[The Mutations on embA, embB and embC Gene Region in Ethambutol-Resistant and Susceptible Clinical Mycobacterium tuberculosis Complex Isolates]. / Etambutole Dirençli ve Duyarli Mycobacterium tuberculosis Kompleksi Klinik Izolatlarinda embA, embB ve embC Gen Bölgesi Mutasyonlari.

Ethambutol (EMB) is one of the first-line drugs used in the standard combination therapy for tuberculosis (TB) caused by Mycobacterium tuberculosis complex (MTC), and resistance to drugs that play a key role in treatment is increasing worldwide. Mutations in the embCAB operon that have been confirmed to be associated with resistance are responsible for EMB resistance. In this study, it was aimed to determine the frequency and patterns of mutations in embA, embB and embC gene regions in clinical MTC isolates found to be phenotypically resistant and susceptible to EMB. A total of 64 MTC isolates, 44 of resistant to EMB and 20 of susceptible to EMB, isoniazid, rifampicin, and streptomycin by conventional phenotypic drug susceptibility test, were included in the study. Following the DNA isolation, embA, embB and embC gene regions associated with EMB resistance were amplified with specific primer sequences. The PCR products were cycle sequenced using the Bigdye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, USA) and electrophoretically separated on the ABI PRISM 3130XL Genetic Analyzer (Applied Biosystems, USA). Mutated gene regions were identified by aligning sequence analysis data in multiple sequence analysis programs. In the study, genomic mutations in the embCAB operon were detected in 68.2% (30/44) of the EMB resistant isolates. Mutations in the embB gene region were detected in 66% (29/44) of the resistant isolates, 76% (22/29) of these mutations were at codon 306 and the most common mutation patterns in this codon were determined as ATG→GTG (M306V; 58.6%; 17/29), ATG→ATA, ATC or ATT (M306I; 17.2%; 5/29). Other mutations in the embB gene region were determined as Y334H (3.4%; 1/29), D354A (6.9%; 2/29), E378A (3.4%; 1/29), G406C (3.4%; 1/29), M423I (3.4%; 1/29) and E521A (3.4%; 1/29). Of the 44 EMB-resistant isolates, mutations were detected in one (2.3%) of the isolate in the embA gene region (L330L) and in two (4.5%) of the isolates in the embC gene region (T270I in one isolate and T270I and E305E in the other isolate). Of the phenotypically EMB susceptible isolates, mutation was detected in only one (5%) of the isolates in the embA gene region (E180G). In our study, it was determined that mutations frequently occur in codon 306 of the embB gene in EMB-resistant MTC isolates and this mutation has a potential role in the development of EMB resistance. However, it was concluded that the absence of mutations does not exclude phenotypic EMB resistance. Our results will shed light on the molecular epidemiology of embCAB operon mutations that cause EMB resistance in our country.

[Investigation of The Efficacy of Three Different Protein Extraction Protocols and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for the Identification of Nontuberculous Mycobacteria]. / Tüberküloz Disi Mikobakterilerin Tanimlanmasinda Üç Farkli Protein Ekstraksiyon Protokolü ve Matriks Destekli Lazer Desorpsiyon IyonizasyonUçus Zamanli Kütle Spektrometresi Etkinliginin Arastirilmasi.

There are more than 160 defined nontuberculous mycobacteria (NTM) species within Mycobacterium genus. In recent years, the number of NTM species associated with human infections and the infections caused by them have been reported at increasing rates. The identification of these species by phenotypic methods is difficult, laborious, and unlikely to obtain reliable results. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) MALDI-TOF MS has proven to be a good method for the identification of bacteria and yeasts in routine laboratory practices. However, Mycobacterium species differ from other bacteria by their cell wall structures, less ribosomal protein content, and slower growth rates. A standardized and efficient protein extraction protocol for MALDI-TOF MS analysis of mycobacteria is essential. The aim of our study was to investigate the efficacy of different protein extraction protocols and the MALDI-TOF MS method in the diagnosis of NTM species. A total of 73 NTM isolates, grown in both solid and liquid media and previously identified with line probe assay, were evaluated with MALDI-TOF MS (Bruker Daltonics GmbH and Co. KG, Germany). Stock isolates were homogenized and decontaminated by N-Acetyl L-cysteine (NALC)/Sodium hydroxide (NaOH) method. For solid media, isolates were inoculated on Löwenstein-Jensen medium and incubated at 35˚C in a normal atmosphere. For liquid media culture, BD BACTEC MGIT 960 automated system (Becton, Dickinson, Sparks, MD, USA) was performed according to the manufacturer's instructions. For the identification of all isolates by MALDI-TOF MS, the manufacturer's recommended protein extraction protocol (Protocol 1) was compared with the two other protocols, using a simplified extraction procedure (Protocol 2), and freezing temperature (Protocol 3). In the liquid media analysis, the rates of the isolates identified by MALDI-TOF MS (score≥ 2.0) for Protocol 1, 2, and 3 were found as 84.93% (n= 62), 63.01% (n= 46), and 43.83% (n= 32), respectively. In the solid media analysis, the rates of the isolates with an identification score of ≥ 2.0 for the protocols with the same order were determined as 87.67% (n= 64), 52.05% (n= 38), and 31.50% (n= 23), respectively. Isolates grown in both solid and liquid media were identified in the same species level in all three protocols, regardless of the identification values and misidentification was not presented. When the reliable identification score was evaluated as ≥ 2.0 in our study, the manufacturer's recommended MYCOEX IVD procedure was found to be the most effective method for the isolates grown in both liquid and solid media. In conclusion, MALDI-TOF MS has the potential to be a reliable, easy-to-use and fast method that can be used in routine practice for the identification of NTM species with its standardized protein extraction protocols.

[Investigation of the presence of Mycobacterium tuberculosis in the lymph node aspirates of the suspected tularemia lymphadenitis cases]. / Tularemi lenfadeniti süphesi ile alinan lenf aspirati örneklerinde Mycobacterium tuberculosis s varliginin arastirilmasi

Recently reports of cervical tuberculous lymphadenitis and oropharyngeal tularemia which are the most common infectious causes of granulomatous lymphadenitis, have been significantly increased in Turkey. The differentiation of cervical tuberculous lymphadenitis and oropharyngeal tularemia is usually confusing on the basis of clinical and histopathological findings. Thus, in tularemia endemic areas, the patients are more commonly evaluated in terms of tularemia lymphadenitis leaving tuberculosis out. The aim of this study was to investigate the presence of Mycobacterium tuberculosis in cervical lymph node aspirates, obtained from tularemia suspected cases. A total of 105 oropharyngeal tularemia-suspected cases which were found negative for Francisella tularensis by bacteriological (culture), molecular (PCR) and serological (microagglutination) methods, were included in the study. The samples had been previously studied at National Tularemia Reference Laboratory, Turkish Public Health Institution, between 2009-2011. The study samples were evaluated in terms of M.tuberculosis by culture and real-time PCR (rtPCR) methods in the National Tuberculosis Reference Laboratory. Both Lowenstein-Jensen (LJ) medium and liquid-based MGIT (BD, USA) automated culture system were used for mycobacterial culture. Samples that yielded mycobacterial growth were identified as M.tuberculosis by immunochromotographic test (BD, USA). The lymph node aspirates of 65 patients who were F.tularensis PCR negative but antibody positive, were used as the control group. As a result, M.tuberculosis was found to be positive in 9 (8.6%) of 105 tularemia-negative lymph node aspirates, sent to our laboratory from different geographic regions for the investigation of tularemia. Six of the M.tuberculosis positive cases were male and the age range of the patients was 26-85 years. The presence of M.tuberculosis was detected only by culture in two samples, only by rtPCR in five samples and both by culture and rtPCR in two samples. M.tuberculosis was not identified in the control group specimens. Three of the samples which revealed tuberculosis, were from the tularemia endemic areas. In conclusion, the data of this preliminary study indicated that tuberculous lymphadenitis should be kept in mind in suspected tularemia cases and those patients should also be investigated simultaneously for the presence of tuberculous lymphadenitis.

[Evaluation of the distribution of non-tuberculous mycobacteria strains isolated in National Tuberculosis Reference Laboratory in 2009-2010, Turkey]. / Ulusal Tüberküloz Referans Laboratuvarinda 2009-2010 Yillarinda Tespit Edilen Tüberküloz Disi Mikobakterilerin Dagilimlarinin Irdelenmesi.

Non-tuberculous mycobacteria (NTM) are commonly encountered environmental bacteria, and most of them are associated with lung diseases. Diagnosis of infections caused by NTM is based on clinical, radiological and microbiological findings. The aim of this study was to investigate the distribution of non-tuberculous mycobacterial species isolated from clinical specimens as etiologic agents. The NTM strains isolated from clinical specimens in National Tuberculosis Reference Laboratory (NTRL), together with the strains that were sent to NTRL for the advanced identification of non-tuberculous mycobacterial species that have clinical or microbiological significance, were analysed retrospectively. The strains belonged to January 2009 - December 2010 period. If the same NTM type was isolated more than once in the clinical specimens of a patient, then it was defined microbiologically as a causative agent. Identification of mycobacteria species was performed by using a commercial line-probe assay (GenoType Mycobacterium CM/AS; Hain Lifescience, Germany). In our study, pulmonary and non-pulmonary samples obtained from 206 patients yielded mycobacterial growth in their cultures, and of them 24 (11.7%) were identified as NTM. On the other hand, 51 of the 101 samples sent to NTRL for identification were confirmed as NTM. Of the patients who were found to be infected with NTM (n= 75), 59 (78.7%) were male and the mean age was 50.9 ± 18.8 years. The most frequently identified NTM species was M.fortuitum (33.3%, n= 25), followed by M.abscessus (18.7%, n= 14), M.gordonae (10.7%, n= 8) and M.avium (%8; n= 6). The other types of NTM species identified in our laboratory were M.chelonae (n= 3), M.intracellulare (n= 3), M.kansasii (n= 3), M.peregrinum (n= 2), M.scrofulaceum (n= 2), M.szulgai (n= 2), M.celatum (n= 1), M.haemophilum (n= 1), M.smegmatis (n= 1) and M.xenopi (n= 1). Rapidly growing NTM species (M.fortuitum and M.abscessus) were the most frequent (52%) species isolated in our laboratory as the cause of non-tuberculous mycobacterial infection. Interestingly, the majority of M.fortuitum isolates (n= 21) which was the most common species identified in our laboratory, were those received from the peripheral laboratories. The most common species identified in our laboratory were rapidly growing NTM, however the countrywide distribution of the NTM species was found different than previously reported. In conclusion, further investigation of the non-tuberculous mycobacteria profile in adjunct with epidemiological data seems to be essential in our country.

Multiple-locus variable-number tandem-repeat analysis genotyping of human Brucella isolates from Turkey.

A multiple-locus variable-number tandem-repeat analysis (MLVA) was applied to investigate the epidemiological relationship and genetic diversity among 162 human Brucella isolates collected from all geographic regions of Turkey in an 8-year period (2001 to 2008). The isolates were genotyped by using an MLVA assay developed in Orsay, France (MLVA-16(Orsay)) including eight minisatellite (panel 1) and eight microsatellite (panel 2, subdivided into 2A and 2B) markers. Panels 1 and 2A distinguish 14 genotypes; two of these represented 85% of the strains. Panel 2B displayed a very high discriminatory power. Three loci from panel 2B had diversity index values higher than 0.74. MLVA-16(Orsay) yielded 105 genotypes; 73 were represented by a unique isolate, and 32 included two to eight isolates. The isolates from different patients within the same outbreak or from the same patient before first-line therapy and after relapse showed identical genotypes. A number of MLVA genotypes appeared to be partially restricted to some geographic areas and displayed no annual variation, possibly reflecting persistence of genotypes in certain areas for a time span of at least a decade. This study, representing the first molecular typing results of human Brucella isolates from Turkey, indicated that Turkish human Brucella melitensis isolates were most closely related to the neighboring countries' isolates included in the East Mediterranean group.