Sam Ogwang

A lipoprotein modulates activity of the MtrAB two-component system to provide intrinsic multidrug resistance, cytokinetic control and cell wall homeostasis in Mycobacterium

The MtrAB signal transduction system, which participates in multiple cellular processes related to growth and cell wall homeostasis, is the only two‐component system known to be essential in Mycobacterium. In a screen for antibiotic resistance determinants in Mycobacterium smegmatis, we identified a multidrug‐sensitive mutant with a transposon insertion in lpqB, the gene located immediately downstream of mtrA–mtrB. The lpqB mutant exhibited increased cell–cell aggregation and severe defects in surface motility and biofilm growth. lpqB cells displayed hyphal growth and polyploidism, reminiscent of the morphology of Streptomyces, a related group of filamentous Actinobacteria. Heterologous expression of M. tuberculosis LpqB restored wild‐type characteristics to the lpqB mutant. LpqB interacts with the extracellular domain of MtrB, and influences MtrA phosphorylation and promoter activity of dnaA, an MtrA‐regulated gene that affects cell division. Furthermore, in trans expression of the non‐phosphorylated, inactive form of MtrA in wild‐type M. smegmatis resulted in phenotypes similar to those of lpqB deletion, whereas expression of the constitutively active form of MtrA restored wild‐type characteristics to the lpqB mutant. These results support a model in which LpqB, MtrB and MtrA form a three‐component system that co‐ordinates cytokinetic and cell wall homeostatic processes.

Rate and Amplification of Drug Resistance among Previously-Treated Patients with Tuberculosis in Kampala, Uganda.

BACKGROUND Drug-resistant Mycobacterium tuberculosis has emerged as a global threat. In resource-constrained settings, patients with a history of tuberculosis (TB) treatment may have drug-resistant disease and may experience poor outcomes. There is a need to measure the extent of and risk factors for drug resistance in such patients. METHODS From July 2003 through November 2006, we enrolled 410 previously treated patients with TB in Kampala, Uganda. We measured the prevalence of resistance to first- and second-line drugs and analyzed risk factors associated with baseline and acquired drug resistance. RESULTS The prevalence of multidrug-resistant TB was 12.7% (95% confidence interval [95% CI], 9.6%-16.3%). Resistance to second-line drugs was low. Factors associated with multidrug-resistant TB at enrollment included a history of treatment failure (odds ratio, 23.6; 95% CI, 7.7-72.4), multiple previous TB episodes (odds ratio, 15.6; 95% CI, 5.0-49.1), and cavities present on chest radiograph (odds ratio, 5.9; 95% CI, 1.2-29.5). Among a cohort of 250 patients, 5.2% (95% CI, 2.8%-8.7%) were infected with M. tuberculosis that developed additional drug resistance. Amplification of drug resistance was associated with existing drug resistance at baseline (P < .01) and delayed sputum culture conversion (P < .01). CONCLUSIONS The burden of drug resistance in previously treated patients with TB in Uganda is sizeable, and the risk of generating additional drug resistance is significant. There is an urgent need to improve the treatment for such patients in low-income countries.

Low-cost rapid detection of rifampicin resistant tuberculosis using bacteriophage in Kampala, Uganda.

BackgroundResistance to anti-tuberculosis drugs is a serious public health problem. Multi-drug resistant tuberculosis (MDR-TB), defined as resistance to at least rifampicin and isoniazid, has been reported in all regions of the world. Current phenotypic methods of assessing drug susceptibility of M. tuberculosis are slow. Rapid molecular methods to detect resistance to rifampicin have been developed but they are not affordable in some high prevalence countries such as those in sub Saharan Africa. A simple multi-well plate assay using mycobacteriophage D29 has been developed to test M. tuberculosis isolates for resistance to rifampicin. The purpose of this study was to investigate the performance of this technology in Kampala, Uganda.MethodsIn a blinded study 149 M. tuberculosis isolates were tested for resistance to rifampicin by the phage assay and results compared to those from routine phenotypic testing in BACTEC 460. Three concentrations of drug were used 2, 4 and 10 μg/ml. Isolates found resistant by either assay were subjected to sequence analysis of a 81 bp fragment of the rpoB gene to identify mutations predictive of resistance. Four isolates with discrepant phage and BACTEC results were tested in a second phenotypic assay to determine minimal inhibitory concentrations.ResultsInitial analysis suggested a sensitivity and specificity of 100% and 96.5% respectively for the phage assay used at 4 and 10 μg/ml when compared to the BACTEC 460. However, further analysis revealed 4 false negative results from the BACTEC 460 and the phage assay proved the more sensitive and specific of the two tests. Of the 39 isolates found resistant by the phage assay 38 (97.4%) were found to have mutations predictive of resistance in the 81 bp region of the rpoB gene. When used at 2 μg/ml false resistant results were observed from the phage assay. The cost of reagents for testing each isolate was estimated to be 1.3US

Protein Kinase G Is Required for Intrinsic Antibiotic Resistance in Mycobacteria

when testing a batch of 20 isolates on a single 96 well plate. Results were obtained in 48 hours.ConclusionThe phage assay can be used for screening of isolates for resistance to rifampicin, with high sensitivity and specificity in Uganda. The test may be useful in poorly resourced laboratories as a rapid screen to differentiate between rifampicin susceptible and potential MDR-TB cases.

Elucidating emergence and transmission of multidrug-resistant tuberculosis in treatment experienced patients by whole genome sequencing

ABSTRACT Antibiotic resistance and virulence of pathogenic mycobacteria are phenotypically associated, but the underlying genetic linkage has not been known. Here we show that PknG, a eukaryotic-type protein kinase previously found to support survival of mycobacteria in host cells, is required for the intrinsic resistance of mycobacterial species to multiple antibiotics.

Bacterial conversion of folinic acid is required for antifolate resistance.

Background Understanding the emergence and spread of multidrug-resistant tuberculosis (MDR-TB) is crucial for its control. MDR-TB in previously treated patients is generally attributed to the selection of drug resistant mutants during inadequate therapy rather than transmission of a resistant strain. Traditional genotyping methods are not sufficient to distinguish strains in populations with a high burden of tuberculosis and it has previously been difficult to assess the degree of transmission in these settings. We have used whole genome analysis to investigate M. tuberculosis strains isolated from treatment experienced patients with MDR-TB in Uganda over a period of four years. Methods and Findings We used high throughput genome sequencing technology to investigate small polymorphisms and large deletions in 51 Mycobacterium tuberculosis samples from 41 treatment-experienced TB patients attending a TB referral and treatment clinic in Kampala. This was a convenience sample representing 69% of MDR-TB cases identified over the four year period. Low polymorphism was observed in longitudinal samples from individual patients (2-15 SNPs). Clusters of samples with less than 50 SNPs variation were examined. Three clusters comprising a total of 8 patients were found with almost identical genetic profiles, including mutations predictive for resistance to rifampicin and isoniazid, suggesting transmission of MDR-TB. Two patients with previous drug susceptible disease were found to have acquired MDR strains, one of which shared its genotype with an isolate from another patient in the cohort. Conclusions Whole genome sequence analysis identified MDR-TB strains that were shared by more than one patient. The transmission of multidrug-resistant disease in this cohort of retreatment patients emphasises the importance of early detection and need for infection control. Consideration should be given to rapid testing for drug resistance in patients undergoing treatment to monitor the emergence of resistance and permit early intervention to avoid onward transmission.

Comparison of time to positive and colony counting in an early bactericidal activity study of anti-tuberculosis treatment

Antifolates, which are among the first antimicrobial agents invented, inhibit cell growth by creating an intracellular state of folate deficiency. Clinical resistance to antifolates has been mainly attributed to mutations that alter structure or expression of enzymes involved in de novo folate synthesis. We identified a Mycobacterium smegmatis mutant, named FUEL (which stands for folate utilization enzyme for leucovorin), that is hypersusceptible to antifolates. Chemical complementation indicated that FUEL is unable to metabolize folinic acid (also known as leucovorin or 5-formyltetrahydrofolate), whose metabolic function remains unknown. Targeted mutagenesis, genetic complementation, and biochemical studies showed that FUEL lacks 5,10-methenyltetrahydrofolate synthase (MTHFS; also called 5-formyltetrahydrofolate cyclo-ligase; EC 6.3.3.2) activity responsible for the only ATP-dependent, irreversible conversion of folinic acid to 5,10-methenyltetrahydrofolate. In trans expression of active MTHFS proteins from bacteria or human restored both antifolate resistance and folinic acid utilization to FUEL. Absence of MTHFS resulted in marked cellular accumulation of polyglutamylated species of folinic acid. Importantly, MTHFS also affected M. smegmatis utilization of monoglutamylated 5-methyltetrahydrofolate exogenously added to the medium. Likewise, Escherichia coli mutants lacking MTHFS became susceptible to antifolates. These results indicate that folinic acid conversion by MTHFS is required for bacterial intrinsic antifolate resistance and folate homeostatic control. This novel mechanism of antimicrobial antifolate resistance might be targeted to sensitize bacterial pathogens to classical antifolates.

Methylfolate Trap Promotes Bacterial Thymineless Death by Sulfa Drugs

SETTING Patients with smear-positive, newly diagnosed pulmonary tuberculosis (TB) presenting to the out-patient TB clinic in Kampala, Uganda. OBJECTIVE To compare colony-forming unit (cfu) counting and time to positive (TTP) in Mycobacteria Growth Indicator Tube (MGIT) culture as measures of early bactericidal activity (EBA). DESIGN Patients were enrolled in an EBA feasibility study of standard TB chemotherapy. Sixteen-hour overnight sputum collections were obtained before and on days 2, 4, 7, 10, 12 and 14 of treatment for quantitative culture on selective Middlebrook 7H11 agar media and TTP in the MGIT liquid culture system. RESULTS Log cfu and TTP were correlated over all time points (r(s) = -0.71, P < 0.001). Within-subject (day to day) variation as a percentage of total variation was very similar between the two measures: 25.7% for cfu and 25% for TTP. Mean EBA 0-14, 0-2 and 2-14 measured by TTP were similar to those previously reported. CONCLUSION TTP measured by an automated, standardized, commercially available culture system correlates with cfu determinations. EBA measured by TTP provides similar information to cfu counting, and is reproducible across sites and in different patient populations. These findings support replacing cfu counting with TTP as the primary measurement in EBA studies.

Comparison of MGIT and Myco/F lytic liquid-based blood culture systems for recovery of Mycobacterium tuberculosis from pleural fluid.

The methylfolate trap, a metabolic blockage associated with anemia, neural tube defects, Alzheimer’s dementia, cardiovascular diseases, and cancer, was discovered in the 1960s, linking the metabolism of folate, vitamin B12, methionine and homocysteine. However, the existence or physiological significance of this phenomenon has been unknown in bacteria, which synthesize folate de novo. Here we identify the methylfolate trap as a novel determinant of the bacterial intrinsic death by sulfonamides, antibiotics that block de novo folate synthesis. Genetic mutagenesis, chemical complementation, and metabolomic profiling revealed trap-mediated metabolic imbalances, which induced thymineless death, a phenomenon in which rapidly growing cells succumb to thymine starvation. Restriction of B12 bioavailability, required for preventing trap formation, using an “antivitamin B12” molecule, sensitized intracellular bacteria to sulfonamides. Since boosting the bactericidal activity of sulfonamides through methylfolate trap induction can be achieved in Gram-negative bacteria and mycobacteria, it represents a novel strategy to render these pathogens more susceptible to existing sulfonamides.

Incubation time of Mycobacterium tuberculosis complex sputum cultures in BACTEC MGIT 960: 4weeks of negative culture is enough for physicians to consider alternative diagnoses.

ABSTRACT The specificities and sensitivities of the Bactec mycobacterial growth indicator tube (MGIT) system for the recovery of Mycobacterium tuberculosis from pleural fluid are not statistically different than those of the Myco/F lytic liquid culture system. The time to positivity is shorter in the MGIT system (12.7 versus 20.7 days, respectively; P = 0.007). The Myco/F lytic culture system may be an alternative to the MGIT system for diagnosing pleural tuberculosis.