JAC Advance Access originally published online on April 4, 2008
Journal of Antimicrobial Chemotherapy 2008 62(1):149-152; doi:10.1093/jac/dkn144
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Original research |
Time–kill studies investigating the killing activity of caspofungin against Candida dubliniensis: comparing RPMI-1640 and antibiotic medium 3
1 Faculty of Dentistry, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary 2 Department of Medical Microbiology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary
* Corresponding author. Tel: +36-52-411-717/54501; Fax: +36-52-414-948; E-mail: major{at}med.unideb.hu
Received 19 December 2007; returned 29 January 2008; revised 6 March 2008; accepted 10 March 2008
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Objectives: We evaluated the in vitro activity of caspofungin against Candida dubliniensis strains using MIC and minimum fungicidal concentration (MFC) measurements and time–kill methodology.
Methods: We used six C. dubliniensis clinical isolates and the CD 36 type strain. MICs and MFCs of caspofungin were determined using the standard broth microdilution method with normal (103 cells/mL) and elevated (105 cells/mL) starting inocula in RPMI-1640 and antibiotic medium 3 (AM3). MIC was determined after 24 h, and plating for MFC determination was performed after 48 h. In time–kill tests, all strains were tested at 0.06–16 mg/L caspofungin concentrations in RPMI-1640 and AM3.
Results: In RPMI-1640, the MIC range was 0.06–8 mg/L. Trailing growth was observed regardless of the starting inoculum after 48 h, but not after 24 h. In AM3 regardless of starting inoculum, MICs were 0.03 mg/L. After 48 h, trailing was not detected; two isolates grew at a concentration of 8 mg/L using 105 cells/mL as the starting inoculum [paradoxical growth (PG)]. All MFCs in RPMI-1640 and AM3 were >8 and 
0.12 mg/L, respectively. In AM3, all but a single isolate showed PG in the MFC tests. Time–kill tests confirmed the results obtained by MFC tests both in RPMI-1640 and AM3.
Conclusions: In vitro activity of caspofungin against C. dubliniensis depended on the starting inoculum and medium used. Using AM3 eliminated trailing from MIC determinations but not PG in MIC, MFC and time–kill tests.
Keywords: paradoxical growth , Eagle effect , fungicidal
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
The CLSI (formerly the NCCLS) proposed a reference standard methodology (M27-A2) for antifungal susceptibility testing of yeasts with azoles, flucytosine and amphotericin B in 2002.1 The method recommends well-defined solvents to dissolve antifungals, starting inoculum (103 cfu/mL), test medium (RPMI-1640 supplemented with glutamine but without bicarbonate) and incubation time (48 h). This method has not yet been standardized for the recently developed echinocandin antifungal class.
However, due to lack of a standard method specifically designed for echinocandins, CLSI recommendations are widely used for the determination of echinocandin MIC values for the time being. It was shown by several groups that echinocandin MIC values show strong dependency on inoculum and medium used;2–4 MIC values are generally lower in antibiotic medium 3 (AM3) than in RPMI-1640 medium.2 Other observable in vitro phenomena are trailing effect and, in the case of echinocandins but not in the case of other antifungal classes, paradoxical growth (PG; or Eagle effect).3–7
PG is a reduced in vitro echinocandin activity at high concentrations (but not at concentrations immediately above the MIC) observed when using normal (103 cfu/mL)4–6 as well as increased (105 cfu/mL)3,7 starting inocula. PG was originally detected for caspofungin in the case of some Candida albicans isolates,5 but newer data suggest that many Candida species may exhibit PG (i.e. Candida parapsilosis, Candida dubliniensis, Candida tropicalis and Candida krusei).3,4,6,7 Trailing effect occurs when yeasts show reduced, but observable, growth in supra-MIC concentrations.
In contrast to PG, where growth occurs at low and high, but not at intermediate concentrations (at least two clear wells are observed between wells showing growth), trailing growth is characterized with full growth in wells until the MIC, followed uninterruptedly by wells with reduced growth. Consequently, trailing may blur the endpoint considerably.
PG was recently found to be associated with altered cell wall composition,8 but these data remain to be confirmed by other studies.
C. dubliniensis, a species closely related and phenotypically similar to C. albicans, was first described as a novel species >10 years ago. Both species show low MIC values against all available antifungal classes. In the case of C. albicans, the above-mentioned trailing growth and, in the case of echinocandins, PG may lead to problems in evaluation and interpretation of susceptibility results. These phenomena are less well documented in the case of C. dubliniensis. Jacobsen et al.3 observed both trailing and PG in MIC determinations using RPMI-1640 in the case of all three currently available echinocandins; Fleischhacker et al.4 noticed PG in 90% of the tested 127 C. dubliniensis isolates, whereas only a minority (14%) of C. albicans strains grew at high caspofungin concentrations.
The aim of our study was to compare the occurrence of PG of C. dubliniensis in the presence of caspofungin using MFC and time–kill tests in RPMI-1640 and AM3 media.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Six C. dubliniensis clinical isolates identified by molecular methods were derived from a previous study.9 C. dubliniensis CD 36 strain was also included for reference. MIC and MFC values were determined according to the CLSI method both in RPMI-16401 (Sigma) and AM37 (Fluka). In MIC and MFC tests, starting inocula were 103 and 105 cfu/mL, respectively.1,7 The caspofungin concentration range was 0.015–8 mg/L. Caspofungin MICs were read after 24 h using the partial inhibition criterion.7 Trailing and PG were evaluated both after 24 and 48 h.
In MFC tests after 48 h of incubation, the entire content of each well containing supra-MIC concentrations was plated onto Sabouraud dextrose agar.7 PG was also evaluated.
Time–kill studies were performed as described previously using RPMI-1640 and AM3 as test media.7,10 All isolates were tested in the two media simultaneously. Caspofungin concentrations ranged from 0.5 to 128 (0.06–16 mg/L) and 2 to 512 (0.06–16 mg/L) times MIC in RPMI-1640 and AM3, respectively. Aliquots were removed at 0, 2, 4, 8, 12, 24 and 48 h and plated onto Sabouraud dextrose agar. Plates were incubated at 35°C for 48 h both in MFC and time–kill tests, and fungicidal activity was defined as a 99.9% reduction in viable cell count compared with the starting inoculum. Tests were performed twice with all strains.
In MFC and time–kill tests, PG was defined as killing activity (99.9% reduction of the starting inoculum) observed at least at two supra-MIC concentrations, but consistent lack of killing by higher concentrations.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Caspofungin MIC values of C. dubliniensis isolates and the CD 36 type strain are listed in Table 1. In AM3, MIC values were uniformly 0.03 mg/L regardless of the starting inoculum. In RPMI-1640, MICs were generally higher, especially in the case of isolate number 4 (MIC = 8 mg/L). In AM3, we did not observe trailing growth regardless of the starting inoculum and incubation time. In contrast, in RPMI-1640, trailing growth was noticed in all wells after 48 h but not after 24 h regardless of the starting inoculum.
|
In AM3, PG was detected in two isolates using elevated (105 cfu/mL) but not normal (103 cfu/mL) starting inocula after 48 h, but not after 24 h (Table 1). In RPMI-1640, PG was not detected.
MFC values in RPMI-1640 were >8 mg/L for all C. dubliniensis strains. In contrast, in AM3, all MFCs were 0.12 mg/L. However, all but isolate number 4 showed PG.
We obtained two types of killing curves in RPMI-1640 (Figure 1). Five C. dubliniensis clinical isolates as well as the type strain were inhibited, but not killed by any caspofungin concentration used in the study (fungistatic effect) in RPMI-1640 (a representative plot is shown in Figure 1a). In AM3, the same five clinical isolates as well as the type strain showed PG, i.e. caspofungin proved to be rapidly fungicidal at 0.12–4 mg/L (4–128 times MIC) within 12 h, but was only fungistatic at 8–16 mg/L (256–512 times MIC) (Figure 1b).
|
In contrast, isolate number 4 was killed at 8–16 mg/L (1–2 times the MIC values) concentrations (Figure 1c) and inhibited at lower caspofungin concentrations in RPMI-1640. In AM3, caspofungin was fungicidal after 24 h at all concentrations (Figure 1d). PG was not observed.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
Our results regarding caspofungin MICs concur with previous data obtained by other investigators. Muller et al.2 compared micafungin MICs of 21 C. dubliniensis in RPMI-1640 and AM3. They found seven times higher geometric mean MICs in RPMI-1640 than in AM3. Our results correspond well, and caspofungin MICs in AM3 were two to three dilutions lower than in RPMI-1640 medium. This was especially marked in the case of isolate number 4 (MIC = 8 and 0.03 mg/L in RPMI-1640 and AM3, respectively).
Jacobsen et al.3 found that using a higher starting inoculum in RPMI-1640 may provoke PG or trailing growth in the case of all echinocandins. When tested against caspofungin, 13 and 2 of 20 C. dubliniensis strains showed PG and trailing growth, respectively. A more recent study on echinocandin MICs in RPMI-1640 showed that PG was common in the case of caspofungin and micafungin but not in the case of anidulafungin.4 Interestingly, the majority of isolates showed a trailing effect when tested against anidulafungin. In our study, true PG was noted in AM3, but not in RPMI-1640; in RPMI-1640, only trailing growth was observed.
Our study extended the search for PG to MFC and time–kill studies. Caspofungin killing activity significantly decreased in RPMI-1640 compared with AM3, both in MFC and time–kill experiments; in RPMI-1640, only fungistatic effects were observed. The killing activity of caspofungin in AM3 was confined to lower (up to 4 mg/L) concentrations; at higher concentrations (8–16 mg/L), except for a single isolate, PG was observed both in MFC and time–kill tests.
To our knowledge, this study is the first to report caspofungin killing activity against C. dubliniensis. Fungicidal activity strongly depended on the medium and starting inoculum used. Interestingly, PG and trailing growth of C. dubliniensis also showed strong medium and inoculum dependency. Our findings together with the difference in frequency of PG and trailing between different members of the echinocandin drug class observed previously3,4 raise the possibility that trailing growth and PG may have a similar or linked physiological background.
|
Funding |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
The study was funded by the standard research allowance of the University of Debrecen and did not receive financial support from third parties.
|
Transparency declarations |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
None to declare.
|
Acknowledgements |
|---|
We thank Ferenc Somogyvári for providing C. dubliniensis isolates. Caspofungin pure powder was kindly provided by Merck Research Laboratories, Rahway, NJ, USA.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionFundingTransparency declarationsReferences |
|---|
1 National Committee for Clinical Laboratory Standards. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts—Second Edition: Approved Standard M27-A2 (2002) Wayne, PA, USA: NCCLS.
2
Muller FM, Kurzai O, Hacker J, et al. Effect of the growth medium on the in vitro antifungal activity of micafungin (FK-463) against clinical isolates of Candida dubliniensis. J Antimicrob Chemother (2001) 48:713–5.
3
Jacobsen MD, Whyte JA, Odds FC. Candida albicans and Candida dubliniensis respond differently to echinocandin antifungal agents in vitro. Antimicrob Agents Chemother (2007) 51:1882–4.
4 Fleischhacker M, Radacke C, Schulz B, et al. 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.[CrossRef][Web of Science][Medline]
5
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.
6
Chamilos G, Lewis RE, Albert N, et al. 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.
7
Soczo G, Kardos G, Varga I, et al. In vitro study of Candida tropicalis isolates exhibiting paradoxical growth in the presence of high concentrations of caspofungin. Antimicrob Agents Chemother (2007) 51:4474–6.
8
Stevens DA, Ichinomiya M, Koshi Y, et al. Escape of Candida from caspofungin inhibition at concentrations above the MIC (paradoxical effect) accomplished by increased cell wall chitin; evidence for β-1,6-glucan synthesis inhibition by caspofungin. Antimicrob Agents Chemother (2006) 50:3160–1.
9 Somogyvari F, Doczi I, Serly I, et al. Rapid discrimination between Candida albicans and Candida dubliniensis by using real-time polymerase chain reaction. Diagn Microbiol Infect Dis (2007) 58:367–9.[CrossRef][Web of Science][Medline]
10
Klepser ME, Ernst EJ, Lewis RE, et al. Influence of test conditions on antifungal time–kill curve results: proposed for standardized methods. Antimicrob Agents Chemother (1998) 42:1207–12.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  I. Varga, G. Soczo, G. Kardos, A. Borbely, Zs. Szabo, A. Kemeny-Beke, and L. Majoros Comparison of killing activity of caspofungin against Candida parapsilosis, Candida orthopsilosis and Candida metapsilosis J. Antimicrob. Chemother., December 1, 2008; 62(6): 1466 - 1468. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||