[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.

[Longitudinal Monitoring of Seroconversion Status in SARS-CoV-2 RT-PCR Positive Healthcare Workers]. / SARS-CoV-2 RT-PCR Pozitif Saglik Çalisanlarinda Serokonversiyon Durumunun Boylamsal Izlenmesi.

The impact of the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome 2 (SARS-CoV-2) still continues. The duration of the immune response in individuals recovering from COVID-19 and its protection against future SARS-CoV-2 infection are not fully understood. This study aimed to longitudinally evaluate anti-SARS-CoV-2 seroconversion status in healthcare workers with positive SARS-CoV-2 Real-time reverse transcription polymerase chain reaction (rRT-PCR), test in Mersin University Hospital. A total of 68 healthcare workers with positive SARS-CoV-2 rRT-PCR test between 19 April and 27 November 2020 were included in the study. Blood samples were collected from healthcare workers for SARS-CoV-2 antibody testing in the 1st, 3rd and 5th months following PCR positivity. Healthcare workers were classified as symptomatic, asymptomatic and reinfected according to their clinical findings, and rRT-PCR cycle thresholds (Ct) were recorded. Elecsys Anti-SARS-CoV-2 (Roche Diagnostics, Germany) kit was used for antibody testing. Of the 68 healthcare workers; 46 were classified as symptomatic, 15 as asymptomatic, and seven as reinfected. Twenty-seven (39.7%) of the healthcare workers were male and 41 (60.3%) were female, and the mean age was 36.4 ± 9.04. Seroconversion was detected in 45 (66.2%) of 68 healthcare workers in the study, and only one person had sero-negative result at the end of the 5th month. While seroconversion was detected in 78.3% (n= 36/46) of symptomatic healthcare workers, it was observed in 26.7% (n= 4/15) of the asymptomatic healthcare workers. Seroconversion was detected in only one of the seven reinfected healthcare workers after primary infection. After reinfection, seroconversion was observed in five of seven reinfected healthcare workers. Antibody response was not detected in two of them after both infections. According to the rRT-PCR Ct values; the median of Ct value was found significantly lower in healthcare workers with seroconversion (23.26, IQR= 18.45-27.30), than the ones without seroconversion (36.20, IQR= 33.09-37.56) (p< 0.001). In those who had reinfection, the mean Ct value (31.77 ± 6.62) detected during the primary infection period was statistically higher than the Ct value (22.44 ± 5.54) detected during reinfection (p= 0.008). The most frequently recorded symptoms in healthcare workers were myalgia (57.3%), fatigue (51.5%), headache (51.5%) followed by sore throat (36.7%), fever (33.8%), cough (27.9%), diarrhea (23.5%) and dyspnea (16.2%). In addition, fever (52%) and fatigue (80.6%) were found to be significantly higher in seroconversion-positive healthcare workers than in those without seroconversion (p= 0.028; p= 0.005, respectively). As a result, a higher rate of antibody response was detected in healthcare workers who had symptomatic infection than those who were asymptomatic. It has been observed that patients with asymptomatic primary infection and without antibody response were more susceptible to reinfection. In addition, it was observed that the probability of immune response increased when the viral load increased (Ct value decreased) in symptomatic infections. Although these findings provide important information about the short-term seroconversion status of healthcare personnel; longer-term and larger-scale studies are needed to evaluate the long-term effectiveness of seroconversion and to better understand the effectiveness of the immune response developed after SARS-CoV-2 vaccine administrations.

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.