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 Table of Contents  
Year : 2021  |  Volume : 10  |  Issue : 1  |  Page : 43-50

The biofilm formation of nontuberculous mycobacteria and its inhibition by essential oils

1 Institute of Ecology and Genetics of Microorganisms, Perm Federal Research Center Ural Branch of RAS; Faculty of Soil Science, Agrochemistry, Ecology and Merchandising, Perm State Agro-Technological University, Perm, Russia
2 Institute of Ecology and Genetics of Microorganisms; Institute of Technical Chemistry, Perm Federal Research Center Ural Branch of RAS, Perm, Russia
3 Faculty of Soil Science, Agrochemistry, Ecology and Merchandising, Perm State Agro-Technological University, Perm, Russia

Date of Submission02-Dec-2020
Date of Acceptance13-Jan-2021
Date of Web Publication28-Feb-2021

Correspondence Address:
Daria V Eroshenko
Perm Federal Research Center Ural Branch of RAS, Institute of Ecology and Genetics of Microorganisms, Goleva 13, Perm 614010
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmy.ijmy_228_20

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Background: Nontuberculous mycobacteria (NTM) form two types of biofilms: Bottom biofilm and pellicle. The spatial distribution of cells between these types of biofilms and their dispersion into the liquid medium depends on the ratio of the nutrient components of the growth medium. The inhibition of biofilm formation by NTM can be achieved through the use of lipophilic compounds, such as essential oils (EOs). Method: The biofilm and pellicle formation of Mycobacterium smegmatis and Mycobacterium avium on four nutrient media under static conditions and in the vapors of six EOs was evaluated by conventional method. The antimycobacterial effect of EOs was also studied by the disc diffusion method. Results: The bottom biofilm and pellicle formation of NTM largely depended on the composition and availability of nutrients. Nutrient media in which NTM form powerful bottom biofilm or pellicle or both have been determined. The growth of studied NTM strains on agar was highly sensitive to the EOs of Scots pine, Atlas cedar, bergamot, and a mixture of EO of different plants. The cultivation of bacteria in the EO vapors also resulted in total suppression of the pellicle for all studied NTM strains. Conclusions: Our data clearly indicate that the carbon-nitrogen ratio is involved in the regulation of the spatial distribution of the biofilm. The preventing effect of EOs vapors, especially the synergistic action of mixture of EOs on the biofilm and pellicle formation by NTMs can be observed.

Keywords: Mycobacteria, biofilm, carbon-nitrogen ratio, essensial oil, vapor

How to cite this article:
Polyudova TV, Eroshenko DV, Pimenova EV. The biofilm formation of nontuberculous mycobacteria and its inhibition by essential oils. Int J Mycobacteriol 2021;10:43-50

How to cite this URL:
Polyudova TV, Eroshenko DV, Pimenova EV. The biofilm formation of nontuberculous mycobacteria and its inhibition by essential oils. Int J Mycobacteriol [serial online] 2021 [cited 2022 Oct 6];10:43-50. Available from: https://www.ijmyco.org/text.asp?2021/10/1/43/310511

  Introduction Top

Mycobacterium species that are widely distributed in the environment but can cause soft-tissue infections in animals and humans usually are surrounded by a remarkably elaborated lipid-rich outer membrane. The hydrophobic structure of the cell wall is responsible for the innate antibiotic resistance of Mycobacterium species.[1] Since it is impermeable for commonly used antibiotics and can be attacked by substances with a high affinity for lipid-rich cell surface. Apparently, one successful route to overcoming the hydrophobic barrier of the mycobacterial outer membrane is to use hydrophobic biologically active compounds, such as essential oils (EOs).

EOs and plant extracts are widely used in the pharmaceutical and cosmetic industries. For example, EOs are used as an adjunct therapy for infectious diseases of the upper respiratory tract.[2] The EOs are used in the agricultural and food industries as an alternative to synthetic food additives to suppress the development of unwanted microflora.[3] In addition, the use of EOs is proposed to prevent food spoilage during transportation and storage.[4] The previous studies of plant EOs as inhibitors of bacterial growth have shown that antibacterial activity depends on the dose, plant and microorganism type, major and minor compounds in the chemical composition of plant EOs, antioxidant ability, and total phenolic content.[4],[5] The inhibitory effect of these compounds has been shown against Gram-positive bacteria of the genera Bacillus, Listeria, Staphylococcus,[6] and Gram-negative such as Escherichia, Klebsiella, Proteus, Salmonella, and Yersinia.[3],[6] The effect of the EOs of some plants has also been studied against Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM). Hence, the oil of the Guinean plant of chalchal (Allophylus edulis) has shown moderate antimycobacterial activity in vivo experiments.[7] EOs obtained from different types of peppers (genus Piper) also have exhibited moderate antibacterial activity against Mycobacterium tuberculosis.[8] The EO of the fruits of the tropical tree Pterodon emarginatus has shown a weak inhibitory effect on Mycobacterium bovis at a concentration of 2.5 mg/ml.[9] A more pronounced antibacterial effect was revealed in the EO of the alpine meadow plant Mutellina purpurea: The inhibition of bacterial growth of M. tuberculosis was observed at a concentration of 64 μg/ml.[10] However, most studies have predominantly demonstrated the antibacterial effects of EOs on planktonic forms of mycobacteria. At the same time, studies of the effect of EOs and their vapors on biofilm formation of NTM are very rare. Thus, Peruč et al. recently established the inhibition of adhesion and biofilm formation of Mycobacterium avium and M. intracellulare on abiotic and biotic surface under the influence of subinhibitory concentrations of Juniperus communis EO and Helichrysum italicum Eos.[11] In most natural environments, including the human body, biofilm is the prevailing microbial lifestyle. Nontuberculous mycobacteria encompass more than 150 Mycobacterium species also prefer surface attachment and growth rather than replicating in aqueous suspension.[12] Hence, mycobacteria are capable of forming powerful biofilms both on a solid surface and at the air-liquid interface.[13],[14] The polymeric matrix consisting of lipid, polysaccharide, DNA, and protein protects cells within the biofilm from water-soluble antibiotics and disinfectants.[15] The biofilm formation and the intensity of matrix components synthesis are regulated by the balance of nutritional components along with other factors.[16] We also suggest that the biofilm formation by mycobacteria can be inhibited by low-molecular hydrophobic compounds comprising the biologically active base of EOs. Therefore, the aim of this study was to investigate the biofilm formation of NTM depending on the availability of the nutrients and the effect of EOs on this process.

  Method Top

Bacterial culture conditions

We use the following strains of nontuberculous mycobacteria: M. avium GISK 168, Mycobacterium smegmatis GISK 107, M. smegmatis mc2 155. Bacteria were grown on Middlbrook 7H9 broth (Difco) at 37°C with shaking for 48 h. Then, the bacteria cells were washed with 0.1% Tween 80 (v/v; Sigma, USA), re-suspended into the appropriate medium at the final concentration of 1-3 × 107 CFU/ml and used as an inoculum for biofilm formation.

To analyze biofilm formation Sauton medium, Luria Bertani (LB) medium, Lactate broth without peptone,[17] and M63 medium supplemented with 0.2% glycerol and 1 mM MgSO4 were used.

Essential oils

The following commercial EOs were used: Essential EO (Eucalyptus globulus Leaf Oil), pine EO (Pinus silvestris Leaf Oil) (Mirrolla LLC, Russia), bergamot EO (Citrus bergamia Risso), Atlas cedar EO (Cedrus atlantica), Scots pine EO (Pinus silvestris) (Botanika LLC, Russia) and the EOs mixture “Dyshi” (Akvion, Russia), including peppermint oil (Mentha piperita) with levomenthol-39.55%, eucalyptus oil (Eucalyptus globulus)-35.45%, cayeput oil (Melaleuca leucadendron)-18.5%, wintergin oil (Gaultheria progumbens)–3.7%, oil juniper (Juniperus communis)–2.7%, and clove oil (Eugenia caryophyllata)–0.1%.

Biofilm and pellicle assay

The assay was performed in triplicate using 40-mm polystyrene Petri dishes. Briefly, 3 ml of NTM suspensions at a final concentration of 1–3 × 107 CFU/ml in the different medium were added into Petri dishes and incubated 5 days at 37°C to allow biofilm and pellicle formation. The Petri dish with medium only was used as a negative control.

Then, the contents of each Petri dish were divided into three subpopulations (pellicle, planktonic cells, and bottom biofilm) as previously described.[18] Briefly, to remove pellicle at the air-liquid interface Phosphate-buffered saline were added to the liquid medium under the pellicle until one was raised above. Then, the lid of the Petri dish was carefully applied to the pellicle surface, so that the pellicle completely adhered to the lid surface due to their hydrophobicity. The liquid part containing the planktonic cells was transferred to a test tube and centrifuged (5 min, 12,000 rpm), the supernatant was removed, and the pellet was used for the staining. The bottom biofilm was stained in the original Petri dish. The biomass of each type of subpopulations was determined by the staining with 3 ml of 0.1% violet crystal for 20 min at room temperature, washed with distilled water at least three times to remove excess dye and allowed to dry at room temperature. The extraction of violet crystal was carried out with 95% ethanol. The optical density at 570 nm was read using a PD-303 spectrophotometer (Apel, Japan).

Antimycobacterial activity of essential oils

The antibacterial effect of EOs was studied by the disc diffusion method. Briefly, 1 ml of NTM suspensions at a final concentration of 108 CFU/ml was spread over 90 mm Petri plate with Middlebrook 7H11 agar (BD, USA), the excess liquid was removed and plates were dried. Under aseptic conditions, a 6 mm empty sterilized paper discs (Munktell, Germany) were impregnated with 5 μl of the investigated EO and placed on the agar surface. Dry paper disc was placed on the seeded Petri plate as a negative control. The plates were incubated at 37°C for 5 days. After the incubation period, the effectiveness of the oils was evaluated based on the diameter of the growth inhibition zones.

Determination of phenolic compounds in essential oils

The content of phenolic compounds in EOs in terms of tannin was determined by the method of permanganatometric titration according to “Determination of the content of tannins in medicinal plant materials and herbal preparations” guidelines.[19]

Antibiofilm effect of essential oils

To study the effect of EOs on the biofilm and pellicle formation mycobacteria (1–3 × 107 CFU/ml) were grown in Lactate Broth in 40-mm polystyrene Petri dishes. EOs (10 μl) were applied to the inner side in the center of the lid of the Petri dish for evaporation. The dishes were sealed with Parafilm M and incubated at 37°C for 5 days. The biomass of each type of subpopulations was determined as described above.


All studies were performed in triplicate, and the mean value was calculated. The means were analyzed by one-way analysis of variance followed by Tukey's post hoc multiple comparison test using Prism 6.0 software for Windows (GraphPad, San Diego, CA, USA) for Windows. The results were expressed as mean ± standard deviation P < 0.05 were considered as significant.

  Results Top

NTMs biofilm spatial distribution depends on carbon-nitrogen ratios

When grown in liquid nutrient media without stirring, mycobacteria form a floating biofilm or pellicle at the air-liquid interface.[14] At the same time, NTM can form biofilms on solid surfaces due to their high affinity for some abiotic materials, such as polyvinyl chloride or polycarbonate.[20] The ratio of carbon and nitrogen in nutrient media can be a determining factor in the coaggregation of mycobacteria and the biofilm formation.[16] Therefore, we decided to conduct a comparative analysis of the ability of NTM to form biofilms in nutrient media with different carbon-nitrogen ratios [Table 1].
Table 1: Carbon-nitrogen composition of nutrient media

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All studied strains of NTMs were capable of forming biofilms both on a solid surface and at the air-liquid interface. The intense growth was observed on LB, Lactate Broth and Sauton media, while the M63 medium did not result in the intensive development of mycobacteria of all the studied strains [Figure 1]. The NTMs biofilm spatial distribution varies in different culture media after 5 days of cultivation. In LB, all the studied strains form an abundant bottom biofilm and weak pellicle. Hence, biomasses of M. smegmatis mc2 155 planktonic cells and pellicle were 10 and 15% of total biomass, respectively. The planktonic cells of M. avium GISK 168 and M. smegmatis GISK 107 grown in LB medium amounted to 5.5 and 9.5%, whereas the pellicle biofilm contained about 21 and 16% of the biomass, respectively [Figure 1]. Lactate broth contributed to the formation of the equally abundant bottom and pellicle biofilms, while the plankton cells were practically not detected (<1% for M. smegmatis mc2 155 and M. smegmatis GISK 107 and 2% for M. avium GISK 168). Contrary, the M63 medium promoted the growth of all studied NTMs attached to the solid surface, and no pellicle biofilm was observed at all. In Sauton, the biomass of all studied NTMs strains was distributed almost evenly in the bottom biofilm, the plankton cells, and pellicle.
Figure 1: Spatial distribution of Mycobacterium smegmatis GISK 107 (a), Mycobacterium smegmatis mc2 155 (b) and Mycobacterium avium GISK 168 (c) subpopulations (bottom biofilm, pellicle and planktonic cells) in different culture media after 5 days of incubation

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It should be noted that the growth pattern and the morphology of mycobacterial cells within the different types of biofilms were significantly different. A more detailed view of the arrangement of the cells within biofilms was obtained by microscopical analysis [Figure 2]. After adhesion of cells to polystyrene surface, their asymmetric division and distribution over the surface occur due to the sliding mechanism.[21] As a result, the bundles and mycelium-like structures were formed in bottom biofilm [Figure 2]a, while pellicle at the air-liquid interface was on the contrary formed by aggregates of shorter cells [Figure 2]b.
Figure 2: Bottom biofilm (a) and pellicle (b) of Mycobacterium smegmatis mc2 155 (5 days, lactate broth) after crystal violet staining (×1000)

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The effect of essential oils on NTMs growth on agar

Before studies of the effect of EOs and their vapors on biofilm formation by NTM, we evaluated the effect of EOs on the growth of mycobacteria on agar using the conventional disco-diffusion method. It has been experimentally shown that all studied EOs inhibit the NTM growth on a solid medium. Zones of suppressed bacterial growth by EOs were regular in shape with sharp edges, which characterizes the uniform diffusion of active substances in agar plate [Figure 3].
Figure 3: The zone of inhibition of Mycobacterium smegmatis mc2 155 growth around the discs with essential oils s: 1 – essential oils of bergamot, 2 – essential oils of eucalyptus, 3 – essential oils of pine

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So, NTMs were highly sensitive to EOs of bergamot, Atlas cedar, Scots pine, and the EOs mixture [Figure 4]. Slight inhibition of bacterial growth was observed when exposed to pine and eucalyptus EOs (Mirroll).
Figure 4: The antimycobacterial activity and the phenolic compounds content of essential oils on: 1 – essential oils of eucalyptus, 2 – essential oil of pine, 3 – essential oil of Scots pine, 4 – essential oils of Atlas cedar, 5 – mixture of essential oils (“Dyshi”), 6 – essential oil of bergamot

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The conventional disco-diffusion method to determine the antibacterial activity of various compounds is based on the diffusion of the substance into the agar layer. The diffusion of the substance, in turn, depends on their solubility in water. Therefore, the content of hydrophilic phenolic compounds in the studied EOs was determined by the method of permanganatometric titration. It turned out that the content of phenolic compounds varies significantly among studied EOs but there was a correlation between the antibacterial activity and the content of hydrophilic phenolic compounds of EOs [Figure 4].

The effect of essential oils on the pellicle of NTMs

However, soluble phenolic compounds represent only a small fraction of biologically active substances in EOs and most EO components can evaporate. In this connection, we studied the effect of EO vapors on NTM biofilm formation in lactate broth. An inhibition of bacterial growth in an atmosphere of EO vapors (1 μl/cm3) was observed for all studied NTM strains. So, the complete absence of NTMs pellicle on the air-liquid interface was observed for all studied EOs [Figure 5]. Moreover, biomass decreased not only in the pellicle but also in the bottom biofilm. It should be noted that the oils of Atlas cedar, Scots pine, and pine contributed to an increase in the number of plankton cells. At the same time, all EOs inhibited the bottom biofilm formation of NTM, except for the pine EO, the vapors of which did not prevent the formation of the bottom biofilm by M. smegmatis GISK 107.
Figure 5: Spatial distribution of Mycobacterium smegmatis GISK 107 (a), Mycobacterium smegmatis mc2 155 (b) and Mycobacterium avium GISK 168 (c) subpopulations (bottom biofilm, pellicle and planktonic cells) in the atmosphere of essential oils vapors

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In our study, bottom biofilms formed on the polystyrene surface in an atmosphere of EOs had lower biomass than biofilms grown under optimal conditions. Microscopy of biofilms revealed that the constituent cells sorbed the dye (gentian violet) to a lesser extent and were distinguished by an elongated filiform shape [Figure 6].
Figure 6: Bottom biofilm of Mycobacterium smegmatis mc2 155 (5 days, Lactate broth): (a) Growth control, (b) In the presence of bergamot essential oils vapors

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  Discussion Top

The development of multicellular structures, biofilms, is based on the ability of mycobacterial cells to adhere to each other and to hydrophobic substrates, such as polystyrene, due to the high cell wall hydrophobicity. Coaggregation of mycobacteria regulate due to a large number of genes, which are usually under the control of the transcriptional regulator GlnR, that is also responsible for the nitrogen nutrition of bacteria and is activated by limiting this element.[22] The global role of GlnR extends far beyond the assimilation of nitrogen as it also controls the expression of carbohydrate ATP-dependent transport systems, and promotes the absorption of carbohydrates in response to nitrogen starvation.[16],[23]

Analysis of the carbon-nitrogen ratios of the studied nutrient media has shown that the smallest amount of nitrogen in the form of an ammonium salt can be found in the minimal M63 medium. As mentioned above, in the M63 medium, the pellicle was practically absent, and the main biomass was concentrated within the bottom biofilm. The low amount of nutrients in this medium probably activated the adhesion of mycobacteria to solid substrates. In attachment to a solid surface, the essential role is usually assigned to cell wall lipids containing glycopeptidolipids in their structure, which contribute to cell disaggregation and spread in response to nitrogen and carbon starvation. Increasing the surface of plankton cells due to disaggregation allows contact with solid substrates with a high probability. This process can be often observed for NTM in nature, such as M. avium, which forms abundant biofilm in water supply systems with fluid flows and oligotrophic environment.[24]

In the Sauton medium, which is designed for the cultivation of mycobacteria, citric acid and glycerin act as carbon sources, but L-asparagine and ammonium ferric citrate act as carbon and nitrogen sources simultaneously. The composition of the Sauton medium suggests a deficiency of ammonia nitrogen in the initial stages of cultural development and thereby, stimulates bacterial adhesion to polystyrene. At the same time, a sufficient number of carbon sources contribute to cell aggregation and the synthesis of trehalozodimycolates and free mycolic acids.[16],[23] Carbon in this medium is represented by compounds that are readily available for mycobacteria and quickly utilized. Therefore, when there is a lack of carbon and an appearance of ammonium ions due to the deamination of asparagine, mycobacterial cells receive a signal for disaggregation and transition to the plankton state.[16] In fact, the appearance of planktonic cells on Sauton medium was observed starting from 2 to 3 days of cultivation. As a result, after 5 days, the bacterial biomass was distributed almost uniformly in the bottom biofilm and planktonic cells with a slight decrease in the pellicle [Figure 1].

The rich LB medium, containing carbon in abundance in the form of amino acids, peptides, and peptones, contributed to the NTM growth, in particular, those attached to the polystyrene surface. Although yeast extract and tryptone are sources of nitrogen and provide the necessary vitamins and minerals, nitrogen in these sources is believed to be inaccessible for NTM, since deamination by mycobacteria was shown only for asparagine, arginine, glutamate, and serine. Moreover, transport systems for the absorption of peptides are rare in mycobacterial cell membrane.[22],[25] Thus, the content of the total nitrogen in the LB medium was similar to one in the M63 medium. The lack of nitrogen stimulated both the bacterial adhesion and cell coaggregation in the pellicle and combined with a sufficient level of carbon, prevented cell dispersion and their transition to the plankton state. Since starvation is a signal for cell disaggregation, the planktonic culture accounted for an insignificant part of bacterial population grown in the LB medium (5%–10%). It should be noted that the biomass of the planktonic subpopulation of M. smegmatis GISK 107 and M. avium GISK 168 was represented by bacterial aggregates without noticeable turbidity of the growth medium; uniform turbidity of the liquid medium was observed only for M. smegmatis mc2 155.

Usually, Lactate broth is used for propionic bacteria only,[26] but it had recently been established that mycobacteria able to use lactate as the sole carbon source.[27] To our surprise, Lactate broth was also well suitable for the robust growth of NTM pellicles, while plankton cells were practically not detected. The insignificant presence of plankton cells in the liquid medium was probably due to the dispersion of individual cell aggregates upon mechanical action on biofilm. We also found that the exclusion of tryptone from the medium composition did not reduce its nutritional properties for the NTMs growth. It is possible that the adhesion on a solid surface was triggered by a deficiency of ammonium nitrogen, similar to the nutrient medium with LB and Sauton mediums. Meanwhile, excess carbon sources promoted cell aggregation and pellicle formation. The Lactate broth was most favorable for the growth of the pellicles for all studied NTMs, as we observed the surface areas coated with the bacterial mass as well as the biofilm thickness was significantly higher for the pellicle biofilm than bottom biofilm [Figure 2]. This may be because NTM cells within the pellicle have access to both nutrients and oxygen due to contact with the gaseous and liquid phases. In addition, pellicle formation is associated with the presence of trehalozodimycolates in the cell wall of mycobacteria (cord-factor). The amphiphilic properties of these compounds allow to orient the cell clusters in such a way that they contact with trehalose sites and mycolate groups mediate the outer hydrophobic layer [Supplementary Figure 1]a, or face the inside of cell aggregates, providing a strong attachment between cells.[28] An excess of trehalozodimycolates in the intercellular space exerts pressure on nearby clusters of cells, causing them to spread over the surface and even form of a ring of biofilm on the Petri dish walls at air–solid interface [Supplementary Figure 1]b.

Thus, the comparative testing of the culture media was necessary to create biofilm models in vitro. According to our data, LB and M63 media can be recommended for the study of the mycobacterial bottom biofilms. They can also be used to screen compounds that inhibit the adhesion of mycobacteria to solid surfaces to reduce microbial colonization on medical devices. Whereas the lactate broth was most favorable for the pellicle formation by NTM and so, to study the pellicle model, we used this medium.

It had been shown that EOs inhibit the growth of tuberculous and nontuberculous mycobacteria on a solid medium.[7],[8],[9],[10],[11] The EOs usually includes terpenoids, among which there are phenols, aldehydes, ketones, alcohols, ethers. The antibacterial activity of terpenoids depends on their functional groups, in particular the number of phenolic and hydroxyl groups, hydrophobicity, the number of acceptor atoms capable of forming hydrogen bonds, and environmental conditions.[29] Therefore, we firstly evaluated the effect of EOs on the growth of NTM on agar using the conventional disco-diffusion method. All the studied EOs had an inhibiting effect on the growth of NTMs on agar.

However, soluble phenolic compounds represent only a small fraction of biologically active substances in EOs. Mycobacterial pellicles have a unique structure in that cells are embedded in a lipid-rich extracellular matrix containing free mycolic acids. In addition, cells in such biofilm are drug-tolerant and can survive at higher concentrations of antibacterial agents.[30] It had been known that the pellicle formation by NTMs can be inhibited by the lipophilic compounds, such as essential plant oils and their biologically active compounds. In this study, we observed an inhibition of bacterial growth when biofilms were formed by mycobacteria in Lactate Broth in an atmosphere of EO vapors (1 μl/cm3). The observed complete suppression of both types of biofilm (pellicle and bottom biofilm) for all the studied strains in the presence of vapors of the EO mixture “Dyshi” most likely was due to the eucalyptus oil, whose inhibitory effect against mycobacteria has been repeatedly shown.[31] Cajuput oil in this composition is also assumed to contribute to the high antimicrobial activity of the composition,[32] as well as Gaultheria procumbens L. oil.[33] The antibacterial activity of Juniper oil against M. avium has been shown recently; in addition, the synergistic effect of EOs of different plants has been proven.[11] Therefore, we supposed the high antimycobacterial and antibiofilm effect of the “Dyshi” composition is due to the synergistic effect of the Eos, but this phenomenon requires further research.

EOs are complex natural mixtures that can contain about 20–60 components in completely different ratios. However, they are dominated by several basic compounds in fairly high concentrations (20%–70%) compared with other substances that are only present in trace amounts. According to various authors, terpenes (limonene, β-pinene, and γ-terpinene) that dominate in EO of bergamot and terpenoids (linalool, linalyl acetate, and limonene) possess the highest antibacterial activity.[29],[34],[35] EOs of Atlas cedar and pine have a similar composition with the main components: ά and β-pinene, and terpenes-limonene and myrcene.[36] Heterocyclic sesquiterpenes were also found in the composition of EO of cedar.[37] A slightly different composition of biologically active compounds is shown for EO of eucalyptus (Eucalýptus), in which cinolol (eucalyptol) predominates; whereas citronellal, citronellol, and a small amount of linalool and ά-pinene are also present.[38] Most of the components in EOs are highly volatile. Inouye et al. shown that a significant part of the compounds constituting EO vapor (linalool, limonel, ά-pinene, carveol, etc.) can diffuse unchanged into the nutrient medium, while some compounds undergo chemical modifications.[34] This can explain the biological activity of EO vapors, manifested in the inhibition of the biofilm formation at the solid-liquid interface.

It is known that the active components of EO affect on the membranes of bacteria, damaging them, and facilitating the leakage of biopolymers from the cell.[39] In addition, it has been shown that the bacterial cell wall, which maintains the cell shape, strength, osmotic stability and determines adhesion properties, is also attacked by biologically active molecules from EO. Meanwhile, the morphology of bacterial cells changes under the action of terpenes and terpenoids.[10] We also observed some alteration in the biofilm structure after EOs treatment. Microscopy of bottom biofilm revealed that the constituent cells sorbed the dye (gentian violet) to a lesser extent and were distinguished by an elongated filiform shape [Figure 6]. Cell elongation without signs of active division can be the result of oxidative stress, which occurs under the action of both individual components of EO and oils in general. Li et al.[40] demonstrated the appearance of reactive oxygen species (ROS) in bacterial cells upon exposure to biologically active compounds from EO of Monarda punctata, but the most intense formation of ROS occurred upon exposure to whole EO. The combined effects of phenols, terpenes, and terpenoids on the bacterial cell wall and outer membrane make them more permeable. The absence of a native form, structural integrity, and osmotic stability after the combined treatment by EO confirms that the outer membrane or bacterial cell wall is most likely the target for terpenes.

  Conclusions Top

All studied NTM strains are capable of forming biofilms in vitro. This property is not species-and strain-specific in this group of mycobacteria. In this report, the influence of medium composition on the type of biofilm formed by M. smegmatis and M. avium was studied. The biofilm formation on different surfaces is significantly affected by the nutrient composition of the medium, varying which one can get durable biofilm on a solid surface or at the air-liquid interface, or robust yielding of plankton. Further studies will be required to fully understand the impact of the nutritional status on the regulation of biofilm formation. Our data clearly indicate the involvement of carbon-nitrogen and amino acid composition in the control of the spatial distribution of the biofilm.

The results obtained in the study indicate a high antimycobacterial activity of natural EOs, which are represented by a huge number of commercial mixtures with impressive antimicrobial potential. The most important conclusion of this work is the revealed effect of EO vapors, which manifests itself in the absolute suppression of the development of mycobacterial pellicle. In this regard, the high volatility of the oils is their advantage, since, in addition to the antibacterial effect of the vapors, there is no accumulation of the negative effect of the intense aroma characteristic of most essential substances. EOs can be used as a safe and natural alternative to antibiotics and preservatives with a lower frequency of formation of multidrug resistance in bacteria. The effect of biologically active agents of EOs on the outer shells of mycobacteria, increasing their permeability, can become the basis for the search for synergistic combinations of oils with traditional antibiotics.

Financial support and sponsorship

RF Ministry of Science and Higher Education (AAAA-A19-119112290009-1).

Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1]


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