Mutations in 23S rRNA are associated with clarithromycin resistance in Helicobacter pylori.

Twelve clarithromycin-resistant Helicobacter pylori isolates (100% of resistant isolates examined) from seven different patients each contained an A-->G transition mutation within a conserved loop of 23S rRNA. A-->G transition mutations at positions cognate with Escherichia coli 23S rRNA positions 2058 and 2059 were identified. Clarithromycin-susceptible H. pylori isolates from 14 different patients displayed no polymorphisms in a conserved loop within domain V of 23S rRNA. The study is the first to report mutations in H. pylori associated with resistance to an antimicrobial agent used in established peptic ulcer treatment regimens.     

Explaining the Bias in the 23S rRNA Gene Mutations Associated with Clarithromycin Resistance in Clinical Isolates of Helicobacter pylori

A single point mutation in the 23S rRNA gene of Helicobacter pylori is known to confer resistance to clarithromycin. Most prevalent among clarithromycin-resistant clinical H. pylori isolates are the mutations from A-2142 to G and A-2143 to G in the 23S rRNA gene. The bias in the 23S rRNA gene mutations conferring clarithromycin resistance may result from the higher MIC, stability of resistance, and growth rate found for the strains with the above-mentioned mutations.     

Detection of macrolide resistance in Mycoplasma pneumoniae by real-time PCR and high-resolution melt analysis

Mycoplasma pneumoniae is a significant cause of community-acquired pneumonia, which is often empirically treated with macrolides or azalides such as erythromycin or azithromycin. Recent studies have discovered the existence of macrolide-resistant strains within the population that have been mapped to mutations within the domain V region of the 23S rRNA gene. Currently, identification of these resistant strains relies on time-consuming and labor-intensive procedures such as restriction fragment length polymorphism, MIC studies, and sequence analysis. The current study reports two distinct real-time PCR assays that can detect the A2063G or A2064G base mutation (A2058G or A2059G by Escherichia coli numbering) conferring macrolide resistance. By subjecting the amplicon of the targeted domain V region of the 23S rRNA gene to a high-resolution melt curve analysis, macrolide-resistant strains can quickly be separated from susceptible strains. Utilizing this method, we screened 100 clinical isolates and found 5 strains to possess mutations conferring resistance. These findings were concordant with both sequencing and MIC data. This procedure was also used successfully to identify both susceptible and resistant genotypes in 23 patient specimens. These patient specimens tested positive for the presence of M. pneumoniae by a separate real-time PCR assay, although the bacteria could not be isolated by culture. This is the first report of a real-time PCR assay capable of detecting the dominant mutations that confer macrolide resistance on M. pneumoniae, and these assays may have utility in detecting resistant strains of other infectious agents. These assays may also allow for clinicians to select appropriate treatment options more rapidly and may provide a convenient method to conduct surveillance for genetic mutations conferring antibiotic resistance.     

Ribosomal mutations in Streptococcus pneumoniae clinical isolates

Eleven clinical isolates of Streptococcus pneumoniae, isolated in Finland during 1996 to 2000, had an unusual macrolide resistance phenotype. They were resistant to macrolides and streptogramin B but susceptible, intermediate, or low-level resistant to lincosamides. No acquired macrolide resistance genes were detected from the strains. The isolates were found to have mutations in domain V of the 23S rRNA or ribosomal protein L4. Seven isolates had an A2059C mutation in two to four out of the four alleles encoding the 23S rRNA, two isolates had an A2059G mutation in two alleles, one isolate had a C2611G mutation in all four alleles, and one isolate had a 69GTG71-to-69TPS71 substitution in ribosomal protein L4.     

PCR-restriction fragment length polymorphism analysis for detection of point mutations associated with macrolide resistance in Campylobacter spp

A 23S rRNA gene fragment in domain V was sequenced from 30 clinical isolates of Campylobacter spp., including 22 resistant to macrolides. Two point mutations associated with erythromycin resistance were identified at positions 2074 and 2075 on the 23S rRNA gene (homologous to A2142C and A2143G mutations in Helicobacter pylori) in which an adenine residue is also replaced with a cytosine and a guanine residue, respectively. A combined PCR-restriction fragment length polymorphism technique was developed to detect these mutations by using the BsaI and BceAI enzymes.     

Characterization and molecular analysis of macrolide-resistant Mycoplasma pneumoniae clinical isolates obtained in Japan

In recent years, Mycoplasma pneumoniae strains that are clinically resistant to macrolide antibiotics have occasionally been encountered in Japan. Of 76 strains of M. pneumoniae isolated in three different areas in Japan during 2000 to 2003, 13 strains were erythromycin (ERY) resistant. Of these 13 strains, 12 were highly ERY resistant (MIC, > or =256 microg/ml) and 1 was weakly resistant (MIC, 8 microg/ml). Nucleotide sequencing of domains II and V of 23S rRNA and ribosomal proteins L4 and L22, which are associated with ERY resistance, showed that 10 strains had an A-to-G transition at position 2063 (corresponding to 2058 in Escherichia coli numbering), 1 strain showed A-to-C transversion at position 2063, 1 strain showed an A-to-G transition at position 2064, and the weakly ERY-resistant strain showed C-to-G transversion at position 2617 (corresponding to 2611 in E. coli numbering) of domain V. Domain II and ribosomal proteins L4 and L22 were not involved in the ERY resistance of these clinical M. pneumoniae strains. In addition, by using our established restriction fragment length polymorphism technique to detect point mutations of PCR products for domain V of the 23S rRNA gene of M. pneumoniae, we found that 23 (24%) of 94 PCR-positive oral samples taken from children with respiratory infections showed A2063G mutation. These results suggest that ERY-resistant M. pneumoniae infection is not unusual in Japan.     

AIDS-Related Mycobacterium kansasii Infection with Initial Resistance to Clarithromycin

Clarithromycin is a promising drug for the treatment of Mycobacterium kansasii infection. We report a patient with AIDS and severe M. kansasii infection who had previously received a short course of clarithromycin for sinusitis. He had clinical failure of treatment using clarithromycin plus ethambutol, and the initial isolate was found to be highly resistant to clarithromycin. Nucleotide sequencing of the 23S rRNA gene of this isolate demonstrated a single base mutation at position 2058, the same as that found in clarithromycin-resistant Mycobacterium avium.     

Mechanisms of resistance to telithromycin in Streptococcus pneumoniae

Reports of ketolide resistance remain scarce, however, a few laboratory-derived and clinical isolates of resistant Streptococcus pneumoniae have been documented. Mutations in key telithromycin-binding sites such as domains II and V of the 23S rRNA and ribosomal proteins L4 and L22, as well as mutations of the resistance determinant erm(B) are associated with elevated telithromycin MICs. Mutations in the secondary binding site of domain II coupled with ribosomal methylation may have serious resistance consequences should the domain II binding site be lost. Although ketolides are purported to maintain excellent activity against efflux-positive isolates, laboratory-derived telithromycin-resistant strains have been generated. As telithromycin usage increases, ketolide-resistant isolates of S. pneumoniae may well increase.     

T2182C mutation in 23S rRNA is associated with clarithromycin resistance in Helicobacter pylori isolates obtained in Bangladesh

Twelve clarithromycin-resistant (MIC, > or = 1 microg/ml) Helicobacter pylori isolates were analyzed for point mutations in the 23S rRNA gene. Sequence analysis of all of the resistant isolates revealed a T-to-C transition mutation at position 2182. Transformation experiments confirmed that a single T-to-C transition mutation at position 2182 is associated with clarithromycin resistance.     

Resistance to macrolides in clinical isolates of Streptococcus pyogenes due to ribosomal mutations

OBJECTIVE: Two clinical strains of Streptococcus pyogenes, 237 and 544, one isolated in Slovakia and the other in Croatia, that were resistant to azithromycin (MIC 8 and 2 mg/L, respectively) but susceptible to erythromycin (MIC 0.5 and 0.12 mg/L, respectively) did not contain any gene known to confer macrolide resistance by ribosomal modification (erm gene) or efflux [mef(A) and msr(A) genes]. The aim of the study was to determine the mechanisms of macrolide resistance in both strains. METHODS: Portions of genes encoding ribosomal proteins L22 and L4, and 23S rRNA (domains II and V) in the two macrolide-resistant strains and in control strains susceptible to macrolides, were analysed by PCR and single-strand conformational polymorphism, to screen for mutations. The DNA sequences of amplicons from resistant strains that differed from those of susceptible strains, in terms of their electrophoretic migration profiles, were determined. RESULTS: S. pyogenes 237 displayed a KG insertion after position 69 in ribosomal protein L4. S. pyogenes 544 contained a C2611U mutation in domain V of 23S rRNA. CONCLUSION: Mutations at a similar position in ribosomal protein L4 and 23S rRNA have been reported previously in macrolide-resistant pneumococci. This report shows that similar mutations can be found in macrolide-resistant S. pyogenes.     

Molecular characterization of resistance to macrolides in Bartonella henselae

We selected in vitro erythromycin-resistant strains of Bartonella henselae. The mutants obtained had point mutations in domain V of 23S rRNA and/or in ribosomal protein L4. One lymph node of a patient with cat-scratch disease had such a mutation in 23S rRNA, suggesting that natural resistant strains may infect humans.     

Genetic basis for clarithromycin resistance among isolates of Mycobacterium chelonae and Mycobacterium abscessus.

Resistance to clarithromycin among isolates of Mycobacterium chelonae and M. abscessus was observed in 18 of 800 (2.3%) patients tested between 1990 and 1995. Patients whose isolates were resistant had either disseminated disease or chronic lung disease, and the resistant isolates were recovered after clarithromycin monotherapy. Sequencing of the gene coding for the 23S rRNA peptidyltransferase region revealed a point mutation involving adenine at position 2058 (38%) or adenine at position 2059 (62%) in 20 of 20 relapse isolates from the first 13 patients identified. By pulsed-field gel electrophoresis or random amplified polymorphic DNA PCR, initial and relapse isolates were shown to have identical DNA patterns. M. chelonae and M. abscessus isolates were found to have only a single chromosomal copy of the rRNA operon, thus making them susceptible to single-step mutations. Thus, clarithromycin resistance in these species of rapidly growing mycobacteria relates to a point mutation in the gene coding for 23S rRNA and occurs in limited clinical situations, but was identified in almost 5% of isolates tested in 1995.     

Transition mutations in the 23S rRNA of erythromycin-resistant isolates of Mycoplasma pneumoniae.

Erythromycin is the drug of choice for treatment of Mycoplasma pneumoniae infections due to its susceptibility to low levels of this antibiotic. After exposure of susceptible strains to erythromycin in vitro and in vivo, mutants resistant to erythromycin and other macrolides were isolated. Their phenotypes have been characterized, but the genetic basis for resistance has never been determined. We isolated two resistant mutants (M129-ER1 and M129-ER2) by growing M. pneumoniae M129 on agar containing different amounts of erythromycin. In broth dilution tests both strains displayed resistance to high levels of several macrolide-lincosamide-streptogramin B (MLS) antibiotics. In binding studies, ribosomes isolated from the resistant strains exhibited significantly lower affinity for [14C]erythromycin than did ribosomes from the M129 parent strain. Sequencing of DNA amplified from the region of the 2S rRNA gene encoding domain V revealed an A-to-G transition in the central loop at position 2063 of M129-ER1 and a similar A-to-G transition at position 2064 in M129-ER2. Transitions at homologous locations in the 23S rRNA from other organisms have been shown to result in resistance to MLS antibiotics. Thus, MLS-like resistance can occur in M. pneumoniae as the result of point mutations in the 23S rRNA gene which reduce the affinity of these antibiotics for the ribosome. Since they involve only single-base changes, development of resistance to erythromycin in vivo by these mechanisms could be relatively frequent event.     

Increased macrolide resistance of Mycoplasma pneumoniae in pediatric patients with community-acquired pneumonia

Among 380 Mycoplasma pneumoniae isolates from 3,678 pediatric patients with community-acquired pneumonia, 50 macrolide-resistant strains had an A2063G transition in domain V of the 23S rRNA, whereas 5 had an A2064G transition. These resistant strains increased rapidly from April 2002 to December 2006.     

Mutations in domain V of Mycoplasma pneumoniae 23S rRNA and clinical characteristics of pediatric M. pneumoniae pneumonia in Nanjing,China

ObjectiveTo investigate the prevalence of mutations in domain V of Mycoplasma pneumoniae (MP) 23S ribosomal RNA (rRNA) and the clinical characteristics of pediatric MP pneumonia (MPP) in Nanjing, China.MethodsDomain V of 23S rRNA was sequenced in MP strains collected from children diagnosed with MPP in Nanjing. Clinical and laboratory data were obtained.ResultsAmong the 276 MP strains, 255 (92.39%) harbored mutations, primarily A2063G in domain V of MP 23S rRNA. When children were stratified according to the presence or absence of mutations, no significant differences were found in sex, age, the MP DNA load at enrollment, lymphocyte counts, pulmonary complications, immunomodulator levels, fever duration, the duration of fever after macrolide therapy, and hospital stay. The prevalence of refractory MPP in the two groups was similar. Children with refractory MPP exhibited higher MP DNA loads than those with non-refractory MPP.ConclusionsDespite the high prevalence of the A2063G mutation in domain V of MP 23S rRNA, mutations were not associated with the clinical characteristics of MPP. The MP DNA load significantly differed between refractory and non-refractory MPP.     

多中心耐利奈唑胺凝固酶阴性葡萄球菌耐药机制及同源性分析

目的:了解江苏地区利奈唑胺耐药凝固酶阴性葡萄球菌耐药机制及菌株流行性。方法:收集2017—2018年江苏省3家三级医院临床和环境分离利奈唑胺耐药凝固酶阴性葡萄球菌共38株(头状葡萄球菌32株、人葡萄球菌4株、表皮葡萄球菌1株和缓慢葡萄球菌1株),采用微量肉汤稀释法检测常规药物的敏感性,并采用E-test试验检测菌株对利奈唑胺的最低抑菌浓度(MIC);采用PCR扩增和测序技术检测cfr、optrA、23S rRNA第V功能区基因;采用脉冲场凝胶电泳(PFGE)对分离菌株进行同源性分析。结果:38株凝固酶阴性葡萄球菌对利奈唑胺、青霉素和苯唑西林均耐药,对万古霉素均敏感;对克林霉素、左氧氟沙星和环丙沙星的耐药率大于95%;38株菌株中检出cfr基因31株;23S rRNAⅤ功能区检出G2576T突变35株,其中2株人葡萄球菌同时检出C2319T突变,另检出C2319T突变人葡萄球菌和表皮葡萄球菌各1株;所有菌株均未检出optrA基因;32株头状葡萄球菌PFGE具有高度的相似性,均为同一个克隆株;4株人葡萄球菌为同一克隆株。结论:江苏地区流行的耐利奈唑胺凝固酶阴性葡萄球菌主要是由cfr基因和23S rRNAⅤ功能区基因突变造成,并存在头状葡萄球菌和人葡萄球菌的克隆播散。     

Comparison of phenotypic and genotypic methods for the detection of clarithromycin resistance in Mycobacterium avium

The MICs of clarithromycin for 10 clinical isolates of Mycobacterium avium were determined using three methods: Bactec 460-TB, broth microdilution and Etest. The results were compared with the presence of resistance mutations in the 23S rRNA gene. Isolates were obtained from five AIDS patients who were treated with clarithromycin. Five isolates were recovered before and five during treatment. MICs were reproducible and comparable between the three methods. They were < or = 4 mg/L for pre-treatment isolates and > or = 128 mg/L for strains recovered during treatment. An MIC > or = 128 mg/L was associated with the presence of mutations in the 23S rRNA gene that were absent in the isolates exhibiting MIC < ro = 4 mg/L.     

Macrolide resistance in Campylobacter jejuni and Campylobacter coli: molecular mechanism and stability of the resistance phenotype

A collection of 23 macrolide-resistant Campylobacter isolates from different geographic areas was investigated to determine the mechanism and stability of macrolide resistance. The isolates were identified as Campylobacter jejuni or Campylobacter coli based on the results of the hippurate biochemical test in addition to five PCR-based genotypic methods. Three point mutations at two positions within the peptidyl transferase region in domain V of the 23S rRNA gene were identified. About 78% of the resistant isolates exhibited an A-->G transition at Escherichia coli equivalent base 2059 of the 23S rRNA gene. The isolates possessing this mutation showed a wide range of erythromycin and clarithromycin MICs. Thus, this mutation may incur a greater probability of treatment failure in populations infected by resistant Campylobacter isolates. Another macrolide-associated mutation (A-->C transversion), at E. coli equivalent base 2058, was detected in about 13% of the isolates. An A-->G transition at a position cognate with E. coli 23S rRNA base 2058, which is homologous to the A2142G mutation commonly described in Helicobacter pylori, was also identified in one of the C. jejuni isolates examined. In the majority of C. jejuni isolates, the mutations in the 23S rRNA gene were homozygous except in two cases where the mutation was found in two of the three copies of the target gene. Natural transformation demonstrated the transfer of the macrolide resistance phenotype from a resistant Campylobacter isolate to a susceptible Campylobacter isolate. Growth rates of the resulting transformants containing A-2058-->C or A-2059-->G mutations were similar to that of the parental isolate. The erythromycin resistance of six of seven representative isolates was found to be stable after successive subculturing in the absence of erythromycin selection pressure regardless of the resistance level, the position of the mutation, or the number of the mutated copies of the target gene. One C. jejuni isolate showing an A-2058-->G mutation, however, reverted to erythromycin and clarithromycin susceptibility after 55 subcultures on erythromycin-free medium. Investigation of ribosomal proteins L4 and L22 by sequence analysis in five representative isolates of C. jejuni and C. coli demonstrated no significant macrolide resistance-associated alterations in either the L4 or the L22 protein that might explain either macrolide resistance or enhancement of the resistance level.     

Rapid differentiation between clinically relevant mycobacteria in microscopy positive clinical specimens and mycobacterial isolates by line probe assay

The Inno LiPA Mycobacteria assay, based on PCR amplification of the 16-23S rRNA spacer region of Mycobacterium species, has been designed for identification of mycobacteria grown in culture media and discrimination between Mycobacterium tuberculosis complex, M. avium, M. intracellulare, M. kansasii, M. gordonae, M. xenopi, scrofulaceum and M. chelonae group including M. abscessus. In order to evaluate the system as a fast diagnostic tool, the assay was for the first time used directly on 14 microscopy positive clinical specimens and 71 isolates and the results were compared to those of conventional identification using 16S rDNA analysis and biochemical properties. The assay only misidentified one strain, which was found to be M. avium complex instead of M. intracellulare as found by the conventional tests. The assay allows rapid discrimination of the eight most clinically relevant mycobacteria in microscopy positive clinical specimens and isolates within 6 h in the same procedural run.