Method for phenotypic identification of Acinetobacter nosocomialis bacteria

Cover Page

Cite item

Abstract

The study's purpose was the developing of the method for Acinetobacter nosocomialis bacteria identification by the totality of phenotypic characteristics of the Acinetobacter baumannii (Ab) group based on the urease activity trait.

The research's objects were clinical strains of the Ab group, isolated in 2018—2019 in the bacteriological laboratory of Kirov Military Medical Academy (St. Petersburg), of which were A. baumannii (n = 85), A. nosocomialis (n = 12), A. pittii (n = 10). In addition, 9 strains of A. nosocomialis were isolated from water samples from the Neva River and 2 strains of A. calcoaceticus from the cyanobacterial mats. The belonging of the strains to Ab group was determined by the combination of the metabolic and physiological characteristics of the taxonomic tests for this group. The species identification of the studied strains was determined by the matrix-associated laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The urease activity of bacteria was determined using the microvolume method. We used a medium with urea in the following composition (g/l): Na2HPO4 — 1.1; KH2PO4 — 1.1; NaCI — 5.0; 40% alkaline solution phenol red — 5 ml; urea — 10—15; distilled water — 1 l. The ingredients, without urea, were dissolved in distilled water, dispensed to vials, sterilized for 20 minutes at 121°C, urea was added in the calculated amount. The medium with urea was added in 0.1 ml to wells of the plate, then inoculated the daily agar culture of the studied and control strains by full loop (2 mm in diameter) and incubated under aerobic conditions at 37°C. The reaction results for a rapid urease activity were determined after 3 hours by a change in the initial color of the medium. The reactions were accounted after 7—24 hours to detect activity urease. It was found that 100% of A. nosocomialis strains and 18.82% of A. baumannii strains had urease activity. At the same time, high activity urease was found only in one strain of A. baumannii (1.17%) and in all strains of A. nosocomialis. Therefore, the presence of high urease activity is of taxonomic importance for the species A. nosocomialis as the marker of distinguishing this species from other bacteria species of the Ab group. The sensitivity and specificity of the identifying A. nosocomialis strains by suggested approach compared to the studied strains identification by MALDI-TOF MS were 100 and 98.83% (x2 = 103.2; p < 0.0001), respectively.

About the authors

E. P. Sivolodskii

Military Medical Academy named after S.M. Kirov; St. Petersburg Pasteur Institute

Email: es279@yandex.ru

PhD, MD (Medicine), Professor of the Department of Microbiology, Military Medical Academy named after S.M. Kirov; Senior Researcher, Laboratory of Molecular and Biological Technologies, St. Petersburg Pasteur Institute.

197101, St. Petersburg, Mira str., 14.

Russian Federation

E. V. Zueva

St. Petersburg Pasteur Institute

Author for correspondence.
Email: elenazueva9@gmail.com

Elena V. Zueva - PhD (Biology), Senior Researcher, Laboratory of Molecular Immunology, St. Petersburg Pasteur Institute.

197101, St. Petersburg, Mira str., 14.

Phone: +7 (921) 382-50-07

Russian Federation

References

  1. Патент № 2712895 Российская Федерация, МПК C12Q 1/04 (2006.01), C12N 1/20 (2006.01), C12R 1/01 (2006.01). Способ идентификации бактерий Acinetobacter nosocomialis: № 2019129885; заявлено 2019.09.23: опубликовано 2020.01.31 / Сиволодский Е.П. Патентообладатель: Сиволодский Евгений Петрович. 9 с.
  2. Сиволодский Е.П. Пероксидводородный микрообъемный метод ОФ-теста: экспрессное определение окисления и ферментации глюкозы грамотрицательными бактериями // Журнал микробиологии, эпидемиологии и иммунобиологии. 1991. № 1. С. 14—17.
  3. Сиволодский Е.П., Кудакаев М.Т. Ускоренное определение нитратредуктазы неферментирующих грамотрицатель-ных бактерий // Лабораторное дело. 1991. № 1. С. 61—63.
  4. Bouvet P.J., Grimont P.A. Identification and biotyping of clinical isolates of Acinetobacter. Ann. Inst. Pasteur Microbiol., 1987, vol. 138, no. 2, pp. 569—578.
  5. Espinal P., Seifert H., Dijkshoorn L., Vila J., Roca I. Rapid and accurate identification of genjmic species from the Acinetobacter baumannii (Ab) group by MALDI-TOF MS. Clin. Microbiol. Infect., 2012, vol. 18, no. 11, pp. 1097—1103. doi: 10.1111/j.1469-0691.2011.03696.x
  6. Gerner-Smidt P., Tjernberg I., Ursing J. Reliablity of phenotypic tests for identification of Acinetobacter species. J. Clin. Microbiol, 1991, vol. 29, no. 2, pp. 277-282. doi: 10.1128/JCM.29.2.277-282.1991
  7. Nemec A., Krizova L., Maixnerova M., Van der Reijden T.J., Deschaght P., Passet V., Veneechoutte M., Brisse S., Dijkshoorn L. Genotypic and phenotypic characterization of the Acinetobacter calcoaceticus — Acinetobacter baumannii complex with the proposal of Acinetobacter pittii sp. nov. (formeily Acinetobacter genomic species 3) and Acinetobacter nosocomialis sp. nov. (formerly Acinetobacter genomic species 13 TU). Res. Microbiol., 2011, vol. 162, pp. 393-404. doi: 10.1016/j.resmic.2011.02.006
  8. Park G.N., Kang H.S., Kim H.R., Jung B.K., Kim D.H., Chang K.S. A comparison of genospecies of clinical isolates in the Acinetobacter spp. complex obtained fuom hospitalized patiens in Busan, Korea. Biomed. Sci. Letters, 2019, vol. 25, no. 1, pp. 4053. doi: 10.15616/bsl.2019.25.1.40
  9. Sedo O., Nemec A., Krizova L., Kacalova M., Zdrahal Z. Improvement of MALDI-TOF MS hrofilling for the differtntiation of species within the Acinetobacter calcoaceticus—Acinetobacter baumannii complex. Syst. Appl. Microbiol., 2013, vol. 36, no. 8, pp. 572-578. doi: 10.1016/j.syapm.2013.08.001
  10. Wang J., Ruan Z., Feng Y., Fu Y., Jiang Y., Wang H., Yu Y. Species distribution of clinical Acinetobacter isolates revealed by different identification techniques. PLoS One, 2014, vol. 9, no. 8: e104882. doi: 10.1371/journal.pone.0104882

Supplementary files

There are no supplementary files to display.


Copyright (c) 2020 Sivolodskii E.P., Zueva E.V.

Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 International License.

This website uses cookies

You consent to our cookies if you continue to use our website.

About Cookies