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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 ​< ​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 ​= ​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., (Copyright © 2023 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

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 qRt‑PCR 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.

Tuberculosis (TB) remains the leading cause of deaths from infectious disease worldwide. Nowadays, the tendency of Mycobacterium tuberculosis complex (MTBC) to spread between continents due to uncontrolled migration movements shows that TB is a global health problem. The number of studies for the detection of MTBC strains' epidemiological features in areas with TB spread risk using molecular-based methods such as spoligotyping and Mycobacterial Interspersed Repetitive Unit (MIRU) Variable Number Tandem Repeats (VNTR) at the clonal level is insufficient. In this study, it was aimed to determine the phylogenetic relationships of MTBC strains at the species level by spoligotyping and 15 locus MIRU-VNTR (MIRU-VNTR 15 ) molecular methods of 96 multidrug-resistant (MDR) MTBC strains isolated from sputum samples of patients with a preliminary diagnosis of pulmonary TB or suspected contact history those sent to National Tuberculosis Reference Laboratory from the centers that are members of the Tuberculosis Laboratory Surveillance Network. The phylogenetic relationship between 96 MDR-TB strains was investigated with the combination of bead-based spoligotyping and MIRU-VNTR 15 methods on the MAGPIX® Milliplex Map device. In this study, it was determined that the T1 family is more common in our country and LAM7-TUR family is less common than the Beijing family unlike other studies. It was determined that the strains in the same cluster had different locus profiles, and there was no transmission from the same clone in the clonal typing we performed with spoligotyping and MIRU-VNTR 15 .

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.

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.

Rapid identification of resistant Mycobacterium tuberculosis complex isolates is quite important for the establishment of early and appropriate therapy. The Genotype MTBDR (Hain Lifescience, Nehren, Germany) is a commercially available DNA strip assay designed for the rapid detection of rpoB and katG gene mutations in clinical isolates. This study was conducted to determine the mutation types of phenotypically drug resistant 26 M. tuberculosis complex clinical isolates [15 isoniazid (INH), 1 rifampin (RMP) and 10 INH and RMP resistant] by Genotype MTBDR (G-MTBDR) DNA strip assay and to compare the diagnostic performance of this test. Sixteen of 25 (64%) INH-resistant and 9 of 11 (81.8%) RMP-resistant clinical isolates were correctly identified with the presence of hybridization in mutation probe or lack of hybridization at least by one of the wild type probes, by G-MTBDR assay. Hybridization with mutation probes was detected in only 5 of the RMP resistant isolates. We observed rpoB MUT3 (S531L, Ser-->4Leu) mutation in 4 and rpoB MUT1 (D516V) in one of these isolates. In 56% (14/25) of the INH-resistant isolates, katG T1 (S315T1) hybridization pattern was observed at katG mutation probe. G-MTBDR assay couldn't identify two of the 11 (18.2%) RMP-resistant isolates and one of these iSolates was shown to have a mutation at codon 531 (TCG-GCG) and the other at codon 545 (CTG-->ATG), possibly not associated with resistance, by sequence analysis. In four of the eight (8/25; 32%) INH-resistant isolates not identified by G-MTBDR assay, DNA cycle sequencing revealed different nucleotide changes outside the most common mutation zone. One of these were at codon 293 (GCT-->ACT) in katG, one with dual mutation at 279 (GGC-->ACC) in katG and at 15th C-->T in inhA gene, one at 15th C-->T in inhA gene and one at 279 (GGC-->ACC) in katG gene region. DNA membrane strip assay can be a use ful tool for the rapid detection of resistant M. tuberculosis complex isolates and therefore accelerate th initiation of the appropriate treatment. However, due to its restrictions in the determination of relatively rare mutations, this rapid screening test should be used together with conventional susceptibility tests in routine laboratory practices.

Streptomycin (SM) displays antimicrobial activity via inhibition of protein synthesis in bacteria and it is one of the major antituberculous drugs used in chemotherapy of tuberculosis (TB). The aim of this study was to investigate the presence of mutations in rpsL and rrs gene regions responsible for SM resistance using DNA sequencing system in SM resistant Mycobacterium tuberculosis complex (MTC) isolates detected by a phenotypic drug susceptibility test. Ten SM resistant and 10 SM sensitive MTC strains isolated between 2003-2007, in Mersin University Medical Faculty Department of Medical Microbiology Laboratory, were included to this study. The identification of the strains and their susceptibility to primary antituberculous drugs were performed by BACTEC 460 TB system (Becton Dickinson, USA). The SM susceptibility was retested by agar proportion method at low (2 microg/ml) and high (10 microg/ml) concentrations of SM. For genotypic analysis, isolated DNA samples were amplified with polymerase chain reaction (PCR) using primers specific for rpsL and rrs gene regions associated with SM resistance. PCR products were analyzed for the presence of nucleotide changes by silver stained DNA sequence analysis (Silver Sequence DNA Sequencing System, Promega) and the data were compared to sequences encoded as L08011 for rpsL gene region and X52917 for rrs gene region in GeneBank database. Of the 10 SM resistant isolates 8 were resistant to SM at both low and high drug concentrations (2 microg/ml and 10 microg/ml) while two were resistant to SM at low drug concentration (2 microg/ml) using agar proportion method. A point mutation as AAG-->AGG (Lys-->Arg) was detected at codon 43 of rpsL gene region in eight isolates resistant to SM at both low and high drug concentrations. Nucleotide changes associated with mutation were not detected in 10 sensitive isolates and two low drug concentration resistant isolates. It was concluded that molecular analysis of MTC isolates might aid to the success of TB treatment and larger scale nationwide studies should be conducted to enlighten the molecular basis of drug resistance in MTC isolates.

Genitourinary tuberculosis presents a challenge in diagnosis and treatment due to variations in clinical and radiological signs, insufficient patient history and difficulty in the isolation of the bacilli. The aim of this study was to isolate and identify Mycobacterium tuberculosis from the urine samples obtained from patients with suspected urinary tuberculosis admitted to our hospital by using Ehrlich-Ziehl-Neelsen (EZN), culture and polymerase chain reaction-restriction analysis (PCR-RFLP) methods. A total of 1004 urine samples collected from 437 patients who were admitted to our hospital between January 2004-July 2006, were inoculated on Löwenstein-Jensen (LJ) and/or BACTEC 12B (Becton Dickinson, USA) after decontamination and, direct preparations stained with EZN method were evaluated microscopically. M. tuberculosis complex (MTC) and mycobacteria other than tuberculosis (MOTT) were differentiated by nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) test and the susceptibility testing for the MTC strains to primary antituberculosis drugs were performed by BACTEC 460 TB (Becton Dickinson, USA) system. PCR-RFLP method was performed for the identification of Mycobacterium spp. Twenty-two (5%) patients have yielded positive results by at least one of the conventional methods (EZN, LJ and/or BACTEC). Fifteen samples were positive for acido-resistant bacilli (ARB) by EZN method, and 17 samples were positive for mycobacterial growth in the cultures. Ten of 22 patients were found positive by both of the methods, while seven were culture positive but ARB negative and five were culture negative but ARB positive. These five patients received BCG treatment because of the presence of bladder tumor. Twelve (70.5%) of 17 strains isolated from culture were identified as MTC, while five (29.4%) were identified as M. fortuitum. Of 12 MTC isolates, eight (66.7%) were found susceptible to all of the antituberculosis agents, while one was found resistant to isoniazide (INH) and ethambutole (ETB), one was resistant to INH and rifampicin (RIF), and two were resistant to only INH. It is concluded that, in order to identify mycobacteria and to perform antituberculous susceptibility tests, cultivation of mycobacteria is a prerequisite.

Tuberculosis (TB) poses significant health challenges globally, contributing significantly to illness and mortality. Approximately one-fourth of the world's population is believed to carry the tuberculosis bacterium, with a 5-10% lifetime risk of developing active TB disease. Timely identification of TB and rapid detection of drug resistance are crucial in mitigating its global impact. Clinical, radiological, bacteriological, and molecular methods are used to screen and diagnose TB. Microscopy, culture, and immunological methods are commonly employed. Although microscopy has been used for approximately a century, it has limitations, necessitating its use and evaluation alongside other diagnostic techniques for a complete and accurate diagnosis. Bacterial culture in solid and liquid media is still the gold standard recommended by the World Health Organization (WHO) for the diagnosis of TB, as it enables the isolation of Mycobacterium tuberculosis (MTB) as well as the detection of drug resistance. In recent years, promising modern solutions have emerged that aim to overcome the limitations of culture and microscopic examination methods in molecular diagnostic testing. In routine laboratories in resource-wealthy countries, methods such as Mycobacterium Tuberculosis Drug Resistance Plus (MTBDRplus), loop-mediated isothermal amplification (LAMP), line probe assay (LPA), GeneXpert, and whole genome sequencing are increasingly being used to diagnose and characterize TB and determine anti-TB drug susceptibilities. Our goal is to explore and assess the evolution, advancements, and future prospects in TB diagnosis, focusing particularly on the current laboratory methods used for diagnosing the disease. [ABSTRACT FROM AUTHOR]

Mycobacterium tuberculosis (M. tuberculosis), which is the etiological agent of tuberculosis (TB), has become one of the most successful pathogens in human history with its ability to cause active/latent infection even in the face of a perfectly functioning immune system, thanks to its mutual evolution with the host cell over thousands of years. While one-fourth of the world’s population are estimated to be infected by M. tuberculosis, only 5-15% of this population fall ill with active TB disease throughout life. The main reason for this situation is the effective mechanisms of host immunity. After the TB bacillus is taken into the body by inhalation, it first encounters the natural immune cells. Acquired immunity develops against bacilli that cannot be eliminated by natural immunity. M. tuberculosis has developed quite a variety of strategies to evade the immune system. These strategies include escape from pathogen recognition receptors, inhibition of phagolysosome formation, inhibition of apoptosis and autophagy, immune escape with granuloma formation, inhibition of oxidative stress, and reactive oxygen and reactive nitrogen intermediates. Unraveling the mechanisms by which M. tuberculosis alters or modulates host immune function is probably of great importance for the development of more effective new vaccines and immunotherapy against TB. In this review, we aim to reveal new developments about the host immune response against M. tuberculosis and the escape mechanisms of bacillus from this response. [ABSTRACT FROM AUTHOR]

Objective: All resources of the health system have been allocated to the pandemic to combat the coronavirus disease (COVID-19) epidemic that has affected the world. In addition, measures implemented during the pandemic, such as isolation, lockdown and restricted mobility, adversely affected tuberculosis (TB) control programs in our country and around the world. In this study, it was aimed to investigate the effect of the COVID-19 pandemic on TB patients and TB control program in Mersin province. Methods: In this study, a total 6328 samples of 3731 patients with a preliminary diagnosis of TB, from Mersin University Hospital and Mersin tuberculosis dispensaries, between January 2019 and December 2020, were included. In the study, how much the number of samples decreased during the 2020 pandemic period, the number of patients diagnosed with TB, the change in the age distribution of the patients, and the number of control samples from the treatment follow-up of TB patients were examined. Results: In the study, a decrease of 42.2% in the number of patients and a decrease of 45.4% in the number of samples were observed during the pandemic period of 2020.The highest decrease in the number of patients by months were observed in March, April and May (respectively 44.2%, 67.2% and 69.8%) when the pandemic started in our country and the restrictions were applied intensively. During the pandemic period, a decrease of 28.2% was observed in the number of TB positive patients. While the mean age of TB positive patients was 53.5±16.2 in 2019, it decreased to 40.3±19.6 years in 2020, and the decrease in the mean age was statistically significant (p <0.001). When the age groups between years are compared; While the rate of patients over 65 years of age was higher in 2019, the rate of patients under the age of 18 was found to be higher in 2020 (p=0.003). In the study, control samples came during treatment follow-up from 73% of TB patients in 2019 and 52.9% in 2020. The rate of decrease in the number of patients with control samples from TB positive patients during the pandemic period was found to be statistically significant (p=0.021). Conclusion: The findings of our study revealed that the restrictions applied during the pandemic period adversely affected the diagnosis and follow-up of TB in our region. In addition, the decrease in the average age of TB patients and the tendency towards younger age and the increase in the number of positive patients under the age of 18 suggest that intra-familial transmission increased in this period. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is an infectious disease that has deeply affected societies throughout human history due to the serious effects of Mycobacterium tuberculosis complex. The Bacille Calmette-Guérin (BCG) vaccine, which has a historical importance in the prevention of TB, has been administered to more than 4 billion people with its 100-year history. While the studies to develop a more effective vaccine than BCG in the prevention of TB continue, BCG has surprisingly came to the fore again on its 100th birthday with the studies on the protection of BCG against the coronavirus disease pandemic. In addition to the non-specific/non-TB effect of BCG on infectious diseases, it has been used as a ‘‘Food and Drug Administration’’ approved immunomodulator in the treatment of bladder cancer in humans since 1976. In addition, studies on its use as an immunotherapy agent in topical treatment for common genital warts are ongoing. Although the fact that the BCG vaccine reduces the risk of TB infection by approximately 50% is actually a limited efficacy, the non-specific effects of heterologous and trained immunity in protection against other infections keep the interest in the vaccine alive. This study was planned to compile the discovery and development history of BCG vaccine, its history and its effect on immunological mechanisms in non-TB infectious diseases. [ABSTRACT FROM AUTHOR]

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