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.     

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.     

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.     

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。     

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。     

Native valve endocarditis and femoral embolism due to Granulicatella adiacens: a rare case report

The fastidious Gram-positive cocci Granulicatella adiacens, previously known as nutritionally variant streptococci (NVS) are unusual but significant cause of endocarditis due to increased mortality and morbidity. Difficulties in reaching correct bacteriological identification, increased resistance to beta-lactam antibiotics and macrolides and complicated clinical course have contributed to problems in management of cases of infective endocarditis caused by this bacterium. We present the first Indian case of endocarditis with arterial embolus by G. adiacens in an elderly male with no pre-existing cardiac abnormality.     

脓肿分枝杆菌复合群对克拉霉素耐药基因的初步研究

目的 分析脓肿分枝杆菌复合群对克拉霉素的耐药特征,为临床正确选用克拉霉素及有效控制其耐药提供依据和新思路。方法 选取中国疾病预防控制中心国家结核病参比实验室保存的2016—2017年全国耐药监测菌株中,鉴定为脓肿分枝杆菌复合群的385株临床分离株作为研究对象。采用多靶位基因测序法进行菌种鉴定和耐药基因检测;采用微孔板稀释法对菌株进行药物敏感性试验(简称“药敏试验”),检测其对克拉霉素和阿米卡星的耐药情况,并分析耐药菌株的耐药基因突变情况。结果 385株脓肿分枝杆菌复合群中,218株为脓肿分枝杆菌[包括185株为erm(41)T28基因型和33株erm (41)C28基因型],163株为马赛分枝杆菌,4株为博莱分枝杆菌。脓肿分枝杆菌、马赛分枝杆菌对阿米卡星和克拉霉素的耐药率分别为2.8%(6/218)和6.4%(14/218)、4.3%(7/163)和10.4%(17/163)。其中,erm(41)C28基因型脓肿分枝杆菌培养3d和14d对克拉毒素的耐药率(均为9.1%,3/33)不变,未发生诱导耐药,而erm(41)T28基因型脓肿分枝杆菌的耐药率则由培养3d时的5.9%(11/185)上升为培养14d的98.4%(182/185),171株发生诱导耐药。31株获得性耐药菌株中,10株脓肿分枝杆菌及10株马赛分枝杆菌均在rrl基因的2058/2059位点突变,最常见突变类型分别为A2058C(6株脓肿分枝杆菌,3株马赛分枝杆菌)和A2058G (3株脓肿分枝杆菌,5株马赛分枝杆菌);另有9株对阿米卡星耐药的菌株在rrs基因的A1408G位点发生突变,其中7株同时对克拉霉素耐药。结论 erm(41)T28基因型脓肿分枝杆菌更易诱导克拉霉素耐药。脓肿分枝杆菌和马赛分枝杆菌最常见的获得性耐药突变位点是rrl基因的A2058C和A2058G。     

Crystal structure of RlmAI: implications for understanding the 23S rRNA G745/G748-methylation at the macrolide antibiotic-binding site

The RlmA class of enzymes (RlmA(I) and RlmA(II)) catalyzes N1-methylation of a guanine base (G745 in Gram-negative and G748 in Gram-positive bacteria) of hairpin 35 of 23S rRNA. We have determined the crystal structure of Escherichia coli RlmA(I) at 2.8-A resolution, providing 3D structure information for the RlmA class of RNA methyltransferases. The dimeric protein structure exhibits features that provide new insights into its molecular function. Each RlmA(I) molecule has a Zn-binding domain, responsible for specific recognition and binding of its rRNA substrate, and a methyltransferase domain. The asymmetric RlmA(I) dimer observed in the crystal structure has a well defined W-shaped RNA-binding cleft. Two S-adenosyl-l-methionine substrate molecules are located at the two valleys of the W-shaped RNA-binding cleft. The unique shape of the RNA-binding cleft, different from that of known RNA-binding proteins, is highly specific and structurally complements the 3D structure of hairpin 35 of bacterial 23S rRNA. Apart from the hairpin 35, parts of hairpins 33 and 34 also interact with the RlmA(I) dimer.     

梅毒螺旋体阿奇霉素耐药的分子生物学检测方法的建立

目的建立梅毒螺旋体阿奇霉素耐药的分子生物学检测方法。方法为了提高检测的敏感性和特异性,应用套式聚合酶链反应（Nested-PCR）扩增样本DNA;限制性酶切反应检测阿奇霉素耐药突变;并对PCR产物和酶切产物进行测序以验证实验结果。结果套式PCR能很好地扩增样本DNA,耐阿奇霉素的菌株经酶切后形成188bp和440bp两个片段,而不耐药者不发生酶切反应。测序结果显示,耐药者的2058位DNA从A突变为G,而野生型者无此突变。结论利用套式PCR和限制性酶切分析法,成功建立了梅毒螺旋体阿奇霉素耐药的分子生物学检测方法 ,为梅毒螺旋体的耐药监测以及进一步的分子流行病学研究,提供了实验室基础。     

解脲脲原体对大环内酯类抗生素耐药机制的研究

目的探索解脲脲原体对大环内酯类抗生素的耐药机制。方法自行设计引物,对已知药敏结果的解脲脲原体进行编码核糖体蛋白L4/L22的基因以及23S rRNA的II区和V区的扩增并测序,比对分析其碱基和氨基酸的改变。结果在耐药株X01核糖体蛋白L4上,出现第67位谷氨酰胺→赖氨酸和71位甘氨酸→丝氨酸的改变;敏感株Y292经阿奇霉素诱导耐药后,L22核糖体蛋白上出现9个氨基酸的改变;耐药株Y187 23S rRNAV区出现第2566位碱基T→C、第2569位缺失碱基G和第2621位碱基A→T的变化。结论核糖体蛋白L4、L22以及23S rRNA上相应区域碱基、氨基酸的改变,可能是导致解脲脲原体对大环内酯类抗生素出现耐药的机制之一。     

Novel agents for resistant Gram-positive infections—a review

Gram-positive infections have increased in recent years, particularly those that are of nosocomial origin, leading to a broad use of agents with activity against these pathogens. Concomitantly, antimicrobial resistance of these pathogens also became widespread. Among the most common Gram-positive resistant pathogens are: Streptococcus pneumoniae, resistant to penicillin and macrolides, methicillin-resistant Staphylococcus aureus (MRSA), glycopeptide-intermediately-resistant S. aureus (GISA), methicillin-resistant S. epidermidis, glycopeptide-resistant enterococci and vancomycin-resistant enterococci (VRE). The response of the pharmaceutical industry to this challenge was the development of new antibiotics active against these pathogens. Among these antibiotics, this review will focus on: linezolid, an oxazolidinone; GAR-936, a tetracycline derivative; daptomycin, a lipopeptide; and ortivancin (LY333328), a glycopeptide related to vancomycin. Except for linezolid, which has been recently launched in many countries, all other agents referred to in this review are still at various developmental stages. It is hoped that in the near future most of these agents will be approved and thus the grim outlook of patients infected with resistant Gram-positive bacteria may improve.     

Annual changes in antimicrobial susceptibility and macrolide resistance of Streptococcus pyogenes from 1995 through 2004

This aim of this study was to reveal annual changes in antibiotic susceptibility, especially the macrolide susceptibility of Streptococcus pyogenes. A total of 755 strains of S. pyogenes were clinicaly isolated from throat swabs of children from 1995 through 2004 in Chiba Municipal Kaihin Hospital. All isolates were fully susceptible to benzylpenicillin, cefotaxim and cefaclor. The rate of resistance to erythromycin (EM) was over 10% every year after 2001 and 19% in 2004, and the rate of high resistance (MIC > or =16 microg/mL) has been increasing. A significant increase in EM resistance was observed over a 10-year period. There were 118 strains (15.6%) that persisted after treatment with beta-lactams. In the past few years it has been discovered that some S. pyogenes can be internalized by human cells of respiratory tract origin and survive within them. Since beta-lactams do not reach high intracellular concentrations, this ability of S. pyogenes may be related to treatment failure. Since macrolides can enter eukaryotic cells and remain active in intracellular compartments, they will be effective for these S. pyogenes. In case of pharyngitis which againist treatment with beta-lactams, there is a possibility macrolides are effective. Macrolides may be effective in pharyngitis resistant to treatment with beta-lactams. However, macrolide resistance is not rare, susceptibility must be tested.     

The use of macrolides in treatment of upper respiratory tract infections

Antimicrobial resistance is a growing problem among upper respiratory tract pathogens. Resistance to β-lactam drugs among Streptococcus pneumoniae, Haemophilus influenzae, and Streptococcus pyogenes is increasing. As safe and well-tolerated antibiotics, macrolides play a key role in the treatment of community-acquired upper respiratory tract infections (RTIs). Their broad spectrum of activity against gram-positive cocci, such as S. pneumoniae and S. pyogenes, atypical pathogens, H. influenzae (azithromycin and clarithromycin), and Moraxella catarrhalis, has led to the widespread use of macrolides for empiric treatment of upper RTIs and as alternatives for patients allergic to β-lactams. Macrolide resistance is increasing among pneumococci and recently among S. pyogenes, and is associated with increasing use of the newer macrolides, such as azithromycin. Ribosomal target modification mediated by erm(A) [erm(TR)] and erm(B) genes and active efflux due to mef(A) and mef(E) are the principal mechanisms of resistance in S. pneumoniae and S. pyogenes. Recently, ribosomal protein and RNA mutations have been found responsible for acquired resistance to macrolides in S. pneumoniae, S. pyogenes, and H. influenzae. Although macrolides are only weakly active against macrolide-resistant streptococci species producing an efflux pump (mef) and are inactive against pathogens with ribosomal target modification (erm), treatment failures are uncommon. Therefore, macrolide therapy, for now, remains a good alternative for treatment of upper RTIs; however, continuous monitoring of the local resistance patterns is essential.     

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.     

The use of macrolides in treatment of upper respiratory tract infections

Antimicrobial resistance is a growing problem among upper respiratory tract pathogens. Resistance to β-lactam drugs among Streptococcus pneumoniae, Haemophilus influenzae, and Streptococcus pyogenes is increasing. As safe and well-tolerated antibiotics, macrolides play a key role in the treatment of community-acquired upper respiratory tract infections (RTIs). Their broad spectrum of activity against gram-positive cocci, such as S. pneumoniae and S. pyogenes, atypical pathogens, H. influenzae (azithromycin and clarithromycin), and Moraxella catarrhalis, has led to the widespread use of macrolides for empiric treatment of upper RTIs and as alternatives for patients allergic to β-lactams. Macrolide resistance is increasing among pneumococci and recently among S. pyogenes, and is associated with increasing use of the newer macrolides, such as azithromycin. Ribosomal target modification mediated by erm(A) [erm(TR)] and erm(B) genes and active efflux due to mef(A) and mef(E) are the principal mechanisms of resistance in both S. pneumoniae and S. pyogenes. Recently, ribosomal protein and RNA mutations have been found to be responsible for acquired resistance to macrolides in S. pneumoniae, S. pyogenes, and H. influenzae. Although macrolides are only weakly active against macrolide-resistant streptococci species, producing an efflux pump (mef), and are inactive against pathogens with ribosomal target modification (erm), treatment failures are uncommon. Therefore, macrolide therapy, for now, remains a good alternative for treatment of upper RTIs; however, continuous monitoring of the local resistance patterns is essential.     

Microbiological aspects of antibiotic resistant Helicobacter pylori strains

Resistance of Helicobacter pylori to antibiotics has been described for macrolides, nitroimidizoles, and fluoroquinolones. In 1996, the mechanism of resistance to macrolides was determined to be a point mutation on the 23S rRNA which leads to decreased binding of macrolides to the ribosome. Recently, mutations in the gene coding for nitroreductase have been linked to resistance to nitroimidazoles but more work will be necessary to determine whether this is the only mechanism involved. Point mutations have also been associated with resistance to fluoroquinolones. A decreased susceptibility to amoxicillin has been observed and may be linked to changes in the penicillin binding proteins. The same phenotypic methods generally used to test antibiotic susceptibility can be applied to Helicobacter pylori. The disk diffusion method can be used for macrolides, the E-test for amoxicillin, and the point limit method for nitroimidazoles but the reference method of all of these is the agar dilution method. Molecular methods such as polymerase chain reaction-RFLP and various techniques using hybridization can also be employed but to date they have only been used for macrolides. These techniques have the advantage that they can be applied directly to the biopsy specimen.     

Molecular mechanisms of resistance to commonly used non-betalactam drugs in Streptococcus pneumoniae.

This article reviews the molecular mechanisms of resistance to fluoroquinolones, erythromycin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole in Streptococcus pneumoniae. Resistance to fluoroquinolones primarily involves mutations in the DNA gyrase gene, gyrA, and in the topoisomerase IV genes, parC and parE, although in vitro studies have indicated that some strains may use an efflux mechanism for resistance to certain fluoroquinolones. Ciprofloxacin resistance results from initial and necessary mutations in ParC leading to low-level resistance and subsequent mutations in GyrA leading to high-level resistance. Sparfloxacin resistance results from initial mutations in GyrA, with ParC mutations occurring subsequently. A single amino acid substitution in ParE has also been associated with low-level resistance in S pneumoniae. Two mechanisms have been described for resistance to erythromycin. Coresistance to macrolides, lincosamides, and streptogramin B type antibiotics is a result of modification of the ribosome through methylation of an adenine residue in domain V of the 23S rRNA. This methylation is encoded by the methylase gene, ermAM. Resistance only to 14-and 15-membered macrolides is a result of efflux of the antibiotic from the cell, encoded by the gene, mefE, in S pneumoniae, and appears to be rapidly emerging as the predominant mechanism of resistance to erythromycin in many countries. The production of chloramphenicol acetyltransferase, an enzyme capable of catalyzing the conversion of chloramphenicol to its nonfunctional 1-acetoxy, 3-acetoxy, and 1,3-diacetoxy derivatives, leads to chloramphenicol resistance in S pneumoniae. Chloramphenicol acetyltransferase is encoded by a cat gene identical to the cat gene from the Staphylococcus aureus plasmid, pC194. Tetracycline resistance occurs through ribosomal protection encoded by the genes tet(M) and tet(O). It is possible that the Tet(M) and Tet(O) proteins cause tetracycline to be released from the ribosome, although the precise mechanism remains unclear. Resistance to trimethoprim is mediated through a single amino acid substitution in the chromosomal dihydrofolate reductase gene of S pneumoniae, which is thought to disrupt the bond with trimethoprim without affecting the action of the dihydrofolate reductase. Sulphonamide resistance appears to result from repetitions of one or two amino acids in the chromosomal dihydropteroate synthase. Although resistance exists to nearly all antimicrobial agents used in the treatment of S pneumoniae infections, ongoing research into new or alternative therapies is encouraging.     

2004-2005年度全国革兰阳性菌耐药监测(Mohnarin)

目的 监测2004-2005年度全国临床分离革兰阳性菌耐药状况.方法 选定全国17家医院作为成员单位,收集特定病房2004年10月1日至2005年9月30日分离的革兰阳性致病菌,用标准平皿二倍稀释法测定35种药物体外抗菌活性,计算最低抑菌浓度(MIC)50、MIC90,依据2004年美国国家临床实验标准委员会制订的标准计算细菌对抗菌药物的耐药率、中介率和敏感率.结果 共收集革兰阳性菌925株,包括葡萄球菌536株、肠球菌249株、链球菌137株和其他革兰阳性菌3株.苯唑西林耐药金黄色葡萄球菌与苯唑西林耐药表皮葡萄球菌的检出率分别为62.9%和82.9%;肺炎链球菌对青霉素的耐药率为10.5%,中介率为30.2%,不敏感率合计为40.7%;未发现对替考拉宁中介或耐药的肠球菌,5株肠球菌对万古霉素中介,分别为1株粪肠球菌、2株屎肠球菌、1株鹑鸡肠球菌和1株鸟肠球菌;术发现糖肽类不敏感葡萄球菌.结论 革兰阳性菌耐药呈明显上升趋势,青霉素不敏感肺炎链球菌、甲氧西林耐药的葡萄球菌比例高;细菌对大环内酯类耐药率高;未发现对万占霉素耐药的革兰阳性细菌.     

Novel imatinib-sensitive PDGFRA-activating point mutations in hypereosinophilic syndrome induce growth factor independence and leukemia-like disease

The FIP1L1-PDGFRA fusion is seen in a fraction of cases with a presumptive diagnosis of hypereosinophilic syndrome (HES). However, because most HES patients lack FIP1L1-PDGFRA, we studied whether they harbor activating mutations of the PDGFRA gene. Sequencing of 87 FIP1L1-PDGFRA-negative HES patients revealed several novel PDGFRA point mutations (R481G, L507P, I562M, H570R, H650Q, N659S, L705P, R748G, and Y849S). When cloned into 32D cells, N659S and Y849S and-on selection for high expressors-also H650Q and R748G mutants induced growth factor-independent proliferation, clonogenic growth, and constitutive phosphorylation of PDGFRA and Stat5. Imatinib antagonized Stat5 phosphorylation. Mutations involving positions 659 and 849 had been shown previously to possess transforming potential in gastrointestinal stromal tumors. Because H650Q and R748G mutants possessed only weak transforming activity, we injected 32D cells harboring these mutants or FIP1L1-PDGFRA into mice and found that they induced a leukemia-like disease. Oral imatinib treatment significantly decreased leukemic growth in vivo and prolonged survival. In conclusion, our data provide evidence that imatinib-sensitive PDGFRA point mutations play an important role in the pathogenesis of HES and we propose that more research should be performed to further define the frequency and treatment response of PDGFRA mutations in FIP1L1-PDGFRA-negative HES patients.     

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.