Journal of microbiology, epidemiology and immunobiologyJournal of microbiology, epidemiology and immunobiology0372-93112686-7613Central Research Institute for Epidemiology112810.36233/0372-9311-218Research ArticleEffect of trypsin, lidase and fluimucil on the growth of <i>Bordetella pertussis</i> biofilms on an abiotic substrateZaуtsevEugene M.<p>D. Sci. (Med.), Head, Laboratory of immunomodulators</p>pertussis@yandex.ruhttps://orcid.org/0000-0002-4813-9074BritsinaMarina V.<p>Cand. Sci. (Biol.), leading researcher, Laboratory of immunomodulators</p>britsinamarina@yandex.ruhttps://orcid.org/0000-0002-3044-0790OzeretskovskayaMaria N.<p>Cand. Sci. (Biol.), leading researcher, Laboratory of immunomodulators</p>manja33@yandex.ruhttps://orcid.org/0000-0001-9809-4217BazhanovaIrina G.<p>Cand. Sci. (Biol.), senior researcher, Laboratory of immunomodulators</p>ibajanowa@yandex.ruhttps://orcid.org/0000-0003-1404-1498I. Mechnikov Research Institute for Vaccines and Sera0712202299554555115122021Copyright © 2022, Zaуtsev E.M., Britsina M.V., Ozeretskovskaya M.N., Bazhanova I.G.2022<p><strong>Aim.</strong> Study the effect of trypsin, lidase (hyaluronidase) and fluimucil (N-acetyl-L-cysteine) on the growth of biofilms of <em>Bordetella pertussis</em> strains on the abiotic substrate.</p>
<p><strong>Materials and methods.</strong> In the experiments, the strains of the main <em>B. pertussis</em> serotypes isolated in the Russian Federation from whooping cough patients in 20012010 were used: No. 178 (serotype 1.2.0), No. 287 (serotype 1.0.3) and No. 317 (serotype 1.2.3), grown on a dense nutrient medium. The intensity of biofilm formation in a liquid nutrient medium in the presence of trypsin, lidase and fluimucil in round-bottomed polystyrene 96-well plates was estimated by staining with 0.1% gentian-violet solution.</p>
<p><strong>Results.</strong> Trypsin suppressed the growth of biofilms and destroyed the formed biofilms. Lidase suppressed the growth of biofilms less actively, without affecting the formed biofilms. Fluimucil did not affect both the growth of biofilms and the formed biofilms. The growth of colonies typical for <em>B. pertussis</em> was noted when planting fluids from cultures in the presence of preparations, as well as from culture control wells on a dense nutrient medium.</p>
<p><strong>Conclusion.</strong> The different effect of the drugs studied by us may be related to the different quantitative content of targets for trypsin (proteins), lidase (mucopolysaccharides, containing uronic acids), fluimucil (acid mucopolysaccharides) in the biofilm matrix. The growth of the typical morphological properties of the colony of <em>B. pertussis</em> during the sowing of culture seedlings on a dense nutrient medium testifies to the destruction of the biofilm matrix by trypsin and lidase in the absence of influence on plankton cells.</p>B. pertussis strainsbiofilmstrypsinfluimucillidasematrixштаммы B. pertussisбиоплёнкитрипсинфлуимуциллидазаматрикс[Nguyen V.T.N., Simon L. Pertussis: The whooping cough. Prim. Care. 2018; 45(3): 423–31. https://doi.org/10.1016/j.pop.2018.05.003][Polinori I., Esposito S. Clinical findings and management of pertussis. Adv. Exp. Med. Biol. 2019; 1183: 151–60. https://doi.org/10.1007/5584_2019_410][Lomonosova A.V. Causes and consequences of delayed vaccination against pertussis infection in the Russian Federation. Zhurnal mikrobiologii, epidemiologii i immunobiologii. 2020; 97(5): 492–52. https://doi.org/10.36233/0372-9311-2020-97-5-11 (in Russian)][Cattelan N., Dubey P., Arnal L., Yantorno O.M., Deora R. Bordetella biofilms: a lifestyle leading to persistent infections. Pathog. Dis. 2016; 74(1): ftv108. https://doi.org/10.1093/femspd/ftv108][Cattelan N., Yantorno O.M., Deora R. Structural analysis of Bordetella pertussis biofilms by confocal laser scanning microscopy. Bio Protoc. 2018; 8(15): e2953. https://doi.org/10.21769/BioProtoc.295][Conover M.S., Mishra M., Deora R. Extracellular DNA is essential for maintaining Bordetella biofilm integrity on abiotic surfaces and in the upper respiratory tract of mice. PLoS One. 2011; 6(2): e16861. https://doi.org/10.1371/journal.pone.0016861][Guidelines MUK 4.2.2317-08. Selection, verification and storage of production strains of pertussis, parapertussis and bronchisepticose bacteria. Moscow; 2009. (in Russian)][Zaytsev E.M., Britsina M.V., Ozeretskovskaya M.N., Mertsalova N.U., Bazhanova I.G. Cultivation of Bordetella pertussis biofilms on abiotic substrate. Zhurnal mikrobiologii, epidemiologii i immunobiologii. 2019; 96(1): 49–53. https://doi.org/10.36233/0372-9311-2019-1-49-53 (in Russian)][Ashmarin I.P., Vorob'ev A.A. Statistical Methods in Microbiological Research [Statisticheskie metody v mikrobiologicheskikh issledovaniyakh]. Leningrad; 1962. (in Russian)][Uruén C., Chopo-Escuin G., Tommassen J., Mainar-Jaime R.C., Arenas J. Biofilms as promoters of bacterial antibiotic resistance and tolerance. Antibiotics (Basel). 2020; 10(1): 3. https://doi.org/10.3390/antibiotics10010003][Kovach K.N., Fleming D., Wells M.J., Rumbaugh K.P., Gordon V.D. Specific disruption of established Рseudomonas aeruginosa biofilms using polymer-attacking enzymes. Langmuir. 2020; 36(6): 1585–95. https://doi.org/10.1021/acs.langmuir.9b02188][Fleming D., Rumbaugh K. The consequences of biofilm dispersal on the host. Sci. Rep. 2018; 8(1): 10738. https://doi.org/10.1038/s41598-018-29121-2][Okulich V.K., Kabanova A.A., Plotnikov F.V. Microbial Biofilms in Clinical Microbiology and Antibacterial Therapy [Mikrobnye bioplenki v klinicheskoy mikrobiologii i antibakterial'noy terapii]. Vitebsk; 2017. (in Belarusian)][Wille J., Coenye T. Biofilm dispersion: The key to biofilm eradication or opening Pandora's box? Biofilm. 2020; 2: 100027. https://doi.org/10.1016/j.bioflm.2020.100027][Kayumov A.R., Trizna E.Yu., Sharafutdinov I.S., Baydamshina D.R., Ryzhikova M.N. Biofilms as a Pathogenicity Factor of Staphylococcus Aureus: Approaches to Therapy [Bioplenki kak faktor patogennosti Staphylococcus aureus: podkhody k terapii]. Kazan'; 2017. (in Russian)][Bosch A., Serra D., Prieto C., Schmitt J., Naumann D., Yantorno O. Characterization of Bordetella pertussis growing as biofilm by chemical analysis and FT-IR spectroscopy. Appl. Microbiol. Biotechnol. 2006; 71(5): 736–47. https://doi.org/10.1007/s00253-005-0202][Duvanova O.V., Mishan'kin B.N., Titova S.V., Korneeva L.A. The effect of N-acetyl-L-cysteine on biofilm of Vibrio cholerae. Zhurnal mikrobiologii, epidemiologii i immunobiologii. 2018; 95(2): 83–7. https://doi.org/10.36233/0372-9311-2018-2-83-87 (in Russian)]