Candida albicans: Kluyveromyces B0399 direct inhibitory activity

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:
-    in the modulation of the immune system
-    in the prevention and in the treatment of intestinal dismicrobisms  that can provoke diarrhea or syndromes caused by degenerations of inflammatory reactions (e.g. Crohn’s disease, irritable bowel syndrome) (Castagliuolo M.S. et al., Gorbach S.L. 2000),
-    in the reduction of the development of allergic phenomena such as asthma and eczema  in children (Benn C. et al. 2002) if used mothers during pregnancy,
-    in the reduction of the risk of infection in the genitourinary tract (Senok A.C. et al. 2005, Reid G. et al. 2003, Reid G. et al. 2004, Reid G. 2001).  

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.

Materials and methods

The test took place in the microbiological laboratory of the Azienda Speciale Ambiente  of the Chamber of Commerce of Udine.
The interaction of the yeasts was evaluated by setting up several trials, in agar medium.

Kluyveromyces marxianus B0399 was produced and provided by Turval laboratories Ltd. Candida albicans ATCC 1023 was acquired from Oxoid.

K. marxianus B0399 was maintained on MV1 medium (lactose 2%, hydrolized casein 1%, yeast extract 0.1%, peptone 0.5 %, solidified with agar 1.5% when required), while Candida strain was maintained on YEPD (glucose 2%, yeast extract 1%, peptone 2% solidified with agar 1.8% when required).

All strains were maintained at 4 °C and cultivated at 37°C unless otherwise stated.
The medium used for the tests is Chromalbicans agar (Biolife).  It was chosen for the opportunity of distinguishing the two yeast species:
Candida ATCC 10231 appears blue, thanks to the chromogen present in the medium, while K. marxianus B0399 has milky-white colonies.
The suspensions were inoculated on dishes by the spread- plate technique.
Concentrations of yeast inocula (CFU/mL) were determined by using a Petroff-Hausser counting chamber. All tests were done in triplicate.

TESTS

TEST A

Set-up of trial:
From one overnight culture on a Petri dish, C. albicans ATCC 10231 was re-suspended in a 0.85% physiological solution at a concentration of 3 x 10^5 ufc/ml.  100 µl of the suspension were spread onto a Chromabicans agar plate and incubated at 37° C for 12 hours.
At the end of the incubation, 10 µl of the overnight K. marxianus B0399, MV1 broth (3 x 10^6 ufc/ml) at 37°C for 48h before being scored for the inhibition of C.albicans. 

Comments and Results:
The results from Test A were compared with the results of the other tests (Fig.).

The left half of figure 1a, indicated as A1, demonstrated that in the circular area spotted with the K. marxianus B0399 suspension, indicated with the radius “r”, colonies of C. albicans ATCC 10231 grew smaller in size (fig.1a - A 1) compared to the colonies grown outside of the K. marxianus B0399 zone (larger C. albicans colonies, numerous and evident on the rest of the agar surface area indicated with the radius “R”).  The morphology of the two types of colonies was identical.

With respect to the surface densities (CFU/mm2) of the two yeast strains inside of the area of interference, the concentration (surface density) of K. marxianus B0399 was 10-fold higher than the one of C. albicans. Precisely 3 x 10³ CFU/mm² (10 x 10^-3 mL x 3 x 10⁶ CFU/mL divided by 10 mm²) for K. marxianus and 3 x 10² CFU/mm² for C. albicans (100 x 10^-3 mL x 3 x 10⁵ CFU/mL divided by 100 mm²).


TEST B
 

Set-up of trial: 

A variation of the test A was done in the following way: C. albicans inoculated, and 12h incubated Chromalbicans plate (as previously described) was poured with and left to absorb 500 µl of a K. marxianus B0399 PBS suspension. Two different concentrations of K. marxianus B0399 PBS suspensions were tested, suspension i) 6 x 10⁵ CFU/mL and ii) 6 x 10⁴ CFU/mL. The plates were then incubated again at 37°C for 48 hours. 
 
Comments and Results:
The 10-fold higher surface density/concentration of the “inhibitor” strain seems to be a relevant determinant of the inhibition since the “small colony phenotype” of the target strain was repeated in the variation of the test A in which 500 μl of a 6 x 10⁵ CFU/mL K. marxianus B0399 PBS suspension was poured over the whole plate in order to obtain a 10-fold higher surface density with respect to the target strain; conversely the “small colony phenotype” was lacking when 6 x 10⁴ CFU/mL K. marxianus PBS suspension was used and the surface densities of the two strains were equal (data not shown). 

TEST C

Set-up of trial:
A Petri dish of Chromalbicans agar was inoclulated by the spread-plate technique with 100 µl of a PBS suspension of K. marxianus B0399 (3 x 10^6  ufc/ml) and is incubated at 37 °C for 12 hours.
The plate was then poured and left to absorb 500 µl of the C. albicans ATCC 10231 PBS suspension (6 x 10^4 ufc/ml).

The Petri dish was incubated again at 37° C for 48 hours.

Comments and Results:
The growth and development of the Candida resulted as being altered in the presence of K. marxianus B0399.  We can observe stronger inhibition phenotype in this case where the “inhibitor” strain was plated before the target strain. The relative concentrations of the two strains as well as the absolute cell number plated were kept the same as in test A.

TEST D

Set-up of trial:
The YEPD broth was simultaneously inoculated with C. albicans ATCC 10231 and the K. marxianus B0399 overnight, liquid culture, so that the final concentrations of C.albicans ATCC 10231 and K. marxianus B0399 in the suspension were 3 x 10⁴ CFU/mL and 3 x 10⁵ CFU/mL, respectively.  The mixed culture was incubated at 37°C for 48 hours. After the incubation, a dish of Chromalbicans agar was inoculated with 100 µl of the mixed microbial suspension and incubated at 37°C for 48 hours.

Comments and Results:
C. albicans ATCC 10231 developed with difficulty, which included smaller colonies compared to the controlled Petri dish. This situation where the inhibitor and the target strains were first incubated together in liquid media and then plated on Chromalbicans agar resulted in the strongest inhibition. The relative concentrations of the two strains, as well as the absolute cell number plated, were kept the same as those in test A.  This might indicate that the strongest inhibition phenotype (small and isolated C. albicans ATCC 10231 developed with difficulty) is a direct consequence of longer inhibition time (two days in liquid medium plus two days on the agar plate) and/or production of the putative bioactive molecule (“killer toxin”) released by the “killer strain”, K. marxianus B0399, and dispersed in the liquid medium.

TEST E
Set-up of trial:
100 µl of mixed microbial 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.

Comments and Results:
The C. albicans ATCC 10231 colonies developed with a reduced diameter (Photo E1). Strong inhibition phenotype, similar to the tests C, was obtained now that the “inhibitor” strain was plated simultaneously with the target strain.

Conclusions:

The total results for the different trials performed show how on all the examined conditions in this study, C. albicans ATCC 10231 grown in the presence of K. marxianus B0399 presents colonies of inferior diameter compared to those grown in the absence of K. Marxianues B0399.
It can be pointed out that K. marxianus B0399 has the capability to influence 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), and when there is a disturbance while K. marxianus B0399 is inoculated afterwards—(test A and test B).
Considering that the morphology of the C. albicans ATCC 10231 colonies does not change in the presence of  K. marxianus Bo399, 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 results show an effect of disturbance/inhibition of K. marxianux B0399 on C. albicans ATCC 10231, but the mechanisms of action that pertain to these observations are unclear and therefore more examination is necessary.

PHOTOS:

- Comparison of Photos A1 – C1

A1: A situation where candida colonizes intestinal mucosa and where it develops without any interferences on an uncontaminated Petri dish (large colonies, numerous and evident on the agar); then K. marxianus is added and the Candida colonies regress and their diameter is minimized.
CURATIVE ACTION

C1: A situation where K. marxianus first colonizes the intestinal mucosa; then it reaches the Candida, but its development is characterized as having small and isolated colonies.

ASSUMPTION OF THE PREVENTIVE ACTION OF LACTIC YEAST

- Comparison of Photos A1 – C1:

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).

PREVENTATIVE ACTION

- Comparison of Photos A1 – E1:

E1: 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.

PREVENTATIVE ACTION

 

- Comparison of Photos A1 – D1:

D1: A situation where two yeasts reach the intestinal mucosa simultaneously after previous contact: the Candida does not attach onto the intestinal mucosa—the  colonies  are small and far away from each other. 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. 

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