Comparative molecular-genetic characterization Comparative molecular-genetic characterization of collections of tubercle bacilli from distant of collections of tubercle bacilli from distant geographical regions of prime Soviet Uniongeographical regions of prime Soviet Union

Filipenko M.L., Rot M.A., Voronina E.N., Vikhrova M.A., Khrapov E.A., Ljashenko A.A., Krut’ko V.S., Poluektov E.I., Gorbunova V.V.,

Shabaldin A.V., Kushugulova A.R., Rakhimova S.E., Bekbolatova J.T.

Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, RussiaKharkiv Medical Academy of Postgraduate Education, Phtiziatry and Pulmonology Department, Kharkiv, UkraineMLPU “Tuberculosis clinic”, Central Bacteriological Lab, Nizhnyi Novgorod, RussiaKemerovo Tuberculosis Clinic, Kemerovo, RussiaCancer Immunology Department, Kemerovo Scientific Center SB RAS, Kemerovo, RussiaRGKP “Republic Collection of Microorganisms”, Astana, Kazakhstan

What kind of TB strains is What kind of TB strains is endemic for territory studiedendemic for territory studied

What kind of TB strains is What kind of TB strains is epidemic for territory studiedepidemic for territory studied

Directions of TB strains evolution Directions of TB strains evolution in clinical practice viewin clinical practice view

Genotypic fingerprinting utilizes Genotypic fingerprinting utilizes slight differences in the total slight differences in the total

chromosome that could be or not chromosome that could be or not be related to phenotypic differencesbe related to phenotypic differences

..AGCATTCAG.. ..AGAATTCAG..

..CCTCGGGAAA

....CCCCGGGAAA..

..GCTGGCTT…..AATTCCGAT..

..GCTAGCTT…..AATTCCGAT..

..GCTGGCTT…..AATTCTGAT..

..GCTAGCCT…..AATTCCGAT..

TIME

EVOLUTIONARY

RATE

MethodsMethods

VNTR typingVNTR typing RFLP IS6110RFLP IS6110 RFLP Ser315Thr KatG RFLP Ser315Thr KatG RFLP Ser531Leu rpoB RFLP Ser531Leu rpoB Direct sequencing rpoBDirect sequencing rpoB

Collections testedCollections tested

Kharkiv (Ukraine)Kharkiv (Ukraine) Astana (Kazakhstan)Astana (Kazakhstan) Nizhny Novgorod (Russia, West)Nizhny Novgorod (Russia, West) Kemerovo (Russia, East)Kemerovo (Russia, East)

TypeTypeA,C,EA,C,E KazakhstanKazakhstan NovgorodNovgorod KharkivKharkiv KemerovoKemerovo

222222 6%6% 1, 4%1, 4% 23%23%   15%15%

243243 1%1% -- 5, 6%5, 6%   

333333 -- -- 9%9%   4%4%

343343 4, 8%4, 8% -- 7%7%   

345345 3, 6%3, 6% -- --   

442442 -- -- 3%3%   

443443 1%1% -- 4%4%   

445445 41%41% 75%75% 24%24%   37%37%

452452 1%1% 1, 4%1, 4% 4%4%   

455455 1%1% 1, 4%1, 4% --   

totaltotal 5151 2323 8989   5454

Древо филогенетического родства штаммов M.tuberculosis,

полученных из г.Харькова

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

0,40

31 126

94 56 46 29 67 51 122

105

102

98 78 76 75 74 61 44 41 38 37 36 35 27 24 23 11 7 14 32 84 15 12 125

42 40 43 79 69 64 52 13 123

115

110

109

103

100

92 86 85 83 71 65 62 57 53 34 17 10 58 128

113 54 127

63 45 101

47 26 18 9 811

2 49 87 22 48 20 124

90 19 5 4 73 21 6 3 2

222 452 445 333 343443 243

38 41 44 73 74 75 76 85 86

222 222 445 243 222 222 222 445 445 А B A2

RFLP IS6110 analysis of TB strains from Kharkov

mutmut wildwild

INHINHRR 4141 44 91%91%INHINHSS 44 3434

RIFRIFRR 3131 1919 62%62%RIFRIFSS 11 2929

mutmut wildwild

INHINHRR 6767 44 94%94%INHINHSS 88 2211

RIFRIFRR 2929 1616 64%64%RIFRIFSS 00 88

mutmut wildwild

INHINHRR 2424 66 80%80%INHINHSS 11 66

RIFRIFRR 1414 1313 53%53%RIFRIFSS 00 55

mutmut wildwild

INHINHRR 1515 22 88%88%INHINHSS 22 22

RIFRIFRR 1414 22 87.5%87.5%RIFRIFSS 00 55

Харьков- Ukraine Нижний Новгород- RUS

Кемерово- RUS Астана - Kazakhstan

Ser315Thr KatG and Ser531Leu rpoB mutationsSer315Thr KatG and Ser531Leu rpoB mutations

CodonCodon NovosibirskNovosibirsk KharkivKharkiv

512 Ser->Gly512 Ser->Gly AGC->GGCAGC->GGC 11 --

516 Asp->Val516 Asp->Val GAC->GTCGAC->GTC 77 44

516 Asp->Leu516 Asp->Leu GAC->CTCGAC->CTC -- 11

522 Ser->Leu522 Ser->Leu TCG->TTGTCG->TTG 33 --

526 His->Asp526 His->Asp CAC->GACCAC->GAC 22 --

526 His->Asn526 His->Asn CAC->AACCAC->AAC 11 --

526 His->Tyr526 His->Tyr CAC->TACCAC->TAC -- 44

526 His->Leu526 His->Leu CAC->CTTCAC->CTT -- 11

531 Ser->Leu531 Ser->Leu TCG->TTGTCG->TTG 2121 3333

533 Leu->Pro533 Leu->Pro CTG->CCGCTG->CCG -- 11

513 Gln->term513 Gln->term526 His->Tyr526 His->Tyr

CAA->TAACAA->TAACAC->TACCAC->TAC

-- 11

509 Ser->Thr509 Ser->Thr511 Leu->Pro511 Leu->Pro

AGC ->ACC AGC ->ACC CTG -> CCGCTG -> CCG

-- 11

No mutationNo mutation 55 33

totaltotal 4040 4949

Comparison of rpoB mutations spectrum in Comparison of rpoB mutations spectrum in Novosibirsk and KharkivNovosibirsk and Kharkiv

Древо филогенетического родства штаммов M. tuberculosis,

полученных из Харькова.

Lin

ka

ge

Dis

tan

ce

0,00

0,05

0,10

0,15

0,20

0,25

0,30

0,35

0,40

12

69

4.m 46

12

21

05

10

2.m 98

78

.m7

6.m 75

74

61

44

.m 41

38

37

36

.m3

5.m 27

24

.m2

3.m 11

7.m 8

41

51

21

25

42

.m 40

12

311

511

0.m

10

91

03

.m1

00

92

.m 86

85

.m8

3.m

71

.m 65

62

.m 57

53

.m 34

17

.m1

0.m 54

10

14

72

61

8 9 811

3.m 31

87

.m 22

12

49

01

9 5 4 73

21 6 3 2

44

5

44

2

Beijing strains

445

S42 strains

222

West East

ConclusionsConclusions

S42 (222) is probably endemic for S42 (222) is probably endemic for west territory of prime SU and moves west territory of prime SU and moves to eastto east

This strain possesses different This strain possesses different mechanism of genome evolutionmechanism of genome evolution

Beijing (445) is probably endemic for Beijing (445) is probably endemic for Kazakhstan and acquire the drug Kazakhstan and acquire the drug resistance laterresistance later

Thank you for attentionThank you for attention