Direct detection of heteroresistance in Mycobacterium tuberculosis using molecular techniques.
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Codon↗
Biomedical subjects
Timothy D McHugh
Publications and source records attributed to Timothy D McHugh.
Early bactericidal activity of a moxifloxacin and isoniazid combination in smear-positive pulmonary tuberculosis.
BACKGROUND: In vitro and animal studies have shown that moxifloxacin-containing combinations may improve the bactericidal efficacy of antituberculosis regimens. PATIENTS AND METHODS: We measured the decline in the sputum viable count of 13 patients who were given a combination of moxifloxacin 400 mg daily and isoniazid 300 mg daily. RESULTS: The time required to reduce the viable count by 50% (vt50) was 0.38 days (95% CI -0.03-0.78 days, SEM 0.13) and the mean early bactericidal activity (EBA) was 0.60 log10 cfu/day (95% CI 0.23-0.97, SEM 0.14). This compares with the vt50 calculated for isoniazid and moxifloxacin alone in the same population of 0.48 and 0.88 days, respectively. The EBA values for isoniazid and moxifloxacin alone were 0.77 and 0.53 log10 cfu/day, respectively. CONCLUSIONS: The combination of moxifloxacin and isoniazid is not antagonistic, but the combination does not significantly enhance bactericidal activity above that of isoniazid alone.
Antibiotics, Antitubercular↗
Effect of subinhibitory concentrations of ciprofloxacin on Mycobacterium fortuitum mutation rates.
OBJECTIVES: Fluoroquinolones have found a place in the management of mycobacterial diseases including tuberculosis. It has been previously shown that subinhibitory concentrations of quinolones increase the mutation rate in Escherichia coli and staphylococci. The purpose of this study is to extend this observation to mycobacteria and to quantify mutation rates. METHODS: The mutation rate in Mycobacterium fortuitum to ciprofloxacin, levofloxacin, moxifloxacin, rifampicin, erythromycin and gentamicin resistance was determined when grown with and without various sub-MIC concentrations of ciprofloxacin. RESULTS: M. fortuitum exposed to 1/2 MIC ciprofloxacin had an increase in the mutation rate of between 72- and 120-fold when selected on quinolones or other antimycobacterial antibiotics. Smaller, but significant increases in mutation rate were seen when the organism was exposed to lower concentrations (1/4 MIC and 1/8 MIC). CONCLUSIONS: These data show that sub-MIC concentrations of fluoroquinolone significantly increase mutation rates and these data suggest that care must be taken to ensure that bacteria are not exposed to subinhibitory concentrations when adding quinolones to a regimen used to treat mycobacterial infection.
Anti-Bacterial Agents↗
Analysis of rpoB and pncA mutations in the published literature: an insight into the role of oxidative stress in Mycobacterium tuberculosis evolution?
INTRODUCTION: It is perceived wisdom that within the host macrophage, Mycobacterium tuberculosis frequently encounters oxidative stress. Exposure of bacteria to reactive oxygen intermediates can have a mutagenic effect on the DNA. Various mutations are thought to arise as a consequence, including the oxidation of guanine residues, leading to G?C-->T?A substitution, and oxidation of cytosine resulting in a G?C-->A?T substitution. METHODS: We measured the relative contribution of oxidative stress by recording the percentage of single nucleotide substitutions reported in the genes rpoB and pncA that confer resistance to the antimicrobials rifampicin and pyrazinamide, respectively, and determined whether there is an excess of G?C-->T?A or G?C-->A?T substitutions. RESULTS: Out of 840 clinical isolates reported with single nucleotide mutations in the rpoB gene, 67% were G?C-->A?T changes, and 3% were G?C-->T?A substitutions. These figures were compared to the pncA gene, where out of 114 isolates, 30% of the single nucleotide mutations were G?C-->A?T transitions and 9% were G?C-->T?A changes. CONCLUSIONS: While there is an excess of G?C-->A?T changes in the rpoB gene, this was not the case in the pncA gene. Fifty-three percent of mutations within the rpoB gene were C-->T mutations of the type S531L. Although this mutation gives a fitness disadvantage, it is less than other common mutations, so it is more likely that that fitness is the determinant of surviving mutation rather than oxidative stress because of the small numbers of other C-->T and G-->A mutations at other sites (12%). There was no evidence of oxygen free radicals damaging the guanine bases in either gene.
Amidohydrolases↗
Origins and properties of Mycobacterium tuberculosis isolates in London.
Using similarities of IS6110 banding patterns, isolates of Mycobacterium tuberculosis from a population-based study in London were assigned to 12 large groups termed 'superfamilies' (sfams). Analysis of patient data showed a marked geographical association in the distribution of these sfams. In particular, isolates from patients born in Europe were from different sfams than those born elsewhere, indicating that there had been relatively little transmission of tuberculosis in London from immigrant communities into the endogenous population. Multivariate analysis showed that certain sfams were significantly associated with pulmonary rather than extrapulmonary disease, or with sputum smear negativity, independently of country of birth or ethnicity, suggesting that the properties of the infecting organism play a role in the nature of the disease process.
Adolescent↗
Novel PCR-restriction fragment length polymorphism method for determining serotypes or serogroups of Streptococcus pneumoniae isolates.
Serotyping Streptococcus pneumoniae is a technique generally confined to reference laboratories, as purchasing pneumococcal antisera is a huge investment. Many attempts have been made to modify serological agglutination techniques to make them more accessible, and more recently developments in serotyping have focused on molecular techniques. This paper describes a PCR assay which amplifies the entire capsulation locus between dexB and aliA. Amplicons are digested to produce serotype-specific patterns. We have shown, using 81 epidemiologically unrelated strains representing 46 different serotypes, that the patterns correlate with a 90 to 100% similarity range for the same serotype or serogroup. Prospective testing of 73 isolates of unknown serotype confirmed reliable serotype attribution, and serotype profiles are reproducible on repeated testing. Once our database contains all 90 serotypes, this technique should be fully portable, cost-effective, and useful in any laboratory with sufficient molecular experience.
Bacterial Typing Techniques↗
Tuberculosis: amplification-based clinical diagnostic techniques.
Tuberculosis (TB) is one of the major infectious causes of morbidity and mortality worldwide. TB is difficult to control due to the time taken for the microbiological diagnosis; typically culture on solid media takes 6-8 weeks. There are number of rapid molecular methods that have been developed to diagnose new cases of tuberculosis, detect drug resistance and identify the type of mycobacteria. These assays are based on recognition of mycobacterial DNA sequences and the subsequent amplification of nucleic acid sequences to facilitate detection. This review will describe some of the molecular assays that are in use for TB diagnosis and the considerations in designing and performing such assays. Early diagnosis of tuberculosis is critical for the successful management of patients allowing informed use of chemotherapy ensuring that the right patients are treated with the right antimicrobials.
Bacterial Typing Techniques↗
Evolutionary relationships among strains of Mycobacterium tuberculosis with few copies of IS6110.
Molecular typing of Mycobacterium tuberculosis by using IS6110 shows low discrimination when there are fewer than five copies of the insertion sequence. Using a collection of such isolates from a study of the epidemiology of tuberculosis in London, we have shown a substantial degree of congruence between IS6110 patterns and both spoligotype and PGRS type. This indicates that the IS6110 types mainly represent distinct families of strains rather than arising through the convergent insertion of IS6110 into favored positions. This is supported by identification of the genomic sites of the insertion of IS6110 in these strains. The combined data enable identification of the putative evolutionary relationships of these strains, comprising three lineages broadly associated with patients born in South Asia (India and Pakistan), Africa, and Europe, respectively. These lineages appear to be quite distinct from M. tuberculosis isolates with multiple copies of IS6110.
Africa↗
Multiple drug-resistant Mycobacterium tuberculosis: evidence for changing fitness following passage through human hosts.
Recent studies have shown a difference in the genotype of resistant bacteria following passage in animals compared to those passaged in vitro. This suggests that organisms rapidly adapt to their environment of growth. We sought to investigate whether this phenomenon occurred in human infection and whether changes could be detected in the fitness (growth velocity) of isolates transmitted between human hosts. Isogenic strains of Mycobacterium tuberculosis were obtained from a well-documented hospital outbreak. The subjects included those who were HIV seropositive and immunocompromised. The relative fitness of each sample was measured using growth competition in vitro. The results confirmed that our method of measuring fitness was not influenced by the storage conditions of the isolates, and demonstrated that the fitness of genetically similar isolates obtained from different patients in the outbreak differed significantly, as reflected in the growth velocity of the strains. This study provides the first evidence that multiple drug resistant M. tuberculosis strains adapt to the environment of their human host.
Adult↗