General patternsof Aspergillus spp. biofilm development dynamics

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Introduction

Given a statistically significant trend of increasing incidence of socially significant mycoses mediated by excessive growth and adventitious sporulation in tissues, the dominance of the etiological significance of Aspergillus spp. fungi has been established [1–3]. The duration of mycological studies in the differentiation of chronic phthisiatric processes reaches from 46 to 110 days, which is associated with the use of antifungal therapy [4–6]. Azole-resistant strains are classified as the first category of critical priority for research in accordance with the WHO Fungal Priority Pathogens List ^[1] [7, 8]. Territorial rankings of the representative sample make it possible to predict a further increase in resistance to drugs and disinfectants in isolates identified from pathological material of mammals, birds, reptiles, amphibians, fish, and bees [9–15]. Contamination of food products and feed with fungal metabolites at concentrations hazardous to human and animal health can reach 40.0 to 100.0% [16–19]. During the development of local and systemic infections in humans and animals, phytopathology, as well as degradation of natural and synthetic polymers, residential and industrial structures, the morphogenesis of micromycetes is initiated by the presentation of Quorum Sensing (QS) signaling molecules [20–23]. To optimize long-term and retrospective schemes of mycological research, to develop effective drugs that reduce the adhesive properties of microorganisms, block synthesis, or destroy the polymer matrix, studies of the mechanisms of initiation, development, formation, and dispersal of biofilms are of relevance and scientific novelty.

The aim of the study was to analyze the dynamics of changes in morphofunctional parameters during the initiation, development, and dispersal of Aspergillus spp. fungi.

Materials and methods

Strains. The reference strains (ATCC) used in the experiments were Aspergillus flavus 16883, Aspergillus fumigatus 1028, and Aspergillus niger 16404 ^[2].

Nutrient media. The following media were used for cultivation: meat-peptone broth (MPB), meat-peptone agar (MPA), corn agar; Czapek agar with yeast extract (Obolensk, Federal Budgetary Institution of Science “State Scientific Center for Applied Microbiology and Biotechnology”, Russia); chocolate agar (BioMedia, Russia); Sabouraud Fluid Medium and Sabouraud Dextrose Agar with Chloramphenicol & Cycloheximide (Condalab, Spain).

The microorganisms were cultivated at 35 ± 2.0 °C for 168 ± 1 h. Phenotypic characteristics were studied using standard mycological methods. For morphometric studies, samples were fixed with 30.0% NaOH solution for 30 min; stained with aqueous gentian violet solution 1:2000 and by Gram staining (BioVitrum, Russia). Crushed drop slides were made from the fungal mycelium by lightly touching the surface of the microbial colony with dissecting needles. The mycelial hyphae were carefully separated using dissecting needles, then a drop of glycerol was applied and covered with a coverslip ^[3]. To preserve the natural architecture of the biofilms, the preparations were fixed: in vapors of 25.0% glutaraldehyde solution for 8 h; and in 1.0% aqueous osmium tetroxide solution for 4 h. Dehydration of the samples was performed with increasing concentrations of ethanol (30, 50, 96, and 100°) [24]. Optical density of the samples was assessed using a photometric analyzer Immunochem‑2100 (HTI, USA) at OD₅₈₀. Morphometric parameters were evaluated using a Trinocular Unico optical microscope (Unico, USA).

The results were processed using statistical analysis with Student's test. Differences were considered significant at p ≤ 0.05.

Results and Discussion

When cultivated in liquid and solid media at 35 ± 2.0 °C for 168 ± 1 h, the studied microorganisms A. flavus, A. fumigatus, and A. niger exhibited characteristic growth, which proved to be highly informative. The microbial cultures were fast-growing: the growth of micromycetes was observed on the surface of liquid media in test tubes and at the periphery of Petri dishes. This is attributed to the aerobic properties of the fungi. Depending on the composition of the nutrient media and the cultivation time, the micromycetes differed both in the volume of aerial mycelium — ranging from extremely scant to abundant — and in the pigmentation of the reverse side of the colonies (reverse): from barely visible to intensely colored pinkish-­brown tones. The central part of the colony is very dense and more intensely colored, while the peripheral part is thinner, and a pale pigment diffusing into the culture medium was observed (Fig. 1).

 Fig. 1. Cultures of microorganisms A. flavus (35 ± 2.0) °C, 48 ± 1 h: a — Sabouraud's medium; b — chocolate agar
Source: compiled by E.M. Lenchenko, N.P. Sachivkina, D.A. Bannikova.

Cultures of A. fumigatus were smoky, grayish green, with a light yellow reverse. The colony texture ranged from woolly to cottony with some granularity. At the apical poles of the finest thread-like structures, rounded grain-like formations of light gray or intense brown color were observed. Cultures of A. flavus formed woolly green or white fluffy cottony colonies, diffusing into the depth of the medium (2.0–3.0 mm). Initially light green, yellow-­green, or lemon in color, they became dark green, dark yellow-­green, or brown-­green in the center as sporulation progressed. The reverse of the colonies was white cream. Sclerotia composed of tightly interwoven mycelial hyphae were white; they became cream-­colored with increasing cultivation time and turned brown after 168 h. Exudate was typically observed, the drops of which were initially colorless but gradually acquired a light brown color. Differential characteristics of the colonies included a dark green center and radial furrows, with a thin openwork (lace-like) periphery. Cultures of A. niger initially formed fluffy colonies, then granular colonies; the reverse was colorless or yellowish. With sporulation, the mycelial color varied from dark brown to black.

At 35 ± 2 °C for 168 ± 1 h under static cultivation conditions, depending on the stage of the cell cycle, general patterns of biofilm development were observed in the studied fungi. The dynamics of heterogeneous population development were mediated by adhesion, synthesis of extracellular molecules, proliferation, coaggregation, and differentiation of mycelial structures depending on the stage of the cell cycle. The implementation of intercellular communication processes was initiated by adhesion of conidia to the substrate surface (in our studies, glass). Due to the apical growth of germ tubes, thread-like structures — hyphae — differentiated. As cultivation time increased, areas of significant hyphal consolidation emerged, and conidiogenic structures differentiated. The hyphae of the studied fungal species were septate and colorless.

Conidiophores of A. flavus were coarsely roughened, colorless, 400.0–800.0 ± 0.8 µm in length; vesicles were rounded, 20.0–45.0 ± 0.7 µm, the surface of which was almost completely covered with phialides, 8.0–12.0 ± 0.6 µm, arranged on metulae. Conidia were smooth, rounded, d = 3.0–6.0 ± 0.5 µm.

Conidiophores of A. fumigatus were smooth, colorless, 100.0–300.0 ± 0.4 µm in length; vesicles were dome-shaped, 20.0–30.0 ± 0.3 µm, with densely arranged phialides, 5.0–10.0 ± 0.6 µm. Conidia were smooth or finely roughened, rounded, d = 2.0–3.5 ± 0.8 µm.

Conidiophores of A. niger were smooth, colorless, dark near the vesicle, 300.0–400.0 ± 0.2 µm in length. Vesicles were spherical, 30.0–75.0 ± 0.5 µm; the surface was covered with phialides, 8.0–12.0 ± 0.6 µm, arranged on metulae. Conidia were roughened, rounded, d = 4.0–5.0 ± 0.5 µm (Fig. 2).

Fig. 2. Morphology of A. niger microorganisms, cultivated on MPA at 35 ± 2 °C for 168 ± 1 h. Fixation with 30.0% NaOH solution for 30 min: approx. ×10, vol. ×40, H604 Trinocular Unico, USA
Source: performed by E.M. Lenchenko, N.P. Sachivkina, D.A. Bannikova.

Population immobilization of biofilms is a genetically and phenotypically determined form of intercellular communication — quorum sensing — mediated by the characteristics of growth cycles and population adaptation to ecological and anthropogenic factors [5, 25, 26]. In the development and rotation of new drugs and disinfectants, broad-­spectrum anti-adhesive composite preparations are recognized as promising [11, 24, 27, 28]. The adaptive potential of a heterogeneous multicellular population is inhibited by QS molecules that block the synthesis of intercellular communication inducers, thereby reducing adhesion and, consequently, the degree of microbial contamination [24, 29, 30].

Conclusion

The present study has revealed fundamental aspects of the formation and development of biofilms in representatives of the genus Aspergillus under optimal temperature conditions of 35 ± 2 °C for 168 ± 1 hours. During the experiment, the ability of fungi for self-organization was demonstrated, manifested in the formation of spatially ordered multicellular structures. Each stage of biofilm development — from primary adhesion to final dispersal — demonstrates remarkable population coordination and adaptability. The complex dynamics of population immobilization, involving a successive sequence of phases — proliferation, coaggregation, filamentation, and other key closely interrelated processes — deserve special attention.

The obtained results open new horizons in understanding the mechanisms of micromycete vital activity and can be utilized in several promising areas: improving the methodology of long-term mycological studies; developing innovative strategies to combat persistent biofilms; optimizing biotechnological processes using fungi; and advancing bioremediation methods.

The study of the mechanisms of formation and functioning of fungal biofilms represents a significant contribution to modern mycology. Understanding these processes not only expands fundamental knowledge about fungal vital activity but also opens practical prospects for addressing pressing problems in medicine, veterinary science, and biotechnology.

 

^{1 WHO fungal priority pathogens list to guide research, development and public health action. Geneva: World Health Organization; 2022. Available at: https://www.who.int/publications/i/item/9789240060241 (accessed on: 20.01.2026).}

^{2 ATCC. The Global Bioresource Center. Available at: https://www.lgcstandards-atcc.org (accessed on: 05.05.2023).}

^{3 Sutton D, Fothergill A, Rinaldi M. Identifier of pathogenic and opportunistic fungi. Moscow: Mir; 2001.}

About the authors

Ekaterina M. Lenchenko

All-Russian Research Institute of Veterinary Sanitation, Hygiene, and Ecology - Branch of the Federal Scientific Center VIEV RAS

Author for correspondence.
Email: lenchenko.ekaterina@yandex.ru
ORCID iD: 0000-0003-2576-2020
SPIN-code: 9417-0889

Doctor of Veterinary Sciences, Professor, Scientific Consultant of the Laboratory of Sanitary Microbiology and the Sector for Preparation of Nutrient Media

5 Zvenigorodskoe Shosse, Moscow, 123022, Russian Federation

Nadezhda P. Sachivkina

RUDN University

Email: sachivkina@yandex.ru
ORCID iD: 0000-0003-1100-929X
SPIN-code: 1172-3163

Candidate of Biological Sciences (PhD in Biology), Associate Professor, Department of Veterinary Medicine

8 Miklukho-Maklaya St., bldg. 2, Moscow, 117198, Russian Federation

Daria A. Bannikova

All-Russian Research Institute of Veterinary Sanitation, Hygiene, and Ecology - Branch of the Federal Scientific Center VIEV RAS

Email: andreevna.07@mail.ru
ORCID iD: 0000-0002-4766-0183
SPIN-code: 1618-0317

Candidate of Veterinary Sciences (PhD in Veterinary Medicine), Leading Rese- archer of the Laboratory of Sanitary Microbiology and the Sector for Preparation of Nutrient Media

5 Zvenigorodskoe Shosse, Moscow, 123022, Russian Federation

Marina I. Shopinskaya

RUDN University

Email: shopinskaya-mi@rudn.ru
ORCID iD: 0000-0002-3823-3737
SPIN-code: 2550-4781

Candidate of Veterinary Sciences (PhD in Veterinary Medicine), Associate Professor, Department of Veterinary Medicine

8 Miklukho-Maklaya St., bldg. 2, Moscow, 117198, Russian Federation

References

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