Ahmet Yilmaz Coban

Antimicrobial activity of plant extract Ankaferd Blood Stopper.

The in vitro antimicrobial activity of Ankaferd Blood Stopper (ABS) was assessed on 102 clinical isolates from both Gram negative and Gram positive bacteria and four standard strains, including MRSA ATCC 43300, MSSA ATCC 25923, P. aeruginosa ATCC 27853 and E. coli ATCC 35218. ABS was significantly active against all bacteria investigated.

T helper 1 type cytokines polymorphisms: association with susceptibility to Behçet’s disease

The pathogenesis of Behçet’s disease (BD) is not fully understood and immunological abnormalities and genetic factors have been investigated. Because serum concentrations of mainly T helper 1 (Th1) type cells have been reported to be increased in BD, we aimed to investigate whether certain cytokine polymorphisms might represent a risk factor for developing BD. We genotyped 80 patients with BD and 105 healthy controls for interleukin (IL)-1 α (T/C −889), IL-1 β (C/T −511, T/C +3962), IL-1R (C/T pst11970), IL-1RA (T/C mspa111100), IL-2 (T/G −330), IL-12 (C/A −1188), interferon (IFN)-γ (A/T UTR 5644), and TNF-α (G/A −238) polymorphisms. Analyses of cytokine polymorphisms were performed with PCR-SSP. The genotype and allele frequencies of the patients and controls were compared and the association between the polymorphisms of the cytokines with the clinical findings was investigated. Genotype distribution showed significant differences between the patients and the controls for the IL-1 α −889, IL-1 β −511, IL-1 β +3962, IL-1R, IL-12, IFN- γ, and TNF-α cytokines. We didn’t observe significant difference in genotypic frequencies of IL-1RA and IL-2 in our study. Comparison of the IL-1 α −889, IL 1 β −511, and IL 1 β +3962 genotype frequencies showed significant increase in CC genotype between the patients and the controls. The individuals with IL-1R TT polymorphism had a higher risk for BD compared to patients with CT/CC polymorphism. Comparison of IL-12, IFN- γ, and TNF-α, genotype frequencies showed significant increase in CA, AA, and AA genotypes between the patients and controls, respectively. The frequencies of genotypes according to the clinical features of the patients with BD did not show a significant difference (p > 0.05). Our study suggests that development of BD might be determined by various cytokine gene polymorphisms. However, further studies on larger numbers of cases are needed before definite conclusions can be drawn.

A Multidrug Efflux Pump Inhibitor Reduces Fluoroquinolone Resistance in Pseudomonas aeruginosa Isolates

In general, resistance to fluoroquinolones (FQs) in gram-negative bacteria is acquired either by mutations in DNA gyrase and topoisomerase IV or by active export of the agents via antibiotic efflux pumps. Reduced porin expression is also proposed to be another mechanism leading to resistance. In this study, interaction between levofloxacin, ofloxacin, and ciprofloxacin with MC-207,110 (multidrug efflux pump inhibitor) was investigated by a checkerboard assay using Pseudomonas aeruginosa. Levofloxacin, ofloxacin, and ciprofloxacin were tested at different concentrations (0.06–64 µg/ml) and MC-207,110 was tested at a concentration range of 4–128 µg/ml. In the presence of MC-207,110 (at 128, 64, 32, 16 µg/ml) resistance to FQs was inhibited significantly and MIC values were decreased, except at 8 and 4 µg/ml of MC-207,110. When MC-207,110 was used, resistance of P. aeruginosa to FQs in vitro was inhibited significantly, suggesting that MC-207,110 may be useful for use in clinical treatment protocols to overcome FQs resistance.

Drug susceptibility testing of Mycobacterium tuberculosis by the broth microdilution method with 7H9 broth

In this study, we have evaluated the broth microdilution method (BMM) for susceptibility testing of Mycobacterium tuberculosis. A total of 43 clinical isolates of M. tuberculosis and H37Rv as a control strain were studied. All isolates were tested by the proportion method and the BMM for isoniazid (INH), rifampicin (RIF), streptomycin (STR), and ethambutol (ETM). The proportion method was carried out according to the National Committee for Clinical Laboratory Standards (NCCLS) on Löwenstein-Jensen (LJ) medium. The BMM was carried out using 7H9 broth with 96 well-plates. All strains were tested at 3.2-0.05 micro g/ml, 16-0.25 micro g/ml, 32-0.5 micro g/ml, and 32-0.5 micro g/ml concentrations for INH, RIF, STR, and ETM, respectively. When the BMM was compared with the proportion method, sensitivity was 100, 100, 96.9, and 90.2%, while specificity was 100, 85.7, 90.9, and 100% for INH, RIF, STR, and ETM, respectively. The plates were examined 7, 10, 14, and 21 days after incubation. The majority of the result were obtained at 14th days after incubation, while the proportion method result were ended in 21-28 days. According to our results, it may be suggested that the BMM is suitable for early determining of multidrug-resistance-M. tuberculosis strains in developed or developing countries.

Two New Colorimetric Methods for Early Detection of Vancomycin and Oxacillin Resistance in Staphylococcus aureus

ABSTRACT We developed two colorimetric methods for the detection of vancomycin- and oxacillin-resistant Staphylococcus aureus in ≤6 h: (i) a nitrate reductase assay and (ii) a resazurin microplate method. MICs agreed with results obtained by CLSI methods for oxacillin. However, detection of vancomycin resistance required a larger inoculum. These methods may be recommended for the detection of vancomycin- and oxacillin-resistant S. aureus.

Rapid determination of methicillin resistance among Staphylococcus aureus clinical isolates by colorimetric methods

ABSTRACT In the present study, the effectiveness of a rapid and colorimetric nitrate reductase analysis (NRA) method and resazurin microplate assay (REMA) for rapid determination of methicillin resistance among Staphylococcus aureus was investigated. A total of 275 clinical isolates of S. aureus were included in the present study. Among these isolates, 151 had the mecA gene and were resistant to methicillin. The remaining 124 isolates were methicillin susceptible and did not have the mecA gene. Cefoxitin MICs of all isolates were detected by NRA, REMA, and reference broth microdilution methods. Category and essential agreement were determined as 100% and 99.6%, respectively, comparing both NRA and REMA with the reference method. No minor, major, or very major discrepancy was observed for either of the methods. The time necessary to have the MIC results was 5 h for NRA, whereas it was 6 h for REMA. Early detection of MRSA is an important public health concern, and the results of this study showed that both of the colorimetric methods are easy to perform and save time in the determination of MRSA. These methods have a potential use for early detection of MRSA for laboratories unable to use molecular techniques.

Human leucocyte antigens and cytokine gene polymorphisms and tuberculosis

PURPOSE Several genes encoding different cytokines and human leucocyte antigens (HLA) may play crucial roles in host susceptibility to tuberculosis (TB). Our objective was to investigate whether these genes might be associated with protection from or susceptibility to TB. MATERIALS AND METHODS Genomic DNA from patients with TB (n = 30) and ethnically matched controls (n = 30) was genotyped by using sequence-specific primers-polymerase chain reaction and sequence-specific oligonucletid methods. RESULTS Our results demonstrated that HLA-CwFNx0101 [P = 0.05, odds ration (OR) (95% confidence interval) = 2.269 (1.702-3.027)] allele frequency was significantly more common in TB patients than in healthy controls, and HLA-CwFNx0101 may be associated with susceptibility to TB. Analysis of cytokine allele frequencies showed that interleukin (IL)-10, -819 C and -592 C alleles was significantly more common in TB patients than in controls (pc: 0.038 and 0.017, respectively). From the IL-10 cluster, a positive significant difference was found at positions -1082 and -592 C/C (pc: 0.027 and 0.054, respectively) genotypes. Although these differences could be explained by the highest frequency of C/C and G/G homozygous patients with TB, in contrast to the control group, statistically significant differences for the C/C genotype however were lost after Bonferroni correction of the P-values. CONCLUSION Altogether, our results suggest that the polymorphisms in HLA (class I) and cytokine (IL-10) genes may affect the susceptibility to TB and increase the risk of developing the disease.

Prospective multicentre evaluation of the direct nitrate reductase assay for the rapid detection of extensively drug-resistant tuberculosis

OBJECTIVES To perform a multicentre study evaluating the performance of the direct nitrate reductase assay (NRA) for the detection of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis in sputum samples. METHODS The study was conducted in six laboratories performing tuberculosis diagnosis that were located in six different countries. The NRA was performed directly on sputum samples in parallel with the reference method used at each site. Detection of resistance was performed for rifampicin, isoniazid, ofloxacin and kanamycin. RESULTS Excellent agreement was obtained for all drugs tested at the majority of sites. The accuracy was 93.7%-100% for rifampicin, 88.2%-100% for isoniazid, 94.6%-100% for ofloxacin and 100% for kanamycin. The majority of NRA results were available at day 21 for sites 1, 2 and 5. Site 3 had a turnaround time of 13.9 days, at site 4 it was 18.4 days and at site 6 it was 16.2 days. The contamination rate ranged between 2.5% and 12%. CONCLUSIONS Rapid detection of drug resistance by the direct NRA on sputum smear-positive samples was accurate and easy to implement in clinical diagnostic laboratories, making it a good alternative for rapid screening for MDR and XDR tuberculosis.

Nitrate Reductase Assay for Rapid Detection of Isoniazid, Rifampin, Ethambutol, and Streptomycin Resistance in Mycobacterium tuberculosis: a Systematic Review and Meta-Analysis

ABSTRACT Colorimetric phenotypic tests recently gained interest because traditional primary drug susceptibility testing of Mycobacterium tuberculosis isolates takes a long time. We used meta-analysis techniques to review the reliability and accuracy of the nitrate reductase assay (NRA), which is one of the most popular colorimetric methods to detect resistance to first-line drugs. Medline, PubMed, ISI Web, Web of Science, and Google Scholar were used to search for studies enrolled in the meta-analysis. The analysis included 35 studies for isoniazid (INH), 38 for rifampin (RIF), and 22 for ethambutol (EMB) and streptomycin (STR). Summary receiver operating characteristic (SROC) curves were applied to summarize diagnostic accuracy. The meta-analyses were performed by the use of Meta-DiSc software (version 1.4) and were focused on sensitivity and specificity values for measurements of accuracy. The pooled sensitivities were 96% for INH, 97% for RIF, 90% for EMB, and 82% for STR. The pooled specificities for INH, RIF, EMB, and STR were 99%, 100%, 98%, and 96%, respectively. The times required to obtain results were between 5 and 28 days by the direct NRA and between 5 and 14 days by the indirect test. In conclusion, the present meta-analysis showed that the NRA is a reliable low-cost rapid colorimetric susceptibility test that can be used for the detection of multidrug-resistant (MDR) tuberculosis, including detection of EMB resistance. However, the test appears to have a relatively low sensitivity for STR and needs further improvement.

Effect of 1-(1-Naphtylmethyl)-piperazine, an Efflux Pump Inhibitor, on Antimicrobial Drug Susceptibilities of Clinical Acinetobacter baumannii Isolates

In this study, the effects of 1-(1-naphtylmethyl)-piperazine (NMP), an efflux pump inhibitor, on antimicrobial drug susceptibilities of 42 clinical Acinetobacter baumannii isolates were investigated by the disc diffusion method. The inhibition zone diameters of antibiotic discs were tested in the presence and absence of NMP and then these zone diameters were compared. Presence of NMP restored ciprofloxacin susceptibility in 15 intermediate and 2 resistant isolates. One ciprofloxacin resistant isolate became intermediate in the presence of NMP. One isolate resistant to gentamicin became intermediate with NMP. Interestingly, one isolate susceptible to meropenem became resistant in the presence of NMP. Although NMP increased the inhibition zone diameters of some of the tested antibiotics against the resistant isolates, the increase was not enough to restore susceptibility. In conclusion, the presence of NMP increases the zone diameters of ciprofloxacin and levofloxacin. Intermediate strains become susceptible but the resistant isolates do not.