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Candida albicans: Kluyveromyces B0399 active competitor

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.
Several 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 by the mother while pregnant.
-    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 mechanisms of action connected to these effects are quite varied and were highlighted by numerous scientific studies:  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 – a  homofermentative yeast of human origin utilized as a probiotic in persons affected by intestinal problems deriving from diysbiosis (meteorism, constipation alternating with diarrhea, difficulty in assimilation, etc.) and/or  by lactose intolerance.  The yeast, in fact,  produces the enzyme β galactosidase.
Also, observations were made (A. Miclavez 2006 communication) that seem to demonstrate a positive in vivo activity of K.  marxianus BO399 on patients suffering from candidiasis.  In photos 1 and 2, colonies of K.  marxianus BO399 on medium used for the test  can be observed.


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, in which each is given an indication of the method used and the results obtained.
The yeast strain was preserved at a temperature of 4° C .  At the moment of utilization, it was sub-cultured  on  agar medium  MV1, and incubated at 37° C for24-48 hours.

The strain of C. albicans ATCC 10231 acquired at Oxoid, revitalized and maintained at a temperature of 4° C in a suitable medium.

The medium used for the tests is Chromalbicans agar (Biolife).  It was chosen for the opportunity it gave of distinguishing the two species of yeast.
Candida ATCC 10231 appears blue, thanks to the chromogen present in the medium, while K. Marxianus B0399 has milky-white colonies.

The pH (6.2) of the medium is maintained at the level indicated by the producing company.  The suspensions were inoculated on dishes for spatulation and not with agar-germi methodology so as to better mimic the conditions of vaginal mucosa.
These procedures and general conditions of treatment and incubation of the strains are the same in all testing unless otherwise specified.

 

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 1.25 x 10^5 ufc/ml.  100 µl of the suspension were inoculated on a Petri dish of Chromabicans agar for the spatulation, and we incubated at 37° C for 6 hours.
At the end of the incubation on the Petri dish, 50 µl of the K. marxianus B0399 strain was deposited, which had been inoculated in MV1 broth 24 hours before.
The Petri dish was incubated at 37°C for 48 hours. The trial was done twice.  The calculation of the UFC/ml of the K. Marxianus B0399 suspension at the moment of inoculation was 4.7 x 10^6 .

Comments and Results:

In the area where a drop of K. Marxianus B0399 suspension was deposited, the colonies of C. albicans ATCC 10231 are smaller (photo A 1) compared to the colonies of yeast that grew without K. Marxianus B0399: the morphology of the two types of colonies resulted as being identical, but the diameter of the first colonies is inferior to that of the second colonies.


TEST B
 
Set-up of trial:

The preceding trial was repeated with the following variation: a colony of K. Marxianus B0399 and just one drop of suspension were deposited onto the Petri dish containing C. albicans ATCC 10231.
 
Comments and Results:
The colonies of C. albicans ATCC 10231 were found to be morphologically identical to those grown in the absence of K. marxianus B0399, but had a significantly inferior diameter. The effect that K. Marxianus B0399 has on the development of C. albicans can be clearly observed in pictures B1 and B2.

 

 

TEST C


Set-up of trial:

A Petri dish of Chromalbicans agar is inoclulated for spatulation with 100 µl of a suspension in a physiological solution (0.85% NaCl) of K. Marxianus B0399 (7.4 x 10^5  ufc/ml) and is incubated at 37 °C for 18 hours.
Then they were poured and left to absorb 500 µl of a suspension in a C. albicans ATCC 10231 physiological solution (2 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.  The C. albicans were small, as previously shown (photo C1).

 

 

TEST D


Set-up of trial:

The MV1 broth is simultaneously inoculated with the C. albicans ATCC 10231 and the K. marxianus B0399 and then incubated at 37°C for 48 hours.  After the incubation, a dish of Chromalbicans agar is inoculated with 100 µl of microbial suspension and then 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.  The photo D1 shows the development of C. albicans ATCC 10231 with the presence of K. Marxianus B0399.


TEST E
Set-up of trial:
100 µl of MV1 broth, inoculated like in the previous test, was immediately inoculated in a Chromalbicans agar Petri dish.  The Petri dish was incubated at 37°C for 48 hours.

Comments and Results:
The C. albicans ATCC 10231 colonies developed ubiquitously, but with a reduced diameter (Photo E1).


TEST F

Set-up of trial:

A Chromalbicans agar Petri dish was inoculated for spatulation with 100 µl of C. albicans ATCC 10231 suspension in a physiological solution (0.85% NaCl) and then incubated at 37°C for 18 hours.
They were then poured and left to absorb 500 µl of a K. Marxianus B0399 suspension in a physiological solution (0.85% NaCl).
The Petri dish was then incubated again at 37°C for 48 hours.  
 
Comments and Results:

The growth of C. albicans ATCC 10231 was ubiquitous in the Petri dish, but its colonies resulted small, as was shown in the preceding tests (Photo F1).  

 

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 pre-emptively in regards to Candida -- test C), and when there is a disturbance while K. Marxianus B0399 is inoculated afterwards—(test A, test B, test F).
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 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.

PREVENTIVE ASSUMPTION OF LACTIC YEAST

 

 

 

- Comparison of Photos A1 – E1:

E1: A situation where two yeasts reach the intestinal mucosa at the same time; the one which has the best adhering capacity, attaches more quickly and develops on the majority of the surface, at 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.
After 18 hours of union between Kluyveromyces marxianus B0399 and the Candida albicans, almost a complete inactivation of the proliferative capacity is present: As can be noted in the photo below, on the right side, the Candida colonies, after a DIRECT contact with the Kluyveromyces B0399, develop with notable difficulty due to the preceding action exercised by the lactic yeast.

PREVENTATIVE ACTION


prova 96 confronto A1 e D1.jpg


 

 


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