1. In vitro determination of baicalein and chitosan action on Candida parapsilosis, Candida krusei and Trichosporon cutaneum biofilm
Good morning dear committee, dear colleagues. My name is Eva Kvasničková and I would like to short present my work, which name is „Effect of natural substances on Candida spp. and Trichosporon cutaneum biofilms.
Eva Kvasničková, Jan Masák
University of Chemistry and Technology Prague, Czech Republic
Department of Biotechnology

2. Aims of study In vitro determination of baicalein and chitosan action on Candida parapsilosis, Candida krusei and Trichosporon cutaneum biofilm
20. 8.
2015
Eva Kvasničková
To evaluate the effect of antibiotic amphotericin B on Candida parapsilosis, C. krusei and Trichosporon cutaneum biofilms.
To evaluate the effect of natural substances baicalein and chitosan on Candida parapsilosis, C. krusei and Trichosporon cutaneum biofilms.
Using new method for evaluation – Cellavista automatic microscope with software for image analysis.
The main aim of this study was the evaluation of the effect of antibiotic amphotericin B and also natural substances - baicalein and chitosan - on Candida parapsilosis, C. krusei and Trichosporon cutaneum biofilms. (And) Out of our knowledge, the first time using cellavista automatic microscope with software for image analysis for evaluation. 2

3. Introduction In vitro determination of baicalein and chitosan action on Candida parapsilosis, Candida krusei and Trichosporon cutaneum biofilm
20. 8.
2015
Eva Kvasničková
Biofilms
Infections
C. parapsilosis (e.g. fungemia, ocular infections, vulvovaginitis, joint diseases)
C. krusei (e.g. arthritis, endocarditis)
T. cutaneum (e.g. onychomycosis)
Resistance to antibiotics
Amphotericin B
Natural substances
Baicalein
Chitosan
Biofilms are highly organized matrix-enclosed microbial communities that are irreversibly attached to a surface, interface or to each other. These microbial communities often contaminate medical instruments or industrial devices and cause a wide range of human infections.1 Candida spp. are one of the most common isolated yeasts from nosocomial infections (or from biofilms which are formed on medical instruments). C. parapsilosis causes many types of diseases particularly in immunodeficient patients. Examples of these illnesses include fungemia, endocarditis, meningitis, arthritis, ocular infections, vulvovaginitis, urinary tract infections and joint diseases.6 C. krusei is able to cause a spectrum of clinical manifestations such as fungemia, endophthalmitis, arthritis and endocarditis.8 Trichosporon spp. are a normal part of gastrointestinal and oral cavity microbiota, but can cause superficial biofilm associated infections, such as onychomycosis. The huge problem of treating these infections is the high antimicrobial resistance of biofilms to the typically used antibiotics (e.g. amphotericin B). Therefore attention is turning to the use of natural substances with antibacterial, antifungal and antibiofilm properties that may help in the treatment of biofilm infections. These substances can be used in low concentrations and no resistance against them is developed. Baicalein, a flavonoid obtained from Scutellaria baicalensis, has an antioxidative, neuroprotective, antibacterial, antiviral and antifungal properties.16 (And) Chitosan is a hydrophilic biopolymer, polysaccharide, which is present in the outer shell of crustaceans and has a broad antimicrobial activity.17 3

4. Methodology In vitro determination of baicalein and chitosan action on Candida parapsilosis, Candida krusei and Trichosporon cutaneum biofilm
20. 8.
2015
Eva Kvasničková
Candida parapsilosis DBM 2165
Candida krusei DBM 2163
Trichosporon cutaneum CCY
Cellavista
Effect of selected substances on
Microbial adhesion
Eradication of pre-formed biofilm A B
Candida parapsilosis DBM 2165 and Candida krusei DBM 2163 were kindly provide by the collection of microorganisms UCT, Prague. Trichosporon cutaneum CCY was obtained from the Collection of Yeast, Institute of Chemistry, Slovak Academy of Sciences. Yeasts were cultivated in 96-wells polystyrene microtiter plates. The colonized areas on bottom of the wells were measured using Cellavista automatic microscope with subsequent image analysis. On this picture you can see the evaluation process of biofilm photographs taken by Cellavista device. (A) Picture of sessile cells in biofilm. (B) Colonized area of sessile cells in biofilm evaluated by Cellavista software for image analysis. (C) Colonized area of sessile cells in biofilm on black backround. (D) Inversion view of colonized area of sessile cells in biofilm. The effect of selected subtances on microbial adhesion and eradication of pre-formed biofilm was investigated. To test the effect on microbial adhesion the yeasts were cultivated in the presence of either one of the natural substances or antibiotic from the very beginning and after 24 h (in the case of Candida) or 48 hours (Trichosporon) of cultivation the colonized area of the bottom of each well was measured. To test eradication the yeasts were cultivated and after 24 (in the case of Candida) or 48 hours (Trichosporon) of cultivation, either one of the natural substances or one antibiotic was added to the well.
Scale bar = 400 µm 4

5. Results and Discussion In vitro determination of baicalein and chitosan action on Candida parapsilosis, Candida krusei and Trichosporon cutaneum biofilm
20. 8.
2015
Eva Kvasničková A B
And there you can see the finally results. In this table is shown the minimum inhibitory concentration (according to definition it is the lowest concentration of an antimicrobial substances that will inhibit the visible growth of a microorganism after overnight incubation) for suspension cells, which was our starting concentration for finding the effective concentration for inhibition or eradication of much more resistant sessile cells in biofilm. In this case the important concentration is 1.5 mg/l of amphotericin B for C. parapsilosis.
And on this figure we can see, that amphotericin B is able to inhibit microbial adhesion maximally about 60 % in concentration 3 mg/l, which is two times higher than MIC for suspension cells of CP, but it promote the biofilm formation instead of eradicate it.
Scale bar = 400 µm
 MIC (mg/l)
antibiotic control
natural substances
amphotericin B
baicalein
chitosan
C. parapsilosis
1.5
350 10
C. krusei
2.5
400 25
T. cutaneum
5.0
300 15 5

6. Results and Discussion In vitro determination of baicalein and chitosan action on Candida parapsilosis, Candida krusei and Trichosporon cutaneum biofilm
20. 8.
2015
Eva Kvasničková A B
On the other hand amphotericin B has the ability to reduce the biofilm formation of CK sessile cells even in concentration 5 mg/l. It‘s inhibiton effect on microbial adhesion is just about 35 % in the higher tested concentration (20 mg/l).
Scale bar = 400 µm
 MIC (mg/l)
antibiotic control
natural substances
amphotericin B
baicalein
chitosan
C. parapsilosis
1.5
350 10
C. krusei
2.5
400 25
T. cutaneum 5
300 15 6

7. Results and Discussion In vitro determination of baicalein and chitosan action on Candida parapsilosis, Candida krusei and Trichosporon cutaneum biofilm
20. 8.
2015
Eva Kvasničková A B
TC biofilm is not sensitive on amph B. This antibiotic increases the biofilm formation of TC and is not able to eradicate pre-formed biofilm.
Scale bar = 400 µm
 MIC (mg/l)
antibiotic control
natural substances
amphotericin B
baicalein
chitosan
C. parapsilosis
1.5
350 10
C. krusei
2.5
400 25
T. cutaneum
5.0
300 15 7

8. Results and Discussion In vitro determination of baicalein and chitosan action on Candida parapsilosis, Candida krusei and Trichosporon cutaneum biofilm
20. 8.
2015
Eva Kvasničková A B
Interesting question for us was the efficiancy of natural substances on inhibition of microbial adhesion or eradication of pre-formed sessile cells in biofilm. Baicalein proved significant effect on reduction of biofilm formation of pre-formed biofilm in concentration more than ten times lower than minimum inhibitory concentration is, but it inhibited the microbial adhesion of CP maximally about 30 % in concentration 50 mg/l.
Scale bar = 400 µm
 MIC (mg/l)
antibiotic control
natural substances
amphotericin B
baicalein
chitosan
C. parapsilosis
1.5
350 10
C. krusei
2.5
400 25
T. cutaneum 5
300 15 8

9. Results and Discussion In vitro determination of baicalein and chitosan action on Candida parapsilosis, Candida krusei and Trichosporon cutaneum biofilm
20. 8.
2015
Eva Kvasničková A B
Similar effect was confirmed in the case of CK.
Scale bar = 400 µm
 MIC (mg/l)
antibiotic control
natural substances
amphotericin B
baicalein
chitosan
C. parapsilosis
1.5
350 10
C. krusei
2.5
400 25
T. cutaneum
5.0
300 15 9

10. Results and Discussion In vitro determination of baicalein and chitosan action on Candida parapsilosis, Candida krusei and Trichosporon cutaneum biofilm
Eva Kvasničková A B
And biofilm of TC was extremely sensitive to the baicalein action in both cases.
Scale bar = 400 µm
 MIC (mg/l)
antibiotic control
natural substances
amphotericin B
baicalein
chitosan
C. parapsilosis
1.5
350 10
C. krusei
2.5
400 25
T. cutaneum
5.0
300 15 10

11. Results and Discussion In vitro determination of baicalein and chitosan action on Candida parapsilosis, Candida krusei and Trichosporon cutaneum biofilm
Eva Kvasničková A B
The second studied natural substances was chitosan, which is able to inhibit microbial adhesion ober than 60 %, but it has not so significant effect on eradication of pre-formed biofilm.
Scale bar = 400 µm
 MIC (mg/l)
antibiotic control
natural substances
amphotericin B
baicalein
chitosan
C. parapsilosis
1.5
350 10
C. krusei
2.5
400 25
T. cutaneum
5.0
300 15 11

12. Results and Discussion In vitro determination of baicalein and chitosan action on Candida parapsilosis, Candida krusei and Trichosporon cutaneum biofilm
20. 8.
2015
Eva Kvasničková A B
Chitosan inhibits around 80 % of CK microbial adhesion and also hasn‘t got significant effect on eradication of pre-formed biofilm.
Scale bar = 400 µm
 MIC (mg/l)
antibiotic control
natural substances
amphotericin B
baicalein
chitosan
C. parapsilosis
1.5
350 10
C. krusei
2.5
400 25
T. cutaneum
5.0
300 15 12

13. Results and Discussion In vitro determination of baicalein and chitosan action on Candida parapsilosis, Candida krusei and Trichosporon cutaneum biofilm
20. 8.
2015
Eva Kvasničková A B
And the similar effect is also on biofilm of TC.
Scale bar = 400 µm
 MIC (mg/l)
antibiotic control
natural substances
amphotericin B
baicalein
chitosan
C. parapsilosis
1.5
350 10
C. krusei
2.5
400 25
T. cutaneum
5.0
300 15 13

14. Conclusion In vitro determination of baicalein and chitosan action on Candida parapsilosis, Candida krusei and Trichosporon cutaneum biofilm
20. 8.
2015
Eva Kvasničková
Amphotericin B - not able to eradicate the sessile cells of C. parapsilosis and T. cutaneum
Baicalein - reduction of pre-formed biofilms of all tested yeast strains
antimicrobial activity in high concentrations
antibiofilm activity in low concentrations
Chitosan - able to significantly decrese microbial adhesion of these yeast strains
To find the synergistic effect of baicalein, chitosan or amphotericin B in combination on biofilm forming inhibition
So the conclusion is, that …
…. and our plan to the future is to find the synergistic effect of baicalein, chitosan or amphotericin B in combination, that may help to fight against various infections. 14

15. Acknowledgement 20. 8. 2015 Eva Kvasničková
prof. Ing. Alena Čejková CSc.
prof. Ing. Jan Masák CSc.
Ing. Olga Maťátková PhD.
Financial support from specific university research (MSMT No 20/2015) and by GACR grant S.