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 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 35  |  Issue : 2  |  Page : 252-255
 

Identification of the paradoxical effect of caspofungin and micafungin on Candida albicans growth in vitro using a growth curve methodology


1 Department of Laboratory Medicine, Union Hospital, Huazhong University of Science and Technology, Guangzhou, China
2 Department of Clinic Laboratory, The First Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China
3 Department of Clinic Laboratory, Wuhan Pu Ai Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

Date of Web Publication5-Jul-2017

Correspondence Address:
Zeng Ji
Department of Clinical Laboratory, Wuhan Pu Ai Hospital, Tongji Medical College, Huazhong University of Science and Technology, Hanzheng Road 473#, Wuhan 430033
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_15_171

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 ~ Abstract 

Background: The paradoxical effect of fungi, (which is a special phenomenon that certain Candida spp. might exhibit augmented growth in the presence of echinocandin at some concentration higher than the minimum inhibitory concentration) would be better illustrated through growth curves. Materials and Methods: We investigated the paradoxical effect of caspofungin and micafungin on the growth and cell viability of 38 clinical isolates of Candida albicans using a microbial sensitivity test and growth curve methodology. Results: When cells were incubated in the presence of antifungal agents for 48 h, the paradoxical effect was observed in 50% and 47.4% of the isolates incubated with caspofungin and micafungin, respectively. Growth curves indicated the occurrence of the paradoxical effect varied between different echinocandins. Moreover, increased turbidity in the sensitivity test did not correlate with increased fungal cell growth. For caspofungin, the paradoxical effect appeared earlier than in the microbial sensitivity test, while for micafungin, the effect observed in the microbial sensitivity test was not a true paradoxical effect. Conclusion: The growth curve methodology is more accurate for evaluating the occurrence of the paradoxical effect in comparison with the microbial sensitivity test.


Keywords: Candida albicans, echinocandins, growth curve, microbial sensitivity test, paradoxical effect


How to cite this article:
Zhouying H, Chen L, Ruiqi W, Kang L, Ji Z. Identification of the paradoxical effect of caspofungin and micafungin on Candida albicans growth in vitro using a growth curve methodology. Indian J Med Microbiol 2017;35:252-5

How to cite this URL:
Zhouying H, Chen L, Ruiqi W, Kang L, Ji Z. Identification of the paradoxical effect of caspofungin and micafungin on Candida albicans growth in vitro using a growth curve methodology. Indian J Med Microbiol [serial online] 2017 [cited 2017 Sep 26];35:252-5. Available from: http://www.ijmm.org/text.asp?2017/35/2/252/209564



 ~ Introduction Top


Echinocandins can specifically kill fungal cells without adversely affecting human cells and have been recommended by the several guidelines of the first-line antifungal therapy.[1],[2],[3],[4],[5],[6] However, several recent studies reported the paradoxical effect that certain Candida spp. exhibited augmented growth in the presence of echinocandin at concentrations higher than the minimum inhibitory concentration (MIC).[7],[8],[9] In these studies, the paradoxical effect was identified using an in vitro microbial sensitivity test, in which increased cell growth was determined by increased turbidity. Therefore, we initiate the present study with the aim of exploring the paradoxical effect of caspofungin and micafungin on Candida albicans using a growth curve methodology.


 ~ Materials and Methods Top


Stains and reagents

Thirty-eight clinical strains of C. albicans, collected from January 2011 to April 2013, were isolated from patients in our laboratory. The quality control strain Candida parapsilosis ATCC 22019 was from the American Type Culture Collection (Manassas, VA, USA). Roswell Park Memorial Institute (RPMI) 1640 medium was from Sigma-Aldrich (St Louis, MO, USA). Caspofungin and micafungin were from Merck (White House Station, NJ, USA) and Astellas Toyama Co., Ltd., Takaoka Plant (Toidesakaemachi, Takaoka City, Japan), respectively; both drugs were dissolved to 1.6 mg/ml in distilled water as stock solutions and stored at −20°C until use. Other reagents were of the highest quality available and obtained from the commercial sources.

Detection of the paradoxical effect

Preparation of cell suspensions

Each isolate was subcultured twice and grown for 24 h at 35°C on 2-3-5-triphenyl tetrazolium chloride (TTC) plates. Colonies of each strain were suspended in 1 ml of 0.85% sterile normal saline (NS), and the turbidity was adjusted visually to that of a 0.5 McFarland standard (approximately 1 × 106–5 × 106 colony-forming units [CFU]/ml); yeast cell numbers were determined using a hemocytometer. Then, the cells were further diluted in the standard RPMI 1640 medium buffered to pH 7.0 with 0.165 M 3-(N-morpholino)propanesulfonic acid (MOPS) (Sigma-Aldrich). Cell densities were adjusted by the optical density measured at 660 nm to a final concentration of 0.5–2.5 × 103 yeast cells/ml.

Microbial sensitivity test

The paradoxical effect of caspofungin and micafungin was determined by broth microdilution susceptibility testing according to the Clinical and Laboratory Standards Institute – approved document M27-A3.[10],[11] In brief, the cells were incubated in the standard RPMI 1640 medium containing serial two-fold dilutions of each antifungal agent (caspofungin and micafungin) in 96-well microtitration plates for 48 h. The final concentrations of all drugs ranged from 0.031 to 256 mg/l. The MIC of each antifungal agent was determined as the lowest concentration in which a prominent decrease in turbidity (>80%) was observed.[8],[10],[11] The paradoxical effect was defined as a progressive increase in turbidity (cell growth) occurring after treatment with drug concentrations at least four-fold higher than the MIC.[8]C. parapsilosis ATCC 22019 was used as a quality control strain.[11]

Growth curve methodology

A growth curve methodology was adapted from the time-kill methods described previously.[12],[13] Five paradoxical effect – positive C. albicans strains were selected for caspofungin or micafungin treatment. In this experiment, each selected isolate was subcultured twice and grown for 24 h at 35°C on TTC plates. Colonies of each strain were suspended in 1 ml of 0.85% sterile NS, and the turbidity was adjusted to approximately 1 × 106–5 × 106 CFU/ml; yeast cell counts were determined using a haemocytometer. Then, the cells were further diluted in the standard RPMI 1640 medium buffered to pH 7.0 with 0.165 M MOPS. Cell densities were adjusted by the optical density measured at 660 nm to a final concentration of 0.5–2.5 × 103 yeast cells/ml. Afterwards, the cells were incubated for 48 h with the lowest concentrations of caspofungin or micafungin for which a paradoxical effect was observed in each selected isolate. Sampling times for each isolate were at 0, 1, 2, 4, 8, 24 and 48 h. Samples (10 μl) were taken from each well at each designated time point and then inoculated on TTC plates at 35°C for 48 h. Forty-eight hours later, the colonies on each plate were counted to calculate the growth curve, and the morphology of the cells was observed under the microscope. Growth curve experiments were performed in triplicate for each strain. C. parapsilosis ATCC 22019 was used as a quality control strain.[11]


 ~ Results Top


Paradoxical effect of caspofungin and micafungin on Candida albicans evaluated using a microbial sensitivity test

As shown in [Table 1], following a 48 h incubation, the percentage of the 38 C. albicans isolates which observed paradoxical effect was 50% in the group with caspofungin treatment while the group with micafungin treatment was 47.4%. The concentration ranges of caspofungin and micafungin that showed a paradoxical effect on the growth of C. albicans were 8–32 mg/l and 4–16 mg/l, respectively [Table 1]. Caspofungin showed a higher MIC (0.125 mg/l) than micafungin (0.031 mg/l); correspondingly, the start point concentration for the paradoxical effect of caspofungin was higher than that of micafungin (8 vs. 4 mg/l, respectively). However, no correlation between the MIC and paradoxical effect was observed. These results indicate that the prevalence of the paradoxical effect in clinical C. albicans isolates is high and that the occurrence of the paradoxical effect varies among different echinocandins.
Table 1: Paradoxical effect and minimum inhibitory concentrations of caspofungin and micafungin on Candida albicans

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Cell viability of Candida albicans detected by growth curves and cell morphologies

To further investigate the dynamic change in cell number and viability of C. albicans exhibiting the paradoxical effect, we selected five paradoxical effect – positive C. albicans strains and performed growth curves in the presence of caspofungin and micafungin. The results demonstrated that for caspofungin treatment at concentrations showing a paradoxical effect in the microbial sensitivity test. The number of living cells started to increase at 8 h, peaked at 24 h and then decreased dramatically. At the end of the incubation (48 h), even though the paradoxical effect was obvious in the microbial sensitivity test, few colonies were detected on the plates, indicating that most cells were dead at this time point [Figure 1]a. Under the microscope, clumps of fungal spores were observed [Figure 2]a, indicating that the paradoxical effect, as evidenced by the increased turbidity observed at 48 h in the microbial sensitivity test, may be due to the aggregation of dead cells and fungal spores. In contrast, when fungal cells were incubated with micafungin at concentrations that exhibited a paradoxical effect in the microbial sensitivity test, the number of living cells decreased slowly but steadily from the very beginning, and no growth peak was observed at any time point. At the end of the incubation (48 h), few surviving cells were detected [Figure 1]b, suggesting that there was no actual paradoxical growth of C. albicans during the incubation with micafungin. Under the microscope, dead fungal cells and numerous hyphae were observed [Figure 2]b, indicating that the paradoxical increased turbidity observed at 48 h in the microbial sensitivity test was caused by the accumulation of dead fungal cells and hyphae.
Figure 1: Time-growth curves of Candida albicans after caspofungin and micafungin treatments. Time-growth curve methodology was performed on Candida albicans strains. Drug concentrations were set to the concentrations in which showing a paradoxical effect in microbial sensitivity test. (a) Time-growth curve of Candida albicans after caspofungin treatment; (b) Time-growth curve of Candida albicans after micafungin treatment.

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Figure 2: Cell morphology of Candida albicans after caspofungin and micafungin treatments for 48 h. Paradoxical effect-positive Candida albicans strains were incubated with caspofungin or micafungin in the concentrations which paradoxical effect was observed for 48 h. (a) Cell morphology of Candida albicans after caspofungin treatment; (b) Cell morphology of Candida albicans after micafungin treatment.

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 ~ Discussion Top


The paradoxical effect of echinocandins on the growth of certain Candida spp. has been observed in in vitro microbial sensitivity tests by several groups.[7],[8],[9] Researchers found an abnormal increase in the turbidity of the medium when fungal cells were incubated with echinocandins at concentrations higher than their MIC. Normally, this phenomenon implies increased cell growth in the medium. Thus, it is defined as the 'paradoxical effect.' However, whether there is a true cell growth in the paradoxical effect and how this condition influences cell viability remains unclear. In the present study, we explored the paradoxical effect of two classic echinocandins, caspofungin and micafungin, on 38 clinical C. albicans isolates. We determined the viability of the cells using a growth curve methodology, and we observed the morphology of the cells under the microscope when the paradoxical effect arose.

The results of susceptibility testing showed that the MICs of both caspofungin and micafungin on clinical C. albicans isolates were very low, and no drug resistance was observed consistent with the high sensitivity of C. albicans to caspofungin and micafungin. Through susceptibility testing, we observed the paradoxical effect in 50% of the isolates in response to caspofungin treatment and in 47.4% of the isolates following micafungin treatment. Similar to a previous report,[8] we did not observe any relationship between the MICs of echinocandins and the occurrence of paradoxical growth. The prevalence of the paradoxical effect in this study was not consistent with the data reported by Stevens et al.,[7],[8],[9] which may be explained by different sample numbers and different sources of isolates.

To explore the dynamic changes in the viability of C. albicans under conditions that induced the paradoxical effect and to further study the underlying mechanism, we selected paradoxical effect-positive C. albicans strains in this study and examined them using a growth curve methodology. To our surprise, we found that caspofungin and micafungin showed different growth curves, even though both drugs showed the same paradoxical effect at 48 h in the susceptibility test. The abnormal growth peak of the cells incubated with caspofungin appeared at 24 h, but not at 48 h. The turbidity that we observed at 48 h was possibly caused by the accumulation of dead cells and fungal spores. Furthermore, the growth curve during micafungin treatment showed no growth peak at any time point, suggesting that the turbidity at 48 h in the susceptibility test was not induced by the cell growth, but by the accumulation of dead cells and hyphae, as observed under the microscope. Since cell numbers decreased more slowly at the beginning of the micafungin incubation than in the caspofungin incubation, at the end of the incubation (48 h), the accumulation of dead cells was sufficient to increase the turbidity in the microbial sensitivity test, even though the growth curves demonstrated that there was no abnormal cell growth at 24 or 48 h. Thus, the paradoxical effect of micafungin on the studied C. albicans isolates might not be a true paradoxical effect. Based on these findings, we concluded that the identification of the paradoxical effect using the microbial sensitivity test should be interpreted with caution.

According to the characteristics of the growth curve during caspofungin treatment, paradoxical cell growth at higher drug concentrations does exist. The growth peak appeared at 24 h, then the number of viable cells decreased sharply. Rueda et al. have reported that the paradoxical growth of C. albicans in the presence of caspofungin is not due to antifungal degradation or instability, but to fungal adaptation to caspofungin.[14] We hypothesised that this adaptation may decrease cell survival.

In summation, the occurrence of the paradoxical effect varies among different echinocandins. Increased turbidity in the microbial sensitivity test does not correlate with increased fungal cell growth. Thus, the identification of the paradoxical effect should be interpreted carefully when evaluated by the microbial sensitivity. Compared with the microbial sensitivity test, the growth curve methodology is more accurate for the evaluation of the occurrence of the paradoxical effect.

Financial support and sponsorship

Nil

Conflicts of interest

This work was partially supported by the National Natural Science Foundation of China (No. 81471304).



 
 ~ References Top

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Denning DW. Echinocandin antifungal drugs. Lancet 2003;362:1142-51.  Back to cited text no. 1
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Hatano K, Morishita Y, Nakai T, Ikeda F. Antifungal mechanism of FK463 against Candida albicans and Aspergillus fumigatus. J Antibiot (Tokyo) 2002;55:219-22.  Back to cited text no. 2
    
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Deresinski SC, Stevens DA. Caspofungin. Clin Infect Dis 2003;36:1445-57.  Back to cited text no. 3
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Chandrasekar PH, Sobel JD. Micafungin: A new echinocandin. Clin Infect Dis 2006;42:1171-8.  Back to cited text no. 4
    
5.
Pappas PG, Kauffman CA, Andes D, Benjamin DK Jr., Calandra TF, Edwards JE Jr., et al. Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis 2009;48:503-35.  Back to cited text no. 5
    
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Walsh TJ, Anaissie EJ, Denning DW, Herbrecht R, Kontoyiannis DP, Marr KA, et al. Treatment of aspergillosis: Clinical practice guidelines of the Infectious Diseases Society of America. Clin Infect Dis 2008;46:327-60.  Back to cited text no. 6
    
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Stevens DA, Espiritu M, Parmar R. Paradoxical effect of caspofungin: Reduced activity against Candida albicans at high drug concentrations. Antimicrob Agents Chemother 2004;48:3407-11.  Back to cited text no. 7
    
8.
Fleischhacker M, Radecke C, Schulz B, Ruhnke M. Paradoxical growth effects of the echinocandins caspofungin and micafungin, but not of anidulafungin, on clinical isolates of Candida albicans and C. dubliniensis. Eur J Clin Microbiol Infect Dis 2008;27:127-31.  Back to cited text no. 8
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Chamilos G, Lewis RE, Albert N, Kontoyiannis DP. Paradoxical effect of Echinocandins across Candida species in vitro: Evidence for echinocandin-specific and candida species-related differences. Antimicrob Agents Chemother 2007;51:2257-9.  Back to cited text no. 9
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National Committee for Clinical Laboratory Standards (NCCLS). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard. Document M27-A. Wayne, PA: National Committee for Clinical Laboratory Standards; 1997.  Back to cited text no. 10
    
11.
Clinical and Laboratory Standards Institute (CLSI). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard. CLSI Document M27-A3. 3rd ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.  Back to cited text no. 11
    
12.
Bizerra FC, Melo AS, Katchburian E, Freymüller E, Straus AH, Takahashi HK, et al. Changes in cell wall synthesis and ultrastructure during paradoxical growth effect of caspofungin on four different Candida species. Antimicrob Agents Chemother 2011;55:302-10.  Back to cited text no. 12
    
13.
Córdoba S, Afeltra J, Vitale RG. Evaluation of the in vitro activity of amphotericin B by time-kill curve methodology against large and small capsulate C. neoformans isolates. Diagn Microbiol Infect Dis 2011;71:260-2.  Back to cited text no. 13
    
14.
Rueda C, Cuenca-Estrella M, Zaragoza O. Paradoxical growth of Candida albicans in the presence of caspofungin is associated with multiple cell wall rearrangements and decreased virulence. Antimicrob Agents Chemother 2014;58:1071-83.  Back to cited text no. 14
    


    Figures

  [Figure 1], [Figure 2]
 
 
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