Journal
POLYMERS
Volume 13, Issue 22, Pages -Publisher
MDPI
DOI: 10.3390/polym13223889
Keywords
hydrogels; oligonucleotide microarray; DNA hybridization; antimicrobial resistance determinants; mutations; Neisseria gonorrhoeae
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Funding
- Ministry of Science and Higher Education of the Russian Federation [075-15-2019-1660]
- Russian Science Foundation [17-75-20039]
- Russian Science Foundation [17-75-20039] Funding Source: Russian Science Foundation
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A multiplex assay using a low-density hydrogel microarray is developed to identify genomic substitutions in N. gonorrhoeae determining resistance to treatment agents. The microarray accurately identifies drug resistance determinants and single-nucleotide substitutions, with results consistent with Sanger sequencing. Analysis of clinical isolates in Russia over 5 years demonstrates the utility of hydrogel microarrays in monitoring antibiotic resistance spread.
A multiplex assay based on a low-density hydrogel microarray was developed to identify genomic substitutions in N. gonorrhoeae that determine resistance to the currently recommended treatment agents ceftriaxone and azithromycin and the previously used drugs penicillin, tetracycline, and ciprofloxacin. The microarray identifies 74 drug resistance determinants in the N. gonorrhoeae penA, ponA, porB, gyrA, parC, rpsJ, mtrR, bla(TEM), tetM, and 23S rRNA genes. The hydrogel elements were formed by automated dispensing of nanoliter-volume droplets followed by UV-induced copolymerization of NH2-containing oligonucleotides with gel-forming monomers. Polybutylene terephthalate plates without special modifications were used as microarray substrates. Sequences and concentrations of immobilized oligonucleotides, gel composition, and hybridization conditions were carefully selected, and the median discrimination ratio ranged from 2.8 to 29.4, allowing unambiguous identification of single-nucleotide substitutions. The mutation identification results in a control sample of 180 N. gonorrhoeae isolates were completely consistent with the Sanger sequencing results. In total, 648 clinical N. gonorrhoeae isolates obtained in Russia during the last 5 years were analyzed and genotyped using these microarrays. The results allowed us to draw conclusions about the present situation with antimicrobial susceptibility of N. gonorrhoeae in Russia and demonstrated the possibility of using hydrogel microarrays to control the spread of antibiotic resistance.
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