CC BY
22
Section 1. Biology
Abdurakhimov Abrorjon Akramovich, researcher, Institute of Bioorganic Chemistry of the Academy of Sciences of Republic Uzbekistan, Tashkent E-mail: Abror1978@mail.ru Dalimova Dilbar Akbarovna, Senior Researcher, Center for Advanced Technologies of the Republic of Uzbekistan, Tashkent E-mail: dalimova@gmail.com Turdikulova Shakhlo Utkurovna, doctor of biological science, Center for Advanced Technologies of the Republic of Uzbekistan, Tashkent E-mail: shahlo.ut@gmail.com
STUDY OF MUTATIONS IN THE 23 S rRNA GENE ARE ASSOCIATED WITH CLARITHROMYCIN RESISTANCE IN H.PYLORI
Abstract: Uzbekistan living in patients diagnosed with atrophic gastritis and stomach ulcers caused by H.pylori bacteria clarithromycin 23S rRNA gene that provides resistance to the domain V 2142 A/G/C, 2143 A/G mutations Multiplex Real-Time PCR method. 114 atrophic gastritis and gastric ulcer patients out of 109 (95,6%) caused by H.pylori bacteria and 114 samples, 19 (16,6%) caused by H.pylori 23S rRNA gene isolated from the domain V 2142 A/G/C, 2143 A/G mutations. Of them, 15 (78,9%) A2142G, 3 (15,79%), A214G and 1% (5,268) A2142C mutation was present.
Keywords: H.pylori, clarithromycin, 23S rRNA, multiplex Real-time PCR, gene ureC.
Helicobacter pylori is one of the most studied bacteria in eases, provides for anti-Helicobacter therapy, which, as a rule, the world today. Approximately 60% of the world's popula- is carried out without determining the sensitivity of H. pylori tion is infected with H.pylori. For example, the infection of to antibacterial agents.
the adult population of Russia ranges from 50 to 80%, and However, to increase the effectiveness of treatment of pa-
in some regions it approaches 100% [1, p. 2-12; 2, p. 41]. tients with this pathology, it is necessary to take into account In infected people, H.pylori causes a disease associated with the resistance of H.pylori to antibacterial drugs. It is known asymptomatic carriage. At the same time, epidemiological that the Maastricht Agreement-5 is the main advisory docu-data obtained in various countries show that almost 100% ment on the diagnosis and treatment of diseases associated of duodenal ulcers and more than 80% of gastric ulcers are with H.pylori infection. This scientific work provides for the associated with persistence of H.pylori [1, 2-12; 2, 41]. To- empirical use of antibacterial drugs for therapy at a known day, it is proved that H.pylori infection is the most important level of H.pylori resistance and makes it possible to accurately etiopathogenetic factor not only of a peptic ulcer, but also of predict the effectiveness of therapy [3, 1-25.]. The resistance chronic gastritis associated with H.pylori in 75-92% of cases of H. pylori to antibacterial drugs is a major factor in the fail-of gastroduodenitis, stomach cancer [2, 41]. ure of treatment of Helicobacter-associated diseases. This pa-
H.pylori has a wide range of genetic factors that provide it per presents data on the resistance of H.pylori strains to clar-with the ability to adapt and survive in an acidic environment, ithromycin in the region, which is associated with a decrease colonizing the mucous membrane of the stomach and duode- in the effectiveness of standard therapy regimens. num, and cause disease. It is known that in modern medicine The aim of this work is to determine the prevalence
the treatment of patients with Helicobacter-associated dis- of A2142G/C and A2143G point mutations that form
Section 1. Biology
clarithromycin resistance in Uzbekistan based on Real-Time polymerase chain reaction (PCR-Real-time PCR).
Materials and methods
In 2016-2017 in the department of Gastroenterology of the Republican Specialized Scientific and Practical Medical Center for Therapy and Medical Rehabilitation of the Ministry of Health of the Republic of Uzbekistan, endoscopy collected samples from epithelial cells and gastric juice in 114 patients diagnosed with atrophic gastritis and gastric ulcer. DNA samples were isolated from biopsy samples using the AmpliPrim RIBO-prep reagent kit (interLabService, Russia), according to the manufacturer's instructions. The concentration and purity of the isolated DNA samples were checked on a BioSpec-nano spectrophotometer (Shimadzu Biotech, Japan). The presence of H. pylori in the samples was confirmed by PCR using specific primers to the nucleotide sequence of the ureC gene described in [4, 1-6].
A multiplex Real-Time PCR with specially selected primers and probes (scorpion primers) was conducted to identify point mutations A2142G/C, A2143G, in the functional domain V 23S rRNA-gene responsible for the formation of HP resistance to clarithromycin [5, 2-7]. In each sample, an amplifier 7500 Fast Real-Time PCR System (Applied Biosystems) reads 4 channels (FAM, Cy3, ROX, and Cy5) fluorescence.
Results and its discussion
In all 114 samples of biopsy collected, the presence of the bacterium H.pylori was detected, the infection rate was 109 (95.6%). Of these, the resistance of H.pylori to clarithromycin was determined in 19 cases, which was 17.4%. The most common mutation was A2142G, which was detected in 15 patients (78.95%). The A2143G mutation was noted in 3 patients (15.79%).
To a large extent, resistance to clarithromycin in Uzbekistan is due to the presence of a point mutation A2142G. This mutation is present in 78,95% of resistant strains, and its presence in most cases explains the ineffectiveness of eradication therapy. The remaining 95 patients were infected by H.pylori bacteria in his stomach all of 2142 and 2143 precincts mutation detected in these patients ET scheme clarithromycin.
Patients who do not have point mutations, i.e. The resistance of H.pylori to clarithromycin in the appointment of the scheme can be recommended antibiotic clarithromycin. In the next series of experiments, sequencing of the strains of
the bacterium H. pylori was carried out. Sequencing of the strains of the bacterium H.pylori resistant to clarithromycin in the 23S rRNA gene revealed several mutations, of which
2142 A/G/C, 2143 A/G mutations were most common, and the mutation 2142 A/C was not detected [6, 1-6; 7, e29694].
The sequencing of resistant strains of the bacterium H.pylori to clarithromycin revealed mutations A2147 G (79.8%), A2146G (17.2%) and A2146C (2%). Bacteria H.pylori resistant to clarithromycin were not detected [8, 2-5]. From the discussions of the above literature data one can see that, even in one infected population, the indicators of bacterial resistance to antibiotics are different [9, 164-174]. During the sequence of strains of the bacterium H.pylori resistant to clarithromycin in the 23 S rRNA gene, several mutations were identified, ofwhich 2142 A/G/C and
2143 A/G mutations were the most common. For example, in the countries of Bulgaria [6, 1-6], Northern Iran [7, e29694] mainly identified mutations 2142 A/G, 2143 A/G, and mutation 2142 A/C was not identified. In Northern Spain, from 1986, cultures of isolates of H. pylori 349 (17.6%) were resistant to clarithromycin.
The sequencing of resistant bacteria H.pylori to clar-ithromycin revealed mutations A2147G (79.8%), A2146G (17.2%) and A2146C (2%) [10, 43-46]. In Italy, according to E-test, 82 cultures of H.pylori isolates were studied, of which 42 patients showed resistance of H.pylori to clarithromycin. In addition to the mutations A2142C, A2142G and A2143G, new mutations A2115G, G2141A and A2144T were detected in these isolates of cultures [11, 453-457]. Resistant bacteria H.pylori to clarithromycin that were not detected by the microbiological method E-test was determined by the method of Scorpion Real time PCR [12, 2320-2326].
Thus, on the basis of the obtained results, it can be concluded that in Uzbekistan it is necessary to conduct a study of the resistance of H.pylori not only to clarithromycin, but also to other antibiotics, since this will give the opportunity to choose the right ET regimens, which will lead to an increase in efficiency and monitoring therapy. In the future, for a broader study of molecular genetic aspects of the resistance of the bacterium H.pylori to clarithromycin, it is necessary to sequence the 23 S rRNA gene in domain V, as well as implement the Real Time PCR test systems for the most frequently encountered H.pylori mutations in Uzbekistan.
References:
1. Hunt R. H., Xiao S. D., Megraud F., Leon-Barua R., Bazzoli F., van der Merwe S., Vaz Coelho L. G., Fock M., Fedail S., Cohen H., Malfertheiner P., Vakil N., Hamid S., Goh K. L., Wong B. C.Y., Krabshuis J., Le Mair A. Helicobacter pylori в развивающихся странах // World Gastroenterology Organization. 2010.- P. 2-12.
2. Кудрявцева Л. В., Щербаков П. Л., Иваников И. О., Говорун В. М. Helicobacter pylory-инфекция: современные аспекты диагностики и терапии. Пособие для врачей.- М. 2004.- 41 c.
3. Malfertheiner P., Megraud F., Morain CAO', Gisbert J. P., Kuipers E. J., Axon A. T., Bazzoli F., Gasbarrini A., Atherton J., Graham D. Y., Hunt R., Moayyedi P., Rokkas T., Rugge M., Selgrad M., Suerbaum S., Sugano K., El-Omar E. M. Management of Helicobacter pylori infection-the Maastricht V/Florence Consensus Report // Gut 2016.- P. 1-25.
4. Rune Johannessen, Kere Bergh, Constantin Jianu, Per Martin Kleveland. Polymerase chain reaction versus culture in the diagnosis of Helicobacter pylori infection // Gastroenterology Insights. 2013.- V. 5.- P. 1-6.
5. Khansa Ben Mansour, Christophe Burucoa, Meriem Zribi, Afef Masmoudi, Sami Karoui, Lamia Kallel, Soufiune Chouaib, Samira Matri, Monia Fekih, Sonia Zarrouk, Mounir Labbene, Jalel Boubaker, Imed Cheikh, Mongi Ben Hriz, Nadia Siala, Abdelkarim Ayadi, Azza Filali, Nabil Ben Mami, Taoufik Najjar, Ahmed Maherzi, Mohamed Tahar Sfar, Chedlia Fendri. Primary resistance to clarithromycin, metronidazole and amoxicillin of Helicobacter pylori isolated from Tunisian patients with peptic ulcers and gastritis: a prospective multicentre study // Annals of Clinical Microbiology and Antimicrobials. 2010.- No. 9.- P. 2-7.
6. Boyanova Lyudmila, Markovska Rumyana, Yordanov Daniel, Gergova Galina, and Mitov Ivan. Clarithromycin Resistance Mutations in Helicobacter pylori in Association with Virulence Factors and Antibiotic Susceptibility of the Strains // Microbial Drug Resistance. 2015.- Vol. 22.- No. 3.- P. 1-6.
7. Zahra Eghbali, Ali Mojtahedi, Malek Moien Ansar, Saba Fakhrieh Asl and Keyvan Aminian. Detection of 23 SrRNA Mutations Strongly Related to Clarithromycin Resistance in Helicobacter pylori Strains isolated from patents in the North of Iran // Jundishapur J Microbiol. 2016.- No. 9 (2).- P. e29694.
8. Abdoulaye Seck, Christophe Burucoa, Daouda Dia, Mouhamadou Mbengue, Manuella Onambele, Josette Raymond and Sebastien Breurec. Primary antibiotic resistance and associated mechanisms in Helicobacter pylori isolates from Senegalese patients // Annals of Clinical Microbiology and Antimicrobials. 2013.- No. 12:3.- P. 2-5.
9. Reza Ghotaslou, Hamed Ebrahimzadeh Leylabadlo, and Yalda Mohammadzadeh Asl. Prevalence of antibiotic resistance in Helicobacter pylori: A recent literature review // World J Methodol. 2015.-No. 5 (3).- P. 164-174.
10. Esther Tamayoa, Milagrosa Montesa, Mama Fernöndez-Reyesb, Jacobo Lizasoainc, Begoca Ibarrac, Usua Mendartec, Eva Zapatad, Josune Mendiolae, Emilio Pnrez-Tralleroa. Clarithromycin resistance in Helicobacter pylori and its molecular determinants in Northern Spain, 2013-2015 // Journal of Global Antimicrobial Resistance. 2017.- No. 9.- P. 43-46.
11. Vincenzo De Francesco, Angelo Zullo, Floriana Giorgio, Ilaria Saracino, Cristina Zaccaro, Cesare Hassan, Enzo Ierardi, Alfredo Di Leo, Giulia Fiorini, Valentina Castelli, Giovanna Lo Re and Dino Vaira. Change of point mutations in Helicobacter pylori rRNA associated with clarithromycin resistance in Italy // Journal of Medical Microbiology. 2014.-No. 63.- P. 453-457.
12. Christophe Burucoa, Martine Garnier, Christine Silvain, and Jean-Louis Fauch'ere. Quadruplex Real-Time PCR Assay Using Allele-Specific Scorpion Primers for Detection of Mutations Conferring Clarithromycin Resistance to Helicobacter pylori // Journal of Clinical Microbiology.- 2008.- P. 2320-2326.
