New insertion sequences and a novel repeated sequence in the genome of Mycobacterium tuberculosis H37Rv.
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The genome sequence of Mycobacterium tuberculosis H37Rv was found to contain 56 loci with homology to insertion sequences (ISs). As well as the previously described IS6110, IS1081, IS1547 and IS-like elements, new ISs belonging to the IS3, IS5, IS21, IS30, IS110, IS256 and ISL3 families were identified. In addition, six ISs created a grouping of their own to form a new family (the IS1535 family). Elements with similarity to ISs in other actinomycetes were identified, suggesting the movement of ISs between related genera. The location of ISs on the chromosome revealed that an approximately 600 kb region close to the origin of replication lacks ISs, pointing to the possible detrimental effect of insertions in this area. Analysis of the distribution of ISs through the tubercle strains Mycobacterium africanum, M. microti, M. bovis, M. bovis BCG Pasteur, M. tuberculosis H37Ra, M. tuberculosis CSU#93 and 29 clinical isolates revealed that only IS1532, IS1533, 1S1534, and IS1561' were absent from some of the strains tested. A novel repeated sequence, the REP13E12 family, is described that is present in seven copies on the M. tuberculosis H37Rv chromosome and which contains a probable phage attachment site. This study therefore offers an insight into the possible role of ISs and repetitive elements in the evolution of the M. tuberculosis genome, as well as identifying genetic markers that may be useful for phylogenetic and epidemiological analysis of the tubercle complex. (+info)
Evaluation of reverse transcription-PCR and a bacteriophage-based assay for rapid phenotypic detection of rifampin resistance in clinical isolates of Mycobacterium tuberculosis.
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New rapid phenotypic assays for the detection of rifampin resistance in Mycobacterium tuberculosis have recently been described, but most of these require liquid cultures, which reduces the utility of many tests in terms of turnaround times. In the United Kingdom, over 90% of rifampin-resistant isolates are also resistant to isoniazid, so rifampin resistance can be used as a sensitive marker for multidrug-resistant tuberculosis. In this study, two new rapid phenotypic assays were compared to the standard resistance ratio method on 91 clinical isolates of M. tuberculosis. One, the phage amplified biologically (PhaB) assay, has been described previously and is based on the inability of susceptible isolates of M. tuberculosis to support the replication of bacteriophage D29 in the presence of inhibitory doses of rifampin. The other employed reverse transcription (RT)-PCR to demonstrate a reduction in inducible dnaK mRNA levels in susceptible isolates treated with rifampin. After incubation for 18 h with 4 microg of rifampin per ml, the PhaB assay showed concordance with the resistance ratio method for 46 of 46 (100%) susceptible and 31 of 31 (100%) resistant isolates, while RT-PCR showed concordance for 46 of 48 (96%) susceptible and 35 of 36 (97%) resistant isolates. We believe these assays provide a reliable rapid means of susceptibility testing with a total turnaround time of only 48 h, although the PhaB assay is better in terms of its lower technical demand and cost and its applicability to tuberculosis susceptibility testing in developing countries. (+info)
Evaluation of a bacteriophage-based assay (phage amplified biologically assay) as a rapid screen for resistance to isoniazid, ethambutol, streptomycin, pyrazinamide, and ciprofloxacin among clinical isolates of Mycobacterium tuberculosis.
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Rapid molecular assays for the detection of mutations associated with rifampin resistance in Mycobacterium tuberculosis are commercially available. However, they are complex and expensive and have predictive values of 90 to 95%. Molecular assays for other drugs are less predictive of resistance. Ideally, assays based on phenotypic markers should be used for susceptibility testing, but these can take weeks to complete. We previously described a rapid phenotypic assay, the phage amplified biologically (PhaB) assay, for the rapid determination of rifampin and isoniazid susceptibility in clinical isolates of M. tuberculosis. In this study, we extended the assay to the study of ethambutol, pyrazinamide, streptomycin, and ciprofloxacin. After the optimization of antibiotic concentrations and incubation conditions, the assay was applied to each drug for a total of 157 isolates. The correlations between the results of the PhaB assay and the resistance ratio method were 94% for isoniazid, 96% for streptomycin, 100% for ciprofloxacin, 88% for ethambutol, and 87% for pyrazinamide. For ciprofloxacin, ethambutol, and pyrazinamide, significantly better correlations were found when a 90% reduction in plaque count was used as the cutoff. Turnaround times for the PhaB assay were 2 to 3 days, compared with 10 days for the resistance ratio method. We believe that this low-cost assay may have widespread applicability for the rapid screening of drug resistance in M. tuberculosis isolates, especially in developing countries. (+info)
Biophysical properties of mycobacteriophages.
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Biophysical characterization of two mycobacteriophages (Phlei phage and Butyricum phage) was carried out. Biophysical parameters obtained were: (i) buoyant densities in CsCl of I . 51 g/ml for both phages; (ii) sedimentation coefficient of 490 S and 410 S; (iii) DNA content of 42 and 34% and (iv) mol. wt. calculated by electron microscopic dimensions to be 123 x 10(6) and 116 x 10(6) for Butyricum and Phlei phage, respectively. (+info)
The nature and incidence of lysogeny in Mycobacterium fortuitum.
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Ten of 28 strain of Mycobacterium fortuitum (ranae) were found to be associated with bacteriophage; three were pseudolysogenic, one liberated a phage that lysed a sensitive indicator strain, two liberated morphologically complete phages that did not lyse any of the strains used in this study and four liberated morphologically defective phages. The lysogenic and defectively lysogenic strains showed anomalies in cultural, biochemical and antigenic properties and in susceptibility to superinfecting phages. In view of the high frequency of lysogeny found in M. fortuitum, the role of bacteriophage in the variation of properties, including pathogenicity, of mycobacteria of greater clinical importance merits further consideration. (+info)
Luciferase reporter mycobacteriophages for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis in Mexico.
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The utility of luciferase reporter mycobacteriophages (LRPs) for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis was prospectively evaluated in a clinical microbiology laboratory in Mexico City, Mexico. Five hundred twenty-three consecutive sputum samples submitted to the laboratory during a 5-month period were included in this study. These specimens were cultivated in Middlebrook 7H9 (MADC), MGIT, and Lowenstein-Jensen (LJ) media. Of the 71 mycobacterial isolates recovered with any of the three media, 76% were detected with the LRPs, 97% were detected with the MGIT 960 method, and 90% were detected with LJ medium. When contaminated specimens were excluded from the analysis, the LRPs detected 92% (54 of 59) of the cultures. The median time to detection of bacteria was 7 days with both the LRPs and the MGIT 960 method. LRP detection of growth in the presence of p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) was used for selective identification of M. tuberculosis complex (MTC) and compared to identification with BACTEC 460. Using the LRP NAP test, 47 (94%) out of 50 isolates were correctly identified as tuberculosis complex. The accuracy and speed of LRP antibiotic susceptibility testing with rifampin, streptomycin, isoniazid, and ethambutol were compared to those of the BACTEC 460 method, and discrepant results were checked by the conventional proportion method. In total, 50 MTC isolates were tested. The overall agreement between the LRP and BACTEC 460 results was 98.5%. The median LRP-based susceptibility turnaround time was 2 days (range, 2 to 4 days) compared to 10.5 days (range, 7 to 16 days) by the BACTEC 460 method. Phage resistance was not detected in any of the 243 MTC isolates tested. Mycobacteriophage-based approaches to tuberculosis diagnostics can be implemented in clinical laboratories with sensitivity, specificity, and rapidity that compare favorably with those of the MGIT 960 and BACTEC 460 methods. The phages currently provide the fastest phenotypic assay for susceptibility testing. (+info)
Ultrastructure of L-phase variants isolated from a culture of Mycobacterium phlei.
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Relatively stable L-phase colonies were isolated from old cultures of a selected clone of Mycobacterium phlei. The colonies grew at 52 degrees C and were composed of rod-shaped, oval or spherical cells. Large amoeba-like cells were occasionally present. These were usually limited by a double-layered membrane and devoid of normal cell-wall components such as bacteriophage receptors. The large amoeba-like bodies sometimes showed both outer and inner double-layered membranes, especially in pseudopodium-like cellular extensions. An unusual feature of rod-shaped cells was retention of the original shape despite the loss of their cell walls. Two types of walled cells occurred during successive transfers of L colonies. One was the true revertant which had characteristics in common with the wild-type M. phlei, such as growth at 52 degrees C and ultrastructural organisation. The other, designated as the "atypical-cell-wall variant", was characterised by growth at 52 degrees C, thick cell walls, and disordered septation. Wild-type M. phlei, L variants, revertants and atypical-cell-wall variants released mycobacteriophage particles. These bacteriophages were almost identical in respect to morphology, host range, and neutralisation by antiserum. The results obtained suggest strongly that all types of cells examined were derived from M. phlei. (+info)
Expression of Mycobacteriophage Ms6 lysis genes is driven by two sigma(70)-like promoters and is dependent on a transcription termination signal present in the leader RNA.
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A mycobacteriophage Ms6 strong promoter region (P(lys)) was isolated by using transcriptional fusions with the lacZ reporter gene. Two tandem sigma(70)-like promoter sequences (P1 and P2) were found in this region. DNA sequencing of the promoter downstream region revealed a 214-bp leader sequence followed by five adjacent coding regions of 231 bp (ORF1), 1,152 bp (ORF2), 996 bp (ORF3), 231 bp (ORF4), and 372 (ORF5). ORF1 has the potential to encode a 77-amino-acid protein which revealed similarity to mycobacteriophage TM4 gp90, a predicted protein with unknown function. ORF2 encodes a 384-amino-acid protein which is related to several bacteriophage amidases. This protein induced cell lysis upon addition of chloroform, confirming its mureinolytic activity. ORF3 encodes a 332-amino-acid protein which is related to TM4 gp30, a protein with sequence similarity to amidases. ORF4 encodes a 77-amino-acid holin-like protein with significant similarity to the holin of Lactococcus lactis r1t bacteriophage. ORF5 encodes a 124-amino-acid protein which is related to mycobacteriophage L5 gp30, a protein with unknown function. These data indicate that the promoter region P(lys) drives the transcription of the Ms6 lysis genes. An intrinsic transcription termination signal was identified in the leader sequence. Experiments using lacZ fusions showed that beta-galactosidase synthesis is inhibited when this transcription termination signal is present in the leader sequence. In conclusion, mycobacteriophage Ms6 cell lysis genes are expressed by their own promoter region, independently of virion structure and assembly protein genes. Moreover, an antitermination mechanism might be involved in their transcription regulation. (+info)