Biological microchip for assessing tetracycline-resistance in Neisseria gonorrhoeae clinical isolates in Russian Federation

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Abstract

A total of 399 Neisseria gonorrhoeae clinical isolates collected in different regions of the Russian Federation in 20152017 were analyzed for tetracycline susceptibility and genetic markers of resistance. Drug susceptibility testing was performed by serial dilution method in agar and minimum inhibitory concentration (MIC) was measured according to the Russian “Guidelines for microbial susceptibility testing for antibacterial agents No. 4.2.1890-04”. Tetracycline resistance determinants were studied by using hydrogel microarray with immobilized oligonucleotide probes able to identify a series of chromosomal mutations and detect plasmid tetM gene. Different resistance determinants were found in 193 isolates (48.4%). Mutation in codon 57 in the rpsJ gene (41.2%) was most common that decreases tetracycline affinity to ribosome 30S subunit, mainly due to Val57Met substitution both as a point mutation as well as in combination with others. Mutations in the rpsJ gene were found in strains with the intermediate tetracycline susceptibility. Mutations in the porB gene (lower tetracycline influx) held the se cond place in prevalence pattern (23.1%); the Gly120Lys substitution usually led to emergence of tetracycline resistance either as a point mutation or in combination with other substitutions. Substitutions of Gly120 for other residues (Asp, Asn, and Thr) and Ala121 for Asp, Asn, and Gly had much less effect on resistance level. The –35 delA deletion in the promoter region of mtrR gene (increased expression of MtrC-MtrD-MtrE efflux pump) was observed in 11.3% strains. The tetM gene was found in 27 strains including 17 American and 10 Dutch type tetM determinants. Evolutionary tree was constructed for the tetM genes with the estimation of their homology with similar genes in genera Streptococcus, Enterococcus and Mycoplasma. Mutations in chromosomal genes resulted in increase of tetracycline MIC up to 2–4 mg/L; 4 mg/L MIC was observed in case of simultaneous presence of several mutations. Strains bearing tetM gene-containing plasmid showed extremely high resistance level: MIC ≥ 8 mg/L (64 mg/L for the two samples). Thus, long-lasting withdrawal of tetracycline use for treatment of gonococcal infections in Russia (since 2003) resulted in decreased percentage of resistant strains (including strains with intermediate susceptibility) from 75% down to 45.4%. However, currently tetracycline resistance in Russia remains elevated that is explained by the presence of different resistance determinants in the half of isolates under study.

About the authors

E. I. Dementieva

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Author for correspondence.
Email: kdem@biochip.ru
ORCID iD: 0000-0002-7623-2217

Ekaterina I. Dementieva, PhD (Chemistry), Researcher, Laboratory of Molecular Diagnostics Technologies

119991, Moscow, Vavilova str., 32.

Phone: +7 (499) 135-98-46 (office).

Россия

B. L. Shaskolskiy

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: b.shaskolskiy@biochip.ru
ORCID iD: 0000-0002-0316-2262

PhD (Chemistry), Researcher, Laboratory of Molecular Diagnostics Technologies

Moscow

Россия

A. T. Leinsoo

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: aequorin@yandex.ru
ORCID iD: 0000-0003-4006-6787

Junior Researcher, Laboratory of Molecular Diagnostics Technologies

Moscow 

Россия

D. A. Gryadunov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: grad@biochip.ru
ORCID iD: 0000-0003-3183-318X

PhD (Biology), Head of the Laboratory of Molecular Diagnostics Technologies, Deputy Director for Science

Moscow

Россия

N. P. Petrova

State Research Center of Dermatovenerology and Cosmetology, Russian Ministry of Health

Email: petrova.natasha.p@gmail.com

Junior Researcher, Department of Laboratory Diagnostics of STD and Dermatoses

Moscow

Россия

A. V. Chestkov

State Research Center of Dermatovenerology and Cosmetology, Russian Ministry of Health

Email: chestkov@cnikvi.ru

PhD (Biology), Senior Researcher, Department of Laboratory Diagnostics of STD and Dermatoses

Moscow 

Россия

A. A. Kubanov

State Research Center of Dermatovenerology and Cosmetology, Russian Ministry of Health

Email: alex@cnikvi.ru

RAS Corresponding Member, PhD, MD (Medicine), Professor, Chief Researcher

Moscow

Россия

D. G. Deryabin

State Research Center of Dermatovenerology and Cosmetology, Russian Ministry of Health

Email: dgderyabin@yandex.ru

PhD, MD (Medicine), Professor, Head of the Department of Laboratory Diagnostics of STD and Dermatoses

Moscow

Россия

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Copyright (c) 2019 Dementieva E.I., Shaskolskiy B.L., Leinsoo A.T., Gryadunov D.A., Petrova N.P., Chestkov A.V., Kubanov A.A., Deryabin D.G.

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