Effect of inoculum form on in vitro antifungal susceptibilities of Aspergillus spp.

Isabel Pujola, Joan Fernández-Ballartb and Josep Guarroc,d,*

a Laboratori de Microbiologia, Hospital Universitari de Sant Joan de Reus, Reus, Tarragona; b Unitat de Medicina Preventiva, Facultat de Medicina, Universitat Rovira i Virgili, Reus, Tarragona; c Unitat de Microbiologia, Facultat de Medicina, Universitat Rovira i Virgili, Carrer Sant Llorenç 21, Reus, Tarragona; d Institut d'Estudis Avançats, Universitat Rovira i Virgili, Tarragona, Spain


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The effect of inoculum form, i.e. ungerminated conidia, germinated conidia and hyphae on the in vitro antifungal susceptibility of 12 strains belonging to Aspergillus spp. was compared using a broth microdilution method. The isolates were tested three times on different days against amphotericin B, itraconazole and ketoconazole. There were no significant differences between MICs obtained with the three types of inoculum (P > 0.05) for any antifungal tested. The degree of reproducibility of the tests was high (>=75%) for all antifungals with each type of inoculum, except for itraconazole (58.3%) when the inoculum was prepared with ungerminated conidia.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The incidence of severe fungal infections caused by opportunistic moulds has risen in recent years, especially in immunocompromised patients. The National Committee for Clinical Laboratory Standards (NCCLS) has devoted a great deal of effort to developing a reference method for in vitro antifungal susceptibility testing of moulds that could serve as guidance for clinical treatment. Recently, a reference method was proposed in which the inoculum consists of conidial suspensions.1 However, considering that the invasive form of mould infection is hyphal, the use of conidia as the starting inoculum may be questioned. Some comparative studies on MICs obtained with hyphae and conidia have been performed and the reported data are controversial.26 Guarro et al.5 found important discrepancies between MICs obtained with both forms of inoculum. On the other hand, Manavathu et al.6 reported practically identical results with germinated and ungerminated conidia of Aspergillus fumigatus. In our previous study, the differences were attributed to differences in inoculum size.5 The present study evaluated the influence of three different forms of inoculum (ungerminated conidia, germinated conidia and hyphae), with a standardized inoculum size, on the MICs of three antifungal agents against different species of Aspergillus.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Twelve clinical isolates of Aspergillus spp. (seven of A. fumigatus, three of Aspergillus niger and one isolate each of Aspergillus oryzae and Aspergillus flavus) were used in this study. Paecilomyces variotii ATCC 36257 was included as a control. The following antifungal agents were assessed: amphotericin B (Fungizone, Squibb & Sons, Barcelona, Spain), itraconazole (Janssen Pharmaceutica, Beerse, Belgium) and ketoconazole (Roig-Farma, Barcelona, Spain). A broth microdilution method, described previously,7 was used to determine the MICs. Dilutions of all antifungal agents, except itraconazole, were prepared at 10 times the final concentration with sterile distilled water, and further diluted 1:5 with RPMI 1640 medium. The dilutions of itraconazole were prepared at 100 times the final concentration with DMSO (100%), and further diluted 1:50 with RPMI. Volumes of 100 µL of each drug concentration were dispensed into the wells of a sterile microplate to provide final test dilutions in the range 0.03–16 mg/L.

The isolates were subcultured on slants of potato dextrose agar (PDA) at 30°C for 7 days. Three types of inoculum were prepared for each strain tested, which included ungerminated conidia, germinated conidia and hyphal fragments. The inoculum consisting of ungerminated conidia (FigureGo, a) was prepared by flooding the surface of the agar slant with sterile distilled water and gently scraping the surface of the sporing surface with a loop. The resulting suspension was then filtered once through sterile gauze to remove hyphae, to produce a suspension with >90% conidia. Germinated conidia and hyphal fragments were prepared from this conidial suspension as follows: for each strain, two Erlenmeyer flasks containing 10 mL of Czapek broth medium (Difco, Detroit, IL, USA) supplemented with 2% Tween 80 and 0.07% agar4 were inoculated with 2 mL of the conidial suspension, adjusted spectrophotometrically to 50% transmittance (T) at a wavelength of 530 nm. The inoculated Erlenmeyer flasks were incubated at 30°C without agitation. After 4–17 h incubation (depending on the species tested) the cultures were examined microscopically for the presence of germinated forms and hyphal elements. Germinated conidia were classified as those in which the germ tubes had a maximum length of three times the diameter of the conidia (FigureGo, b). Hyphae were classed as those structures in which the germ tubes were at least five times as long as the diameter of the conidia (FigureGo, c). The densities of these suspensions were read and adjusted with a spectrophotometer (at 530 nm), and adjusted by adding sterile distilled water to obtain suspensions of 80–82% T for A. fumigatus, 78–82% T for A. flavus and A. oryzae, 60–62% T for A. niger and 74–76% T for P. variotii. Inoculum quantification was performed for each strain and inoculum type by plating 10 µL of serial dilutions of the adjusted suspensions on to PDA plates to determine the number of colony-forming units per millilitre (cfu/mL). The adjusted suspensions were further diluted 1:50 with RPMI medium to obtain the working suspension. Each well was inoculated with 100 µL of the appropriate fungal inoculum. The microplates were incubated without agitation at 30°C and readings were taken after 48 h.



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Figure. A. niger FMR 5944: (a) ungerminated conidia; (b) germinated conidia; (c) hyphae. Magnification x1400.

 
Ketoconazole and itraconazole MICs were defined as the lowest drug dilution causing a prominent (>=50%) decrease in turbidity as compared with the control growth. The amphotericin B MIC was defined as the lowest drug dilution at which there was no fungal growth. Each strain was tested three times on different days. A test was considered reproducible when the highest and the lowest of the three MICs obtained for each strain differed by no more than one dilution. Differences among mean MICs obtained with the three types of inoculum were evaluated by analysis of variance. The modal MIC or median MIC was calculated for each strain, and each antifungal and type of inoculum tested. P values <0.05 were considered statistically significant.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The incubation time required to obtain inoculum suspensions with >=90% of germinated conidia and >=90% of hyphal fragments was variable and depended on the species tested. For germinated conidia it was 10–12 h for A. fumigatus, 6–8 h for A. niger and A. flavus, and 4–5 h for A. oryzae. For hyphal suspensions it was 15–17, 10–12 and 7–8 h, respectively.

The number of cfu/mL corresponding to the adjusted inoculum suspensions were variable and depended on the type of inoculum. In >80% of the suspensions they were 5 x 105 to 4 x 106 for ungerminated conidia, 1 x 105 to 9 x 105 for germinated conidia, and 5 x 104 to 4 x 105 for hyphal fragments.

The TableGo shows the modal MIC for each isolate. In general, the MICs obtained with the three types of inoculum were similar. Differences were greater than one dilution in only one isolate for ketoconazole, two isolates for amphotericin B and three for itraconazole. There were no significant differences between the mean MICs obtained with the three types of inoculum (P > 0.05). The reproducibility of MICs from all the combinations of antifungal drugs and type of inoculum was high (>=75%), except for itraconazole (58.3%) when ungerminated conidia were used.


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Table. Modal MICs of four antifungal agents obtained with different types of inoculum, ungerminated conidia (UC), germinated conidia (GC) and hyphae (H), against 12 isolates of Aspergillus spp.a
 
Our results disagree with a previous study in which other fungal species were examined and where differences were consistently observed between the MICs obtained with conidia and hyphae.5 This disagreement may be explained by differences in inoculum preparation. In the previous study we used the same number of conidia/mL and hyphal fragments/mL, thus producing differences in densities of the two forms of inoculum, which have been shown to affect MICs.7 In the present study, despite the variability in cfu/mL counts for the different types of inoculum, the same density was used for all three.

Little information is available regarding the influence of the form of fungal inoculum on in vitro antifungal activity results. This is probably due to problems in the standardization of germinated conidia or hyphae. The more uniform shape of conidia makes them more amenable to standardization, although they are not the vegetative form of moulds. In this study, depending on the species tested, different incubation times were required to obtain the different inoculum forms. Although other studies have been published, it is difficult to compare results due to the variability in the conditions under which they were conducted. Bezjak4 used a broth microdilution method and Regli et al.3 used an agar diffusion method for testing Aspergillus spp., and both found that MICs were very similar when they used either conidial or hyphae inocula. In contrast, Koenig & Kremer2 demonstrated large differences in MICs produced by the two types of inoculum for Aspergillus spp. using an agar diffusion method. Our results agree with those of Manavathu et al.,6 who examined a broth macrodilution method and reported no significant differences in MICs for A. fumigatus obtained with ungerminated or germinated conidia. It seems that, at least in Aspergillus spp., there are no significant differences in MICs when a conidial inoculum is compared with a hyphal inoculum using the NCCLS reference method with an identical inoculum density. The walls of the conidia of Aspergillus, however, have approximately the same thickness as those of vegetative hyphae. It is not known whether the results would also be similar if other fungi with thicker conidial walls were studied, as this may affect drug penetration into the conidial cell.


    Acknowledgments
 
This work was supported by CICYT (Ministerio de Educación y Ciencia of Spain) grant PM98-0059 and the Fundació Ciència i Salut, Reus, Spain.


    Notes
 
* Correspondence address. Unitat de Microbiologia, Facultat de Medicina, Universitat Rovira i Virgili, Carrer Sant Llorenç 21, 43201 Reus, Tarragona, Spain. Tel: +34-977-759359; Fax: +34-977-759322; E-mail: umb{at}astor.urv.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . National Committee for Clinical Laboratory Standards. (1998). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Conidium-Forming Filamentous Fungi: Proposed Standard M38-P. NCCLS, Wayne, PA.

2 . Koenig, H. & Kremer, M. (1979). A propòs des discordances observees dans les resultats des CMI faites sur spores ou filaments d'Aspergillus. Bulletin de la Société Française de Mycologie Médicale 8, 237–42.

3 . Regli, P., Ferrari, H. & Goudard, M. (1980). Incidence de l'ensemencement sur les resultats de l'antibiogramme antifongique des champignons filamenteux du genre Aspergillus. Bulletin de la Société Française de Mycologie Médicale 9, 269–73.

4 . Bezjak, V. (1985). Standardization of a hyphal inoculum of aspergilli for amphotericin B susceptibility testing. Journal of Clinical Microbiology 21, 509–12.[ISI][Medline]

5 . Guarro, J., Llop, C., Aguilar, C. & Pujol, I. (1997). Comparison of in vitro antifungal susceptibilities of conidia and hyphae of filamentous fungi. Antimicrobial Agents and Chemotherapy 41, 2760–2.[Abstract]

6 . Manavathu, E. K., Cutright, J. & Chandrasekar, P. H. (1999). Comparative study of susceptibilities of germinated and ungerminated conidia of Aspergillus fumigatus to various antifungal agents. Journal of Clinical Microbiology 37, 858–61.[Abstract/Free Full Text]

7 . Pujol, I., Guarro, J., Sala, J. & Riba, M. D. (1997). Effects of incubation temperature, inoculum size, and time of reading on broth microdilution susceptibility test results for amphotericin B against Fusarium. Antimicrobial Agents and Chemotherapy 41, 808–11.[Abstract]

Received 23 June 2000; returned 3 October 2000; revised 17 November 2000; accepted 2 January 2001