a Laboratoire de Botanique, Cryptogamie et Biologie cellulaire (EA 864) Université de la Méditerranée, Faculté de Pharmacie, 13 385 Marseille Cedex 5 b Laboratoire de Biogénotoxicologie et Mutagénèse environnementale (EA 1784), Université de la Méditerranée, Faculté de Pharmacie, 13 385 Marseille Cedex 5; c Laboratoire de Bactériologie et Parasitologie, CHU Nord, 13 015 Marseille; d Service de Parasitologie-Mycologie, CHU Pitié-Salpêtrière, 75 651 Paris, Cedex 13, France
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Abstract |
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Introduction |
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Flow cytometry, which allows for high-speed, multiparametric analysis of individual cells in suspension, could overcome this drawback. This technique has a broad range of applications, and is currently used in haematology and cancer cytology, for example. Continuing improvements in sensitivity and specificity of instrumentation and techniques and the development of new dyes have led to a wide range of microbiological applications. 12 Flow cytometry for evaluating the activity of antifungal agents was initiated in 1990 by Pore, 13 yet few studies have addressed this approach. 14,15 Flow cytofluorometric susceptibility tests (FCSTs) require specific fluorescent probes suitable for monitoring the interactions between the antifungal agent and a target site of the fungal cell. Recently, our laboratory developed a rapid and accurate FCST using 3,3'-dipentyloxacarbocyanine iodide (DiOC5(3)), a cationic membrane potential-sensitive fluorescent dye, to assess the amphotericin B susceptibility of yeast isolates. 16
The aim of this study was to evaluate this flow cytofluorometric method with a series of C. lusitaniae isolates including some that were known to be amphotericin B-resistant on the basis of in-vivo and/or in-vitro data. 10 The results were compared with those obtained by the NCCLS broth macrodilution method and the Etest method, both with AM3.
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Materials and methods |
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A total of 58 C. lusitaniae isolates were tested. Fifty-five isolates were from patients in
oncology or intensive care units. They were recovered from upper respiratory tract (28 isolates),
mouth (six isolates), stool (seven isolates), urine (five isolates), skin (one isolate), vagina (one
isolate) and blood (seven isolates) cultures. They were identified by standard methods
17 and the ID 32C system (BioMérieux, Marcy
l'Etoile, France). The 55 isolates included two recent isolates (94-6856 and 6-103) that
were putatively resistant to amphotericin B on the basis of clinical data. Three C. lusitaniae isolates were kindly provided by J. Rex, University of Texas Medical School, Houston, TX,
USA. One of these (CL 2887) was proven to be resistant to amphotericin B in an animal model
of disseminated candidiasis, and the other two (Y533 and Y534) were presumed resistant after
in-vitro testing.
10 Candida parapsilosis CL 524, previously
identified as being susceptible to amphotericin B in an animal model, was also studied.
10 Isolates were maintained on Sabouraud glucose agar
(BioMérieux) slants and stored at 4°C. Before being tested, they were subcultured
on Sabouraud glucose agar (BioMérieux) and incubated at 35°C for 24 h. C.
parapsilosis ATCC 22019 (MIC = 0.251 mg/L) and Candida tropicalis IP 1275-81 (MIC 2 mg/L) were included in each series of experiments as internal
controls.
Antifungal agent
Amphotericin B (Sigma, St Louis, MO, USA) was dissolved in reagent-grade dimethyl sulphoxide (DMSO; Sigma) at a starting concentration of 12,800 mg/L. The stock solution was stored at -70°C until use. Amphotericin B Etest strips (AB Biodisk, Solna, Sweden) were kept at -20°C until use.
Standard antifungal susceptibility testing
The broth macrodilution susceptibility test was done according to the NCCLS M27-A method 7 with AM3 broth buffered to pH 7.0 (BioMérieux) and supplemented with glucose (2%). 10 The incubation period was 48 h at 35°C. The MIC was defined according to the NCCLS criteria (the MIC endpoint was read as the lowest amphotericin B concentration preventing any discernible growth).
Etest
The Etest was done according to the manufacturer's recommendations with solidified (2% agar) AM3 buffered to pH 7.0 (BioMérieux) and supplemented with glucose (2%). 11 The inoculum suspensions were adjusted spectrophotometrically at 530 nm to match the turbidity of a 0.5 McFarland standard. Agar plates were inoculated with a cotton swab, and they were allowed to dry for 15 min before the Etest strips were applied. The plates were incubated at 35°C and read at 24 and 48 h. The Etest MIC endpoint was the drug concentration at which the inhibition ellipse intercepted the scale on the antifungal strip.
FCST conditions
The conditions for the FCST were as previously described. 16 Briefly, the assay medium was RPMI 1640 with L-glutamine (Sigma) buffered to pH 7.0 with 0.165 M morpholinepropanesulphonic acid (MOPS). Drug dilutions were prepared to ten times the strength of the final concentration with medium as the diluent (e.g. 0.3160 mg/L). Stock inoculum suspensions were made in sterile 0.85% saline and matched with a 0.5 McFarland standard. The adjusted yeast suspensions were diluted 1:10 with the medium to achieve a final concentration of 45 x 10 5 cfu/mL. The final inoculum was added in 0.9 mL volumes to glass tubes containing 0.1 mL of drug dilutions, except the growth control. Final concentrations of amphotericin B ranged from 0.03 to 16 mg/L. Tubes were incubated at 27°C for 30 min. DiOC5(3) (Molecular Probes, Eugene, OR, USA) was then added to all tubes at a final concentration of 0.5 mM. Flow cytofluorometric analysis was initiated 5 min after the dye had been added to the first tube.
Flow cytometry
Samples were run on a FACSort cytometer (Becton Dickinson, Sunnyvale, CA, USA) with a 15 mW, 488 nm argon ion laser, with a Consort 32 workstation combined with Lysis II software. Instrumental parameters were as follows: forward light scatter (FSC, log), sideways light scatter (SSC, log, 304 PMT voltage, 488/10 filter), and green fluorescence (FL1, log, 443 PMT voltage, 530/30 filter). An untreated control of each isolate was sampled first. A gate that excluded cell clusters and debris was adjusted from a cytogram obtained by plotting FSC against SSC. Five thousand cells per sample were analysed, and data were recorded as density plots of DiOC5(3) fluorescence (FL1) versus FSC and as histograms of fluorescence. The geometric mean of fluorescence for the defined population was calculated using Becton Dickinson software.
Study design and statistical analysis
A total of 60 Candida spp. isolates were tested once by the three methods. The same commercial lot of AM3 was used in the Etest and in the NCCLS broth macrodilution method. A set of putatively amphotericin B-resistant and -susceptible isolates was tested in a second experiment to assess the reproducibility of the results. Regarding the FCST, dose response relationships were determined with a non-linear model: F = ab/(b + C) where F is the mean fluorescence, a and b are coefficients calculated by non-linear regression analysis, and C is the amphotericin B concentration (mg/L). For each isolate, the drug concentration producing an 80% decrease in fluorescence mean relative to the fluorescence mean of the drug-free control (IC80 = 4b) was calculated. The correlation between the IC80 and the M27-A MIC, and that between the IC80 and the Etest MIC, were determined. For each linear model, analysis of variance was performed. All calculations were performed with `Table Curve 2D' software (Jandel Scientific, version 3.0).
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Results |
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Discussion |
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Acknowledgments |
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Notes |
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References |
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Received 20 April 1998; returned 15 June 1998; revised 6 July 1998; accepted 17 September 1998