MYCOBACTERIUM TUBERCULOSIS : AGENT POTENTIEL DE BIOTERRORISME

Transcription

1 MYCOBACTERIUM TUBERCULOSIS : AGENT POTENTIEL DE BIOTERRORISME Michel DRANCOURT, MD, Ph.D. Unité des Rickettsies URMITE UMR CNRS 7872 Faculté de Médecine Aix-Marseille Université

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5 Pulmonary tuberculosis

6 Ecrouelles : tuberculose ganglionnaire

7 EPIDEMIOLOGY Tuberculosis = major public health problem 8 millions of new cases and 2 millions of deaths by year Etiologic agent = Mycobacterium tuberculosis

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11 Mycobacterium tuberculosis : diagnostic microbiologique Prélèvements Examen direct : coloration de Ziehl Isolement et culture Détection moléculaire Antibiogramme

12 Direct microscopy : presence of acid-fast bacilli

13 M. tuberculosis : cords

14 M. tuberculosis : Isolement et culture Prélèvements : classe 2 Souches : classe 3

15 Pathogènes de Groupe C Pathogènes émergents : Disponibles Faciles à cultiver Faciles à disséminer Induisent une forte mortalité

16 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

17 Isolation and culture Manual: Solid media Automatic: Liquid media

18 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 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 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 Middlebrook 7H11 - Lowenstein-Jensen Lee et al. (2003), Int J Tuberc Lung Dis 5 Respiratory tract (7153) - Bactec Lowenstein-Jensen Rodrigues et al.(2007), Ind J Med Microbiol 6 Respiratory tract (2566) - Bactec MGIT 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 Lowenstein-Jensen 10.3 (2-34) 26.1 (14-56) Otu et al.(2008), J Infect Developing Countries - Bactec MGIT (11-38) Aono et al.(2009), Diagn Microbiol Infect Dis - Bactec MGIT Lowenstein-Jensen - BacT/Alert 3D - Lowenstein-Jensen - Gottsacker medium (6-48) 19 (10-60) 19 (10-60) Muyoyeta et al.(2009), Int J Tuberc Lung Dis Tessema et al.(2011), JCM

19 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

20 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

21 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)

22 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)

23 2 Optimization of culture media and detection tools : optimal detection tools - GFP3 filter -Zoom = 0,8-1,6 - Maximum magnification (230x) -wavelength = nm -Camera LeicaDC300F Micro-colony: mm (Joshiet al., 2012, Patiño et al, 2008) 2 mm

24 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) * 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 ph 6.5 0

25 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< 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

26 Colonies de Mycobacterium tuberculosis sur gélose au sang

27 Isolement et culture sur cellules Entre Mai 1998 et Août 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

28 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

29 Rapid identification: GenExpert Identification of M. tuberculosis isolmates Detection of M. tuberculosis in sputum Detection of rifampicin - resistance

30 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

31 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 -

32 M. tuberculosis complex : génomes publiés M. tuberculosis CDC 1551 NC M. tuberculosis H37 RV NC M. tuberculosis F11 NC M. tuberculosis H37 Ra NC M. bovis AF NC M. bovis BCG Pasteur NC

33 Molecular Identification 1-16S rrna 94% Mycobacteria genus 96% Pathogenic RGM 2- rpob -Ringet et al Kim et al. 1999

34 Molecular Identification 1-16S rrna (gold standard) % 2- rpob (alternative tool) 2.1. Identification of mycobactial species by comparative sequence analysis of the RNA polymerase (rpob). Kim et al rpob-based identification of nonpigmentated and latepigmenting rapidly growing mycobacteria. Adekambi et al. 2003

35 Strictly human disease Tuberculosis Transmission through air Reactivation (Immunodepression) Prevalence unchanged Families regroupement Student group Hospital / Laboratory Homeless and prison Promiscuity Immunocompromized

36 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

37 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

38 Materials and Methods Choice of spacers Genome H37Rv GenBank accession number AE Genome CDC1551 GenBank accession number AL Data Difseq Software Data of differences between the two genomes Filtre Data of differences located on the spacer of H37Rv/CDC1551 Alignment Dotplot

39 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

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41 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 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 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

42 Strictly human disease Tuberculosis Transmission through air Reactivation (Immunodepression) Prevalence unchanged Families regroupement Student group Hospital / Laboratory Homeless and prison Promiscuity Immunocompromized

43 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

44 Methodology M.tb + soil M.tb + soil + 0.1% cellulose control soil control soil + 0.1% cellulose Monthly sampling of 250 mg soil ml PBS, serial dilution and plating on 7H10 agar Check colonies by Ziehl staining & identify each species by MALDI-TOF

45 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).

46 Preliminary results of MTC organisms in soil Negative controls (soil alone) Negative controls (soil + 0.1% cellulose) Mycobacterium bovis Mycobacterium canettii Mycobacterium tuberculosis

47 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

48 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: Os

49 Mycobacterium organisms escape from the amoebal trophozoite Carlson F. et al. Science 2009, vol 323, 1678

50 What is known about the interactions of MTC and amoeba Taylor J.S. et al., Applied & Environmental Microbiology. 2003; 69: Hagedorn, M. et al., Science. 2009; 323:

51 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

52 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

53 Summary Bacteria size (nm) avium bovis M. tb * Ma Mb Mt Mc Ma Mb Mt Mc canettii Uptake Culture CFU/mL Ma 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 Intracystic bacteria * Ma Mt Mb M. avium M. tuberculosis M. bovis Culture Ma, Mt, Mb Excystment Visible bacillis o cyst Visible into cyst * Amoebal Cysts Ejectosome, [Hagedorn, M., et al. Science. 2009; 323: ] 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

54 Structure of Acanthamoeba cyst

55 Ultrastructure and composition of Acanthamoeba polyphaga cyst wall

56 Cellulose: The target for GH and CBM In plants n up to 14000

57 Profil of cellulolytic bacteria Among mycobacteria, the combination of MtCel6, MtCel12, MtCBM2 is unique to MTC members 12

58 Organization of MtCel6, MtCel12 & MtCBM2 in mycobacteria M. tuberculosis H37Rv M. tuberculosis H37Ra M. bovis GH bp GH bp GH bp GH bp GH bp 105bp Mb CelA2a Mb CelA2b 456bp 105bp Mb CelA2a Mb CelA2b 105bp 456bp Mb CelA2a CelA2b 456bp M. tuberculosis F Mb M.tuberculosis CDC1551 M. bovis BCG Tokyo M. bovis BCG Pasteur GH bp GH bp 105bp Mb CelA2a Mb CelA2b 105bp 456bp Mb CelA2a Mb CelA2b 105bp 456bp Mb CelA2a Mb CelA2b 105bp 456bp 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

59 Acanthamoeba polyphaga cysts, Congo red staining

60 Acanthamoeba polyphaga cysts, calcofluor staining

61 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