Mutations In 23S Ribosomal RNA Research Articles

Exploring the Molecular Mechanisms of Macrolide Resistance in Laboratory Mutant Helicobacter pylori.

Resistance to clarithromycin, a macrolide antibiotic used in the first-line treatment of Helicobacter pylori infection, is the most important cause of treatment failure. Although most cases of clarithromycin resistance in H. pylori are associated with point mutations in 23S ribosomal RNA (rRNA), the relationships of other mutations with resistance remain unclear. We examined possible new macrolide resistance mechanisms in resistant strains using next-generation sequencing. Two resistant strains were obtained from clarithromycin-susceptible H. pylori following exposure to low clarithromycin concentrations using the agar dilution method. Sanger sequencing and whole-genome sequencing were performed to detect resistance-related mutations. Both strains carried the A2142G mutation in 23S rRNA. Candidate mutations (T1495A, T1494A, T1490A, T1476A, and G1472T) for clarithromycin resistance were detected in the Mutant-1 strain. Furthermore, a novel mutation in the gene encoding for the sulfite exporter TauE/SafE family protein was considered to be linked to clarithromycin resistance or cross-resistance, being identified as a target for further investigations. In the Mutant-2 strain, a novel mutation in the gene that encodes DUF874 family protein that can be considered as relevant with antibiotic resistance was detected. These mutations were revealed in the H. pylori genome for the first time, emphasizing their potential as targets for advanced studies.

Evaluation of clarithromycin and levofloxacin resistance of Helicobacter pylori strains isolated from patients at the Hospital for Tropical Diseases, Ho Chi Minh City

Clarithromycin (CLA) and levofloxacin (LFX) have been recommended as the most effective antibiotics for treating Helicobacter pylori infection. However, the increase in H. pylori’s resistance to antibiotics is an alarming and growing challenge. The study aimed to determine the antimicrobial resistance profiles of H. pylori strains isolated from patients at the Hospital for Tropical Diseases and to detect point mutations in 23S ribosomal RNA (23S rRNA) and gyraseA (gyrA) genes. Point mutations in 23S rRNA and gyrA were detected using Sanger sequencing. Antibiotic resistance was tested by the microdilution method. Out of the 45 isolates, 44 (97.8%) were found to be resistant to at least one antibiotic, 38 (84.4%) resistant to metronidazole (MTZ), and all were sensitive to amoxicillin (AMX) and tetracycline (TET). Sixty percent of the isolated strains were resistant to 3-6 antibiotics; among them, multiple drug resistance (MDR) strains presented twenty percent resistance to more than two classes of antibiotics. Eleven strains (24.4%) carried two mutations associated with CLA and LFX but only nine of them were dual resistant to CLA and LFX, and twenty-four (53.3%) carried either CLA or LFX mutation. The point mutations A2143G in the 23S rRNA and N87K (Asn87Lys) in the gyrA were detected in the CLA and LFX resistant strains, respectively. The prevalence of MDR, especially CLA, MTZ, and LFX resistance, in the H. pylori isolates suggests that the use of these antibiotics need to be more considerable and cautious.

Efficacies of Genotypic Resistance-Guided vs Empirical Therapy for Refractory Helicobacter pylori Infection

We aimed to compare the efficacy of genotypic resistance-guided therapy vs empirical therapy for eradication of refractory Helicobacter pylori infection in randomized controlled trials. We performed 2 multicenter, open-label trials of patients with H pylori infection (20 years or older) failed by 2 or more previous treatment regimens, from October 2012 through September 2017 in Taiwan. The patients were randomly assigned to groups given genotypic resistance-guided therapy for 14 days (n= 21 in trial 1, n= 205 in trial 2) or empirical therapy according to medication history for 14 days (n= 20 in trial 1, n= 205 in trial 2). Patients received sequential therapy containing esomeprazole and amoxicillin for the first 7 days, followed by esomeprazole and metronidazole, with levofloxacin, clarithromycin, or tetracycline (doxycycline in trial 1, tetracycline in trial 2) for another 7 days (all given twice daily) based on genotype markers of resistance determined from gastric biopsy specimens (group A) or empirical therapy according to medication history. Resistance-associated mutations in 23S ribosomal RNA or gyrase A were identified by polymerase chain reaction with direct sequencing. Eradication status was determined by 13C-urea breath test. The primary outcome was eradication rate. H pylori infection was eradicated in 17 of 21 (81%) patients receiving genotype resistance-guided therapy and 12 of 20 (60%) patients receiving empirical therapy (P= .181) in trial 1. This trial was terminated ahead of schedule due to the low rate of eradication in patients given doxycycline sequential therapy (15 of 26 [57.7%]). In trial 2, H pylori infection was eradicated in 160 of 205 (78%) patients receiving genotype resistance-guided therapy and 148 of 205 (72.2%) patients receiving empirical therapy (P= .170), according to intent to treat analysis. The frequencies of adverse effects and compliance did not differ significantly between groups. Properly designed empirical therapy, based on medication history, is an acceptable alternative to genotypic resistance-guided therapy for eradication of refractory H pylori infection after consideration of accessibility, cost, and patient preference. ClinicalTrials.gov ID: NCT01725906.

Decreased Susceptibility to Macrolide-Lincosamide in Mycoplasma synoviae Is Associated with Mutations in 23S Ribosomal RNA.

The mechanism responsible for acquired decreased susceptibility to macrolides (14-membered erythromycin [Ery], 16-membered tylosin [Ty] and tilmicosin [Tm]) and to lincosamides (lincomycin [Ln]) was investigated in Mycoplasma synoviae, a pathogen that causes respiratory infections and synovitis in chicken and turkey. Sequence analysis of domains II and V of the two 23S rRNA alleles and ribosomal proteins L4 and L22 was performed on 49 M. synoviae isolates, M. synoviae type strain WVU1853, and reference strain FMT showing minimal inhibitory concentrations (MICs) to Ty (≤ 0.015 to 2 μg/ml), Tm (0.03 to ≥ 8 μg/ml), and Ln (0.125 to 8 μg/ml); MICs to Ery ranged from 32 to ≥ 128 μg/ml. Our results showed that the nucleotide substitution G748A (Escherichia coli numbering) in domain II of one or both 23S rRNA alleles may account for a slight increase in MICs to Ty and Tm (up to 0.5 and 2 μg/ml, respectively). No correlation between the presence of G748A and decreased susceptibility to Ln was found. However, the presence of the point mutations A2058G or A2059G in domain V of one or both alleles of the 23S rRNAs was correlated with a more significant decrease in susceptibility to Ty (1-2 μg/ml), Tm (≥ 8 μg/ml), and Ln (≥ 8 μg/ml). All M. synoviae isolates tested had a G2057A transition in the 23S rRNAs consistent with previously described intrinsic resistance to Ery. Mutations G64E (one isolate) and Q90K/H (two isolates) were identified in the L4 and L22 proteins, respectively, but their impact on decreased susceptibility to macrolides and lincomycin was not clear.

Dissecting the Mechanisms of Linezolid Resistance in a Drosophila melanogaster Infection Model of Staphylococcus aureus

Mini-host models are simple experimental systems to study host-pathogen interactions. We adapted a Drosophila melanogaster infection model to evaluate the in vivo effect of different mechanisms of linezolid (LNZ) resistance in Staphylococcus aureus. Fly survival was evaluated after infection with LNZ-resistant S. aureus strains NRS119 (which has mutations in 23S ribosomal RNA [rRNA]), CM-05 and 004-737X (which carry cfr), LNZ-susceptible derivatives of CM-05 and 004-737X (which lack cfr), and ATCC 29213 (an LNZ-susceptible control). Flies were then fed food mixed with LNZ (concentration, 15-500 µg/mL). Results were compared to those in mouse peritonitis, using LNZ via oral gavage at 80 and 120 mg/kg every 12 hours. LNZ at 500 µg/mL in fly food protected against all strains, while concentrations of 15-250 µg/mL failed to protect against NRS119 (survival, 1.6%-20%). An in vivo effect of cfr was only detected at concentrations of 30 and 15 µg/mL. In the mouse peritonitis model, LNZ (at doses that mimic human pharmacokinetics) protected mice from challenge with the cfr+ 004-737X strain but was ineffective against the NRS119 strain, which carried 23S rRNA mutations. The fly model offers promising advantages to dissect the in vivo effect of LNZ resistance in S. aureus, and findings from this model appear to be concordant with those from the mouse peritonitis model.

Activity of telithromycin and seven other agents against 1034 pediatric Streptococcus pneumoniae isolates from ten central and eastern European centers

To test the activity of telithromycin against 1034 Streptococcus pneumoniae isolates from pediatric patients in ten centers from ten central and eastern European countries during 2000-2001, and to compare it with the activities of erythromycin A, azithromycin, clarithromycin, clindamycin, and quinupristin-dalfopristin. The minimum inhibitory concentrations (MICs) of telithromycin, erythromycin A, azithromycin, clarithromycin, clindamycin, levofloxacin, quinupristin-dalfopristin and penicillin G were tested by the agar dilution method with incubation in air, and mechanisms of resistance to macrolides and quinolones were investigated. Strains were isolated from sputum, tracheal aspirates, ear, eye, blood, and cerebrospinal fluid. Among S. pneumoniae strains tested, 36% had raised penicillin G MICs (>/= 0.12 mg/L). Susceptibilities were as follows: telithromycin, quinupristin-dalfopristin and levofloxacin, >/= 99%; clindamycin, 83%; and erythromycin A, azithromycin and clarithromycin, 78%. Of 230 (22.3%) erythromycin A-resistant S. pneumoniae strains, 176 (79.6%) had erm(B), 38 (16.1%) had mef(A), and 10 (4.3%) had mutations in 23S ribosomal RNA or in ribosomal protein L4. The rates of drug-resistant S. pneumoniae are high in all centers except Kaunas, Riga, and Prague. Telithromycin had low MICs against all strains, irrespective of macrolide, azalide or clindamycin resistance. Ribosomal methylation was the most prevalent resistance mechanism among all resistant strains, except in Sofia, where the prevalence of the efflux mechanism was higher.