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。     

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.     

Evidence for functional interaction between domains II and V of 23S ribosomal RNA from an erythromycin-resistant mutant.

A mutation affording low levels of erythromycin resistance has been obtained by in vitro hydroxylamine mutagenesis of a cloned ribosomal RNA operon from Escherichia coli. The site of the mutational event responsible for antibiotic resistance was localized to the gene region encoding domain II of 23S rRNA by replacement of restriction fragments in the wild-type plasmid by corresponding fragments from the mutant plasmid. DNA sequencing showed that positions 1219-1230 of the 23S rRNA gene are deleted in the mutant. Since all previously characterized rRNA mutations conferring resistance to erythromycin show changes exclusively in domain V, our present findings provide direct evidence for functional interaction between domains II and V of 23S rRNA.     

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。     

幽门螺杆菌对克拉霉素耐药的分子机制研究

目的：研究幽门螺杆菌（Hp)对克拉霉素耐的分子机制。方法：用E－test进行克拉霉素药敏试验,选取治疗前敏感、治疗后耐药的配对菌株及原发耐药Hp菌株进行研究;应用随机扩增多态性DNA（RAPD）分析,确定治疗前后菌株的同一性;用PCR－限制性片段长度多态性（RFLP）分析探讨克拉霉素耐药机制。结果9株克拉霉素耐药菌23SrRNA基因功能区V PCR扩增片段,8株被BsaI酶切,9株均未被BbsI酶切,提示8株在2144位点有A→G突变。结论上海地区大多数克拉霉素耐药Hp菌株存在23SrRNA基因功能区V2144位点A→G突变。     

23S rRNA base pair 2057-2611 determines ketolide susceptibility and fitness cost of the macrolide resistance mutation 2058A-->G

The 23S rRNA A2058G alteration mediates macrolide, lincosamide, and streptogramin B resistance in the bacterial domain and determines the selectivity of macrolide antibiotics for eubacterial ribosomes, as opposed to eukaryotic ribosomes. However, this mutation is associated with a disparate resistance phenotype: It confers high-level resistance to ketolides in mycobacteria but only marginally affects ketolide susceptibility in streptococci. We used site-directed mutagenesis of nucleotides within domain V of 23S rRNA to study the molecular basis for this disparity. We show that mutational alteration of the polymorphic 2057-2611 base pair from A-U to G-C in isogenic mutants of Mycobacterium smegmatis significantly affects susceptibility to ketolides but does not influence susceptibility to other macrolide antibiotics. In addition, we provide evidence that the 2057-2611 polymorphism determines the fitness cost of the 23S rRNA A2058G resistance mutation. Supported by structural analysis, our results indicate that polymorphic nucleotides mediate the disparate phenotype of genotypically identical resistance mutations and provide an explanation for the large species differences in the epidemiology of defined drug resistance mutations.     

Linezolid resistance in sequential Staphylococcus aureus isolates associated with a T2500A mutation in the 23S rRNA gene and loss of a single copy of rRNA

Linezolid is an important therapeutic option for infections caused by resistant gram-positive bacteria. We report the characterization of sequential methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates that developed resistance in a patient treated with a prolonged course of linezolid. Analysis of this series of clinical MRSA isolates detected, in the resistant isolates, the presence of a T2500A mutation in the domain V region of the 23S rRNA gene. In addition, the loss of a single copy of the 23S rRNA gene was found in 2 of the resistant isolates. As a result of these 2 factors, the proportion of mutant : wild-type 23S rRNA genes increased in association with an increase in the minimum inhibitory concentration of linezolid. The most recent isolate of this series was recovered 7 months after the patient discontinued linezolid and demonstrated reversion to a susceptible phenotype associated with a loss of the T2500A mutation.     

幽门螺杆菌临床菌株克拉霉素耐药性及耐药基因突变位点分析

摘要：目的：了解贵阳地区幽门螺杆菌（Helicobacter pylori,Hp）临床菌株对克拉霉素耐药性及克拉霉素耐药相关基因突变情况,为耐药性的快速检测提供依据。方法：采用琼脂稀释法对临床分离鉴定的Hp菌株,进行体外抗生素敏感试验,了解贵阳地区Hp临床株对克拉霉素耐药状况。选取Hp克拉霉素耐药的临床菌株10株、克拉霉素敏感的临床菌株4株和质控菌株2株,进行23S rRNA基因功能区V区片段的PCR扩增和测序,与GenBank中公布的Hp菌株相关序列进行比对分析。结果：贵阳地区Hp临床分离株对克拉霉素的耐药率达30.9%。贵阳地区10株Hp耐药菌的23S rRNA基因片段的碱基突变包括T2183C（10/10）、T2245C（9/10）、 A2144G（6/10）、C2196G（1/10）、A2204G（1/10）,4株敏感菌株在2183、2245、2196和2204位点也存在碱基差异,2144位点的基因突变仅存于耐药菌株中。结论：贵阳地区Hp克拉霉素耐药率较高,耐药菌株23SrDNA与耐药性相关的基因突变主要为A2144G。     

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.     

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.     

Characterization of clarithromycin resistance in Malaysian isolates of Helicobacterpylori

AIM： To characterize the types of mutations present in the 23S rRNA genes of Malaysian isolates of clarithromycin-resistant Helicobacter pylori （H pylorl~. METHODS： Clarithromycin susceptibility of H pylori isolates was determined by E test. Analyses for point mutations in the domain V of 23S rRNA genes in clarithromycin-resistant and -sensitive strains were performed by sequence analysis of amplified polymerase chain reaction products. Restriction fragment length polymorphism was performed using Bsa I and MboI enzymes to detect restriction sites that correspond to the mutations in the clarithromycin- resistant strains. RESULTS： Of 187 isolates from 120 patients, four were resistant to clarithromycin, while 183 were sensitive. The MIC of the resistant strains ranged from 1.5 to 24 pg/mL. Two isolates had an A2142G mutation and another two had A2143G mutations. A T2182C mutation was detected in two out of four clarithromycin-resistant isolates and in 13 of 14 clarithromycin-sensitive isolates. Restriction enzyme analyses with Bsa I and Mbo I were able to detect the mutations. CONCLUSION： Clarithromycin resistance is an uncommon occurrence among Malaysian isolates of Hpylori strains and the mutations A2142G and A2143G detected were associated with low-level resistance.     

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.     

Clarithromycin resistance and point mutations in the 23S rRNA gene in Helicobacter pylori isolates from Malaysia

OBJECTIVE: To determine the prevalence of primary clarithromycin resistance amongst Helicobacter pylori (H. pylori) strains in Malaysian patients with gastroduodenal diseases, by using restriction fragment length polymorphism (RFLP) in domain V of 23S rRNA. METHODS: Gastric biopsies were obtained from H. pylori positive patients undergoing gastroscopy. DNA extraction was followed by PCR amplification using the primers Hp23-1 and Hp23-2 flanking a region of 425bp within the bacterial 23S rRNA peptidyltranferase (Hp23S fragment). Analysis of the 23S rRNA gene mutations is based on the generation of restriction sites for two restriction enzymes: BbsI and BsaI, which correspond to the base substitutions characteristic of clarithromycin resistance from A to G at positions 2142 and 2143, respectively. RESULTS: Gastric biopsy samples were obtained from 107 patients. A fragment of size 425bp corresponding to that expected from amplification of domain V of 23S rRNA was PCR-amplified from only 105 samples. The amplicon was subsequently subjected to restriction by BbsI and BsaI. Only 1 sample (0.95%) had the BbsI mutation (base substitution at A2142G) and 2 samples (1.90%) the BsaI mutation (base substitution at A2143G). Thus 3 of 105 (2.9%) samples harbored clarithromycin resistant strains. CONCLUSION: In our experience, PCR-RFLP is a rapid and precise method to detect the resistance of H. pylori to clarithromycin. Using this method, a low prevalence of clarithromycin resistance was detected in our local Malaysian strains. This augurs well for the continued use of clarithromycin as a first line drug in the treatment and eradication of H. pylori infection.     

肉鸡源多重耐药空肠、结肠弯曲菌的耐药分子特征

目的了解肉鸡源空肠和结肠弯曲菌的耐药谱特征,检测多重耐药菌株Ⅰ类整合子/基因盒、gyrA基因突变位点、tetO基因、23S rRNA突变位点的分子特征。方法利用PCR检测弯曲菌Ⅰ类整合子/基因盒的存在情况;利用MAMA PCR技术检测弯曲菌gyrA基因第257位碱基的突变情况;针对弯曲菌23S rRNA的V区2075突变位点检测突变菌株。结果多重耐药菌株占分离株的94.5%。146株多重耐药空肠和结肠弯曲菌中Ⅰ类整合子检出率为98.6%,有78株菌株检出3种基因盒,1 000 bp+750 bp+500 bp+250 bp为主要谱型,所占比例为92.3%;有131株在gyrA喹诺酮类耐药决定区发生突变,突变率为92.9%。127株四环素耐药弯曲菌tetO基因的检出率为95.3%。81株红霉素耐药菌株中,23S rRNA的V区2075处突变发生率为96.3%。结论空肠和结肠弯曲菌分离株携带aadA2耐药基因盒,与氨基糖苷类药物的耐药性相关;gyrA基因突变、tetO基因的携带以及23S rRNA突变,与弯曲菌对喹诺酮、四环素和大环内酯类耐药密切相关。     

Mutation frequency and biological cost of antibiotic resistance in Helicobacter pylori

Among the several factors that affect the appearance and spread of acquired antibiotic resistance, the mutation frequency and the biological cost of resistance are of special importance. Measurements of the mutation frequency to rifampicin resistance in Helicobacter pylori strains isolated from dyspeptic patients showed that approximately 1/4 of the isolates had higher mutation frequencies than Enterobacteriaceae mismatch-repair defective mutants. This high mutation frequency could explain why resistance is so frequently acquired during antibiotic treatment of H. pylori infections. Inactivation of the mutS gene had no substantial effect on the mutation frequency, suggesting that MutS-dependent mismatch repair is absent in this bacterium. Furthermore, clarithromycin resistance conferred a biological cost, as measured by a decreased competitive ability of the resistant mutants in mice. In clinical isolates this cost could be reduced, indicating that compensation is a clinically relevant phenomenon that could act to stabilize resistant bacteria in a population.     

New mutation points in 23S rRNA gene associated with Helicobacter pylori resistance to clarithromycin in northeast China

AIM: To investigate the resistance rate of Helicobacter pylori (H pylori ) to clarithromycin, metronidazole, amoxicillin and tetracycline to guide clinical practice, and to study the mechanism of H pylori resistant to clarithromycin. METHODS: Thirty H pylori strains were isolated from the mucosa of peptic ulcer, gastric tumor and chronic gastritis patients, then the minimal inhibitory concentration (MIC) to clarithromycin, metronidazole, amoxicillin and tetracycline was evaluated by E-test method. The sequence analysis of PCR fragments was conducted in 23S rRNA gene of H pylori resistant to clarithromycin to get the resistance mechanism of the bacteria. RESULTS: Among 30 H pylori strains, 7 cases were resistant to clarithromycin, 12 to metronidazole, 2 to tetracycline and no strain was found to be resistant to amoxicillin. The resistance rates were 23.3%, 40%, 6.7% and 0%, respectively. Three new mutation points were found to be related to the clarithromycin resistance in H pylori isolates, which were G2224A, C2245T and T2289C. CONCLUSION: In northeast China, H pylori shows high resistance to metronidazole, while sensitive to amoxicillin. The mechanism of resistance to clarithromycin may be related to the mutation of G2224A, C2245T and T2289C in the 23S rRNA gene.     

Prevalences of metronidazole‐ and clarithromycin‐resistant Helicobacter pylori isolates in Shanghai and molecular mechanism of resistance to clarithromycin

OBJECTIVE : To investigate the prevalences of metronidazole‐ and clarithromycin‐resistant Helicobacter pylori over the period from 1995 to 1999 in Shanghai, and the molecular mechanism of resistance to clarithromycin. METHODS : A total of 150 H. pylori strains were randomly selected from the isolates collected in 1995, 1997 and 1999, and tested for sensitivity against metronidazole and clarithromycin by using the E‐test. The mechanism of resistance was studied by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP). RESULTS : It was found that 42% (21/50), 57% (27/50) and 70% (35/50) of the tested strains were resistant to metronidazole among the isolates collected in 1995, 1997 and 1999, respectively. In 1995, there was no strain (0/50) resistant to clarithromycin, of which the prevalence rose to 2% (1/50) in 1997, and to 10% (5/50) in 1999. The prevalences of metronidazole‐ and clarithromycin‐resistant H. pylori in 1999 were significantly higher than those in 1995 (P < 0.05). Of nine clarithromycin‐resistant H. pylori strains, eight were found to have an A→G mutation at position 2144 of domain V of the 23S rRNA. CONCLUSIONS : These results suggest a significant increase in the prevalences of metronidazole‐ and clarithromycin‐resistant H. pylori in Shanghai during the 1995–1999 period. The majority (88.8%) of clarithromycin‐resistant H. pylori isolates have an A2144G mutation in domain V of the 23S rRNA.     

耐异烟肼结核分枝杆菌临床分离株耐药相关基因突变研究

目的阐明结核分枝杆菌耐异烟肼临床分离株katG、inhA、ahpC、kasA及oxyR基因突变特点。方法对144株结核分枝杆菌临床分离株(耐异烟肼菌株101株;异烟肼敏感株43株)的katG、inhA、kasA、ahpC及oxyR基因进行DNA片断扩增及DNA序列分析,与GeneBank中结核分枝杆菌标准序列进行比较。结果(1)耐异烟肼菌株中未发现katG完全缺失,81株耐药株(80.2%)katG存在点突变、缺失或插入,其中16个突变位点未见报道;39株(38.6%)耐药株第315位点突变,低耐药菌株(1μg/ml)第315位点突变率显著高于高耐药菌株(10μg/ml;χ2=9.31,P<0.05);58株(57.4%)耐药株第463位点突变。23株(53.3%)敏感株第463位点突变。(2)5株(4.9%)耐药株inhA发生突变。敏感株inhA无突变。(3)3株(2.9%)耐药株ahpC发生突变。敏感株ahpC无突变。(4)17株(16.8%)耐药株kasA发生突变。敏感株中3株菌株Gly312Ser突变。(5)在全部菌株中未发现oxyR基因突变。(6)综合本项研究中各基因的突变情况,共有91株耐异烟肼菌株发生与异烟肼耐药相关的突变。结论本项研究进一步证实了结核分枝杆菌耐异烟肼与katG、inhA、ahpC及kasA基因突变之间的关系,并且提示还有其他机制参与异烟肼耐药。     

中国耐利福平结核分支杆菌rpoB基因突变特点

目的 为了阐明中国结核分支杆菌耐利福平析rpoB基因突变特点。方法 对242株结核分支杆菌临床分离包括rpoB基因核心区域81个碱基在内的588个碱基进行序列测定,其中耐利福平株193株,利福平敏感株46株,人工诱导的耐利福平株3株。结果 89．1％（172．193）的临床分离耐药株存在rpoB基因突变,而46株敏感株无突变。531位氨基酸突变率为46．1％;526位氨基酸突变率为17．1％;联合突变发生率为12．4％;还有4株细菌发生同义突变;未检测到发生缺失或插入突变的菌株。高耐药组（耐250μg/ml利福平）531位氨基酸的突变率显著高于低耐药组（耐50μg/ml利福平）,P＜0．05。结论 中国结核分支杆菌耐利福平株的rpoB基因突变的发生率约为90％,其中最常见的突变位点是531位丝氨酸和526位组氨酸,两者突变率之和约为63％;利福平高耐药组531位氨基酸发生突变的几率高于低耐药组;受度蓖株未发现搬运入或缺失突变;DNA序列分析对临床用药有指导意义。     

Occurence of resistance mutation and clonal expansion in Helicobacter pylori multiple-strain infection: a potential risk in clarithromycin-based therapy.

In a previous study, a patient was shown by means of DNA fingerprinting to be infected with different Helicobacter pylori strains before and after clarithromycin treatment. The strain isolated before treatment was susceptible (ClaS), whereas the strain isolated after 3 months of treatment was resistant (ClaR). Eighty H. pylori colonies from primary isolates were analyzed by DNA fingerprinting and restriction enzyme digestion of the 23S rRNA product of polymerase chain reaction in order to distinguish between ClaS and ClaR strains. One of the 40 colonies from isolates recovered before treatment showed a DNA fingerprint similar to that of the 40 from after treatment. However, a notable difference was that this isolate was not ClaR before treatment. Two ClaS H. pylori strains were present in the patient before treatment. The underrepresented strain gained a resistance mutation in the 23S rRNA gene and underwent clonal expansion during treatment. Recrudescence of the H. pylori infection therefore was the result.