Thai J Vet Med Suppl.

2017, 47 : 131-132

In vitro antifungal susceptibilities of Cryptococcus neoformans from pigeon

droppings, cats, and human sources in Bangkok, Thailand
S. Panapruksachat1, P. Krangvichain1, S. Surachetpong2, S. Assarasakorn2, N. Prapasarakul1,*

1
Department of Veterinary Microbiology, 2Department of Veterinary Medicine, Faculty of Veterinary Science,
Chulalongkorn University, Bangkok 10330, Thailand
*Corresponding author: Nuvee.P@chula.ac.th

Keywords: Cryptococcus neoformans, pigeon droppings, cat, drug susceptibility, Vitek 2

Introduction saline (0.45% w/v NaCl) and adjusted to a density of

Cryptococcus neoformans has emerged as a clinically
important opportunistic pathogen worldwide due to the
AIDS pandemic. This pathogenic yeast causes
pulmonary and systemic cryptococcosis in
immucompromised human and susceptible animals
such as cats and dogs (8, 10). C. neoformans is
globally distributed and found to associate with soil,
pigeon excreta, and decaying wood. Cryptococcal
infections are proposed to be caused by inhalation of
yeast spores in environment and these infectious
propagules are deposited into the pulmonary alveoli of
the hosts leading to meningitis or disseminated disease
in immunodeficiency hosts (4, 7). Previously, there are
several reports on isolation of C. neoformans from
pigeon droppings in several provinces of Thailand such
as Bangkok, Phayao, Chonburi, and Chiang Mai
(5,6,11,12). Therefore it is possible that
immunocompromised and immunocompetent patients
could be infected with C. neoformans from the
environmental sources. However, little is known about
the antifungal susceptibility of C. neoformans isolated
from environment even though the increased drug
resistance has become a clinical concern. In this study,
we aimed to determine the drug susceptibility of C.
neoformans isolated from pigeon fecal droppings and
cats in Bangkok, Thailand to amphotericin B,
caspofungin, fluconazole, flucytosine, micafungin, and
voriconazole using an automated susceptibility system.
Materials and Methods
A total of 27 C. neoformans isolates from pigeon
droppings and cats were collected during 2011-2012
and 2016 from Bangkok, Thailand and kept as glycerol
stock at -80C (5). Two C. neoformans clinical
isolates from patients were obtained from Mycology
laboratory, King Chulalongkorn Memorial Hospital,
Faculty of Medicine, Chulalongkorn University,
Thailand and Kajiwara laboratory, Graduate School of
Bioscience and Biotechnology, Tokyo Institute of
Technology, Japan and used as reference strains in this
study. All yeast strains were cultivated on Sabouraud
dextrose agar and incubated at 37C for 2-3 days
before subjected to determine the antifungal
susceptibility testing by the automated machine (Vitek
2®, bioMérieux, Marcy l'Etoile, France). The
cryptococcal colonies were suspended in sterile normal
1.8-2.2 McFarland standards by using the
DensiChekTM plus (bioMérieux). Then 280 ml of the
standardized McFarland inoculum was transfer to a
new sterile polystyrene tube containing 3 mL of 0.45%
w/v NaCl. This tube was later placed into the cassette
array along with an AST-YS07 susceptibility test card
and loaded into the detection instrument. The
antifungal array panel was automatically filled with the
inoculum suspension and incubated at 35.5C for 16-
32 hours. The optical density was read every 15
minutes during incubation period and the results were
reported as minimum inhibitory concentrations (MICs)
in g/ml.
Results and Discussion
In this study, 27 strains of C. neoformans isolated from
pigeon droppings and cats were determined their MICs
against amphotericin B, caspofungin, fluconazole,
flucytosine, micafungin, and voriconazole using the
commercial available drug susceptibility system. C.
neoformans isolates from pigeon excreta, cats, and
humans had the same MIC values for caspofungin,
flucytosine, micafungin, and voriconazole (modes 4, 4,
1, and ≤0.12 g/ml, respectively) while amphotericin
B, drug of choice for cryptococcosis, showed a bit
lower MIC values at 0.5g/ml in pigeon source. For
fluconazole, C. neoformans isolated from human
exhibited a slightly increased in MIC value (2 g/ml)
(Table 1). To date, even though C. neoformans is
recognized as life-threatening mycoses in
immunocompromised hosts, there are still no clinical
breakpoints for antifungal substances against C.
neoformans (1,2). However, epidemiological cutoff
values (ECVs) of amphotericin B (1g/ml),
flucytosine (16 g/ml), fluconazole (16 g/ml), and
voriconazole (0.25 g/ml) for this important
pathogenic yeast have been proposed in several studies
(2, 9, 3). We found that the MICs of our C. neoformans
isolates are still similar or lower than those ECVs. This
might indicate that the resistant strains have not yet
emerged in our areas.

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Thai J Vet Med Suppl. 2017, 47 : 131-132

Acknowledgements 4. Kaocharoen et al. 2013. PLoS Negl Trop

This research is supported by Rachadapisek Sompote Dis. 7: e2297.
Fund for Postdoctoral Fellowship, CE; Center of 5. Krangvichain et al. 2016. J Vet Med Sci.
Emerging and Re-emerging Infectious Diseases in 78: 2391-396.
Animals and STAR; Detection and Monitoring Animal 6. Kuroki et al. 2004. Yeast. 21: 809-812.
Pathogen, Chulalongkorn University. The authors 7. Meyer et al. 2003. Emerg Infect Dis. 9:
189-195.
would like to thank Assoc. Prof. Ariya Chindamporn
8. O’Brien et al. 2004. Med Mycol. 42:
and Prof. Susumu Kajiwara for kindly providing C.
449-460.
neoformans isolated from humans. 9. Pfaller et al. 2013. J Clin Microbiol. 51:
2571-258
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Table 1 MIC values among Cryptococcus neoformans isolates from pigeon droppings, cats, and humans.

Source of
Antifungal
isolation MIC50b MIC90c Mode
druga ≤0.12 0.25 0.5 1 2 4 8
(No. tested)
Pigeon droppings
AMB 4 14 1 0.5 0.5 0.5
(19)
CFG 19 4 4 4
FLZ 16 3 1 2 1
FC 17 2 1 2 1
MFG 19 4 4 4
VCZ 19 ≤0.12 ≤0.12 ≤0.12
Cats (8) AMB 2 6 1 1 1
CFG 1 7 4 4 4
FLZ 4 1 1 2 1 8 1
FC 8 1 1 1
MFG 1 7 8 8 4
VCZ 7 1 ≤0.12 ≤0.12 ≤0.12
Humans (2) AMB 2 1 1 1
CFG 2 4 4 4
FLZ 2 2 2 2
FC 2 1 1 1
MFG 2 4 4 2
VCZ 2 ≤0.12 ≤0.12 ≤0.12

a
AMB, amphotericin B; CFG, caspofungin; FLZ, fluconazole; FC, flucytosine; MFG, micafungin; VCZ,
voriconazole.
b
MIC50: MIC at which 50% of the isolates tested are inhibited.
c
MIC90: MIC at which 90% of the isolates tested are inhibited.

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