[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 Efflux Pump Genes in Resistant Mycobacterium tuberculosis Complex Clinical Isolates Exposed to First Line Antituberculosis Drugs and Verapamil Combination]. / Birinci Seçenek Antitüberküloz Ilaçlara ve Verapamil Kombinasyonuna Maruz Birakilan Dirençli Mycobacterium tuberculosis Kompleks Klinik Izolatlarindaki Efluks Pompa Genlerinin Arastirilmasi.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis, still one of the most common life-threatening infectious diseases worldwide. Although drug resistance in M.tuberculosis is mainly due to spontaneous chromosomal mutations in genes encoding drug target or drug activating enzymes, the resistance cannot be explained only by these mutations. Low permeability of the cell wall, drug inactivating enzymes and especially efflux pumps (EPs) are other mechanisms of drug resistance in mycobacteria. Efflux pump inhibitors (EPIs) binding to M.tuberculosis EPs were shown to inhibit efflux of anti-TB drugs, to enhance M.tuberculosis killing, to reduce drug resistance and to produce synergistic effects with first line anti-TB drugs. In this study, we aimed to determine the minimum inhibitory concentration (MIC) of first-line anti-TB drugs in the presence of verapamil (VER) and the expression of 21 putative EP genes belonged to the ATP-binding cassette (ABC), major facilitator superfamily (MFS) and resistance-nodulation-division (RND) families which might have caused the resistance in nine M.tuberculosis complex clinical isolates resistant to all of the first line anti-TB drugs. MIC values of the isolates were determined in 96-well U-bottom plates by the resazurin microtiter test (REMA) method based on the color change principle. According to the determined MIC values of each isolate, freshly grown cultures in Middlebrook 7H9 broth were exposed to first-line anti-TB drugs and MIC of first-line anti-TB drugs in the presence of VER (½ MIC) at 37°C for 48 hours for RNA extraction. The non-drug exposed cultures were used as control. Total RNA was extracted using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and then treated with DNase I (Thermo Fischer Scientific Inc., Waltham, MA). Complementary DNA (cDNA) from the extracted RNAs was synthesized with the "First strand cDNA synthesis kit" (Thermo Fischer Scientific Inc., Waltham, MA) using oligo primers. The expression levels of efflux pump genes by quantitative realtime polymerase chain reaction (qRt-PCR) were performed using the QuantiTect SYBR Green Rt-PCR Kit (Qiagen, Germany). The housekeeping sigma factor gene sigA (Rv2703) was used as internal control in qRt­PCR assays. Relative quantification of the clinical isolates was determined by the 2-∆∆Ct method by comparing the expression levels of efflux genes in cultures exposed to primary anti-TB drugs and VER with those of non-drug exposed cultures. MIC values of nine isolates by REMA method was determined between 32-512 µg/mL, 1-128 µg/mL, 2-32 µg/mL, 4-16 µg/mL and 15.62-250 µg/mL for streptomycin (SM), isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and VER, respectively. In the presence of ½ MIC VER, it was determined that the MIC of SM decreased 2-32 fold in eight isolates, the MIC of INH decreased by 2-8 fold in nine isolates, the MIC of RIF decreased by 2-16 fold in eight isolates, and the MIC of EMB decreased 2-4 fold in only five isolates. There was an increase in the expression of Rv1273c, Rv1456c, Rv1457 and Rv1819 efflux pump genes from the ABC family, Rv1634 and Rv0842 from the MFS family and Rv3823 efflux from the RND family in isolates exposed to ½ MIC of first-line anti-TB drugs stress. Rv1456c and Rv1819 were found to be associated with SM resistance, Rv1273c with EMB resistance, Rv1457, Rv0842 and Rv3823 with both RIF and EMB resistance, and Rv1634 with INH, RIF and EMB resistance. It was determined that there was a decrease in the expression levels of eight efflux pump genes from the ABC family (Rv1456c, Rv1457c, Rv1458c, Rv0194, Rv1272c, Rv1686c, Rv1687c, Rv1819c), six from MFS family (Rv0842, Rv0849, Rv1634, Rv2265, Rv2456c, Rv0876c) and two from RND family (Rv0507, Rv0676c) in isolates exposed to MIC of first-line anti-TB drugs in the presence of VER (½ MIC). Further studies with clinical isolates are needed to investigate the EPIs that can be used in alternative therapy and to determine the contribution of EPs to the development of resistance due to the increasing TB resistance.

[Investigation of pncA, rpsA and panD Gene Mutations Associated with Resistance in Pyrazinamide-Resistant Mycobacterium tuberculosis Isolates and Spoligotyping]. / Pirazinamid Dirençli Mycobacterium tuberculosis Izolatlarinda Direnç ile Iliskili pncA, rpsA ve panD Gen Mutasyonlari ve Spoligotiplerin Arastirilmasi.

Pyrazinamide (PZA) is one of the first-line anti-tuberculous drugs used in the treatment of tuberculosis (TB). Considering the ability of PZA to shorten the treatment period from 9-12 months to six months by eliminating persistent bacilli, it appears to be an important cornerstone of TB therapy. While the main mechanism causing the PZA resistance is pncA mutations at a rate of 70-97%, it has been determined that rpsA and panD mutations can also cause resistance. In this study, we aimed to investigate the pncA, rpsA and panD gene mutations, the efficiency of the pyrazinamidase (PZAse) enzyme test in determining PZA resistance, the drug susceptibility and their families in PZA-resistant Mycobacterium tuberculosis isolates. Totally 46 PZA resistant M.tuberculosis isolates were included in the study. The pncA, rpsA and panD mutations caused by PZA resistance were investigated by in-house PCR followed by DNA sequencing method. Drug susceptibility was determined with Bactec MGIT 960 (Becton Dickinson, USA) system, the presence of PZAse was evaluated by colorimetric PZAse enzyme assay and the families were determined by the spoligotyping method. Of the 46 PZA-resistant isolates, 24 (52.2%) were identified as PZA monoresistant, 11 (23.9%) multidrug resistant (MDR)-TB and 11 (23.9%) poly drug resistant (PDR)-TB. Gene mutations associated with resistance were detected in 73.9% (34) of PZA-resistant M.tuberculosis isolates. The pncA, rpsA and panD mutations were found in 71.7% (33), 28.2% (12) and 4.3% (2) of the isolates, respectively. The coexistence of pncA/rpsA and pncA/panD gene mutations were determined in 12 and two isolates, respectively. The pncA gene mutations were observed in 3 (33.3%) of 9 (19.6%) isolates whose enzyme presence was detected by the colorimetric PZAse test. In the pncA gene, eight different point mutations in the form of missense mutation;A226C (27.3%), A152C (24.2%), C169G (21.2%) A422C (9.1%), G145A (6.1%), A29G (6.1%), A424G (3%) and T464G (3%) were detected. In the rpsA gene, A636C (42.9%) silent and G1318A (42.9%) missense mutations and in the panD gene, C66G (50%) nonsense and A145G (50%) missense mutations were the most common mutations detected. As a result of genotyping of PZA resistant isolates, the most common genotypes were found in T1 cluster with 17 (36.9%) isolates; followed by the families of Beijing with 7 (15.2%) isolates, H3 with 6 (13%) isolates, TUR with 5 (10.9%) isolates, and LAM 9 with 4 (8.7%) isolates, respectively. In addition, 2 (4.3%) isolates belonging to the ORPHAN family and one isolate belonging to each of LAM TUR, LAM 2, LAM 7, T2, T5-RUS1 families were identified. Our study is the first to investigate all pncA, rpsA and panD gene mutations that have been found to cause PZA resistance in Turkey. Epidemiological studies on PZA resistance will make important contributions to the determination of resistance mechanism and the development of methods that will provide rapid diagnosis for the detection of resistance.

[Legionella Species Isolated in the Water Systems of Ships and Their Molecular Characterization]. / Gemilerin Su Sistemlerinden Izole Edilen Legionella Türleri ve Moleküler Karakterizasyonu.

Legionella species are generally found in nature and in water resources, and they are gram negative bacilli that can cause pneumonia by being transmitted from water systems to humans via aerosol or aspiration. Legionnaires' disease caused by this agent continues to be a public health problem in cruise ships. In this study, it was aimed to determine the prevalence of the colonization of Legionella species by culture method and to determine the molecular characterization of the isolated Legionella in water samples taken from the water systems of the ships docking in Mersin International Port. A total of 158 cold water samples were taken from 18 ferry and/or cargo ships docking in Mersin International Port between December 2014 and June 2015. Fifty-four of the samples were obtained from tanks, 68 from taps and 36 from shower heads. All samples were centrifuged and inoculated from the pellet onto "Buffered Coal Yeast Extract" (BCYE) (Oxoid, CM0655, UK) agar medium supplemented with iron pyrophosphate, L-cysteine and α-ketoglutarate (Oxoid, SR0110, UK). The culture plates were incubated for 10-15 days in microaerophilic environment in a desiccator at 37°C. The suspicious colonies grown in cultures were serogrouped by latex agglutination test (Oxoid, DR0800M, UK) and fluorescent antibody method (m-Tech Monoclonal Technologies, Inc., USA). For the molecular analysis of Legionella species grown in culture, DNA isolation was made from Legionella colonies and then polymerase chain reaction amplification was performed using specific primer sequences targeting the rpoB gene region of the Legionella genome. Direct DNA sequencing of rpoB gene products was performed in the "ABI PRISM 3130XL Genetic Analyzer" (Applied Biosystems, USA). The DNA sequences were typed by BLAST analysis and the determined types, and NCBI (National Center for Biotechnology Information) reference Legionella sequences were phylogenetically compared with the Neighbor-Joining comparison method by using the Mega 7 program. Legionella spp. was isolated in 18 (11.4%) of 158 samples. Of these, four (7.4%, 4/54) were detected from the tank, 11 (16.2%, 11/68) from the tap and three (8.33%, 3/36) from the shower head. After the latex agglutination test performed from the growing bacterial colonies, five (27.8%) were serogrouped as Legionella spp., four (22.2%) as Legionella pneumophila sg 5, two (11.1%, each) as L.pneumophila sg 1,L.pneumophila sg 8 and Legionella bozemanii and one (5.6%) as L.pneumophila sg 3. Two (11.1%) of the isolates grown in culture could not be serogrouped. Molecular characterization of 12 Legionella isolates could be performed. One of them was serologically serogrouped as L.bozemanii, and it was found to be 99% similar to Legionella rubrilucens when compared with NCBI Legionella sequence data in the BLAST program. One isolate that could not be differentiated by serogrouping was identified as Legionella erytra in the BLAST program after DNA sequence analysis. The remaining 10 isolates (55.6%, n= 18) were confirmed as L.pneumophilia after the comparison with reference NCBI sequences. In this study, it was determined that 11.4% of the water samples collected from the water systems of the ships docking in Mersin International Port were contaminated with Legionella species. The detected Legionella species have an important potential source of infection for the captain, ship workers and passengers travelling on the ships. In this respect, this study reveals the necessity of establishing studies to improve the risk management of Legionella in the water systems of ships.

Biological properties and conformational studies of amphiphilic Pd(II) and Ni(II) complexes bearing functionalized aroylaminocarbo-N-thioylpyrrolinate units.

A series of novel palladium(II) and nickel(II) complexes of multifunctionalized aroylaminocarbo-N-thioylpyrrolinates were synthesized and characterized by analytical and spectroscopic techniques. The biological properties of the freshly prepared compounds were screened against S. aureus, B. subtilis, A. hydrophila, E. coli, and A. baumannii bacteria and antituberculosis activity against M. tuberculosis H37Rv strains. Also, the antifungal activity was studied against C. albicans, C. tropicalis, and C. glabrata standard strains. A deep conformational survey was monitored using DFT calculations with the aim to explain the importance of the final conformation in the biological experimental results.

Association of cytokine gene polymorphisms with susceptibility to cutaneous leishmaniasis in a Turkish population.

AIMS: The objective of this study was to determine the association of TNF-α -308 G/A, IFN-γ +874 T/A, IL-12B + 1188 A/C, IL-10 -1082 G/A and IL-4 -590 C/T polymorphisms with susceptibility to CL. METHODS AND RESULTS: A total of 55 CL patients and 110 controls from Sanliurfa province of Turkey were included to this study. Polymorphisms were genotyped by 'polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)' and 'amplification refractory mutation system-PCR (ARMS-PCR)' methods. A statistically significant difference was noted in the allele (P < .001, P = .002) and genotype (P < .001, P = .001,) frequencies of TNF-α -308 G/A and IL-4 -590 C/T, respectively. TNF-α 308 GG versus GA genotype (OR = 19.556 [95% CI 8.310-46.019] P < .001), GG versus GA + AA genotype (OR = 20.444 [95% CI 8.707-48.004] P < .001) and G versus A allele (OR = 6.968 [95% CI 3.903-12.440] P < .001) revealed significant association with CL. IL-4 -590 CC versus TT + CT genotype (OR = 2.049 [95% CI 1.025-4.096], P = .041) and C versus T allele (OR = 2.441 [95% CI 1.355-4.396], P = .002) revealed significant association with CL. CONCLUSION: Our study indicates that TNF-α 308 G/A and IL-4-590 C/T polymorphisms are significantly associated with susceptibility to CL. Individuals carrying A allele at TNF-α promoter -308 position and T allele at IL-4 promoter -590 position are at a higher risk for CL.

Serological evidence of Rift Valley fever virus among humans in Mersin province of Turkey.

BACKGROUND & OBJECTIVES: Rift Valley fever virus (RVFV) is a vector-borne pathogen that causes serious outbreaks among livestock, and severe symptoms and mortality in humans. The virus is known to be widespread throughout African countries and Arabian peninsula. The aim of the present study was to investigate the seroprevalence of RVFV infection among human populations of Mersin province, Turkey. METHODS: A region-wide serological survey was conducted on humans residing in rural and urban areas of Mersin province located in the subtropical mediterranean region of Turkey from July 2011- January 2014. Plasma samples were tested for the presence of anti-RVFV antibodies using commercially available indirect immunofluorescence assay. RESULTS: The overall past infections were detected in 48 (4.9%) of the 977 human blood samples. The RVF virus- specific IgG positivity was detected in 33 (4.9%) of the 677 blood samples obtained from the urban area and in 15 (5%) of the 300 samples obtained from the rural area. There was no statistically significant difference in the distribution of RVFV IgG positivity rates between urban and rural areas (p = 0.933); though difference was significant between the rural areas (p = 0.029). INTERPRETATION & CONCLUSION: The study confirmed for the first time, the presence of the RVFV antibody in the urban and rural areas of mediterranean province of Mersin in Turkey, suggesting wide circulation of RVFV in the human population.

Synthesis, antimicrobial activity and acid dissociation constants of methyl 5,5-diphenyl-1-(thiazol-2-yl)pyrrolidine-2-carboxylate derivatives.

In this study, a series of polysubstituted methyl 5,5-diphenyl-1-(thiazol-2-yl)pyrrolidine-2-carboxylate derivatives were designed and synthesized by the cyclization reaction of methyl 1-(benzoylcarbamothioyl)-5,5-diphenylpyrrolidine-2-carboxylates and 2-bromo-1-(4-substituted phenyl)ethanones in 70-96% yield. The starting pyrrolidine derivatives were synthesized via a 1,3-dipolar cycloaddition reaction in 83-88% yield. The stereochemistry of one of these methyl 5,5-diphenyl-1-(thiazol-2-yl)pyrrolidine-2-carboxylate derivatives was characterized by a single crystal X-ray diffraction study and the acid dissociation constants of these compounds were determined. An antimicrobial screening was performed against different bacterial and fungal strains and against the M. tuberculosis H37Rv strain. Interesting antibacterial activity was observed for two compounds against the A. baumannii strain with MIC values of 31.25 µg/mL (Ampicillin: 125 µg/mL) and against the M. tuberculosis H37Rv strain with MIC values of 0.98-1.96 µg/mL (Isoniazid: 0.98 µg/mL, Ethambutol: 1.96 µg/mL). Therefore, these structures can be considered as good starting points for the development of new powerful antimycobacterial agents.

Design of potent fluoro-substituted chalcones as antimicrobial agents.

Owing to ever-increasing bacterial and fungal drug resistance, we attempted to develop novel antitubercular and antimicrobial agents. For this purpose, we developed some new fluorine-substituted chalcone analogs (3, 4, 9-15, and 20-23) using a structure-activity relationship approach. Target compounds were evaluated for their antitubercular efficacy against Mycobacterium tuberculosis H37Rv and antimicrobial activity against five common pathogenic bacterial and three common fungal strains. Three derivatives (3, 9, and 10) displayed significant antitubercular activity with IC50 values of ≤16,760. Compounds derived from trimethoxy substituent scaffolds with monofluoro substitution on the B ring of the chalcone structure exhibited superior inhibition activity compared to corresponding hydroxy analogs. In terms of antimicrobial activity, most compounds (3, 9, 12-14, and 23) exhibited moderate to potent activity against the bacteria, and the antifungal activities of compounds 3, 13, 15, 20, and 22 were comparable to those of reference drugs ampicillin and fluconazole.

[Serological investigation of phlebovirus exposure in blood donors from the Mediterranean Province of Mersin, Turkey]. / Mersin bölgesi kan donörlerinde fl ebovirus maruziyetinin serolojik olarak arastirilmasi.

Phleboviruses are enveloped segmented RNA viruses, capable of inducing febrile disease and/or meningoencephalitis in exposed individuals, according to the infecting strain, following transmission via arthropods. Prototype medically-important phlebovirus strains responsible for sandfly fever are sandfly fever Sicilian virus (SFSV) and sandfly fever Naples virus (SFNV), where the SFSV variant sandfly fever Cyprus virus (SFCV) is also detected in individuals with febrile disease. Toscana virus (TOSV) is unique among phleboviruses as the cause of infections involving central nervous system. In this seroepidemiological study, human exposure to selected medically-important phleboviruses was investigated in healthy adult residents of the Mersin province, Mediterranean Anatolia, Turkey, where the current data on phlebovirus epidemiology is scarce. A total of 1784 healthy individuals (mean age: 34.7±9.6 years; 97.3% were male), accepted as blood donors at the Mersin University Center for Health Research and Application Blood Bank were included in the study after informed consent during a seventeen month period between July 2011 to November 2012. All participants were requested to fill out a questionnaire to reveal risk factors for vector exposure. SFSV, SFNV, SFCV and TOSV IgG antibodies in serum were investigated via a commercial indirect immunofluorescence test (IIFT) (Sandfly Fever Virus IgG Mosaic I; Euroimmun, Germany). Sera interpreted as positive or strong positive for TOSV or SFNV+TOSV in IIFT were evaluated via TOSV virus neutralization test (VNT) for specificity confirmation. IIFT seroreactivity for at least one of the tested phleboviruses was present in 66.8% (1192/1784) of the samples. The most frequently-detected phlebovirus strain was SFSV (51.6%; 920/1784), followed by SFNV (46.4%; 827/1784), TOSV (43.7%; 779/1784) and SFCV (47.3%; 843/1784). Among the reactive sera, 6.6% (79/1192) were positive for a single virus serotype, whereas in 39.8% (475/1192) antibodies reacting with all tested virus serotypes were revealed. A total of 187 sera was included in the TOSV VNT and neutralizing antibodies were detected in 13.9%. According to the IIFT reactivity, residing in rural areas was observed as a statistically significant risk factor for exposure in all phleboviruses tested (p values for SFSV, SFNV, TOSV and SFCV were 0.002, 0.001, <0.001 and 0.003, respectively). TOSV exposure is more frequently detected via IIFT in individuals having pets or domestic farm animals around the living quarters (p=0.005). As a result, frequent exposure to SFSV/SFCV or antigenically similar phlebovirus strains and viruses of the SFNV species were determined in healthy blood donors in Mersin province, located in the Mediterranean region of Turkey. Furthermore, TOSV neutralizing antibodies were detected in selected samples with IIFT reactivity, confirming previous reports suggesting TOSV activity in the region. TOSV and other phleboviruses must be included in the diagnostic work-up in cases with febrile diseases and viral central nervous system infections during the sandfly-active months.

[Characterization of basal core promoter/precore gene mutations in chronically infected patients with hepatitis B virus genotype D in Mersin Province, Turkey]. / Mersin ilinde hepatit B virus genotip D ile kronik enfekte hastalarda bazal kor promotor/prekor gen bölgesi mutasyonlarinin karakterizasyonu.

The basal core promoter (BCP) and precore (PC) gene regions of hepatitis B virus (HBV) genome are important for the viral replication and synthesis of "e" antigen. Genetic variability has been described in PCP and PC gene regions, commonly in HBeAg negative patients. The aim of this study was to determine the frequency of the predominant mutation patterns of BCP/PC gene regions and their correlations with HBeAg status, HBV-DNA levels, and liver biochemical profiles in chronic hepatitis B (CHB) patients infected with genotype D, in Mersin province which is located at Mediteranean part of Turkey. A total of 54 CHB patients (33 male, 21 female; mean age: 40.05±12.91 years) infected with HBV genotype D were enrolled in the study. Serum HBV-DNA levels, serological markers (HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc) and biochemical profiles (ALT and AST) were analyzed in all patients. BCP and PC gene regions were determined by polymerase chain reaction (PCR) and mutations of these regions were determined by direct sequencing of PCR products then aligned with known wild-type HBV sequences. BCP [nucleotide (nt.) 1753-1762/1764] and/or PC (nt. 1896) mutations were detected in 87.75% (43/49) of the patients. Mutation rates were detected as 97.1% (33/34) and 66.7% (10/15) in the HBeAg negative and in HBeAg positive patient groups, respectively (p=0.008). PC nt. G1896A mutation was more common in HBeAg negative samples than in HBeAg positive samples (73.5% vs. 20%, p=0.001), however there was no significant differences in the occurrence of BCP mutations between the two groups (p=0.331). No correlation was found between the presence of BCP and/or PC mutations and serum HBV-DNA or ALT-AST levels. Our study reveals that significant number of chronically infected patients with genotype D HBV have BCP and PC variants. G1896A stop codon mutation in precore region seems to have a significant role in the loss of HBeAg in our patients. The results of our study provided important data about the frequency and the genetic heterogeneity of different kinds of mutations occurring at BCP and PC gene regions.

Profiles of serum microRNAs; miR-125b-5p and miR223-3p serve as novel biomarkers for HBV-positive hepatocellular carcinoma.

Recently, circulating miRNAs have been reported as promising biomarkers for various pathologic conditions including cancer. Certain microRNAs (miRNAs) have been shown early diagnostic potential for many types of cancer. The objective of this study was to investigate the potential of certain serum/plasma miRNAs as novel non-invasive biomarkers for early diagnosis of hepatitis B virus (HBV) related hepatocellular carcinoma (HCC). For this reason, the expression levels of 24 miRNA (let-7c, miR-92a-3p, 423-5p, 150-5p, 223-3p, 125b-5p, 342-3p, miR-206, 122-5p, 375, 223-5p, 10a-5p, 23b-5p, 99a-5p, 23a-5p, 10a-3p, 122-3p, 125b-1-3p, 23b-3p, 125b-2-3p, 23a-3p, 92a-1-5p, 92a-2-5p, 99a-3p) were analyzed in plasma of patients with chronic hepatitis B, HBV-positive cirrhosis and HBV-positive HCC and compared with control group samples. Totally 94 plasma samples; 28 control and 66 patient plasma (24 CHB, 22 HBV-positive cirrhosis, 20 HBV-positive HCC) and were included in this study. The expression levels of 24 miRNAs were detected for all control and patient group plasma samples by qRT-PCR using BioMark™ 96.96 Dynamic Array (Fluidigm Corporation) system. The expression levels of miR-125b-5p were detected 2.85 fold, 2.46 fold and 1.89 fold (p = 0.01513, p = 0.0009440, p = 0.0001446) up regulated in CHB, HBV-positive cirrhosis and HBV-positive HCC, respectively when compared versus control group individually by Mann-Whitney U test. The expression levels of miR-223-3p were detected 5.55 fold, 13.88 fold and 12.65 fold (p = 0.01513, p = 0.0009440, p = 0.0001446) down regulated in same comparisons. When all groups were compared versus control group by one-way ANOVA test, the expression levels of miR-223-3p were also found statistically significant (p < 0.05). Although not statistically significant, miR-125b-5p tended to be upregulated. (p = 0.07192). These results significantly imply that miR-125b-5p and miR223-3p could be used as novel non-invasive biomarkers of HBV-positive HCC in very early, even at CHB stage of liver disease.

[Investigation of the clonal relationship between nosocomial Acinetobacter baumannii isolates by Rep-PCR]. / Hastane enfeksiyonu etkeni olan Acinetobacter baumannii izolatlari arasindaki klonal iliskinin Rep-PCR ile arastirilmasi

Acinetobacter spp. are opportunistic bacterial pathogens primarily associated with hospital-acquired infections and the spread of multidrug resistant Acinetobacter strains is a growing problem in terms of infection control. The aim of this study was to determine the clonal relationship between strains of nosocomial Acinetobacter baumannii by using rep-PCR method. A total of 75 Acinetobacter strains isolated from various clinical samples of the hospitalized patients between October 2011-May 2012 were included in the study. Antibiotic susceptibilities of Acinetobacter isolates were investigated by Kirby-Bauer disk diffusion method according to CLSI guidelines. According to disk diffusion test, the resistance rates for piperacillin, piperacillin-tazobactam, cefepime, ceftazidime, imipenem, meropenem, gentamicin, amikacin, tetracycline, levofloxacin, ciprofloxacin and trimetoprim-sulfamethoxazole were 96%, 96%, 97.3%, 89.3%, 96%, 94.6%, 66.7%, 85.3%, 68%, 82.7%, 97.3% and 89.3% respectively. In this study, 73 (97%) strains were found resistant to three or more than three antibiotics (multidrug resistant). Rep-PCR analysis have shown the presence of eight clones, including two major clones [A (7subtypes), B (3 subtypes)] and six unique clones (C-H). Clone A was found to be the dominant type. Fifty-four (72%) of the 75 Acinetobacter strains belonged to clone A, 13 (17.3%) to clone B, two strains to clone C, D, and one of each to the other clones (E, F, G, H). Clone A was isolated from 71% (20/28), 70% (7/10) and 100% (6/6) of the samples sent from reanimation intensive care unit, surgery ward and internal diseases intensive care units, respectively. The time interval between the first and last strain was eight months. The results of this study indicated an increase in the resistance rates of Acinetobacter strains in our hospital and this increase was attributed to the clonal dissemination of the strains. Strains of the clone A were found to be dominant at the intensive care and other clinics of our hospital. It is contemplated that Acinetobacter strains were scattered as a result of cross transmission and patient transfer among clinics. The rep-PCR method which was used in this study was evaluated as a rapid, easily applicable and successful procedure for epidemiological studies. Clonal distribution of resistant strains in the hospital environment emphasizes the significance of infection control measures.

[Flavivirus seroepidemiology in blood donors in Mersin province, Turkey]. / Mersin ili kan donörlerinde flavivirus seroepidemiyolojisi.

Among the vector-borne flaviviruses, West Nile virus (WNV), tick-borne encephalitis virus (TBEV) and Dengue virus (DENV) constitute the most frequently-observed pathogens with significant public health impact in endemic regions throughout the globe. This seroepidemiological study was undertaken to investigate human exposure to DENV, WNV and TBEV, as well as other flaviviruses via various serological assays in the Mediterranean province of Mersin, Turkey, where scarce data is currently present for the circulation of these agent. A total of 920 sera were collected after informed consent from asymptomatic blood donors (all were male; age range: 18-63 yrs, mean age: 35.17 ± 9.56 yrs) were taken between August 2010 and April 2011. All samples were initially screened via a commercial ELISA kit for DENV IgM and IgG. Reactive samples were further evaluated via commercial indirect immunofluorescence tests (IIFTs) for yellow fever virus (YFV) IgG, TBEV IgG and via ELISA for WNV IgG. Moreover, presence of neutralizing antibodies were investigated in all reactive samples via plaque reduction neutralization (PRNT) assay for WNV, whose activity has been detected previously in the region. Samples interpreted as positive for TBEV IgG were further evaluated for specificity by TBEV PRNT assay. DENV IgM reactive samples were also assessed for NS1 antigens and IgM/IgG antibodies via a commercial immunochromatographic assay (ICA). DENV IgM and IgG antibodies were detected in 0.9% (8/920) and 16.6% (153/920) of the samples, respectively. One sample was simultaneously positive for IgM and IgG. WNV PRNT revealed positive results in 85.6% (137/160) of the reactive samples, which indicated frequent WNV exposure and frequent development of cross-reactions in the screening assay. Positive or borderline DENV IgM reactivity was identified in 0.43% (4/920) of the samples, which remained negative for NS1 antigen and antibodies in the ICA. Antibody specificity in two samples, positive for DENV and TBEV IgG in IIFT could not be confirmed by TBEV PRNT. A total of 19 reactive samples (19/920, 2.1%), that comprise seven borderline and six positive DENV IgG positivities as well as six samples with IgG positivity for different virus combinations remained negative after DENV confirmatory and WNV/TBEV PRNT assays. When the samples with borderline results were omitted from the evaluation, 12 samples (12/920, 1.3%) were considered to represent exposure to DENV or an antigenically-similar flavivirus. These findings indicated the activity of and frequent exposure (137/920, 14.9%) to WNV, as previously suggested in the study region. In 1.3% of the samples, probable exposure to DENV or other flaviviruses was revealed and this requires further serosurveillance efforts. WNV must be considered in the etiology of febrile diseases or viral neuroinvasive infections of unexplained etiology in the study area.

Design, synthesis, biological evaluation and docking analysis of pyrrolidine-benzenesulfonamides as carbonic anhydrase or acetylcholinesterase inhibitors and antimicrobial agents.

The synthesis and biological assessment of novel multi-functionalized pyrrolidine-containing benzenesulfonamides were reported along with their antimicrobial, antifungal, CAs inhibition, and AChE inhibition as well as DNA-binding effects. The chemical structure of the compounds was elucidated by using FTIR, NMR, and HRMS. Compound 3b, which had Ki values of 17.61 ± 3.58 nM (hCA I) and 5.14 ± 0.61 nM (hCA II), was found the be the most potent CAs inhibitor. Compounds 6a and 6b showed remarkable AChE inhibition effects with Ki values 22.34 ± 4.53 nM and 27.21 ± 3.96 nM in comparison to tacrine. Compounds 6a-6c had moderate antituberculosis effect on M. tuberculosis with a MIC value of 15.62 µg/ml. Compounds had weaker antifungal and antibacterial activity in the range of MIC 500-62.5 µg/ml against standard bacterial and fungal strains. Besides these above, molecular docking studies were performed to examine and evaluate the interaction of the remarkable compounds (3b, 6a and 6b) against the current enzymes (CAs and AChE). Novel compounds gained interest in terms of enzyme inhibitory potencies. Therefore, the most potent enzyme inhibitors may be considered lead compounds to be modified for further research.Communicated by Ramaswamy H. Sarma.

[Determination of the cytokine gene polymorphism and genetic susceptibility in tuberculosis patients]. / Tüberküloz hastalarinda sitokin gen polimorfizmi ve genetik yatkinligin belirlenmesi.

Tuberculosis (TB) is a complicated disease in which biological, socioeconomical and environmental factors play role. Since only 10% of the individuals infected with Mycobacterium tuberculosis develop active disease, it has been suggested that host genetic factors may influence the risk for the development of TB. In this study, we aimed to investigate the presence and role of single nucleotide polymorphisms in the gene regions responsible for cytokine production, since these factors are considered to be associated with susceptibility or resistance to disease development. Single nucleotide polymorphisms were investigated by Amplification Refractory Mutational System (ARMS) Polymerase Chain Reaction (PCR) and PCR-Restriction Fragment Length Polymorphism (RFLP) methods. The presence of single nucleotide polymorphisms were analyzed in tumor necrosis factor alpha (TNF-α) gene promoter -308 G>A (rs1800629) region, interferon gamma (IFN-γ) gene +874 T>A (rs61923114) region, interleukin (IL)-12B p40 gene 1188 A>C (rs3212227) region, IL-10 gene promoter -1082 G>A (rs1800896) region and IL-4 gene promoter -590 C>T (rs2243250) region. A total of 84 patients (71 male, 13 female; mean age: 32.57 ± 15.94 years) whose clinical samples yielded M.tuberculosis complex growth, and 110 healthy blood donors (93 male, 17 female; mean age: 29.40 ± 11.56 years) as control group were included in this study. Of the patients, 76 (90.5%) were diagnosed as pulmonary and 8 (9.5%) as extrapulmonary TB. While 79 (94.1%) patients were newly diagnosed as TB, 5 (5.9%) patients had a TB history (relapsed TB). It was detected that acid-fast bacilli (AFB) were positive in 58 (69%) patients. According to the single nucleotide polymorphism results, gene frequencies could not be compared for TNF-a gene promoter -308 G>A region since healthy controls were in Hardy-Weinberg equilibrium while the patients were not. There were no statistically significant differences in allele and genotype distribution between the patients and healthy controls in IFN-γ gene +874 T>A region, IL-12B p40 gene 1188 A>C region, IL-10 gene promoter -1082 G>A region and IL-4 gene promoter -590 C>T region (p> 0.05). There were also no statistically significant differences between AFB positive (n= 58) and negative (n= 26) patients, and AFB positive (n= 56) and negative (n= 20) pulmonary TB patients (p> 0.05). In conclusion, no statistically significant differences were found associated with the susceptibility or resistance to TB with single nucleotide polymorphisms in the gene regions responsible for cytokine production in the study population. Only some of the single nucleotide polymorphisms of the gene regions responsible for cytokine release were investigated in our study. Therefore further detailed studies to investigate the polymorphisms in the genes that control the cytokine release and receptors specific for these cytokines, should be conducted. Although this study was performed in a relatively small sized population, these findings might provide a significant contribution to the epidemiologic data about the molecular immunology of TB in Turkey.

[Determination of hepatitis C virus genotype distribution in Mersin province, Turkey]. / Mersin ilinde hepatit C virusu genotip dagiliminin belirlenmesi.

Hepatitis C virus (HCV) is a member of the Flaviviridae family and the RNA genome e x hibit high genetic heterogeneity. Six major genotypes were phylogenetically determined and each genotype contains different subtypes. The distribution of HCV genotypes varies geographically throughout the world. Determination of viral genotype has great importance in the selection of antiviral therapy, treatment duration and monitoring the response to treatment. The aim of this study was to determine the distribution of HCV genotypes in Mersin province located at the Southern part of Turkey. A total of 236 patients (137 females, 99 males; mean age: 53.28 ± 14.99 years) with chronic HCV infection who were admitted to Mersin University Hospital Microbiology Laboratory during March 2010-May 2012 period were included in the study. The patients were anti-HCV (ELISA; Abbott Laboratories, USA) and HCV-RNA (Cobas TaqMan 48, Roche Diagnostic, USA) positive. HCV genotype analysis was determined by using a commercial LiPA kit (Line Probe Assay; AMPLIQUALITY HCV-TS; AB Analitica, Italy) based on the reverse hybridization of amplification products of viral 5'-UTR region. Out of the 236 patients, genotype 1b was observed in 84.7% (n= 200), genotype 3a in 4.2% (n= 10), genotype 1 in 3.8% (n= 9), genotype 1a/1b in 2.1% (n= 5), genotype 4a in 2% (n= 2), genotype 1a in 1.7% (n= 4), genotype 2b in 1.3% (n= 3), genotype 2 in 0.4% (n= 1), genotype 2a/2c in 0.4% (n= 1) and genotype 6 in 0.4% (n= 1). In the cases infected with genotype 1b, statistically significant differences were detected between gender distribution with the mean serum ALT (46.14 IU/L in females, 63.9 IU/L in males; p= 0.029) and HCV-RNA (634 x 103 IU/L in females, 20 x 105 IU/L in males; p= 0.005) levels. This was the first study that reflected the distribution of HCV genotypes in southern Turkey region. Genotype 1b, associated with poor prognosis and which had the highest prevalence in Turkey, was also determined as the most common genotype with a rate of 84.7% in our region. In addition, low rates of genotype 1a, 2b, 3a and 4a which were identified with low frequency in our country and newly introduced genotype 6 were also demonstrated.

Investigation of efflux pump genes in isoniazid resistant Mycobacterium tuberculosis isolates.

BACKGROUND: Tuberculosis (TB) is one of the most important infectious diseases worldwide. Resistance to antituberculosis drugs develops because of genetic mutations that render drug-activating enzymes inactive, changes in cell wall permeability, and increased expression of efflux pump genes and also combination therapy with efflux pump inhibitors may be more effective in drug-resistant TB patients. AIMS: To investigate the effect of verapamil (VR) on isonicotinic acid hydrazide (INH) resistance and the expression of 21 efflux pump genes in INH monoresistant MTBC clinical isolates. STUDY DESIGN: In vitro study. METHODS: In our mycobacteriology laboratory, 10 INH monoresistant and 10 primary anti-TB drug-susceptible MTBC clinical isolates were selected. Drug susceptibilities for INH and VR were studied by resazurin microtiter plate method and minimum inhibitory concentration (MIC) was determined. Additionally, mRNA gene expressions were investigated by quantitative Real Time Polymerase Chain Reaction for 21 efflux gene regions. RESULTS: While no change was observed in INH MICs of susceptible isolates under VR effect, 6 (60%) of the 10 INH-resistant isolates showed a decrease of less than one dilution in INH MIC under VR effect. VR significantly reduced resistance in resistant isolates (p â€‹< â€‹0.05). INH monoresistant MTBC isolates showed a 2.85-fold expression increase in the Rv1634 region of the Major Facilitator Superfamily efflux family under INH stress (p â€‹= â€‹0.029). No statistically significant change was observed in other efflux gene regions. Herein, increased expression was observed in the Rv1634 region, consistent with other studies in the literature, and this was associated with drug resistance. No significant change in expression was detected in other gene regions. CONCLUSION: The effect of efflux pump inhibitor VR on INH MIC levels is promising for the treatment of resistant TB. However, studies with more resistant strains are needed to evaluate the efficacy of efflux pump genes.

Design, synthesis, and computational studies of benzimidazole derivatives as new antitubercular agents.

The increase in the drug-resistant strains of Mycobacterium tuberculosis has led researchers to new drug targets. The development of new compounds that have effective inhibitory properties with the selective vital structure of Mycobacterium tuberculosis is required in new scientific approaches. The most important of these approaches is the development of inhibitor molecules for Mycobacterium cell wall targets. In this study, first of all, the antitubercular activity of 23 benzimidazole derivatives was experimentally determined. And then molecular docking studies were carried out with 4 different targets: Arabinosyltransferase C (EmbC), Filamentous Temperature Sensitive Mutant Z (FtsZ), Protein Tyrosine Phosphatase B (PtpB), and Decaprenylphosphoryl-ß-D-ribose-2'-oxidase (DprE1). It has been determined that benzimidazole derivatives show activity through the DprE1 enzyme. It is known that DprE1, which has an important role in the synthesis of the cell envelope from Arabinogalactan, is also effective in the formation of drug resistance. Due to this feature, the DprE1 enzyme has become an important target for drug development studies. Also, it was chosen as a target for this study. This study aims to identify molecules that inhibit DprE1 for the development of more potent and selective antitubercular drugs. For this purpose, molecular docking studies by AutoDock Vina, and CDOCKER and molecular dynamics (MD) simulations and in silico ADME/Tox analysis were implemented for 23 molecules. The molecules exhibited binding affinity values of less than -8.0 kcal/mol. After determining the compound's anti-TB activities by a screening test, the best-docked results were detected using compounds 20, 21, and 30. It was found that 21, was the best molecule with its binding affinity value, which was supported by MD simulations and in silico ADME modeling results.Communicated by Ramaswamy H. Sarma.

[Identification of filamentous fungi isolated from clinical samples by two different methods and their susceptibility results]. / Klinik Örneklerden Izole Edilen Filamentöz Mantarlarin Iki Farkli Yöntemle Tanimlanmasi ve Duyarlilik Sonuçlari

Molds are widely distributed in nature. Aspergillus spp. represent the most frequently observed causative agents, however less frequent pathogens Fusarium, Scedosporium and Zygomycetes have also been considered the most important causes of morbidity and mortality in profoundly immunosuppressed hosts. The aims of this study were to identify filamentous fungi isolated from clinical specimens by conventional and molecular methods, and to detect their antifungal susceptibilities. A total of 6742 clinical specimens obtained from hospitalized patients at critical units of Mersin University Medical Faculty Hospital and sent to our laboratory between April 2008-January 2010 were included in the study. The isolates were identified by classical mycological methods and polymerase chain reaction-based DNA sequencing. Susceptibilities to fluconazole and voriconazole were tested by disk diffusion method and to fluconazole, voriconazole, amfoterisin B, caspofungin and posaconazole by E-test. Filamentous fungi were isolated from 71 (1.05%) samples (13 sputum, 4 wound, 4 peritoneal fluid, 3 extrenal ear discharge, 3 abscess and one of each cerebrospinal fluid, blood, tissue biopsy, nasal swab and conjunctival swab) which belonged to 32 patients (13 female, 19 male; age range 7 months-77 years, mean age: 46.6 years). Of the patients 62.3% presented one or more risk factors such as chronic renal failure (n= 8), chronic obstructive lung disease (n= 6), malignancy (n= 6), diabetes mellitus (n= 5) and peripheral vascular disease (n= 5). Of the isolates six were identified as Aspergillus niger, six as Aspergillus flavus, five as Aspergillus fumigatus, four as Aspergillus terreus, five as Fusarium spp., two as Bipolaris spp., and one of each as Acremonium spp., Aurebasidium spp., Mucor spp., and Scedosporium spp. By conventional methods. Three isolates exhibited different identities by DNA sequencing. All Aspergillus isolates were correctly identified at species level by both methods, Other fungi were identified at genus level by conventional methods and at species level by DNA sequencing. Fluconazole minimum inhibitory concentration (MIC) values were determined as > 256 mg/L in all strains, except Scedosporium; voriconazole MIC values were < 0.38 mg/L in all Aspergillus spp. Caspofungin MIC values were > 32 mg/L for Fusarium, Scedosporium, Rhizopus and Bipolaris strains and ≤ 0.006-0.125 mg/L in all Aspergillus isolates, In three strains (Fusarium equiseti, Cylindrocarpon lichenicola and Rhizopus oryzae) posaconazole minimum inhibitory concentration (MIC) values were > 32 mg/L, however it was < 1.5 mg/L, for the other strains. Amphotericin B MIC values were > 32 mg/L for Fusarium, Scedosporium, Rhizopus and all A.terreus strains and < 2 mg/L for the others. E-test and disk diffusion test results were compatible with each other for all the antifungal agents tested. In conclusion, the identification of filamentous fungi such as Aspergillus and Fusarium spp. is easily and reliably achieved by conventional methods. Since the rate of invasive fungal infections is increasing currently, filamentous molds should be searched especially in the clinical specimens of immunocompromised patients for accurate and prompt diagnosis of such infections and to decrease the related mortality risk.