Trial 96: In vitro test on the effect of the typified lactic yeast (Kluyveromyces marxianus B0399) on the development of Candida albicans ATCC10231.
Authors:
Dr. Tiziana Cettolo, Dr. Lorena Riul – Specialized Laboratory of Microbiology of the ASA – CCIAA of Udine
INTRODUCTION
Probiotic organisms are defined by the guidelines of the FAO/WHO (Cordona, Argentina 2001) as living microorganisms which give beneficial effects when taken in an adequate quantity.
Different studies highlight the efficacy of probiotics:
The proposed mechanisms of action connected to these effects are quite varied: competition for space and nutrients, activation of the immune system of the host and production of antimicrobic catabolites ( short-chained fatty acids and, in particular, lactic acid, hydrogen peroxide…). It was also demonstrated that many yeasts show a significant partial or total killer-type activity against pathogenic fungi of clinical importance (Sugisaki Y. et al. 1983, Walzer G.M. et al. 1995,Cerikcioglu N. 2003). This activity was attributed to the production of protein-type toxins.
In particular, a possible antimicotic action of Kluyveromyces marxianus B0399 was noted by MIclavez et all. (2006, personal communication) that seem to demonstrate a positive in vivo effect of K. marxianus B0399 on patients suffering from candidiasis. This homofermentative yeast, commonly found in fermented diary products, is utilized as a probiotic in persons affected by intestinal problems deriving from diysbiosis (meteorism, constipation alternating with diarrhea, difficulty in assimilation, etc.) and/or lactose intolerance, taken that this yeast produces the enzyme β galactosidase.
TEST A
Test A was designed to assess the inhibition of existing biofilms of C. albicans by a K. marxianus, probiotic as might occur during an active infection. An overnight culture of C. albicans ATCC 10231 grown in YEPD was resuspended in a 0.85% physiological solution (PBS) at a concentration of 3 x 105 CFU/mL and 100 μL of the suspension (3 x 104 CFU) was spread onto Chromalbicans agar plates. Plates were incubated at 30° C for 12 hours. At the end of the 12-hour incubation, 10 μL of the overnight culture of K. marxianus B0399 broth (3 x 106 CFU/mL; 3 x 104 CFU) was spotted on the plate (Figure 1A) and left to absorb. The Petri dish was incubated at 30°C for 48 hours and then scored for inhibition of C. albicans.
TEST C
A Chromalbicans agar plate was inoculated using the spread-plate technique with a 100 μL PBS suspension of K. marxianus B0399 (3 x 106 CFU/mL; 3 x 105 CFU) and incubated at 30 °C for 12 hours. The plate was then poured and left to absorb 500 μl of a C. albicans ATCC 10231 PBS suspension (6 x 104 CFU/mL; 3 x 104 CFU). The plate was incubated again at 30° C for 48 hours.
TEST D
YEPD broth was simultaneously inoculated with the equal amounts of C. albicans ATCC 10231 and K. marxianus B0399 overnight liquid cultures. The final concentration of albicans ATCC 10231 and K. marxianus B0399 in the suspension was 3 x 104 CFU/mL and 3 x105 CFU/mL, respectively. The resulting K. marxianus/C. albicans microbial suspension was then incubated at 30°C for 48 hours. After the incubation, 100 μL of the K. marxianus/C. albicans broth was plated on Chromalbicans agar and incubated at 30°C for 48 hours.
TEST E
100 µl of mixed, K. marxianus/C. albicans, YEPD culture, inoculated like in the previous test, was immediately plated on a Chromalbicans agar Petri dish (without previous incubation). The Petri dish was incubated at 37°C for 48 hours.
RESULTS AND CONCLUSIONS
Several variations of “agar overlay” assay, designated as test A-D in the text, were designed in order to provide insight into the antagonistic capacity of K. marxianus B0399 strain against C. albicans as well as into the physiological conditions required for maximal anti-Candida activity by K. marxianus B0399. All the tests revealed that the growth of C. albicans ATCC 10231 colonies in the presence of K. Marxianus B0399 was negatively influenced by the presence of K. marxianues B0399. Candida colonies appeared inferior in diameter compared to those grown in the absence of the probiotic yeast (test A). It can also be observed that K. Marxianus B0399 has the capability to inhibit the development of the C. albicans ATCC 10231 colonies when K. Marxianus B0399 is inoculated preemptively in regards to Candida -- test D, C and E).
Test A mimics the situation where Candida colonizes intestinal/vaginal mucosa in the absence of K. marxianus, where it develops without any interference or inhibition of the latest. The successive administration of K. marxianus should have restrained the growth of the pathogen; Similarly to this assumption, after addition of K. marxianus and co-incubation of the two strains, Candida colonies regressed and their diameter minimized. Test C mimics the situation where K. marxianus first colonizes the intestinal/vaginal mucosa (“prevention - type utilisation”) and competitively hinders Candida colonisation (small and isolated colonies in our in vitro model). Test E mimics the situation where two yeasts reach the intestinal/vaginal mucosa at the same time; the one with better adhering capacity would develop on the expense of the other yeast. Finally, Test D represents a situation where the two yeasts reach the intestinal mucosa simultaneously after previous contact – it might be the “direct” contact between K.marxianus and C.albicans that have led to almost complete inhibition of Candida growth whose colonies appeared small and distant between them.
Considering that the morphology of the C. albicans ATCC 10231 colonies does not change in the presence of K. Marxianus B0399, it is presumed that there are no variations of the superficial structure of the Candida cellular wall, but that there are other mechanisms that intervene to alter the development. The possible mechanisms of action connected to anti-Candida effects could be quite varied going from competition for space and nutrient, over killer toxin production. The results show an effect of inhibition of K. marxianux B0399 against C. albicans ATCC 10231, but the mechanisms of action that pertain to these observations are unclear and therefore more examination is necessary.
