Mikrobiyoloji bulteni最新文献

Cow's milk, which is one of today's most important food sources, can be a reservoir for many pathogens that create a risk to public health. One of these pathogens is Cryptosporidium parvum. The oocysts of C.parvum, an obligate intracellular parasite, cause infection when ingested orally. The oocysts scattered around with the feces of infected cows or calves can contaminate raw milk and this is frequently seen in dairy farms. The aim of this study was to investigate the viability of C.parvum by propidium monoazide (PMA)-quantitative polymerase chain reaction (qPCR) method after heat treatment applied to contaminated raw cow's milk. For the study, 50 ml of unpasteurized cow's milk was contaminated with 5 X 105 C.parvum oocysts and portioned into 1.5 ml microcentrifuge tubes. Three groups, namely the control group, pasteurization group and boiling group were formed. No warming procedure was applied to the control group. In the pasteurization group, the milks in microcentrifuge tubes were poured into the wells of the dry block heater set to 71.7 °C and incubated for five seconds. At the end of the period, the milks were transferred to the wells of the cold metal tube, which was removed at -20 °C with the help of a micropipette, and incubated for five seconds. The milks in the boiling group were incubated for two minutes in a dry block heater set to 95 °C. After the heat treatment, the milks in microcentrifuge tubes were transferred to 10 ml centrifuge tubes, PBS was added to make the final volume 10 ml, and centrifuged at 4000 rpm for 20 minutes. After this process was repeated twice, 400 µl of PBS was added to the precipitate remaining at the bottom, and the precipitate was homogenized. One sample of each group was applied with PMA, while PMA was not applied to the other sample. PMA-applied samples were incubated for five minutes at room temperature and in the dark, and then exposed to UV light for five minutes in the device with cooling feature. The oocysts were collected by centrifugation at 5000 g for five minutes. After DNA isolation from oocysts, SYBR Green real time PCR (Rt-PCR) was performed using primers amplifying the COWP gene region. As a result of SYBR Green Rt-PCR, the mean Ct values of the control without PMA, pasteurization and boiling groups were determined as 25 ± 1.24, 23 ± 0.98 and 26 ± 1.03, respectively. While no peak was obtained in the boiling group after PMA application, the mean Ct values of the control and pasteurization groups were 28 ± 1.38 and 31 ± 1.46, respectively. As a result, it was concluded that live C.parvum cysts in milk could be detected by PMA-qPCR method and live oocysts could be found in pasteurized milk.

In this study, it was aimed to investigate the antimalarial activity of cinnamaldehyde (CIN) and cannabidiol (CBD) which have shown various biological activities such as potent antimicrobial activity and eravacycline (ERA), a new generation tetracycline derivative, in an in vivo malaria model. The cytotoxic activities of the active substances were determined by the MTT method against L929 mouse fibroblasts and their antimalarial activity were determined by the four-day test in an in vivo mouse model. In this study, five groups were formed: the CIN group, the CBD group, the ERA group, the chloroquine group (CQ) and the untreated group (TAG). 2.5 x 107 parasites/mL of P.berghei-infected erythrocyte suspension was administered IP to all mice. The determined doses of active substances were given to the mice by oral gavage in accordance with the four-day test and the parasitemia status in the mice was controlled for 21 days with smear preparations made from the blood taken from the tail end of the mice. The IC50 values, which express the cytotoxic activity values of the active substances were determined as 27.55 μg/mL, 16.40 μM and 48.82 μg/mL for CIN, CBD and ERA, respectively. The mean parasitemia rate in untreated mice was 33% on day nine and all mice died on day 11. On the ninth day, when compared with the TAG group, no parasites were observed in the CIN group, while the average parasitemia was 0.08% in the CBD group and 17.8% in the ERA group. Compared to the mice in the TAG group, the life expectancy of the other groups was prolonged by eight days in the CIN group, 12 days in the CBD group and eight days in the ERA group. It has been determined that all three active subtances tested in this study suppressed the development of Plasmodium parasites in an in vivo mouse model and prolonged the life span of the mice. It is thought that the strong antimalarial activity of CIN and CBD shown in the study and the possible positive effect of ERA on the clinical course can be improved by combining them with the existing and potential antimalarial molecules.

In recent years, it has been shown that some bacteria may be associated with colorectal cancer (CRC). In this study, it was aimed to investigate the role of Fusobacterium nucleatum and enterotoxigenic Bacteroides fragilis (ETBF) in the etiology of CRC by comparing the amounts of these bacteria in colon biopsy tissues of patients with CRC and healthy individuals. The amounts of F.nucleatum and ETBF were determined by quantitative polymerase chain reaction (qPCR) in colon biopsy samples taken from 35 CRC and 35 healthy individuals, and the results were compared in the patient and control groups. The detection rate and amounts of F.nucleatum were found to be statistically significantly higher in tissues of female patients with CRC compared to male patients (p= 0.003, p= 0.013, respectively). There was no statistically significant difference between the tissues of female and male patients with CRC in terms of detection rate and amount of ETBF (p= 0.521, p= 0.515, respectively). It was found that in the 50-74 age group, the amount of ETBF was statistically significantly higher in women and men with CRC compared to the controls (p= 0.005, p= 0.047, respectively), while the amount of F.nucleatum was statistically significantly higher in female patients compared to controls. However, no difference was found between male patients and controls (p= 0.009, p= 0.083). It was determined that the detection rate and amount of F.nucleatum in the tissues of patients with CRC, regardless of age and gender, were not statistically different from the controls (p= 0.473, p= 0.995, respectively), however, the detection rate of ETBF and the amount of ETBF were found to be statistically significantly higher (p= 0.002, p= 0.004, respectively). It has been determined that ETBF can play a role in the etiology of CRC in both men and women, and F.nucleatum only in women, in the age range of 50-74 years, when routine screenings for CRC are performed.

Tuberculosis causes serious mortality and morbidity worldwide each year. A lot of effort and money is spent for the diagnosis and treatment of tuberculosis all over the world. The importance that countries give to health policies and public health is inversely proportional to the incidence of tuberculosis and multidrug resistant tuberculosis. The aim of our study was to evaluate the resistance profiles of Mycobacterium tuberculosis complex strains which were isolated from sputum samples, collected by World Health Organisation from patients living in the northern region of Syria, where health services were disrupted due to the civil war. According to the protocol signed between the World Health Organization and our hospital; sputum samples taken from tuberculosis patients living in Afrin, Azez and Idlib regions or suspected of being resistant to anti-tuberculosis drugs were studied in our hospital. The cultivation process was performed in our laboratory using Löwenstein Jensen media and MGIT-960 system. The susceptibility tests for primary anti-tuberculosis drugs were performed using MGIT-960 system for M.tuberculosis complex isolates. The isolates identified as MDR/RD-TB (multi-drug-resistant-rifampicin-resistant tuberculosis) were sent to National Tuberculosis Reference Laboratory of Public Health Institution of Türkiye for susceptibility testing to first and second line drugs. Mutation and wild-type determination were studied by "Line Probe Assay (LPA)" method to investigate the susceptibility of the isolates to isoniazid, rifampicin, fluoroquinolone and aminoglycoside/cyclic peptide. The results obtained from the patients were collected and evaluated retrospectively from the records. Growth was observed in 18 samples out of 171 sputum samples from 67 patients; 13 isolates were detected as MDR-TB while one isolate was detected as mono RR-TB. The rate of mono RR-TB was 1.5% and the rate of MDR-TB was 19.4%. MUT3 causing rifampicin resistance was detected in 17.9% of the patients, katG/MUT1 causing isoniazid resistance in 17.9% and WT loss causing aminoglycoside/cyclic peptide resistance were detected in 19.4% of the patients. Neither fluoroquinolone resistance nor a mutation leading to fluoroquinolone resistance was detected in the study. When the sputum samples taken from the patients living in Northern Syria were examined, the frequency of MDR-TB was found to be quite high. MDR-TB, which is an important public health problem, was found at high rates due to the internal turmoil in the region and poor accessibility to health services. Since the gene mutations causing drug resistance with the LPA method differ with the conducted studies, it is important to evaluate the dominant gene mutations for determining the TB treatment strategies in the region.

Ganciclovir-resistant cytomegalovirus (CMV) strains are reported following long-term antiviral agent use, especially for immune-suppressive patients. In this study, it was aimed to investigate the mutations in the UL97 gene of CMV, which causes ganciclovir (GCV) resistance by genotypic and phenotypic methods in patients who developed CMV infection following hematopoietic cell (HCT) or solid organ transplantation (SOT). Thirty patients who had HCT or SOT in Mediterranean University Hospital and developed CMV infection during routine follow-up with a viral load of CMV over 1000 copies/mL were included in the study. CMV DNA was analyzed by an automated system (Cobas Ampliprep/COBAS TaqMan CMV Test, Roche Diagnostics, Germany) quantitatively. DNA sequence analysis of the regions including codons 420-664 in the UL97 gene region was done by the Sanger sequencing method to detect mutations causing antiviral resistance and compared with defined mutations. In order to investigate antiviral resistance by phenotypic methods, heparinized blood samples of the patients were collected, 'buffy coat (leukocyte layer)' was inoculated into MRC-5 cells by centrifugation method and CMV growth in these cells was controlled with monoclonal antibodies when growth was detected, virus titer was determined and plaque reduction test was applied as recommended. It was determined that 22 of the 30 patients were HCT recipients and eight were SOT (five kidney, three liver) recipients. When the CMV serology pattern of the patients was evaluated before transplantation, 29 (96.7%) patients were found to be seropositive and one (3.3%) patient was found to be seronegative. Totally, nine CMV UL97 mutations were detected in seven (23.3%) pediatric patients who had HCT, including six seropositive and one seronegative case. In addition, one mutation (D605E) not known to cause GCV resistance was detected in a seronegative recipient and three previously unidentified mutations were detected (1474T, F499S, V559A) in a seronegative recipient. Five of the mutations defined were UL97 mutations with a defined clinical resistance against GCV in each of the five recipients (C603W, C592G, H520Q, M460V, A594T). In the plaque reduction test using 3 µM, 12 µM, 48 µM and 96 µM concentrations of GCV in CMV strains, the IC50 value was determined to be ≥ 8 µM for the five CMV strains, and the phenotypic presence of GCV resistance was shown. Clinical resistance associated with CMV UL97 mutation was detected in five (22.7%) of 22 patients who had HCT. GCV resistance was also demonstrated in these patients by phenotypic methods. No UL97 mutation was detected in the patients who had SOT.

The Malassezia yeast species colonize on the skin immediately after birth and could be found on the healthy skin flora for life. Although they are more frequently involved in the etiology of common skin infections in the community, particularly Malassezia furfur could cause life-threatening infections such as fungemia. Detection of biofilm during the colonization of these yeasts on the skin is an important criterion for its virulence. Since they are lipophilic yeasts, commonly used biofilm detection methods are not applicable to the Malassezia strains. The aim of the study was to describe the growth and measurement of M.furfur isolates on a polypropylene membrane to demonstrate their biofilm-forming capacities. Twenty-seven M.furfur strains colonized in the newborns were included in the study. Basically, sterile polypropylene membranes were placed on different polysorbates (tween 20, 40, and 80) which were spread on Sabouraud dextrose agar. Ten µl saline suspension of M.furfur was dropped on the polypropylene membrane and incubated in standard growth conditions for three days. Later, the visible colony was removed gently by washing with running water and the biofilm structure formed on the membrane was stained with safranin. The stained biofilm was photographed. Performing image analysis, the values obtained against background activity were digitized according to the specified protocol. Moreover, XTT reduction test was performed and the measured metabolic activity results were compared with the safranin-stained biofilm data. The safranin hydrolysis of the strains was measured spectrometrically. Twenty-five (92.6%) of the strains included in the study were stained with safranin, which indicated the presence of biofilm on the polypropylene membrane. The strains grown with tween 20 and tween 80 formed a higher biofilm layer density than those supplied with tween 40. Isolates with low and high biofilm-forming capacity were clearly separated by tween 20 (p< 0.05). XTT activity was detected in 26 (96.3%) isolates. No correlation was found between biofilm density obtained by the described method and XTT reduction. It was observed that hydrolysis of safranin did not affect the biofilm evaluation method. In this study, it was shown that as a result of sufficient diffusion through hydrophobic membranes, polysorbate-based growth factors could maintain measurement of the biofilm layer formed by lipophilic M.furfur strains. The best grouping properties for M.furfur were obtained with tween 20 which could determine low and high level of biofilm formation. Image analysis was used with high performance for this method. As conclusion, the utilization of different hydrophobic membranes and dyes would lead to the development of new techniques for the application in other lipophilic yeasts.

Influenza virus-induced autophagy is often accompanied by apoptosis and results in cell death in virus-infected cells. It is well known that autophagy is modulated by the mTOR/PI3K/Akt pathway, which plays an important role in the response to the presence of energy sources and external stimuli. This pathway is modulated by mucin 1 (MUC1), which has extracellular and intracellular components and plays an important role in metastasis and chemotherapeutic resistance. In this study, it was aimed to investigate the expression of MUC1 after the inoculation of influenza viruses into the cancer-derived cell cultures and, accordingly, the changes in autophagy markers such as mTOR and LC3B. In this study, MCF-7, HeLa and A-549 cell lines were used which have adenocarcinoma origin. To control the growth of influenza virus in these cells, the MDCK cell line was also inoculated. Centrifuge-enhanced shell-vial cell culture method was used in all experiments. Influenza A (H1N1) pdm09 strain was inoculated into these cell lines then the expressions of viral nucleic acid and cycle threshold (Ct) of MUC1, mTOR, LC3B associated genes were investigated by quantitative real-time reverse transcriptase polymerase chain reaction (qRTPCR) method in the samples taken from the supernatants of all cells at the end of the 48-hour incubation period. To investigate whether these markers were present in cells, after all cells were permeabilized with paraformaldehyde, cell-coated infected coverslips were stained with fluorescent labeled monoclonal antibodies developed against MUC1, mTOR, LC3B and influenza virus antigens. In the examination of fluorescence microscopy, all of the cell cultures (MCF-7, He-La and A-549) infected with influenza virus yielded positive results in terms of LC3B, mTOR and MUC1 monoclonal antibody staining, whereas all of the non-infected cells were found negative. Cycle threshold values of MUC1, LC3B and mTOR associated genes were found to be lower in A-549 cell line inoculated with influenza virus. Although protein expression was demonstrated in MCF-7 and He-La cell lines, similar changes were not detected in the 1/Ct values of genes in the autophagy pathway. The Ct value of the MUC1 gene was found to be higher only in the MCF-7 cell line after inoculation. In conclusion, it was observed that the specific expression pattern for influenza virus-induced autophagy was formed only in the A-549 cell line among the adenocarcinoma cells. It was thought that this relationship could constitute a dataset in further research on lung adenocarcinoma. However, in future studies, the determination of the expression of these genes at the protein level by using further tests will provide better comparison of the results.