Yeast: It’s Not Only Found in Food

By October 21, 2014Blog

Yeast is used to make bread rise and ferments sugar to make wine or beer. The most commonly studied yeast is Saccharomyces cerevisiae which has historically been used for baking, winemaking and brewing. S. cerevisiae is also a model organism, used to study molecular biology and genetics. But there are many other yeast species, some of which can spoil wine (Brettanomyces) or cause infections (Candida).

MIC-and-MFC-Testing-against-Yeast

A well-known infection is vaginal yeast infection caused most commonly by Candida albicans and sometimes by other Candida species. Ironically, C. albicans is a commensal yeast found in our mouth, gut flora and vaginal area at small amounts, but can become invasive due to environmental cues such as changes in pH. The vagina normally has a pH of 3.5-4.5.  The pH can rise during menstruation, intercourse, douching, pregnancy and menopause. Unfortunately, some of these natural occurrences will slightly increase the pH, inducing an optimal environment for Candida to proliferate.

Yeast infection of the mouth and/or esophagus is known as thrush. Thrush is regularly found in newborns and immunocompromised people. Naturally, the bacteria in your mouth prevent further yeast growth. The flora of newborns are still developing and do not have the protection against yeast. Immunocompromised people on the other hand have poor health and could be on medication such as antibiotics or corticosteroids that works against some of the mouth and esophagus bacteria, allowing Candida to flourish.

Oral-thrush-newbors

Emeryville Pharmaceutical Services offers antifungal susceptibility testing using the Clinical and Laboratory Standards Institute’s (CLSI) method of broth microdilution.[1] In short, yeasts are grown and cultured from Sabouraud dextrose agar (SDA) and diluted into Roswell Park Memorial Institute (RPMI) 1640 media containing 3-(N-morhpholino)propanesulfonic acid (MOPS). The antifungal agents are prepared in DMSO or water and diluted to start at 8µg/mL, or higher if requested. Two-fold dilutions are performed thereafter into the buffer/media. The inoculum is added and then the 96-well plate is incubated at 35°C and read at 48 hours (72 hours for Cryptococcus neoformans).  Control anifungal agents will include one of the following: Amphotericin B, Ketoconazole, Itraconazole, or Fluconazole, unless specified otherwise.  Unlike the bacterial broth microdilutions, the yeast broth microdilutions are scored using the following compared to the untreated wells:

Scores Visibility

0

Optically clear

1

Slightly hazy

2

Prominent decrease in turbidity

3

Slight reduction in turbidity

4

No reduction of turbidity

This scoring is irrelevant unless the test article falls into the same group as the azoles or Flucytosine. In that instance, the minimum inhibition concentration (MIC) is defined as the lowest score of 2 observed. In all other cases, the MIC is defined with the first lowest concentration with a score of 0.

At this point, we can continue to perform a minimum fungicidal concentration (MFC) assay. We will plate the clear wells onto SDA plates and incubate the plates for 24-48 hours prior to colony enumerations. The MFC is defined as the lowest concentration of drug to kill ~99% of yeast (≤ 1 colony).[2]

Emeryville Pharmaceutical Services offers these MIC and MFC assays for testing potential antimycotic drugs. Emeryville Pharmaceutical Services also provides services for biofilm and resistance profiling. For more information and a quote, please contact us at 510-899-8828.

References

[1] “M27-A2 Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard—Second Edition.” Clinical Laboratory Standards Institute. 22.15 (2002).

[2] Canton, E., Pemán, J., Valentín, A., Espinel-Ingroff, A. and Gobernado, M. “In Vitro of Echinocandins against Candida krusei Determined by Three Methods: MIC and Minimal Fungicidal Concentration Measurements and Time-Kill Studies.” Antimicrobial Agents and Chemotherapy. 53.7 (2009).

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