结核分枝杆菌gyrA基因突变与氟喹诺酮类药物体外最低抑菌浓度的关系

目的探讨结核分枝杆菌gyrA基因突变与氟喹诺酮类药物体外最低抑菌浓度(MIC)的关系。方法收集640例痰结核分枝杆菌阳性临床分离株,用探针熔解曲线法筛选gyrA基因耐药决定区突变株,对其gyrA基因耐药决定区进行测序,并检测氧氟沙星、左氧氟沙星、莫西沙星及加替沙星对不同突变位点菌株的MIC,分析其差异。结果探针熔解曲线法筛选出gyrA突变株共45株(7.03%),其中90位点突变15株(33.33%)、94位点突变26株(57.78%)、91位点突变4株(8.89%)。90位点突变菌株中,氧氟沙星高水平耐药2株,左氧氟沙星无耐药,莫西沙星低水平耐药3株、高水平耐药2株,加替沙星高水平耐药3株; 91位点突变菌株中,氧氟沙星、左氧氟沙星无耐药,莫西沙星高水平耐药2株,加替沙星高水平耐药1株; 94位点突变菌株中,氧氟沙星低水平耐药4株、高水平耐药7株,左氧氟沙星低水平耐药7株,莫西沙星低水平耐药2株、高水平耐药13株,加替沙星低水平耐药3株、高水平耐药14株。除左氧氟沙星外, 90位点突变菌株对其他3种药物易出现高水平耐药; 91位点突变菌株对莫西沙星易出现高水平耐药; 94位点突变菌株对莫西沙星、加替沙星多为高水平耐药,其中天冬氨酸(Asp)→天冬酰胺(Asn)和Asp→甘氨酸(Gly)突变类型的菌株更易出现。结论结核分枝杆菌gyrA基因突变类型与氟喹诺酮类药物MIC相关,可为临床用药提供重要参考。     

肺炎支原体对大环内酯类抗生素耐药性及耐药机制研究

目的 了解肺炎支原体(mycoplasma pneumoniae,MP)对大环内酯类抗生素的耐药情况及耐药机制.方法 对370份咽拭子标本进行MP分离培养,应用套式PCR扩增MP种特异16S核蛋白体RNA(16SrRNA)基因对临床分离株进行分子鉴定;通过药物敏感实验测定MP分离株对大环内酯类药物的MIC并筛选出耐药株;设计套式PCR扩增红霉素作用靶位23S核蛋白体RNA(23SrRNA)基因,扩增产物进行全自动DNA测序,测得序列与NCBI已登录的MP标准株M129(登录号X68422)23SrRNA基因作比对.结果 370份临床标本中分离MP 50株,分离阳性率为13.5%.50株中敏感株4株,耐药株46株(占92%).耐药菌株的红霉素、阿奇霉素、交沙霉素MIC值均升高.4株敏感株和肺炎支原体国际标准株FH的23SrRNA基因序列与基因库的MP基因序列相同,46株耐药株的23SrRNA基因发生点突变,41株突变位点在23SrRNA V区中心环的2063位,其中40株发生了A→G的点突变,1株发生了A→C的点突变;另5株突变位点在2064位,A→G.结论 MP对大环内酯类抗生素耐药率高,耐药性的分子基础是23SrRNA基因的点突变,其中2063位点突变占主导地位.23SrRNA基因发生点突变的肺炎支原体对红霉素、阿奇霉素及交沙霉素的MIC值均升高.     

2012－2017年天津市百日咳鲍特菌耐药情况分析

目的 了解2012 — 2017年天津市百日咳鲍特菌菌株对抗生素敏感性的分布情况及耐药位点变异情况,为百日咳防治提供依据。 方法 以2012年2月至2017年7月天津市分离的18株百日咳鲍特菌菌株作为研究对象,采用纸片扩散法对培养的阳性菌株进行红霉素、阿奇霉素、克拉霉素、左氧氟沙星及克林霉素敏感性进行检测,同时采用E-test法检测菌株对红霉素、阿奇霉素、克拉霉素、左氧氟沙星及复方新诺明的敏感性;对红霉素结合区域23S rRNA domain V基因片段进行扩增、序列测定及分析。 结果 纸片扩散法结果显示,18株菌株中仅1株对5种抗生素均敏感,其余17株对红霉素、阿奇霉素、克拉霉素及克林霉素均耐药,红霉素耐药率最高,达94.44%（17/18）,18株菌对左氧氟沙星均敏感;E-test法结果显示,仅1株菌对红霉素、阿奇霉素、克拉霉素的最低抑制浓度（MIC）在敏感范围内,其余17株对红霉素、阿奇霉素、克拉霉素的MIC均＞256 μg/ml,18株菌对左氧氟沙星的MIC为0.19～0.38 μg/ml,复方新诺明的MIC为0.047～1.500 μg/ml;23S rRNA扩增产物序列分析发现,17株菌发生A2047G位点突变。 结论 纸片扩散法和E-test法对红霉素、阿奇霉素、克拉霉素和左氧氟沙星的耐药结果完全符合,17株菌对红霉素、阿奇霉素、克拉霉素同时耐药,耐药株23S rRNA序列均发生A2047G红霉素耐药位点改变。      

幽门螺杆菌对抗生素耐药的分子机制研究进展

随着幽门螺杆菌(Helicobacter pylori,H.pylori)耐药率的上升,对其耐药的分子机制的研究日益受到人们的重视并取得了很大进展.甲硝唑的耐药主要与rdxA和frxA基因突变有关;克拉霉素的耐药主要与23SrRNA V区上的点突变有关;阿莫西林的耐药主要与青霉素结合蛋白( PBPs)的突变有关;喹诺酮类的耐药主要与gyrA 基因喹诺酮类药物耐药决定区(QRDR)的突变有关;四环素的耐药主要与编码 16SrRNA的基因突变有关;呋喃唑酮的耐药可能与porD和oorD 基因突变有关.     

铜绿假单胞菌对喹诺酮类药物耐药机制的研究

目的研究30株铜绿假单胞菌对喹诺酮类药物的耐药机制。方法用PCR及测序法研究拓扑异构酶Ⅱ和Ⅳ的gyrA、gyrB、parC和parE基因;同时用琼脂稀释法测定环丙沙星、左氧氟沙星的MIC和加入羰基氢氯苯腙(CCCP)后的MIC,以确定存在外排机制。结果菌株中23株(76.7%)有gyrA突变,主要为Thr-83→Ile,第87位突变3株;10株(33.3%)parC基因突变,其中5株为Ser-87→Leu,3株第91位突变;gyrB和parE突变较少见。CCCP能显著降低环丙沙星和左氧氟沙星的MIC。结论铜绿假单胞菌的耐药性日趋严重,两类拓扑异构酶基因突变和外排泵机制是铜绿假单胞菌耐喹诺酮类药物的主要机制。     

基于可视化基因芯片技术指导幽门螺杆菌个体化治疗的临床研究

目的利用可视化基因芯片检测技术检测结果指导幽门螺杆菌"个体化"治疗,评价其优越性。方法 244例初诊幽门螺旋杆菌感染患者的胃黏膜组织,检测组织标本中幽门螺旋杆菌gyrA基因上喹诺酮耐药相关的Asn87(N87K)、Asp91(D91G/Y/N)突变位点和23s rRNA基因上克拉霉素耐药相关的A2142G、A2143G突变位点,以及人CYP2C19上与PPI代谢相关的等位基因2C19*2、2C19*3突变位点。根据检测结果喹诺酮和克拉霉素的敏感性分为敏感及耐药;PPI药物代谢活性分为强代谢型(EM)、中代谢型(IM)和弱代谢型(PM);结合检测结果给予幽门螺杆菌"个体化"四联根除治疗,并统计根除率。结果 244例中检出63例对抗生素耐药,耐药率25.80%(63/244),其中左氧氟沙星和克拉霉素耐药率耐药率2.86%(7/244)和20.49%(50/244),两者双重耐药率2.45%(6/244)。244例中检出CYP2C19代谢类型有242例(99.18%),其中EM、IM、PM、分别占40.08%(97/242)、58.26%(141/242)和1.65%(4/242)。242例检测结果阳性患者根据检测结果给予幽门螺杆菌个体化的"四联"治疗,240例完成治疗,初次根除率达到91.67%。EM、IM、PM的根除率分别为89.58%、92.86%和100%,差异无统计学意义(P>0.05)。结论可视化基因芯片技术能够同时提供准确的CYP2C19代谢类型和克拉霉素、左氧氟沙星耐药情况,指导临床用药,具有较好的临床应用价值。     

淋病奈瑟菌的耐药性及氟喹诺酮耐药基因突变的分析

目的 了解本地区淋病奈瑟菌(NG)流行株对5种抗生素的敏感性,分析NG的耐药性特点,为淋病的治疗提供实验依据。方法 纸片扩散法检测108株NG对5种抗生素的敏感性,用产色头孢硝噻吩法检测β-内酰胺酶,用聚合酶链反应(PCR)扩增NG gyrA和parC基因的氟喹诺酮耐药决定区(QRDR)相关序列并作测序分析。结果 108株NG中检出84株对青霉素耐药(77．8％),由质粒介导的产青霉素酶NG(PPNG)为60株(55．6％);四环素耐药率为61．1％,其中质粒介导高度耐四环素NG(TRNG)为28株,占25．9％;环丙沙星耐药率为83．3％;未发现对大观霉素和头孢曲松的耐药菌株。耐氟喹诺酮的22株NG均发生gyrA基因的91、95氨基酸位点突变,原91位氨基酸全部由丝氨酸转变为苯丙氨酸,parC基因的氨基酸突变位点分别为86、87和91。结论 青霉素、四环素以及氟喹诺酮类药物已不宜作为本地区治疗淋病的常规药物;大观霉素、头孢曲松可作为治疗淋病的首选药物,基因检测分析证明了NG gyrA和parC基因突变与氟喹诺酮耐药性有关。     

耐左氧氟沙星结核分枝杆菌gyrA基因上新的突变位点

目的:分析耐左氧氟沙星结核分枝杆菌临床菌株gyrA基因的突变情况及其耐药机制.方法:用聚合酶链反应(PER)和DNA直接测序技术(DS)测定64株耐左氧氟沙星结核分枝杆菌gyrA基因的QRDR(quinolone resistance-determining regions)序列.结果:64株耐药菌株有47株QRDR序列发生突变,其中45株为单位点突变,另2株为双位点突变;突变分布为70位突变2株、89位1株、90位12株、91位4株、94位30株,其中70位和89位为新发现的突变位点.结论:结核分枝杆菌喹诺酮类药物耐受现象与gyrA基因QRDR的突变有关,包括新发现的70位和89位突变.     

幽门螺杆菌对左氧氟沙星、克拉霉素耐药性及基因突变分析

目的 分析幽门螺杆菌(Hp)对左氧氟沙星、克拉霉素的耐药表型及与耐药相关基因突变(基因型)的相关性,为临床制定Hp个体化根除治疗方案提供实验依据。方法 分别选择2021年6月至12月和2018年8月至2019年3月在南京医科大学附属南京医院就诊的未经治疗的292例和350例Hp阳性患者的胃黏膜组织进行分离培养,用E-test药敏试验法检测左氧氟沙星、克拉霉素的耐药性,用卡方检验分析不同性别、年龄患者耐药表型的差异;用基因测序方法检测耐药相关基因型,并分析与耐药表型的一致性。结果 在左氧氟沙星耐药性研究的292例病例中,有120例(41.10%)表型耐药,不同性别(P=0.031)、年龄(P<0.01)患者间耐药率差异均有统计学意义;耐药相关基因型中有123例(42.12%)突变,与表型的一致性很高(Kappa=0.951,P<0.01),且主要耐药基因gyrA的第87位突变与表型的一致性略高于第91位突变。在克拉霉素耐药性研究的350例病例中,有168例(48.00%)表型耐药,不同性别(P=0.165)、年龄(P=0.192)患者间耐药率差异均无统计学意义;耐药相关基因型...     

舟山群岛渔民幽门螺杆菌克拉霉素耐药菌株23S rRNA全基因生物信息学研究

目的:通过对舟山群岛渔民胃镜检查患者分离胃黏膜幽门螺杆菌(Hp),对Hp克拉霉素的耐药情况和耐药基因进行研究,指导合理使用克拉霉素治疗Hp。方法:取188份胃镜活检标本,Hp培养后对188株Hp临床分离株采用E-test法行克拉霉素药敏试验。对17株克拉霉素耐药和3株敏感的Hp23SrRNA全基因序列和3株标准序列进行比对和生物信息学分析。结果:胃炎患者Hp培养阳性率为26.5%(27/102),溃疡患者培养阳性率为48.7%(38/78),胃癌患者培养阳性率为25.0%(2/8),溃疡患者Hp培养阳性率明显高于胃炎患者和胃癌患者。67例Hp菌株克拉霉素耐药23株,占34.3%(23/67),敏感株44株,占65.7%(44/67)。胃炎患者、溃疡患者和胃癌患者克拉霉素耐药率分别为29.6%(8/27)、36.8%(14/38)和50.0%(1/2)。17株克拉霉素耐药Hp菌株2143位点A>G突变率为29.4%,2182位点C>T突变率为11.8%;新的突变点C588U在6株耐药株中出现,而且该6株菌未见其他已知突变。结论:舟山群岛渔民溃疡患者Hp培养阳性率和克拉霉素耐药率明显高于胃炎患者。本地区分离的克拉...     

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.     

Distribution of spontaneous gyrA mutations in 97 fluoroquinolone-resistant Helicobacter pylori isolates collected in France

We determined the prevalence of gyrA mutations conferring fluoroquinolone resistance in 97 Helicobacter pylori isolates collected in France from 2007 to 2010. Ninety-four harbored one or two mutations already found in the quinolone resistance determining region (QRDR) of gyrA (for T87I, n = 23; for N87K, n = 32; for D91N, n = 30; for D91G, n = 7; for D91Y, n = 6), 2 harbored a mutation never previously described (D91H and A88P), and one strain was resistant (ciprofloxacin MIC of 8 mg/liter) without a detected mutation conferring this resistance in gyrA or gyrB genes.     

Distribution of fluoroquinolone MICs in Helicobacter pylori strains from Korean patients

OBJECTIVES: The aim of this study was to assess the prevalence rate of primary fluoroquinolone resistance in Helicobacter pylori isolates from Korean patients over the past 16 years. METHODS: One hundred and thirty-five strains of H. pylori (34 strains in 1987, 36 in 1994 and 65 in 2003) were isolated from antral gastric mucosal biopsy specimens. The determination of MICs for the H. pylori isolates of ciprofloxacin, levofloxacin and moxifloxacin was examined by using the serial 2-fold agar dilution method. DNA sequences of the gyrA gene in fluoroquinolone-resistant strains were determined. RESULTS: The distribution of fluoroquinolone MICs (ciprofloxacin, levofloxacin and moxifloxacin) shifted to higher concentrations during 1987-2003. All of the levofloxacin- or moxifloxacin-resistant strains were resistant to ciprofloxacin. Sequence analysis in fluoroquinolone-resistant strains showed point mutation of the gyrA gene at A272G and G271A, indicating mutations of the codon Asp-91 in the fluoroquinolone-resistance-determining region of the DNA gyrase. CONCLUSIONS: These results suggest that resistance to fluoroquinolones has been increasing in the Korean population and the resistance is most likely mediated through point mutation in gyrA.     

Genotypic resistance in Helicobacter pylori strains correlates with susceptibility test and treatment outcomes after levofloxacin- and clarithromycin-based therapies

The accuracy of genotypic resistance to levofloxacin (gyrA mutations) and its agreement with treatment outcomes after levofloxacin-based therapy have not been reported. We aimed to assess the correlation. Helicobacter pylori strains isolated from patients who received levofloxacin-based and clarithromycin-based triple therapies in a previous randomized trial were analyzed for point mutations in gyrA and 23S rRNA. PCR followed by direct sequencing was used to assess the gyrA and 23S rRNA mutations. An agar dilution test was used to determine the MICs of clarithromycin and levofloxacin. We found that the agreement between genotypic and phenotypic resistance to levofloxacin was best when the MIC breakpoint was >1 μg/ml (kappa coefficient, 0.754). The eradication rates in patients with and without gyrA mutations were 41.7% and 82.7%, respectively (P = 0.003). The agreement between genotypic and phenotypic resistance to clarithromycin was best when the MIC breakpoint was >2 μg/ml (kappa, 0.694). The eradication rates in patients with and without 23S rRNA mutations were 7.7% and 93.5%, respectively (P < 0.001). The agreements (kappa coefficient) between therapeutic outcomes after clarithromycin-based triple therapy and genotypic and phenotypic resistance were 0.671 and 0.356, respectively. The agreements (kappa coefficient) between therapeutic outcomes after levofloxacin-based triple therapy and genotypic and phenotypic resistance were 0.244 and 0.190, respectively. In conclusion, gyrA and 23S rRNA mutations in H. pylori strains appeared to be better markers than phenotypic resistance in the prediction of treatment outcomes. The optimal breakpoints for levofloxacin and clarithromycin resistance appeared to be >1 μg/ml and >2 μg/ml, respectively.     

石家庄地区幽门螺杆菌临床分离株耐药性分析

目的了解石家庄地区幽门螺杆菌(Helicobacter pylori,H.pylori)临床分离株对多种抗生素的耐药状况,为选择有效的H.pylori根除方案提供临床指导。方法收集200例H.pylori感染的患者,对其胃黏膜活检组织进行H.pylori分离培养,采用Kirby-Bauer法检测H.pylori临床分离株对甲硝唑、克拉霉素、阿莫西林、左氧氟沙星、呋喃唑酮、四环素等6种抗生素的耐药率。结果从200例患者中分离出121株H.pylori,阳性率60.5%,不同性别、年龄的培养阳性率差异无统计学意义(P0.05);H.pylori对甲硝唑、克拉霉素、阿莫西林、左氧氟沙星、呋喃唑酮和四环素的耐药率分别为95.0%(115/121)、26.4%(32/121)、2.5%(3/121)、5.8%(7/121)、0和3.3%(4/121)。不同性别、年龄的耐药率差异无统计学意义(P0.05)。结论石家庄地区H.pylori对甲硝唑、克拉霉素的耐药率较高,这些药物不宜作为治疗H.pylori的一线药物;阿莫西林、左氧氟沙星、呋喃唑酮和四环素的耐药率较低,可作为治疗H.pylori感染的主要药物。     

gyrA、gyrB和外排系统共同介导铜绿假单胞菌对喹诺酮类耐药机制研究

目的探讨临床分离的对环丙沙星和左氧氟沙星均耐药的铜绿假单胞菌耐药机制。方法收集临床分离经VITEK-2（C0mpact细菌鉴定仪检测环丙沙星和左氧氟沙星均耐药的铜绿假单胞菌,琼脂稀释法测定环丙沙星和左氧氟沙星的MIC值,PCR扩增DNA促旋酶基因gyrA和gyrB以及DNA拓扑异构酶Ⅳ的parC和parE基因,实时-RT-PCR分析细菌外排系统表达情况。结果琼脂稀释法检测结果与VITEK-2 Compact细菌鉴定仪检测结果相符。PCR扩增测序发现以DNA促旋酶基因gyrA（在位点941处插入碱基C）和gyrB（3株在位点1588处缺失碱基A,其他菌株在位点1543处插入碱基T）基因缺失或者插入导致移码突变为主,parE基因有3株在位点1895处插入碱基C。实时定量PCR检测发现以mexA和mexC表达增加为主。结论检出的耐环丙沙星和左氧氟沙星铜绿假单胞菌是由于DNA促旋酶基因gyrA和gyrB基因的突变和mexAB-OprM和mexCD-OprJ表达增加共同作用的结果。     

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.     

Accurate prediction of macrolide resistance in Helicobacter pylori by a PCR line probe assay for detection of mutations in the 23S rRNA gene: multicenter validation study

Helicobacter pylori strains from 299 patients were tested in six laboratories in different countries. Macrolide susceptibility of the strains was determined by agar dilution (17.4%) or the epsilometer test (82.6%). Mutations in the 23S ribosomal DNA (rDNA) that are associated with macrolide resistance were analyzed by PCR and reverse hybridization (PCR-line probe assay [LiPA]). This method identifies A2115G, G2141A, A2142G, A2142C, A2142T, A2143G, and A2143C mutations in the 23S rDNA. vacA s-region (s1a, s1b, s1c, and s2) and m-region (m1, m2a, and m2b) genotypes and cagA status were also determined using another PCR-LiPA system. Of the 299 strains investigated by MIC testing, 130 (43.5%) were resistant and 169 (56.5%) were susceptible to clarithromycin. Of the 130 resistant strains, 127 (97.7%) contained 23S rDNA mutations, whereas 167 (98.8%) of the 169 susceptible strains contained wild-type sequences. The predominant mutations were A2143G (45.2%) and A2142G (33.3%). Twenty-eight (19.8%) strains contained multiple 23S rDNA mutations. Only five resistant strains contained the A2142C mutation (three of these in combination with the A2142G mutation), and the A2115G, G2141A, A2142T, and A2143C mutations were not found. MICs of clarithromycin for the A2142G mutant strains were significantly higher than MICs for the A2143G strains. Although there was no significant association between 23S rDNA mutations and the vacA and cagA status, clarithromycin-susceptible strains more often contained mixed vacA genotypes, indicating the presence of multiple H. pylori strains. In conclusion, our data confirmed the very strong association between 23S rDNA mutations and macrolide resistance and showed that the PCR-LiPA permits accurate and reliable diagnosis of macrolide resistance in 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.