MYCOBACTERIUM TUBERCULOSIS : AGENT POTENTIEL DE BIOTERRORISME Michel DRANCOURT, MD, Ph.D. Unité des Rickettsies URMITE UMR CNRS 7872 Faculté de Médecine Aix-Marseille Université
http://www.mediterranee-infection.com/
Pulmonary tuberculosis
Ecrouelles : tuberculose ganglionnaire
EPIDEMIOLOGY Tuberculosis = major public health problem 8 millions of new cases and 2 millions of deaths by year Etiologic agent = Mycobacterium tuberculosis
Mycobacterium tuberculosis : diagnostic microbiologique Prélèvements Examen direct : coloration de Ziehl Isolement et culture Détection moléculaire Antibiogramme
Direct microscopy : presence of acid-fast bacilli
M. tuberculosis : cords
M. tuberculosis : Isolement et culture Prélèvements : classe 2 Souches : classe 3
Pathogènes de Groupe C Pathogènes émergents : Disponibles Faciles à cultiver Faciles à disséminer Induisent une forte mortalité
M. tuberculosis Belongs to Actinobacteria high GC% gram positive bacteria) M. tuberculosis complex of 6 species Two complete genome sequences available Acid fast bacilli Grown on cultured cells M. komossense M. farcinogenes M. sphagni M. aichiense. M. gilvum M. diernhoferi M. gadium M. obuense M. chubuense M. avium M. leprae M. paratuberculosis M. africanum M. microti M. tuberculosis M. bovis 0.005
Isolation and culture Manual: Solid media Automatic: Liquid media
omparative performance of different isolation systems for the culture of M. tuberculosis rom respiratory specimens Study Samples tested (Number) Culture media used Time to detection in days (range) References Smear positive Smear negative 1 Respiratory tract (2832) - Bactec MGIT 960 - Middlebrook 7H11 - Lowenstein-Jensen - Colstsos medium 11 (3-39) 10.4 (3-35) 21.4 (6-60) 21.3 (6-60) 16.1 (16-34) 20.7 (10-47) 26.7 (14-66) 26.8 (11-60) Idigoras et al.(2000), Eur J Clin Microbiol Infect Dis 2 Respiratory tract (462) - BacT/Alert MB - Bactec 460 - Lowenstein-Jensen 15.3 (7-30) 11.5 (4-35) 19.1 (14-28) Harris et al. (2000), J Clin Pathol 3 Respiratory tract (915) - BacT/Alert 3D - Lowenstein-Jensen 14.3 (6-24) 19.4 (13-30) 17.4 (7-35) 24.6 (16-35) Carricajo et al.(2001), JCM 4 Respiratory tract (1396) - Bactec MGIT 960 - Middlebrook 7H11 - Lowenstein-Jensen 9.1 16.1 17.6 16.2 25.2 23.5 Lee et al. (2003), Int J Tuberc Lung Dis 5 Respiratory tract (7153) - Bactec 460 - Lowenstein-Jensen 13.3 31.2 Rodrigues et al.(2007), Ind J Med Microbiol 6 Respiratory tract (2566) - Bactec MGIT 960 - Lowenstein-Jensen 9 (7-12) 26 (21-33) 14 (12-17) 35 (29-42) Srisuwanvilai et al. (2008), Diagn Microbiol Infect Dis 7 Respiratory tract (147) 8 Respiratory tract (79) 9 Respiratory tract (1916) 10 Respiratory tract (319) - Bactec MGIT 960 - Lowenstein-Jensen 10.3 (2-34) 26.1 (14-56) Otu et al.(2008), J Infect Developing Countries - Bactec MGIT 960 16.6 (11-38) Aono et al.(2009), Diagn Microbiol Infect Dis - Bactec MGIT 960 - Lowenstein-Jensen - BacT/Alert 3D - Lowenstein-Jensen - Gottsacker medium 9.5 21 14 34 14 (6-48) 19 (10-60) 19 (10-60) Muyoyeta et al.(2009), Int J Tuberc Lung Dis Tessema et al.(2011), JCM
Le temps de croissance est le premier élément de classification des mycobactéries Croissance rapide < 7 jours : - M. chelonae-abscessus group - M. fortuitum group - M. mucogenicum group - M. mageritense group - M. porcinum group - M. smegmatis group - M. septicum group - M. moriokaense group Croissance lente > 7 jours : - M. tuberculosis - M. marinum / ulcerans - M. leprae - M. xenopi - M. avium - intracellulare - M. haemophilum
Isolement et culture de Mycobacterium tuberculosis sur gélose au sang M. tuberculosis cultive sur milieux à l œuf Mise en évidence fortuite de la croissance sur gélose au sang Etude systématique de la croissance sur gélose au sang
Etude de croissance de M. tuberculosis sur gélose au sang 38 souches calibrées à 10 6 mycobactéries/ml Inoculées en parallèle sur GS / LJ Culture significativement plus riche sur GS à 6 jours (p < 0,001, test ANOVA)
Etude de croissance de M. tuberculosis sur gélose au sang 15 prélèvements respiratoires et 8 prélèvements ganglionnaires Inoculation en parallèle GS / LJ Isolement apparemment plus rapide sur GS mais p = 0,11 (test ANOVA)
2 Optimization of culture media and detection tools : optimal detection tools - GFP3 filter -Zoom = 0,8-1,6 - Maximum magnification (230x) -wavelength = 460-540 nm -Camera LeicaDC300F Micro-colony: 0.335 mm (Joshiet al., 2012, Patiño et al, 2008) 2 mm
Optimization of culture media and detection tools : optimal condition 2 Temperature effect Atmosphere * * * * ph effect in 7-day culture Time to detection in Days Times to detection (Days) 7 6 5 4 3 2 70 1 * 60 0 50 1 Anaerobic 5% CO2, 2.5 % O2 Micoraerophilic Atmosphere 40 M.tuberculosis M.bovis DSMZ 30 nbr of colony 5% CO2 Ambient atmosphere M.bovis BCG Pasteur 20 10 5 6 7 8 9 ph 6.5 0
2 Optimization of culture media : solid medium 55 M. tuberculosis clinical isolates tested in solid media days * Subculturing for clinical isolates including reference strain M. tuberculosis H37Rv was 3±0.days in MOD4 vs 4.5±1.2 days in Middelbrook 7H10 (p=0.023) MOD4 7H10 * P<0.05 40 sputum specimens including 16 specimens exhibiting acid-fast bacilli on microscopy observation. days culturing of clinical specimens was 6.3±2.4 days in MOD4 vs 8.5±2.8 days in Middelbrook 7H10 (p=0.0031) 26 % time saving. MOD4 7H10 P<0.05
Colonies de Mycobacterium tuberculosis sur gélose au sang
Isolement et culture sur cellules Entre Mai 1998 et Août 2004 70 prélèvements positifs: 48 prélèvements ganglionnaires 7 prélèvements d abcès 2 liquides péricardiques 7 liquides céphalo-rachidiens 2 liquides articulaires 1 biopsie ostéomédullaire 3 biopsies cutanées
Isolement et culture sur cellules Parmi les 70 prélèvements positifs: 53 M. tuberculosis 11 M. avium - intracellulare 1 BCG 1 M. xenopi 3 M. abcessus / M. gordonae 1 M. fuerthensis
Rapid identification: GenExpert Identification of M. tuberculosis isolmates Detection of M. tuberculosis in sputum Detection of rifampicin - resistance
MALDI-TOF identification (a) 70% ethanol inactivation (b) without Tween-20 (c) ß-mercaptoethanol (d) acetone (e) absolute ethanol (f) 0.1 M NaOH (g) 10 % TFA (h) 0.1 M HCl (i) final protocol
Mycobacterium tuberculosis complex Source Infections humaines M. tuberculosis Homme +++ M. canettii Homme ++ M. bovis BCG Vaccin ++ M. bovis Bovins + M. microti Rongeurs + M. caprae Caprins - M. pinnipedii Phoques -
M. tuberculosis complex : génomes publiés M. tuberculosis CDC 1551 NC 002755 M. tuberculosis H37 RV NC 000962 M. tuberculosis F11 NC 009565 M. tuberculosis H37 Ra NC 009525 M. bovis AF 212297 NC 002945 M. bovis BCG Pasteur NC 008769
Molecular Identification 1-16S rrna 94% Mycobacteria genus 96% Pathogenic RGM 2- rpob -Ringet et al. 1998 -Kim et al. 1999
Molecular Identification 1-16S rrna (gold standard) 94-100% 2- rpob (alternative tool) 2.1. Identification of mycobactial species by comparative sequence analysis of the RNA polymerase (rpob). Kim et al. 1999 2.2. rpob-based identification of nonpigmentated and latepigmenting rapidly growing mycobacteria. Adekambi et al. 2003
Strictly human disease Tuberculosis Transmission through air Reactivation (Immunodepression) Prevalence unchanged Families regroupement Student group Hospital / Laboratory Homeless and prison Promiscuity Immunocompromized
M. tuberculosis genotyping : current methods Profile Analysis Restriction profile Fragment Amplification profile Fragment BET staining Probe staining BET staining Probe staining PFGE RFLP-IS6110 VNTR MIRU Spoligotyping
MST approach Spacer A Spacer B Spacer C Orf 1 Orf 2 Orf 3 Orf 4 Seq 1 Seq 2 Seq 3 Seq 1 Seq 2 Seq 3 Seq 1 Seq 2 Seq 3 Strain Spacer A Spacer B Spacer C MST type Strain 1 SeqA1 SeqB1 SeqC1 MST 1 type Strain 2 SeqA2 SeqB2 SeqC2 MST 2 type Strain 3 SeqA3 SeqB3 SeqC3 MST 3 type SeqA1 differs from SeqA2 by : - Mutation - Deletion / Insertion These 2 events have the same weight Strain 4 SeqA1 SeqB1 SeqC1 MST 1 type
Materials and Methods Choice of spacers Genome H37Rv GenBank accession number AE 000516 Genome CDC1551 GenBank accession number AL 123456 Data Difseq Software Data of differences between the two genomes Filtre Data of differences located on the spacer of H37Rv/CDC1551 Alignment Dotplot
Phylogenic analysis MST and VNTR ST1 Black Africa (9) ST17 Tunisia (1) ST23 Tunisia (1) ST3 Algeria (1) ST11 Algeria (5) ST8 Algeria (2) ST4 Algeria (14) ST16 Algeria (1) ST12 Thailland (2) ST20 Philippines (1) ST7 Thailland (1) ST2 Vietnam (1) ST14 Vietnam (3) ST26 Kurdistan (1) ST6 Poland (1) ST9 Poland (1) ST5 Italia, France (3) ST15 Spain (2) ST10 France (3) ST19 France (2) ST25 France (1) ST22 France (1) ST13 Algeria (1) ST24 France (1) ST18 France (1) ST21 Turkey (1) VNTR 1 VNTR 4 VNTR 2 VNTR 3
Genomic regions associated with decreased susceptibility to antituberculosis agents Antituberculosis agent Gene Product Mutation frequency among Drug-resistant clinical isolates (%) Streptomycin rpsl Ribosomal protein S12 ~ 60 rrs 16SrRNA < 10 Rifampin rpob β subunit of RNA polymerasme > 95 Isoniazid katg Catalase-peroxidase 60-70 oxyr-ahpc Alkylhydroreductase ~ 20 inha Enoyl ACP reductase < 10 kasa β-ketoacyl-acp synthase < 10 ndh NADH dehydrogenase NA Ethambutol embcab Arabinosyltransferases ~ 70 Pyrazinamide pnca Amidase 70-100 Ethionamide inha Enoyl ACP reductase < 10 etha Flavoprotein monooxygenase NA Kanamycin rrs 16SrRNA ~ 65 Fluoroquinolone gyra DNA gyrase α subunit > 90 gyrb DNA gyrase β subunit NA Capreomycin tlya rrna methyltransferase NA rrs 16SrRNA NA Para-aminosalicylic acid thya Thymidylate synthase NA
Strictly human disease Tuberculosis Transmission through air Reactivation (Immunodepression) Prevalence unchanged Families regroupement Student group Hospital / Laboratory Homeless and prison Promiscuity Immunocompromized
M. tuberculosis in animals M. tuberculosis in captive mammals : Cattle, elephants, dogs, seals M. tuberculosis in wild mammals : mongooses (Mungos mungo), suricates (Suricata suricatta). M. tuberculosis in birds : canary (Serinus canaria forma domestica), amazons parrots. M. tuberculosis in non-human primates : old world monkeys like rhesus macaques (Macaca mulatta). Many non-human cases of infection with M. tuberculosis M. tuberculosis in amoeba : Acanthamoeba polyphaga M. tuberculosis persitance in biofilm and soil
Methodology 1 2 3 1 2 3 1 2 3 1 2 3 M.tb + soil M.tb + soil + 0.1% cellulose control soil control soil + 0.1% cellulose Monthly sampling of 250 mg soil + 250 ml PBS, serial dilution and plating on 7H10 agar Check colonies by Ziehl staining & identify each species by MALDI-TOF
1 M. tuberculosis Study of the survival of M. tuberculosis complex in soil (with Felix M Ba Medie) CFU (every month) M. bovis Sterilized soil M. canetti Long-term survival of tuberculosis mycobacteria in soil Ramzi Ghodbane, Felix Mba Medie, Bernard Henrissat and Michel Drancourt (in preparation) 13 months Infection in on BALB/c mice CFU Electronic microscopy WEIGHT (gr) Electronic microscopy Immuno-glod (in progress) Incubation in ambient temperature on NSB3 (18-25 C).
Preliminary results of MTC organisms in soil Negative controls (soil alone) Negative controls (soil + 0.1% cellulose) Mycobacterium bovis Mycobacterium canettii Mycobacterium tuberculosis
Preliminary results of MTC organisms in soil P = 0.31 P = 0.19 P = 0.03 Mba Medie, F. et al: Persistence of tuberculosis mycobacteria in soil In preparation
Mycobacteria-amoeba: Background Environmental mycobacteria & amoeba, Acanthamoeba polyphaga (2) Multiplication Trophozoite Trophozoites 7 days Excystment Encystment 1-3 days Cr Ed Ec Cyst composition Mature cysts Op 2 µm 2- Adékambi, T. et al., Applied & Environmental Microbiology. 2006; 72: 5974-5981 Os
Mycobacterium organisms escape from the amoebal trophozoite Carlson F. et al. Science 2009, vol 323, 1678
What is known about the interactions of MTC and amoeba Taylor J.S. et al., Applied & Environmental Microbiology. 2003; 69: 4316 4319 Hagedorn, M. et al., Science. 2009; 323: 1729 733
Microscopy analysis of infected A. polyphaga A B 2 µm M. tuberculosis in the trophozoite M. canettii in trophozoite 2 µm M. bovis in trophozoite 2 µm M. avium in trophozoite 2 µm MTC mycobacteria are indeed internalized
Microscopy analysis of infected A. polyphaga cysts 2 µm 2 µm M. tuberculosis in the cyst M. avium in the cyst D 2 µm M. tuberculosis in the cyst 2 µm M. tuberculosis in the cyst
Summary Bacteria size (nm) 3000 2500 2000 1500 1000 500 0 avium bovis M. tb * Ma Mb Mt Mc Ma Mb Mt Mc canettii Uptake Culture CFU/mL Ma 400 300 200 100 Bacteria uptake by A. polyphaga 0 M. avium M. bovis M. t b M. canet t ii Mb * Ma Mb Mt Mc Mt Mc EM Amoebal trophozoite Encystment Mt, Mb Ma CFU/mL 400 300 200 100 0 Intracystic bacteria * Ma Mt Mb M. avium M. tuberculosis M. bovis Culture Ma, Mt, Mb Excystment Visible bacillis o cyst Visible into cyst * 14 12 Amoebal Cysts 10 8 6 Ejectosome, [Hagedorn, M., et al. Science. 2009; 323: 1729-1733 ] 4 2 0 Ma Mt Mb M. avium M.bovis M. tb Out Mba Medie, F. et al: Mycobacterium tuberculosis complex mycobacteria EMas amoeba-resistant organisms. PloS ONE. 2011; 6: e20499
Structure of Acanthamoeba cyst
Ultrastructure and composition of Acanthamoeba polyphaga cyst wall
Cellulose: The target for GH and CBM In plants n up to 14000
Profil of cellulolytic bacteria Among mycobacteria, the combination of MtCel6, MtCel12, MtCBM2 is unique to MTC members 12
Organization of MtCel6, MtCel12 & MtCBM2 in mycobacteria M. tuberculosis H37Rv M. tuberculosis H37Ra M. bovis GH6 1142 bp GH6 1142 bp GH6 1143 bp GH6 1143 bp GH6 1142 bp 105bp 1.148 Mb CelA2a 1.015 Mb CelA2b 456bp 105bp 1.148 Mb CelA2a 1.023 Mb CelA2b 105bp 456bp 1.152 Mb CelA2a CelA2b 456bp M. tuberculosis F11 1.019 Mb M.tuberculosis CDC1551 M. bovis BCG Tokyo M. bovis BCG Pasteur GH6 1143 bp GH6 1142 bp 105bp 1.149 Mb CelA2a 1.012 Mb CelA2b 105bp 456bp 1.148 Mb CelA2a 1.015 Mb CelA2b 105bp 456bp 1.148 Mb CelA2a 1.015 Mb CelA2b 105bp 456bp 1.149 Mb CelA2a CelA2b 456bp Conclusion: Similar organization for all MTC organisms CBM2 428 bp CBM2 429 bp CBM2 429 bp CBM2 429 bp CBM2 428 bp CBM2 428 bp 26
Acanthamoeba polyphaga cysts, Congo red staining
Acanthamoeba polyphaga cysts, calcofluor staining
Summary 1,500 bacteria surveyed 62% without cellulases 38% with cellulases 1 13% saprophyte only 6% both 11% cellulose biosynthesisers only 8% others with unknown function M. tuberculosis organisms are among this 8% of bacteria with unknown function Mba Medie, F. et al: Genome analyses highlight the biological roles of cellulases Under revision in Nature Review Microbiology 11