Mechanisms of resistance and multiresistance in tuberculosis

Transcription

1 Mechanisms of resistance and multiresistance in tuberculosis Ying Zhang, MD, PhD Professor Department of Molecular Microbiology & Immunology Bloomberg School of Public Health Johns Hopkins University

2 Historical Perspective Old infectious disease, since antiquity, Egyptian mummies 4000 years ago had typical spine TB (Pott s disease) Phthisis ( wasting, Hippocrates), Consumption, White Plague Association with art, literature unique in human civilization Robert Koch discovered M. tuberculosis in 1882 Calmette and Guerin developed BCG vaccine in % all deaths in 18 th and 19 th century due to TB (5% now) Improved sanitation and nutrition lowered the incidence of TB even before chemotherapy in 1950s Introduction of chemotherapy in 1950s made TB curable and led to further decrease in TB cases TB declined in developed countries until 1970s In the late 1980s incidence in developed countries increased, with emergence of drug-resistant strains

3 Complacency is costly MDR-TB outbreak in New York City , in HIV-positive persons 1 billion $ to control Developed versus developing countries Worldwide, TB is on the increase in recent years due to HIV, movement of people

4 TB: A leading infectious killer - Top 3 infectious killer One-third of world population is infected with TB bacillus 2 billion people TB kills about million people each year 8-9 million people become sick with TB each year TB is the leading killer of HIV/AIDS patients WHO proposed a Global Plan to Stop Tuberculosis which aims to save 14 million lives between

5 Drug Resistant TB 50 million people infected with MDR-TB, or 5.3% MDR globally (all cases) Each year there are 500,000 cases MDR-TB (110,000 deaths), and 40,000 cases XDR, 25,000 deaths) Highest rates ever recorded of MDR-TB (WHO survey ( Highest rates are in countries of the former Soviet Union and China China, India, Russia carry the highest MDR-TB cases Former Soviet Union countries, Baku, Azerbaijan had 22.3% new TB cases MDR, Moldova (19.4%), Ukraine (16%), Tomsk (15%) China, Inner Mongolia and Heilongjiang have 7.25% MDR-TB

6 The New Tuberculosis HIV and Drug-resistant TB A lethal combination and a major threat to TB control WHO declared TB a global emergency in 1993 TB emergency declared in Africa - August 25, 2005

7 Extensively Drug-Resistant TB (XDR-TB) Virtually untreatable" form of TB XDR-TB: defined as MDR-TB (resistance to INH, RIF), plus resistance to 2 major second-line drugs (quinolone and aminoglycoside), reported in 45 countries

8 TB Chemotherapy: THE Effective TB Control Pre-antibiotic era: before 1940s (e.g., cod liver oils, bed rest, fresh air) Drugs used to treat TB: Streptomycin first TB drug (1944), followed by PAS (1946), isoniazid (1952), pyrazinamide (1952), rifampin (1963) (a) Front-line Drugs: isoniazid (INH) rifampicin (RIF), pyrazinamide (PZA), streptomycin, ethambutol (b) Second-line Drugs: PAS, kanamycin, cycloserine, ethionamide, thiacetazone, ciprofloxacin/ofloxacin, rifapentine, amikacin, viomycin, capreomycin

9 DOTS - The Best TB Therapy since early 1990s DOTS: 6 month therapy - The best therapy against TB (78%-96% cure rate) Initial phase (daily, 2 months) with 4 drugs: Isoniazid, Rifampin, Pyrazinamide, Ethambutol Continuation phase (3 times a week, 4 months) with 2 drugs: Isoniazid, Rifampin

10 Lengthy TB chemotherapy makes patient compliance difficult

11 TB Chemotherapy-cont Treatment principle: Drug combination, for other infections (e.g. HIV-HAART), and cancer (e.g., MOPP, lymphoma) Why drug combination? (a) prevent drug resistance: Spontaneous mutations (calculations, e.g., R to INH occur at 1 in a million bacilli, R to RMP at 1 in 100 million) (b) enhance the efficacy of the therapy (Mitchison hypothesis)

12 Special Bacterial Populations Theory (Mitchison Hypothesis) High Speed of bacterial growth Low A. Continuous growth D. Dormant INH (RMP, SM, EMB) RMP Semi-dormant PZA B. Spurts of C. Acid metabolism inhibition

13 Yin and Yang of Bacterial Life Cycle: Effect of Drugs Reverters RIF, J Compound PZA INH, EMB, SM Persisters (Y. Zhang, Clin Pharmacol Ther. 2007; 82: )

14 Drug Resistance Mechanisms Two Types of Drug Resistance: Genetic drug resistance: due to chromosomal mutations or acquisition of antibiotic resistance genes on plasmids or transposons Phenotypic drug resistance: due to changes in bacterial physiological state as in stationary phase, antibiotic persisters, dormant state

15 Feature of Drug-Resistant TB Drug resistance in M. tuberculosis is NOT mediated by plasmids or transposons as in many other bacteria, but due to mutations in chromosomal genes

16 Virulence of Drug-resistant TB INH-resistant strains are often attenuated for virulence in guinea pigs KatG-negative INH-resistant strains with high level of resistance may be attenuated or less transmissible for humans Resistance to other drugs is not associated with attenuation of virulence: e.g., PZA-mono-resistant strain is still fully virulent and cause active transmission

17 Mechanisms of Drug Resistance in M. tuberculosis Drugs Physiologic Molecular target Genes associated with effect/inhibition resistance INH cell wall mycolic acid enoyl acyl carrier catalase-peroxidase synthesis; protein reductase (InhA) (katg) (KatG %); inha (10-20%) oxygen radicalassociated damage; NAD metabolism? RIF RNA synthesis RNA polymerase rpob (95%) PZA membrane function Membrane energy PZase (pnca) (84%) EMB cell wall component arabinosyl transferase embb (EmbB306 50%) arabinogalactan synthesis SM protein synthesis ribosome S12 protein; rpsl (60%); 16S rrna; rrs (20%); rrna methyltransferase (G527 in 530 loop) gidb Amk, Kan, Cap :: 16S rrna rrs (1400A->G) Quinolone DNA synthesis DNA gyrase gyra (95%), gyrb

18 Isoniazid (INH) One of the most important front-line TB drug INH is a cheap drug with a simple structure First chemically synthesized in 1912, but its antituberculosis activity was not recognized until 1952 by Roche, Bayer, Bristol Meyer Squibb - Euphoria Highly active against MTB, MIC= ug/ml

19 Euphoria - Dancing in the Wards Patients dancing at the Sea View Hospital, Staten Island, New York, 1953, at the startling improvement following INH treatment (Times 1953, March 13)

20 INH Resistance Mechanism 1. katg (catalase-peroxidase gene) INH-resistant TB strains often lose catalase/peroxidase activity (an enzyme involved in detoxifying peroxide) and often lose virulence in guinea pigs (Middlebrook, 1952) Cloning of catalase-peroxidase gene (katg) and demonstration that katg deletion or point mutations cause INH resistance (Zhang et al. 1992, Nature, 358: ) (Fig.) Restoration of INH sensitivity by katg gene (Zhang et al. 1993, Mol. Microbiol)

21 katg Deletion in INH-Resistant TB Strains (Zhang et al., 1992, Nature, 358: )

22 Mechanisms of Drug Resistance 1. Reduced permeability/uptake 2. Enhanced efflux 3. Enzymatic inactivation (beta-lactamase) 4. Alteration of drug target 5. Loss of enzymes involved in drug activation e.g. isoniazid resistance-katg (1992), pyrazinamide resistance-pnca (1996), metronidazole resistance in H. pylori RdxA (1998)

23 INH Resistance-cont 2. inha (enoylacyl carrier protein reductase) encoding an enzyme involved in mycolic acid synthesis, is a target for INH (Jacobs, Bloom and colleagues, 1994) Among katg, inha genes, mutations in katg are the most frequent cause of INH resistance, accounting for some 60-90% resistant strains, whereas inha mutations accounting for 10-30% strains. There are some R- strains without mutations in katg, inha, suggesting new mechanism of resistance

24 INH induces its own resistance in MTB (S. Siddiqi, Y. Zhang, AAC, June, 2007) A. Growth Index (GI ) values (days) exposed to 0.4 u g/ml INH Final CFU count Control Week /ml Week x10 3 /ml Week x10 4 /ml Week x10 4 /ml B. Growth Index ( GI) values (days) exposed to 2 u g/ml RIF Final CFU count Control Week Week Week Week

25 Characterization of INH-resistant subcultures derived from INH-exposed culture of H37Rv Subcultures AST by MIC Catalase Mutation in BACTEC!g/ml activity katg H37Rv Control +++ WT SC1 R WT SC2 R >2 + WT SC3 R >2 + D329H SC4 R >2 - WT SC5 R WT SC6 R >2 - W328R SC7 R >2 - W328R SC8 R >2 - WT

26 O NH NH 2 C INH N Passive diffusion KatG activation Efflux Reactive oxygen/ organic radicals Antagonists NAT? AhpC? Multiple targets DNA damage? Mycolic acid synthesis InhA NAD metabolism

27 Pyrazinamide (PZA): An unconventional and paradoxical drug One of the most important front-line TB drug, that plays a key role in shortening the therapy, because PZA kills persister TB bacilli that are not killed by other TB drugs Despite its powerful in vivo activity in shortening the therapy, curiously, PZA has no activity against TB bacilli in vitro in normal culture medium

28 Paradoxical Features of PZA PZA is active at acidic ph (ph 5.5) (McDermott 1954) MIC is high = mg/ml at acid ph , and kills MTB slowly (50-70% kill in two weeks) PZA kills old, dormant bacilli more effectively than actively growing bacilli (Zhang et al., 2002) PZA kills non-replicating TB bacilli more effectively under hypoxic/anaerobic conditions (Wade and Zhang, 2004) In vivo (mice or humans), it has impressive sterilizing activity against persister bacilli and is involved shortening the therapy Correlation with EBA studies in humans: INH has high EBA in first 3 days, PZA has poor in first 2 weeks

29 Walsh McDermott ( ) Founding father of IOM, National Academies Clinical evaluation of INH(Lasker Award, 1955) Cornell mouse model of TB Persistence Work on pyrazinamide (PZA): (a) acid ph, (b) PZA-resistant TB lose PZase, (c) unique sterilizing activity of PZA

30 PZA achieves high sterilizing activity with INH - Basis for SCC From McCune R M, Tompsett R, McDermott W J Exp Med 1956; 104:

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32 History of PZA Unconventional discovery: In 1944, Chorine found nicotinamide (niacinamide), a vitamin B3 derivative (structure Fig.) had antimycobacterial activity in animal models-> in 1948, Lederle Lab made analogs of nicotinamide -> PZA Used in clinical treatment in 1952 (same year as INH), mainly used as a second-line drug for drug-resistant cases or relapse because of liver toxicity due to high dose and long term use In late 1970s and early 80s, through clinical trials by British MRC, PZA was found to shorten the therapy from previously 9-12 months to 6 months -> now used as firstline drug -> Basis for modern short-course TB therapy

33 PZA Resistance In 1967, McDermott and colleagues showed that PZA-resistant strains lose pyrazinamidase/nicotinamidase enzyme activity

34 Cloning of TB pnca gene Cloning of the pyrazinamidase gene (pnca) and found mutation in pnca gene is major mechanism of PZA resistance (Scorpio and Zhang, 1996, Nature Medicine)

35 PZA Mechanism of Resistance Mutations in pnca gene are the major mechanism of PZA resistance M. bovis strains including BCG are naturally resistant to PZA and lack PZase enzyme, due to a single characteristic point mutation in pnca gene, at nt. 169 changing from C (MTB pnca) to G, causing Histidine to Aspartic Acid at position 57. This point mutation is the cause for the natural PZA resistance to PZA of M. bovis strains Rapid differentiation of M. bovis from MTB strains based on detecting the C to G point mutation in the pnca gene

36 The number and percentage of PZA-resistant strains/pnca mutations (data collected from ) (sensitivity ranges from %) (Yanqiu Zhang, Ying Zhang) PZA-R MutationsPoint M insertion Frameshift deletion strains times percentage - 84% 81% 8% 11%

37 pnca mutation distribution at nucleotide level 12)3+'4 (5 )2*6*7(189*(*6:;/%# %" %! $" $! #" #! "! &'()(*+' $#,$ -%., #!" #$- #," #-- #./ $!. $$0 $"! $/# $0$ %#% %%, %"" %/- %0/,#.,%0,-!,.# "!$ "$% ",, nucleotide position

38 Detection of pnca mutations as a rapid test for PZA resistance PZA susceptibility testing is not performed routinely because of the difficulty and problem of DST at acid ph, as a result drug resistance survey does not have PZA resistance data Acid ph inhibits growth of MTB (about 25-30% TB strains are acid sensitive to ph 5.5 on LJ medium) BACTEC PZA test at ph 6.0 with MIC cutoff of 100 ug/ml tend to have false resistance problem, takes 2 weeks, expensive and not widely used We propose to sequence pnca gene for all drug resistant TB strains, since pnca gene is quite small (560 bp), and since there is pretty good correlation between pnca mutations and PZA resistance (84%), and since the current PZA susceptibility testing is time-consuming, unreliable and expensive

39 Mode of Action of PZA PZA PZase conversion pka = 2.9 N N Passive diffusion O C Passive diffusion NH 2 [POA - ] Model can explain unusual properties of PZA: Acid ph, preferential activity for non-replicating persisters, hypoxic conditions POA - + H + HPOA Acid ph Defective efflux H HPOA NAD metabolism? Acidification of cytoplasm Disruption of membrane function

40 Enhanced PZA activity by energy inhibitors Zhang et al. J. Antimicrob. Chemother Young H37Ra cell; PZA=100 µg/ml; 5 day incubation at ph5.5

41 Synergy Between Diarylquinoline (J) and PZA (Andries et al., 2005, Science, 307: 223-7) Like DCCD, J compound Inhibits F1F0 H-ATPase

42 Importance of Drug Resistance Detection Timely detection of drug resistance is important for the outcome of treatment Detection of drug resistance is a measure of quality of treatment CDC and British Thoracic Society recommend drug susceptibility tests (DST) be performed on isolates from all new TB patients

43 The Problem-Unique Lengthy chemotherapy (6-8 months) selects drug resistant TB bacteria Unlike detection of drug resistance in other bacteria, TB drug resistance detection is time-consuming due to slow growth of TB bacteria Unique challenge for molecular detection of TB drug resistance: mutations in diverse chromosomal drug resistance genes, and mutations in some cases are not clustered

44 Molecular Methods for Detecting Drug Resistance Divided into Two Categories: Both use PCR -Methods for detecting known mutations in resistance genes: -Methods for detecting unknown mutations in resistance genes: DNA sequencing/pyrosequencing, Microarray

45 Correlation between Mutations and Drug Resistance INH resistance: KatG315 (80-95%), inha (10-20%) (- 15C->T), KatG315 and inha 15 C-to-T (80-95%) RIF resistance: rpob (95%), 81 bp, 531, 526, 516 PZA resistance: pnca (84%), scattered EMB resistance: EmbB306 (50%) Fluoroquinolone resistance: gyra(95%) SM resistance: RpsL43/88(60%), rrs (20%) amikacin, kanamycin, capreomycin: rrs 1400A->G

46 Diagnostic Methods for Detecting Known Mutations in Drug Resistance Genes PCR, followed by PCR-RFLP (HapII-katG 315), SSCP, heteroduplex formation, WAVE-DHPLC System (Transgenomics Inc. Omaha), FRET (fluorescence resonance energy transfer) probes, molecular beacons, ARMS (Amplification Refractory Mutation System), hybridization (Line-Probe assay) tests in microarray and macroarray, real-time PCR (TaqMan), isothermal amplification assay Line-Probe assay (GenoType MTBDRplus); Real-time PCR; (loopmediated isothermal amplification (LAMP), are currently being evaluated in the field with promising results

47 Methods For Detecting Unknown Mutations In Resistance Genes DNA sequencing/pyrosequencing (expensive and cumbersome, not appropriate for high throughput large number of samples) Microarray -High Density Oligo Arrays by Affymetrix (Re-Sequencing) (Gingeras et al., Genome Res. 1998;8:435 48; Troesch et al., 1999, JCM, 37:49-55): speciation, RIF rpob mutations (too expensive as this array requires too many probes) -Sliding-Frame Oligo Array: 79 overlapping oligos: pnca mutations in PZAresistant strains (Wade et al., 2004, Diagn Micro Inf Dis)

48 Bacterial Persisters The phenomenon of bacterial persisters was first described by Joseph Bigger in 1944 Penicillin could not completely sterilize Staphylococcal culture in vitro. The residual persisters (about 1%) not killed by antibiotic were still susceptible to the same antibiotic upon subculture The resistance (tolerance) in persisters is phenotypic and distinct from the genetic resistance

49 Dormant or Persistent Bacilli Cornell model: Mice infected with TB bacilli are treated for 3 months with INH and PZA --> No bacilli found in infected organs (spleens/lungs) by plating --> stop treatment, 3 months later -->1/3 mice relapse with TB (drug susceptible) and all mice relapse with TB if treated with immunosuppressing steroids --> suggest existence of dormant bacilli or persisters (phenotypic resistance) When one realizes that, even though the bacilli vanish and there is truly latent infection, the bacilli are, nevertheless, still there drug susceptible, I think you will agree with me that it shows that you can t win. ---Walsh McDermott

50 New TB Drug Candidates FDA approved drugs: -New rifamycin: rifapentine in 1998, first TB drug in 40 years; Rifapentine at higher dose with PZA and moxifloxacin can shorten TB therapy to 3 months in mice (Grosset, Nuermberger) Under clinical development: -New fluoroquinolones (Bayer): moxifloxacin, gatifloxacin, Phase II trials, shorten therapy to 4 months -Ethambutol analog, SQ-109 (Sequella), Phase I trial -Nitroimidazoles: nitroimidazolepyran-pa-824, activity in mice -> Phase I trial (GATB) -OPC (Otsuka), a nitro-dihydro-imidazooxazole derivative, OPC+RIF+PZA shorten therapy to 2 months in mice, ->Phase I, II trial -Diarylquinoline drugs (Johnson & Johnson): diarylquinoline showing good activity in mice and shorten treatment with INH+PZA to 2 month-> Phase I, II trial

51 Why is TB therapy this long (6 months)? Dormant or persistent tubercle bacilli (as in Cornell model). The current TB drugs are not good enough!!!. All the TB drugs are only active against actively growing bacilli, excep PZA and RMP, thus they cannot kill persisters/dormant bacilli. Drugs that can kill persisters/dormant bacilli could shorten the therapy. New challenge: Develop more effective drugs that can shorten the therapy from 6 months to a few weeks. TB Control in Perspective:

52 TB Control Strategy Factors that make TB worse Rapid Diagnostic Tools BCG Vaccine Political and Socioeconomic factors Persisters HIV DOTS Drug-resistant TB