gyrB Gene Mutations Research Articles

Molecular analysis of bedaquiline resistance and genetic variations in clinical isolates of multidrug and fluoroquinolones-resistant Mycobacterium tuberculosis

BackgroundMycobacterium tuberculosis developing resistance to anti-tubercular therapy leads to pioneering innovative treatment protocols featuring carefully curated combinations of potent medications. The aim of this study was to analyze the frequency and pattern of fluoroquinolone (FQ) resistant mutations among the first-line drug-resistant specimens. By employing an elective sampling strategy, we deliberately focused our investigation on a targeted subset of multidrug resistant (MDR) and FQ resistant (FQR) TB cases. MethodSputum samples from first-line drug-resistant patients were subjected to a GenoType MTBDRsl VER 2.0 assay to determine FQR. To investigate the effect of bedaquiline (BDQ) on these mutation-conferring genes (Rv0678, pepQ, and atpE), sanger sequencing was carried out on the MDR + FQR subset that was obtained. We have also conducted a computational analysis of the gene products of Rv0678 and pepQ with BDQ using molecular docking methods. ResultsEighty-three samples were observed to have FQR, and the majority of these specimens were associated with naïve and treatment failure cases. Among them, 64 isolates exhibited mutations in gyrA gene, while 19 isolates showed mutations in gyrB gene. The predominant occurrence of genetic variations, particularly in D94G and A90V, was observed within the specimens displaying resistance in gyrA gene, and the same has been confirmed by PCR-based detection. And the sequence analysis of BDQ genes among MDR + FQR subset indicated that the majority of the samples did not exhibit mutations in pepQ and atpE genes. However, only 2 samples were found to have the genetic variant G337A, which involves replacing the amino acid glutamic acid (E) at codon position 113 of Rv0678 gene with lysine (K). ConclusionsMost of the mutations were harbored in rpoB + katG + gyrA gene pattern. These findings pave the way for the development of databases and rapid diagnostic probes.

Characterization of Genetic Mutations in Multi-Drug-Resistant Isolates of Mycobacterium tuberculosis Bacilli Conferring Resistance to a Second-Line Anti-tuberculosis Drug.

Multi-drug-resistant tuberculosis (MDR-TB) has become a major public health concern globally. Mutations in first- and second-line drug targets such as katG, inhA, rpoB, rrs, eis, gyrA, and gyrB have been associated with drug resistance. Monitoring predominant mutations in the MDR-TB patient population is essential to monitor and devise future therapeutic regimes. The present study is aimed to characterize genetic mutations in MDR isolates of Mycobacterium tuberculosis (MTB)bacilli conferring resistance to a second-line anti-tuberculosis drug in the Eastern Indian population. This cross-sectional study was conducted in the Department of Microbiology, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, and in the Tuberculosis Demonstration & Training Centre, Agamkuan, Patna. A total of 3270 patients suspected to have MDR-TB were recruited in the study. Two sputum samples, one on the spot, and the other in the morning were collected from each patient and the diagnosis ofrifampicin-sensitive (RS)/rifampicin-resistant (RR/MDR) TB was done by Gene-Xpert test. One hundred fifty RS-TB samples and 150 RR/MDR-TBsamples were considered for line probe assay (LPA). RSsamples were subjected to first-line LPA using Genotype® MTBDR Plus ver 2.0 and RR/MDRsamples were considered for second-line LPA using Genotype®MTBDRslver 2.0. All sputum samples were subjected to sputum smear microscopy using the Ziehl-Neelsen staining method.Statistical analysis was done using Statistical Package for Social Sciences (SPSS) version 26.0 (IBM Corp. Armonk, NY) and R (version 4.1; R Core Team 2021). In the present study, out of 3270 patients, we detected RR/MDR-TB in 235 patients (7.19%),RS-TB in 812 patients (24.83%), the rest of the patients negative for MTB (2223, 67.98%). Out of 150 RR/MDR-TB sputum samples tested, resistance to fluoroquinolone (FQ) was observed in 41 samples. The selected patients had predominantly FQ resistance due to the gyrA gene mutations (97.56%, n=40) compared to the gyrB gene mutations (2.44%, n=1). We observed >60% of the mutations in the gyrA gene in codon 94 (MUT3C (D94G), MUT3A (D94A), and MUT3D (D94H).In addition, we found the mutations MUT1 (A90V) and MUT2 (S91P)in the codons 90 and 91 of the gyrA gene in the considered MTB patient population. The identified genes can be further validated to be considered as therapeutic targets, but more therapeutics and advanced strategies should be applied in the management of MTB.

Correlating clinical breakpoint concentration of moxifloxacin with gyrA mutations using the GenoType MTBDRsl assay Version 2.0

IntroductionWidespread use of Fluoroquinolones (FQs) has led to the development of its resistance in clinical isolates of Mycobacterium tuberculosis. However, in Mycobacterium tuberculosis, phenotypic resistance to FQs has been shown to be heterogeneous, ranging from low-level resistance to high-level resistance. This stratification in resistance has important implications for the inclusion of moxifloxacin (Mfx) in the treatment regimen. The World Health Organization recommends the use of GenoType MTBDRsl assay as the initial test for detecting resistance conferring mutations (both high and low) to FQs in patients with confirmed MDR-RR TB. The present study was conducted to explore the relationship of MTBDRsl Version 2.0 detected mutations in gyrA gene and genotypic DST of Mfx at WHO defined Clinical Breakpoint (CB). Materials and methodsA total of 200 sputum samples from Confirmed MDR/RR TB patients were included in this study. All of these samples had mutations conferring resistance to FQ confirmed by GenoType MTBDRsl assay. These samples were further subjected to Phenotypic DST against moxifloxacin using the Bactec MGIT-960 system. ResultsAll of the 200 representative FQ resistant isolates had mutations in gyrA gene only with no detectable mutation in gyrB gene. 109 (54.5%) of the isolates had mutations associated with high-level increase in MIC while 91 (45.5%) isolates had mutations associated with low-level increase in MIC. Phenotypic DST of these 200 isolates against Mfx at CB (1.0μg/ml) revealed that of the 109 isolates with mutations associated with high-level increase in MIC and expected to be resistant at CB, only 34 (31.2%) were resistant and the remaining 75 (68.8%) were sensitive. ConclusionMoxifloxacin is an important drug in the regimen for treating Drug-resistant TB and the decision to exclude this drug from the regimen should not be taken merely on the basis of mutational patterns. It should rather be taken after considering the combined results of mutational analysis and phenotypic DST.

Genotyping and Molecular Characterization of Fluoroquinolone's Resistance Among Multidrug-Resistant Mycobacterium tuberculosis in Southwest of China.

Although fluoroquinolones (FQs) are the backbone drugs for the treatment of multidrug-resistant tuberculosis (MDR-TB), the knowledge about the resistance pattern and molecular characterization of new-generation FQs in Chongqing is limited. This study aimed to investigate the resistance rate and mutation types of later-generation FQs against MDR-TB in Chongqing, and further to explore the relationship between different genotypes and phenotypes. A total of 967 clinical strains were characterized using multilocus sequence typing and drug susceptibility testing, followed by analysis of genotype/phenotype association. The 229 (23.7%, 229/967) isolates were identified as MDR-TB. The most effective agent against MDR-TB was gatifloxacin (GFX) (20.1%, 46/229), and the highest resistant rate was observed in ofloxacin (OFX) (41.0%, 94/229). Of the 190 strains (83.0%) identified as Beijing genotype, 111 isolates were modern Beijing genotype (58.4%) and 79 isolates were ancient Beijing genotype (41.6%). By analyzing 94 OFX-resistant isolates, 13 isolates were clustered with the cumulative clustering rate of 13.8% (13/94). Of the 91 isolates (39.7%, 91/229) with a mutation in gyrA gene, mutation in codon 94 was the most prevalent. Only 15 isolates (6.6%, 15/229) harbored a mutation in gyrB gene. There was no significant difference in the mutation rate of gyrA gene between Beijing and non-Beijing genotype, clustered isolates, and nonclustered isolates (p > 0.05).

DESAIN IN SILICO DNA PROBE PENDETEKSI MUTASI DAERAH RESISTENSI QUINOLON Gen gyrA DAN gyrB Mycobacterium tuberculosis

Fluoroquinolone (FQ) is the main drug used in MDR-TB therapy resistance to FQ can cause death and increase the risk of treatment failure in MDR-TB patients. Mutations in gyrA gene and gyrB gene from Mycobacterium tuberculosis are responsible for the occurrence of FQ resistance. The highest mutation of gyrA gene in QRDR was found in codon 94, while mutations in gyrB gene was found in codon 500. M. Tuberculosis which resistant to FQ can be detected using the Real Time Polymerase Chain Reaction (RT-PCR) method with DNA probe. 
 This study will design the nucleotide sequence of the TaqMan type probe using the Clone Manager Suite 9.2 program. The results of the DNA probe design were then analyzed in two stages, which is based on the probe criteria in general and based on the TaqMan probe labeling criteria. The design of the mutant probe DNA using the program produced 1 probe for Asp94Ala specific mutations in the gyrA gene and 33 probes for Asp500Ala specific mutations in the gyrB gene. After being analyzed by the two criteria, it was obtained the A94MA1 probe with the 5 '-TCGATCTACGCCAGCCTGGT-3' sequence and B500MA12 probe with the order of 5 '-TACCACAAGCTCGTGCTGATGGC-3'. The results of these probes meet both criteria and can be used to detect mutations in codon 94 gyrA genes and codons 500 gyrB genes of Mycobacterium tuberculosis.

Mutational analysis of gyrB at amino acids: G481A & D505A in multidrug resistant (MDR) tuberculosis patients

BackgroundThe MDR (multidrug resistance) tuberculosis is a serious public health concern. Fluoroquinolones are in use to treat tuberculosis, but M. tuberculosis strains have now become resistant due to several mutations in different genes. We evaluated mutations in gyrB gene at amino acid positions G481A and D505A of M. tuberculosis by semi-multiplex allele specific (MAS) PCR. MethodsThe information on gender, age, type of tuberculosis (TB), positive/negative for MDR-TB and HIV infection was gathered. The genomic DNA isolation from sputum culture samples (n=53) was carried out by non-column based method. The gyrB mutations were investigated by using self-designed primers in semi MAS-PCR, at mentioned amino acid positions. ResultsThere were 38% male patients and 62% were female patients. Most of MDR-TB patients (58.5%) were in the age between 16–30years. There were 90.5% cases of pulmonary TB and 9.4% cases of extra pulmonary TB. Only 1.8% patients were co-infected with HIV. The 24 samples had mutation in gyrB gene out of 53 (45.28%), on both of positions of amino acids Gly481Ala and Asp505Ala. All samples had mutations at Gly481Ala, whereas, 24 samples (45.28%) had mutations at Asp505Ala. ConclusionMutations at amino acids positions 481 and 505 were involved in MDR-TB, which could further develop into an extensively-drug resistance (XDR) TB. Therefore, there is a need to explore all mutations in gyrB gene in MDR-TB, because it can result in a Fluoroquinolones resistance.

Single-nucleotide polymorphisms related to fluoroquinolone and aminoglycoside resistance in Mycobacterium avium isolates.

ObjectiveThe relationships between fluoroquinolone and aminoglycoside resistance and single-nucleotide polymorphisms (SNPs) in gyrA, gyrB, and rpsL genes were investigated in 95 clinical isolates of Mycobacterium avium from China.MethodsFluoroquinolone and aminoglycoside resistance were determined by the broth microdilution method. GyrA, gyrB, and rpsL were sequenced, SNPs were identified, and the corresponding amino acid mutations were recorded.ResultsThe M. avium isolates displayed high levels of ofloxacin (93.68%), ciprofloxacin (92.63%), and streptomycin (65.26%) resistance. Moxifloxacin (18.95%) and amikacin (2.11%) were highly active against the strains. Fluoroquinolone resistance involving gyrA and gyrB gene mutations was identified. For gyrA, the most frequent SNPs were T→C (71/95, 74.74%), followed by A→G (64/95, 67.37%) and T→C (62/95, 65.26%). The amino acid mutations occurred mainly at Gly2444Asp (GGT→GAT) (20/95, 21.05%), Ala2445Ser (GCC→TCC) (20/95, 21.05%), Ala2447Val (GCC→GTC) (20/95, 21.05%), Val2449Ile (GTC→ATC) (20/95, 21.05%), and Glu2450Gln (GAA→CAA) (20/95, 21.05%). Prominent SNPs in gyrB included A→C (69/95, 72.63%), C→T (51/95, 53.68%), and T→G (29/95, 30.53%), and their amino acid substitutions were Ile2160Val (ATT→GTT) (21/95, 22.11%), Ile2160Met (ATT→ATG) (20/95, 21.05%), and Ile2273Leu (ATC→CTC) (11/95, 11.58%). Among the strains with aminoglycoside resistance, SNPs in rpsL were identified mostly at position G→A (73/95, 76.84%). G→C (21/95, 22.11%) was commonly seen. The amino acid mutations primarily involved Ala1539985Thr (GCC→ACC) (19/95, 20.00%), His1539992Asp (CAC→GAC) (19/95, 20.00%), and Gln-1539983Glu (CAG→GAG) (18/95, 18.95%).ConclusionOur study provides valuable information that could be used for the future diagnosis and treatment of M. avium disease.

Characterization of non-classical quinolone resistance in Salmonella enterica serovar Typhi: Report of a novel mutation in gyrB gene and diagnostic challenges

ObjectiveTo establish the relative importance of Salmonella enterica serovar Typhi with non-classical quinolone resistance. MethodsEight hundred and ninety-one isolates of S. Typhi, isolated between 2004 and 2011, were tested for antibiotic susceptibility determination using disc diffusion and E-test. The mechanisms of fluoroquinolone resistance were studied in a sub-set of the NALS (nalidixic acid susceptible) isolates by wave nucleic acid fragment analysis of PCR products from gyrA, gyrB, parC and parE and from the plasmid borne determinants: qnrA,B,S; aac(6′)-Ib-cr and qepA. To assess genetic relatedness multi-locus variable number tandem repeat analysis was carried out using five loci. ResultsEighty isolates with a nalidixic acid MIC of <32mg/L (NALS) and a ciprofloxacin MIC of >0.064mg/L CIPI (ciprofloxacin reduced susceptibility) were found. In 36 NALS CIPI isolates two distinct genotypes were identified when compared with 16 susceptible controls: Group B (n=34), mutation in gyrB at codon 464, NAL MIC of 3–12mg/L and CIP MIC of 0.064–0.5mg/L.; and Group C, mutation in gyrA at codon 83 (n=2) NAL MIC of 16mg/L and CIP MIC of 0.25–0.38mg/L. Group B isolates were found in different strain backgrounds as defined by MLVA. ConclusionThe use of nalidixic acid to screen for reduced susceptibility to fluoroquinolones in S. Typhi misses CIPI-NALS isolates, an established phenotype in India.

Characterization of multiple and extensively drug resistant Mycobacterium tuberculosis isolates with different ofloxacin-resistance levels

The goal of the study was to determine different mutation types in gyrA and gyrB genes in Mycobacterium tuberculosis strains with low-level (2 μg/ml) and high-level (10 μg/ml) ofloxacin (OFL) resistance and to compare genetic diversity of ofloxacin-resistant and susceptible M. tuberculosis isolates. M. tuberculosis isolates were collected in Leningrad Region in 2011. DNA sequencing showed that 54.3% of isolates with low-level and 76.9% of isolates with high-level OFL-resistance had mutations in gyrA gene. Few isolates carried mutations in gyrB gene - five among isolates with low-level resistance and two among high-level resistant isolates. Altogether, detection of point mutations in both DNA gyrase genes allows to identify 66.9% of mycobacterial isolates with low-level and 84.5% of isolates with high-level of OFL-resistance. Novel mutations S91L in gyrA gene and S512L in gyrB gene described in this study were detected in OFL-resistant isolates and may play role in M. tuberculosis fluoroquinolone resistance. M. tuberculosis Beijing family spoligotypes were identified among 70.8% of isolates with low-level resistance, 84.6% of isolates with high-level resistance and 50% of strains susceptible to all tuberculosis drugs. Fisher's exact test revealed significant difference between Beijing prevalence in groups of drug-susceptible or high-level OFL-resistant M. tuberculosis strains (p-value = 0.032).

GyrAandgyrBGene Mutation in Ciprofloxacin-ResistantMycobacterium massilienseClinical Isolates from Southern Brazil

Fluoroquinolones (FQs) have been increasingly used for effective treatment of infections caused by rapidly growing mycobacteria, and resistance to this drug has been predominantly attributed to gyrA and gyrB mutations. Accordingly, this study investigated a total of 36 Mycobacterium massiliense clinical isolates for their susceptibility to ciprofloxacin and presence of gyrA and gyrB gene mutations. The minimal inhibitory concentration (MIC) values, determined by broth microdilution method, of 35 ciprofloxacin-resistant isolates ranged between 4 and 16 μg/mL and a single susceptible isolate was obtained. A total of 31 of 35 (88.5%) ciprofloxacin-resistant isolates presented an amino acid substitution at codon 90 (Ala-90→Val) and no isolate presented mutation at position Asp-94. Moreover, 4 of 35 (11.4%) ciprofloxacin-resistant and one susceptible isolate had no mutation in Ala-90 and Asp-94. No gyrB mutation was observed in all tested M. massiliense isolates. In conclusion, our results have shown that mutations of gyrA codon 90 are frequent and may constitute an important mechanism of resistance to FQ in M. massiliense.

Detection of gyrA and gyrB mutations in quinolone-resistant clinical isolates of Escherichia coli by single-strand conformational polymorphism analysis and determination of levels of resistance conferred by two different single gyrA mutations.

Twelve quinolone-resistant clinical isolates of Escherichia coli (nalidixic acid MICs, 64 to 512 micrograms/ml; norfloxacin MICs, 0.25 to 8 micrograms/ml) were transformed with plasmid pJSW101 carrying the gyrA+ gene and with plasmid pJB11 carrying the gyrB+ gene to examine the proportion of gyrA and gyrB mutations. Transformation with pJSW101 resulted in complementation (nalidixic acid MICs, 4 to 32 micrograms/ml; norfloxacin MICs, 0.06 to 0.25 micrograms/ml). In contrast, no change in MICs were observed after transformation with pJB11. A 418-bp fragment of gyrA from the 12 strains was amplified by PCR. Direct DNA sequencing of that fragment identified the causes of quinolone resistance in eight strains as a single point mutation leading to a substitution of the serine at position 83 (Ser-83) to Leu and in four strains as a single point mutation leading to a substitution of Asp-87 to Gly. Exchange of the fragment from one of these strains with that of gyrA+ and transformation of resistance with the hybrid gyrA plasmid indicated the contribution of Gly-87 to resistance and the stabilities of mutants containing GyrA (Gly-87). Thus, gyrA gene mutations are probably encountered more often than gyrB gene mutations in clinical isolates of E. coli. In addition, the substitution of Asp-87 to Gly can be encountered in such strains. On the basis of the level of resistance found in the fragment exchange experiment, the quinolone resistance attributable to Gly-87 appears to be comparable to that attributable to Leu-83. The levels of resistance found in the clinical isolates shown to have a Gly-87 mutation (nalidixic acid MICs, 64 to 512 micrograms/ml; norfloxacin MICs, 0.5 to 4 micrograms/ml) suggest that the Gly-87 mutation causes resistance at the level of the nalidixic acid MIC (64 micrograms/ml) or the norfloxacin MIC (0.5 micrograms/ml or less) and that the additional increments in resistance seen in the other strains with higher levels of resistance may be attributable to additional mutations. The single-strand conformational polymorphism analysis with PCR products readily detected te Leu-83 and Gly-87 mutations.