Genotyping and Identification of Antigen B Gene Polymorphism of Echinococcus granulosus in Edirne, Thrace, and the First Report of Genotype G2 (Tasmanian Sheep Strain) in Turkey

Background: Echinococcus granulosus is the causative agent of cystic echinococcosis in humans and livestock. It is common worldwide. Cystic echinococcosis is still an important public health problem in Turkey, which is an endemic region. Aims: To genotype Echinococcus granulosus isolates and investigate antigen B gene polymorphism in Thrace, Turkey. Study Design: A cross-sectional study. Methods: Seventy-five hydatid cyst materials obtained between June 2020 and May 2021 were included in the study. Hydatid cyst materials were collected from 12 humans from various hospitals in Edirne and 63 from slaughterhouse animals during the same period. Cyst materials were localized in 8 livers and 4 lungs in humans, 23 livers and 17 lungs in cattle, and 13 livers and 10 lungs in sheep. In the first step, the 12S ribosomal RNA gene was amplified by polymerase chain reaction for all samples and run on an agarose gel. Band patterns were used for strain typing. Then, the selected samples that represented each of the band patterns obtained by single-strand conformation polymorphism analysis were sequenced for AgB1, AgB2, mt-CO1, and mt-ND1 genes. Results: Three different genotypes in Edirne, Thrace, Turkey, were observed for Echinococcus granulosus: G1 (domestic sheep strain), G2 (Tasmanian sheep strain), and G3 (buffalo strain). G1 was the dominant genotype in Edirne, and G3 was the second most common. Additionally, polymorphism in AgB1 and AgB2 gene regions was found. Conclusion: This study is the first to report on Echinococcus granulosus G2 (Tasmania sheep strain) in Turkey and G3 (buffalo strain) and antigen B polymorphism in Thrace. The study results will contribute to the prevention and control programs for cystic echinococcosis in Turkey and worldwide.

[A case of Infective Endocarditis Caused by Pseudoclavibacter species]. / Pseudoclavibacter spp.'nin Etken Oldugu Enfektif Endokardit Olgusu.

Infective endocarditis is an infectious disease usually caused by bacteria, including streptococci, staphylococci, and enterococci. In this report, a case of infective endocarditis in which Pseudoclavibacter spp. detected as the causative agent was presented. A 66-year-old female patient was admitted to our hospital with weight loss, malaise, and lumbar pain. A 2/6 murmur was detected in the physical examination of the patient, who had a history of mitral valve surgery nine years earlier. Blood sample was collected for culture and vancomycin [1 g/24 hours, intravenously (IV)] and gentamicin (80 mg/8 hours, IV) therapy were started. Gram-positive bacilli were detected on the second day of incubation in the blood culture bottle incubated in the BacT/ALERT 3D microbial detection system (bioMerieux, France). High uptake in the focal area of the mitral valve on positron emission tomography-computerized tomography (PET-CT) was interpreted as infective endocarditis. The patient was considered to be at very high risk for surgery and she was discharged after the vancomycin treatment completed for 42 days. For bacterial identification, DNA was isolated using a commercial kit (Invitrogen PureLink Genomic DNA Mini Kit; ThermoFisher Scientific, Waltham, MA, USA), and the 16S rRNA gene was amplified by a polymerase chain reaction (PCR) assay using universal primers 27F and 1492R. Sequence analysis of the PCR product was carried out on an Applied Biosystems 3730XL DNA Analyzer with an Applied Biosystems BigDye Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific, Waltham, MA, USA). A 1366 bp long sequence of the isolate was analyzed using the Basic Local Alignment Search Tool (BLAST) in GenBank and the sequence was 99% compatible with Gulosibacter spp. (GenBank sequence ID: LR884222.1, identity 1365/1366 bp) and Pseudoclavibacter spp. (GenBank sequence ID: FJ375951.1, identity 1364/1366 bp). Upon the negative result of nitrate reduction test of bacterium that was oxidasepositive, the bacterium was considered as Pseudoclavibacter spp. Linezolid, clindamycin, and tetracycline susceptibilities were determined by using the disc diffusion method. The isolate was susceptible to linezolid and resistant to clindamycin and tetracycline. It was also susceptible to penicillin [minimum inhibitory concentration (MIC) = 0.002 µg/ml], vancomycin (MIC= 0.25 µg/ml), and rifampicin (MIC= 0.003 µg/ml) and was categorized as "susceptible, increased exposure" to ciprofloxacin (MIC= 0.25 µg/ml), as determined by using a gradient strip test. Pseudoclavibacter species are non-spore-forming, non-motile, catalase-positive, aerobic gram-positive bacilli belonging to the Microbacteriaceae family. A limited number of clinical cases due to Pseudoclavibacter species have been reported. In the present case, gram-positive bacilli were considered to be the causative agent of infective endocarditis because of the growth of the same bacteria in six bottles of blood cultures taken at different times and increased focal involvement on PET-CT. To our knowledge, this is the second reported case of infective endocarditis due to Pseudoclavibacter species. In conclusion, in cases of clinical compliance in patients with prosthetic heart valves, gram-positive bacilli should be considered causative agents of infective endocarditis and in such cases, a range of microbiological assays should be used for identification.

Investigation of antimicrobial resistance and virulence genes of Campylobacter isolates from patients in a tertiary hospital in Edirne, Turkey.

Purpose: Campylobacter is one of the most common pathogens that cause food-borne infections worldwide. The aim of this study was to determine the antimicrobial resistance rates and the presence of multiple virulence genes in Campylobacter isolates obtained from humans. Materials and Methods: In this study, 71 Campylobacter isolates obtained from human faecal samples were used. Antimicrobial susceptibility tests were performed through the gradient strip method. The presence of virulence genes was investigated by monoplex and multiplex polymerase chain reaction. Results: The rate of resistance of the 66 Campylobacter jejuni isolates was 12.1% for erythromycin, 40.9% for tetracycline and 68.2% for ciprofloxacin. Only one of five Campylobacter coli isolates was resistant to these three antimicrobial agents. The flaB, pldA, cdtA, cadF, cdtC and ceuE genes were found in all 66 of the C. jejuni isolates. In the C. jejuni isolates, positivity rates of 92.4% for flaA, 96.7% for cdtB, 98.5% for ciaB, 90.9% for dnaJ and 96.7% for racR were observed. The flaA, flaB, ciaB, cdtA and cdtC genes were present in all C. coli isolates. Conclusions: It was detected that there is an increase in antimicrobial resistance of Campylobacter strains in our region, and most of the isolates harbour virulence genes.

Caffeine Increases Apolipoprotein A-1 and Paraoxonase-1 but not Paraoxonase-3 Protein Levels in Human-Derived Liver (HepG2) Cells.

BACKGROUND: Apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 are antioxidant and anti-atherosclerotic structural high-density lipoprotein proteins that are mainly synthesized by the liver. No study has ever been performed to specifically examine the effects of caffeine on paraoxonase enzymes and on liver apolipoprotein A-1 protein levels. AIMS: To investigate the dose-dependent effects of caffeine on liver apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 protein levels. STUDY DESIGN: In vitro experimental study. METHODS: HepG2 cells were incubated with 0 (control), 10, 50 and 200 µM of caffeine for 24 hours. Cell viability was evaluated by 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. Apolipoprotein A-1, paraoxonase-1 and paraoxonase-3 protein levels were measured by western blotting. RESULTS: We observed a significant increase on apolipoprotein A-1 and paraoxonase-1 protein levels in the cells incubated with 50 µM of caffeine and a significant increase on paraoxonase-1 protein level in the cells incubated with 200 µM of caffeine. CONCLUSION: Our study showed that caffeine does not change paraoxonase-3 protein level, but the higher doses used in our study do cause an increase in both apolipoprotein A-1 and paraoxonase-1 protein levels in liver cells.

Effect of lipoic acid on paraoxonase-1 and paraoxonase-3 protein levels, mRNA expression and arylesterase activity in liver hepatoma cells.

Paraoxonase-1 (PON1) and PON3 (PON3) are anti-atherosclerotic enzymes, synthesized primarily in liver and bound to HDL in circulation. The aim of the present study was to investigate the effects of therapeutic doses of lipoic acid on PON1 and PON3 protein levels, mRNA expression and arylesterase activity in liver. We treated HepG2 cells with 10, 40 and 200 µM lipoic acid for 72 h. Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. PON1 and PON3 protein levels were measured by Western blotting, their mRNA expression was measured by quantitative PCR and arylesterase activity was measured spectrophotometrically. 200 µM lipoic acid caused a significant increase on PON1 and PON3 protein levels and arylesterase activity as compared with control, 10 µM and 40 µM lipoic acid-treated cells. 200 µM lipoic acid also caused a significant decrease on PON1 mRNA expression whereas on a significant increase PON3 mRNA expression as compared with control, 10 µM and 40 µM lipoic acid-treated cells. Our study showed that although lipoic acid up-regulates PON3 but down-regulates PON1 mRNA expression, it increases both PON1 and PON3 protein levels and arylesterase activity in HepG2 cells. We can report that lipoic acid may be useful for preventing atherosclerosis at therapeutic doses.

Effects of permissible maximum-contamination levels of VOC mixture in water on total DNA, antioxidant gene expression, and sequences of ribosomal DNA of Drosophila melanogaster.

In this study, we aimed to investigate the mutagenic and carcinogenic potential of a volatile organic compound (VOC) mixture with references to the response of D.melanogaster using selected antioxidant gene expressions, RAPD assay and base-pair change of ribosomal 18S, and the internal transcribed spacer, ITS2 rDNA gene sequences. For this purpose, Drosophila melanogaster Oregon R, reared under controlled conditions on artificial diets, were treated with the mixture of thirteen VOCs, which are commonly found in water in concentrations of 10, 20, 50, and 75 ppb for 1 and 5 days. In the random amplified polymorphic DNA (RAPD) assay, band changes were clearly detected, especially at the 50 and 75 ppb exposure levels, for both treatment periods, and the band profiles exhibited clear differences between the treated and untreated flies with changes in band intensity and the loss/appearance of bands. Quantitative real-time PCR (qRT-PCR) analysis of Mn-superoxide dismutase (Mn-SOD), catalase (CAT) and glutathione-synthetase (GS) expressions demonstrated that these markers responded significantly to VOC-induced oxidative stress. Whilst CAT gene expressions increased linearly with increasing concentrations of VOCs and treatment times, the 50- and 75-ppb treatments caused decreases in GS expressions compared to the control at 5 days. Treatment with VOCs at both exposure times, especially in high doses, caused gene mutation of the 18S and the ITS2 ribosomal DNA. According to this research, we thought that the permissible maximum-contamination level of VOCs can cause genotoxic effect especially when mixed.

17p Deletion is associated with resistance of B-cell chronic lymphocytic leukemia cells to in vitro fludarabine-induced apoptosis.

We explored the relationship between the cytogenetic/biologic characteristics of B-chronic lymphocytic leukemia (B-CLL) cells and their tendency to undergo spontaneous or fludarabine-induced apoptosis in vitro. B cells from 36 B-CLL patients were incubated with or without fludarabine for 48 h. Apoptosis was determined by two assays: annexin V staining and DNA staining. Fluorescence in situ hybridization was used for detection of trisomy 12, 11q deletion, and 17p deletion. Bcl-2 and CD38 expressions were determined by flow cytometry. Five patients had 17p deletion, 6 had trisomy 12, and another 6 had 11q deletion. B-CLL cells with 17p deletion had significant resistance to apoptosis induced by fludarabine and a slight spontaneous resistance to apoptosis. Bcl-2 and CD38 were not associated with in vitro spontaneous and fludarabine-induced apoptosis. In conclusion, 17p deletion, which causes loss of p53 gene, is associated with resistance to fludarabine-induced apoptosis in vitro. New treatment modalities should be tried in B-CLL patients with 17p deletion.