- Email: [email protected]
Susceptibility assays of Candida tropicalis to miconazole
ELSEVIER
Journal of MicrobiologicalMethods 30 (1997) 221-229
Susceptibility assays of Cundida tropic& N. Simonetti”‘“,
Journal OfMicrobiological Methods
to miconazole
G. Simonettia, V. Strippolib, A. Callarib, M. Teccab
“Institute of Microbiology, Faculty of Pharmacy, University of Rome ‘La Sapienza’, V; le Regina Margherita, 255 Rome, Italy ‘Institute of Microbiology, Faculty of Medicine and Surgery, University of Rome ‘La Sapienza ‘, P. le Aldo Moro, 5 Rome, Italy
Received 10 May 1997; received in revised form 23 July 1997; accepted 4 August 1997
Abstract In vitro evaluation of antifungal activity of imidazole derivatives is made difficult by the inhibitory effects of several factors such as inoculum size, growth form of the fungus, incubation temperature, the presence of complex substances, including divalent ions, which strongly influence final results. This is particularly evident when testing clinical isolates of C. tropicalis strains resistant to imidazole drugs. Our data based on assays of miconazole nitrate and miconazole sulfosalicylate against C. tropic& show that it is possible to abolish various interference activities on the antimicrobial activity by suitable modifications of some cultural conditions. Thus, a study has been carried out to assess miconazole sulfosalicylate activity on C. tropic&is throughout experiments performed by contact test and agar diffusion test. The use of these techniques made it possible to display some activity of the imidazoles even against strains of C. tropicalis, which were defined as resistant using usual susceptibility assay conditions. Experimental conditions which cause the increase of susceptibility of C. tropicalis are related to factors that modify the barrier function and cellular permeability as demonstrated mainly by the effect of electrical conductivity (E.C.), pH of the medium and pretreatment of fungal inoculum with sodium dioctylsulfosuccinate (SDSS). Our results suggest that the correlation between drug dosage md inhibitory activity in vitro can be improved by the modifications proposed in this paper. 0 1997 Elsevier Science B.V. Keywords:
Candida
tropicalis;
Imidazole drugs; Resistance
1. Introduction The frequency of isolation of pathogenic Candida species other than albicans is increasing [l]. Notably, C. tropicalis has become a prominent pathogen with a remarkable role in neutropenic patients [2]. Interest in C. tropicalis is also increased because of the high frequency of strains with low susceptibility to imidazoles [3]. In the ‘in vitro’ susceptibility studies of 545 isolates of Can&h sp. to azoles, C. tropicah was the most resistant species *Corresponding author. Tel.: +39 6 4402628; fax: +39 6 4404130.
0167-7012/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO 167-7012(97)00072-9
[4]. Diminution of susceptibility of Candidu sp. is correlated to the lack of ergosterol in their cytoplasmic membrane [4,5]. However, ergosterol is not essential to the resistance of the fungus, because bacteria, particularly the Gram-positive ones [6], may show a degree of susceptibility to azole compounds despite the absence of ergosterol in their membrane [7,8]. Simonetti et al. [8] demonstrated that the Gram-negative bacteria also ,resistant to imidazoles [9], may become susceptible to azoles under particular conditions [lo] considering that the azolic activity could not be related to cell membrane sterols in microorganisms. Imidazoles exert mainly an inhibition of mem-
222
N. Simonetti et al. I Jounuzl of Microbiological
brane sterol synthesis in fungi; consequently, the accumulation of methylated sterols disrupt the organization of the lipidic bilayer of membranes. Furthermore, some imidazole drugs, at high concentrations, could exert direct inhibitory effects on membranes, without interference with sterols and sterol esters [lo,1 I]. Evaluation of imidazole derivative activity ‘in vitro’ against fungi is made variable by inhibitory effects of several factors (inoculum size, fungal growth form, incubation temperature, complex substances, divalent ions), particularly when testing some clinical isolates of C. tropicalis. The aim of our work was to study the most suitable ‘in vitro’ conditions to evaluate the susceptibility of C. tropicalis towards miconazole and to its derivative sulfosalicylate miconazole, keeping in mind that the reduced sensitivity ‘in vitro’ to the imidazoles could be due to unsuitable conditions, such as a high electric conductivity (E.C.) of the media and others. Furthermore, it is well known that sodium dioctylsulfosuccinate (SDSS) added to Cundida albicans cultures at non-antimicrobial concentrations, increases azole antifungal activity in agar diffusion assays [ 12,131. Environmental conditions and pretreatment of inoculum with SDSS could affect the methods of evaluation of susceptibility of C. tropicalis to imidazoles, bringing a more correct evaluation of susceptibility assays. Modification and a better assessment of the above conditions could lead to an improved estimate and interpretation of the susceptibility tests ‘in vitro’.
Methods 30 (1997) 221-229
(SDSS) (Aldrich Chimica, Milan, Italy), an anionic surfactant, was dissolved either in distilled water at concentrations of 5 g/l and then added to yield the final concentration in different media, or in NaH,PO,/Na,HPO, phosphate buffers (at pH 5.6, 0.002 M or 0.2 M) for Cundida inocula. 2.2. Microorganism Sixty strains of Candida tropicalis were isolated from out-patients. All strains were identified by Microscan panels (Baxter, Milan, Italy) and by conventional methods [14]. The inoculum was obtained by a shaken Sabouraud liquid medium overnight at 37°C. Cell suspensions of C. tropicalis were diluted for experiments at final concentrations of lo4 cells ml-’ (estimated by a direct count using a Thoma-Zeiss Camera? and also controlled by determination of the Colony Forming Units (CFUs)). 2.3. Electric conductivity (E.C.) The media conductance cm-’ (Hanna instruments
has been measured HI 9032).
2.4. Cell surface hydrophobic&
in &S
(CSH)
2. Materials and methods
This was determined using a modified method of the Rosenberg procedure [ 15,161. Hydrophobicity was expressed as hydrophobicity index (HI.) related to the number of Candidu cells before the xylene was added minus the number of Cundidu cells after the xylene addition divided by the pre-added number.
2.1. Drugs
2.5. Culture inhibition tests
Imidazole drugs such as miconazole nitrate (Mz) and miconazole-5sulfosalicylate (MSS) (Manetti and Roberts, Florence, Italy) were used. The drugs were dissolved in dimethylsulphoxide (DMSO, Merck, Germany) at 5 g/l and diluted in the medium of the inhibition test as required for experiments. Ethylenediaminetetracetic acid tetrasodium salt (EDTA) (Sigma, USA) was dissolved in distilled water and then added to different media to yield the final concentration. Sodium dioctylsulfosuccinate
In culture inhibition test in solid medium was used as a multipoint inoculator [17]. The inocula were prepared from Candida cultures grown in Sabouraud glucose broth for 24 h at 37°C; the dilutions were carried out in the same medium of the cultural inhibition test. For the experiments we have used different media: Brain Heart Infusion (BHI, BBL USA), Sabouraud dextrose (Becton Dickinson) and diluted Sabouraud dextrose broth (1:3), Nutrient broth (BBL, USA), diluted Nutrient broth (1:3)+2%
N. Simonetti et al. ! Journal of Microbiological Methods 30 (1997) 221-229
glucose; diluted (1:40) Yeast Nitrogen Base supplemented with 2% dextrose (YNB, Difco USA). Ethylenediaminetetraacetic acid (EDTA) and sodium dioctylsulfosuccinate (SDSS) have been used at a not-antibacterial concentration (2000-200 lug ml-‘) dissolved in phosphate buffer (pH 5.6, 0.002 M or 0.2 M) for 3 h at 37°C in contact with the Candida suspensions. Then the suspensions were washed with the same phosphate buffer without EDTA or SDSS and inoculated into the media. According to the literature data [18], strains are regarded as resistant (R) when growing at a concentration 212.5 ,ug ml-’ of drugs; and data are expressed as percentage (R%) of resistant strains.
223
with 2% dextrose and purified agar (Difco) were used. The inoculum ( lo6 cells) was incorporated into melted medium (30 ml) and dispensed in Petri dishes (diameter 12 cm) in a thin seeded layer according to Bauer et al. Four replicates for each dilution were prepared; experiments were repeated five times. Wells of 8 mm diameter were bored in the medium and loaded with 90 ~1 serial miconazole sulfosalicylate dilutions. The inoculated plates were incubated and the inhibition zone (diameter) was measured in mm with a calliper after a 24-h incubation at 37°C. The values are the average of zone diameter for four replicates, standard deviations (S.D.) and regression line analyses were evaluated in all experiments.
2.6. Contact experiments 2.8. Cell permeability Contact experiments with MSS were carried out at 22°C. Candida suspensions of lo8 cells ml-’ were obtained from cultures in Sabouraud broth incubated for 24 h at 37”C, then diluted in phosphate buffer at pH 5.6 in different molarity and used in the experiments. The cell suspension concentration was calculated by measuring the optical density at 540 nm and then verified by CFU counts. In some experiments fungal inocula were pre-treated with EDTA or SDSS for 3 h and then washed with the same phosphate buffer used in the tests before the contact with MSS. The effects of short-term contact (ranging between 60 s and 15 min) of C. tropicalis with the imidazole were evaluated at a temperature of 22°C. After contact, the suspensions were diluted 1000 times, thus removing the activity of the antimicrobial agent, and seeded in the culture medium (Sabouraud agar) with the ‘triple stratum’ technique. After an incubation period of 48 h at 37°C the colony forming units (CFU) were evaluated. The survival/time curves analysis gave the measure of the drug activity: the survival percentage gave the percentage of surviving of lo4 bacteria after 15 min.
2.7. Diffusion agar experiments The diffusion agar experiments were performed in pH 5.6 Sabouraud dextrose (diluted 1:3) agar, diluted (1:40) yeast nitrogen base (YNB) and supplemented
studies
I. After 18 h of growth the cells were harvested, washed three times with a 0.01 M phosphate buffer pH 5.6 and suspended in the same medium. Suspensions after treatment with EDTA or SDSS for 3 h have been used. The cells were exposed to miconazole at 22°C at various concentrations. At different time intervals samples were removed and the cell supernatants were obtained by centrifugation at 3000 rev./min. Samples were examined by measuring the absorbance at 260 nm. 2. K+ release tests were performed with the aid of a suitable electrode connected to a microion instrument 2008 CRISON type with calibration curves adjusted to between 1O-6 mol/l and 5X10e3 molll. The C. tropicalis strain 13 was used. MSS were tested at the concentration of 100 mgfml and Candida at lo6 cells/ml. Determinations of K+ released were performed after 1, 3, 5, 10, and 15 min. 2.9. Statistics In contact experiments, K gave the complete hilling time of lo4 yeast in min=Time T; E shows the efficiency measure of the killing time of lo4 cells suspension= 1 it log Ni IN, where t = time; Ni =initial number of cells; N,=final number of cells (
224 Table 1 MSS activity
N. Simonetti et al. / Journal of Microbiological
in culture media with different
EC. against
C. tropic&
Methods 30 (1997) 221-229
strains
Media”
E.Ch
nH
MIC’
CId
R%’
BHI
15 500 15 500 2500 2500 2500 1500 1500 1500
1.2 5.6 1.2 5.6 4.5 7.2 5.6 4.5
105.5 25 18.4 10.4 6.25 1.6 6.25 5.0
60.6-150.3 2.95-47.0 7.1-29.6 5.6-15.2 6.2-6.2
100 loo 40 20 -
5.5-9.7 6.2-6.2 3.8-6.7
20 -
Sabouraud
Nutrient broth
“Sabouraud and nutrient broth media were diluted 1:3 and supplemented with 2% dextrose. bE.C. expressed as PS cm-‘. ‘MIC are mean values of nine strains expressed in pg ml-‘. d95% CI (confidence intervals). “Percentage of Cum&da rropicalis strains with MIC? 12.5 (see Section 2). Readings carried out after incubation
95% CI. R% are percentage of strains with MIC? 12.5 pg ml-‘. F tests with values of F,,Ol
3. Results Since in previous assays C. tropicalis was found to be more susceptible to MSS than to MZ [19], subsequent tests have been carried out with MSS only. C. tropicalis strains that exhibited ‘in vitro’ MIC values 212.5 ,ug ml-’ were defined as resistant
U81. Antifungal activity of MSS toward C. tropicalis was found to be affected by E.C. and by the pH of the cultural medium (Table 1). The results demon-
Table 2 Influence of EDTA-treated
inocula of C. tropicalis
strate a decrease of MIC and of R% values when the E.C. of culture media was reduced. A further reduction would be obtained by lowering the pH of the culture media. Growth inhibition tests of C. tropicalis with the inocula pretreated for 3 h with EDTA show an increased activity of MSS only at low culture medium E.C. (Table 2). Table 3 shows an increased activity of MSS when the inocula were treated with SDSS before exposure to MSS. The treated inocula show a low H.I. The data reported in Table 4 show that time of contact with SDSS modifies the susceptibility of C. tropicalis to MSS. Moreover, MSS activity was concentration-dependent (Table 5). Fig. 1 shows that cytocidal activity by contact of MSS is also correlated to phosphate buffer concentration. With MSS in 0.002 M pH 5.6, cytocidal activity was complete in 15 min.
on MSS activity in E.C. different
Media”
E.C.h
PH
MIC with EDTA-treated
BHI Sabouraud Nutrient broth
15 500 2500 1500
5.6 5.6 5.6
25 10 0.19
Inocula: (lo4 cells/ml) were treated for 3 h with EDTA 2000 pg ml-’ “Sabouraud and nutrient broth media were diluted I:3 and supplemented bE.C. expressed as /_LScm-‘. ‘MIC are mean values of five strains expressed in pg ml-‘. ‘F,,,z11.26. For other details see Section 2.
of cultures for 24 h at 37°C.
culture media
inocula‘
MIC with untreated 25 10 6.25
at 37°C. with 2% dextrose.
inocula”
F test*
37.6**
225
N. Simonetti et al. / Journal of Microbiological Methods 30 (1997) 221-229 Table 3 Influence of SDSS-treated
inocula of C. tropicalis on MSS activity
in E.C. different
culture media
Media”
EC!
pH
H.I.
MIC’
CId
H.I.’
MIC with SDSS-treated
BHI Sabouraud Nutrient broth
15500 2500 1500
5.6 5.6 5.6
22-74 3-62
25 10.4 6.25
2.9-47.0 5.6-15.2 6.2-6.2
O-27 0 O-30
0.7 0.9 0.39
58-81
CId
cells’
0.42- I .O 0.48-1.32 0.24-0.58
Inoculum lo4 cells ml-‘. Inocula treated for 3 h with SDSS 2000 pg ml-’ at 37°C. “Sabouraud and nutrient broth media were diluted 1:3 and supplemented with 2% dextrose. bE.C. expressed as PS cm-‘. ‘MIC are mean values of nine strains expressed in pg ml-‘. d95% CI (confidence intervals). “H.I.: see Section 2.
Table 4 MSS activity related to SDSS treatment
time
Treatment time
MIC”
CP
0 min 10 min 30 min lh 3h
6.25 3.12 1.56 1.56 0.39
2.64-9.84 1.32-4.92 0.67-2.45 0.67-2.45 0.17-0.61
Medium: nutrient broth (diluted 1:3) and supplemented with 2% glucose at pH 7.2. Inocula of C. tropicalis ( lo4 cells ml- ‘) treated for 3 h with SDSS 2000 pg ml-’ at 37°C. “MIC are mean values of six strains expressed in pg ml-‘. h95% CI (confidence intervals). For other details see Section 2.
Table 5 MSS activity related to SDSS concentration SDSS concentration’
MICh
CI’
0 200 500 1000 2000
3.12 0.78 0.78 0.78 0.39
1.32-4.92 0.34-l .22 0.12-1.44 0.12-1.44 0.19-0.59
Medium: nutrient broth diluted I:3 supplemented with 2% glucose at pH 7.2. Inocula of C. tropicalis ( lo4 cells ml-‘) treated for 3 h with SDSS at 37°C. “Expressed as pg ml-’ of medium. ‘MIC are mean values of six strains expressed in pg ml-‘. ‘95% CI (confidence intervals). For other details see Section 2.
01
3
5
1t
15
min Fig. 1. Cytocidal contact activity of MSS against C. tropicalis. The contact experiments were carried out with MSS at a concentration of 1000 pg ml _’ in 0.2 M and 0.002 M Na,Na phosphate buffer, at 22°C pH 5.6. C. tropicalis strain 13 at a concentration of lo* cells ml-‘. After contact time the suspensions were diluted lo4 times and seeded in triple layer Sabouraud agar for CFU determination. Reading after 48 h at 37°C. K, killing time in mins; E, efficiency measure.; 0 0.2 M buffer; 0 0.002 M buffer; D 0.002 M buffer, SDSS @eated inoculum; A-+0 1 min F=862** (F 001 ~11.26); O+O 5 min F=980** (F,,,,=11.26).
SDSS treatment of the Candida suspension increases the cytocidal effect of MSS reducing the killing time, so as to markedly improve MSS effectiveness (E= 1,32). The SDSS-treated cells turned out to be more permeable to azoles than untreated cells, as shown by Kt release tests (Fig. 2). In diffusion tests (Figs. 3 and 4), SDSS treatment of inoculum showed effectiveness in increasing the activity of drug up to 16 times with respect to the
-----+
226
N. Simonetti et al. I Journal of Microbiological Methods 30 (1997) 221-229
mV
-60 -80
Oasl 2 3
10
5
min
15
Fig. 2. K’ ion release from Candida tropicalis induced by miconazole sulfosalicylate. 0 MSS; A MSS-treated inocula with SDSS 2000 pg ml-‘. MSS: 100 pg ml-‘. Inoculum of C. tropicalis 13 ( IO6 cells ml-‘) in 0.002 M Na,Na phosphate buffer at pH 5.6. Temperature, 22°C. .-+A 10 min F= 112.5** (F,, ,), = 11.26).
Fig. 4. MSS diameters of inhibition without SDSS (2000 pg ml-‘) 3-h-treated inocula Candida tropicalis 424 in Sabouraud dextrose pg ml-‘; 2, MSS 100 pg ml-‘; 3, MSS 50 pg pg ml-‘. For more details see Section 2.
(A) and with (B) at 22°C against agar. 1, MSS 200 ml-‘; 4, MSS 25
controls. The diameters of the inhibition zone correlated well with MSS dosage. No microcolonies of growth were visible (Fig. 4), resulting in a better evaluation of diameters of inhibition zones.
4. Discussion and conclusions
04 0.19
o.f.9
Ix85
1.21
1.57
1.93
2.29
MSS (log cont. mg/Ll
Fig. 3. Increased zones of inhibition plotted against SDSS-treated inocula of Candida tropicalis in YNB agar pH 5.6. Diameter of inhibition (in mm) vs. MSS (log concentrations in mg/l). Treated inocula with SDSS for 3 h. The experiments were carried out in YNB broth pH 5.6 diluted 1:40 and supplemented with 2% dextrose and purified agar. Reading carried out after 24 h incubation at 37°C. A C. tropicalis 178 untreated; 0 C. tropicalis 424 untreated; A C. tropicalis 178 treated with SDSS 2000 pg ml-‘; 0 C. tropicalis 424 treated with SDSS 2000 pg ml-‘. A+& F=162** (F o o, = 11.26; MSS concentration is 1.93 mgll); O+O: F=2000** (F o_(,,=11.26; MSS concentration is 1.93 mg/ 1).
The definition of resistant Candida isolates is difficult and does not necessarily reflect the clinical response to treatment [18,20,21]. The ‘in vitro’ susceptibility cannot always be extrapolated into predicting ‘in vivo’ activity or clinical success even when an agent is applied locally, resulting in high concentrations of the antifungal agent [22]. Moreover, with all azoles the rate of microbiological cure is substantially lower than the rate of clinical response [23]. The choice of MIC values 212.5 to define the resistant strains is somewhat arbitrary but has been made by others and is the result of statistical considerations [ 18,20,21,24,25]. Many isolates of Candida have the ability to readily generate variants that may either show decreased susceptibility to antifungal drugs or contribute to increased pathogenicity [26]. Since the sterols are one of the must important constituents of plasma membrane and they are the main target of antifungal drugs, it is also true that a membrane sterol rearrangement causing fluidity change gives a drastic
N. Simonetti er al. I Journal of MicrobiologicalMethods 30 (1997) 221-229 reduction of intracellular drug transport [27,28]. The changes that occurred when resistance emerged in a variant were closely related to changes in cell membrane functions [5]. There are considerable phenotypic variations in the sensitivity of Candida to imidazole-induced release of K+ [29] and the phenotypic variations responsible for the development of drug resistance appear to be related to functional changes in the plasma membranes [29]. We have demonstrated [8] that the phenotypic resistance can be overcome by addition of compounds which modify imidazole’s ability to penetrate the lipid bilayer of the outer membrane. It must be considered that the major source of variation in susceptibility test and their lack of reproducibility are due mainly to the endpoints of MICs which are less sharply defined in agar diffusion [30]. Moreover, the presence of microcolonies are an obstacle in the correct measurement of the diameters of the inhibition zone [ 121 due to phenotypic variation and to the switching of Candida strains [26]. C. tropicalis is naturally less susceptible to the imidazole drugs than other Candida species [4]. In addition, with these strains, the assessment of susceptibility to azoles ‘in vitro’ is more difficult. We thought it to be of some advantage to exploit ‘in vitro’ conditions and allow for a better evaluation of the activity of antifungal drugs against clinical isolates of C. tropicalis. Candida resistance correlates with loss of cell membrane sterols [4]. On the other hand, it should be considered that Gram-negative bacteria (lacking, as all others bacteria, sterols) are resistant to imidazoles [9], although some experimental conditions can modify their resistance [8]. Similarly, it may be possible to improve the ‘in vitro’ activity of miconazole sulfosalicylate (MSS) against C. tropicalis. The antifungal activity of imidazoles critically depends on the media used [24] (YNB, TC, MEM) [31] as also demonstrated by the results found with the NCCLS methods to assess susceptibility to azoles [32]. This activity can be increased by the addition of antibiotics which inhibit protein synthesis [33], although these additions may not be sufficient to totally overcome resistance of some C. tropicalis strains. On this assumption, we tried to improve the activity of miconazole using inocula of C. tropicalis
227
sodium dioctylsulfosuccinate pre-treated with (SDSS) [17], an anionic surfactant used at a nonantimicrobial concentration. This was mostly performed in the light of obtaining further insight into the conditions affecting MIC determination of fungi ‘in vitro’ than with the purpose of proposing a new method for the clinical laboratory, as for the NCCLS method. The results of our studies indicate that the main factor of MSS activity is the alteration of membrane permeability correlated mainly to the electrical conductivity (E.C.) and, secondarily, to the acid pH of the environment [8]. On the other hand, SDSS seems to affect imidazole resistance when inocula of Candida cells are treated for 3 h before being tested in MSS. Inhibition culture tests, cytocidal contact tests and agar diffusion experiments were equally affected by these conditions. In Candida, correlation between resistance to imidazoles and E.C. of the media may be due to membrane permeability [34]: a low electrolyte concentration can promote both the imidazole uptake [35] and the increase of Kf leakage caused by imidazoles [36]. The anionic surfactant SDSS increased anti-C. albicans fluconazole activity ‘in vitro’, possibly helping drug penetration [17]. The barrier functions of the cells are important for imidazole uptake into the lipidic bilayer of the membrane, and the consequent impairment of the membrane function. The modifications of the membrane due to SDSS (at concentrations below a direct antimicrobial activity) may modify the imidazole resistance of C. tropicalis. The modifications of the superficial cell wall layer of imidazole-resistant Candida cells suggest that a prolonged treatment of cells with SDSS may be useful to increase MSS activity [37]. The greater permeability to azoles of SDSS-treated cells [38] has also been demonstrated by K+ release curves. The phospholipids may provide a suitable electrostatic interaction with anionic surfactants [39], also provoking a change of cell permeability [38]. It should be stressed here that the ratio of total phospholipids to non-esterified sterols was found to be approximately two-fold lower in azole-resistant strains [40]. The fluctuation in phospholipid composition can
228
N. Simonetti et al. I Journal of Microbiological Methods 30 (1997) 221-229
lead to altered properties of plasma membranes, namely membrane fluidity, transport activities and drug sensitivity [38]. The phospholipids form electronegative reactive sites capable of binding metal cations [41]. Calcium has been proposed as the main ligand metal cation in bacteria, as supported by studies with metal chelators. Calcium is regarded as an important accessory component which stabilizes the cell membrane by bridging polyanionic molecules. In EDTA-treated Candida cells, metal cations may be important, and susceptibility to azoles can improve in cultural media with a low E.C. In general, the susceptibility assay methods which take into account the regulation of cellular permeability are more predictive of the clinical efficacy of imidazole compounds [42]. Our methods allowed us to demonstrate an increased sensitivity and a better evaluation of antifungal susceptibility of C. tropicalis in the presence of MSS. Important in this context seems to be the inhibition of the growth of microcolonies and a more sensitive evaluation of diameters of inhibition zones. Our modifications could be tested for assessing other azoles as well as other Candida species.
Acknowledgements This work was partially tion of CNR (Italy).
supported
by a contribu-
References N.H. Georgopapadakou, T.J. Walsh, Antifungal agents: chemotherapeutic targets and immunologic strategies, Antimicrob. Agents Chemother. 40 (1996) 279-291. PI D.W. Warnock, Azole drug resistance in Candida species, J. Med. Microbial. 37 (1992) 225-226. S.W. Edelstein, L. Lippman, Candida 131 B.J. Horowitz, tropicalis vulvovaginitis, Obstet. Gynecol. 2 (1985) 229232. [41 A. Arias, M.P. Arevalo, A. Andreu, C. Rodriguez, A. Sierra, In vitro susceptibility of 545 isolates of Candida spp. to four antifungal agents, Mycoses 37 (1994) 285-289. of [51 W.G. Merz, G.R. Sandford, Isolation and characterization a polyene resistant variant of Candida tropicalis, J. Clin. Microbial. 9 (1979) 677-680. and Gilman’s The Pharmacological Basis of [61 Goodman Therapeutics, VI ed., McMillan, London, 1980, 982 p.
[7] F.C. Odds, Candida and Candidosi, II ed., Balliere Tindall, London, 1988, 294 p. [S] G. Simonetti, S. Baffa, N. Simonetti, Overcoming imidazole resistance in E. co/i, J. Chemother. 8 (1996) 201-207. r91 D. Kerridge, Mode of action of clinically important antifungal drugs, Adv. Microbial. Physiol. 27 (1986) l-72. [lOI H. Yamaguchi, Antagonistic action of lipid components of membranes from Candida albicans and various other lipids on two imidazole antimycotics, clotrimazole and miconazole, Antimicrob. Agents Chemother. 12 (1977) 16-25. 1111 H. Yamaguchi, Protection by unsaturated lecithin against the imidazole antimycotics, clotrimazole and miconazole, Antimicrob. Agents Chemother. 13 (1978) 423-426. N. Simonetti, V Strippoli, Increased it[I21 F.D. D’Auria, raconazole antifungal activity by agar diffusion test, J. Microbial. Methods 20 (1994) 47-54. 1131 F.D. D’Auria, N. Simonetti, V Strippoli, Sodium dioctylsulfosuccinate (SDSS) increases the sensitivity of Candida albicuns to miconazole sulfosalicylate, Eur. Bull. Drug Res. I (1992) 32-36. 1141 B.H. Cooper, M. Silva Hutnor, Yeast of medical importance, in: E.H. Lennette, A. Balows, W.J. Hausler, H.J. Shadomy (Eds.), Manual of Clinical Microbiology, 4th edn., American Society for Microbiology, Washington, DC, 1985, pp. 526542. [I51 K.C. Hazen, B.J. Plotkin, D.M. Klimas, Influence of growth conditions on cell surface hydrophobicity of Candida albicans and Candida glabrata, Infect. Immun. 54 (1986) 269-271. Adherence of [I61 M. Rosenberg, D. Gutnick, E. Rosenberg, bacteria to hydrocarbons: a simple method for measuring cell surface hydrophobicity. FEMS Microbial. Lett. 9 (1980) 29-33. [I71 N. Simonetti, F.D. D’Auria, V Strippoli, Increased in vitro sensitivity of Candidu albicans to fluconazole, Chemotherapy 37 (1991) 32-37. A. Maeland, the Norwegian [I81 P. Sandven, A. Bjomeklett, Yeast Study Group, Susceptibilities of Norwegian Candida albicans strains to fluconazole: emergence of resistance, Antimicrob. Agents Chemother. 37 (1993) 2443-2448. [I91 V Strippoli, F.D. D’Auria, N. Simonetti, In vitro antimicrobial activity of econazole and miconazole sulfosalicylate, Drugs Exp. Clin. Res. 16 (1990) 237-242. WI M. Ruhnke, A. Eigler, I. Tennagen, B. Geiseler, E. Engelmann, M. Trautmann, Emergence of fluconazole-resistant strains of Candida albicans in patients with recurrent oropharyngeal candidosis and human immunodehciency virus infection, J. Clin. Microbial. 32 (1994) 2092-2098. [21] D.A. Goff, S.L. Koletar, W.J. Buesching. J. Barnishan, R.J. Fass, Isolation of fluconazole-resistant Candidu albicans from human immunodeficiency virus-negative patients never treated with azoles, Clin. Infect. Dis. 20 (1995) 77-83. [22] M.E. Lynch, J.D. Sobel, Comparative in vitro activity of antimycotic agents against pathogenic vaginal yeast isolates, J. Med. Vet. Mycol. 32 (1994) 267-274. [23] A. White, M.B. Goetz, Azole-resistant Candida albicans: report of two cases of resistance to fluconazole and review, Clin. Infect. Dis. 19 (1994) 687-692.
N. Simonetti et al. I Journal of Microbiological Methods 30 (1997) 221-229 [24] A. Favel, M. Liebennann, A. Michel-Nguyen, P. Regli, Fluconazole susceptibility testing of Candida species: a comparative study of RPMI, high resolution and casitone media, J. Mycol. Med. 5 (1995) 7-12. [25] E.M. Johnson, D.W. Wamock, J. Luker, S.R. Porter, C. Scully, Emergence of azoles drug resistance in Candida species from HIV-infected patients receiving prolonged fluconazole therapy for oral candidosis. J. Antimicrob. Chemother. 35 (1995) 103-I 14. [26] P.J. Gallagher, D.E. Bennett, M.C. Henman, R.J. Russel, S.R. Flint, D.B. Shanley, D.C. Coleman, Reduced azole susceptibility of oral isolates of Candida albicans from HIVpositive patients and a derivative exhibiting colony morphology variation, J. Gen. Microbial. 138 (1992) 1901-1911. [27] HV. Bossche, P. Marichal, F.C. Odds, L. Le Jeune, M.C. Coene, Characterization of an azole-resistant Candida glabrata isolate, Antimicrob. Agents Chemother. 36 (1992) 2602-2610. [28] S. Ansari, R. Prasad, Effect of miconazole on the structure and function of plasma membrane of Candida albicans. FEMS Microbial. L&t. 114 (1993) 93-98. [29] D. Kerridge, Mode of action of clinically important antifungal drugs, Adv. Microbial Physiol. 27 (1986) l-72. [30] J.L. Rodriguez-Tudela, J.V. Martinez-Suarez, Improved medium for fluconazole susceptibility testing of Candida albicans, Antimicrob. Agents Chemother. 38 (1994) 45-48. [31] MS. Marriot. K. Richardson, The discovery and mode of action of fluconazole, in: R.A. Fromtling, J.R. Prous (Eds.), Recent Trends in the Discovery, Development and Evaluation of Antimicrobial Agents, Barcellona, 1987, pp. 81-92. [32] J.N. Galgiani, Susceptibility testing of fungi: current status of the standardization process, Antimicrob. Agents Chemother. 37 (1993) 25 17-252 1, [33] F.C. Odds, A.B. Abbott, G.W. Pye et al., Improved method
229
for estimation of azole antifungal inhibitory concentrations against Candida species, based on azoleslantibiotics interaction, J. Med. Vet. Mycol. 24 (1986) 305-311. [34] H.R. Shmeeda, E.B. Golden, Y. Barenholz, Membrane lipids and aging, in: M. Shinitzky (Ed.), Biomembranes, Structural and Functional Aspects,VCH Balabam, Weinheim, 1994. pp. l-82. [35] V. Strippoli, F.D. D’Auria, N. Simonetti, Investigations of the activity by contact of miconazole sulfosalicylate, J. Chemolher. 2 (1990) 371-376. [36] A. Retico, G. Apuzzo, N. Simonetti, The action of imidazole against bacteria, J. Chem. 1 (Suppl. 4) (1989) 235-237. [37] 0. Shimokawa, H. Nakayama, Increased sensitivity of Candida albicans cells accumulating 14 wmethylated sterols to active oxygen: possible relevance to in vivo efficacies of azole antifungal agents, Antimicrob. Agents Chemother. 36 ( 1992) 1626- 1629. [38] A. Obayashi, Actions of surface active agents upon lactic acid bacteria. The germicidal action and the effect on the permeability, J. Gen. Appl. Microbial. 7 (1961) 233-242. [39] G.R. Dychdala, Surface-active agents: acid-anionic compounds, in: S.S. Block (Ed.), Disinfection, Sterilization and Preservation, III ed., Lea and Febiger, PA. 1983, pp. 330334. [40] P. Mishra, J. Bolard, R. Prosad, Emerging role of lipids of Candida albicans, a pathogenic dimorphic yeast, B&him. Biophys. Acta 1127 (1992) l-14. [41] C. Pelletier, P. Bourlioux, J. Van Heijenoort. Effects of subminimal inhibitory concentrations of EDTA on growth of E. coli and the release of lipopolysaccharide, FEMS Microbiol. Lett. 117 (1994) 203-206. [42] J.F. Ryley. R.G. Wilson, K.J. Barret-Beer, Azole resistance in Candido albicans, Sabour Med. Vet. Mycol. 22 (1984) 53-63.
Journal of MicrobiologicalMethods 30 (1997) 221-229
Susceptibility assays of Cundida tropic& N. Simonetti”‘“,
Journal OfMicrobiological Methods
to miconazole
G. Simonettia, V. Strippolib, A. Callarib, M. Teccab
“Institute of Microbiology, Faculty of Pharmacy, University of Rome ‘La Sapienza’, V; le Regina Margherita, 255 Rome, Italy ‘Institute of Microbiology, Faculty of Medicine and Surgery, University of Rome ‘La Sapienza ‘, P. le Aldo Moro, 5 Rome, Italy
Received 10 May 1997; received in revised form 23 July 1997; accepted 4 August 1997
Abstract In vitro evaluation of antifungal activity of imidazole derivatives is made difficult by the inhibitory effects of several factors such as inoculum size, growth form of the fungus, incubation temperature, the presence of complex substances, including divalent ions, which strongly influence final results. This is particularly evident when testing clinical isolates of C. tropicalis strains resistant to imidazole drugs. Our data based on assays of miconazole nitrate and miconazole sulfosalicylate against C. tropic& show that it is possible to abolish various interference activities on the antimicrobial activity by suitable modifications of some cultural conditions. Thus, a study has been carried out to assess miconazole sulfosalicylate activity on C. tropic&is throughout experiments performed by contact test and agar diffusion test. The use of these techniques made it possible to display some activity of the imidazoles even against strains of C. tropicalis, which were defined as resistant using usual susceptibility assay conditions. Experimental conditions which cause the increase of susceptibility of C. tropicalis are related to factors that modify the barrier function and cellular permeability as demonstrated mainly by the effect of electrical conductivity (E.C.), pH of the medium and pretreatment of fungal inoculum with sodium dioctylsulfosuccinate (SDSS). Our results suggest that the correlation between drug dosage md inhibitory activity in vitro can be improved by the modifications proposed in this paper. 0 1997 Elsevier Science B.V. Keywords:
Candida
tropicalis;
Imidazole drugs; Resistance
1. Introduction The frequency of isolation of pathogenic Candida species other than albicans is increasing [l]. Notably, C. tropicalis has become a prominent pathogen with a remarkable role in neutropenic patients [2]. Interest in C. tropicalis is also increased because of the high frequency of strains with low susceptibility to imidazoles [3]. In the ‘in vitro’ susceptibility studies of 545 isolates of Can&h sp. to azoles, C. tropicah was the most resistant species *Corresponding author. Tel.: +39 6 4402628; fax: +39 6 4404130.
0167-7012/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PII SO 167-7012(97)00072-9
[4]. Diminution of susceptibility of Candidu sp. is correlated to the lack of ergosterol in their cytoplasmic membrane [4,5]. However, ergosterol is not essential to the resistance of the fungus, because bacteria, particularly the Gram-positive ones [6], may show a degree of susceptibility to azole compounds despite the absence of ergosterol in their membrane [7,8]. Simonetti et al. [8] demonstrated that the Gram-negative bacteria also ,resistant to imidazoles [9], may become susceptible to azoles under particular conditions [lo] considering that the azolic activity could not be related to cell membrane sterols in microorganisms. Imidazoles exert mainly an inhibition of mem-
222
N. Simonetti et al. I Jounuzl of Microbiological
brane sterol synthesis in fungi; consequently, the accumulation of methylated sterols disrupt the organization of the lipidic bilayer of membranes. Furthermore, some imidazole drugs, at high concentrations, could exert direct inhibitory effects on membranes, without interference with sterols and sterol esters [lo,1 I]. Evaluation of imidazole derivative activity ‘in vitro’ against fungi is made variable by inhibitory effects of several factors (inoculum size, fungal growth form, incubation temperature, complex substances, divalent ions), particularly when testing some clinical isolates of C. tropicalis. The aim of our work was to study the most suitable ‘in vitro’ conditions to evaluate the susceptibility of C. tropicalis towards miconazole and to its derivative sulfosalicylate miconazole, keeping in mind that the reduced sensitivity ‘in vitro’ to the imidazoles could be due to unsuitable conditions, such as a high electric conductivity (E.C.) of the media and others. Furthermore, it is well known that sodium dioctylsulfosuccinate (SDSS) added to Cundida albicans cultures at non-antimicrobial concentrations, increases azole antifungal activity in agar diffusion assays [ 12,131. Environmental conditions and pretreatment of inoculum with SDSS could affect the methods of evaluation of susceptibility of C. tropicalis to imidazoles, bringing a more correct evaluation of susceptibility assays. Modification and a better assessment of the above conditions could lead to an improved estimate and interpretation of the susceptibility tests ‘in vitro’.
Methods 30 (1997) 221-229
(SDSS) (Aldrich Chimica, Milan, Italy), an anionic surfactant, was dissolved either in distilled water at concentrations of 5 g/l and then added to yield the final concentration in different media, or in NaH,PO,/Na,HPO, phosphate buffers (at pH 5.6, 0.002 M or 0.2 M) for Cundida inocula. 2.2. Microorganism Sixty strains of Candida tropicalis were isolated from out-patients. All strains were identified by Microscan panels (Baxter, Milan, Italy) and by conventional methods [14]. The inoculum was obtained by a shaken Sabouraud liquid medium overnight at 37°C. Cell suspensions of C. tropicalis were diluted for experiments at final concentrations of lo4 cells ml-’ (estimated by a direct count using a Thoma-Zeiss Camera? and also controlled by determination of the Colony Forming Units (CFUs)). 2.3. Electric conductivity (E.C.) The media conductance cm-’ (Hanna instruments
has been measured HI 9032).
2.4. Cell surface hydrophobic&
in &S
(CSH)
2. Materials and methods
This was determined using a modified method of the Rosenberg procedure [ 15,161. Hydrophobicity was expressed as hydrophobicity index (HI.) related to the number of Candidu cells before the xylene was added minus the number of Cundidu cells after the xylene addition divided by the pre-added number.
2.1. Drugs
2.5. Culture inhibition tests
Imidazole drugs such as miconazole nitrate (Mz) and miconazole-5sulfosalicylate (MSS) (Manetti and Roberts, Florence, Italy) were used. The drugs were dissolved in dimethylsulphoxide (DMSO, Merck, Germany) at 5 g/l and diluted in the medium of the inhibition test as required for experiments. Ethylenediaminetetracetic acid tetrasodium salt (EDTA) (Sigma, USA) was dissolved in distilled water and then added to different media to yield the final concentration. Sodium dioctylsulfosuccinate
In culture inhibition test in solid medium was used as a multipoint inoculator [17]. The inocula were prepared from Candida cultures grown in Sabouraud glucose broth for 24 h at 37°C; the dilutions were carried out in the same medium of the cultural inhibition test. For the experiments we have used different media: Brain Heart Infusion (BHI, BBL USA), Sabouraud dextrose (Becton Dickinson) and diluted Sabouraud dextrose broth (1:3), Nutrient broth (BBL, USA), diluted Nutrient broth (1:3)+2%
N. Simonetti et al. ! Journal of Microbiological Methods 30 (1997) 221-229
glucose; diluted (1:40) Yeast Nitrogen Base supplemented with 2% dextrose (YNB, Difco USA). Ethylenediaminetetraacetic acid (EDTA) and sodium dioctylsulfosuccinate (SDSS) have been used at a not-antibacterial concentration (2000-200 lug ml-‘) dissolved in phosphate buffer (pH 5.6, 0.002 M or 0.2 M) for 3 h at 37°C in contact with the Candida suspensions. Then the suspensions were washed with the same phosphate buffer without EDTA or SDSS and inoculated into the media. According to the literature data [18], strains are regarded as resistant (R) when growing at a concentration 212.5 ,ug ml-’ of drugs; and data are expressed as percentage (R%) of resistant strains.
223
with 2% dextrose and purified agar (Difco) were used. The inoculum ( lo6 cells) was incorporated into melted medium (30 ml) and dispensed in Petri dishes (diameter 12 cm) in a thin seeded layer according to Bauer et al. Four replicates for each dilution were prepared; experiments were repeated five times. Wells of 8 mm diameter were bored in the medium and loaded with 90 ~1 serial miconazole sulfosalicylate dilutions. The inoculated plates were incubated and the inhibition zone (diameter) was measured in mm with a calliper after a 24-h incubation at 37°C. The values are the average of zone diameter for four replicates, standard deviations (S.D.) and regression line analyses were evaluated in all experiments.
2.6. Contact experiments 2.8. Cell permeability Contact experiments with MSS were carried out at 22°C. Candida suspensions of lo8 cells ml-’ were obtained from cultures in Sabouraud broth incubated for 24 h at 37”C, then diluted in phosphate buffer at pH 5.6 in different molarity and used in the experiments. The cell suspension concentration was calculated by measuring the optical density at 540 nm and then verified by CFU counts. In some experiments fungal inocula were pre-treated with EDTA or SDSS for 3 h and then washed with the same phosphate buffer used in the tests before the contact with MSS. The effects of short-term contact (ranging between 60 s and 15 min) of C. tropicalis with the imidazole were evaluated at a temperature of 22°C. After contact, the suspensions were diluted 1000 times, thus removing the activity of the antimicrobial agent, and seeded in the culture medium (Sabouraud agar) with the ‘triple stratum’ technique. After an incubation period of 48 h at 37°C the colony forming units (CFU) were evaluated. The survival/time curves analysis gave the measure of the drug activity: the survival percentage gave the percentage of surviving of lo4 bacteria after 15 min.
2.7. Diffusion agar experiments The diffusion agar experiments were performed in pH 5.6 Sabouraud dextrose (diluted 1:3) agar, diluted (1:40) yeast nitrogen base (YNB) and supplemented
studies
I. After 18 h of growth the cells were harvested, washed three times with a 0.01 M phosphate buffer pH 5.6 and suspended in the same medium. Suspensions after treatment with EDTA or SDSS for 3 h have been used. The cells were exposed to miconazole at 22°C at various concentrations. At different time intervals samples were removed and the cell supernatants were obtained by centrifugation at 3000 rev./min. Samples were examined by measuring the absorbance at 260 nm. 2. K+ release tests were performed with the aid of a suitable electrode connected to a microion instrument 2008 CRISON type with calibration curves adjusted to between 1O-6 mol/l and 5X10e3 molll. The C. tropicalis strain 13 was used. MSS were tested at the concentration of 100 mgfml and Candida at lo6 cells/ml. Determinations of K+ released were performed after 1, 3, 5, 10, and 15 min. 2.9. Statistics In contact experiments, K gave the complete hilling time of lo4 yeast in min=Time T; E shows the efficiency measure of the killing time of lo4 cells suspension= 1 it log Ni IN, where t = time; Ni =initial number of cells; N,=final number of cells (
224 Table 1 MSS activity
N. Simonetti et al. / Journal of Microbiological
in culture media with different
EC. against
C. tropic&
Methods 30 (1997) 221-229
strains
Media”
E.Ch
nH
MIC’
CId
R%’
BHI
15 500 15 500 2500 2500 2500 1500 1500 1500
1.2 5.6 1.2 5.6 4.5 7.2 5.6 4.5
105.5 25 18.4 10.4 6.25 1.6 6.25 5.0
60.6-150.3 2.95-47.0 7.1-29.6 5.6-15.2 6.2-6.2
100 loo 40 20 -
5.5-9.7 6.2-6.2 3.8-6.7
20 -
Sabouraud
Nutrient broth
“Sabouraud and nutrient broth media were diluted 1:3 and supplemented with 2% dextrose. bE.C. expressed as PS cm-‘. ‘MIC are mean values of nine strains expressed in pg ml-‘. d95% CI (confidence intervals). “Percentage of Cum&da rropicalis strains with MIC? 12.5 (see Section 2). Readings carried out after incubation
95% CI. R% are percentage of strains with MIC? 12.5 pg ml-‘. F tests with values of F,,Ol
3. Results Since in previous assays C. tropicalis was found to be more susceptible to MSS than to MZ [19], subsequent tests have been carried out with MSS only. C. tropicalis strains that exhibited ‘in vitro’ MIC values 212.5 ,ug ml-’ were defined as resistant
U81. Antifungal activity of MSS toward C. tropicalis was found to be affected by E.C. and by the pH of the cultural medium (Table 1). The results demon-
Table 2 Influence of EDTA-treated
inocula of C. tropicalis
strate a decrease of MIC and of R% values when the E.C. of culture media was reduced. A further reduction would be obtained by lowering the pH of the culture media. Growth inhibition tests of C. tropicalis with the inocula pretreated for 3 h with EDTA show an increased activity of MSS only at low culture medium E.C. (Table 2). Table 3 shows an increased activity of MSS when the inocula were treated with SDSS before exposure to MSS. The treated inocula show a low H.I. The data reported in Table 4 show that time of contact with SDSS modifies the susceptibility of C. tropicalis to MSS. Moreover, MSS activity was concentration-dependent (Table 5). Fig. 1 shows that cytocidal activity by contact of MSS is also correlated to phosphate buffer concentration. With MSS in 0.002 M pH 5.6, cytocidal activity was complete in 15 min.
on MSS activity in E.C. different
Media”
E.C.h
PH
MIC with EDTA-treated
BHI Sabouraud Nutrient broth
15 500 2500 1500
5.6 5.6 5.6
25 10 0.19
Inocula: (lo4 cells/ml) were treated for 3 h with EDTA 2000 pg ml-’ “Sabouraud and nutrient broth media were diluted I:3 and supplemented bE.C. expressed as /_LScm-‘. ‘MIC are mean values of five strains expressed in pg ml-‘. ‘F,,,z11.26. For other details see Section 2.
of cultures for 24 h at 37°C.
culture media
inocula‘
MIC with untreated 25 10 6.25
at 37°C. with 2% dextrose.
inocula”
F test*
37.6**
225
N. Simonetti et al. / Journal of Microbiological Methods 30 (1997) 221-229 Table 3 Influence of SDSS-treated
inocula of C. tropicalis on MSS activity
in E.C. different
culture media
Media”
EC!
pH
H.I.
MIC’
CId
H.I.’
MIC with SDSS-treated
BHI Sabouraud Nutrient broth
15500 2500 1500
5.6 5.6 5.6
22-74 3-62
25 10.4 6.25
2.9-47.0 5.6-15.2 6.2-6.2
O-27 0 O-30
0.7 0.9 0.39
58-81
CId
cells’
0.42- I .O 0.48-1.32 0.24-0.58
Inoculum lo4 cells ml-‘. Inocula treated for 3 h with SDSS 2000 pg ml-’ at 37°C. “Sabouraud and nutrient broth media were diluted 1:3 and supplemented with 2% dextrose. bE.C. expressed as PS cm-‘. ‘MIC are mean values of nine strains expressed in pg ml-‘. d95% CI (confidence intervals). “H.I.: see Section 2.
Table 4 MSS activity related to SDSS treatment
time
Treatment time
MIC”
CP
0 min 10 min 30 min lh 3h
6.25 3.12 1.56 1.56 0.39
2.64-9.84 1.32-4.92 0.67-2.45 0.67-2.45 0.17-0.61
Medium: nutrient broth (diluted 1:3) and supplemented with 2% glucose at pH 7.2. Inocula of C. tropicalis ( lo4 cells ml- ‘) treated for 3 h with SDSS 2000 pg ml-’ at 37°C. “MIC are mean values of six strains expressed in pg ml-‘. h95% CI (confidence intervals). For other details see Section 2.
Table 5 MSS activity related to SDSS concentration SDSS concentration’
MICh
CI’
0 200 500 1000 2000
3.12 0.78 0.78 0.78 0.39
1.32-4.92 0.34-l .22 0.12-1.44 0.12-1.44 0.19-0.59
Medium: nutrient broth diluted I:3 supplemented with 2% glucose at pH 7.2. Inocula of C. tropicalis ( lo4 cells ml-‘) treated for 3 h with SDSS at 37°C. “Expressed as pg ml-’ of medium. ‘MIC are mean values of six strains expressed in pg ml-‘. ‘95% CI (confidence intervals). For other details see Section 2.
01
3
5
1t
15
min Fig. 1. Cytocidal contact activity of MSS against C. tropicalis. The contact experiments were carried out with MSS at a concentration of 1000 pg ml _’ in 0.2 M and 0.002 M Na,Na phosphate buffer, at 22°C pH 5.6. C. tropicalis strain 13 at a concentration of lo* cells ml-‘. After contact time the suspensions were diluted lo4 times and seeded in triple layer Sabouraud agar for CFU determination. Reading after 48 h at 37°C. K, killing time in mins; E, efficiency measure.; 0 0.2 M buffer; 0 0.002 M buffer; D 0.002 M buffer, SDSS @eated inoculum; A-+0 1 min F=862** (F 001 ~11.26); O+O 5 min F=980** (F,,,,=11.26).
SDSS treatment of the Candida suspension increases the cytocidal effect of MSS reducing the killing time, so as to markedly improve MSS effectiveness (E= 1,32). The SDSS-treated cells turned out to be more permeable to azoles than untreated cells, as shown by Kt release tests (Fig. 2). In diffusion tests (Figs. 3 and 4), SDSS treatment of inoculum showed effectiveness in increasing the activity of drug up to 16 times with respect to the
-----+
226
N. Simonetti et al. I Journal of Microbiological Methods 30 (1997) 221-229
mV
-60 -80
Oasl 2 3
10
5
min
15
Fig. 2. K’ ion release from Candida tropicalis induced by miconazole sulfosalicylate. 0 MSS; A MSS-treated inocula with SDSS 2000 pg ml-‘. MSS: 100 pg ml-‘. Inoculum of C. tropicalis 13 ( IO6 cells ml-‘) in 0.002 M Na,Na phosphate buffer at pH 5.6. Temperature, 22°C. .-+A 10 min F= 112.5** (F,, ,), = 11.26).
Fig. 4. MSS diameters of inhibition without SDSS (2000 pg ml-‘) 3-h-treated inocula Candida tropicalis 424 in Sabouraud dextrose pg ml-‘; 2, MSS 100 pg ml-‘; 3, MSS 50 pg pg ml-‘. For more details see Section 2.
(A) and with (B) at 22°C against agar. 1, MSS 200 ml-‘; 4, MSS 25
controls. The diameters of the inhibition zone correlated well with MSS dosage. No microcolonies of growth were visible (Fig. 4), resulting in a better evaluation of diameters of inhibition zones.
4. Discussion and conclusions
04 0.19
o.f.9
Ix85
1.21
1.57
1.93
2.29
MSS (log cont. mg/Ll
Fig. 3. Increased zones of inhibition plotted against SDSS-treated inocula of Candida tropicalis in YNB agar pH 5.6. Diameter of inhibition (in mm) vs. MSS (log concentrations in mg/l). Treated inocula with SDSS for 3 h. The experiments were carried out in YNB broth pH 5.6 diluted 1:40 and supplemented with 2% dextrose and purified agar. Reading carried out after 24 h incubation at 37°C. A C. tropicalis 178 untreated; 0 C. tropicalis 424 untreated; A C. tropicalis 178 treated with SDSS 2000 pg ml-‘; 0 C. tropicalis 424 treated with SDSS 2000 pg ml-‘. A+& F=162** (F o o, = 11.26; MSS concentration is 1.93 mgll); O+O: F=2000** (F o_(,,=11.26; MSS concentration is 1.93 mg/ 1).
The definition of resistant Candida isolates is difficult and does not necessarily reflect the clinical response to treatment [18,20,21]. The ‘in vitro’ susceptibility cannot always be extrapolated into predicting ‘in vivo’ activity or clinical success even when an agent is applied locally, resulting in high concentrations of the antifungal agent [22]. Moreover, with all azoles the rate of microbiological cure is substantially lower than the rate of clinical response [23]. The choice of MIC values 212.5 to define the resistant strains is somewhat arbitrary but has been made by others and is the result of statistical considerations [ 18,20,21,24,25]. Many isolates of Candida have the ability to readily generate variants that may either show decreased susceptibility to antifungal drugs or contribute to increased pathogenicity [26]. Since the sterols are one of the must important constituents of plasma membrane and they are the main target of antifungal drugs, it is also true that a membrane sterol rearrangement causing fluidity change gives a drastic
N. Simonetti er al. I Journal of MicrobiologicalMethods 30 (1997) 221-229 reduction of intracellular drug transport [27,28]. The changes that occurred when resistance emerged in a variant were closely related to changes in cell membrane functions [5]. There are considerable phenotypic variations in the sensitivity of Candida to imidazole-induced release of K+ [29] and the phenotypic variations responsible for the development of drug resistance appear to be related to functional changes in the plasma membranes [29]. We have demonstrated [8] that the phenotypic resistance can be overcome by addition of compounds which modify imidazole’s ability to penetrate the lipid bilayer of the outer membrane. It must be considered that the major source of variation in susceptibility test and their lack of reproducibility are due mainly to the endpoints of MICs which are less sharply defined in agar diffusion [30]. Moreover, the presence of microcolonies are an obstacle in the correct measurement of the diameters of the inhibition zone [ 121 due to phenotypic variation and to the switching of Candida strains [26]. C. tropicalis is naturally less susceptible to the imidazole drugs than other Candida species [4]. In addition, with these strains, the assessment of susceptibility to azoles ‘in vitro’ is more difficult. We thought it to be of some advantage to exploit ‘in vitro’ conditions and allow for a better evaluation of the activity of antifungal drugs against clinical isolates of C. tropicalis. Candida resistance correlates with loss of cell membrane sterols [4]. On the other hand, it should be considered that Gram-negative bacteria (lacking, as all others bacteria, sterols) are resistant to imidazoles [9], although some experimental conditions can modify their resistance [8]. Similarly, it may be possible to improve the ‘in vitro’ activity of miconazole sulfosalicylate (MSS) against C. tropicalis. The antifungal activity of imidazoles critically depends on the media used [24] (YNB, TC, MEM) [31] as also demonstrated by the results found with the NCCLS methods to assess susceptibility to azoles [32]. This activity can be increased by the addition of antibiotics which inhibit protein synthesis [33], although these additions may not be sufficient to totally overcome resistance of some C. tropicalis strains. On this assumption, we tried to improve the activity of miconazole using inocula of C. tropicalis
227
sodium dioctylsulfosuccinate pre-treated with (SDSS) [17], an anionic surfactant used at a nonantimicrobial concentration. This was mostly performed in the light of obtaining further insight into the conditions affecting MIC determination of fungi ‘in vitro’ than with the purpose of proposing a new method for the clinical laboratory, as for the NCCLS method. The results of our studies indicate that the main factor of MSS activity is the alteration of membrane permeability correlated mainly to the electrical conductivity (E.C.) and, secondarily, to the acid pH of the environment [8]. On the other hand, SDSS seems to affect imidazole resistance when inocula of Candida cells are treated for 3 h before being tested in MSS. Inhibition culture tests, cytocidal contact tests and agar diffusion experiments were equally affected by these conditions. In Candida, correlation between resistance to imidazoles and E.C. of the media may be due to membrane permeability [34]: a low electrolyte concentration can promote both the imidazole uptake [35] and the increase of Kf leakage caused by imidazoles [36]. The anionic surfactant SDSS increased anti-C. albicans fluconazole activity ‘in vitro’, possibly helping drug penetration [17]. The barrier functions of the cells are important for imidazole uptake into the lipidic bilayer of the membrane, and the consequent impairment of the membrane function. The modifications of the membrane due to SDSS (at concentrations below a direct antimicrobial activity) may modify the imidazole resistance of C. tropicalis. The modifications of the superficial cell wall layer of imidazole-resistant Candida cells suggest that a prolonged treatment of cells with SDSS may be useful to increase MSS activity [37]. The greater permeability to azoles of SDSS-treated cells [38] has also been demonstrated by K+ release curves. The phospholipids may provide a suitable electrostatic interaction with anionic surfactants [39], also provoking a change of cell permeability [38]. It should be stressed here that the ratio of total phospholipids to non-esterified sterols was found to be approximately two-fold lower in azole-resistant strains [40]. The fluctuation in phospholipid composition can
228
N. Simonetti et al. I Journal of Microbiological Methods 30 (1997) 221-229
lead to altered properties of plasma membranes, namely membrane fluidity, transport activities and drug sensitivity [38]. The phospholipids form electronegative reactive sites capable of binding metal cations [41]. Calcium has been proposed as the main ligand metal cation in bacteria, as supported by studies with metal chelators. Calcium is regarded as an important accessory component which stabilizes the cell membrane by bridging polyanionic molecules. In EDTA-treated Candida cells, metal cations may be important, and susceptibility to azoles can improve in cultural media with a low E.C. In general, the susceptibility assay methods which take into account the regulation of cellular permeability are more predictive of the clinical efficacy of imidazole compounds [42]. Our methods allowed us to demonstrate an increased sensitivity and a better evaluation of antifungal susceptibility of C. tropicalis in the presence of MSS. Important in this context seems to be the inhibition of the growth of microcolonies and a more sensitive evaluation of diameters of inhibition zones. Our modifications could be tested for assessing other azoles as well as other Candida species.
Acknowledgements This work was partially tion of CNR (Italy).
supported
by a contribu-
References N.H. Georgopapadakou, T.J. Walsh, Antifungal agents: chemotherapeutic targets and immunologic strategies, Antimicrob. Agents Chemother. 40 (1996) 279-291. PI D.W. Warnock, Azole drug resistance in Candida species, J. Med. Microbial. 37 (1992) 225-226. S.W. Edelstein, L. Lippman, Candida 131 B.J. Horowitz, tropicalis vulvovaginitis, Obstet. Gynecol. 2 (1985) 229232. [41 A. Arias, M.P. Arevalo, A. Andreu, C. Rodriguez, A. Sierra, In vitro susceptibility of 545 isolates of Candida spp. to four antifungal agents, Mycoses 37 (1994) 285-289. of [51 W.G. Merz, G.R. Sandford, Isolation and characterization a polyene resistant variant of Candida tropicalis, J. Clin. Microbial. 9 (1979) 677-680. and Gilman’s The Pharmacological Basis of [61 Goodman Therapeutics, VI ed., McMillan, London, 1980, 982 p.
[7] F.C. Odds, Candida and Candidosi, II ed., Balliere Tindall, London, 1988, 294 p. [S] G. Simonetti, S. Baffa, N. Simonetti, Overcoming imidazole resistance in E. co/i, J. Chemother. 8 (1996) 201-207. r91 D. Kerridge, Mode of action of clinically important antifungal drugs, Adv. Microbial. Physiol. 27 (1986) l-72. [lOI H. Yamaguchi, Antagonistic action of lipid components of membranes from Candida albicans and various other lipids on two imidazole antimycotics, clotrimazole and miconazole, Antimicrob. Agents Chemother. 12 (1977) 16-25. 1111 H. Yamaguchi, Protection by unsaturated lecithin against the imidazole antimycotics, clotrimazole and miconazole, Antimicrob. Agents Chemother. 13 (1978) 423-426. N. Simonetti, V Strippoli, Increased it[I21 F.D. D’Auria, raconazole antifungal activity by agar diffusion test, J. Microbial. Methods 20 (1994) 47-54. 1131 F.D. D’Auria, N. Simonetti, V Strippoli, Sodium dioctylsulfosuccinate (SDSS) increases the sensitivity of Candida albicuns to miconazole sulfosalicylate, Eur. Bull. Drug Res. I (1992) 32-36. 1141 B.H. Cooper, M. Silva Hutnor, Yeast of medical importance, in: E.H. Lennette, A. Balows, W.J. Hausler, H.J. Shadomy (Eds.), Manual of Clinical Microbiology, 4th edn., American Society for Microbiology, Washington, DC, 1985, pp. 526542. [I51 K.C. Hazen, B.J. Plotkin, D.M. Klimas, Influence of growth conditions on cell surface hydrophobicity of Candida albicans and Candida glabrata, Infect. Immun. 54 (1986) 269-271. Adherence of [I61 M. Rosenberg, D. Gutnick, E. Rosenberg, bacteria to hydrocarbons: a simple method for measuring cell surface hydrophobicity. FEMS Microbial. Lett. 9 (1980) 29-33. [I71 N. Simonetti, F.D. D’Auria, V Strippoli, Increased in vitro sensitivity of Candidu albicans to fluconazole, Chemotherapy 37 (1991) 32-37. A. Maeland, the Norwegian [I81 P. Sandven, A. Bjomeklett, Yeast Study Group, Susceptibilities of Norwegian Candida albicans strains to fluconazole: emergence of resistance, Antimicrob. Agents Chemother. 37 (1993) 2443-2448. [I91 V Strippoli, F.D. D’Auria, N. Simonetti, In vitro antimicrobial activity of econazole and miconazole sulfosalicylate, Drugs Exp. Clin. Res. 16 (1990) 237-242. WI M. Ruhnke, A. Eigler, I. Tennagen, B. Geiseler, E. Engelmann, M. Trautmann, Emergence of fluconazole-resistant strains of Candida albicans in patients with recurrent oropharyngeal candidosis and human immunodehciency virus infection, J. Clin. Microbial. 32 (1994) 2092-2098. [21] D.A. Goff, S.L. Koletar, W.J. Buesching. J. Barnishan, R.J. Fass, Isolation of fluconazole-resistant Candidu albicans from human immunodeficiency virus-negative patients never treated with azoles, Clin. Infect. Dis. 20 (1995) 77-83. [22] M.E. Lynch, J.D. Sobel, Comparative in vitro activity of antimycotic agents against pathogenic vaginal yeast isolates, J. Med. Vet. Mycol. 32 (1994) 267-274. [23] A. White, M.B. Goetz, Azole-resistant Candida albicans: report of two cases of resistance to fluconazole and review, Clin. Infect. Dis. 19 (1994) 687-692.
N. Simonetti et al. I Journal of Microbiological Methods 30 (1997) 221-229 [24] A. Favel, M. Liebennann, A. Michel-Nguyen, P. Regli, Fluconazole susceptibility testing of Candida species: a comparative study of RPMI, high resolution and casitone media, J. Mycol. Med. 5 (1995) 7-12. [25] E.M. Johnson, D.W. Wamock, J. Luker, S.R. Porter, C. Scully, Emergence of azoles drug resistance in Candida species from HIV-infected patients receiving prolonged fluconazole therapy for oral candidosis. J. Antimicrob. Chemother. 35 (1995) 103-I 14. [26] P.J. Gallagher, D.E. Bennett, M.C. Henman, R.J. Russel, S.R. Flint, D.B. Shanley, D.C. Coleman, Reduced azole susceptibility of oral isolates of Candida albicans from HIVpositive patients and a derivative exhibiting colony morphology variation, J. Gen. Microbial. 138 (1992) 1901-1911. [27] HV. Bossche, P. Marichal, F.C. Odds, L. Le Jeune, M.C. Coene, Characterization of an azole-resistant Candida glabrata isolate, Antimicrob. Agents Chemother. 36 (1992) 2602-2610. [28] S. Ansari, R. Prasad, Effect of miconazole on the structure and function of plasma membrane of Candida albicans. FEMS Microbial. L&t. 114 (1993) 93-98. [29] D. Kerridge, Mode of action of clinically important antifungal drugs, Adv. Microbial Physiol. 27 (1986) l-72. [30] J.L. Rodriguez-Tudela, J.V. Martinez-Suarez, Improved medium for fluconazole susceptibility testing of Candida albicans, Antimicrob. Agents Chemother. 38 (1994) 45-48. [31] MS. Marriot. K. Richardson, The discovery and mode of action of fluconazole, in: R.A. Fromtling, J.R. Prous (Eds.), Recent Trends in the Discovery, Development and Evaluation of Antimicrobial Agents, Barcellona, 1987, pp. 81-92. [32] J.N. Galgiani, Susceptibility testing of fungi: current status of the standardization process, Antimicrob. Agents Chemother. 37 (1993) 25 17-252 1, [33] F.C. Odds, A.B. Abbott, G.W. Pye et al., Improved method
229
for estimation of azole antifungal inhibitory concentrations against Candida species, based on azoleslantibiotics interaction, J. Med. Vet. Mycol. 24 (1986) 305-311. [34] H.R. Shmeeda, E.B. Golden, Y. Barenholz, Membrane lipids and aging, in: M. Shinitzky (Ed.), Biomembranes, Structural and Functional Aspects,VCH Balabam, Weinheim, 1994. pp. l-82. [35] V. Strippoli, F.D. D’Auria, N. Simonetti, Investigations of the activity by contact of miconazole sulfosalicylate, J. Chemolher. 2 (1990) 371-376. [36] A. Retico, G. Apuzzo, N. Simonetti, The action of imidazole against bacteria, J. Chem. 1 (Suppl. 4) (1989) 235-237. [37] 0. Shimokawa, H. Nakayama, Increased sensitivity of Candida albicans cells accumulating 14 wmethylated sterols to active oxygen: possible relevance to in vivo efficacies of azole antifungal agents, Antimicrob. Agents Chemother. 36 ( 1992) 1626- 1629. [38] A. Obayashi, Actions of surface active agents upon lactic acid bacteria. The germicidal action and the effect on the permeability, J. Gen. Appl. Microbial. 7 (1961) 233-242. [39] G.R. Dychdala, Surface-active agents: acid-anionic compounds, in: S.S. Block (Ed.), Disinfection, Sterilization and Preservation, III ed., Lea and Febiger, PA. 1983, pp. 330334. [40] P. Mishra, J. Bolard, R. Prosad, Emerging role of lipids of Candida albicans, a pathogenic dimorphic yeast, B&him. Biophys. Acta 1127 (1992) l-14. [41] C. Pelletier, P. Bourlioux, J. Van Heijenoort. Effects of subminimal inhibitory concentrations of EDTA on growth of E. coli and the release of lipopolysaccharide, FEMS Microbiol. Lett. 117 (1994) 203-206. [42] J.F. Ryley. R.G. Wilson, K.J. Barret-Beer, Azole resistance in Candido albicans, Sabour Med. Vet. Mycol. 22 (1984) 53-63.