2. Rhamnolipids as a Tool for Eradication of Trichosporon cutaneum Biofilm
Rhamnolipids have many advantageous properties, which are used in solving problems of environmental pollution; in the food, cosmetics and agricultural industries; and finally, in pharmacy or medicine. In these industries, emulsifying, solubilizing or wetting properties are mainly used, as well as metal sequestration
[11] and possibly antimicrobial activity in medicine
[12]. These properties are underlined by the environmental friendliness of rhamnolipids. Rhamnolipids are biodegradable substances and, therefore, when applied, we do not encounter toxicity and accumulation in the environment, and thus have huge potential for use in bioremediation
[13].
The antimicrobial and antibiofilm activity of rhamnolipids has been reported in many publications
[14][15][16][17]. The rhamnolipid antibacterial activity against a wide variety of microorganisms has been reported in many studies
[12][17][18]. The mechanism of action of rhamnolipids is complex and mainly involves interactions with cell surface structures such as lipopolysaccharides, phospholipids and proteins
[19][20][21]. Rhamnolipids interact with lipopolysaccharides
[20], the phospholipid membrane and protein structures
[19]. The action of rhamnolipids results in a change in the character of the cell surface, for example, a change in hydrophobicity
[20] and / or the surface charge of the cell
[22].
Additionally, the effectiveness of action is dependent on the composition of the rhamnolipid mixture, as well as on the type of exposed microorganism
[20]. Studied rhamnolipid mixtures produced by four different strains of
P. aeruginosa showed significantly different representations of congeners, comprising RhaFA, RhaFAFA, RhaRhaFA and RhaRhaFAFA. The presence of unsaturated FA was also determined for the properties of rhamnolipid mixtures. In studied mixtures, the abundance of unsaturated FA varied from 5.4% to 21.6%. Rooney et al.
[23] reported similar differences between rhamnolipid mixtures produced by several
P. aeruginosa strains. The content of unsaturated FA in these mixtures varied between 0% to 12.7%.
The composition of rhamnolipids is crucial for the value of the critical micelle concentration (CMC). Among other factors, CMC increases with the amount of unsaturated FA
[24]. On the other hand, the higher content of congeners containing only one molecule of rhamnose (RhaFA and RhaFAFA) results in a decrease in CMC
[25]. This correlates with the results, in which the lowest content of unsaturated FA (5.4%) was found in rh3776, as well the lowest value of CMC (15 mg L
−1). On the contrary, the highest content of unsaturated FA was found in rh3777 (21.6%), but the value of CMC (55.4 mg L
−1) was lower than in the rh3774 mixture (unsaturated FA 18.8%, CMC 75.5 mg L
−1). This was probably influenced by the higher abundance of mono-rhamnolipid congeners in rh3777 in comparison with rh3774.
From the screening experiments under static conditions, it is obvious that the composition of rhamnolipids influenced the response of
T. cutaneum biofilm to the treatment. Interactions between cell surface structures and rhamnolipids are probably a crucial step for the mechanism of action. In addition, the production or presence of rhamnolipids has an important role in the development of biofilm, including the maintenance of open channels and void spaces, as well as the facilitation of cell detachment from the biofilm structure
[26]. It was found that a concentration higher than CMC must be used to produce a significant decrease in the colonized area. When the biofilm was exposed to the highest concentration of surfactants (1000 mg L
−1), removal of more than 90% was achieved. However, the effectiveness of concentrations between CMC and 1000 mg L
−1 varied. Obviously, the suitability of rhamnolipid mixtures and the used concentration must be studied before concrete application. For example, rh3777 was proven to have a significant impact already at CMC concentration (55.4 mg L
−1), and the biofilm was reduced by almost 33%. Moreover, the treatment by 250 mg L
−1 resulted in a decrease of almost 80%. Conversely, rh3776 showed the same effect after biofilm exposition to 100 mg L
−1, and from this point of view, rh3776 seemed to be the most effective mixture. However, at the CMC, it had a very low impact (9–18%), which may be attributed to the low CMC value (15 mg L
−1), showing that the absolute concentration value was a more important factor than the CMC. Singh et al.
[27] also showed the dependency of rhamnolipid action on concentration when biofilm of
Candida albicans was treated by rhamnolipids produced by
P. aeruginosa in a concentration range of 40–5000 mg L
−1.
Kim et al.
[28] reported that the CMC (240 μg mL
−1) of used rhamnolipids demonstrated efficacy on
P. aeruginosa biofilm. The anti-adhesive activity of rhamnolipid produced by
P. aeruginosa against several bacterial and yeast strains isolated from voice prostheses was evaluated in
[29]. The experiments were performed under dynamic conditions in a parallel plate flow chamber. The best results for the reduction in the adhesion rate occurred for
Streptococcus salivarius GB 24/9 and
Candida tropicalis GB 9/9 (an average of 66%). The potential of rhamnolipids to prevent biofilm formation was reported by Gomes and Nitschke
[30]. The treatment by rhamnolipids (1.0% solution) reduced adhesion to the polystyrene of
Listeria monocytogenes (by 57.8%) and
Staphylococcus aureus by (67.8%). Dusane et al.
[15] showed that rhamnolipid disrupted the pre-formed biofilm of
Yarrowia lipolytica in a more effective manner than chemical surfactants (cetyl-trimethyl ammonium bromide and SDS).
The conduction of pilot experiments under static conditions was chosen due to its simplicity, cost efficiency and multiplicity. On the other hand, these conditions also have several limitations, including problems with the separation of attached and loosely attached cells, the definition of the washing process and the quantification of washed-off cells
[9]. Concurrently, it must be taken into account that the process of biofilm development is very stochastic; therefore, the independent repetition of biofilm cultivation may vary, even if the cultivation conditions are kept constant
[31]. In addition, culture conditions are changed over the duration of the experiment (substrate utilization and cell metabolism). These factors could have a significant effect on biofilm stability and further eradication. Therefore, the antibiofilm activity of rhamnolipids was also investigated under dynamic conditions conducted in a single-channel flow cell. The behavior of rhamnolipids and synthetic surfactants did not correspond with those obtained from static conditions in all cases. Rh3774, rh3775 and rh3777 had the same ability to reduce biofilm colonization, up to 95% (16 h). The same effect on biofilm eradication was found after SDS treatment, almost of 86% (16 h). However, rh3776 and Tween 80 showed a different effect; the colonized area was reduced by only 41% and 59%, respectively. These differences in surfactant effectiveness support the necessity to perform experiments under both static and dynamic conditions and highlight the importance of rhamnolipid mixture composition determination and characterization in relation to their intended application. Performed experiments showed that dynamic conditions had no impact on the biological activity of rhamnolipids with higher CMC as well SDS, in contrast to rh3776 and Tween 80, with very low CMC (15 and 13 mg L
−1, respectively), which were very effective in biofilm eradication.
Rhamnolipids with low CMC have hydrophobic characteristics, as their molecules are formed predominantly by mono-rhamnolipid congeners with a low abundance of unsaturated FA
[20]. The amphiphilicity of rhamnolipid molecules is crucial for the interactions with the structures of the cell surface or colonized area. Adsorbed rhamnolipids thus change the surface charge, resulting in varied microbial adhesion ability
[15]. In addition, interactions with proteins or lipids forming the surface of cells can lead to the alteration of cell permeability
[32], resulting in a direct impact on cell viability. The treatment of
T. cutaneum by rhamnolipids caused a decrease in the hydrophobicity of cells. Chrzanowski et al.
[33] reported a decrease in the hydrophobicity of yeast
Candida maltosa after treatment by rhamnolipids (150 mg L
−1). The same effect of rhamnolipids on food pathogenic bacteria was found by Gomes and Nitschke
[30]. Similarly, the conditioning of a glass microscope slide led to a significant decrease in surface hydrophobicity depending on rhamnolipid CMC, and thus rhamnolipid composition.
Differences between results obtained under static and dynamic conditions suggest that biofilm eradication under static conditions is mostly a function of rhamnolipid properties (composition), whereas under the dynamic condition, the eradication is influenced by rhamnolipid properties and medium flow rate. The flow rate of the medium can detach weakly bound cells, which are able to adhere under static condition.