突变敏感性分子开关检测肺炎支原体对大环内酯类抗生素的耐药性研究

目的 应用突变敏感性分子开关检测肺炎支原体对大环内酯类抗生素的耐药性。方法 采用微量稀释法检测5种常用大环内酯类抗生素对40株Mp临床分离株的药物敏感性;建立高保真聚合酶和3'硫化修饰引物的分子开关,用分子开关进行Mp临床分离株的PCR扩增,检测其是否存在Mp 23S rRNA 2063、2064和2617 3个热点突变,并通过基因测序进一步确定是否存在点突变,分析点突变与大环内酯类抗生素敏感性之间的关系。结果 5种大环内酯类抗生素中,Mp对14元环的红霉素和克拉霉素耐药程度最高,其MIC≥128 mg/L;对15元环的大环内酯类抗生素阿奇霉素和交沙霉素相对敏感,其中交沙霉素抗Mp活性最高,其MIC≤4 mg/L。用高保真聚合酶和3'硫化修饰引物的分子开关行PCR扩增,检测出35株发生了2063位点基因突变,3株发生2064位点基因突变,未检测出2617位点突变。用基因测序检测到35株Mp发生A2063G的突变,3株发生A2064G的突变,未检测到2617位点突变,与分子开关的检测结果一致,并且2063、2064位点突变Mp株均对大环内酯类药物高度耐药。结论 分子开关可识别23S rRNA基因突变,可用于分析Mp对大环内酯类抗生素的敏感性。     

Simultaneous colonisation of Helicobacter pylori with and without mutations in the 23S rRNA gene in patients with no history of clarithromycin exposure

BACKGROUND: It was recently reported that A to G transition mutations at positions 2143 and 2144 in the 23S rRNA gene are associated with clarithromycin resistance in Helicobacter pylori. AIMS: To study the incidence and mechanism of development of clarithromycin resistance by analysing these mutations. SUBJECTS: Eighty two H pylori positive patients who had an endoscopic examination and no history of treatment with macrolide antibiotics. METHODS: Clarithromycin resistance was screened for by polymerase chain reaction-restriction fragment length polymorphism of the 23S rRNA gene coupled with antibiotic susceptibility testing. In clinical isolates with mutations or resistance, mutations in individual colonies were analysed by direct sequencing. RESULTS: Of the 79 amplicons (DNA fragments amplified by polymerase chain reaction), Alw26I and MboII digestion disclosed the mutation in four (5%) and one (1%) respectively. However, the Alw26I cleavage was incomplete in two of the four amplicons, as was the MboII cleavage. Individual colony analysis of the isolates with incomplete cleavage patterns showed the presence of both wild type and mutated strains in the 23S rRNA genes. CONCLUSIONS: Both clarithromycin sensitive and resistant strains colonised in some patients with no history of exposure to macrolides. The results suggest that resistant strains may not be formed but selected by clarithromycin administration.     

The use of ketolides in treatment of upper respiratory tract infections

Recent surveillance studies suggest that the incidence of resistance to macrolide antibiotics in common communityacquired respiratory tract pathogens, particularly Streptococcus pneumoniae and Streptococcus pyogenes, is increasing and limiting the usefulness of these drugs. The ketolides, of which telithromycin is the first to be available for clinical use (but not yet in the United States), represent a new class of antibacterials developed specifically to combat respiratory tract pathogens that have acquired resistance to macrolides. The ketolides possess innovative structural modifications, a 3-keto group and a large N-substituted C11, C12-carbamate side chain. This novel structure allows ketolides, which are inhibitors of protein synthesis, to exert a more effective interaction with domain II of the 23S rRNA, enhancing binding to bacterial ribosomes and allowing binding to macrolide-lincosamidestreptogramin B-resistant ribosomes. This novel chemical structure also promotes greater stability of telithromycin in acid conditions, providing the potential for greater stability in gastric fluid and at cellular/tissue levels. Early clinical trials support the bacteriologic and clinical efficacy of telithromycin in the treatment of upper respiratory tract infections (RTIs) such as streptococcal pharyngitis and acute sinusitis, including infections caused by macrolide-resistant S. pneumoniae and S. pyogenes. Common adverse side effects associated with telithromycin are predominantly gastrointestinal, usually of mild to moderate severity, and rarely involve withdrawal of the drug. Telithromycin represents an attractive option for the empiric treatment of upper RTIs, especially as resistance to macrolides is likely to continue to increase.     

Erythromycin resistance due to a mutation in a ribosomal RNA operon of Escherichia coli.

There are seven ribosomal RNA operons (rrn operons) in Escherichia coli. A single rrn operon was amplified by use of a multicopy recombinant plasmid containing a complete rrnH operon. rrnH thereby has the potential to contribute a greater fraction of the rRNA found in ribosomes. Erythromycin-resistant mutants were isolated from cells containing the plasmid, and at least one mutation to resistance was shown to reside in rrnH on the plasmid. Erythromycin resistance was retained when a major deletion was introduced into the 16S rRNA gene and was abolished by deletions that affect the 16S and 23S rRNA genes but do not alter the 5S rRNA gene or non-rrnH DNA. Cell-free S30 protein-synthesizing extracts from cells containing the mutant plasmid have an increased resistance to erythromycin. The selection procedure used to isolate erythromycin-resistance mutations in rrnH may allow, with minor modifications, the isolation of mutations in rrn operons that change resistance of the ribosome to other antibiotics or that alter other properties of ribosomes.     

Emergence of resistance to clarithromycin during treatment of disseminated cutaneous Mycobacterium chelonae infection: case report and literature review.

Results of in vitro susceptibility studies and one clinical trial have led to recommendations of clarithromycin monotherapy for the treatment of disseminated cutaneous Mycobacterium chelonae infections. We describe the case of a 65-year-old woman, immunocompromised by the use of chronic steroid therapy, who developed disseminated cutaneous infection with M. chelonae and failed clarithromycin monotherapy due to the development of drug resistance. In the relapse isolate we document the presence of a single point mutation at position 2058 in the gene coding for 23S rRNA peptidyltransferase regions, a mutation previously implicated in the development of resistance to clarithromycin. Two susceptible control isolates lacked the mutation. Three additional reports in the literature of patients developing recurrent skin lesions with clarithromycin-resistant M. chelonae following initial response to monotherapy are summarized. We demonstrate that clarithromycin monotherapy in patients with disseminated cutaneous infections can lead to clarithromycin resistance and therapeutic failure associated with a single point mutation at position 2058 of 23S rRNA.     

39株对克拉霉素耐药的结核分枝杆菌临床分离株23SrRNA检测结果分析

目的 分析对克拉霉素耐药的结核分枝杆菌临床分离株23S rRNA的A2058位点的变化。 方法 选择我院菌株库的结核分枝杆菌临床分离株64株,其中10株为对全部抗结核药物敏感的结核分枝杆菌临床分离株;14株为单耐克拉霉素的结核分枝杆菌临床分离株;15株为耐多药,同时耐克拉霉素的结核分枝杆菌临床分离株;15株为耐多药,同时对克拉霉素敏感的结核分枝杆菌临床分离株;10株为广泛耐药菌株,同时对克拉霉素耐药的结核分枝杆菌临床分离株;此外,还有结核分枝杆菌标准株H37Rv 1株。对结核分枝杆菌23S rRNA行PCR检测和测序。 结果 经检测,H37Rv标准株没有A2058突变,只有1株广泛耐药临床分离株检测有A2058A-G的突变,其他临床分离株均没有突变,在耐克拉霉素的结核分枝杆菌临床分离株中占2.56%（1/39）,在广泛耐药结核分枝杆菌临床分离株中占1/10。 结论 结核分枝杆菌临床分离株对克拉霉素耐药的机制中, A2058突变可能不是产生对克拉霉素耐药的主要机制。结核分枝杆菌产生对克拉霉素耐药的机制有待进一步研究。     

A possible mechanism of macrolide resistance among multiple resistant Campylobacter jejuni and Campylobacter coli isolated from Thai children during 1991-2000

A total of 495 Campylobacterjejuni and 122 C. coli isolated from Thai children were screened for macrolide (erythromycin and azithromycin) resistance by disk diffusion assay. Minimum inhibitory concentrations for erythromycin, azithromycin, nalidixic acid, ciprofloxacin, tetracycline, streptomycin, gentamicin and chloramphenicol were further determined for these macrolide-resistant Campylobacter isolates. Presence of known point mutations resulting in reduced susceptibility to macrolides was investigated by PCR and DNA sequencing. Seventeen percent (23/122) of C. coli and 2.4% (12/495) of C. jejuni isolates were resistant to macrolides. By sequencing domain V of the 23S ribosomal DNA from all 35 macrolide-resistant isolates, a known point mutation of 23S rRNA associated with reduced susceptibility to macrolides was detected in all isolates except one. Among the macrolide-resistant isolates, all were multiply resistant to nalidixic acid and ciprofloxacin, of which the latter is the preferred antimicrobial used for diarrheal treatment in Thailand. Furthermore, most macrolide-resistant isolates were also resistant to tetracycline and streptomycin. The spread of macrolide and quinolone resistant Campylobacter should be monitored closely in Thailand and elsewhere as these antimicrobials are preferred drugs for treatment of diarrhea.     

Azithromycin resistance in Treponema pallidum

PURPOSE OF REVIEW: Although the recommended treatment for syphilis is penicillin, azithromycin has been used as an alternative. We discuss azithromycin-related treatment failures and resistance in Treponema pallidum, and propose ways to meet the resulting clinical and public health challenges. RECENT FINDINGS: Azithromycin treatment failures in syphilis were first noted in San Francisco in 2002 and result from an A-->G mutation at position 2058 of the 23S rRNA gene of T. pallidum. This mutation confers resistance by precluding macrolide binding to the bacterial 50S ribosomal subunit, of which 23S rRNA is a structural component. Azithromycin resistance has also been identified in T. pallidum specimens from elsewhere in the United States, Ireland, and Canada, and the amount of resistant specimens has increased with time. Treatment with azithromycin or other macrolides appears to be a risk factor for presenting with a resistant T. pallidum strain. SUMMARY: Although T. pallidum remains sensitive to penicillin and certain other antibiotics, azithromycin resistance in T. pallidum has emerged and is increasing in the United States, Canada, and Ireland. This poses clinical and public health challenges, and indicates a need for further antibiotic drug development and surveillance for resistance in T. pallidum. If azithromycin is used to treat syphilis, clinicians and public health practitioners should remain vigilant for treatment failures.     

Resistance to the macrolide antibiotic tylosin is conferred by single methylations at 23S rRNA nucleotides G748 and A2058 acting in synergy

The macrolide antibiotic tylosin has been used extensively in veterinary medicine and exerts potent antimicrobial activity against Gram-positive bacteria. Tylosin-synthesizing strains of the Gram-positive bacterium Streptomyces fradiae protect themselves from their own product by differential expression of four resistance determinants, tlrA, tlrB, tlrC, and tlrD. The tlrB and tlrD genes encode methyltransferases that add single methyl groups at 23S rRNA nucleotides G748 and A2058, respectively. Here we show that methylation by neither TlrB nor TlrD is sufficient on its own to give tylosin resistance, and resistance is conferred by the G748 and A2058 methylations acting together in synergy. This synergistic mechanism of resistance is specific for the macrolides tylosin and mycinamycin that possess sugars extending from the 5- and 14-positions of the macrolactone ring and is not observed for macrolides, such as carbomycin, spiramycin, and erythromycin, that have different constellations of sugars. The manner in which the G748 and A2058 methylations coincide with the glycosylation patterns of tylosin and mycinamycin reflects unambiguously how these macrolides fit into their binding site within the bacterial 50S ribosomal subunit.     

Peptide inhibitors of peptidyltransferase alter the conformation of domains IV and V of large subunit rRNA: a model for nascent peptide control of translation.

Peptides of 5 and 8 residues encoded by the leaders of attenuation regulated chloramphenicol-resistance genes inhibit the peptidyltransferase of microorganisms from the three kingdoms. Therefore, the ribosomal target for the peptides is likely to be a conserved structure and/or sequence. The inhibitor peptides "footprint" to nucleotides of domain V in large subunit rRNA when peptide-ribosome complexes are probed with dimethyl sulfate. Accordingly, rRNA was examined as a candidate for the site of peptide binding. Inhibitor peptides MVKTD and MSTSKNAD were mixed with rRNA phenol-extracted from Escherichia coli ribosomes. The conformation of the RNA was then probed by limited digestion with nucleases that cleave at single-stranded (T1 endonuclease) and double-stranded (V1 endonuclease) sites. Both peptides selectively altered the susceptibility of domains IV and V of 23S rRNA to digestion by T1 endonuclease. Peptide effects on cleavage by V1 nuclease were observed only in domain V. The T1 nuclease susceptibility of domain V of in vitro-transcribed 23S rRNA was also altered by the peptides, demonstrating that peptide binding to the rRNA is independent of ribosomal protein. We propose the peptides MVKTD and MSTSKNAD perturb peptidyltransferase center catalytic activities by altering the conformation of domains IV and V of 23S rRNA. These findings provide a general mechanism through which nascent peptides may cis-regulate the catalytic activities of translating ribosomes.     

Isolation of antibiotic resistance mutations in the rRNA by using an in vitro selection system

Genetic, biochemical, and structural data support an essential role for the ribosomal RNA in all steps of the translation process. Although in vivo genetic selection techniques have been used to identify mutations in the rRNAs that result in various miscoding phenotypes and resistance to known ribosome-targeted antibiotics, these are limited because the resulting mutant ribosomes must be only marginally disabled if they are able to support growth of the cell. Furthermore, in vivo, it is not possible to control the environment in precise ways that might allow for the isolation of certain types of rRNA variants. To overcome these limitations, we have developed an in vitro selection system for the isolation of functionally competent ribosomal particles from populations containing variant rRNAs. Here, we describe this system and present an example of its application to the selection of antibiotic resistance mutations. From a pool of 4,096 23S rRNA variants, a double mutant (A2058U/A2062G) was isolated after iteration of the selection process. This mutant was highly resistant to clindamycin in in vitro translation reactions and yet was not viable in Escherichia coli. These data establish that this system has the potential to identify mutations in the rRNA not readily accessed by comparable in vivo systems, thus allowing for more exhaustive ribosomal genetic screens.     

Linezolid resistance in Staphylococcus aureus: characterization and stability of resistant phenotype

Linezolid is an important therapeutic option for treatment of infections caused by glycopeptide- and beta-lactam-resistant gram-positive organisms. Linezolid resistance is caused by mutations within the domain V region of the 23S ribosomal RNA (rRNA) gene, which is present in multiple copies in most bacteria. Among clinical Staphylococcus aureus isolates, there has been only 1 reported case of linezolid resistance. In the present study, this isolate was further characterized by determination of the number of mutant 23S rRNA copies, assessment of the stability of the resistant phenotype, and comparison of its growth characteristics with those of linezolid-susceptible S. aureus. All 5 copies of the 23S rRNA gene contained a G2576U mutation in the domain V region. After serial passage on antibiotic-free medium, the isolate maintained resistance to high concentrations of linezolid. Compared with 2 linezolid-susceptible S. aureus isolates, the linezolid-resistant S. aureus isolate demonstrated no significant differences in in vitro growth characteristics.     

Molecular basis of clarithromycin-resistance in Mycobacterium avium intracellulare complex.

Nucleotide sequences of domain V and domain II regions of the 23S rRNA gene were determined in both in vitro-made mutants and clinical isolates of Mycobacterium avium and M. intracellulare conferring clarithromycin-resistance. All laboratory-made mutants showed high level resistance to clarithromycin (> 150 micrograms ml-1) and mutation at position 2058 (cognate with Escherichia coli base) in domain V region. In the clinical isolates, while the susceptible ones had no mutation in domain V, the resistant strains showed mutation at 2058 or 2059. Six isolates with low level of resistance exhibited no mutation in domain V. All strains tested had no mutation in domain II region. These results suggested that most of the resistance arose from the mutation in domain V of the 23S rRNA gene, but other unknown mechanisms evidently exist in mycobacteria.     

Analysis of mutations at residues A2451 and G2447 of 23S rRNA in the peptidyltransferase active site of the 50S ribosomal subunit

On the basis of the recent atomic-resolution x-ray structure of the 50S ribosomal subunit, residues A2451 and G2447 of 23S rRNA were proposed to participate directly in ribosome-catalyzed peptide bond formation. We have examined the peptidyltransferase and protein synthesis activities of ribosomes carrying mutations at these nucleotides. In Escherichia coli, pure mutant ribosome populations carrying either the G2447A or G2447C mutations maintained cell viability. In vitro, the G2447A ribosomes supported protein synthesis at a rate comparable to that of wild-type ribosomes. In single-turnover peptidyltransferase assays, G2447A ribosomes were shown to have essentially unimpaired peptidyltransferase activity at saturating substrate concentrations. All three base changes at the universally conserved A2451 conferred a dominant lethal phenotype when expressed in E. coli. Nonetheless, significant amounts of 2451 mutant ribosomes accumulated in polysomes, and all three 2451 mutations stimulated frameshifting and readthrough of stop codons in vivo. Furthermore, ribosomes carrying the A2451U transversion synthesized full-length beta-lactamase chains in vitro. Pure mutant ribosome populations with changes at A2451 were generated by reconstituting Bacillus stearothermophilus 50S subunits from in vitro transcribed 23S rRNA. In single-turnover peptidyltransferase assays, the rate of peptide bond formation was diminished 3- to 14-fold by these mutations. Peptidyltransferase activity and in vitro beta-lactamase synthesis by ribosomes with mutations at A2451 or G2447 were highly resistant to chloramphenicol. The significant levels of peptidyltransferase activity of ribosomes with mutations at A2451 and G2447 need to be reconciled with the roles proposed for these residues in catalysis.     

Ribosomal resistance: Emerging problems and potential solutions

Many systemic antibiotics use ribosomal inhibition to suppress the replication of bacteria. Current research suggests that resistance to macrolide, lincosamide, and streptogramin B (MLS_B) antibiotics is emerging among clinical isolates of Streptococcus pyogenes and Streptococcus pneumoniae. Erythromycin methylases, encoded by erm genes, modify an essential adenine residue in 23S rRNA and confer cross-resistance to MLS_B antibiotics. More recently, macrolide efflux (mef) genes were identified in isolates of S. pyogenes and S. pneumoniae that show resistance to 14- and 15-membered macrolides (M phenotype). Resistance to MLSB has been associated with the increased use of erythromycin, and the recent emergence of the M phenotype has coincided with the marketing of newer macrolides. However, despite increasing macrolide resistance among clinical isolates of S. pneumoniae, convincing data on treatment failures directly attributable to MLSB or M phenotypes are limited. Possible solutions to emerging MLS_B and M phenotype resistance include the introduction of alternative antibiotics, the more prudent use of antibiotics, combination therapy, molecular diagnostics, enhanced understanding of pharmacodynamic variables, and redefined resistance breakpoints.     

A novel site of antibiotic action in the ribosome: interaction of evernimicin with the large ribosomal subunit

Evernimicin (Evn), an oligosaccharide antibiotic, interacts with the large ribosomal subunit and inhibits bacterial protein synthesis. RNA probing demonstrated that the drug protects a specific set of nucleotides in the loops of hairpins 89 and 91 of 23S rRNA in bacterial and archaeal ribosomes. Spontaneous Evn-resistant mutants of Halobacterium halobium contained mutations in hairpins 89 and 91 of 23S rRNA. In the ribosome tertiary structure, rRNA residues involved in interaction with the drug form a tight cluster that delineates the drug-binding site. Resistance mutations in the bacterial ribosomal protein L16, which is shown to be homologous to archaeal protein L10e, cluster to the same region as the rRNA mutations. The Evn-binding site overlaps with the binding site of initiation factor 2. Evn inhibits activity of initiation factor 2 in vitro, suggesting that the drug interferes with formation of the 70S initiation complex. The site of Evn binding and its mode of action are distinct from other ribosome-targeted antibiotics. This antibiotic target site can potentially be used for the development of new antibacterial drugs.     

Direct Killing of Patients in Humanitarian Situations and Armed Conflicts: The Profession of Medicine Is Losing Its Meaning

Treponema pallidum ssp. pallidum (TPA) causes over 10 million new cases of syphilis worldwide whereas T. pallidum ssp. pertenue (TPE), the causative agent of yaws, affects about 2.5 million people. Although penicillin remains the drug of choice in the treatment of syphilis, in penicillin-allergic patients, macrolides have been used in this indication since the 1950s. Failures of macrolides in syphilis treatment have been well documented in the literature and since 2000, there has been a dramatic increase in a number of clinical samples with macrolide-resistant TPA. Scarce data regarding the genetics of macrolide-resistant mutations in TPA suggest that although macrolide-resistance mutations have emerged independently several times, the increase in the proportion of TPA strains resistant to macrolides is mainly due to the spread of resistant strains, especially in developed countries. The emergence of macrolide resistance in TPA appears to require a two-step process including either A2058G or A2059G mutation in one copy of the 23S rRNA gene and a subsequent gene conversion unification of both rRNA genes. Given the enormous genetic similarity that was recently revealed between TPA and TPE strains, there is a low but reasonable risk of emergence and spread of macrolide-resistant yaws strains following azithromycin treatment.     

Requirement for a conserved, tertiary interaction in the core of 23S ribosomal RNA.

A putative base-pairing interaction that determines the folding of the central region of 23S rRNA has been investigated by mutagenesis. Each of the possible base substitutions has been made at the phylogenetically covariant positions adenine-1262 (A1262) and U2017 in Escherichia coli 23S rRNA. Every substitution that disrupts the potential for Watson-Crick base pairing between these positions reduces or abolishes the participation of 23S rRNA in protein synthesis. All mutant 23S rRNAs are assembled into 50S subunits, but the mutant subunits are less able to stably interact with 30S subunits to form translationally active ribosomes. The function of 23S rRNA is largely reestablished by introduction of an alternative G1262.C2017 or U1262.A2017 pair, although neither of these supports polysome formation quite as effectively as the wild-type pair. A 23S rRNA with a C1262.G2017 pair is nonfunctional. In contrast to the considerable effect the mutations have on function, they impart only slight structural changes on the naked rRNA, and these are limited to the immediate vicinity of the mutations. The data show that positions 1262 and 2017 pair in a Watson-Crick manner, but the data also indicate that these nucleotides engage in additional interactions within the ribosome and that these interactions determine what base pairs are acceptable there.     

Dose dependence of emergence of resistance to linezolid in Enterococcus faecalis in vivo

BACKGROUND: The emergence of resistance to antibiotics in vivo, particularly in commensal, potentially pathogenic bacteria, is a factor that is key to the future of antibiotics. To better document the circumstances favoring the emergence of resistance to linezolid (the first of a new class of antibiotics, the oxazolidinones), we modeled the effect of different regimens of linezolid on Enterococcus faecalis in gnotobiotic mice. METHODS: We studied the rate of emergence of linezolid-resistant E. faecalis mutants in the digestive tract of gnotobiotic mice monoassociated with linezolid-susceptible E. faecalis and fed with water containing linezolid (0.5, 0.05, or 0.005 g/L). 23S Ribosomal RNA (rRNA) mutations were characterized by sequencing each of the 4 copies of the rRNA genes individually. RESULTS: Mutants were readily obtained in vivo, but the frequencies, persistence, and type of mutants were all dependent on the linezolid regimen. Mutations conferring resistance, either the G2505A or G2576U mutation, were present in domain V of the 23S rRNA gene of all resistant isolates. Levels of resistance increased with the number of mutated copies of the 23S rRNA gene and with duration of exposure. CONCLUSION: The antibiotic dose appears to be critical in the dynamics and molecular basis of resistance.     

Mutation of a Cytoplasmic Gene in Chlamydomonas Alters Chloroplast Ribosome Function

A mutation, car, determining resistance to several macrolide antibiotics, including carbomycin, has been identified in the alga Chlamydomonas as cytoplasmic, and mapped in the known cytoplasmic linkage group close to genes determining resistance to other antibiotics, including streptomycin, erythromycin, and spectinomycin. The effect of the car mutation on chloroplast ribosome function was demonstrated with an in vitro system incorporating amino acids especially developed to assess activity of 70S chloroplast ribosomes. In an S-30 extract containing both 70S chloroplast and 80S cytoplasmic ribosomes, low concentrations of Mg(++) and spermidine favored 80S ribosome activity, and high concentrations activated 70S ribosomes and reversibly inactivated the 80S component. Under conditions favoring chloroplast ribosome activity, carbomycin inhibited incorporation by an S-30 extract, and by purified 70S ribosomes from wild-type but not from car cells. These results show that cytoplasmic genes are directly involved in chloroplast ribosome function and they suggest that the car gene product is a ribosomal protein; the results further strengthen the evidence that the cytoplasmic linkage group is located in chloroplast DNA.