Mycobacterium kansasiiMycobacterium kansasii,, species or species or complex?complex?Genetic and epidemiological insightsGenetic and epidemiological insights

Enrico TortoliEnrico Tortoli

Regional Reference Center for MycobacteriaRegional Reference Center for Mycobacteria

Florence - ItalyFlorence - ItalyCongress of Japanese Society of Tuberculosis – Kurashiki, April 25, 2003

Brief history of Brief history of M. kansasiiM. kansasii

• 1953: description by Buhler & Pollak1953: description by Buhler & Pollak• 1960-70: most common NTM causing infections 1960-70: most common NTM causing infections

in USA and UKin USA and UK• 1980s: surpassed by 1980s: surpassed by M. M. aviumavium as cause of as cause of

pulmonary disease; isolations on the rise in pulmonary disease; isolations on the rise in JapanJapan

• early 1990s (pre-HAART era): not rare, although early 1990s (pre-HAART era): not rare, although far less common than far less common than M. M. aviumavium as cause of as cause of pulmonary or disseminated disease in AIDSpulmonary or disseminated disease in AIDS

• late 1990s (HAART era): very rare in AIDS, more late 1990s (HAART era): very rare in AIDS, more frequent in immuno-competent patientsfrequent in immuno-competent patients

Phenotypic characteristics of Phenotypic characteristics of M. kansasiiM. 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 Nitrate, Tween 80 hydrolysis, catalase

and urease are positiveand urease are positive• Arylsulfatase and tellurite are negative Arylsulfatase and tellurite are negative

Mycobacterium kansasiiMycobacterium kansasii, a significant , a significant pathogenpathogen

• Annual rate of infection in general population Annual rate of infection in general population ranges from 0.5 to 1 per 100,000ranges from 0.5 to 1 per 100,000

• Significant geographical variability is observedSignificant geographical variability is observed– rare in Australia and Japanrare in Australia and Japan– very frequent in Louisiana (USA) and Czech very frequent in Louisiana (USA) and Czech

RepublicRepublic

• Culturing of Culturing of M. kansasiiM. kansasii from human sources is from human sources is not proof of disease; 1/3 of respiratory isolates not proof of disease; 1/3 of respiratory isolates represent colonization rather than infection represent colonization rather than infection

Diseases inDiseases in immunocompetent immunocompetent patientspatients• Pulmonary diseasePulmonary disease

– preexisting lung diseasepreexisting 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

Other risk factorsOther risk factors

• Exposure to dustExposure to dust• CancerCancer• AlcoholismAlcoholism• SmokeSmoke• Systemic illnessSystemic illness• Exposure to Exposure to M. kansasii M. kansasii -contaminated -contaminated

waterwater• Hyperendemic geographic areasHyperendemic geographic areas

Disease inDisease in immunodeficient immunodeficient patientspatients

• Disseminated diseaseDisseminated disease– low CD4 levellow CD4 level

• Pulmonary diseasePulmonary disease

Therapeutic hintsTherapeutic hints

• The key antimicrobial, The key antimicrobial, rifampin, is mostly used in rifampin, is mostly used in association with ethambutol association with ethambutol and with a 3and with a 3rdrd drug drug (streptomycin, isoniazid or (streptomycin, isoniazid or amikacin)amikacin)

• ATS recommends 18 month ATS recommends 18 month therapytherapy

M. kansasiiM. kansasii in the environment in the environment

• Frequently isolated from both Frequently isolated from both drinking and natural waterdrinking and natural water

• Rarely isolated from soilRarely isolated from soil

First reported variants of First reported variants of M. kansasiiM. kansasii

• Clinically significant strainsClinically significant strains– semiquantitative catalase > 30 mmsemiquantitative catalase > 30 mm– high virulence in guinea pighigh virulence in guinea pig

• Strains with doubtful significanceStrains with doubtful significance– semiquantitative catalase < 30 mmsemiquantitative catalase < 30 mm– low virulence in guinea piglow virulence in guinea pig

(Wayne, 1962)(Wayne, 1962)

Genetic targets of Genetic targets of M. kansasiiM. 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• girAgirA intein-coding sequence intein-coding sequence

DNA probe developed for DNA probe developed for M. kansasiiM. kansasii

• Research toolsResearch tools– pMK1-9pMK1-9

• undetermined DNA regionundetermined DNA region

– p6123p6123• undetermined DNA region undetermined DNA region

• Commercial toolsCommercial tools– AccuProbe (2 formulations)AccuProbe (2 formulations)

• 16S rRNA16S rRNA

– INNO LiPA MYCOBACTERIAINNO LiPA MYCOBACTERIA• ITSITS

pMK1-9 DNA probepMK1-9 DNA probe

• Hybridization with all Hybridization with all M. kansasiiM. kansasii tested tested (Huang (Huang et al.et al., 1991), 1991)

• Hybridization failed by 20 out of 105 Hybridization failed by 20 out of 105 M. kansasiiM. kansasii isolates (Ross isolates (Ross et al.et al.,, 1992) 1992)

p6123 DNA probep6123 DNA probe

• Hybridization with all Hybridization with all M. kansasiiM. kansasii tested tested (Yang (Yang et al.et al., 1993), 1993)

AccuProbeAccuProbe M. kansasiiM. kansasii

• AccuProbe (1AccuProbe (1stst formulation) hybridizes with formulation) hybridizes with all pMK1-9-positiveall pMK1-9-positive M. kansasiiM. kansasii but with only but with only a part of pMK1-9-negative ones (Ross a part of pMK1-9-negative ones (Ross et al.et al., , 1992)1992)

• Only 50, out of 69 Only 50, out of 69 M. kansasiiM. kansasii isolates, isolates, hybridize with the 1hybridize with the 1stst AccuProbe (Tortoli AccuProbe (Tortoli et al.et al., 1994) , 1994)

• The reformulated AccuProbe hybridizes also The reformulated AccuProbe hybridizes also with the strains negative with the previous with the strains negative with the previous version (Tortoli version (Tortoli et al.et al., 1996), 1996)

INNO LiPA MYCOBACTERIAINNO LiPA MYCOBACTERIA

• 3 line-probes are addressed to the 3 line-probes are addressed to the identification of differentidentification of different M. kansasiiM. kansasii strainsstrains– all the strains positive with the 1all the strains positive with the 1stst AccuProbe AccuProbe

hybridize with line MKA-1hybridize with line MKA-1– all the strains negative with the 1all the strains negative with the 1stst and positive and positive

with the 2with the 2ndnd AccuProbe hybridize with line MKA-2 AccuProbe hybridize with line MKA-2– the strains negative with both AccuProbe hybridize the strains negative with both AccuProbe hybridize

with line MKA-3 with line MKA-3

(Tortoli (Tortoli et al.et al., 2001), 2001)

Genetic sequencingGenetic sequencing

• The strains pMK1-9-negative differ 5 The strains pMK1-9-negative differ 5 nucleotides in the 16S rDNA from the pMK1-9-nucleotides in the 16S rDNA from the pMK1-9-positive ones (Ross positive ones (Ross et al.,et al., 1992) 1992)

• 1 to 6 nucleotide diversity differentiates, in 1 to 6 nucleotide diversity differentiates, in the first 500 bp of 16S rDNA, 5 sequevars of the first 500 bp of 16S rDNA, 5 sequevars of M. kansasiiM. kansasii (RIDOM)(RIDOM)

• Extensive diversities (12 to 49 nucleotides) Extensive diversities (12 to 49 nucleotides) characterize, in ITS, 5 sequevars of characterize, in ITS, 5 sequevars of M. kansasiiM. kansasii (RIDOM)(RIDOM)

Repeated DNA sequencesRepeated DNA sequences

• GC-rich repetitive sequence GC-rich repetitive sequence • ISIS16521652 • Major Polymorphic Tandem RepeatMajor Polymorphic Tandem Repeat

Fingerprinting techniquesFingerprinting techniques

• Restriction Fragment Length PolymorphismRestriction Fragment Length Polymorphism– the total bacterial DNA is digested with restriction enzymes the total bacterial DNA is digested with restriction enzymes – the fragments are separated by electrophoresisthe fragments are separated by electrophoresis

• Amplified Fragment Length PolymorphismAmplified Fragment Length Polymorphism– the total bacterial DNA is digested with restriction enzymesthe total bacterial DNA is digested with restriction enzymes– oligonucleotide adapters are added by ligaseoligonucleotide adapters are added by ligase– adapter-containing fragments are selectively amplifiedadapter-containing fragments are selectively amplified– amplification products are separated electrophoreticallyamplification products are separated electrophoretically

• Pulsed Field Gel ElectrophoresisPulsed Field Gel Electrophoresis– large restriction fragments are obtained from total bacterial DNAlarge restriction fragments are obtained from total bacterial DNA– the fragments are separated by pulsed field electrophoresisthe fragments are separated by pulsed field electrophoresis

• PCR Restriction AnalysisPCR Restriction Analysis– a PCR-amplified DNA sequance is digested with restriction enzymes a PCR-amplified DNA sequance is digested with restriction enzymes – the fragments are separated by electrophoresisthe fragments are separated by electrophoresis

GC-rich repetitive sequenceGC-rich repetitive sequence

• A repetitive sequence, with high A repetitive sequence, with high guanosine and cytosine content, is guanosine and cytosine content, is present, in at least 30 copies, in present, in at least 30 copies, in M. kansasiiM. kansasii, , M. M. tuberculosistuberculosis and and M. M. szulgai szulgai and is responsible of and is responsible of polymorphismpolymorphism (Ross (Ross et al.,et al., 1992) 1992)

ISIS16521652

• ISIS16521652 is present in pMK1-9-negative is present in pMK1-9-negative M. kansasiiM. kansasii only (Yang only (Yang et al.,et al., 1993) 1993)

• In such strains the number of copies, In such strains the number of copies, which ranges from 1 to 11, is responsible which ranges from 1 to 11, is responsible for significant polymorphismfor significant polymorphism

• The MPTR consists of short tandemly repeated The MPTR consists of short tandemly repeated sequences of 10 bp separated by 5-bp spacerssequences of 10 bp separated by 5-bp spacers

• About 80 different MPTR-containing regions are About 80 different MPTR-containing regions are present in the mycobacterial genome present in the mycobacterial genome

• MPTR is present in MPTR is present in M. M. tuberculosistuberculosis complex, complex, M. M. gordonaegordonae, , M. M. gastrigastri, , M. kansasiiM. kansasii, , M. M. szulgaiszulgai

• A wide polymorphism characterizes the A wide polymorphism characterizes the MPTRMPTR‑‑containing restriction fragments of containing restriction fragments of M. kansasiiM. kansasii (Hermans (Hermans et al.et al., 1992), 1992)

GCCGGTGTTG

Major Polymorphic Tandem RepeatMajor Polymorphic Tandem Repeat

MPTR-based RFLPMPTR-based RFLP

• pMK1-9-positive pMK1-9-positive M. kansasiiM. kansasii share most bands share most bands in common; pMK1-9-negative strains are more in common; pMK1-9-negative strains are more heterogeneous (Ross heterogeneous (Ross et al.,et al., 1992) 1992)

• AccuProbe-1-positive AccuProbe-1-positive M. kansasiiM. kansasii exhibit a 3kb exhibit a 3kb fragment; AccuProbe-1-negative strains fragment; AccuProbe-1-negative strains present fragments of variable length (Yang present fragments of variable length (Yang et al.,et al., 1993) 1993)

Intein-coding sequencesIntein-coding sequences

• Inteins are protein sequences that are excised Inteins are protein sequences that are excised from the precursor protein during maturationfrom the precursor protein during maturation

• Intein-coding sequences may be present in Intein-coding sequences may be present in gyrAgyrA, the gene encoding for the A subunit of , the gene encoding for the A subunit of mycobacterial DNA gyrasemycobacterial DNA gyrase

• Several mycobacterial species present in Several mycobacterial species present in gyrAgyrA an intein-coding sequence, others species do an intein-coding sequence, others species do notnot

• M. kansasiiM. kansasii, , M. flM. flavescensavescens and and M. gM. gordonaeordonae are are the only species in which strains with and the only species in which strains with and without without gyrAgyrA intein are present intein are present (Sander et al., 1998)(Sander et al., 1998)

Minor genetic heterogeneityMinor genetic heterogeneity

• The gene encoding for the MPB70 protein-The gene encoding for the MPB70 protein-analogue of analogue of M. kansasiiM. kansasii presents sequence presents sequence variations suggesting the heterogeneity of the variations suggesting the heterogeneity of the species (Wooldford species (Wooldford et al.et al.,, 1997) 1997)

• The amplification of ITS from different The amplification of ITS from different M. kansasiiM. kansasii strains reveals three different strains reveals three different product profiles, in other mycobacterial product profiles, in other mycobacterial species the profile is reproducible (Abed species the profile is reproducible (Abed et al.et al.,, 1995)1995)

Tortoli Tortoli et al.et al., 1994, 1994

• AccuProbe-1-positive strains, differently from AccuProbe-1-positive strains, differently from -negative ones, rapidly hydrolyze Tween 80 -negative ones, rapidly hydrolyze Tween 80 and possess and possess alphaalpha‑‑fucosidase fucosidase activityactivity

• AccuProbe-positive and -negative strains AccuProbe-positive and -negative strains cannot be distinguished by means of HPLC cannot be distinguished by means of HPLC analysisanalysis

• The isolation of AccuProbe-1-negative The isolation of AccuProbe-1-negative M. kansasiiM. kansasii is significantly more frequent is significantly more frequent ((PP  ==  0.00007)0.00007) from HIV from HIV‑‑positive than from positive than from HIVHIV‑‑negative patientsnegative patients

Picardeau Picardeau et al.et al., 1997 (1/2), 1997 (1/2)

• RFLP, using MPTR as a probe, reveals 5 RFLP, using MPTR as a probe, reveals 5 clustersclusters

• AFLP reveals multiple patterns clustering in 5 AFLP reveals multiple patterns clustering in 5 minor groups according to the ones of MPTRminor groups according to the ones of MPTR

• RFLP reveals ISRFLP reveals IS1652 1652 in 2 of the above clusters, in 2 of the above clusters, with a single copy and with 4-6 copies with a single copy and with 4-6 copies respectively; ISrespectively; IS16521652 is on the contrary missing is on the contrary missing in the othersin the others

• PFGE reveals a number of patterns clustering PFGE reveals a number of patterns clustering in 5 major groupsin 5 major groups

• PRA of PRA of hsp65hsp65 gene reveals 5 clusters gene reveals 5 clusters

• Cluster I includes typical Cluster I includes typical M. kansasiiM. kansasii and and is AccuProbe-1-positiveis AccuProbe-1-positive

• Clusters II to IV are AccuProbe-1-Clusters II to IV are AccuProbe-1-negativenegative

• 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 Picardeau et al.et al., 1997 (2/2), 1997 (2/2)

Alcaide Alcaide et al.et al., 1997 (1/2), 1997 (1/2)

• PRA of PRA of hsp65hsp65 generate 5 different patterns generate 5 different patterns– type I is AccuProbe-1-positive type I is AccuProbe-1-positive – types II and IV are AccuProbe-1-negativetypes II and IV are AccuProbe-1-negative– types III and V are weakly AccuProbe-1-positivetypes III and V are weakly AccuProbe-1-positive

• gyrAgyrA intein is present in types I, IV and V while it lacks in II intein is present in types I, IV and V while it lacks in II and IIIand III

• Partial 16S rDNA sequence reveals 2 sequevars presented Partial 16S rDNA sequence reveals 2 sequevars presented by types I, IV and V and by types II and III respectivelyby types I, IV and V and by types II and III respectively

• ITS sequence reveals 5 sequevars correlating with the ITS sequence reveals 5 sequevars correlating with the hsp65hsp65 PRA types PRA types

• PFGE of the digested chromosomal DNA generates 10 to 15 PFGE of the digested chromosomal DNA generates 10 to 15 large fragmentslarge fragments– moderate polymorphism in type I (clonal structure)moderate polymorphism in type I (clonal structure)– significant polymorphism in type IIsignificant polymorphism in type II

• Types I and II constitute frequent clinical Types I and II constitute frequent clinical isolatesisolates

• Type I, differently from II, is almost Type I, differently from II, is almost absent in the environmentabsent in the environment

• Types III, IV and V are rare in humans Types III, IV and V are rare in humans and frequent in the waterand frequent in the water

Alcaide Alcaide et al.et al., 1997 (2/2), 1997 (2/2)

The five “types” of The five “types” of M. kansasiiM. kansasii

Type

DNA probe

Sequencing

Molecular typing

gyrA intein

p6123 pMK1-9

AccuProbe INNO LiPA RFLP

PRA PFGEa AFLP

1st vers.

2nd vers. MK-1 MK-2 MK-3 16S rDNA

ITS MPTR IS1652

I + + + + + - - a 1 i - A ib S +

II + - - + - + - b 2 ii + B iic M -

III + - - - - - + b 3 iii + C iiid S -

IV + - - - - - + a 4 iv - D Iv S +

V + - + + - - + a 5 v - E V S +

a S, single pattern; M, multiple patterns b 4 subgroupsc 5 subgroupsd 3 subgroups

The five “types” of The five “types” of M. kansasiiM. kansasii

Type

DNA probe

Sequencing

Molecular typing

gyrA intein

p6123 pMK1-9

AccuProbe INNO LiPA RFLP

PRA PFGEa AFLP

1st vers.

2nd vers. MK-1 MK-2 MK-3 16S rDNA

ITS MPTR IS1652

I + + + + + - - a 1 i - A ib S +

II + - - + - + - b 2 ii + B iic M -

III + - - - - - + b 3 iii + C iiid S -

IV + - - - - - + a 4 iv - D Iv S +

V + - + + - - + a 5 v - E V S +

a S, single pattern; M, multiple patterns b 4 subgroupsc 5 subgroupsd 3 subgroups

The five “types” of The five “types” of M. kansasiiM. kansasii

Type

DNA probe

Sequencing

Molecular typing

gyrA intein

p6123 pMK1-9

AccuProbe INNO LiPA RFLP

PRA PFGEa AFLP

1st vers.

2nd vers. MK-1 MK-2 MK-3 16S rDNA

ITS MPTR IS1652

I + + + + + - - a 1 i - A ib S +

II + - - + - + - b 2 ii + B iic M -

III + - - - - - + b 3 iii + C iiid S -

IV + - - - - - + a 4 iv - D iv S +

V + - + + - - + a 5 v - E v S +

a S, single pattern; M, multiple patterns b 4 subgroupsc 5 subgroupsd 3 subgroups

The five “types” of The five “types” of M. kansasiiM. kansasii

Type

DNA probe

Sequencing

Molecular typing

gyrA intein

p6123 pMK1-9

AccuProbe INNO LiPA RFLP

PRA PFGEa AFLP

1st vers.

2nd vers. MK-1 MK-2 MK-3 16S rDNA

ITS MPTR IS1652

I + + + + + - - a 1 i - A ib S +

II + - - + - + - b 2 ii + B iic M -

III + - - - - - + b 3 iii + C iiid S -

IV + - - - - - + a 4 iv - D iv S +

V + - + + - - + a 5 v - E v S +

a S, single pattern; M, multiple patterns b 4 subgroupsc 5 subgroupsd 3 subgroups

The five “types” of The five “types” of M. kansasiiM. kansasii

Type

DNA probe

Sequencing

Molecular typing

gyrA intein

p6123 pMK1-9

AccuProbe INNO LiPA RFLP

PRA PFGEa AFLP

1st vers.

2nd vers. MK-1 MK-2 MK-3 16S rDNA

ITS MPTR IS1652

I + + + + + - - a 1 i - A ib S +

II + - - + - + - b 2 ii + B iic M -

III + - - - - - + b 3 iii + C iiid S -

IV + - - - - - + a 4 iv - D iv S +

V + - + + - - + a 5 v - E v S +

a S, single pattern; M, multiple patterns b 4 subgroupsc 5 subgroupsd 3 subgroups

The five “types” of The five “types” of M. kansasiiM. kansasii

Type

DNA probe

Sequencing

Molecular typing

gyrA intein

p6123 pMK1-9

AccuProbe INNO LiPA RFLP

PRA PFGEa AFLP

1st vers.

2nd vers. MK-1 MK-2 MK-3 16S rDNA

ITS MPTR IS1652

I + + + + + - - a 1 i - A ib S +

II + - - + - + - b 2 ii + B iic M -

III + - - - - - + b 3 iii + C iiid S -

IV + - - - - - + a 4 iv - D iv S +

V + - + + - - + a 5 v - E v S +

a S, single pattern; M, multiple patterns b 4 subgroupsc 5 subgroupsd 3 subgroups

Taxonomic speculationsTaxonomic speculations

• M. kansasii M. kansasii isis– a single systematic entity? a single systematic entity? – a single species split in several subspecies?a single species split in several subspecies?– complex including multiple species?complex including multiple species?

• Evidence exists that, as a consequence of the Evidence exists that, as a consequence of the high degree of divergence, several high degree of divergence, several M. kansasiiM. kansasii types are more closely related to other species types are more closely related to other species than to each otherthan to each other

M. gordonae

M. marinum

M. ulcerans

M. gastri

M. szulgai

M. tuberculosis

ITS-based phylogenetic treeITS-based phylogenetic tree

M. kansasii iii

M. kansasii iv

M. kansasii v

M. kansasii i

M. kansasii ii}

Epidemiological insightsEpidemiological insights

• Types I and II include all the significantly pathogen Types I and II include all the significantly pathogen isolates isolates

• Type I presents a clonal structureType I presents a clonal structure– adaptation to human host? adaptation to human host? – divergence restricted by virulence properties? divergence restricted by virulence properties? – it includes practically al it includes practically al M. kansasiiM. kansasii pathogens for pathogens for

immunocompetent people (the high catalase producers of immunocompetent people (the high catalase producers of Wayne, and almost all the AccuProbe-1-positive strains)Wayne, and almost all the AccuProbe-1-positive strains)

• Type II is rare in immunocompetent patients and Type II is rare in immunocompetent patients and frequent in HIV-infected onesfrequent in HIV-infected ones– lower ability to overcome natural resistance mechanisms?lower ability to overcome natural resistance mechanisms?– it includes the low catalase producers of Wayne, and it includes the low catalase producers of Wayne, and

almost all the AccuProbe-1-negative strains almost all the AccuProbe-1-negative strains

ConclusionsConclusions

• A more precise definition of various A more precise definition of various M. kansasiiM. kansasii isolates would provide isolates would provide significant contribution to understanding significant contribution to understanding of its biological and epidemiological key of its biological and epidemiological key aspects aspects

• An international task force, including An international task force, including microbiologists, epidemiologists and microbiologists, epidemiologists and clinicians, is needed to efficiently take clinicians, is needed to efficiently take up the challenge of up the challenge of M. kansasiiM. kansasii