Detection of Helicobacter pylori 23S rRNA gene mutation associated with clarithromycin resistance and its clinical applicability

Clarithromycin is one of the most important antibiotics for H. pylori eradication. However, 5-10% was reported to be resistant. It has been shown that one point mutation in the 23S rRNA gene is associated with resistance to clarithromycin. We confirmed that this finding applied to the isolates in Japan. To detect H. pylori infection and the mutation simultaneously, we have designed PCR primers specific for H. pylori, and established assays of PCR-RFLP and PCR-preferential homo-duplex formation (PHFA). Compared with other conventional methods, these assays achieved above 95% sensitivity. It is also demonstrated that the eradication rates achieved by clarithromycin-based regimens significantly differed between mutant and wild type infections. By detecting of 23S rRNA gene mutations associated with clarithromycin resistance, patients can be treated with adequate antibiotics with information about resistance.     

Selection of antibiotics and planning of eradication for H. pylori infection

There is general agreement that H. pylori should be eradicated in patients with peptic ulcers. But the optimal therapeutical regimen to be used still remains a matter for many investigations. An increase in the prevalence of antibiotic-resistant H. pylori strains has been reported recently. The recommended drugs for the eradication in Japan are clarithromycin (CAM) and amoxicillin (AMPC) because metronidazole (MNZ) is anti-parasites drug in Japan. A total of 392 H. pylori strains in the last twelve years were tested for sensitivity to CAM, MNZ, and AMPC. The Primary resistance of H. pylori to CAM, MNZ, and AMPC were found in 10.2%, 26.5%, and 0.3% strains, respectively. The resistant strains to CAM were gradually increasing in the last few years. The eradication therapies which do not increase antibiotics resistant strains after eradication failure were reported. The recommendation for eradication in patients with peptic ulcer disease includes those with bleeding ulcers. The pretreatment with proton pump inhibitors (PPI) does not influence the success of PPI-based triple therapy in eradicating H. pylori.     

Macrolide resistance in Helicobacter pylori: rapid detection of point mutations and assays of macrolide binding to ribosomes.

Resistance of Helicobacter pylori to macrolides is a major cause of failure of eradication therapies. Single base substitutions in the H. pylori 23S rRNA genes have been associated with macrolide resistance in the United States. Our goal was to extend this work to European strains, to determine the consequence of this mutation on erythromycin binding to H. pylori ribosomes, and to find a quick method to detect the mutation. Seven pairs of H. pylori strains were used, the parent strain being naturally susceptible to macrolides and the second strain having acquired an in vivo resistance during a treatment regimen that included clarithromycin. The identity of the strains was confirmed by random amplified polymorphic DNA testing with two different primers, indicating that resistance was the result of the selection of variants of the infecting strain. All resistant strains were found to have point mutations at position 2143 (three cases) or 2144 (four cases) but never on the opposite DNA fragment of domain V of the 23S rRNA gene. The mutation was A-->G in all cases except one (A-->C) at position 2143. Using BsaI and BbsI restriction enzymes on the amplified products, we confirmed the mutations of A-->G at positions 2144 and 2143, respectively. Macrolide binding was tested on purified ribosomes isolated from four pairs of strains with [14C]erythromycin. Erythromycin binding increased in a dose-dependent manner for the susceptible strain but not for the resistant one. In conclusion we suggest that the limited disruption of the peptidyltransferase loop conformation, caused by a point mutation, reduces drug binding and consequently confers resistance to macrolides. Finally, the macrolide resistance could be detected without sequencing by performing restriction fragment length polymorphism with appropriate restriction enzymes.     

Practical advice on eradicating Helicobacter pylori infection

Peptic ulcer disease associated with H pylori infection is curable. The important factors in selecting therapy are efficacy of eradication, prevention of resistance, avoidance or minimization of adverse effects, patient compliance, and cost. The most effective regimens include a bismuth preparation or antisecretory drug (proton pump inhibitor or H2 receptor antagonist) plus two antibiotics administered for 14 days. Dual-drug therapies are not recommended. Triple-drug regimens are more likely to eradicate H pylori and less likely to generate resistant strains among surviving organisms. In general, cure of the infection should be confirmed 4 weeks after completion of the treatment. Antibiotic resistance is an important consideration in choosing therapy, and patients should be taught the importance of compliance. When treatment fails, antibiotic combinations should not be repeated. Considerations for anti-H pylori treatment in a managed care environment mirror those for good medical practice in general, with special attention to stringent cost-control or outcomes-driven measures.     

Frequent association between alteration of the rdxA gene and metronidazole resistance in French and North African isolates of Helicobacter pylori

Mutations in the rdxA gene have been associated with the acquisition of resistance to metronidazole in Helicobacter pylori. This gene encodes an NADPH nitroreductase whose expression is necessary for intracellular activation of the drug. We wished to examine whether mutations in rdxA were present in resistant H. pylori isolates infecting either French or North African patients. We determined the complete nucleotide sequences of the rdxA genes from seven French and six North African patients infected with paired resistant and sensitive strains. Genotyping by random amplified polymorphic DNA analysis confirmed the close genetic relatedness of the susceptible and resistant isolates from individual biopsies. Eight French and five North African individual resistant strains were also studied. For the French strains, an alteration in rdxA most probably implicated in resistance was found in 10 cases (seven frameshift mutations, two missense mutations, and one deletion of 211 bp). One to three putative missense mutations were identified in four cases, and a missense mutation possibly not implicated in resistance was discovered in the last case. For the North African strains, an alteration in rdxA was found in eight cases (three frameshift mutations, three missense mutations, one deletion of 6 bp, and one insertion of a variant of IS605). Two strains contained putative missense mutations, and no change was observed in rdxA of the last strain. Thus, inactivation of the rdxA gene is frequently, but not always, associated with resistance to metronidazole in French and North African clinical isolates of H. pylori. In addition, a variety of alterations of rdxA are associated with the resistant phenotype.     

Antibiotic-resistant H. pylori strains in the last ten years in Japan

An increase in the prevalence of antibiotic-resistant H. pylori strains has been reported. We tested the sensitivity of H. pylori strains to antibiotics before and after the eradication treatment to determine primary and secondary resistance by E-test. A total of 336 H. pylori strains from 1987 to 1998 were tested for sensitivity to clarithromycin (CAM), amoxicillin (AMPC), and metronidazole (MNZ). Primary resistance of H. pylori to CAM, MNZ, and AMPC were found in 9.5%, 26.8%, and 0.3% strains, respectively. The resistant strains to CAM and MNZ were gradually increasing in the last few years. The cure rates of triple therapy with MNZ and CAM for CAM-susceptible and CAM-resistant H. pylori isolates were 83.3% (40/48) and 30.0% (3/10) respectively, and the cure rates of triple therapy with CAM and AMPC for CAM-susceptible and CAM-resistant H. pylori isolates were 79.3% (69/87) and 12.5% (1/8) respectively. After triple therapy, secondary resistant strains to CAM and MNZ were found in 42.1% (8/19) and 31.6% (6/19) of the cases.     

幽门螺杆菌对两种抗生素的耐药性及相关基因突变分析

目的分析浙江地区26株幽门螺杆菌（Helicobacter pylori,H.pylori）对克拉霉素和左氧氟沙星的耐药性,探讨H.pylori对克拉霉素、左氧氟沙星的耐药性,及其与23SrRNA基因、gyrA基因突变的关系。方法采用E-test法测定H.pylori对克拉霉素和左氧氟沙星的最低抑菌浓度（MIC）,提取H.pylori基因组DNA,采用PCR法扩增23SrRNA和gyrA基因片段,并对扩增产物进行测序、分析。结果药敏实验结果显示26例临床分离株中,6例对克拉霉素耐药,6例对左氧氟沙星耐药,8例对克拉霉素和左氧氟沙星双重耐药。所有克拉霉素耐药菌株23SrRNA基因都存在A2143G突变;左氧氟沙星耐药菌株gyrA基因中的喹诺酮类耐药决定区（quinolone-resistance-determining region,QRDR）存在突变,主要在第87和91位氨基酸。结论 23SrRNA基因的A2143G突变是导致H.pylori对克拉霉素耐药的主要原因;gyrA基因的87和91位氨基酸突变是导致H.pylori对左氧氟沙星耐药的主要原因。     

Antimicrobial resistant test of H. pylori

The resistance of Helicobacter pylori (H. pylori) to antibiotics leaves great influence on treatment outcome. Agar and broth dilution techniques are difficult to perform and not practical. E-test has the advantage of allowing visualization of resistant subpopulations of bacteria within zones of inhibitions. We studied point mutation of 23s-rRNA gene for H. pylori strains. (74 clarithromycin (CAM)-resistant, 6 CAM-susceptible) The results of these assays were well correlated with these of E-test. The cure rate of triple therapy with metoronidazole (MTZ) for CAM-resistant H. pylori is 100%(11/11), and that with CAM for MTZ-resistant H. pylori is 94.4% (17/18). It is very difficult to eradicate CAM and MTZ-resistant H. pylori.     

Characterization of rifampicin-resistant clinical Helicobacter pylori isolates from Germany

OBJECTIVES: The aim of this study was to assess the rate of rifampicin resistance in Helicobacter pylori isolated from patients in Germany, to detect rifampicin resistance-associated mutations and to identify non-resistance-associated genetic variants in the rpoB gene. METHODS: Susceptibility to rifampicin in a total of 1585 clinical isolates obtained between January 2003 and July 2006 was tested by disc diffusion and/or by the Etest method. The rpoB genes of a selection of both resistant (n=17) and susceptible (n=100) clinical isolates were sequenced in order to distinguish between resistance- and non-resistance-associated genetic alterations. In vitro mutagenesis experiments such as site-directed mutagenesis were carried out to demonstrate the pivotal role of rpoB mutations in rifampicin resistance. RESULTS: From 1585 clinical isolates examined, 22 (1.4%) showed phenotypic resistance to rifampicin (MIC>4 mg/L). The majority of the resistant strains harboured point mutations in their rpoB genes at codons 530, 540 and 545 and showed cross-resistance to rifabutin. Four clinical isolates with moderate rifampicin resistance (8 mg/L) showed a rifabutin-susceptible phenotype and did not harbour any mutation in the sequenced rpoB fragments. Sequence analysis of 100 rifampicin-susceptible isolates revealed numerous novel silent mutations in the rpoB genes resulting in amino acid exchanges, but not in resistance. CONCLUSIONS: Resistance to rifampicin/rifabutin in H. pylori strains isolated in Germany is still low and is associated with mutations in the rpoB gene. Further surveillance studies analysing the use of rifabutin in H. pylori eradication and its association with the occurrence of rifabutin-resistant strains are required.     

Exploring novel vaccines against Helicobacter pylori: protective and therapeutic immunization

Infection of human stomach by Helicobacter pylori, a gram negative spiral bacterium first isolated in 1983 from a patient with chronic active gastritis (1), causes nearly all duodenal ulcers and most gastric ulcers and is associated with an increased risk of gastric adenocarcinoma (2). Current therapies for gastric infections include combination triple or quadruple therapy of antimicrobial and/or antiulcer agents for eradication of H. pylori infection (3). Development of the resistant strains and ecological niche (habitant) of the bacteria may cause relapse after the termination of the therapy. However, if effective, the high cost, difficulty of patient compliance and risk of selection for resistant strains make these therapeutic regimens impractical on a large scale, though effective on the laboratory trial stages. Studies of the pathogenesis of H. pylori have led to the identification of bacterial antigens and adherin proteins as candidates for inclusion as novel vaccines against these diseases (4-7). Both prophylactic and therapeutic vaccination have been demonstrated in animal models of H. pylori infection (8-10).     

2008－2014年间北京地区幽门螺杆菌耐药变迁分析

目的 分析北京地区2008－2014年幽门螺杆菌（HP）耐药的变化情况。方法 分别收集2008年5月至2010年10月和2013年3月至2014年1月北京地区接受胃镜检查的HP感染者的胃黏膜标本,对分离获得的HP菌株经系统鉴定后,采用E-test方法进行甲硝唑（MZ）、阿莫西林（AC）、克拉霉素（CH）、左氧氟沙星（LE）、莫西沙星（MX）、利福平（RI）、四环素（TC）、庆大霉素（GM）8种抗菌药物的耐药性检测,并进行对比分析。结果 分别收集到461份（A组）和1 848份（B组）胃黏膜标本,标本进行培养后获得412和1 483株HP菌株,菌株分离率分别为89.37%和80.25%。对A组中的412株和B组中随机选择的607株HP菌株进行耐药性检测的结果显示,2013年后HP对CH、LE、MX和RI耐药率明显上升,MZ耐药率略下降,AC、TC和GM耐药率仍保持较低水平。对CH、LE、MX和RI的菌株最低抑菌浓度（MIC）分布特征进行比较发现,2013年后HP的MIC水平不论是敏感菌株还是耐药菌株,都有明显升高。结论 2013年后北京地区HP菌株对CH、LE、MX和RI的耐药率呈上升趋势,同时表现为敏感菌株和耐药菌株MIC的明显增高,是造成HP根除率显著下降的关键因素,对HP耐药判断折点的调整势在必行。     

Distribution of antibiotic MICs for Helicobacter pylori strains over a 16-year period in patients from Seoul, South Korea

Recently, the development of antibiotic resistance emerged as a significant clinical problem in the eradication of Helicobacter pylori. We investigated the MICs of antibiotics for 135 H. pylori isolates from adults in Seoul, South Korea, over the past 16 years. The MICs of amoxicillin, clarithromycin, metronidazole, tetracycline, azithromycin, and ciprofloxacin increased from 1987 to 2003. Rates of primary resistance to clarithromycin increased from 2.8% in 1994 to 13.8% in 2003. The A2144G mutation was frequently observed in the 23S rRNA gene in clarithromycin-resistant isolates. The increase in resistance to clarithromycin seems to result in a decrease in eradication efficacy for H. pylori. These results suggest that the MICs of several antibiotics for H. pylori have increased over the past 16 years in Seoul.     

Exposure to metronidazole in vivo readily induces resistance in Helicobacter pylori and reduces the efficacy of eradication therapy in mice

The Helicobacter pylori SS1 mouse model was used to characterize the development of resistance in H. pylori after treatment with metronidazole monotherapy and to examine the effect of prior exposure to metronidazole on the efficacy of a metronidazole-containing eradication regimen. Mice colonized with the metronidazole-sensitive H. pylori SS1 strain were treated for 7 days with either peptone trypsin broth or the mouse equivalent of 400 mg of metronidazole once a day or three times per day (TID). In a separate experiment, H. pylori-infected mice were administered either peptone trypsin broth or the mouse equivalent of 400 mg of metronidazole TID for 7 days, followed 1 month later by either peptone trypsin broth or the mouse equivalent of 20 mg of omeprazole, 250 mg of clarithromycin, and 400 mg of metronidazole twice a day for 7 days. At least 1 month after the completion of treatment, the mice were sacrificed and their stomachs were cultured for H. pylori. The susceptibilities of isolates to metronidazole were assessed by agar dilution determination of the MICs. Mixed populations of metronidazole-resistant and -sensitive strains were isolated from 70% of mice treated with 400 mg of metronidazole TID. The ratio of resistant to sensitive strains was 1:100, and the MICs for the resistant strains varied from 8 to 64 micrograms/ml. In the second experiment, H. pylori was eradicated from 70% of mice treated with eradication therapy alone, compared to 25% of mice pretreated with metronidazole (P < 0.01). Mice still infected after treatment with metronidazole and eradication therapy contained mixed populations of metronidazole-resistant and -sensitive isolates in a ratio of 1:25. These results demonstrate that H. pylori readily acquires resistance to metronidazole in vivo and that prior exposure of the organism to metronidazole is associated with failure of eradication therapy. H. pylori-infected mice provide a suitable model for the study of resistance mechanisms in H. pylori and will be useful in determining optimal regimens for the eradication of resistant strains.     

浙江省宁波市耐多药结核分枝杆菌吡嗪酰胺耐药特征及与二线抗结核药物耐药关系

目的研究结核分枝杆菌(MTB)对吡嗪酰胺耐药特征及其与二线抗结核药物耐药相关性。方法以2015-2017年浙江省宁波地区结核病耐药监测收集的110例耐多药结核病(MDR-TB)病例作为研究对象,采用1%比例法对其进行5种二线抗结核药物(氧氟沙星、左氧氟沙星、卡那霉素、阿米卡星、卷曲霉素)的耐药检测。同时应用BACTEC MGIT 960系统检测所有MDR-MTB的吡嗪酰胺耐药性,采用PCR DNA直接测序法检测MDR-MTB的pncA基因突变特征。结果110株MDRMTB中吡嗪酰胺耐药率为59.09%(65/110),pncA基因突变率为50.91%(56/110)。吡嗪酰胺耐药株中的基因突变率83.08%(54/65)与吡嗪酰胺敏感株中的基因突变率4.44%(2/45)比较,差异有统计学意义(χ^2=65.787,P<0.001)。pncA基因突变类型为42种,以点突变类型为主92.86%(39/42)。耐链霉素、耐乙胺丁醇、耐氧氟沙星、耐左氧氟沙星及准广泛耐药(PreXDR)与MDR-MTB耐吡嗪酰胺相关。结论本地区MDR-MTB耐吡嗪酰胺形势较为严峻,MDR-MTB耐吡嗪酰胺与二线抗结核药物氧氟沙星、左氧氟沙星及Pre-XDR的耐药相关。     

Rapid detection of clarithromycin resistance in Helicobacter pylori using a PCR-based denaturing HPLC assay

OBJECTIVES: We evaluated a new approach for the rapid detection of clarithromycin resistance in Helicobacter pylori, based on PCR and denaturing HPLC (DHPLC). METHODS: A 180 bp fragment of the 23S rRNA gene was amplified using DNA from 81 clinical H. pylori isolates (51 isolates were shown to be resistant to clarithromycin by Etest), and, directly, from 101 gastric biopsies from patients with digestive diseases, who were infected with H. pylori as assessed by a 13C-urea breath test, histology and/or culture. DHPLC was used to detect mutations in all the PCR products. RESULTS: DHPLC profiles for the 30 susceptible isolates all showed homoduplex peaks; the resistant isolates consistently generated heteroduplex peaks that were easily distinguishable from the wild-type H. pylori reference strain. Sequencing revealed point mutations in all the resistant isolates. Overall, five different mutations were detected. Four of these mutations (A2142G, A2142C, A2143G and T2182C) are known to be associated with clarithromycin resistance; the remaining mutation (C2195T) has not been previously described. This novel single-base substitution was found in combination with the common mutation A2143G. Of the biopsies tested, 25 specimens generated heteroduplexes due to sequence alterations (mutation A2142G, A2142C or A2143G). In one of these specimens, A2143G was found together with the novel mutation T2221C; in another, a mixture of wild-type and mutant (A2143G) sequences was detected. For 20 culture-positive out of the 25 biopsies DHPLC results confirmed the presence of clarithromycin resistance. CONCLUSIONS: Our results suggest that the PCR-DHPLC assay is a valid tool for rapid assessment of clarithromycin resistance in H. pylori and that in the future it could be used directly on biopsy specimens, avoiding the need for culture-based methods.     

A strategy for second-line anti-Helicobacter pylori therapy in eradication-failure patients

Although available H. pylori eradication regimens in Japan fail to cure 10-20% of patients, an optimal re-treatment therapy for eradication-failure patients has still not been established. Since patient compliance, bacterial resistance and genotypic differences in CYP2C19 influence the eradication rate, re-eradication therapy should be selected, taking them into consideration. In the West, meta-analysis of the second-line treatment of H. pylori infection showed therapies comprising ranitidine bismuth and two antimicrobials are very effective re-treatment therapies irrespective of factors influencing H. pylori eradication. However ranitidine bismuth is not available in Japan and re-eradication therapy consisting of PPI, amoxicillin and metronidazole have been often undertaken and have achieved high eradication rate, even including patients with metronidazole resistant H. pylori.     

In vivo selection of a target/efflux double mutant of Pseudomonas aeruginosa by ciprofloxacin therapy

We report the emergence after 4 days of ciprofloxacin monotherapy of a double mutant of Pseudomonas aeruginosa overexpressing the multidrug efflux system MexAB-OprM and harbouring a mutation in the gyrB gene. Compared with its initial susceptible counterpart, this mutant exhibited a significant increase in resistance to most of the beta-lactam antibiotics tested (16 x MIC of ticarcillin) and to ciprofloxacin (128 x MIC). Combined ceftazidime and amikacin therapy finally eradicated the resistant isolate and cured the patient of his infection. This case illustrates how strains of P. aeruginosa may develop high levels of fluoroquinolone resistance by combining efflux mechanisms and target alterations.     

In a view of international consensus conference for the treatment of H. pylori infection

The first guideline of the treatment of H. pylori infection was reported by the working party of the World Congresses of Gastroenterology in 1990. Subsequently, many international consensus statements for the indication and the methods of eradication therapy for H. pylori infection were reported. And, now the standard regimens have been developed in European countries and U.S.A. The other hand, in Japan, the Ministry of Welfare has never been to approve the both of testing and treatment of H. pylori. One of the reasons is that there is a few scientific evidence for the benefit of the treatment of H. pylori infection in Japan. Since the resistant strains of H. pylori against antimicrobial agents are increasing in our country, we have to develop a strong and safety therapeutic regimen in Japan.     

High eradication rate of H. pylori with moxifloxacin-based treatment: a randomized controlled trial

INTRODUCTION: Eradication of Helicobacter pylori remains a problematic treatment issue in clinical practice. The intention is to find a treatment that achieves a high rate of eradication at a low price and treatment options that are now used give us the opportunity to achieve this goal. Recently published results showing a low rate of resistance and better compliance with moxifloxacin-based treatment regimens indicate the need to investigate its efficacy in H. pylori eradication. This study is based on proving the efficacy of moxifloxacin in H. pylori eradication within the triple therapy. AIMS AND METHODS: The aim of the study was to compare the efficacy of one week of moxifloxacin-based treatment with the standard treatment for H. pylori eradication. Patients with H. pylori infection and non-ulcer dyspepsia (n = 277) were randomly divided into four groups to receive: moxifloxacin 400 mg/d, metronidazole 400 mg twice daily, lansoprazole 30 mg twice daily (MML group); moxifloxacin 400 mg/d, amoxicillin 1 g twice daily, lansoprazole 30 mg twice daily (MAL group); clarithromycin 500 mg twice daily, metronidazole 400 mg twice daily, lansoprazole 30 mg twice daily (CML group); clarithromycin 500 mg twice daily, amoxicillin 1 g twice daily, lansoprazole 30 mg twice daily (CAL group). The patients were assessed for prevalence of H. pylori using the CLO test, histology and culture on gastric biopsy samples obtained during upper gastrointestinal endoscopy before randomization and 4-6 weeks after completion of treatment. Bacterial sensitivity to clarithromycin and moxifloxacin was determined with the E-test. RESULTS: 265 (95.6%) patients completed the study forming the basis for PP analysis. Eradication rates of H. pylori in ITT and in PP analyses were: in the MML group 93.5% (58/62) and 96.7% (58/60), respectively; in the MAL group 86.4% (57/66) and 90.5% (57/63); in the CML group 70.4% (50/71) and 75.8% (50/66); and in the CAL group 78.2% (61/78) and 80.2% (61/76). Moxifloxacin treatment protocols were significantly more effective on both ITT and PP analyses than the clarithromycin based protocols with only one exception (MAL vs. CAL on ITT analysis). Among 238 patients (86% of the entire study group), strains showing primary resistance to clarithromycin were found in 10.8% and to moxifloxacin in 5.9%. Eradication of moxifloxacin sensitive/resistant strains was 98.1%/75% for MML (p < 0.01) and 91.1%/66.7% for MAL (p = n.s.); comparison of eradication of sensitive strains in MML and MAL regimens was 98.1%/91.1% (p < 0.05), and for resistant strains 75%/66.7% (p = n.s.). CML and CAL protocols did not differ in efficacy of eradication of clarithromycin sensitive or resistant strains. CONCLUSION: Moxifloxacin-based triple therapies showed higher eradication rates with few side effects and good drug compliance when compared with standard H. pylori treatments. Moreover, the increased prevalence of clarithromycin resistance suggests that moxifloxacin-based regimens could be safe and effective options in treatment of H. pylori infection.     

New site of modification of 23S rRNA associated with clarithromycin resistance of Helicobacter pylori clinical isolates

Resistance of Helicobacter pylori to clarithromycin occurs with a prevalence ranging from 0 to 15%. This has an important clinical impact on dual and triple therapies, in which clarithromycin seems to be the better choice to achieve H. pylori eradication. In order to evaluate the possibility of new mechanisms of clarithromycin resistance, a PCR assay that amplified a portion of 23S rRNA from H. pylori isolates was used. Gastric tissue biopsy specimens from 230 consecutive patients were cultured for H. pylori isolation. Eighty-six gastric biopsy specimens yielded H. pylori-positive results, and among these 12 isolates were clarithromycin resistant. The latter were studied to detect mutations in the 23S rRNA gene. Sequence analysis of the 1,143-bp PCR product (portion of the 23S rRNA gene) did not reveal mutation such as that described at position 2142 to 2143. On the contrary, our findings show, for seven isolates, a T-to-C transition at position 2717. This mutation conferred a low level of resistance, equivalent to the MIC for the isolates, selected using the E-test as well as using the agar dilution method: 1 micro g/ml. Moreover, T2717C transition is located in a highly conserved region of the 23S RNA associated with functional sites: domain VI. This fact has a strong effect on the secondary structure of the 23S RNA and on its interaction with macrolide. Mutation at position 2717 also generated an HhaI restriction site; therefore, restriction analysis of the PCR product also permits a rapid detection of resistant isolates.