developed a quantitative PCR method by using its specific sequences in a putative membrane translocator gene tnaT as primers. According to this method, independent growth of S. thermophilum was first confirmed in a dialyzing culture physically separated from Bacillus strain S with a cellulose membrane. Independent growth of S. thermophilum was also managed by adding conditioned medium prepared from the culture filtrate of the Bacillus strain, but the growth in the conditioned medium stopped at a very limited extent with appearance of filamentous cells, suggesting the uncoupling of cellular growth and cell division. Formation of micro-colonies of S. thermophilum was observed on the conditioned agar medium under both aerobic and anaerobic conditions, but the colony-forming efficiencies remained below 1%. Several other bacterial species, such as Bacillus stearothermophilus, Bacillus subtilis, Thermus thermophilus, and even Escherichia coli, were also found to support the growth of S. thermophilum. These results indicate that S. thermophilum essentially requires some ubiquitous metabolite(s) of low molecular weight produced by various bacterial species as growth factor(s) but coexistence of the living partner cells is still required, probably to maintain an effective level of the putative factor(s) in the medium. (+info)
Influence of a putative ECF sigma factor on expression of the major outer membrane protein, OprF, in Pseudomonas aeruginosa and Pseudomonas fluorescens.
(52/6674)
The gene encoding OprF, a major outer membrane protein in Pseudomonas species (formerly known as type 1 pseudomonads), was thought to be constitutively transcribed from a single sigma 70 promoter immediately upstream of the gene. We now report the identification of a novel putative ECF (extracytoplasmic function) sigma factor gene, sigX, located immediately upstream of oprF in both Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens OE 28.3 and show that disruption of this gene significantly reduces OprF expression. In P. aeruginosa, Northern analysis demonstrated that this reduction was a result of an effect on transcription of monocistronic oprF combined with a polar effect due to termination of a transcript containing sigX and oprF. Comparison of sigX-disrupted and wild-type cell transcripts by primer extension indicated that monocistronic transcription of oprF occurs from two overlapping promoters, one that is SigX-dependent and resembles ECF sigma factor promoters in its minus-35 region and another promoter that is independent of SigX and is analogous to the sigma 70-type promoter previously reported. Complementation of the P. aeruginosa sigX-disrupted mutant with plasmid-encoded OprF did not resolve the phenotypes associated with this mutant, which included a markedly reduced logarithmic-phase growth rate in rich medium (compared to that in minimal medium), further reduction of the growth rate in a low-osmolarity environment, secretion of an unidentified pigment, and increased sensitivity to the antibiotic imipenem. This indicates that SigX is involved in the regulation of other genes in P. aeruginosa. Disruption of the sigX gene in P. fluorescens also had an effect on the logarithmic-phase growth rate in rich medium. A conserved sigX gene was also identified in a Pseudomonas syringae isolate and six P. aeruginosa clinical isolates. Collectively, these data indicate that an ECF sigma factor plays a role in the regulation and expression of OprF and also affects other genes. (+info)
Comparison of the construction of unmarked deletion mutations in Mycobacterium smegmatis, Mycobacterium bovis bacillus Calmette-Guerin, and Mycobacterium tuberculosis H37Rv by allelic exchange.
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Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent of tuberculosis, was hindered by a lack of methods for gene disruptions and allelic exchange. Several groups have described different methods for disrupting genes marked with antibiotic resistance determinants in the slow-growing organisms Mycobacterium bovis bacillus Calmette-Guerin (BCG) and M. tuberculosis. In this study, we described the first report of using a mycobacterial suicidal plasmid bearing the counterselectable marker sacB for the allelic exchange of unmarked deletion mutations in the chromosomes of two substrains of M. bovis BCG and M. tuberculosis H37Rv. In addition, our comparison of the recombination frequencies in these two slow-growing species and that of the fast-growing organism Mycobacterium smegmatis suggests that the homologous recombination machinery of the three species is equally efficient. The mutants constructed here have deletions in the lysA gene, encoding meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step in lysine biosynthesis. We observed striking differences in the lysine auxotrophic phenotypes of these three species of mycobacteria. The M. smegmatis mutant can grow on lysine-supplemented defined medium or complex rich medium, while the BCG mutants grow only on lysine-supplemented defined medium and are unable to form colonies on complex rich medium. The M. tuberculosis lysine auxotroph requires 25-fold more lysine on defined medium than do the other mutants and is dependent upon the detergent Tween 80. The mutants described in this work are potential vaccine candidates and can also be used for studies of cell wall biosynthesis and amino acid metabolism. (+info)
The Avr (effector) proteins HrmA (HopPsyA) and AvrPto are secreted in culture from Pseudomonas syringae pathovars via the Hrp (type III) protein secretion system in a temperature- and pH-sensitive manner.
(54/6674)
We present here data showing that the Avr proteins HrmA and AvrPto are secreted in culture via the native Hrp pathways from Pseudomonas syringae pathovars that produce these proteins. Moreover, their secretion is strongly affected by the temperature and pH of the culture medium. Both HrmA and AvrPto were secreted at their highest amounts when the temperature was between 18 and 22 degrees C and when the culture medium was pH 6.0. In contrast, temperature did not affect the secretion of HrpZ. pH did affect HrpZ secretion, but not as strongly as it affected the secretion of HrmA. This finding suggests that there are at least two classes of proteins that travel the P. syringae pathway: putative secretion system accessory proteins, such as HrpZ, which are readily secreted in culture; and effector proteins, such as HrmA and AvrPto, which apparently are delivered inside plant cells and are detected in lower amounts in culture supernatants under the appropriate conditions. Because HrmA was shown to be a Hrp-secreted protein, we have changed the name of hrmA to hopPsyA to reflect that it encodes a Hrp outer protein from P. syringae pv. syringae. The functional P. syringae Hrp cluster encoded by cosmid pHIR11 conferred upon P. fluorescens but not Escherichia coli the ability to secrete HopPsyA in culture. The use of these optimized conditions should facilitate the identification of additional proteins traveling the Hrp pathway and the signals that regulate this protein traffic. (+info)
In vivo observation of cell division of anaerobic hyperthermophiles by using a high-intensity dark-field microscope.
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To study growth and cell division of anaerobic hyperthermophilic archaea in vivo, a cultivation technique using glass capillaries was developed. At temperatures of 90 to 98 degrees C, at least 10 successive cell divisions of Pyrodictium abyssi TAG 11 were documented. Cells divide by binary fission. Visualized under a modified dark-field microscope, the formation of cannulae, which finally connected all cells, was observed. The cannulae elongated at 1.0 to 1.5 micrometers/min and reached final lengths of between 30 and 150 micrometers. A "snapping division"-like mode of cell fission was discovered for Thermoproteus tenax. (+info)
Evaluation of the one-minute ultra-rapid urease test for diagnosing Helicobacter pylori.
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To determine the diagnostic accuracy of the one-minute ultra-rapid urease test for diagnosing Helicobacter pylori infection, two biopsies were taken from both the gastric corpus and antrum from 1000 patients undergoing upper gastrointestinal endoscopy. All the biopsies were subjected to the one-minute ultra-rapid urease test before imprint smears were prepared from them. Thereafter, the biopsies were fixed in 10% formalin and histological sections were examined for the presence of H pylori by a pathologist who was not aware of the clinical details or the results of the urease test. The prevalence of H pylori in the gastric antrum and corpus was 86.7% and 53.3%, respectively. The sensitivity, specificity, positive and negative predictive value and the overall diagnostic accuracy of the ultra-rapid urease test to diagnose H pylori infection in the gastric antrum were 92%, 100%, 100%, 66%, and 93%, respectively. The corresponding figures for the gastric corpus were 83%, 100%, 100%, 85%, and 91%, respectively. It is concluded that the one-minute ultra-rapid urease test has a high sensitivity and specificity and may be used as a rapid and cheap method to diagnose H pylori infection. (+info)