Microwave-assisted synthesis and antimicrobial activity of 5-trihalomethyl-3-arylisoxazoles

Article abstract of DOI:10.1007/s00706-007-0849-1

A series of twelve 5-trihalomethyl-3-arylisoxazoles was synthesized and screened for antibacterial and antifungal activities. The compounds were synthesized from the cyclondensation of 1,1,1-trihalo-4-alkoxy-3-alken-2-ones [CX ₃C(O)C(R ²

Full text of DOI:10.1007/s00706-007-0849-1

Monatsh Chem 139, 985–990 (2008)
DOI 10.1007/s00706-007-0849-1
Printed in The Netherlands
Microwave-assisted synthesis and antimicrobial activity of 5-trihalomethyl-
3-arylisoxazoles
Marcos A. P. Martins1, Pablo Machado1, Luciana A. Piovesan1, Alex F. C. Flores1,
Marli M. A. de Campos2, Carolina Scheidt1, Helio G. Bonacorso1, Nilo Zanatta1
1
´
´ ^
Nucleo de Quımica de Heterociclos (NUQUIMHE), Centro de Ciencias Naturais e Exatas,
´
Departamento de Quımica, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
´
2
^
Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
´
´ ´
Centro de Ciencias da Saude, Departamento de Analises Clınicas e Toxicologicas,
Received 25 October 2007; Accepted 6 November 2007; Published online 26 June 2008
# Springer-Verlag 2008
Abstract A series of twelve 5-trihalomethyl-3-aryl-
isoxazoles was synthesized and screened for antibac-
terial and antifungal activities. The compounds were
synthesized from the cyclondensation of 1,1,1-trihalo-
4-alkoxy-3-alken-2-ones [CX3C(O)C(R2)¼C(R1)OR,
where X ¼ Cl and F; R ¼ Me; R2 ¼ H; R1 ¼ H, Me,
F, Cl, Br, and NO2] with hydroxylamine hydrochloride
through a rapid one-pot reaction via microwave irra-
diation. Some of the 5-trihalomethyl-3-arylisoxazoles
exhibited good in vitro anti-Cryptococcus activity.
In this context, fungal infections deserve special
attention because fungi are emerging as important
nosocomial pathogens causing severe morbidity and
mortality in immunocompromised patients. Modern
therapies and management such as bone marrow or
solid-organ transplants, and new more aggressive
chemotherapy have resulted in a rapidly expanding
number of immunosuppressed patients. These pa-
tients now survive longer and become highly sus-
ceptible to life-threatening fungal infections [2].
Concomitant with the increased incidence of fungal
infections has been a dramatic increase in the use of
antifungals for the treatment of both systemic and
localized fungal infections. Therefore, the expanded
use of antifungal agents has accelerated the deve-
lopment of antifungal drug resistance, leading to
frequent therapeutic failures and an increasing mor-
tality rate [3].
Keywords Isoxazoles; Microwave irradiation; Antibacterial;
Antifungal.
Introduction
There is no doubt that the existing arsenal of antimi-
crobial agents we have in hand for the treatment of
infectious diseases will be insufficient over the long
term [1]. The primary reason for this state of affairs
is the inexorable drive of evolution that leads to an-
timicrobial resistance culminating with the failure of
current treatments [1].
For this reason, a constant effort toward the syn-
thesis of new antifungal agents has been made over
the last few years. The aim is to obtain novel leads
which act through mechanisms of action distinct from
those of well-known classes of antifungal agents,
allowing for the treatment of many clinically rele-
vant pathogens which are now resistant.
´
´
Correspondence: Marcos A. P. Martins, Nucleo de Quımica de
Heterociclos (NUQUIMHE), Centro de Ciencias Naturais e
^
´
Exatas, Departamento de Quımica, Universidade Federal de
Compounds containing an isoxazole scaffold are
known to possess antiinflammatory [4], antiviral [5],
Santa Maria, 97.105-900 Santa Maria, RS, Brazil. E-mail:
mmartins@base.ufsm.br

986
M. A. P. Martins et al.
antileukemic [6], and antagonist ionotropic glutamate
receptor [7] activities, and are also modulators of the
Multidrug Resistance Protein (MRP1) [8]. Literature
has also reported some isoxazole derivatives en-
dowed with antimicrobial activities [9–12], includ-
ing the class of arylisoxazole derivatives [13, 14],
however these studies have been limited in terms
of microorganism strains.
Thus, the aim of this work is to show the synthesis
of a series of 5-trihalomethyl-3-arylisoxazoles using
environmentally benign microwave induced tech-
niques. Furthermore, this work shows the screening
of the synthesized compounds to observe their
in vitro antibacterial and antifungal activities.
cesses involving sensitive reagents, or when there
is the possibility of compound decomposition under
prolonged reaction conditions. Reactions that require
hours or even days by conventional heating can often
be accomplished in seconds or minutes by micro-
wave heating [18].
The 1,1,1-trihalomethyl-4-methoxy-4-aryl-3-buten-
2-ones 1 and 2 were synthesized from the reaction of
the respective acetal with trichloroacetyl chloride or
trifluoroacetic anhydride [19, 20].
The 5-trihalomethyl-3-arylisoxazoles 3 and 4 were
synthesized in two steps, using a one-pot process, as
shown in Scheme 1. Firstly, enones 1 and 2 reacted
with hydroxylamine hydrochloride in the presence
of pyridine and using methanol as solvent. The solu-
tion was submitted to microwave irradiation (100 W)
for 6 min, at a temperature of 70ꢀC and at 2.2 bar of
pressure, to produce the 5-trihalomethyl-5-hydroxy-
4,5-dihydro-3-arylisoxazole intermediates. Secondly,
after cooling to room temperature, conc. sulfuric acid
was added and the mixture was again heated under
Results and discussion
Chemistry
Over the last decade, we have developed a general
synthesis of a large number of 1,1,1-trihalomethyl-
4-methoxy-4-aryl[alkyl]-3-buten-2-ones, important
halogen-containing building blocks, and demonstrat-
ed their usefulness in heterocyclic preparations [15].
As part of our research program there is an interest
in improving the methodologies for the construction
of heterocycles, and, therefore, we are investigating
alternative methods for the preparation of these com-
pounds, such as microwave irradiation [16] and ul-
trasound applications [17].
The beneficial effects of microwave irradiation
are playing an increasing role in process chemistry,
especially in cases where classical methods re-
quire forcing conditions or prolonged reaction times.
Microwaves have also shown an advantage in pro-
(i) NH2OH ꢁ HCl, MeOH, Py, MW, 100 W, 70ꢀC, 2.2 bar, and
6 min; and (ii) H2SO4 conc., MW, 100 W, 80ꢀC, 3.5 bar, and
10 min
Scheme 1
Table 1 Yieldsa and reaction time used for the microwave-assisted and conventional method for synthesis of 3a–3f and 4a–4f
Product
Microwave method
Conventional methodb
Product
Microwave method
Conventional methodc
Reaction
time=min
Yield=%
Reaction
time=h
Yield=%
Reaction
time=min
Yield=%
Reaction
time=h
Yield=%
3a
3b
3c
3d
3e
3f
16
16
16
16
16
16
85
78
88
80
90
89
21
21
21
21
21
21
84
77
85
81
85
83
4a
4b
4c
4d
4e
4f
16
16
16
16
16
16
87
83
85
80
80
85
48
48
48
48
48
48
67
70
69
73
68
80
a
Yields of isolated products
Reaction conditions: 1) NH2OH ꢁ HCl=pyridine=methanol=70ꢀC=16 h; 2) H2SO4 conc.=35ꢀC=5 h
b
c
Reaction conditions: NH2OH ꢁ HCl=HCl exc.=methanol=70ꢀC=48 h

Microwave-assisted synthesis
987
microwave irradiation (100 W) for 10 min, at a temper-
ature of 80ꢀC to furnish the dehydrated 5-trihalometh-
yl-3-arylisoxazoles 3 and 4 with yields of 78–90%.
The dehydration reaction of 4,5-dihydroazoles in
acidic media is a second order elimination reaction
(E2(E1-like)), where the stability of its activated com-
plex depends on the participation of the electron pair
of the neighboring oxygen atom (O-1) present in the
isoxazole ring and of the electron donating effect of
the group attached to the C-5 of the isoxazole ring.
This new method of forming of isoxazoles 3 and 4
under microwave irradiation offers several advan-
tages: faster reaction rates, fewer byproducts, and
good yields, while the conventional method involves
moderate to good yields and a long tedious process,
Table 1 [20]. For 3a–3f, in the conventional method
the cyclocondensation was carried out under reflux
in methanol for 16 h followed by dehydration with
sulfuric acid for 5 h [20]. Compounds 4a–4f were
obtained in a one-pot procedure using an excess of
concentrate hydrochloric acid and stirring for 48 h
1
[20]. The H and 13C NMR and physical data of all
reported compounds are in full accordance with the
data presented in Ref. [20].
Table 2 The in vitro antimicrobial profile of 5-trihalomethyl-3-arylisoxazoles 3a–3f and 4a–4f (ꢀg=cm3)
Comps.
S. aureus
MICa MBCb
E. coli
P. aeruginosa
MICa MBCb
C. albicans
MICa MFCc
C. tropicalis
MICa MFCc
MICa
MBCb
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
I
>625
312
>625
156
>625
312
>625
>625
>625
>625
312
–
>625
>625
>625
>625
>625
>625
>625
>625
–
–
>625
–
>625
156
>625
>625
312
>625
>625
>625
–
>625
>625
–
>625
>625
>625
–
>625
>625
>625
>625
156
>625
312
312
156
156
312
156
–
–
–
–
–
–
–
312
>625
>625
312
>625
>625
>625
>625
>625
312
–
>625
>625
–
>625
>625
–
–
–
–
–
>625
–
–
–
–
>625
>625
–
–
–
>625
–
>625
312
>625
>625
>625
>625
>625
>625
–
–
>625
–
>625
>625
–
–
>625
–
–
>625
>625
>625
>625
>625
>625
>625
>625
>625
0.06
0.06
2.0
F
4.0
4.0
C. neoformans D
Comps.
C. lusitaniae
MICa MFCc
C. neoformans
var. ‘‘grubii’’ A
C. neoformans var.
‘‘gattii’’ B
C. neoformans var.
‘‘gattii’’ C
MICa
MFCc
MICa
MICa
MFCc
MICa
MFCc
MICa
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
I
>625
156
312
–
–
156
>625
156
39
78
156
156
39
156
39
39
39
39
156
>625
156
312
–
–
156
39
156
156
156
39
156
39
39
39
>625
>625
>625
–
>625
–
>625
>625
–
>625
>625
>625
156
39
78
156
156
39
156
39
39
39
39
156
39
156
156
156
39
156
39
39
39
>625
>625
–
>625
–
>625
>625
–
>625
>625
>625
>625
–
–
–
–
>625
>625
>625
–
>625
–
–
–
–
>625
>625
>625
–
312
312
>625
>625
>625
>625
>625
39
>625
>625
>625
>625
>625
39
>625
312
39
156
>625
312
39
156
>625
>625
F
4.0
2.0
2.0
4.0
2.0
2.0
I Imipenem; F fluconazole; – no activity; nt not tested
a
MIC Minimum inhibitory concentration (ꢀg=cm3)
b
MBC Minimum bactericidal concentration (ꢀg=cm3)
c
MFC Minimum fungicidal concentration (ꢀg=cm3)

988
M. A. P. Martins et al.
Biological studies
tested exhibited a MIC of 39 ꢀg=cm3 against Cryp-
tococcus neoformans var. gattii serotype C. On the
other hand, 3b and 3f showed MICs of >625 and
39 ꢀg=cm3, against Cryptococcus neoformans var.
neoformans serotype D. Thus, the replacement of
methyl by a nitro group at R1 of the 5-trichloro-
methyl-3-arylisoxazoles seems to have improved
activity against this species of fungus. The compari-
son between MICs and MFCs (minimal fungicidal
concentrations) indicated that of 5-trihalomethyl-
3-arylisoxazoles shown only fungistatic activity
against Cryptococcus spp. as MFCs was obtained to
be, in general, higher than MICs by two or more
concentrations.
In conclusion, the synthesized 3a–3f and 4a–4f
were obtained with the use of microwave-assisted
synthesis in a significantly shorter time (16 min)
and with good yields (78–90%). Some of 5-trichloro-
methyl-3-arylisoxazoles showed good anti-Crypto-
coccus activity, thereby deserving additional studies
due to their specificity for this species of fungi.
These studies are in progress and include the de-
termination of their activity against Cryptococcus
neoformans isolates that show resistance to ampho-
tericin B and 5-flucytosine or azole agents. Finally,
the data obtained in this study suggest that the
synthesized compounds could become promising
prototypes for the future development of novel anti-
Cryptococcus agents.
The 5-trihalomethyl-3-arylisoxazoles were evaluated
in vitro for antibacterial activity against represen-
tative human pathogenic Gram-positive bacterium,
such as Staphylococcus aureus ATCC 25923 and
Gram-negative bacterium, such as Escherichia coli
ATCC 25322 and Pseudomonas aeruginosa ATCC
27853. The antifungal profile of the compounds was
evaluated against a panel of fungi including Candida
albicans ATCC 44373, Candida tropicalis ATCC
750, Candida lusitaniae ATCC 66035, Cryptococcus
neoformans var. grubii serotype A ATCC 90012,
Cryptococcus neoformans var. gattii serotype B
ATCC 56990, Cryptococcus neoformans var. gattii
serotype C ATCC 56341, and Cryptococcus neo-
formans var. neoformans serotype D ATCC 28957.
Minimal Inhibitory Concentrations (MIC) were de-
termined by means of a standard twofold dilution
method and are reported in Table 2.
In this study, both Gram-positive cocci and Gram-
negative rod susceptibilities to 5-trihalomethyl-3-
arylisoxazoles only showed poor results. For Candida
species, only 4d exhibited good activity against
Candida lusitaniae, being active at concentrations
of 39 ꢀg=cm3.
The data depicted in Table 2 reveal that in contrast
to the antibacterial behavior shown by these com-
pounds, their antifungal effect was significant and
pronounced. Particularly, Cryptococcus spp. seems
to be more sensitive toward the synthesized 5-tri-
halomethyl-3-arylisoxazoles, as most of the com-
pounds reported exhibited good activity against these
microorganisms.
Cryptococcus neoformans is a yeast-like encapsu-
lated fungus and an etiologic agent of meningoen-
cephalitis affecting from 5 to 30% of AIDS patients,
of which 10–25% die [21]. Therefore, the attainment
of novel compounds able to inhibit cryptococcosis
has been clinically relevant.
In general, anti-Cryptococcus activity was more
pronounced for the 5-trifluoromethyl-3-arylisoxa-
zoles (4a–4f). This differs from previous results
obtained in our laboratories, where the trichloro-
methylated compounds, in general, showed higher
activity than the trifluoromethylated analogues in
carbamate based antimicrobial agents [22]. Another
finding shows that the changes in the R1 group in-
duced only moderate differences in anti-Cryptococ-
cus activity. For example, most of the compounds
Experimental
Chemistry
Unless indicated otherwise, all common reagents were used as
obtained from commercial suppliers without further purifica-
tion. The solvents were dried and purified according to recom-
mended procedures [23]. All melting points were measured
using a Reichert-Thermovar apparatus. Yields listed are of
1
isolated compounds. H and 13C NMR spectra were acquired
on a Bruker DPX 200 or Bruker DPX 400 spectrometer (1H at
200.13 or 400.13 MHz and 13C at 50.32 or 100.63MHz) at
300 K, 5 mm sample tubes, and with a digital resolution of
ꢂ0.01ppm. CDCl3 was used as solvent with TMS as internal
standard. Mass spectra were registered in a HP 5973 MSD
connected to a HP 6890 GC and interfaced by a Pentium PC.
The GC was equipped with a split-splitless injector, auto-
sampler cross-linked HP-5 capillary column (30 m, 0.32 mm
of internal diameter), and helium was used as the carrier gas.
The reactions were performed in a multimode microwave
ETHOS 1 (Milestone Inc.) which possesses a twin magnetron
with a maximum delivered power of 1300 W. The temperature
and the pressure were measured throughout with an ATC-400
CE and APC-55 detector.

Microwave-assisted synthesis
989
(L.A. Piovesan), CAPES (P. Machado), and FAPERGS are
also acknowledged.
General procedure for synthesis of 5-trihalomethyl-3-aryl-
isoxazoles (3a–3f, 4a–4f)
(1) A solution of 1 or 2 (2mmol) and 167.7 mg hydroxylamine
hydrochloride (2.4mmol) in 4 cm3 methanol and 0.5cm3 pyr-
idine was stirred for a few minutes. The mixture was then
irradiated in a microwave ETHOS 1 at 100 W, 2.2 bar of pres-
sure for 6 min, enough time to complete the reaction. The
temperature was set to 70ꢀC and the irradiation was automati-
cally stopped at this temperature.
References
1. a) Brown ED, Wright GD (2005) Chem Rev 105:759;
b) Coates A, Hu Y, Bax R, Page C (2002) Nat Rev Drug
Discov 1:895
(2) After cooling to room temperature, 4 cm3 conc. sulfuric
acid was added to the crude mixture which was again irra-
diated at 100 W, 3.5 bar of pressure for 10 min. The tempera-
ture was set to 80ꢀC and the irradiation was automatically
stopped at this temperature. The solution was then placed in
25cm3 cold water and the resulting solution was extracted
with dichloromethane (3ꢃ20 cm3). The organic phase was
dried with MgSO4, the solution was filtered, and the solvent
was removed in a rotatory evaporator. The 5-trihalomethyl-
3-arylisoxazoles 3a–3f and 4a–4f were obtained in high
purity and, when necessary, the products were recrystallized
from n-hexane. All products were found to be identical
(m.p.s and spectroscopic properties) with those described ear-
lier [20].
2. Espinel-Ingroff A, White T, Pfaller MA (1999) Anti-
fungal Agents and Susceptibility Tests. In: Murray PR,
Baron EJ, Pfaller MA, Tenover FC (eds) Manual of
Clinical Microbiology, 7th edn. ASM Press, Washington,
p 1640
3. Jorgensen JH, Turnidge JD, Washington JA (1999) Anti-
bacterial Susceptibility Tests: Dilution and Disk diffusion
methods. In: Murray PR, Baron EJ, Pfaller MA, Tenover
FC, Yolken RH (eds) Manual of Clinical Microbiology,
7th edn. ASM Press, Washington, p 1526
4. Talley JJ, Brown DL, Carter JS, Graneto MJ, Koboldt
CM, Masferrer JL, Perkins WE, Rogers RS, Shaffer AF,
Zhang YY, Zweifel BS, Seibert KJ (2000) J Med Chem
43:775
5. Lee Y, Kim BH (2002) Bioorg Med Chem Lett 12:1395
6. Raffa D, Daidone G, Maggio B, Cascioferro S, Plescia F,
Schillaci D (2004) Farmaco 59:451
Biological assays
Minimal inhibitory concentrations (MIC) were determined by
means of a standard twofold dilution method using broth me-
dium; the antibacterial activity was based on the NCCLS M7-
A5 document [24] and the evaluation of the antifungal activity
was based on Shadomy and Pfaller [25] and the NCCLS M27-
A2 document [26]. Test compounds were dissolved in DMSO
at an initial concentration of 5000ꢀg=cm3 and were then
7. Frølund B, Greenwood JR, Holm MM, Egebjerg J,
¨
Madsen U, Nielsen B, Brauner-Osborne H, Stensbølc
TB, Krogsgaard-Larsen P (2005) Bioorg Med Chem
13:5391
8. Norman BH, Lander PA, Gruber JM, Kroin JS, Cohen JD,
Jungheim LN, Starling JJ, Law KL, Self TD, Tabas LB,
Williams DC, Paul DC, Dantizig AH (2005) Bioorg Med
Chem Lett 15:5526
9. Zadrozna I, Kurkowska J, Kruszewska H, Mukuch I
(2000) Farmaco 55:499
¨
serially diluted in culture medium (Mueller-Hinton broth for
bacterial and Sabouraud broth for fungal assays).
Cultures of microorganisms were adjusted to 105 CFUs=
cm3 according to the McFarland scale. Antimicrobial assays
were carried out in triplicate and incubated at 35ꢀC during
24h for bacteria and Candida spp. and during 72h for Cryp-
tococcus spp. MIC was defined as the compound concentra-
tion at which no macroscopic sign of microbial growth was
detected. The interpretation of the results was based on flu-
conazole breakpoints for the fungi and based on imipenem
for bacterial pathogens, according to M27-A2 [25] and M7-
A5 [23] techniques. The minimal germicidal concentrations
(MBC or MFC) were determined by subcultivating samples
10. Raffa D, Daidone G, Maggio B, Scillaci D, Plescia F,
Torta L (1999) Farmaco 54:90
´
11. Calı P, Naerum L, Mukhija S, Hjelmencrantz A (2004)
Bioorg Med Chem Lett 14:5997
12. Aiello E, Aiello S, Mingoia F, Bacchi A, Pelizzi G, Musiu
C, Setzu M G, Pani A, La Colla P, Marongiu E (2000)
Bioorg Med Chem 8:2719
13. Davenport JD, Dreikorn BA, Elsasser AF (1976) German
Patent 2723688; (1978) Chem Abstr 88:132015k
14. Barbachyn MR, Cleek GJ, Dolak LA, Garmon SA,
Morris J, Seest EP, Thomas RC, Toops DS, Watt W,
Wishka DG, Ford CW, Zurenko GE, Hamel JC, Schaadt
RD, Stapert D, Yagi BH, Adams WJ, Friis JM, Slatter JG,
Sams JP, Oien NL, Zaya MJ, Wienkers LC, Wynalda MA
(2003) J Med Chem 46:284
¨
from cultures with apparent growth in Mueller-Hinton agar for
bacteria or Sabouraud dextrose agar for fungi.
Acknowledgements
The authors thank Dr. Erico M.D. Flores (Chemistry Depart-
ment, Federal University of Santa Maria, Brazil) for the use
of his laboratory and the multimode microwave ETHOS 1
(Milestone Inc.) used in the synthesis of the compounds. The
authors also thank the Conselho Nacional de Desenvolvimento
15. Martins MAP, Cunico W, Pereira CMP, Flores AFC,
Bonacorso HG, Zanatta N (2004) Curr Org Synthesis
1:391; and references therein
16. a) Martins MAP, Pereira CMP, Beck P, Machado P,
Brondani S, Moura S, Teixeira MVM, Bonacorso HG,
Zanatta N (2003) Tetrahedron Lett 44:6669; b) Martins
MAP, Beck P, Machado P, Brondani S, Moura S, Zanatta
N, Bonacorso HG, Flores AFC (2006) J Braz Chem
´
Cientıfico e Tecnologico (CNPq=PADCT) and Fundac
de Amparo a Pesquisa do Estado do Rio Grande do Sul
´
°
a˜o
`
(FAPERGS) for financial support. The fellowships from CNPq

990
M. A. P. Martins et al.
Soc 17:408; c) Sauzem PD, Machado P, Rubin MA,
Sant’Anna GS, Faber HB, Souza AH, Mello CF, Beck
P, Burrow RA, Bonacorso HG, Zanatta N, Martins MAP
(2007) Eur J Med Chem (in press)
22. Zanatta N, Borchhardt DM, Alves SH, Coellho HS,
Squizani AMC, Marchi TM, Bonacorso HG, Martins
MAP (2006) Bioorg Med Chem 14:3174
23. Perrin DD, Armarego WLF (1998) Purification of
Laboratory Chemicals. Pergamon, Oxford
17. Martins MAP, Pereira CMP, Cunico W, Moura S, Rosa
FA, Peres RL, Machado P, Zanatta N, Bonacorso HG
(2006) Ultrasonics Sonochem 13:364
18. Larhed M, Moberg C, Hallberg A (2002) Acc Chem Res
35:717; and references therein
19. a) Colla A, Martins MAP, Clar G, Krimmer S, Fischer P
(1991) Synthesis 6:483; b) Martins MAP, Bastos GP,
Bonacorso HG, Zanatta N, Flores AFC, Siqueira GM
(1999) Tetrahedron Lett 40:4309; c) Bonacorso HG,
Martins MAP, Bittencourt SRT, Lourega RV, Zanatta
N, Flores AFC (1999) J Fluorine Chem 99:177
20. Martins MAP, Siqueira GM, Bastos GP, Bonacorso HG,
Zanatta N (1996) J Heterocycl Chem 33:1619
21. Viviani MA, Tortorano AM, Ajello L (2003) Crypto-
coccus. In: Anaissie EJ, McGinnis MR, Pfaller MA (eds)
Clinical Mycology. Churchil Livingstone, New York,
p 240
24. National Committee for Clinical Laboratory Standards
(2002) Methods for Dilution Antimicrobial Susceptibility
Tests for Bacteria that Grow Aerobically, Approved Stan-
dard, 5th edn. NCCLS document M7-A5, Wayne, P. A.
National Committee for Clinical Laboratory Standards
25. Shadomy S, Pfaller M (1991) Laboratory studies with
antifungal agents: susceptibility tests and quantitation in
sody fluids. In: Ballours A, Hauster WJ Jr, Herrmanx KL,
Isemberg HD, Shadomy HJ (eds) Manual of Clinical
Microbiology, 5th edn. Americam Society of Micro-
biology, Washington, p 1173
26. National Committee for Clinical Laboratory Standards
(2002) Reference method for broth dilution antifungal
susceptibility testing of yeast: approved standard. NCCLS
document M27-A2. Wayne, P. A. National Committee for
Clinical Laboratory Standards