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Mycobacterium kansasii, species or complex? Genetic and epidemiological insights Enrico Tortoli Regional Reference Center for Mycobacteria Florence -

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Presentation on theme: "Mycobacterium kansasii, species or complex? Genetic and epidemiological insights Enrico Tortoli Regional Reference Center for Mycobacteria Florence -"— Presentation transcript:

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2 Mycobacterium kansasii, species or complex? Genetic and epidemiological insights Enrico Tortoli Regional Reference Center for Mycobacteria Florence - Italy Congress of Japanese Society of Tuberculosis – Kurashiki, April 25, 2003

3 Brief history of M. kansasii 1953: description by Buhler & Pollak1953: description by Buhler & Pollak 1960-70: most common NTM causing infections in USA and UK1960-70: most common NTM causing infections in USA and UK 1980s: surpassed by M. avium as cause of pulmonary disease; isolations on the rise in Japan1980s: surpassed by M. avium as cause of pulmonary disease; isolations on the rise in Japan early 1990s (pre-HAART era): not rare, although far less common than M. avium as cause of pulmonary or disseminated disease in AIDSearly 1990s (pre-HAART era): not rare, although far less common than M. avium as cause of pulmonary or disseminated disease in AIDS late 1990s (HAART era): very rare in AIDS, more frequent in immuno-competent patientslate 1990s (HAART era): very rare in AIDS, more frequent in immuno-competent patients

4 Phenotypic characteristics of M. kansasii Large cross-barred bacilliLarge cross-barred bacilli Prevalently rough coloniesPrevalently rough colonies PhotochromogenicPhotochromogenic Slow growth at 30-40°CSlow growth at 30-40°C Nitrate, Tween 80 hydrolysis, catalase and urease are positiveNitrate, Tween 80 hydrolysis, catalase and urease are positive Arylsulfatase and tellurite are negativeArylsulfatase and tellurite are negative

5 Mycobacterium kansasii, a significant pathogen Annual rate of infection in general population ranges from 0.5 to 1 per 100,000Annual rate of infection in general population ranges from 0.5 to 1 per 100,000 Significant geographical variability is observedSignificant geographical variability is observed –rare in Australia and Japan –very frequent in Louisiana (USA) and Czech Republic Culturing of M. kansasii from human sources is not proof of disease; 1/3 of respiratory isolates represent colonization rather than infectionCulturing of M. kansasii from human sources is not proof of disease; 1/3 of respiratory isolates represent colonization rather than infection

6 Diseases in immunocompetent patients Pulmonary diseasePulmonary disease –preexisting lung disease pneumoconiosispneumoconiosis chronic obstructive pulmonary diseasechronic obstructive pulmonary disease impaired ventilation functionimpaired ventilation function Lymphadenitis (childhood)Lymphadenitis (childhood) Soft tissues infectionSoft tissues infection Cutaneous lesionsCutaneous lesions Bone and joint diseaseBone and joint disease Genitourinary diseaseGenitourinary disease Disseminated diseaseDisseminated disease

7 Other risk factors Exposure to dustExposure to dust CancerCancer AlcoholismAlcoholism SmokeSmoke Systemic illnessSystemic illness Exposure to M. kansasii -contaminated waterExposure to M. kansasii -contaminated water Hyperendemic geographic areasHyperendemic geographic areas

8 Disease in immunodeficient patients Disseminated diseaseDisseminated disease –low CD4 level Pulmonary diseasePulmonary disease

9 Therapeutic hints The key antimicrobial, rifampin, is mostly used in association with ethambutol and with a 3 rd drug (streptomycin, isoniazid or amikacin)The key antimicrobial, rifampin, is mostly used in association with ethambutol and with a 3 rd drug (streptomycin, isoniazid or amikacin) ATS recommends 18 month therapyATS recommends 18 month therapy

10 M. kansasii in the environment Frequently isolated from both drinking and natural waterFrequently isolated from both drinking and natural water Rarely isolated from soilRarely isolated from soil

11 First reported variants of M. kansasii Clinically significant strainsClinically significant strains –semiquantitative catalase > 30 mm –high virulence in guinea pig Strains with doubtful significanceStrains with doubtful significance –semiquantitative catalase < 30 mm –low virulence in guinea pig (Wayne, 1962) (Wayne, 1962)

12 Genetic targets of M. kansasii 16S rRNA gene16S rRNA gene 16S-23S internal transcribed spacer16S-23S internal transcribed spacer 65kD heat shock protein gene65kD heat shock protein gene Repetitive DNA sequencesRepetitive DNA sequences girA intein-coding sequencegirA intein-coding sequence

13 DNA probe developed for M. kansasii Research toolsResearch tools –pMK1-9 undetermined DNA regionundetermined DNA region –p6123 undetermined DNA regionundetermined DNA region Commercial toolsCommercial tools –AccuProbe (2 formulations) 16S rRNA16S rRNA –INNO LiPA MYCOBACTERIA ITSITS

14 pMK1-9 DNA probe Hybridization with all M. kansasii tested (Huang et al., 1991)Hybridization with all M. kansasii tested (Huang et al., 1991) Hybridization failed by 20 out of 105 M. kansasii isolates (Ross et al., 1992)Hybridization failed by 20 out of 105 M. kansasii isolates (Ross et al., 1992)

15 p6123 DNA probe Hybridization with all M. kansasii tested (Yang et al., 1993)Hybridization with all M. kansasii tested (Yang et al., 1993)

16 AccuProbe M. kansasii AccuProbe (1 st formulation) hybridizes with all pMK1-9-positive M. kansasii but with only a part of pMK1-9-negative ones (Ross et al., 1992)AccuProbe (1 st formulation) hybridizes with all pMK1-9-positive M. kansasii but with only a part of pMK1-9-negative ones (Ross et al., 1992) Only 50, out of 69 M. kansasii isolates, hybridize with the 1 st AccuProbe (Tortoli et al., 1994)Only 50, out of 69 M. kansasii isolates, hybridize with the 1 st AccuProbe (Tortoli et al., 1994) The reformulated AccuProbe hybridizes also with the strains negative with the previous version (Tortoli et al., 1996)The reformulated AccuProbe hybridizes also with the strains negative with the previous version (Tortoli et al., 1996)

17 INNO LiPA MYCOBACTERIA 3 line-probes are addressed to the identification of different M. kansasii strains3 line-probes are addressed to the identification of different M. kansasii strains –all the strains positive with the 1 st AccuProbe hybridize with line MKA-1 –all the strains negative with the 1 st and positive with the 2 nd AccuProbe hybridize with line MKA-2 –the strains negative with both AccuProbe hybridize with line MKA-3 (Tortoli et al., 2001)

18 Genetic sequencing The strains pMK1-9-negative differ 5 nucleotides in the 16S rDNA from the pMK1-9-positive ones (Ross et al., 1992)The strains pMK1-9-negative differ 5 nucleotides in the 16S rDNA from the pMK1-9-positive ones (Ross et al., 1992) 1 to 6 nucleotide diversity differentiates, in the first 500 bp of 16S rDNA, 5 sequevars of M. kansasii (RIDOM)1 to 6 nucleotide diversity differentiates, in the first 500 bp of 16S rDNA, 5 sequevars of M. kansasii (RIDOM) Extensive diversities (12 to 49 nucleotides) characterize, in ITS, 5 sequevars of M. kansasii (RIDOM)Extensive diversities (12 to 49 nucleotides) characterize, in ITS, 5 sequevars of M. kansasii (RIDOM)

19 Repeated DNA sequences GC-rich repetitive sequenceGC-rich repetitive sequence IS1652IS1652 Major Polymorphic Tandem RepeatMajor Polymorphic Tandem Repeat

20 Fingerprinting techniques Restriction Fragment Length PolymorphismRestriction Fragment Length Polymorphism –the total bacterial DNA is digested with restriction enzymes –the fragments are separated by electrophoresis Amplified Fragment Length PolymorphismAmplified Fragment Length Polymorphism –the total bacterial DNA is digested with restriction enzymes –oligonucleotide adapters are added by ligase –adapter-containing fragments are selectively amplified –amplification products are separated electrophoretically Pulsed Field Gel ElectrophoresisPulsed Field Gel Electrophoresis –large restriction fragments are obtained from total bacterial DNA –the fragments are separated by pulsed field electrophoresis PCR Restriction AnalysisPCR Restriction Analysis –a PCR-amplified DNA sequance is digested with restriction enzymes –the fragments are separated by electrophoresis

21 GC-rich repetitive sequence A repetitive sequence, with high guanosine and cytosine content, is present, in at least 30 copies, in M. kansasii, M. tuberculosis and M. szulgai and is responsible of polymorphism (Ross et al., 1992)A repetitive sequence, with high guanosine and cytosine content, is present, in at least 30 copies, in M. kansasii, M. tuberculosis and M. szulgai and is responsible of polymorphism (Ross et al., 1992)

22 IS1652 IS1652 is present in pMK1-9-negative M. kansasii only (Yang et al., 1993)IS1652 is present in pMK1-9-negative M. kansasii only (Yang et al., 1993) In such strains the number of copies, which ranges from 1 to 11, is responsible for significant polymorphismIn such strains the number of copies, which ranges from 1 to 11, is responsible for significant polymorphism

23 The MPTR consists of short tandemly repeated sequences of 10 bp separated by 5-bp spacersThe MPTR consists of short tandemly repeated sequences of 10 bp separated by 5-bp spacers About 80 different MPTR-containing regions are present in the mycobacterial genomeAbout 80 different MPTR-containing regions are present in the mycobacterial genome MPTR is present in M. tuberculosis complex, M. gordonae, M. gastri, M. kansasii, M. szulgaiMPTR is present in M. tuberculosis complex, M. gordonae, M. gastri, M. kansasii, M. szulgai A wide polymorphism characterizes the MPTR containing restriction fragments of M. kansasii (Hermans et al., 1992)A wide polymorphism characterizes the MPTR containing restriction fragments of M. kansasii (Hermans et al., 1992) GCCGGTGTTG Major Polymorphic Tandem Repeat

24 MPTR-based RFLP pMK1-9-positive M. kansasii share most bands in common; pMK1-9-negative strains are more heterogeneous (Ross et al., 1992)pMK1-9-positive M. kansasii share most bands in common; pMK1-9-negative strains are more heterogeneous (Ross et al., 1992) AccuProbe-1-positive M. kansasii exhibit a 3kb fragment; AccuProbe-1-negative strains present fragments of variable length (Yang et al., 1993)AccuProbe-1-positive M. kansasii exhibit a 3kb fragment; AccuProbe-1-negative strains present fragments of variable length (Yang et al., 1993)

25 Intein-coding sequences Inteins are protein sequences that are excised from the precursor protein during maturationInteins are protein sequences that are excised from the precursor protein during maturation Intein-coding sequences may be present in gyrA, the gene encoding for the A subunit of mycobacterial DNA gyraseIntein-coding sequences may be present in gyrA, the gene encoding for the A subunit of mycobacterial DNA gyrase Several mycobacterial species present in gyrA an intein-coding sequence, others species do notSeveral mycobacterial species present in gyrA an intein-coding sequence, others species do not M. kansasii, M. flavescens and M. gordonae are the only species in which strains with and without gyrA intein are presentM. kansasii, M. flavescens and M. gordonae are the only species in which strains with and without gyrA intein are present (Sander et al., 1998) (Sander et al., 1998)

26 Minor genetic heterogeneity The gene encoding for the MPB70 protein-analogue of M. kansasii presents sequence variations suggesting the heterogeneity of the species (Wooldford et al., 1997)The gene encoding for the MPB70 protein-analogue of M. kansasii presents sequence variations suggesting the heterogeneity of the species (Wooldford et al., 1997) The amplification of ITS from different M. kansasii strains reveals three different product profiles, in other mycobacterial species the profile is reproducible (Abed et al., 1995)The amplification of ITS from different M. kansasii strains reveals three different product profiles, in other mycobacterial species the profile is reproducible (Abed et al., 1995)

27 Tortoli et al., 1994 AccuProbe-1-positive strains, differently from - negative ones, rapidly hydrolyze Tween 80 and possess alpha fucosidase activityAccuProbe-1-positive strains, differently from - negative ones, rapidly hydrolyze Tween 80 and possess alpha fucosidase activity AccuProbe-positive and -negative strains cannot be distinguished by means of HPLC analysisAccuProbe-positive and -negative strains cannot be distinguished by means of HPLC analysis The isolation of AccuProbe-1-negative M. kansasii is significantly more frequent (P = 0.00007) from HIV positive than from HIV negative patientsThe isolation of AccuProbe-1-negative M. kansasii is significantly more frequent (P = 0.00007) from HIV positive than from HIV negative patients

28 Picardeau et al., 1997 (1/2) RFLP, using MPTR as a probe, reveals 5 clustersRFLP, using MPTR as a probe, reveals 5 clusters AFLP reveals multiple patterns clustering in 5 minor groups according to the ones of MPTRAFLP reveals multiple patterns clustering in 5 minor groups according to the ones of MPTR RFLP reveals IS1652 in 2 of the above clusters, with a single copy and with 4-6 copies respectively; IS1652 is on the contrary missing in the othersRFLP reveals IS1652 in 2 of the above clusters, with a single copy and with 4-6 copies respectively; IS1652 is on the contrary missing in the others PFGE reveals a number of patterns clustering in 5 major groupsPFGE reveals a number of patterns clustering in 5 major groups PRA of hsp65 gene reveals 5 clustersPRA of hsp65 gene reveals 5 clusters

29 Cluster I includes typical M. kansasii and is AccuProbe-1-positiveCluster I includes typical M. kansasii and is AccuProbe-1-positive Clusters II to IV are AccuProbe-1-negativeClusters II to IV are AccuProbe-1-negative Clusters V is AccuProbe-1-positiveClusters V is AccuProbe-1-positive Clusters II and III are closely relatedClusters II and III are closely related Picardeau et al., 1997 (2/2)

30 Alcaide et al., 1997 (1/2) PRA of hsp65 generate 5 different patternsPRA of hsp65 generate 5 different patterns –type I is AccuProbe-1-positive –types II and IV are AccuProbe-1-negative –types III and V are weakly AccuProbe-1-positive gyrA intein is present in types I, IV and V while it lacks in II and IIIgyrA intein is present in types I, IV and V while it lacks in II and III Partial 16S rDNA sequence reveals 2 sequevars presented by types I, IV and V and by types II and III respectivelyPartial 16S rDNA sequence reveals 2 sequevars presented by types I, IV and V and by types II and III respectively ITS sequence reveals 5 sequevars correlating with the hsp65 PRA typesITS sequence reveals 5 sequevars correlating with the hsp65 PRA types PFGE of the digested chromosomal DNA generates 10 to 15 large fragmentsPFGE of the digested chromosomal DNA generates 10 to 15 large fragments –moderate polymorphism in type I (clonal structure) –significant polymorphism in type II

31 Types I and II constitute frequent clinical isolatesTypes I and II constitute frequent clinical isolates Type I, differently from II, is almost absent in the environmentType I, differently from II, is almost absent in the environment Types III, IV and V are rare in humans and frequent in the waterTypes III, IV and V are rare in humans and frequent in the water Alcaide et al., 1997 (2/2)

32 The five types of M. kansasii Type DNA probe Sequencing Molecular typing gyrA intein p6123pMK1-9 AccuProbeINNO LiPARFLP PRAPFGE a AFLP 1 st vers. 2 nd vers. MK-1MK-2MK-316S rDNA ITSMPTRIS1652 I+++++--a1i-Aibib S+ II+--+-+-b2ii+Bii c M- III+-----+b3iii+Ciii d S- IV+-----+a4iv-DIvS+ V+-++--+a5v-EVS+ a S, single pattern; M, multiple patterns b 4 subgroups c 5 subgroups d 3 subgroups

33 The five types of M. kansasii Type DNA probe Sequencing Molecular typing gyrA intein p6123pMK1-9 AccuProbeINNO LiPARFLP PRAPFGE a AFLP 1 st vers. 2 nd vers. MK-1MK-2MK-316S rDNA ITSMPTRIS1652 I+++++--a1i-Aibib S+ II+--+-+-b2ii+Bii c M- III+-----+b3iii+Ciii d S- IV+-----+a4iv-DIvS+ V+-++--+a5v-EVS+ a S, single pattern; M, multiple patterns b 4 subgroups c 5 subgroups d 3 subgroups

34 The five types of M. kansasii Type DNA probe Sequencing Molecular typing gyrA intein p6123pMK1-9 AccuProbeINNO LiPARFLP PRAPFGE a AFLP 1 st vers. 2 nd vers. MK-1MK-2MK-316S rDNA ITSMPTRIS1652 I+++++--a1i-Aibib S+ II+--+-+-b2ii+Bii c M- III+-----+b3iii+Ciii d S- IV+-----+a4iv-D S+ V+-++--+a5v-EvS+ a S, single pattern; M, multiple patterns b 4 subgroups c 5 subgroups d 3 subgroups

35 The five types of M. kansasii Type DNA probe Sequencing Molecular typing gyrA intein p6123pMK1-9 AccuProbeINNO LiPARFLP PRAPFGE a AFLP 1 st vers. 2 nd vers. MK-1MK-2MK-316S rDNA ITSMPTRIS1652 I+++++--a1i-Aibib S+ II+--+-+-b2ii+Bii c M- III+-----+b3iii+Ciii d S- IV+-----+a4iv-D S+ V+-++--+a5v-EvS+ a S, single pattern; M, multiple patterns b 4 subgroups c 5 subgroups d 3 subgroups

36 The five types of M. kansasii Type DNA probe Sequencing Molecular typing gyrA intein p6123pMK1-9 AccuProbeINNO LiPARFLP PRAPFGE a AFLP 1 st vers. 2 nd vers. MK-1MK-2MK-316S rDNA ITSMPTRIS1652 I+++++--a1i-Aibib S+ II+--+-+-b2ii+Bii c M- III+-----+b3iii+Ciii d S- IV+-----+a4iv-D S+ V+-++--+a5v-EvS+ a S, single pattern; M, multiple patterns b 4 subgroups c 5 subgroups d 3 subgroups

37 The five types of M. kansasii Type DNA probe Sequencing Molecular typing gyrA intein p6123pMK1-9 AccuProbeINNO LiPARFLP PRAPFGE a AFLP 1 st vers. 2 nd vers. MK-1MK-2MK-316S rDNA ITSMPTRIS1652 I+++++--a1i-Aibib S+ II+--+-+-b2ii+Bii c M- III+-----+b3iii+Ciii d S- IV+-----+a4iv-D S+ V+-++--+a5v-EvS+ a S, single pattern; M, multiple patterns b 4 subgroups c 5 subgroups d 3 subgroups

38 Taxonomic speculations M. kansasii isM. kansasii is –a single systematic entity? –a single species split in several subspecies? –complex including multiple species? Evidence exists that, as a consequence of the high degree of divergence, several M. kansasii types are more closely related to other species than to each otherEvidence exists that, as a consequence of the high degree of divergence, several M. kansasii types are more closely related to other species than to each other

39 M. gordonae M. marinum M. ulcerans M. gastri M. szulgai M. tuberculosis ITS-based phylogenetic tree M. kansasii iii M. kansasii iv M. kansasii v M. kansasii i M. kansasii ii }

40 Epidemiological insights Types I and II include all the significantly pathogen isolatesTypes I and II include all the significantly pathogen isolates Type I presents a clonal structureType I presents a clonal structure –adaptation to human host? –divergence restricted by virulence properties? –it includes practically al M. kansasii pathogens for immunocompetent people (the high catalase producers of Wayne, and almost all the AccuProbe-1-positive strains) Type II is rare in immunocompetent patients and frequent in HIV-infected onesType II is rare in immunocompetent patients and frequent in HIV-infected ones –lower ability to overcome natural resistance mechanisms? –it includes the low catalase producers of Wayne, and almost all the AccuProbe-1-negative strains

41 Conclusions A more precise definition of various M. kansasii isolates would provide significant contribution to understanding of its biological and epidemiological key aspectsA more precise definition of various M. kansasii isolates would provide significant contribution to understanding of its biological and epidemiological key aspects An international task force, including microbiologists, epidemiologists and clinicians, is needed to efficiently take up the challenge of M. kansasiiAn international task force, including microbiologists, epidemiologists and clinicians, is needed to efficiently take up the challenge of M. kansasii


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