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OBJECTIVES: Disruption of protein synthesis, by drug-mediated restriction of the ribosomal nascent peptide exit tunnel (NPET), may inhibit bacterial growth. Here, we have studied the secondary and tertiary structures of domain V of the 23S rRNA in the wild-type and mutant (resistant) H. pylori strains and their mechanisms of interaction with clarithromycin (CLA). METHODS: H pylori strains, isolated from cultured gastric biopsies, underwent CLA susceptibility testing by E test, followed by PCR amplification and sequencing of domain V of 23S rRNA. The homology model of this domain in H pylori, in complex with L4 and L22 accessory proteins, was determined based on the E. coli ribosome 3D structure. The interactions between CLA and 23S rRNA complex were determined by molecular docking studies. RESULTS: Of the 70 H pylori strains, isolated from 200 dyspeptic patients, 11 (16%) were CLA-resistant. DNA sequencing identified categories with no (A), A2142G (B), and A2143G (C) mutations. Docking studies of our homology model of 23S rRNA complex with CLA showed deviated positions for categories B and C, in reference to category A, with 12.19 Å and 7.92 Å RMSD values, respectively. In both mutant categories, CLA lost its interactions at positions 2142 and 2587 and gained two new bonds with the L4 accessory protein. CONCLUSION: Our data suggest that, in mutant H pylori strains, once the nucleotides at positions 2142 and 2587 are detached from the drug, CLA interacts with and is peeled back by the L4 accessory protein, removing the drug-imposed spatial restriction of the NPET.
Assuntos
Antibacterianos , Claritromicina , Helicobacter pylori , Ribossomos/química , Antibacterianos/química , Antibacterianos/farmacologia , Claritromicina/química , Claritromicina/farmacologia , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , RNA Ribossômico 23SRESUMO
The nimbleness of Neisseria gonorrhoeae to evade the effect of antibiotics has perpetuated the fight against antibiotic-resistant gonorrhea for more than 80 years. The ability to develop resistance to antibiotics is attributable to its indiscriminate nature in accepting and integrating exogenous DNA into its genome. Here, we provide data demonstrating a novel combination of the 23S rRNA A2059G mutation with a mosaic-multiple transferable resistance (mosaic-mtr) locus haplotype in 14 N. gonorrhoeae isolates with high-level azithromycin MICs (≥256 µg/ml), a combination that may confer more fitness than in previously identified isolates with high-level azithromycin resistance. To our knowledge, this is the first description of N. gonorrhoeae strains harboring this novel combination of resistance determinants. These strains were isolated at two independent jurisdictions participating in the Gonococcal Isolate Surveillance Project (GISP) and in the Strengthening the U.S. Response to Resistant Gonorrhea (SURRG) project. The data suggest that the genome of N. gonorrhoeae continues to shuffle its genetic material. These findings further illuminate the genomic plasticity of N. gonorrhoeae, which allows this pathogen to develop mutations to escape the inhibitory effects of antibiotics.
Assuntos
Antibacterianos/farmacologia , Azitromicina/farmacologia , Mutação/genética , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/genética , Proteínas de Bactérias/genética , Sequência de Bases , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Gonorreia/tratamento farmacológico , Gonorreia/microbiologia , Humanos , Testes de Sensibilidade Microbiana/métodos , RNA Ribossômico 23S/genéticaRESUMO
Neisseria gonorrhoeae resistance to ceftriaxone and azithromycin is increasing, which threatens the recommended dual therapy. We used molecular epidemiology to identify N. gonorrhoeae clusters and associations with azithromycin resistance in Amsterdam, the Netherlands. N. gonorrhoeae isolates (n = 143) were selected from patients visiting the Amsterdam STI Outpatient Clinic from January 2008 through September 2015. We included all 69 azithromycin-resistant isolates (MIC ≥ 2.0 mg/liter) and 74 frequency-matched susceptible controls (MIC ≤ 0.25 mg/liter). The methods used were 23S rRNA and mtrR sequencing, N. gonorrhoeae multiantigen sequence typing (NG-MAST), N. gonorrhoeae multilocus variable-number tandem-repeat analysis (NG-MLVA), and a specific PCR to detect mosaic penA genes. A hierarchical cluster analysis of NG-MLVA related to resistance and epidemiological characteristics was performed. Azithromycin-resistant isolates had C2611T mutations in 23S rRNA (n = 62, 89.9%, P < 0.001) and were NG-MAST genogroup G2992 (P < 0.001), G5108 (P < 0.001), or G359 (P = 0.02) significantly more often than susceptible isolates and were more often part of NG-MLVA clusters (P < 0.001). Two resistant isolates (2.9%) had A2059G mutations, and five (7.3%) had wild-type 23S rRNA. No association between mtrR mutations and azithromycin resistance was found. Twenty-four isolates, including 10 azithromycin-resistant isolates, showed reduced susceptibility to extended-spectrum cephalosporins. Of these, five contained a penA mosaic gene. Four of the five NG-MLVA clusters contained resistant and susceptible isolates. Two clusters consisting mainly of resistant isolates included strains from men who have sex with men and from heterosexual males and females. The co-occurrence of resistant and susceptible strains in NG-MLVA clusters and the frequent occurrence of resistant strains outside of clusters suggest that azithromycin resistance develops independently from the background genome.
Assuntos
Azitromicina/uso terapêutico , Epidemiologia Molecular/métodos , Neisseria gonorrhoeae/efeitos dos fármacos , Neisseria gonorrhoeae/patogenicidade , Adulto , Estudos de Casos e Controles , Farmacorresistência Bacteriana/genética , Feminino , Humanos , Masculino , Países Baixos , RNA Ribossômico 23S/genética , Adulto JovemRESUMO
AIMS: To investigate the erythromycin resistance patterns and mechanism for Campylobacter isolates in China. METHODS AND RESULTS: The minimum inhibitory concentrations of erythromycin on 858 Chinese Campylobacter isolates were analysed. PCR and DNA sequencing were used to identify mutations in the 23S rRNA and the presence of the ermB gene in the 158 erythromycin resistance isolates (18·4%). About 83% (131/158) had A2075G mutation in their 23S rRNA; no A2074C/G mutants were found. The ermB gene was identified in 30 Campylobacter coli isolates (19%). Four types of multidrug-resistant gene islands (MDRGIs) were found. Fifty-three types were identified by multilocus sequence typing among the resistant isolates. All isolates of STs 6322 and 1145 had the ermB gene. CONCLUSIONS: The erythromycin resistance rate of Camp. coli (58·56%) was much higher than Campylobacter jejuni (0·67%). The insertion sites between cadF and CCO1582 and between nfsB and cinA on the chromosome might be hot spots for MDRGI transformation. SIGNIFICANCE AND IMPACT OF THE STUDY: Point mutation in domain V of the 23S rRNA and the ermB gene accounted for 100% of the erythromycin resistance of Campylobacter in China.
Assuntos
Antibacterianos/farmacologia , Infecções por Campylobacter/microbiologia , Campylobacter/efeitos dos fármacos , Campylobacter/isolamento & purificação , Farmacorresistência Bacteriana , Eritromicina/farmacologia , Campylobacter/classificação , Campylobacter/genética , China , Humanos , Testes de Sensibilidade Microbiana , Mutação , Reação em Cadeia da PolimeraseRESUMO
Background: Rising clarithromycin resistance undermines Helicobacter pylori (H. pylori) treatment efficacy. We aimed to determine clarithromycin's minimum inhibitory concentration (MIC) levels and identify specific mutation sites in the 23S ribosomal subunit (23S rRNA) that predict treatment outcomes in a 14-day regimen of clarithromycin bismuth quadruple therapy (amoxicillin 1g, clarithromycin 500 mg, rabeprazole 10 mg, and colloidal bismuth pectin 200 mg). Materials and methods: We included adult H. pylori patients who hadn't previously undergone clarithromycin-based treatment, either as initial or rescue therapy. Exclusions were made for penicillin allergy, recent use of related medications, severe illnesses, or inability to cooperate. Patients underwent a 14-day clarithromycin bismuth quadruple therapy. Gastric mucosa specimens were obtained during endoscopy before eradication. MIC against amoxicillin and clarithromycin was determined using the E-test method. The receiver operating characteristic (ROC) curve helped to find the optimal clarithromycin resistance MIC breakpoint. Genetic sequences of H. pylori 23S rRNA were identified through Sanger Sequencing. (ChiCTR2200061476). Results: Out of 196 patients recruited, 92 met the inclusion criteria for the per-protocol (PP) population. The overall intention-to-treat (ITT) eradication rate was 80.00 % (84/105), while the modified intention-to-treat (MITT) and PP eradication rates were 90.32 % (84/93) and 91.30 % (84/92) respectively. No amoxicillin resistance was observed, but clarithromycin resistance rates were 36.19 % (38/105), 35.48 % (33/93), and 34.78 % (33/92) in the ITT, MITT, and PP populations respectively. Compared with the traditional clarithromycin resistance breakpoint of 0.25 µg/mL, a MIC threshold of 12 µg/mL predicted better eradication. Among 173 mutations on 152 sites in the 23S rRNA gene, only the 2143A > G mutation could predict eradication outcomes (p < 0.000). Conclusions: Interpretation of elevated MIC values is crucial in susceptibility testing, rather than a binary "susceptible" or "resistant" classification. The 2143A > G mutation has limited specificity in predicting eradication outcomes, necessitating further investigation into additional mutation sites associated with clarithromycin resistance.
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Objectives: The aim of the study was to characterize phenotypically and genotypically an uncommon mechanism of resistance to macrolides, lincosamides, and streptogramins (MLS) in a Streptococcus milleri group clinical isolate. Materials and Methods: The isolate UCN96 was recovered from an osteoradionecrosis wound, and was identified using the matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and the partial sequencing of the sodA gene. Antimicrobial susceptibility testing were carried out by the disk diffusion method and minimal inhibitory concentrations (MICs) were determined by the broth microdilution technique. PCR screening was performed for MLS resistance genes described in Gram-positive bacteria. Specific mutations in the ribosomal proteins L3-, L4-, and L22-encoding genes were also screened and those in domain V of the 23S rRNA gene (rrl). The number of mutated copies of the rrl gene was determined using amplification-refractory mutation system quantitative-polymerase chain reaction (qPCR) analysis. Results: The clinical isolate UCN96 was unambiguously identified as Streptococcus constellatus. It was susceptible to all macrolides and lincosamides (ML) antibiotics except spiramycin (MIC >256 mg/L) while it was also resistant to streptogramins. Screening for all acquired resistance genes was negative and no mutation was found in genes coding for L3, L4, and L22 ribosomal proteins. Of interest, a single mutation, A2062C (according to Escherichia coli numbering), was detected in the domain V of 23S rRNA. Conclusion: Mutations at the position 2062 of 23S rRNA have been detected once in Streptococcus pneumoniae, and not yet in other Streptococcus spp. This mechanism is very likely uncommon in Gram-positive bacteria because different copies of 23S rRNA operons should be mutated for development of such a resistance pattern.
Assuntos
Antibacterianos/farmacologia , Eritromicina/farmacologia , RNA Ribossômico 23S/genética , Espiramicina/farmacologia , Streptococcus milleri (Grupo)/efeitos dos fármacos , Streptococcus milleri (Grupo)/genética , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Genótipo , Humanos , Lincosamidas/farmacologia , Macrolídeos/farmacologia , Testes de Sensibilidade Microbiana , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Streptococcus constellatus/efeitos dos fármacos , Streptococcus constellatus/genética , Estreptograminas/farmacologia , Superóxido Dismutase/genéticaRESUMO
All the 31 isolates of Alloscardovia omnicolens exhibited low MICs for ß-lactams, glycopeptides, linezolid, tetracyclines, and cotrimoxazole. One strain showed MICs ≥256µg/mL for both erythromycin and clindamycin with a single point mutation in 23S rRNA. One strain likely had acquired fluoroquinolone resistance associated with a unique mutation in ParC.
Assuntos
Actinobacteria/efeitos dos fármacos , Actinobacteria/genética , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Actinobacteria/isolamento & purificação , Sequência de Bases , Infecções por Bactérias Gram-Positivas/diagnóstico , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Dados de Sequência Molecular , Mutação , RNA Ribossômico 23S/química , RNA Ribossômico 23S/genética , Alinhamento de SequênciaRESUMO
Among 323 specimens from male patients with symptoms of non-gonococcal urethritis, Mycoplasma genitalium was detected in 19 samples by real-time PCR. Mutations of 23S rRNA gene associated with macrolide resistance were confirmed in 10 strains. Amino acid changes at positions 81 and 83 of ParC protein were demonstrated indicating quinolone resistance of two strains.