Eurosurveillance

ECDC

Molecular characterisation of mycobacterium tuberculosis strains from the northwest region of Russia

 Rediger
  Published: 30.05.05 Updated: 30.05.2005 11:55:17
O. Narvskaya 1, I. Mokrousov 1, E. Limeschenko 1, T. Otten 2, L. Steklova 3, O. Graschenkova 2, B. Vishnevsky 2
Pasteur Institute 1, Phthisiopulmonology Institute 2, City Tuberculosis Dispencery 3, St. Petersburg, Russia

Mycobacterium tuberculosis Bejing family IS6110 profile and related S1 spoligotype have high prevalence among cluster isolates in the north - west region of Russia including Saint Petersburg. Perhaps, the spread of this genotype reflects an ongoing transmission of multi drug - resistant strains of Mycobacterium tuberculosis even among BCG - vaccinated population in Russia.

Molecular typing provides reliable strain specific differentiation of Mycobacterium tuberculosis at the DNA level. The most widely used method of genotyping is restriction fragment length polymorphism (RFLP) analysis based on the IS6110 insertion element which produces fingerprint diversity among M. tuberculosis isolates (1). Another more rapid and easier to perform PCR-based method for the analysis of polymorphisms in M. tuberculosis complex chromosomal direct repeat region is known as spacer oligotyping (spoligotyping) (2).

Together with conventional methods both techniques are successfully used for the epidemiological study of tuberculosis. A combination of such approaches is used to detect laboratory errors, to confirm transmission in outbreak investigations and to determine risk factors associated with the spread of multi drug-resistant (MDR) tuberculosis in different human populations (3). Molecular epidemiological population based surveillance is a powerful tool to ascertain the origin and dissemination of particular M. tuberculosis clones in some geographic regions (4, 5, 6). Single publications have been devoted to molecular characterisation of a limited number of M. tuberculosis strains isolated in Russia (7, 8).

Objectives

The aim of the present study, as a part of an ongoing molecular epidemiological investigation of tuberculosis in northwest region of Russia, was to characterise the population structure of M. tuberculosis and to define predominant genotypes among the isolates.

Methods

Mycobacterial culture was done on Lowenstein-Jensen medium. The method of absolute concentrations was used for testing drug-susceptibility of isolates to rifampicin, izoniazid, streptomycin and ethambutol. The isolates were considered to be MDR when they were resistant to at least izoniazid and rifampicin. Internationally standardised RFLP analysis of Pvu II cleaved DNA followed by hybridisation with DIG-labelled IS 6110-derived probe and spoligotyping were performed for typing M. tuberculosis strains (1, 2). The GelCompar TM version 4.1 (Applied Maths BVBA, Kortrijk, Belgium) was used for cluster analysis of RFLP and spoligotyping profiles by UPGMA method based on the Dice coefficient.

Results

Among 100 strains isolated from single patients with acute and recurrent pulmonary tuberculosis in 1996-1999 we found 62 MDR, 9 resistant to one drug, 9 resistant to two drugs, 17 drug-sensitive and 2 strains of unknown drug-resistance. The number of bands in RFLP patterns of different strains varied from 8 to 20. 74 distinct banding patterns have been identified, 64 (86%) of which were found only in single patients. Fingerprints of the remaining 36 isolates were represented by 10 different patterns. The number of strains (patients) with shared RFLP patterns varied from 2 to 10. Computer analysis of banding patterns identified a group of 49 isolates that clustered with very similar patterns (similarity coefficient 76%). These isolates shared more than 2/3 of IS6110 containing restriction fragments and thus seemed to belong to a distinct clonal group known as the Beijing family (4).

Table 1. Molecular differentiation of M.tuberculosis isolates from north-west region of Russia

No. of isolates (n=48)

No. of RFLP-IS6110 profiles

IS6110 copy no.

Spoligotype designation

Spoligotype pattern

22

15

14-20

sp1 S1a 1b

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxxxxxxxx

1

1

17

sp2 S169a

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxxxx .xxx

1

1

8

sp3 S29a 53b

xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx . . . .xxxxxxx

1

1

10

sp4

xxxxxxxxxxxxxxxxxxxx .xxxxxxxxxxx . . . .xxxxxxx

1

1

12

sp5

xxx .xxxxxxxxxxxxxxxxxxxx .xxxxxxx . . . .xxxxxxx

1

1

12

sp6

xxxxxxxxxxxxxxxxxxxx .xx . .xxxxxxx . . . .xxxxxxx

1

1

9

sp7

xxxxxxxxxxxxxxxxxxxx .xxxxxxxxx .x . . . .xxxxxxx

5

5

10-11

sp8 42b

xxxxxxxxxxxxxxxxxxxx . . . .xxxxxxxx . . . .xxxxxxx

1

1

13

sp9

xxxxxxx .xxxxxxxxxxxx . . . .xxxxxxx . . . . .xxxxxxx

6

6

11-14

sp10

xxxxxxxxxx . .xxxxxxxx . . . .xxxxxxxx . . . .xxxxxxx

1

1

9

sp11 S187a 35b

xxxxxxxxxxxx .xxxxxxxxxxxxxxx . . .x . . . .xxxxxxx

1

1

8

sp12

xxx . .xxxxxxxxxxxxx . .xxxxxxxxxxxx . . . .xxx .xxx

1

1

10

sp13

xxxxxxxxxxxxxxxxxxxx . . . . . . . . . . . . . . . . .xxxxxx

1

1

10

sp14

xxxxxxxxxxxxxx . . . . . . . . . .xxxxxxxx . . . .xxxxxxx

1

1

10

sp15

xxxxxxx .xxxxx . . . . . . . . . . . .xxxxxxx . . . .xxxxxxx

1

1

9

sp16

xxxxxxxxxxxx . . . . . . . . . . . . . .xxxxxx . . . .xxxxxxx

2

2

9-10

sp17

xxxxxxx . . . . . . . . . . . . . . . . . . . .xxxxx . . . .xxxxxxx

a Spoligotype defined by Soini et al. (6)

b Spoligotype defined by Sola et al. (5)

We identified 17 distinct spoligotypes (table). 22 (17 MDR) of 23 isolates belonging to the Beijing family as defined by RFLP-IS6110 had the same pattern designated sp1 as characterised by hybridisation with probes 35 to 43. It has been reported as S1 or 1 (6, 5). The sp2 (S169) pattern of one isolate was very close to sp1 (S1). The remaining 25 isolates were of 15 different spoligotypes. Of these, two patterns were assigned to recently published S29 and S187 spoligotypes (6). We identified 3 of 15 spoligotypes that were shared by 2 to 5 strains. One of these spoligotypes, shared by 5 MDR isolates, was previously described as type 42 (5). The other patterns, perhaps unique, were not described previously.

Discussion

In the present study 49 isolates that clustered had a specific IS6110 profile associated with the Beijing family of M. tuberculosis. Of these, 22 randomly selected isolates had a corresponding sp1 (S1) pattern that was previously reported as predominant in China, in some countries of East Asia and the USA but uncommon in Europe and the Caribbean (4, 6). The S1 spoligotype is also characteristic of the W family, which is a group of closely, related multi drug-resistant isolates that have recently spread from New York City (9, 10). Our data demonstrate the high prevalence of the Beijing family IS6110 profile and related S1 spoligotype among the isolates at least in the northwest region of European part of Russia including St. Petersburg. Since the clustered Beijing/S1 MDR strains have been isolated from patients with recurrent as well as acute tuberculosis (the earliest available isolate was dated 1996), we consider the spread of this genotype to reflect an ongoing transmission of MDR tuberculosis. Taking into consideration the increase in tuberculosis incidence and the hypothesis that the prevalence of M. tuberculosis of the Beijing family is higher among BCG-vaccinated individuals (4) we consider the transmission of the MDR clone among the fully vaccinated population in Russia to be threatening.

The study was supported by International Atomic Energy Agency (IAEA) Research Contract No. 9924.

References

  1. Van Embden J D A, Cave M D, Crawford J T et al. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardised methodology. J Clin Microbiol 1993; 31: 406-409.
  2. Kamerbeek J, Schouls A, Kolk M et al. Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 1997; 35: 907-914.
  3. Small P, van Embden J D A. Molecular epidemiology of tuberculosis. In B R Bloom (ed). Tuberculosis: pathogenesis, protection and control. Washington, DC. 1994: 569 - 581.
  4. Van Soolingen D L, Qian P E, de Haas J T. Predominance of a single genotype of Mycobacterium tuberculosis in countries of East Asia. J Clin Microbiol 1995; 33: 3234-3238.
  5. Sola C, Devallois A, Horgen L et al. Tuberculosis in the Caribbean: using spacer oligonucleotide typing to understand strain origin and transmission. Emerg Inf Dis 1999; 5: 404-414.
  6. Soini H, Pan X, Amin A et al. Characterization of Mycobacterium tuberculosis isolates from patients in Houston, Texas, by spoligotyping. J Clin Microbiol 2000; 38: 669-676.
  7. Narvskaya O V, Mokrousov I V, Otten T F et al. Genetic marking of polyresistant Mycobacteriun tuberculosis strains isolated in the north-west of Russia. Problemi tuberculjoza 1999; 3: 39-41.
  8. Shaginian I A, Nesterenko L N, Grishina T D et al. Study of the genomic polymorphism of Mycobacterium tuberculosis strains. Zhurnal Epidemiologii, Microbiologii i Immunobiologii 1996; 3: 65-68.
  9. Agerton T B, Valway S E, Blinkhorn R J et al. Spread of strain W, a highly drug-resistant strain of Mycobacterium tuberculosis, across the United States. Clin Infect Dis 1999; 29: 85-92.
  10. Bifani P J, Plikaytis B B, Kapur V et al. Origin and interstate spread of a New York City multi drug-resistant Mycobacterium tuberculosis clone family. JAMA 1996; 275: 452-457.

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