About Resistance

The pathogen C. neoformans developed resistance to 5-fluorocytosine (5-FC), a then-new antifungal medication, in 1973, according to an experiment conducted by Block et al. Although 5-FC had few negative side effects, some of which were treatable through therapy, it was only partially effective in treating Cryptococci infections. Particularly, the limitation was caused by the treatment-related medication resistance found in the infected Cryptococcal organisms. Additionally, utilizing 5-FC to treat 21 individuals with Cryptococcus resulted in the failure of the treatment in six patients due to resistance and the emergence of serious therapeutic problems. Due to the demonstrated resistance, Block et al. were determined to find ways to eliminate as well as predict the development of 5-FC resistance during therapy. In their report, they studied the origin of the 5-FC resistance, the mutation frequency and rate, and the mechanisms under which resistance of 5-FC develops.

How Block et al. (19723 determined the Cause of 5-FC Resistance

Block et al. (1973) carried out an experiment to determine the drug susceptibility, 5-FC resistant mutants, and a fluctuation analysis (to determine whether the appearing 5-FC resistant Cryptococci were drug-induced mutants or not). They also determined the mutant frequency and mutant rate in the test subjects.

To test drug susceptibility, the authors tested 16 susceptible isolates of C. neoformans, with the MIC of 5-FC and that of 5-FU placed but isolated within one tube of each other. In the experiment, al the stains of Cryptococcus neoformans were placed on a serial subculture (Sabouraud agar) and maintained at 30°C. Through broth dilution testing, the minimal inhibitory concentration (MIC) of all isolates (5-FC and 5-fluorouracil (5-FC)). Afterwards, the Block et al. incubated 5 X q0 Cryptococci/ml concentrated isolated on a rotating drum at 32°C and left it for 48 hours. During the drum rotation, two dilutions of 5-FC and 5-FCU were placed in yeast nitrogen base broth with an additional 2% dextrose. The result showed that, of the out of the 16 isolates, 9 developed 5-FC resistant organisms, and were susceptible to both 5-FC and 5-FU resistant-isolates. Out of the 5-FC- resistant isolates, six showed massive resistance compared to just one 5-FU-susceptible.

Block et al. (1973) isolated, from 6 patients, the stains of C. neoformans at the National Institute of Health. They noticed that each of the instances of resistant organisms came from patients who had, or were, receiving 5-FC and failed to respond to the therapy. They determined that the 5-FC resistance among the Cryptococci was both massive and stable. Even after 10 passages on Sabouraud agar,the resistant strains isolated from the subject did not change.

In carrying out the fluctuation analysis, Block et al. (1973) suspended 100 yeast in 20 test tubes containing cloned isolates of C. neoformans and results determined using the Luria and Delbruck method of fluctuation analysis. The results showed that there was a spontaneous appearance of 5-FC-resistant mutant documented in four susceptible isolates of C. neoformans. The MR calculated from the isolates showed that the patients cured by 5-FC were 398 and 407, while those that developed resistance through treatment failures were 436 and 490.

When testing for mutant frequency (MF), the authors analyzed 10 susceptible strains of C. neoformans (Five from those cured by 5-FC and 5 from which the treatment by 5-FC failed) and 6 with in vivo-acquired resistance. After 48 hours at 30°, each of the isolates was harvested in saline. One portion of the isolate was placed in a drug-free Sabouroud agar and another on YNB agar containing 2,000 micrograms of 5-FC/ml; both equal portions were incubated for 72 hours at 30°C. The calculated concluded that the MF values were 63.7 ± 27.6 (mean ± SE) and 76.1 ± 24.2 per 107 Cryptococci respectively, thereby providing no mean of distinguishing them. In comparison, the 6 isolates with in vivo-acquired resistance had a cell division approaching 100% of the cell population.

The experiment showed that the resistance, not drug-induced, but stable, massive, and in almost all cases, accompanied by 5-FU resistance.



Using the Approach to Determine Whether or Not Exposure to A-Aminoadipate Creates Lys- Mutants.

Block et al. (1973) used a form of a negative selection method to analyze the development of a 5-fluorocytosine (5-FC) resistance in the pathogen C. neoformans. It is possible to use the same technique to isolate the mutants in lysine biosynthesis pathway in a media that allows the growth on media containing a chemical capable of killing wild-type yeast. In this experiment, a portion containing lysine mixed with alpha-aminoadipate used to see how the wild-type yeast will behave in the mixture. Additionally, the mixture containing lysine mixed with alpha-aminoadipate is a good ground since wild-type yeast metabolizes alpha-aminoadipate. Overnight, the wildtype yeast can be grown in YEPD. During the experiment, one will look for the mutants that are autotrophic for lysine. In the mixture, the only nitrogen source is the alpha-adipate. Thus, it is expected that the alpha-aminoapipate will be reduced by alpha-aminoadipate reductase to alpha-aminoadipate-semialdehyde rapidly. However, the next phase of the reaction becomes slow in nature, and the high concentration of alpha-aminoadipate-semialdehyde, toxic to yeast, leads to cell death. It is expected that some of the cells would not die, but rather become mutants. Through complementation test, the number of recessive mutation can be determined. The results from the experiment will show whether, or not, the exposure to alpha-aminoadipate creates lys- mutants or not.













Bibliography

Block, Edward R., Anne E. Jennings, and John E. Bennett. "5-Fluorocytosine resistance in Cryptococcus neoformans." Antimicrobial agents and chemotherapy 3, no. 6 (1973): 649-656.

Chattoo, Bharat B., Fred Sherman, Dalia A. Azubalis, Thorsten A. Fjellstedt, David Mehnert, and Maurice Ogur. "Selection of lys2 mutants of the yeast Saccharomyces cerevisiae by the utilization of α-aminoadipate." Genetics 93, no. 1 (1979): 51-65.

Maresca, Bruno, George S. Kobayashi, and Hideyo Yamaguchi, eds. Molecular biology and its application to medical mycology. Vol. 69. Springer Science & Business Media, 2013. 49

Praphanphoj, Verayuth, Katherine A. Sacksteder, Stephen J. Gould, George H. Thomas, and Michael T. Geraghty. "Identification of the α-aminoadipic semialdehyde dehydrogenase-phosphopantetheinyl transferase gene, the human ortholog of the yeast LYS5 gene." Molecular genetics and metabolism 72, no. 4 (2001): 336-342.

Zaret, K. S., and F. Sherman. "alpha-Aminoadipate as a primary nitrogen source for Saccharomyces cerevisiae mutants." Journal of bacteriology 162, no. 2 (1985): 579-583.