MURAT SAYAN, Sevil GÜRBİLEK ERDENLİĞ

Single nucleotide polymorphism analysis of the rpoB gene region for genotyping of Brucella melitensis strains isolated from field in Turkey

Ülkemizde ilk kez Brucella izolatlarının moleküler karakterini ortaya çıkardığımız önceki çalışmada erişkin ve çocuk hastalardan izole edilen B. melitensis izolatlarında rpoB geni tek nükleotit polimorfizm (SNP) analizi ile değerlendirilmişti. Ülkemizde daha once hayvan enfeksiyonlarından izole edilen B. melitensis suşlarında SNP analizi ile genotiplendirme değerlendirilmiş değildir. Bu çalışmada, B. melitensis saha suşlarının moleküler tiplendirmesinde rpoB geninin SNP ile analizinin değerlendirilmesi ve epidemiyolojik çalışmalarda kullanılabilecek uygun tiplendirme yönteminin tanımlanması amaçlanmıştır. Araştırmada referans suşlar ile birlikte Marmara, Ege, Akdeniz, Orta Anadolu ve Güneydoğu Anadolu bölgelerimizden izol edilmiş toplam 32 B. melitensis saha suşu çalışıldı. rpoB geni sekanslarının değerlendirilmesi sonrasında B. melitensis saha suşlarında üç moleküler tip tanımlandı; SNP tip 1, SNP tip 2 ve SNP variant tip 2. SNP analiz tekniği B. melitensis suşlarını biyovar özelliklerinden bağımsız bir şekilde moleküler olarak tiplendirmektedir. Çalışmamız, epidemiyolojik olarak SNP analizinin B. melitensis suşlarını tanımlamada klasik yönteme göre yüksek ayrım gücüne sahip olduğunu göstermektedir. Sonuç olarak SNP analizinin B. melitensis izolatları arasındaki ilişkiyi saptayacak faydalı bir moleküler epidemiyolojik metot olduğu ve bu yöntemin brusellozun kontrolü ve etkili bir surveyansına yönelik hazırlanacak ulusal bir brusella veritabanının oluşturulması halinde katkı sağlayacağı görüşüne varılmıştır.

Türkiye’de sahadan izole edilmiş Brucella melitensis suşlarının genotiplendirilmesinde rpoB gen bölgesi tek nükleotit polimorfizm analizi

We previously described the first molecular characterization of Brucella isolates in Turkey that were examined by single nucleotide polymorphisms (SNPs) at the rpoB locus of B. melitensis strains isolated from adult and pediatric patients. However, the molecular typing of B. melitensis strains causing animal infections in Turkey has not been previously investigated. The aim of this study was to evaluate SNP analysis of rpoB gene of B. melitensis from field isolates in Turkey and to try to find out one of the most appropriate methods other than conventional method for long term evaluation of epidemiological studies. Thirty-two B. melitensis strains isolated from Marmara, Aegean, Mediterranean, Central Anatolia and Eastern Anatolia regions of Turkey were investigated together with 3 reference strains. According to rpoB sequencing results, three distinct genotypes (SNP type 1, type variant 2 and type 2) were recognized. SNP technique characterized the strains at the molecular levels independently from B. melitensis biovars. Our study showed that SNP analysis has a better discriminatory capability in identification of B. melitensis strains compared to classical method. In conclusion, it was suggested that SNP analysis could be useful as a molecular epidemiological method to determine relationships between B. melitensis isolates and might aid in effective surveillance and control method for brucellosis particularly in conjunction with a national databases.

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1. Al Dahouk S, Hagen RM, Nockler K, Tomaso H, Wittig M, Scholz HC: Failure of a short-term antibiotic therapy for human brucellosis using ciprofloxacin. A study on in vitro susceptibility of Brucella strains. Chemotherapy, 51, 352-356, 2005.
2. Kattar MM, Jaafar RF, Araj GF, Le Fleche P, Matar GM, Rached RA: Evaluation of a multilocus variable-number tandem-repeat analysis scheme for typing Human Brucella isolates in a region of brucellosis endemicity. J Clin Microbiol, 46, 3935-3940, 2008.
3. Marianelli C, Petruccab A, Pasquali P, Ciuchini F, Papadopouloub S, Cipriani P: Use of MLVA-16 typing to trace the source of a laboratory- acquired Brucella infection. J Hosp Infect, 68, 274-276, 2008.
4. Al Dahouk S, Nöckler K, Hensel A, Tomaso H, Scholz HC, Hagen RM: Human brucellosis in a nonendemic country: A report from Germany, 2002 and 2003. Eur J Clin Microbiol Infect Dis, 24, 450-456, 2005.
5. Al Dahouk S, Le Flèche P, Nöckler K, Jacques I, Grayon M, Scholz HC: Evaluation of Brucella MLVA typing for human brucellosis. J Microbiol Meth, 69, 137-145, 2007.
6. Whatmore AM, Shankster SJ, Perrett LL, Murphy TJ, Brew SD, Thirlwall RE: Identification and characterization of variable-number tandem-repeat markers for biotyping of Brucella spp. J Clin Microbiol, 44, 1982-1893, 2006.
7. Bricker BJ, Ewalt DR, MacMillan AP, Foster G, Brew S: Molecular characterization of Brucella strains isolated from marine mammals. J Clin Microbiol, 38, 1258-1262, 2000.
8. Al Dahouk S, Tomaso H, Berninghoff PE, Splettstoesser WD, Scholz HC, Neubauer H: Identification of Brucella species and biotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP). Crit Rev Microbiol, 31, 191-196, 2005.
9. Marianelli C, Ciuchini F, Tarantino M, Pasquali P, Adone R: Molecular characterization of the rpoB gene in Brucella species: new potential molecular markers for genotyping. Microbes Infect, 8, 860-865, 2006.
10. Sayan M. Yumuk Z, Bilenoğlu O, Erdenliğ S, Willke A: Genotyping of B. melitensis by rpoB gene analysis and reevaluation of conventional serotyping method. Jpn J Infect Dis, 62, 160-163, 2009.
11. Sayan M: Brucella melitensis biovar 2 can not be detected by serological method in Turkey. Letter to Editor. Turk Klin Tip Bilim Derg, 29, 1351-1352, 2009.
12. Turkish Statistical Institute: Address-based population register system, December 31, 2008 census. Turkish Statistical Institute, Ankara, Turkey. www.turkstat.gov.tr, May, 2012.
13. Bodur H, Balaban N, Aksaray S, Yetener V, Akıncı E, Colpan A: Biotypes and antimicrobial susceptibilities of Brucella isolates. Scand J Infect Dis, 35, 337-338, 2003.
14. Dokuzoguz B, Ergönül O, Baykam N, Esener H, Kiliç S, Celikbas A: Characteristics of B. melitensis versus B. abortus bacteraemias. J Infect, 50, 41-45, 2005.
15. Tanyel E, Coban AY, Koruk ST, Simsek H, Hepsert S, Cirit OS: Actual antibiotic resistance pattern of Brucella melitensis in central Anatolia. An update from an endemic region. Saudi Med J, 28, 1239-1242, 2007.
16. Sayan M, Kılıc S, Uyanık MH: Epidemiological survey of rifampicin resistance in clinic isolates of Brucella melitensis obtained from all regions of Turkey. J Infect Chemother, 18, 41-46, 2012.
17. Kilic S, Ivanov IN, Durmaz R, Bayraktar MR, Ayaslioglu E, Uyanik H: MLVA genotyping of human Brucella isolates from Turkey. J Clin Microbiol, 49, 3276-3283, 2011.
18. Alton GG, Jones LM, Angus RD, Verger JM: Techniques for the brucellosis laboratory. Institut National de la Recherche Agronomique, Paris,1988.
19. Marianelli C, Ciuchini F, Tarantino M, Pasquali P, Adone R: Genetic bases of the rifampicin resistance phenotype in Brucella spp. J Clin Microbiol, 42, 5439-5443, 2004.
20. Whatmore AM, Perret LL, MacMillan A: Characterization of the genetic diversity of Brucella by multilocus sequencing. BMC Microbiol, 7, 34, 2007. DOI: 10.1186/1471-2180-7-34