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© Borgis - Postępy Nauk Medycznych 4/2015, s. 235-241
*Tomasz Jagielski1, Magdalena Klatt2, Zofia Zwolska2, Ewa Augustynowicz-Kopeć2
Struktura populacyjna lekoopornych szczepów Mycobacterium tuberculosis izolowanych z obszaru Polski Wschodniej, na podstawie typowania genetycznego metodą spoligotyping
Spoligotype-defined population structure of drug-resistant Mycobacterium tuberculosis isolates in Eastern Poland
1Department of Applied Microbiology, Institute of Microbiology, Faculty of Biology, University of Warsaw
Head of Department: prof. Jacek Bielecki, PhD
2Department of Microbiology, National Tuberculosis and Lung Diseases Research Institute, Warsaw
Head of Department: prof. Ewa Augustynowicz-Kopeć, MD, PhD
Wstęp. Jednym z istotnych czynników wpływających na sytuację epidemiologiczną gruźlicy w Polsce jest transmisja prątków gruźlicy z krajów sąsiednich. Polska graniczy z byłymi postsowieckimi republikami (Łotwa, Ukraina, Rosja), gdzie wskaźniki zapadalności na gruźlicę, w tym gruźlicę lekooporną są szczególnie wysokie.
Cel pracy. Celem pracy było zdefiniowanie struktury genetycznej lekoopornych szczepów Mycobacterium tuberculosis izolowanych z obszaru Polski Wschodniej.
Materiał i metody. Przedmiotem badania była kolekcja 44 szczepów M. tuberculosis pochodzących od tyluż chorych na gruźlicę płuc z województw lubelskiego, podlaskiego, podkarpackiego i warmińsko-mazurskiego. Typowanie genetyczne szczepów przeprowadzono przy użyciu metody spoligotyping. Otrzymane wzory genetyczne porównywano w międzynarodowej bazie danych SpolDB4. W analizie filogenetycznej wykorzystano również program SpotClust.
Wyniki. Wśród 44 badanych szczepów wyróżniono 25 różnych wzorów genetycznych, z których 5 nie było zarejestrowanych w bazie SpolDB4. Z pozostałych 20 wzorów, 16 (80%) było wcześniej notowanych na terenie Polski. Większość (56%) wykrytych spoligotypów było charakterystycznych dla krajów graniczących z Polską, tj. Niemiec, Czech, Łotwy i Rosji. Blisko 80% wszystkich szczepów sklasyfikowano, na podstawie ich spoligotypów, w dwóch rodzinach molekularnych: T i Haarlem.
Wnioski. Struktura populacyjna szczepów M. tuberculosis z obszaru Polski Wschodniej była typowa dla krajów europejskich. Niewielki udział genotypów o rodowodzie spoza Europy oraz brak cudzoziemców wśród chorych, od których pochodziły badane szczepy, wskazują na autochtoniczny charakter transmisji gruźlicy lekoopornej w województwach Polski Wschodniej.
Introduction. The most detrimental impact on the epidemiology of tuberculosis (TB) in Poland may have the spread of tubercle bacilli from outside the country. This is because Poland is geographically situated in close proximity to the former Soviet Union countries, such as Latvia, Estonia, Ukraine, and Russia, where the incidence of TB, including drug-resistant (DR) TB is exceptionally high.
Aim. To describe the genetic diversity of DR Mycobacterium tuberculosis isolates, circulating in four provinces (Lublin, Podkarpacie, Podlasie, and Warmia-Masuria) constituting the so-called „Eastern Wall” of Poland.
Material and methods. A total of 44 DR M. tuberculosis isolates were spoligotyped. The results were compared with the international spoligotype database (SpolDB4). SpolDB4 and the web-based program SpotClust were used to classify isolates into phylogenetic clades.
Results. Spoligotyping resulted in 25 distinct patterns, of which five were not recorded in the SpolDB4. Of the remaining 20 types, 16 (80%) had already been reported in Poland. Most of the spoligotypes (56%) identified were found to be present in Poland’s neighbors, such as Germany, the Czech Republic, Latvia, and Russia. At the phylogenetic level, the T and Haarlem families accommodated 80% of the isolates.
Conclusions. The M. tuberculosis population in Eastern Poland displayed features characteristic of a European country. Given the low frequency of imported genotypes and the absence of foreign-born patients, DR-TB in Eastern Poland appears to be due to the local transmission of M. tuberculosis strains that have been active in Poland for many years, rather than to the importation of strains from neighboring countries.

Tuberculosis (TB) still represents a significant global health problem, with an estimated 9 million new cases and almost 2 million deaths due to this disease per annum worldwide (1). One of the main reasons for the continuing TB prevalence is the emergence and spread of drug-resistant (DR), and more importantly multidrug-resistant (MDR) (defined as resistance to at least isoniazid [INH] and rifampicin [RMP]) Mycobacterium tuberculosis strains. Based on the most recent report by the World Health Organization (WHO), 20% of all TB cases are resistant to at least one first-line drug, and 5% are MDR cases (2). In Poland, in 2004, the number of DR- and MDR-TB cases was 246 (7.6%) and 51 (1.6%), respectively (2). These figures place Poland among the countries with low to moderate DR-TB rates in the world. The most detrimental impact on the epidemiology of TB in Poland may have the spread of DR-TB from outside the country. This is because Poland is geographically situated in close proximity to the former Soviet Union countries, such as Latvia, Estonia, Ukraine and Russia, where the incidence of DR-TB, including MDR-TB is particularly high (2).
With the advent of molecular biology tools, various genotyping methods have been developed for the differentiation of clinical isolates of M. tuberculosis (3). The use of molecular strain typing methods, in conjunction with traditional contact tracing approaches, has significantly improved our understanding of the epidemiology of TB. Among the genotyping methods available, those targeting different repetitive DNA elements and PCR-based are most widely used and preferred. One such a method is spoligotyping which detects polymorphisms in the M. tuberculosis complex direct repeat (DR) chromosomal locus. The DR locus contains multiple direct repeats, each of 36 bp, interspersed with non-repetitive, unique, spacer sequences (spacers) of 35-41 bp in length. The number of direct repeats as well as the presence or absence of specific spacers reflect the polymorphic structure of the DR region and thus demonstrate variations between the strains (4). Spoligotyping is a simple, rapid, robust, and highly reproducible method, whose results are expressed in an octal code format, so as they can be easily compared between different laboratories. Spoligotyping has successfully been applied for the detection of outbreaks (5, 6) and laboratory cross contaminations (7), as well as for the identification of mycobacterial species within the M. tuberculosis complex (4, 8) and recovery of genotypes of particular epidemiological importance, such as those of W-Beijing family (9).
A significant advantage of the method is the availability of the international spoligotype database, SpolDB4 (www.pasteur-guadeloupe.fr/tb/spoldb4; Institute Pasteur, Guadeloupe) (10). The SpolDB4 database contains a total of 1,939 distinct spoligotypes representing 35,925 strains of M. tuberculosis from 122 isolation countries. The organization of the spoligotype database offers a possibility to recognize the genetic structure of M. tuberculosis strains within particular region in relation to other geographical locations. This in turn provides clues about transmission of specific genotypes and/or genotype families (clades) in different human populations (11, 12).
The objective of this study was to explore the genetic diversity of drug-resistant M. tuberculosis isolates from Eastern Poland, by using spoligotyping, and to compare the data obtained with those from neighbouring countries1 so that the dissemination of major phylogenetic clades of tubercle bacilli within the analysed region could be demonstrated.
The population examined included 44 non-related, pulmonary TB patients residing in four provinces of Eastern Poland: Lublin (20 patients), Podkarpacie (13), Podlasie (10), and Warmia-Masuria (1) (fig. 1). The study group represented 90% of all culture-proven drug-resistant TB cases notified in those four Polish provinces in 2004 and 18% of all culture-proven drug-resistant TB cases notified in the whole country in the same year. Of the patients, 34 (77.3%) were males, and 10 (22.7%) were females. The patients were aged between 14 and 81 years. The median age for all the patients was 53 years.
Fig. 1. A map of Poland and its geographical context. Lightly shaded is the so-called Poland’s Eastern Wall, encompassing four provinces, i.e. Warmia-Masuria, Podlasie, Lublin, and Podkarpacie, from where the M. tuberculosis isolates were obtained.
This study was approved by the Ethics Committee of the National Tuberculosis and Lung Diseases Research Institute.
Bacterial strains
The analyzed 44 M. tuberculosis strains were collected in the National Tuberculosis Reference Laboratory (N.T.R.L.) throughout 2004. The diagnostic specimens were: sputum (39), bronchoscopic material (4), and gastric washing (1). Primary isolation was performed using the Löwenstein-Jensen (L.-J.) medium and the BACTEC 460-TB system (Becton-Dickinson, Sparks, MD, USA), and the species identification was done by means of niacin test, gene probes (AccuProbe; GenProbe, San Diego, CA), and mycolic acid analysis by high-pressure liquid chromatography. Drug susceptibility testing was performed using the proportion method on L.-J. medium. The criterion used for drug resistance was growth of 1% or more of the bacterial population on critical concentrations of the drugs tested (i.e. 0.2 μg/ml for isoniazid [INH], 40 μg/ml for rifampicin [RMP], 4 μg/ml for streptomycin, and 2 μg/ml for ethambutol [EMB]) (13).
DNA isolation
Genomic DNA was obtained from L.-J. slants by the cetyl-trimethyl-ammonium bromide (CTAB) method, as previously reported (14).
Spoligotyping and spoligotype analysis
Spoligotyping was performed with a commercially available kit (Isogen Bioscience BV, Maarssen, The Neetherlands), according to the manufacturer’s specifications and as described earlier (4).
Spoligotypes with 100% similarity were considered clusters, whereas non-clustered spoligotypes were referred to as unique. All spoligotypes obtained were compared to the world spoligotype database (SpolDB4) at the Pasteur Institute of Guadeloupe (www.pasteur-guadeloupe.fr/tb/spoldb4). In this database, SIT (Spoligotype International Type) designates spoligotype pattern shared by two or more patient isolates, as opposed to „orphan” which designates patterns reported for a single isolate.
The spoligotypes absent in SpolDB4, and thus identified for the first time, were designated either as new SITs (if two or more isolates harbored that spoligotype) or ‘orphans’ (if the spoligotype occurred in only one isolate). Major phylogenetic clades were assigned according to signatures provided in SpolDB4, which defined 62 genetic (sub-)lineages (10). Clade assignment of the spoligotypes not found in SpolDB4 (orphan types) was performed with SpotClust, an algorithm based on the SpolDB3 database, described previously15 and available online (http://cgi2.cs.rpi.edu/~bennek/SPOTCLUST.html).
The genotypic diversity index (GDI) was calculated by dividing the total number of spoligotypes identified by the total number of M. tuberculosis isolates tested.
Terms: (sub-)families and (sub-)clades were used interchangeably.
Statistical analysis
The Chi-square test or Fisher’s exact test (if the cell count was less than five) were performed to define any association of drug resistance profiles with specific spoligotype families. A P value that was less than 0.05 was considered statistically significant.

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otrzymano: 2015-01-30
zaakceptowano do druku: 2015-03-05

Adres do korespondencji:
*Tomasz Jagielski
Department of Applied Microbiology Institute of Microbiology
Faculty of Biology
University of Warsaw
ul. I. Miecznikowa 1, 02-096 Warszawa
tel./fax +48 (22) 554-13-12

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