Investigation of drug resistant mycobacterium tuberculosis strains with molecular methods

Tüberküloz modern tanı, tedavi ve kontrol yöntemlerine rağmen tüm dünyada önemli bir sağlık problemidir. Tanıda altın standart halen M. tuberculosis kültürüdür. M. tuberculosis kültürü, etkenin tanımlanması ve ilaç duyarlılığının çalışılması açısından önemlidir. Rifampin bakterilerde protein sentezini etkileyerek, etambutol ise arabinozil transferaz enzim inhibisyonu ile arabinogalaktan sentezini engelleyerek antimikobakteriyal etki gösteren ve tüberküloz tedavisinde kullanılan major antitüberküloz ilaçlardandır. Bu çalışmada fenotipik ilaç duyarlılık testi ile rifampin ve/veya etambutol direnci belirlenen izolatlarda, rpoB ve embB gen bölgelerindeki mutasyon varlığının ve mutasyon paternlerinin DNA dizi analizi ile araştırılması amaçlanmıştır. Çalışmamızda rifampin dirençli 7, etambutol dirençli 15, hem rifampin hem de etambutol dirençli 6 olmak üzere toplam 28 suş incelenmiştir. Rifampin dirençli 13 suşun 11'inde (%84,6) rpoB geninde mutasyon, etambutol dirençli 21 suşun ise 14'ünde (%66,6) embB geninde mutasyon saptanmıştır. RpoB mutasyonu saptanan 11 suşun 10'unda 531. kodonda yer alan Ser531Leu (TCG-TTG) mutasyonu bulunmaktadır. Bir suşta ise His526Asn (CAC-AAC) mutasyonu saptanmıştır. En sık saptanan embB mutasyonu 297. kodonda yer alan Ser297Ala (TCG-GCG) mutasyonudur ve 8 suşta saptanmıştır. Dört suşta ise 306. kodonda Met306Val (ATG-GTG) mutasyonu saptanmıştır. İkişer suşta Gly406Asp (GGC-GAC) ve Glu405Asp (GAG-GAC) mutasyonu 1 suşta ise Glu378Ala (GAG-GCG) saptanmıştır. Anahtar kelimeler: Mycobacterium tuberculosis, dizi analizi, rpoB, embBInvestigation of Drug Resistant Mycobacterium tuberculosis Strains with Molecular Methods Tuberculosis is an important public health problem in all over the world despite of modern diagnostic, treatment and control methods. The gold standard for diagnosis is still M. tuberculosis culture. M. tuberculosis culture is important for identification and drug susceptibility tests. Rifampin and ethambutol which have the antimycobacterial effects, are both of the major anti-tuberculosis drugs used in the treatment of tuberculosis, rifampin affects protein synthesis in bacteria and ethambutol also inhibits the synthesis arabinogalactan with arabinosyl transferase enzyme inhibition, respectively. This study aimed to investigate the presence of mutations in the embB and rpoB gene regions and mutation patterns by DNA sequence analysis on isolates which have determined rifampicin and/or ethambutol resistance with phenotypic drug susceptibility test. In this study, we examined the 28 stains which consist of 7 rifampin resistant strains, 15 ethambutol resistant strains, and 6 both rifampin and ethambutol resistant strains. It has been found that there have been mutations on rpoB gene regions of 11 of the 13 (84.6%) rifampin resistant strains and also on embB gene regions of 12 of 21 (66.6%) etambutol resistant strains. It was determined that the 10 of 11 strains identified in rpoB mutation have Ser531Leu (TCG-TTG) mutation which have located at codon 531 and the 1 of 11 strains has also His526Asn (CAC-AAC) mutation. Ser297Ala (TCG-GCG) mutation is the most frequent embB mutation which was found at codon 297 and it was determined on 8 strains. It was also detected that the 4 strains have Met306Val (ATG-GTG) mutation which was found at codon 306, 2 strains have Gly406Asp (GGC-GAC) and Glu405Asp (GAG-GAC) mutation and finally one strain have Glu378Ala (GAG-GCG) mutation. Key words: Mycobacterium tuberculosis, sequence analysis, rpoB, emb

Development of a Real-Time Polymerase Chain Reaction Method for the Identification of Candida Species

Candida species are one of the major causes of nosocomial infections and are the fourth most common agent involved in bloodstream infections. The impact of non-albicans Candida species is increasing, however C.albicans is still the most common species. Since the antifungal susceptibility pattern among Candida spp. may be different, rapid diagnosis and identification of non-albicans Candida spp. are important for the determination of antifungal agents that will be used for treatment. The aim of the study was to describe a real-time polymerase chain reaction (Rt-PCR) assay that rapidly detects, identifies and quantitates Candida species from blood culture samples. A total of 50 consecutive positive blood culture bottles (BACTEC, Beckton Dickinson, USA) identified at our laboratory between June-November 2013, were included in the study. Reference strains of Candida spp. (C.albicans ATCC 10231, C.glabrata ATCC 90030, C.tropicalis ATCC 1021, C.krusei ATCC 6258, C.parapsilosis ATCC 22019 and C. dubliniensis CD36) grown on Sabouraud dextrose agar were used for quality control. BACTEC bottles that were positive for Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus were also studied to search the cross-reactivity. A commercial kit (Zymo Research, USA) was used for DNA extraction. Real-time PCR was performed on LightCycler 480 (Roche, Germany) with primers and probes specific for 18S rRNA of Candida species. Twenty microlitres of the reaction mix contained 2 mu l of extracted DNA, 2 mu l of LightCycler Fast Start DNA Master Probe (Roche Diagnostics, Germany), 2 mu l of MgCl2 (5 mmol), 2 mu l of 10x PCR buffer (Roche Diagnostics, Germany), 0.5 mu l of each primer (0.01 nmol/mu l) and 1 mu l of each probe (0.1 mu mol/mu l) (TibMolBiol, Germany). Amplification was performed using the following conditions; 95 degrees C for 10 mins and 50 cycles of denaturation at 95 degrees C for 10 secs, annealing at 62 degrees C for 10 secs and polymerisation at 72 degrees C for 20 secs. A melting curve was created by cooling the producs at 50 degrees C for 30 secs and then heating to 80 C at a rate of 0.1 degrees C/sec measuring of the fluorescence simultaneously. For the quantitation of fungal DNA according to the standard curve, serial dilutions of C.albicans ATCC 10231 DNA from 3 x 10(5) to 3 x 10(2) ng/mu l were used. All of the strains were also identified by conventional methods and sequence analysis in order to compare the results obtained by Rt-PCR. In our study, all patient and standard samples could be amplified, identified and quantitated by this developed Rt-PCR method. A total of 50 strains, of them 26 were C.parapsilosis, 15 were C.glabrata, 6 were C.albicans, and 3 were C.tropicalis have been detected and identified among patient samples. The results were completely concordant with the sequencing and conventional methods, so the sensitivity and specificity of this method were estimated as 100 percent. In conclusion, it was novel Rt-PCR developed and evaluated in this study is considered as a rapid, accurate, reproducible, sensitive and specific method for the detection, identification and quantitation of commonly observed Candida spp. strains