Basic study in molecular noninvasive estimation of Clarithromycin resistant Helicobacter pylori: Set up of ASP-PCR in differentiation of infB, and PCR-sequencing to evaluate 23S rRNA, and rpl22 related mutations

Document Type : Original Research

Authors
A. Mohebati Mobarez
A. Abedi Maghami
A. Yadegar
M. Nikkhah
A. Sadeghi
Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University
Abstract
Introduction. Designation of the local profile of Clarithromycin resistant (CAM-R) in Helicobacter pylori (H. pylori) positive patients with phenotypic testes consequently evaluation of probable agreement between resistance phenotypes to genotypes is the necessity of accessing rapid molecular noninvasive tests. So, we designed ASP-PCR and PCR-sequencing methods to evaluate infB (G160A), 23S rRNA (A2142C/G, A2143C/G), and rpl22 (GTG deletion or TTCCATGTA insertion) nucleotide polymorphisms from the stool of patients with symptoms of gastritis. Urea tubes were used to transport 96 gastric biopsies to the laboratory. Methods. The Agar dilution method was performed to assess CAM-R strains. Besides the phenotypical identification, stool samples were collected and stored at -80° C. Molecular identity w:as char:acterized by amplification of the 23S rRNA target gene. In the evaluation of non-invasive genotypical molecular tests in the detection of corresponding mutations, ASP-PCR was performed to isolate infB G160G wild-type strains and PCR-sequencing in determining 23S rRNA and rpl22 polymorphisms.

Results. Molecular isolation of H. pylori positive-patients was reported to be 34/54(62%). Among 35/96 (36%) phenotypically characterized H. pylori-positive infected patients,16/35(45%) were considered for CAM-R strains. The distribution of point mutations between resistant isolates has been revealed to be (1/16) for A2143C, (4/16) for infB G160A (PCR negative patients), (2/16) rpl22 for 3bp deletion, and (16/16) for rpl22 9bp insertion. Conclusion. We are honored to introduce rpl22-related point mutations as the potential marker in designing a noninvasive molecular method in Clarithromycin-resistant infected patients screening.

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Roszczenko-Jasińska P, Wojtyś MI, Jagusztyn-Krynicka EK: Helicobacter pylori treatment in the post-antibiotics era—searching for new drug targets. Appl Microbiol Biotechnol 2020, 104(23):9891-9905.
2. Abadi ATB, Taghvaei T, Mobarez AM, Carpenter BM, Merrell DS: Frequency of antibiotic resistance in Helicobacter pylori strains isolated from the northern population of Iran. J Microbiol 2011, 49(6):987-993.
3. Papastergiou V, Georgopoulos SD, Karatapanis S: Treatment of Helicobacter pylori infection: meeting the challenge of antimicrobial resistance. World J Gastroenterol: WJG 2014, 20(29):9898.
4. Garza-González E, Perez-Perez GI, Maldonado-Garza HJ, Bosques-Padilla FJ: A review of Helicobacter pylori diagnosis, treatment, and methods to detect eradication. World J Gastroenterol: WJG 2014, 20(6):1438.
5. Gonzalez-Hormazabal P, Musleh M, Escandar S, Valladares H, Lanzarini E, Castro VG, Jara L, Berger Z: Prevalence of clarithromycin resistance in Helicobacter pylori in Santiago, Chile, estimated by real-time PCR directly from gastric mucosa. BMC Gastroenterol 2018, 18(1):1-5.
6. Giorgio F, Ierardi E, Sorrentino C, Principi M, Barone M, Losurdo G, Iannone A, Giangaspero A, Monno R, Di Leo A: Helicobacter pylori DNA isolation in the stool: an essential pre-requisite for bacterial noninvasive molecular analysis. Scand J Gastroenterol Suppl 2016, 51(12):1429-1432.
7. Malfertheiner P, Megraud F, O'Morain CA, Atherton J, Axon AT, Bazzoli F, Gensini GF, Gisbert JP, Graham DY, Rokkas T: Management of Helicobacter pylori infection—the Maastricht IV/Florence consensus report. Gut 2012, 61(5):646-664.
8. Khademi F, Sahebkar AH, Vaez H, Arzanlou M, Peeridogaheh H: Characterization of clarithromycin-resistant Helicobacter pylori strains in Iran: A systematic review and meta-analysis. J Glob Antimicrob Resist 2017, 10:171-178.
9. De Francesco V, Zullo A, Hassan C, Giorgio F, Rosania R, Ierardi E: Mechanisms of Helicobacter pylori antibiotic resistance: An updated appraisal. World J Gastrointest Pathophysiol 2011, 2(3):35.
10. Binh TT, Shiota S, Suzuki R, Matsuda M, Trang TTH, Kwon DH, Iwatani S, Yamaoka Y: Discovery of novel mutations for clarithromycin resistance in Helicobacter pylori by using next-generation sequencing. J Antimicrob Chemother 2014, 69(7):1796-1803.
11. Miftahussurur M, Syam AF, Nusi IA, Makmun D, Waskito LA, Zein LH, Akil F, Uwan WB, Simanjuntak D, Wibawa IDN: Surveillance of Helicobacter pylori antibiotic susceptibility in Indonesia: different resistance types among regions and with novel genetic mutations. PloS one 2016, 11(12):e0166199.
12. Farzi N, Yadegar A, Sadeghi A, Asadzadeh Aghdaei H, Marian Smith S, Raymond J, Suzuki H, Zali MR: High prevalence of antibiotic resistance in Iranian Helicobacter pylori isolates: importance of functional and mutational analysis of resistance genes and virulence genotyping. J Clin Med 2019, 8(11):2004.
13. Tuan VP, Narith D, Tshibangu-Kabamba E, Dung HDQ, Viet PT, Sokomoth S, Binh TT, Sokhem S, Tri TD, Ngov S: A next-generation sequencing-based approach to identify genetic determinants of antibiotic resistance in Cambodian Helicobacter pylori clinical isolates. J Clin Med 2019, 8(6):858.
14. De Francesco V, Zullo A, Fiorini G, Saracino IM, Pavoni M, Vaira D: Role of MIC levels of resistance to clarithromycin and metronidazole in Helicobacter pylori eradication. J Antimicrob Chemother 2019, 74(3):772-774.
15. Kuo Y-T, Liou J-M, El-Omar EM, Wu J-Y, Leow AHR, Goh KL, Das R, Lu H, Lin J-T, Tu Y-K: Primary antibiotic resistance in Helicobacter pylori in the Asia-Pacific region: a systematic review and meta-analysis. Lancet Gastroenterol Hepatol 2017, 2(10):707-715.
16. Dekhnich N, Ivanchik N, Kozlov R, Alimov A, Steshits A, Kirsov P, Pandav K: Dynamics of antimicrobial resistance of Helicobacter pylori isolates in the Smolensk region of Russian Federation. Helicobacter 2018, 23(6):e12545.
17. Shamsdin SA, Alborzi A, Ghaderi A, Lankrani KB, Pouladfar GR: Significance of TC9 and TH9 in Helicobacter pylori‐induced gastritis. Helicobacter 2020, 25(1):e12672.
18. Fakheri H, Firoozi MS, Bari Z:Eradication of Helicobacter pylori in Iran: a review,
Middle East J Dig Dis 2018, 10(1):5-17 .
19. Khademi F, Sahebkar A: An updated systematic review and meta-analysis on the Helicobacter pylori antibiotic resistance in Iran (2010–2020). Microb. Drug Resist 2020, 26(10):1186-1194.
20. Kuo C-H, Lu C-Y, Shih H-Y, Liu C-J, Wu M-C, Hu H-M, Hsu W-H, Yu F-J, Wu D-C, Kuo F-C: CYP2C19 polymorphism influences Helicobacter pylori eradication. World J Gastroenterol: WJG 2014, 20(43):16029.
21. Raj DS, Kesavan DK, Muthusamy N, Umamaheswari S: Efflux pumps potential drug targets to circumvent drug Resistance–Multi drug efflux pumps of Helicobacter pylori. Mater. Today: Proc 2021, 45:2976-2981.
22. Cai Y, Wang C, Chen Z, Xu Z, Li H, Li W, Sun Y: Transporters HP0939, HP0497, and HP0471 participate in intrinsic multidrug resistance and biofilm formation in Helicobacter pylori by enhancing drug efflux. Helicobacter 2020, 25(4):e12715.
23. Marques AT, Vítor JM, Santos A, Oleastro M, Vale FF: Trends in Helicobacter pylori resistance to clarithromycin: from phenotypic to genomic approaches. Microb Genom 2020, 6(3).
24. Miftahussurur M, Yamaoka Y: Appropriate first-line regimens to combat Helicobacter pylori antibiotic resistance: an Asian perspective. Molecules 2015, 20(4):6068-6092.
25. Saniee P, HosseinI F, Kadkhodaei S, Siavoshi F, Khalili-Saman S: Helicobacter pylori multidrug resistance due to misuse of antibiotics in Iran. Arch Iran Med 2018, 21(7):283-288.
26. Alavifard H, Mirzaei N, Yadegar A, Baghaei K, Smith SM, Sadeghi A, Zali MR: Investigation of Clarithromycin Resistance-Associated Mutations and Virulence Genotypes of Helicobacter pylori Isolated from Iranian Population: A Cross-Sectional Study. Curr Microbiol 2021, 78(1):244-254.
27. Keshavarz Azizi Raftar S, Moniri R, Saffari M, Razavi Zadeh M, Arj A, Mousavi SGA, Mirzaei Ghazi Kalayeh H, Dastehgoli K: The Helicobacter pylori resistance rate to clarithromycin in Iran. Microb. Drug Resist 2015, 21(1):69-73.
28. Tajbakhsh S, Falahi J, Motamed N, Tabib SM, Bahador A, GHARIBI S: Prevalence OFA2143G AND A2144G point mutations responsible for clarithromycin resistance among Helicobacter pylori strains in Bushehr, IRAN. Avicenna J Clin Microbiol Infect 2016, 3(2): 0-0.
29. Bluemel B, Goelz H, Goldmann B, Grüger J, Hamel H, Loley K, Ludolph T, Meyer J, Miehlke S, Mohr A: Antimicrobial resistance of Helicobacter pylori in Germany, 2015 to 2018. Clin. Microbiol. Infect 2020, 26(2):235-239.
30. Marais A, Monteiro L, Occhialini A, Pina M, Lamouliatte H, Megraud F: Direct detection of Helicobacter pylori resistance to macrolides by a polymerase chain reaction/DNA enzyme immunoassay in gastric biopsy specimens. Gut 1999, 44(4):463-467.
31. Salehi N, Attaran B, Zare‐Mirakabad F, Ghadiri B, Esmaeili M, Shakaram M, Tashakoripour M, Eshagh Hosseini M, Mohammadi M: The outward shift of clarithromycin binding to the ribosome in mutant Helicobacter pylori strains. Helicobacter 2020, 25(6):e12731.