Alexander S. Pym

A new evolutionary scenario for the Mycobacterium tuberculosis complex

The distribution of 20 variable regions resulting from insertion-deletion events in the genomes of the tubercle bacilli has been evaluated in a total of 100 strains of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, and Mycobacterium bovis. This approach showed that the majority of these polymorphisms did not occur independently in the different strains of the M. tuberculosis complex but, rather, resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis specific deletion (TbD1), M. tuberculosis strains can be divided into ancestral and “modern” strains, the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters. Furthermore, successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum, M. microti, and M. bovis that diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the often-presented hypothesis that M. tuberculosis, the etiological agent of human tuberculosis evolved from M. bovis, the agent of bovine disease. M. canettii and ancestral M. tuberculosis strains lack none of these deleted regions, and, therefore, seem to be direct descendants of tubercle bacilli that existed before the M. africanum→M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could well have been a human pathogen already.

The Diarylquinoline TMC207 for Multidrug-Resistant Tuberculosis

BACKGROUND The diarylquinoline TMC207 offers a new mechanism of antituberculosis action by inhibiting mycobacterial ATP synthase. TMC207 potently inhibits drug-sensitive and drug-resistant Mycobacterium tuberculosis in vitro and shows bactericidal activity in patients who have drug-susceptible pulmonary tuberculosis. METHODS In the first stage of a two-stage, phase 2, randomized, controlled trial, we randomly assigned 47 patients who had newly diagnosed multidrug-resistant pulmonary tuberculosis to receive either TMC207 (400 mg daily for 2 weeks, followed by 200 mg three times a week for 6 weeks) (23 patients) or placebo (24 patients) in combination with a standard five-drug, second-line antituberculosis regimen. The primary efficacy end point was the conversion of sputum cultures, in liquid broth, from positive to negative. RESULTS The addition of TMC207 to standard therapy for multidrug-resistant tuberculosis reduced the time to conversion to a negative sputum culture, as compared with placebo (hazard ratio, 11.8; 95% confidence interval, 2.3 to 61.3; P=0.003 by Cox regression analysis) and increased the proportion of patients with conversion of sputum culture (48% vs. 9%). The mean log(10) count of colony-forming units in the sputum declined more rapidly in the TMC207 group than in the placebo group. No significant differences in average plasma TMC207 concentrations were noted between patients with and those without culture conversion. Most adverse events were mild to moderate, and only nausea occurred significantly more frequently among patients in the TMC207 group than among patients in the placebo group (26% vs. 4%, P=0.04). CONCLUSIONS The clinical activity of TMC207 validates ATP synthase as a viable target for the treatment of tuberculosis. (ClinicalTrials.gov number, NCT00449644.)

Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis

The live tuberculosis vaccines Mycobacterium bovis BCG (bacille Calmette-Guérin) and Mycobacterium microti both lack the potent, secreted T-cell antigens ESAT-6 (6-kDa early secretory antigenic target) and CFP-10 (10-kDa culture filtrate protein). This is a result of independent deletions in the region of deletion-1 (RD1) locus, which is intact in virulent members of the Mycobacterium tuberculosis complex. To increase their immunogenicity and protective capacity, we complemented both vaccines with different constructs containing the esxA and esxB genes, which encode ESAT-6 and CFP-10 respectively, as well as a variable number of flanking genes. Only reintroduction of the complete locus, comprising at least 11 genes, led to full secretion of the antigens and resulted in specific ESAT-6–dependent immune responses; this suggests that the flanking genes encode a secretory apparatus. Mice and guinea pigs vaccinated with the recombinant strain BCG::RD1-2F9 were better protected against challenge with M. tuberculosis, showing less severe pathology and reduced dissemination of the pathogen, as compared with control animals immunized with BCG alone.

Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti

Although large human populations have been safely immunized against tuberculosis with two live vaccines, Mycobacterium bovis BCG or Mycobacterium microti, the vole bacillus, the molecular basis for the avirulence of these vaccine strains remains unknown. Comparative genomics has identified a series of chromosomal deletions common to both virulent and avirulent species but only a single locus, RD1, that has been deleted from M. bovis BCG and M. microti. Restoration of RD1, by gene knock‐in, resulted in a marked change in colonial morphology towards that of virulent tubercle bacilli. Three RD1‐encoded proteins were localized in the cell wall, and two of them, the immunodominant T‐cell antigens ESAT‐6 and CFP‐10, were also found in culture supernatants. The BCG::RD1 and M. microti::RD1 knock‐ins grew more vigorously than controls in immunodeficient mice, inducing extensive splenomegaly and granuloma formation. Increased persistence and partial reversal of attenuation were observed when immunocompetent mice were infected with the BCG::RD1 knock‐in, whereas BCG controls were cleared. Knocking‐in five other RD loci did not affect the virulence of BCG. This study describes a genetic lesion that contributes to safety and opens new avenues for vaccine development.

Multidrug-Resistant Tuberculosis and Culture Conversion with Bedaquiline

BACKGROUND Bedaquiline (Sirturo, TMC207), a diarylquinoline that inhibits mycobacterial ATP synthase, has been associated with accelerated sputum-culture conversion in patients with multidrug-resistant tuberculosis, when added to a preferred background regimen for 8 weeks. METHODS In this phase 2b trial, we randomly assigned 160 patients with newly diagnosed, smear-positive, multidrug-resistant tuberculosis to receive either 400 mg of bedaquiline once daily for 2 weeks, followed by 200 mg three times a week for 22 weeks, or placebo, both in combination with a preferred background regimen. The primary efficacy end point was the time to sputum-culture conversion in liquid broth. Patients were followed for 120 weeks from baseline. RESULTS Bedaquiline reduced the median time to culture conversion, as compared with placebo, from 125 days to 83 days (hazard ratio in the bedaquiline group, 2.44; 95% confidence interval, 1.57 to 3.80; P<0.001 by Cox regression analysis) and increased the rate of culture conversion at 24 weeks (79% vs. 58%, P=0.008) and at 120 weeks (62% vs. 44%, P=0.04). On the basis of World Health Organization outcome definitions for multidrug-resistant tuberculosis, cure rates at 120 weeks were 58% in the bedaquiline group and 32% in the placebo group (P=0.003). The overall incidence of adverse events was similar in the two groups. There were 10 deaths in the bedaquiline group and 2 in the placebo group, with no causal pattern evident. CONCLUSIONS The addition of bedaquiline to a preferred background regimen for 24 weeks resulted in faster culture conversion and significantly more culture conversions at 120 weeks, as compared with placebo. There were more deaths in the bedaquiline group than in the placebo group. (Funded by Janssen Pharmaceuticals; TMC207-C208 ClinicalTrials.gov number, NCT00449644.).

Genomic Deletions Classify the Beijing/W Strains as a Distinct Genetic Lineage of Mycobacterium tuberculosis

ABSTRACT Beijing/W strains of Mycobacterium tuberculosis are geographically widespread and hypervirulent. To enhance our understanding of their origin and evolution, we sought phylogenetically informative large sequence polymorphisms (LSPs) within the Beijing/W family. Comparative whole-genome hybridization of Beijing/W strains revealed 21 LSPs, 7 of which were previously unreported. We show that some of these LSPs are unique event polymorphisms that can be used to define and subdivide the Beijing/W family. One LSP (RD105) was seen in all Beijing/W strains and thus serves as a useful marker for the identification of this family of strains. Additional LSPs (RD142, RD150, and RD181) further divided this family into four monophyletic subgroups, demonstrating a deeper population structure than previously appreciated. All Beijing/W strains were also observed to have an intact pks15/1 gene that is involved in the biosynthesis of a phenolic glycolipid, a putative virulence factor. A simple PCR assay using these Beijing/W strain-defining deletions will facilitate molecular epidemiological studies and may assist in the identification of the molecular basis of phenotypes associated with this important lineage of M. tuberculosis.

Randomized Pilot Trial of Eight Weeks of Bedaquiline (TMC207) Treatment for Multidrug-Resistant Tuberculosis: Long-Term Outcome, Tolerability, and Effect on Emergence of Drug Resistance

ABSTRACT The 2-year follow-up results for a randomized placebo-controlled study of 47 patients with multidrug-resistant pulmonary tuberculosis treated with either the new diarylquinoline TMC207, recently renamed bedaquiline, or placebo, added to the first 8 weeks of a background regimen, are presented. Bedaquiline significantly reduced the time to culture conversion over 24 weeks (hazard ratio, 2.253; 95% confidence interval, 1.08 to 4.71; P = 0.031). With the exception of nausea reported in 26% of patients receiving bedaquiline and none receiving placebo, adverse events occurred at similar frequencies in both groups of patients: bilateral hearing impairment, extremity pain, acne, and noncardiac chest pain occurred in 13 and 21%, 17 and 13%, 9 and 17%, and 4 and 17% of patients, respectively, receiving bedaquiline or placebo. Excluding resistance to ethambutol and ethionamide, only one patient receiving bedaquiline acquired resistance to companion drugs, but five patients receiving placebo (4.8% versus 21.7%; P = 0.18) acquired resistance to companion drugs, and resistance to ofloxacin was acquired in four patients receiving placebo and none receiving bedaquiline (0% versus 22%; 0 = 0.066). In all, 23 patients (49%), including 13 receiving placebo (54%) and 10 receiving bedaquiline (44%), discontinued the study prior to its completion, 12 during the first 24 weeks of treatment. Eight subjects were withdrawn for noncompliance or default, and seven withdrew consent, citing the rigorous program of investigations for safety and pharmacokinetic monitoring. Bedaquiline may contribute to the management of multidrug-resistant tuberculosis by effecting more rapid sputum culture negativity and by preventing acquired resistance to companion drugs.

Effect of katG Mutations on the Virulence of Mycobacterium tuberculosis and the Implication for Transmission in Humans

ABSTRACT The usefulness of isoniazid (INH), a key component of short-course chemotherapy of tuberculosis, is threatened by the emergence of drug-resistant strains of Mycobacterium tuberculosis with mutations in the katG gene. It is shown here that the most commonly occurring KatG mutation, where Ser 315 is replaced by Thr (S315T), is associated with clinically significant levels of INH resistance. In contrast to another resistant isolate, in which Pro replaces Thr 275, the S315T mutant produces active catalase-peroxidase and is virulent in the mouse model of the disease, indicating that a significant loss of bacterial fitness does not result from this frequent mutation. The implications of this finding for the transmission and reactivation of multidrug-resistant strains of M. tuberculosis are severe.

The evolution of mycobacterial pathogenicity: clues from comparative genomics

Comparative genomics, and related technologies, are helping to unravel the molecular basis of the pathogenesis, host range, evolution and phenotypic differences of the slow-growing mycobacteria. In the highly conserved Mycobacterium tuberculosis complex, where single-nucleotide polymorphisms are rare, insertion and deletion events (InDels) are the principal source of genome plasticity. InDels result from recombinational or insertion sequence (IS)-mediated events, expansion of repetitive DNA sequences, or replication errors based on repetitive motifs that remove blocks of genes or contract coding sequences. Comparative genomic analyses also suggest that loss of genes is part of the ongoing evolution of the slow-growing mycobacterial pathogens and might also explain how the vaccine strain BCG became attenuated.

Impact of bacterial genetics on the transmission of isoniazid-resistant Mycobacterium tuberculosis.

Understanding the ecology of drug-resistant pathogens is essential for devising rational programs to preserve the effective lifespan of antimicrobial agents and to abrogate epidemics of drug-resistant organisms. Mathematical models predict that strain fitness is an important determinant of multidrug-resistant Mycobacterium tuberculosis transmission, but the effects of strain diversity have been largely overlooked. Here we compared the impact of resistance mutations on the transmission of isoniazid-resistant M. tuberculosis in San Francisco during a 9-y period. Strains with a KatG S315T or inhA promoter mutation were more likely to spread than strains with other mutations. The impact of these mutations on the transmission of isoniazid-resistant strains was comparable to the effect of other clinical determinants of transmission. Associations were apparent between specific drug resistance mutations and the main M. tuberculosis lineages. Our results show that in addition to host and environmental factors, strain genetic diversity can influence the transmission dynamics of drug-resistant bacteria.