Variability of pathogenicity factors representatives of the human microbiome under the influence of γ-Fe2O3 iron oxide nanoparticles published by Ural Federal University eISSN 2411-1414; chimicatechnoacta.ru ARTICLE 2022, vol. 9(4), No. 20229401 DOI: 10.15826/chimtech.2022.9.4.01 1 of 6 Variability of pathogenicity factors representative of the human microbiome under the influence of γ-Fe2O3 nanoparticles Lyubov A. Kokorina a, Yana V. Chernyavskaya b, Tatiana P. Denisova b* , Elena V. Simonova а, Alexander P. Safronov c, Galina V. Kurlyandskaya сd a: Irkutsk State Medical University MOH, Irkutsk 664003, Russia b: Irkutsk State University, Irkutsk 664011, Russia c: Institute of Natural Sciences and Mathematics, Ural Federal University, Ekaterinburg 620002, Russia d: University of the Basque Country UPV/EHU, Department of the Electricity and Electronics, Leioa 48940, Spain * Corresponding author: denis_tp@inbox.ru This paper belongs to the Regular Issue. © 2022, the Authors. This article is published in open access under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Abstract Biomedical applications of nanoparticles require deep understanding of their interaction with normal human microflora. Previously, the toxic and mutagenic properties of iron oxide nanoparticles as well as their effect on the growth and morphology of the microflora were ex- tensively investigated. However, the studies related to the variability of microbial pathogenicity factors induced by iron oxide nanoparti- cles are very limited. Meanwhile, this characteristic of microbes is genetically determined and is important for their survival and distri- bution in the human body. Therefore, pathogenicity factors are sig- nificant indicators of the experimental studies. In this work, the ef- fect of the presence of Fe2O3 nanoparticles obtained by laser target evaporation (LTE) on selected enzymes that demonstrate invasion and aggression factors was evaluated for three reference strains of Candida albicans, Staphylococcus aureus, and Escherichia coli. It was found that the presence of LTE Fe2O3 nanoparticles supplied in the form of water-based suspensions does not induce changes of the above-mentioned parameters. Keywords laser target evaporation iron oxide nanoparticles magnetic nanoparticles biomedical applications eukaryotic and prokaryotic microorganisms normal human microflora pathogenicity factors Received: 17.05.22 Revised: 16.06.22 Accepted: 21.06.22 Available online: 04.07.22 1. Introduction Magnetic nanomaterials are moving from the stage of scien- tific research to the stage of practical implementation in di- agnostics, therapy and biotechnologies [1, 2]. Many studies have been associated with proposals to use nanomaterials for targeted drug delivery, creating bone implants, wearable devices, etc. Magnetic nanoparticles (MNPs) are especially attractive as their movements inside the body can be con- trolled by application of gradient of the external magnetic field [3, 4], which can be also used to induce hyperthermia and thermal ablation [5]. Magnetic nanoparticles can be produced by different chemical and physical techniques. Interestingly, about 92% of MNPs of iron oxides are prepared by chemical routes: co-precipitation, hydrothermal and solvothermal synthesis, sol-gel and polyol microemulsion methods, mi- crowave-assisted synthesis electrochemical deposition and others [2, 6, 7]. About 6% of MNPs of MNPs are fabricated by physical techniques [8, 9], and about 2% by biological synthesis methods [2, 10]. The largest batches of the MNPs can be obtained using electrophysical techniques such as electric explosion of the wire, laser target evaporation or spark discharge [2, 9]. In any case, biomedical applica- tions require synthesis of large batches, since established protocols demand testing with an extended set experi- mental technique [11]. In addition, biological and biomedi- cal applications are only possible with aqueous suspen- sions (not with the air-dry MNPs). Their fabrication also to some extent reduces the amount of available material. On the one hand, the composition of nanoparticles in- cludes natural components: iron, silver, copper, alumi- num, oxygen, etc. On the other hand, their size, shape, exact composition, and physicochemical properties make it possible to classify nanoparticles as xenobiotics, i.e. chemical substances found within an organism that is not http://chimicatechnoacta.ru/ https://doi.org/10.15826/chimtech.2022.9.4.01 mailto:denis_tp@inbox.ru http://creativecommons.org/licenses/by/4.0/ https://orcid.org/0000-0002-1044-2916 https://crossmark.crossref.org/dialog/?doi=https://doi.org/10.15826/chimtech.2022.9.4.01&domain=pdf&date_stamp=2022-7-4 Chimica Techno Acta 2022, vol. 9(4), No. 20229401 ARTICLE 2 of 6 naturally produced or expected to be present in it. The question about the degree of safety of each particular na- nomaterial for organisms and ecosystems in general is a very important one to be answered. An analysis of the experimental results presented in the scientific sources allows to distinguish three groups of effects of nanoparticles on biological systems: modifica- tions, toxicity, and mutagenicity. For example, a change in the color of eukaryotic organisms was established in an experiment with iron-containing nanoparticles [12]. Iron nanoparticles caused reversible changes in the biochemi- cal activity of Pseudomonas aeruginosa: a decrease in car- bohydrate fermentation was observed [13]. The toxic effects of nanoparticles were found for organ- isms of different systematic groups [14]. Copper nanoparti- cles caused the death of bacteria. Nanoparticles based on aluminum oxide reduced the ability of Escherichia coli (E.сoli) to form a biofilm [15]. Iron nanoparticles had a tox- ic effect on Pseudomonas aeruginosa [13]. The low toxicity of nanoparticles of silicon dioxide was shown for some strains of E.coli [16]. Toxic effects were also found in dif- ferent types of eukaryotic organisms [14, 17, 18]. Deoxyribonucleic acid (DNA) damage, including mito- chondrial DNA, and the formation of micronuclei in blood cells can be induced by silicon, nickel, and gold containing nanoparticles. Metal nanoparticles cause excessive pro- duction of reactive oxygen species (peroxides, superoxide, singlet oxygen, etc.), which induce DNA damage [14]. The mutagenicity of aluminum oxide nanoparticles for human cells was also proven [19]. It is important to take into ac- count that, regardless the type of nanoparticles and biotests used in the experiment, the first interaction of the agent occurs with the normal microflora of the body, regardless of the method of administration. Previously, the toxic and mutagenic properties of iron oxide nanoparticles as well as their effect on the growth and morphology of the microflora were extensively inves- tigated. However, up to now, the studies of the microbial variability of pathogenicity factors related to the presence of the iron oxide nanoparticles were quite limited. The variability of pathogenicity factors is both genetically de- termined and important for the microbial survival and distribution in the body. Therefore, pathogenicity factors are significant indicators of the experimental studies. In this work, the effect of the presence of iron oxide nanoparticles obtained by laser target evaporation sup- plied as water-based suspension was studied for selected enzymes that demonstrate invasion and aggression fac- tors. Three reference strains of Candida albicans, Staphy- lococcus aureus, and Escherichia coli were used. 2. Materials and methods Electrophysical techniques allow fabrication of large batch- es of MNPs (of the order of 100 g) required for biomedical research and applications [9]. One of these techniques is the laser target evaporation (LTE) in which the solid target is evaporated by the high-power pulse of a laser beam. The beam energy transform into the kinetic energy of the evap- orated products. In this study, the LTE target was fabricated using the commercial Fe3O4 iron oxide powder (Alfa Aesar). More details of the experimental procedure for the LTE syn- thesis can be found elsewhere [9]. The X-ray diffraction investigation was performed us- ing s DISCOVER D8 Bruker diffractometer and TOPAS software allowing Rietveld full-profile refinement for the quantitative analysis. Transmission electron microscopy (TEM) was performed for the evaluation of the size and shape of MNPs (JEOLJEM2100). For the measurements of the specific surface area (Ssp) of the MNPs ensemble, the low temperature sorption of nitrogen technique was used [1]. Magnetic properties were studied with a MPMSXL-7 SQUID magnetometer. Biomedical applications require supplying magnetic MNPs as water-based suspensions. Electrostatically stabi- lized water-based suspension of MNPs was prepared with sodium citrate in 5 mM concentration via disaggregation by ultrasound treatment on a Cole-Parmer CPX-750 ho- mogenizer at the power output 300 W and centrifuging using a Hermle Z383 apparatus in order to remove the largest aggregates. The final concentration of MNPs in the stock suspension was 47.8 g/l, and the suspension was slightly acidic (pH=4.5) due to the specific adsorption of hydroxide ions on the surface. The stock suspension was then diluted with distilled water to provide the desired concentration of MNPs therein. Hydrodynamic diameters of the MNPs and their aggregates were defined by dynamic light scattering technique (Brookhaven Zeta Plus). The same instrument allowed measuring the electrokinetic zeta-potential of the suspensions by the electrophoretic light scattering. The fungus strain Candida albicans АТСС 10231 [20], Staphylococcus aureus ATCC 25923 [21] and Escherichia coli ATCC 25922 [22] strains, which belong to the normal human microflora, were used as bioassays. In the control group, the bacteria grew in a liquid nutrient medium MPA (meat-peptone agar), the pH of which was 7.4±0.4. The fungi were cultivated in a liquid Sabouraud medium (pH 5.7±0.2). In the experimental group, a suspension of MNPs was introduced into the liquid medium, the concentration of which reached 0.1, 1.0 and 10.0 MPD (maximum per- missive dose) for Fe+3. The microbial suspension was ex- posed for 144 hours for all groups. The experiments included the following stages. Start- ing from the zero point, every 24 hours, microorganisms were inoculated from the culture liquid onto a solid nutri- ent medium (MPA, and Nickerson agar). Several clones were selected by random sampling among the grown colo- nies to determine pathogenicity factors. The clones were placed onto differential diagnostic media and cultivated at a temperature of 37 o C for 24 hours. After that, the pres- ence or absence of pathogenicity factors was recorded Chimica Techno Acta 2022, vol. 9(4), No. 20229401 ARTICLE 3 of 6 similar to the way reported in the literature [23–25]. The analyzed indicators (pathogenicity factors) were provided by the presence of certain enzymes in microbes and their activity. Respiratory, lecithinase, plasmacoagulase, hemo- lytic, and DNAase activities were taken into account. Respiratory activity is assessed by changing the color of the culture in the medium with the dye Congo red. Le- cithinase activity is assessed by the formation of a cloudy "corolla" around the colony on a nutrient yolk medium. The plasmacoagulating activity of S. aureus is determined by the presence of a gelatinous formation. Hemolytic ac- tivity is manifested through the formation of a zone of enlightenment on a nutrient medium. Determination of DNAase activity is associated with the formation of a cleavage zone, i.e. zones of enlightenment of the nutrient medium around the colony. It should be noted that qualitative indicators were col- lected; therefore, their presence “+” or absence “–” was recorded. Depending on the severity of the symptom, the following states were noted: "–" – the indicator is nega- tive, "+" – low activity, "++" – moderate activity, "+++" – pronounced activity, "++++" – maximum activity. 3. Results X-ray diffraction analysis showed that obtained iron oxide powder have the inverse spinel structure (Fd-3m space group). According to the TEM studies, which included the graphical analysis (Figure 1), the shapes of the MNPs were very close to being spherical. The nanoparticles in the en- semble were size-distributed in accordance with the lognormal law with 17±3 nm average mean diameter. Ac- cording to PSD, 99% of MNPs fell within the diameter range of 2–40 nm. The specific surface area was about 69 m2/g. Magnetic measurements confirmed that nanopar- ticles had low coactivity of about 30 Oe at room tempera- ture and the saturation magnetization of about 37±2 emu/g (both numbers are consistent with existing sources for the Fe2O3 MNPs of this size) [1, 9]. The average hydrodynamic diameter, 51.4±0.4 nm, was determined for the species in MNPs suspension. It was higher than that for the air-dry MNPs. It meant that the suspension was in fact a mixture of individual MNPs and their small aggregates. The value of zeta-potential of the suspension was –44±2 mV, which was well above the co- agulation threshold (20 mV regardless of the sign). It meant that the aggregates were stable and no further ag- gregation occurred in the suspension. These parameters of the suspension did not change in storage for over a year. Figure 2 shows the general view of the samples of the control groups of the fungus strain Candida albicans АТСС 10231, Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 strains, belonging to the normal human microflora. In order to determine the effect of MNPs on the varia- bility of pathogenicity factors, 144 clones were isolated from the eukaryotic strain C.albicans ATCC 10231, 144 clones from the gram-positive prokaryotic strain S.aureus ATCC 25923 and 83 clones from the gram- negative prokaryotic strain E.coli ATCC 25922. It was found that the reference strains under physio- logical conditions (control) retain a set of specific aggres- sion factors (Table 1), which they implement in a low viru- lent form. This corresponds to the levels of activity that are capa- ble of being manifested by the microorganisms of these species, which are part of the structure of the normal hu- man microbiome, without leading to the development of pathological processes in internal organs and tissues. This result is evidenced by the absence of DNase and hemolytic activities. Figure 1 Transmission electron microscopy: general view of γ- Fe2O3 LTE nanoparticles (a), TEM microdiffraction confirming spinel structure (b). Chimica Techno Acta 2022, vol. 9(4), No. 20229401 ARTICLE 4 of 6 Figure 2 Candida albicans on Nickerson's nutrient medium in Petri dish (a), Staphylococcus aureus on nutrient medium yolk-salt agar (b), Escherichia coli on ENDO nutrient medium (c). Table 1 Evaluation of pathogenicity factors in reference strains in the control group. Enzymatic activity Тest objects C. albicans St. aureus E. coli. Respiratory ++ ++++ ++++ Lecithinase – + – Plasmocoagulase – +++ – Hemolytic – – – DNАase – – – The presence of lecithinase and plasma-coagulase ac- tivity, due to which they are able to realize their immuno- genic properties in the human body, indicates the normal- ly functioning work of plastic metabolism. Respiratory activity in microorganisms is an indicator of a normally functioning energy metabolism system. The results of the experiments on the variability of factors of invasion and aggression of microorganisms in the presence of nanopar- ticles are shown in Table 2. It was found that the activity of the reference strains of microorganisms after exposure to the nanoparticles does not change in all variants of the experiment. DNase and hemolytic activities were not detected. Respiratory activi- ty also remained unchanged in all variants of the experi- ment (Figure 3a). Lecithinase and plasmacoagulase activities were found only in staphylococcus, being at the same level as in the con- trol group. C. albicans shows only respiratory activity (Figure 3b). Thus, the factors associated with the level of virulence of microorganisms remain stable in the experiment and do not differ from the control level. Perhaps this is due to the fact that nanoparticles cannot affect the metabolic processes of a microbial cell due to their adhesion to the cell wall. 4. Conclusions Iron oxide Fe2O3 nanoparticles were obtained by the LTE technique. They had the inverse spinel structure (Fd-3m space group), close to spherical shapes and the lognormal size-distribution, and they had 17±3 nm average mean diameter and magnetic parameters consistent with their size and composition. Biological experiments resulted in the following findings: 1. DNase and hemolytic activities were not detected in all test samples, neither in the control nor in the experi- mental groups. 2. Lecithinase and plasmacoagulase activities were recorded only in S. aureus in the control and in all exper- imental groups at the same level. 3. C. albicans showed moderate respiratory activity, and S. aureus and E. coli showed pronounced respiratory activity. The control and all experimental groups did not differ from each other. Table 2 Evaluation of pathogenicity factors in reference strains obtained in the experiments. Тest objects MTD calculated on the basis of Fe+3 content in MNPs Enzymatic activity DNАase Respiratory Hemolytic Lecithinase Plasmocoagulase C. albicans 0.1 – ++ – – – 1.0 – ++ – – – 10.0 – ++ – – – St. aureus 0.1 – ++++ – + +++ 1.0 – ++++ – + +++ 10.0 – ++++ – + +++ E. coli. 0.1 – ++++ – – – 1.0 – ++++ – – – 10.0 – ++++ – – – Chimica Techno Acta 2022, vol. 9(4), No. 20229401 ARTICLE 5 of 6 Figure 3 Pronounced respiratory activity of staphylococcus (a), a negative test for plasmocoagulase in C. albicans (no clot is formed) (b). 4. The induced variability of indicators of pathogenici- ty factors in the experiment was not established. Given that pathogenicity factors are the genetically determined traits, it can be assumed that the studied nanoparticles either do not penetrate into the bacterial cell or have no effect on the genetic material. Supplementary materials No supplementary materials are available. Funding This research in part was supported by the University of the Basque Country UPV/EHU Research Groups Funding (GMMM). Acknowledgments Authors would like to thank the Dr. I.V. Beketov (Institute of Electrophysics UD RAS) and Dr. I. Orue for special sup- port. Selected measurements were made at SGIKER ser- vices of UPV/EHU. Author contributions Conceptualization: E.V.S. Data curation: L.A.K., T.P.D., Ya.V.C. Formal Analysis: L.A.K., T.P.D., Ya.V.C. Funding acquisition: E.V.S., T.P.D., G.V.K. Investigation: L.A.K., Ya.V.C., A.P.S., G.V.K. Methodology: E.V.S., L.A.K., A.P.S. Project administration: E.V.S., G.V.K. Resources: E.V.S., L.A.K., T.P.D., G.V.K. Software: L.A.K., E.V.S., A.P.S. Supervision: E.V.S., T.P.D., G.V.K. Validation: E.V.S., G.V.K., L.A.K., T.P.D., A.P.S. Visualization: L.A.K., T.P.D., Ya.V.C., A.P.S. Writing – original draft: L.A.K., T.P.D., Ya.V.C., G.V.K., A.P.S. Writing – review & editing: T.P.D., E.V.S., A.P.S., G.V.K. Conflict of interest The authors declare no conflict of interest. Additional information Author IDs: Lyubov A. Kokorina, Scopus ID 57194266259; Tatyana P. Denisova, Scopus ID 56443729400; Elena V. Simonova, Scopus ID 57194285465; Alexander P. Safronov., Scopus ID 35588399100; Galina V. Kurlyandskaya, Scopus ID 7004350808. Websites of Irkutsk State Medical University MOH, https://www.ismu.baikal.ru/ismu/news.php; Irkutsk State University, https://isu.ru/ru/index.html; Ural Federal University, https://urfu.ru/en; University of the Basque Country, https://www.ehu.eus/es/. References 1. Kurlyandskaya GV, Blyakhman FA, Makarova EB, Buznikov NA, Safronov AP, Fadeyev FA, Shcherbinin SV, Chlenova AA. Functional magnetic ferrogels: from biosensors to regenera- tive medicine. AIP Adv. 2020;10:12512. doi:10.1063/9.0000021 2. Zamani Kouhpanji MR, Stadler BJH. A guideline for effective- ly synthesizing and characterizing magnetic nanoparticles for advancing nanobiotechnology: a review. Sens. 2020;20(9):2554. doi:10.3390/s20092554 3. Zhigachev AO, Golovin YuI, Klyachko NL. A new physical meth- od of localization of nanomechanical action of magnetic nano- particles controlled by low-frequency magnetic field on me- https://www.scopus.com/authid/detail.uri?authorId=57194266259 https://www.scopus.com/authid/detail.uri?authorId=56443729400 https://www.scopus.com/authid/detail.uri?authorId=57194285465 https://www.scopus.com/authid/detail.uri?authorId=35588399100 https://www.scopus.com/authid/detail.uri?authorId=7004350808 https://www.ismu.baikal.ru/ismu/news.php https://isu.ru/ru/index.html https://urfu.ru/en https://www.ehu.eus/es/ https://doi.org/10.1063/9.0000021 https://doi.org/10.3390/s20092554 Chimica Techno Acta 2022, vol. 9(4), No. 20229401 ARTICLE 6 of 6 chanically sensitive biochemical systems. Adv Mater Technol. 2018;3:63–69. doi:10.17277/AMT.2018.03.PP.063-069 4. Sokolov IL. In vitro and in vivo study of the behavior of hy- brid nanostructures with positive magnetic susceptibility for biomaging and targeted drug delivery [dissertation]. Mos- cow, Russia: Moscow Institute of Physics and Technology (National Research University); 2019. 134 р. 5. Alonso J, Khurshid H, Devkota J, Nemati Z, Khadka NK, Sri- kanth H, Pan J, Phan M-H. Superparamagnetic nanoparticles encapsulated in lipid vesicles for advanced magnetic hyper- thermia and biodetection. J Appl Phys. 2016;119:083904. doi:10.1063/1.4942618 6. Khawja Ansari SAM, Ficiara E, Ruffinatti FA, Stura I, Argen- ziano M, Abollino O, Cavalli R, Guiot C, D’Agata F. Magnetic iron oxide nanoparticles: synthesis, characterization and functionalization for biomedical applications in the central nervous system. Mater. 2019;12:465. doi:10.3390/ma12030465 7. Aphesteguy JC, Jacobo SE, Schegoleva NN, Kurlyandskaya GV. Characterization of nanosized spinel ferrite powders synthe- sized by coprecipitation and autocombustion method. J Alloy Compd. 2010;495:509–512. doi:10.1016/j.jallcom.2009.10.037 8. Goiriena-Goikoetxea M, García-Arribas A, Rouco M, Svalov AV, Barandiaran JM. High-yield fabrication of 60 nm Permal- loy nanodiscs in well-defined magnetic vortex state for bio- medical applications. Nanotechnol. 2016;27:175302. doi:10.1088/0957-4484/27/17/175302 9. Kurlyandskaya GV, Safronov AP, Shcherbinin SV, Beketov IV, Blyakhman FA, Makarova EB, Korch MA, Svalov AV. Magnetic nanoparticles obtained by electrophysical technique: focus on biomedical applications. Phys Solid State. 2021;63:1447–1461. doi:10.1134/S1063783421090237 10. Bazylinski DA, Frankel RB. Magnetosome formation in pro- karyotes. Nat Rev Microbiol. 2004;2:217–230. doi:10.1038/nrmicro842 11. Grossman JH, McNeil SE. Nanotechnology in Cancer Medi- cine. Phys Today. 2012;65:38. doi:10.1063/PT.3.1678 12. Denisova TP, Simonova EV, Kokorina LA, Maximova EN, Sa- matov OV. Heterogeneity of population of microorganisms grown in presence of iron oxide maghemite nanoparticles. EPJ Web Conf. 2018;185:10002. doi:10.1051/epjconf/201818510002 13. Babushkina IV, Korshunov GV, Puchinyan DM, Vlasova SP, Fedorova AV, Goroshinskay IA, et al. Antibacterial effect of iron and copper nanoparticles on Pseudomonas aeruginosa. Izvestiya vuzov. The North Caucasus Reg. Nat Sci. 2010;2:82– 84. Available from: https://cyberleninka.ru/article/n/antibakterialnoe-deystvie- nanochastits-zheleza-i-medi-na-klinicheskie-shtammy- rseudomonas-aeruginosa, Accessed on 10 May 2022. 14. Durnev AD. Assessment of the genotoxicity of nanoparticles when used in medicine. Hygiene Sanit. 2014;93(2):76–83. Available from: https://cyberleninka.ru/article/n/otsenka- genotoksichnosti-nanochastits-pri-ispolzovanii-v-meditsine, Accessed on 7 May 2022. 15. Mikhailova EA, Mironov AYu, Fomina MV, Kirgizova SB, Aznabayeva LM, Zherebyatyeva OO. The ability of Escherichia coli to form biofilms in the presence of aluminum oxide na- noparticles. Clin Lab diagn. 2017;6:381–384. Available from: https://cyberleninka.ru/article/n/sposobnost-escherichia- coli-formirovat-bioplenki-v-prisutstvii-nanochastits-oksida- alyuminiya, Accessed on 7 May 2022. 16. Kosyan DB, Makaeva AM, Rusakova EA. Biological effects of silicon dioxide nanoparticles. Mod Probl Sci Educ. 2018;6. Available from: https://science- education.ru/ru/article/view?id=28276, Accessed on 7 May 2022. 17. Castro-Gamboa S, Garcia-Garcia MR, PiñonZarate G, Rojas- Lemus M, Jarquin-Yañez K, Angel Herrera-Enriquez M, et al. Toxicity of silver nanoparticles in mouse bone marrow- derived dendritic cells: Implications for phenotype. J Immu- notoxicol. 2019;16(1):54–62. doi:10.1080/1547691X.2019.1584652 18. Abramenko NB. Investigation and modeling of the toxic effect of silver nanoparticles on hydrobionts [dissertation]. Mos- cow, Russia: State University named after M.V. Lomonosov; 2017. 122 p. 19. Zaitseva NV, Zemlyanova MA, Stepankov MS, Ignatova AM. Assessment of toxicity and potential danger of aluminum ox- ide nanoparticles for human health. Human Ecol. 2018;5:9– 15. Available from: https://cyberleninka.ru/article/n/otsenka-toksichnosti-i- potentsialnoy-opasnosti-nanochastits-oksida-alyuminiya- dlya-zdorovya-cheloveka, Accessed on 7 May 2022. 20. Candida albicans (Robin) Berkhout. Available from: https://www.atcc.org/products/10231-mini-pack, Accessed on 10 May 2022. 21. Staphylococcus aureus subsp. aureus Rosenbach. Available from: https://www.atcc.org/products/25923, Accessed on 10 May 2022. 22. Escherichia coli (Migula) Castellani and Chalmers. https://www.atcc.org/products/25922, Accessed on 10 May 2022. 23. Gruber IM, Egorova NB, Astashkina EA. Pathogenicity factors of Staphylococcus aureus their role in the infectious process and in the formation of post-vaccination immunity. Epidemi- ology and vaccination prevention. 2016;15(3):72–82. doi:10.31631/2073-3046-2016-15-3-72-82. 24. Ivanova EI, Popkova SM, Dzhioev YP, Dolgikh VV, Rakova EB, Bukharova EV. Detection of some genes encoding pathogenic- ity factors in typical isolates of Escherichia coli. Acta Biomed Sci. 2014;5(99):89–94. Available from: https://cyberleninka.ru/article/n/detektsiya-nekotoryh- genov-kodiruyuschih-faktory-patogennosti-u-tipichnyh- izolyatov-escherichia-coli, Accessed on 10 May 2022. 25. Haitovich AB, Gafarova AS. Pathogenicity factors of Candida albicans and determination of their gene determinants. TMBV. 2016;3:121–126. Available from: https://cyberleninka.ru/article/n/faktory-patogennosti- candida-albicans-i-opredelenie-ih-gennyh-determinant, Ac- cessed on 10 May 2022. https://doi.org/10.17277/AMT.2018.03.PP.063-069 https://doi.org/10.1063/1.4942618 https://doi.org/10.3390/ma12030465 https://doi.org/10.1016/j.jallcom.2009.10.037 https://doi.org/10.1088/0957-4484/27/17/175302 https://doi.org/10.1134/S1063783421090237 https://doi.org/10.1038/nrmicro842 https://doi.org/10.1063/PT.3.1678 https://doi.org/10.1051/epjconf/201818510002 https://cyberleninka.ru/article/n/antibakterialnoe-deystvie-nanochastits-zheleza-i-medi-na-klinicheskie-shtammy-rseudomonas-aeruginosa https://cyberleninka.ru/article/n/antibakterialnoe-deystvie-nanochastits-zheleza-i-medi-na-klinicheskie-shtammy-rseudomonas-aeruginosa https://cyberleninka.ru/article/n/antibakterialnoe-deystvie-nanochastits-zheleza-i-medi-na-klinicheskie-shtammy-rseudomonas-aeruginosa https://cyberleninka.ru/article/n/otsenka-genotoksichnosti-nanochastits-pri-ispolzovanii-v-meditsine https://cyberleninka.ru/article/n/otsenka-genotoksichnosti-nanochastits-pri-ispolzovanii-v-meditsine https://cyberleninka.ru/article/n/sposobnost-escherichia-coli-formirovat-bioplenki-v-prisutstvii-nanochastits-oksida-alyuminiya https://cyberleninka.ru/article/n/sposobnost-escherichia-coli-formirovat-bioplenki-v-prisutstvii-nanochastits-oksida-alyuminiya https://cyberleninka.ru/article/n/sposobnost-escherichia-coli-formirovat-bioplenki-v-prisutstvii-nanochastits-oksida-alyuminiya https://science-education.ru/ru/article/view?id=28276 https://science-education.ru/ru/article/view?id=28276 https://doi.org/10.1080/1547691X.2019.1584652 https://cyberleninka.ru/article/n/otsenka-toksichnosti-i-potentsialnoy-opasnosti-nanochastits-oksida-alyuminiya-dlya-zdorovya-cheloveka https://cyberleninka.ru/article/n/otsenka-toksichnosti-i-potentsialnoy-opasnosti-nanochastits-oksida-alyuminiya-dlya-zdorovya-cheloveka https://cyberleninka.ru/article/n/otsenka-toksichnosti-i-potentsialnoy-opasnosti-nanochastits-oksida-alyuminiya-dlya-zdorovya-cheloveka https://www.atcc.org/products/10231-mini-pack https://www.atcc.org/products/25923 https://www.atcc.org/products/25922 https://cyberleninka.ru/article/n/detektsiya-nekotoryh-genov-kodiruyuschih-faktory-patogennosti-u-tipichnyh-izolyatov-escherichia-coli https://cyberleninka.ru/article/n/detektsiya-nekotoryh-genov-kodiruyuschih-faktory-patogennosti-u-tipichnyh-izolyatov-escherichia-coli https://cyberleninka.ru/article/n/detektsiya-nekotoryh-genov-kodiruyuschih-faktory-patogennosti-u-tipichnyh-izolyatov-escherichia-coli https://cyberleninka.ru/article/n/faktory-patogennosti-candida-albicans-i-opredelenie-ih-gennyh-determinant https://cyberleninka.ru/article/n/faktory-patogennosti-candida-albicans-i-opredelenie-ih-gennyh-determinant