Hakemi Vala Mojdeh, Eyvazi Shirin, Goudarzi Hossein, Sarie Hamid Reza, Gholami Mehrdad
Department of Microbiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran.
Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran.
Jundishapur J Microbiol. 2016 Mar 19;9(5):e29839. doi: 10.5812/jjm.29839. eCollection 2016 May.
Helicobacter pylori is an important pathogen of human gastric mucosa. Antibiotic resistance, especially resistance to clarithromycin is a major factor for treatment failure of H. pylori infections. The main mechanism of clarithromycin resistance in these bacteria is related to point mutations in three different locations of 23S rRNA gene.
The aims of this study were to evaluate the resistance rate to clarithromycin among local H. pylori isolates by the E-test method and to determine the profile of point mutation in 23S rRNA by real-time polymerase chain reaction (PCR) method.
Eighty biopsy samples were collected from dyspeptic patients by endoscopy during 2011 - 2012. All samples were homogenized immediately and cultured on supplemented brucella blood agar and incubated under microaerophilic conditions. Further biochemical tests and ureC gene PCR was done for H. pylori confirmation. The H. pylori OC1096 strain was used as the control strain, simultaneously. Frequency of clarithromycin resistance was determined by the E-test method based on the clinical and laboratory standard institute (CLSI) standards. Point mutation profile was determined by real-time PCR and further analysis of melting curve, amplicon sequencing was done continuously.
From 80 biopsy samples, 20 positive H. pylori isolates were detected and confirmed by biochemical tests and PCR method. Overall, 21.7% of the H. pylori isolates, showed clarithromycin resistance phenotype by use of the E-test. Also, the minimal inhibitory concentration of clarithromycin was determined as ≥ 0.5 mg/L by the E-test method. Only point mutation in the location of A2143G with melting temperature of 54.7°C was observed in all resistant isolates.
This study showed that the frequency of H. pylori clarithromycin resistance in Iran is relatively high. Since clarithromycin is not commonly used in Iran for H. pylori eradication, the high rate of resistance could be related to cross-reactivity between other macrolides. Therefore, macrolide antibiotics must be prescribed with precaution in any case of treatment other than H. pylori infections. All resistant isolates showed A2143G mutation in 23S rRNA as the dominant pattern of point mutation at least in Tehran H. pylori isolates.
幽门螺杆菌是人类胃黏膜的重要病原体。抗生素耐药性，尤其是对克拉霉素的耐药性是幽门螺杆菌感染治疗失败的主要因素。这些细菌中克拉霉素耐药的主要机制与23S rRNA基因三个不同位置的点突变有关。
本研究旨在通过E-test法评估当地幽门螺杆菌分离株对克拉霉素的耐药率，并通过实时聚合酶链反应（PCR）法确定23S rRNA中的点突变情况。
2011 - 2012年期间，通过内镜检查从消化不良患者中收集80份活检样本。所有样本立即匀浆，接种于补充有布鲁氏菌血琼脂的培养基上，并在微需氧条件下培养。进一步进行生化试验和ureC基因PCR以确认幽门螺杆菌。同时，将幽门螺杆菌OC1096菌株用作对照菌株。根据临床和实验室标准协会（CLSI）标准，采用E-test法确定克拉霉素耐药频率。通过实时PCR确定点突变情况，并进一步分析熔解曲线，连续进行扩增子测序。
从80份活检样本中，通过生化试验和PCR方法检测并确认了20株幽门螺杆菌阳性分离株。总体而言，21.7%的幽门螺杆菌分离株通过E-test法表现出克拉霉素耐药表型。此外，通过E-test法确定克拉霉素的最低抑菌浓度≥0.5mg/L。在所有耐药分离株中仅观察到A2143G位置的点突变，熔解温度为54.7°C。
本研究表明伊朗幽门螺杆菌对克拉霉素的耐药频率相对较高。由于克拉霉素在伊朗并不常用于根除幽门螺杆菌，高耐药率可能与其他大环内酯类药物的交叉反应有关。因此，在除幽门螺杆菌感染以外的任何治疗情况下，必须谨慎使用大环内酯类抗生素。所有耐药分离株在23S rRNA中均显示A2143G突变，至少在德黑兰的幽门螺杆菌分离株中是主要的点突变模式。
