Mycopathologia (2012) 174:4152

DOI 10.1007/s11046-011-9500-0

Antifungal Susceptibility, Enzymatic Activity,

PCR-Fingerprinting and ITS Sequencing of Environmental
Cryptococcus laurentii Isolates from Uberaba, Minas Gerais,
Brazil
Kennio Ferreira-Paim Leonardo Andrade-Silva Delio Jose Mora

Eliane Lages-Silva Andre Luiz Pedrosa Paulo Roberto da Silva

Anderson Assuncao Andrade Mario Leon Silva-Vergara

Received: 6 June 2011 / Accepted: 21 October 2011 / Published online: 25 November 2011
Springer Science+Business Media B.V. 2011

Abstract Cryptococcus laurentii has been classically
considered a saprophytic species, although several cases
of human infection have been already reported. This
study aimed to evaluate the phospholipase, proteinase
and hemolysins activity, the antifungal susceptibility
profile, the genetic variability by M13 and (GACA)4
fingerprinting and the internal transcribe spacer (ITS)
sequencing of 38 C. laurentii isolates recovered from
captive bird droppings and surrounding hospital areas.
All of them exhibited phospholipase activity, while the
hemolytic activity was evidenced in 34 (89.4%) isolates.
None of them exhibited proteinase activity. Twenty-
seven isolates (71.1%) presented susceptibility dose
dependent to fluconazole. Most isolates (94.7%) were
susceptible to voriconazole, while one (2.65%) was
resistant to this drug. Twenty-one (55.3%) isolates
K. Ferreira-Paim (&)  L. Andrade-Silva 
D. J. Mora  E. Lages-Silva  M. L. Silva-Vergara (&)
Department of Infectious and Parasitic Diseases,
Triangulo Mineiro Federal University, Postal Code 118,
Uberaba, MG 38001-170, Brazil
e-mail: kennio_p@hotmail.com
M. L. Silva-Vergara
e-mail: marioleon.dip@mednet.com.br
showed reduced susceptibility to itraconazole while
nine (23.7%) were resistant. Three (7.9%) and five
(13.1%) isolates exhibited resistance to ketoconazole
and amphotericin B, respectively. Most C. laurentii
fingerprinting obtained with M13 and (GACA)4 showed
high heterogeneity. By using the two primers, seven
(18.4%) isolates grouped as A (CL2, CL7, and CL8), B
(CL35, CL38) and C (CL29, CL30) with 100%
similarity. Different from most variable surrounding
hospital isolates, all but one of the pet shops strains
clustered with the two primers, although they had been
recovered from different neighborhoods. All isolates
were identified as C. laurentii phylogenetic group I by
ITS sequencing. Thus, the presence of virulence factors,
a decreased antifungal susceptibility and a heteroge-
neous molecular pattern of the C. laurentii isolates here
described suggests this species can be a potential
pathogen in the context of the immunocompromised
population.
Keywords Cryptococcus laurentii  Phospholipase
activity  Antifungal susceptibility  M13
fingerprinting  (GACA)4 fingerprinting  Internal
transcribed spacer

A. L. Pedrosa
Department of Molecular Biology, Triangulo Mineiro
Federal University, Uberaba, MG, Brazil Introduction
P. R. da Silva  A. A. Andrade
Department of Microbiology, Triangulo Mineiro Federal Cryptococcus genus includes several species world-
University, Uberaba, MG, Brazil wide distributed and found in different environments

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42
[1, 2]. At present, Cryptococcus neoformans and
Cryptococcus gattii are considered pathogenic [3, 4],
although some non-neoformans species as Crypto-
coccus laurentii and Cryptococcus albidus have
occasionally been described infecting immunocom-
promised hosts [510].
Cryptococcus laurentii has been recovered from
pigeons and captive bird droppings and from trees
hollows with decaying wood [1113]. In the last years,
nearly 20 cases of cryptococcosis by this specie in
patients with underlying diseases were described. The
clinical picture ranged from asymptomatic pulmonary
infection to cutaneous lesions or systemic involve-
ment with fever, hypothermia, hypotension and men-
ingitis. The blood and the cerebrospinal fluid (CSF)
were the most common sites where the fungus was
recovered [1421].
Virulence factors such as the capsular polysaccha-
ride, ability to growth at 37C and the melanin,
phospholipase, proteases, DNase, collagenase, and
urease production were previously characterized to
C. neoformans and C. gattii [2224]. In contrast, few
studies evaluating these factors in non-neoformans
species have been carried out [25, 26]. Moreover, the
hemolytic activity formerly described for bacteria and
Candida sp. [27, 28] was still not reported to
C. laurentii.
The emergence of antifungal resistance of clinical
and environmental isolates of C. neoformans and
C. gattii has been evidenced during the last decades
around the world. This led to the growing necessity to
perform susceptibility tests, aiming to improve the
therapeutic decision in critically ill patients [29, 30].
Currently, both species present in vitro susceptibility
to most common antifungals used in clinical practice,
although different levels of resistance to fluconazole,
itraconazole, and amphotericin have been reported
[31, 32]. Similarly, clinical and environmental
C. laurentii isolates have shown decreased profiles
of sensitivity to several azoles derivates [31, 33]. Due
to the increasing population of critically ill and/or
immunocompromised patients, the antifungals pre-
scription is each time more common, and together
with the indiscriminate use of these drugs in agricul-
ture and veterinary practice, it can partially contribute
to the emergence of resistance and pathogenicity of
saprophytic fungal species [32].
The molecular diversity of environmental and
clinical isolates of C. neoformans and C. gattii around
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Mycopathologia (2012) 174:4152
the world has been evaluated through PCR-finger-
printing using random and repetitive microsatellites
amplification sequences which has also improved the
knowledge about the geographical distribution of
these species [3436]. In contrast, few molecular
studies evaluating environmental C. laurentii strains
using these techniques were carried out so far [37].
The sequencing of different regions of the ribo-
somal DNA (rDNA) genes has been proposed as the
standard technique to characterize C. laurentii isolates
due to the great variability found in these regions [38].
Through the sequencing of D1/D2 regions of the large
subunit of rDNA (LSSU-rDNA) and the internal
transcribe spacer (ITS), C. laurentii species have been
divided into two phylogenetic groups (I and II). The
species C. laurentii, C. flavescens, and C. aureus
belong to the phylogenetic group I, while C. carnes-
cens, C. peneaus, C. victoriae e Cryptococcus sp.
belong to group II [38, 39].
Considering the pathogenic potential of C. laurentii
environmental isolates to humans and the scarcity of
studies about this microorganism, this study aimed to
characterize the activity of virulence factors, the
antifungal susceptibility profile, and the molecular
patterns of C. laurentii environmental isolates from
Brazil, where no clinical isolates of this species were
ever reported to date.
Materials and Methods
Strains Identification
Thirty-eight environmental C. laurentii strains previ-
ously identified by classical mycological methods
such as: India ink test, urease and phenoloxidase
activity, thermotolerance at 37C on Sabouraud dex-
trose agar, nitrate and carbon assimilation assays,
carbohydrate fermentation and microculturing on
cornmeal with Tween 80 were included. Then, they
were preserved on yeast peptone dextrose broth (Difco
Laboratories, USA) with 30% glycerol at -20C. Of
these, seven were recovered from captive bird drop-
pings in pet shops in different neighborhoods, while
the remaining 31 were obtained from bird droppings
and trees hollows debris from surrounding areas at the
teaching hospital in Uberaba, Minas Gerais, Brazil.
Considering the possibility of cross reactivity
between the capsular antigens of C. laurentii with
Mycopathologia (2012) 174:4152
the C. neoformans ones, we extracted the polysaccha-
ride antigen of C. laurentii isolates as follows: the
strains were cultured on Sabouraud dextrose agar
plates for 72 h at 32C. The yeast cells were harvested
in 20% veronal buffered solution (NaCl 0.71 mol L-1,
15 mmol L-1 NaHCO3, 7.27 mmol L-1 Sodium
5,5-diethylbarbiturate, 27.32 mmol L-1 5,5-Diethyl-
barbituric acid) plus 2 mL 0.15 mmol L-1 CaCl2 and
2 mL MgCl2 0.5 mmol L-1, mixed and centrifuged
for 2 min at 2,000 rpm. The pellet was washed with
acetone and sulfuric ether (510 volumes for three
times each). The cell volume was quantified and stored
at 4C until dry. Then, a 15% veronal buffered
solution (v/v) was prepared and added on the dried
cells, autoclaved at 120C for 20 min, and then
centrifuged for 30 min at 2,000 rpm. The supernatant
containing the antigen was stored at -4C.
The agglutination reaction was performed using
Cryptococcus Antigen Test Kit (Remel Inc. Lenexa,
KS, United States) according to the manufacturer
instructions. The C. neoformans ATCC 90112 and
Candida albicans ATCC 90028 strains were used as
positive and negative control, respectively.
This study was approved by the Ethical Board of
Triangulo Mineiro Federal University.
Phospholipase and Proteinase Production
All isolates were cultured on Sabouraud dextrose agar
plates for 48 h at 30C. Then, a single colony was
obtained and streaked again on this medium under
identical conditions. An inoculum of 5 9 104 cells
was added on Phospholipase agar (consisting of
Sabouraud dextrose agar, 1 mol/L NaCl2, 5 mmol/L
CaCl2 and 8% of egg yolk [Oxoid, Basingstoke,
Hampshire, England]), incubated at 30C for 7 days
and after this, the readings were taken [40].
Proteinase medium was prepared as follows: Nine
hundred milliliters of a base solution containing 11.7 g
of yeast carbon base (YCB, HiMedia Laboratories
Put., Mumbai, Maharashtra, India) and 18.0 g of agar
was sterilized at 120C for 15 min. Then, a solution of
100 mL [2 g of BSA (Fraction V, Sigma Chem Co.,
St. Louis, Mo., USA) and 1 mL of Protovit (Roche
SA, Sao Paulo, Sao Paulo, Brazil)] was sterilized by
filtration, mixed with the base solution, and distributed
on Petri dishes. An inoculum of 5 9 104 cells was
added on this medium, incubated at 30C for 7 days
and after this, the readings were taken [41].
43
The phospholipase and proteinase activities were
measured dividing the colony diameter by the colony-
plus precipitation zone (Pz) [40]. The enzyme activity
ranges of Pz were determined as follows: Pz = 1.0,
negative activity, Pz = 0.70.99, low enzymatic
activity, Pz = 0.50.69, moderate enzymatic activity,
and Pz \ 0.5, high enzymatic activity [24]. The C.
albicans ATCC 90028 and the C. neoformans ATCC
90112 strains were used as positive controls, whereas
C. krusei ATCC 6258 strain was the negative control
for phospholipase. The C. albicans ATCC 90028 and
C. krusei ATCC 6258 were the positive and negative
controls for proteinase activity, respectively. All the
experiments were performed in duplicate and in two
different days. The mean values obtained were
considered.
Hemolytic Activity
The strains were cultured on Sabouraud dextrose agar
for 48 h at 30C. Then, a single colony was retired and
newly cultured on this medium under the same
conditions. An inoculum of 5 9 104 cells was added
on Sabouraud dextrose agar supplemented fresh sheep
blood (7%), incubated at 30C for 7 days and after this,
the readings were taken. Hemolytic index (Hi) was
obtained dividing the colony diameter by the colony-
plus translucent halo around the colony, and the results
were interpreted as described previously for phospho-
lipase [27]. The same control strains used for phos-
pholipase activity were included. The experiments
were performed in duplicate and in two different days.
The mean values obtained were considered.
Antifungal Susceptibility
The broth microdilution test was performed according
to the Clinical and Laboratory Standards Institute,
CLSI-M27A3, changing the incubation temperature
from 37 to 33C in order to standardize the strains
growth [42]. Amphotericin B (Bristol-Myers Squibb
Co., Princeton, NJ, USA), voriconazole (Pfizer, Sao
Paulo, SP, Brazil), itraconazole (Janssen S.A., Madrid,
Spain), and ketoconazole (Pfizer, Guarulhos, SP,
Brazil) were dissolved in dimethylsulfoxide (Sigma-
Aldrich, Madrid, Spain), while fluconazole (Pfizer,
Guarulhos, SP, Brazil) was dissolved in sterile distilled
water. The RPMI 1640 medium (with glutamine and
without bicarbonate) buffered to pH 7.0 with
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0.165 mol L-1 MOPS (Sigma-Aldrich, Madrid,
Spain) was used to prepare the final dilutions. The
concentrations intervals ranged from 0.03 to 16 lg/mL
for amphotericin B, voriconazole, itraconazole, keto-
conazole and from 0.125 to 64 lg/mL for fluconazole.
The minimum inhibitory concentration (MIC) end
point for amphotericin B was defined as the lowest
drug concentration in which a score of 0 (optically
clear) was observed compared with the control,
whereas fluconazole, itraconazole, voriconazole, and
ketoconazole had the lowest drug concentration in
which a score of 2 (prominent decrease in turbidity)
was observed. The C. parapsilosis ATCC 22019,
C. krusei ATCC 6258, and C. albicans ATCC 24433
strains were used as controls.
The MIC results in this study were defined in
accordance with CLSI M27-A3 [42] and those used by
several authors to C. neoformans as follows: for
amphotericin B and ketoconazole, MIC C2 lg/mL
was considered resistant [31], for itraconazole,
MIC C1 lg/mL was considered resistant, between
0.25 and 0.5 lg/mL susceptible dose dependent and
B0.125 lg/mL susceptible [43]. For voriconazole,
MIC C4 lg/mL was considered resistant, 2.0 lg/mL
susceptible dose dependent, and B1.0 lg/mL suscep-
tible [42]. For fluconazole, a MIC result C64.0 lg/mL
was defined as resistant, between 16 and 32 lg/mL
susceptible dose dependent and B8.0 lg/mL suscep-
tible [44, 45]. The MIC 50 and MIC 90 values were
obtained by ordering the MIC data for each antifungal
in ascending arrays and selecting the median and 90th
quantile, respectively, of the MIC distribution.
M13 and (GACA)4 Fingerprinting
DNA extraction was performed in accordance with the
formerly described method [46]. The PCR reaction was
based on random microsatellite amplification
sequences of phage M13 (50 GAGGGTGGCGGTTCT
30 ) and repetitive microsatellite sequence (GACA)4 as
the only PCR primer. Both amplification reactions
were independently performed in a volume of 50 lL,
containing 100 ng of genomic DNA, 19 PCR buffer
(10 mmol L-1 TrisHCl pH 8.3, 50 mmol L-1 KCl
and 1.5 mmol L-1 MgCl2), 2.5 U Taq DNA polymer-
ase (Invitrogen, Sao Paulo, SP, Brazil), and 60 pmol of
primer. PTC-100 Thermocycler (MJ Research Inc.,
Watertown, MA, USA) was programmed for 10 min at
94C, followed by 36 cycles of 1 min at 94C, 1 min at
123
Mycopathologia (2012) 174:4152
50C, 1 min at 72C, with a final extension of 10 min at
72C. Amplicons were electrophoresed on 1.5%
agarose gel in TAE 19 buffer at 70 V during 4 h.
The gel was stained with 0.5 mg mL-1 ethidium
bromide and analyzed through an UV transilluminator
[35]. Two strains of Cryptococcus flavus (CF001
GenBank ID: JN627021 and CF002 GenBank ID:
JN627022) which present similar pattern of assimila-
tion (positive lactose and melibiose) and had been
recovered from the same place (surrounding hospital)
of the C. laurentii were included as control.
The PCR-fingerprinting profiles were analyzed
according to the presence or absence of defined bands
in the digitized gel images. The GelComparII soft-
ware, version 5.0 (Applied Maths, Kortrijk, Belgium),
was used to establish the genetic relationships among
the strains. The similarity coefficients were calculated
by the Dice algorithm, and the generated matrixes
were analyzed by UPGMA (Unweighted Pair-Group
Method, Arithmetic averages) grouping coefficient, to
create the phenograms.
Intergenic Spacer (ITS) Sequencing
ITS PCR was performed in final volume of 50 lL. Each
reaction contained 50 ng of genomic DNA, 19 PCR
buffer (10 mmol L-1, TrisHCl pH 8.3, 50 mmol L-1
KCl and 1.5 mmol L-1 MgCl2), 0.25 mmol L-1 each
of dATP, dCTP, dGTP, and dTTP, 1.25 U of Taq DNA
polymerase (Invitrogen, Sao Paulo, SP, Brazil), and
70 pmol of each primer: ITS1 (50 -GTCGTAA
CAAGGTTAACCTGCGG-30 ) and ITS4 (50 -TCCTCC
GCTTATTGATATGC-30 ). Thirty-six PCR cycles were
performed in a PTC-100 Thermocycler (MJ Research
Inc., Watertown, MA, USA) at 94C for 2 min initial
denaturation, followed by 1 min of denaturation at
94C, 1 min of annealing at 52C and 1 min of
extension at 72C, and a final extension cycle of
15 min at 72C. The amplicons were visualized under
UV light after 2 h of electrophoresis at 80 V and
staining with 0.5 mg mL-1 of ethidium bromide [47].
Amplicons were purified adding 4.0 lL of
3.0 mol L-1 sodium acetate, 4.0 lL of cold 100%
ethanol and incubated at -20C for 30 min. Then, the
samples were centrifuged at 8,1509g for 10 min.
Next, 80 lL cold 70% ethanol was added and
centrifuged at 8,1509g for 10 min. The samples were
air-dried, resuspended in 20 lL of Milli-Q water, and
stored at -20C for sequencing reactions.
Mycopathologia (2012) 174:4152 45

Fig. 1 Agarose gel

electrophoresis and
phenogram of polymerase
chain reaction fingerprinting
profiles obtained from 38
environmental
Cryptococcus laurentii
isolates with the single
primer M13 a and GACA4
b, created with the software
GELCOMPAR II (applied
maths), with dice coefficient
and unweighted pair-group
method, arithmetic averages

123
46
PCR products were independently sequenced with
the forward (ITS1) and reverse (ITS4) primer using
the BigDye terminator reagent kit (Applied Biosys-
tems, Foster City, CA, USA) on an automated DNA
sequencer (ABI PRISM 3130 XL Genetic Analyzer,
Applied Biosystems, Foster City, CA, USA), accord-
ing to the protocol recommended by the manufacturer.
The nucleotide sequences here discussed have been
deposited in the GenBank and their accession numbers
are given in Fig. 2 (accession number JN626983 to
JN627020).
Data Analysis
The sequences were edited using the software
Sequence Scanner V. 1.0 (Applied Biosystems). The
sequences included in the study were the consensus
sequences originated with the forward and reverse
primers. The sequences were aligned with the software
Clustal W [48]. The evolutionary distance for the
neighbor-joining method [49] was calculated in
accordance with Kimura [50]. All sites with gaps in
any sequences were excluded. A bootstrap analysis
was performed with 1,000 random resamplings. The
phylogenetic comparison was performed with the
software MEGA 5.0 [51]. The nucleotide sequences of
other strains or species were obtained from GenBank
and were identified by its accession number. BLAST
and phylogenetic analyses enabled the distinction of
C. laurentii sequences from other species.
Results
The C. laurentii environmental strains exhibited
capsule and grew at 37C, although the optimal
Table 1 In vitro activity of fluconazole, itraconazole, voriconazole, ketoconazole, and amphotericin B against 38 environmental
isolates of Cryptococcus laurentii
Antifungal Antifungal susceptibility and minimal inhibitory concentration MIC (lg/mL)
Susceptible SDD
Amphotericin B 33 (86.9%)
Ketoconazole 35 (92.1%)
Itraconazole 8 (21.0%) 21 (55.3%)
Voriconazole 36 (94.7%) 1 (2.65%)
Fluconazole 11 (28.9%) 27 (71.1%)
GM geometric mean, SDD susceptible dose dependent
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Mycopathologia (2012) 174:4152
Fig. 2 Evolutionary relationship of C. laurentii environmental c
isolates from Uberaba, Minas Gerais, Brazil using the ITS
sequencing. The optimal tree with the sum of branch
length = 0.23874285 is shown. The percentage of replicate
trees in which the associated taxa clustered together in the
bootstrap test (1,000 replicates) are shown next to the branches.
The analysis involved 51 nucleotide sequences. There were a
total of 192 positions in the final dataset. Evolutionary analyses
were conducted in the software MEGA5
growth was obtained at 35C. The colonies showed a
beige color on nigerseed (Guizotia abyssinica) agar
plates which suggests a low melanin production. All
isolates presented positive assimilation tests for dex-
trose, galactose, maltose, sucrose, raffinose, rham-
nose, dulcitol, inositol, mannitol, xylose, peptone,
lactose, celobiose, and melibiose. In addition, they
were negative for inulin and potassium nitrate assim-
ilation or gas production by the dextrose fermentation
test. The extracted antigen of the C. laurentii isolates
did not present reactivity to C. neoformans antibodies.
The phospholipase production was evidenced in all
C. laurentii strains (mean Pz of 0.783 0.09), of
which 30 (78.9%) presented low activity, and the
remaining exhibited a moderate one. The hemolytic
activity was evidenced in 34 (89.4%) isolates (mean
Hi of 0.716 0.14). Of these, 14 (36.8%) were low
hemolytic producers and 20 (52.6%) were moderate.
No evidence of proteinase activity was detected
through the used method in all isolates.
Among isolates, 27 (71.1%) and one (2.65%)
presented susceptibility dose dependent to fluconazole
and voriconazole, respectively. None of the isolates
were resistant to fluconazole while one (2.65%)
presented resistance to voriconazole. For itraconazole,
21 (55.3%) were susceptible dose dependent, and nine
(23.7%) were resistant. Moreover, most isolates were
susceptible to ketoconazole and amphotericin B, while
Resistant Range GM MIC50 MIC90
5 (13.1%) 0.124.0 0.50 0.5 2.0
3 (7.9%) 0.064.0 0.41 0.5 1.0
9 (23.7%) 0.122.0 0.34 0.25 1.0
1 (2.65%) 0.124.0 0.44 0.50 1.0
0 832 14.87 16.0 32.0
Mycopathologia (2012) 174:4152 47

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48
three (7.9%) and five (13.1%), respectively, exhibited
resistance. The strain CL 28 which was susceptible
dose dependent to fluconazole (MIC: 16 lg/mL) was
also resistant to other azoles, e.g., itraconazol (MIC:
2 lg/mL), voriconazol (1 lg/mL), ketoconazole
(1 lg/mL) and to amphotericin B (2 lg/mL). The
highest geometric mean MIC was 14.87 lg/mL for
fluconazole, followed by geometric mean MICs of
0.50 lg/mL for amphotericin B and lower geometric
mean MICs for itraconazole, ketoconazole, and the
new triazole voriconazole (0.34, 0.41, 0.44 lg/mL,
respectively) (Table 1).
Cryptococcus laurentii fingerprinting obtained with
M13 and (GACA)4 showed high heterogeneity. Seven
(18.4%) isolates clustered as (CL2, CL7, and CL8),
(CL35, CL38), and (CL29, CL30) showed 100%
similarity between them by the two primers used. Only
the strain CL32 did not amplify using the (GACA)4.
By M13 fingerprinting, the lowest similarity (27.0%)
was observed for the isolates CL19, CL20, CL22, and
CL28 when compared with others. By (GACA)4
fingerprinting, the lowest similarity (44.9%) was
evidenced for the isolates CL15, CL27, CL25, and
CL28. Different from the high variability among the
surrounding hospital isolates, all but one of the pet
shop strains clustered with the two primers, although
they had been recovered from different neighbor-
hoods. The fingerprinting profiles were not associated
with the phospholipase and hemolysins activities and
the antifungal susceptibility patterns (Fig. 1a, b).
All strains were identified as C. laurentii through
the generated sequences using the BLAST. Most of
them had maximum identity of 99% and a query
coverage of 100% with environmental Brazilian
C. laurentii isolates from Rio de Janeiro and with
the C. laurentii ATCC MYA-2946 strain. In addition,
the environmental isolates clustered near the clinical
isolate CBS 2174 recovered from a patient with a
tumor. All isolates presented similar sequences
regardless the place of recovery and were included
in the phylogenetic group I (Fig. 2).
Discussion
Classically C. laurentii has been recognized as
saprophytic species, although several cases of human
infection caused by this species have been reported in
the last years. Most C. laurentii infections occurred in
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Mycopathologia (2012) 174:4152
non-HIV patients who presented diverse underlying
immunodeficiency status. Curiously, most of these
individuals developed fungemia [5254]. Different
from C. neoformans and C. gattii, the tropism of this
species for the central nervous system (SNC) seems to
be uncommon [5557].
The commercial kits to detect capsular antigen of
C. neoformans in serum and CSF have been used for
C. neoformans cryptococcosis diagnosis [5860].
Despite their high specificity, cross-reaction with
other Cryptococcus species was already described
[61, 62]. None of the isolates herein characterized as
C. laurentii by mycological methods and ITS sequenc-
ing presented reactivity to C. neoformans antibodies,
which emphasizes the high specificity of this test for
C. neoformans diagnosis. This fact may be related to
different expression of capsular epitopes in C. laur-
entii which could not be recognized by the commercial
kit antibodies. Also the method herein used to extract
C. laurentii polysaccharides antigen may have some
limitations [62].
Different from C. neoformans and C. gattii, the
virulence factors in C. laurentii have been less
studied. In the present report, the phospholipase
activity was evidenced in all C. laurentii isolates.
This figure is similar to that observed in Italy
evaluating nine strains recovered from bird cloaca
[63], but it is higher than the 64.2% obtained in
Malaysia evaluating 14 environmental isolates [64].
This variability had already been observed for
C. neoformans and C. gattii isolates [24, 26], and it
would be related to the isolation origin, number of
strains and intrinsic conditions of each isolate among
other factors. It is believed that this enzyme is
involved in the phospholipids breakdown during the
infection process. The role of the phospholipase B
gene in C. neoformans virulence was formerly shown
in experimental models [65], and it would be impor-
tant to know its role in C. laurentii pathogenicity.
Despite several attempts, none C. laurentii strains
produced proteinase on BSA agar. The absence of this
enzyme would be associated with the saprophytic
origin of the isolates, but this hypothesis needs to be
tested in animal model. This finding is in line with a
previous report where most of the environmental
isolates did not exhibit proteinase activity [37], and
different from that observed for C. neoformans and
C. gattii [23, 24]. Recently, it was suggested that the
solid BSA medium is not able to detect low enzyme
Mycopathologia (2012) 174:4152
activity and for this, more sensitive methods should be
applied for better performance [37].
In the present study, 89.4% of the C. laurentii
isolates exhibited hemolytic activity. This fact sug-
gests its pathogenic potential and may eventually be
associated with its ability to grow in the blood as
observed in most reported human cases. The hemo-
lytic activity was formerly described to C. albicans
and some bacteria [28]. To our knowledge, this is the
first evidence of the hemolytic activity for C. laurentii.
So far, most of the antifungal susceptibility studies
reported were carried out with clinical and environ-
mental isolates of C. neoformans and C. gattii. In
contrast, few studies with clinical [14, 33] or environ-
mental [66] C. laurentii strains have been published.
According to the adopted criteria, most C. laurentii
isolates exhibited more susceptibility to voriconazole,
amphotericin B, and ketoconazole than to itraconazole
and fluconazole.
The decreased susceptibility to azoles derivates is an
interesting finding considering the environmental origin
of these isolates. Maybe, they could have been more
exposed to antifungals present in aerosols in surround-
ing hospital areas and due to the fact that these drugs are
commonly used to prevent fungal infections in birds.
However, cases of bird yeast infections have been rarely
reported [67]. The detection of five resistant isolates to
amphotericin B is relevant since this drug is considered
the gold standard to treat cryptococcosis infection.
Recently, an amphotericin B resistant C. laurentii strain
was reported infecting an HIV patient with meningoen-
cephalitis from Italy, reinforcing the relevance of this
fact. However, more studies are required in order to
validate and correlate the susceptibility and resistance
patterns of C. laurentii around the world [20].
In addition, 71.1% isolates were susceptible dose
dependent to fluconazole, whereas 2.65% of them
exhibited resistance to voriconazole, one of the latest
antifungal released in the market to treat severe fungal
infections. These results are in line with those
obtained in Spain where eight clinical strains of
C. laurentii were evaluated. Half of them were
susceptible dose dependent to fluconazole (MIC
C16.0), one (12.5%) isolate was resistant to itraco-
nazole and one (12.5%) to voriconazole [33]. These
figures are worrying and must be taken into account in
the C. laurentii infection context.
Due to the base composition of the nuclear DNA
and as reveled by the sequence analysis of the 28S
49
region of rDNA and of ITS regions, C. laurentii has
been considered a heterogeneous species [38]. Herein,
this variability was observed using the M13 and
(GACA)4 fingerprinting. Among the 38 strains eval-
uated, it was possible to observe a more heterogeneous
profile by M13 when compared with that obtained by
(GACA)4. Only seven (18.4%) isolates (CL2, CL7,
and CL8), (CL35, CL38), and (CL29, CL30) showed
100% similarity by the two primers used. Interest-
ingly, both primers clustered most of the pet shop
strains which were recovered from drooping samples
collected within 1 day of environmental exposition
although they were recovered from different neigh-
borhoods. Otherwise, the high variability found in the
surrounding hospital isolates could be related to more
environmental exposition to UV radiation, chemical
agents including antifungals and microorganism com-
petition among others. No relation between the
generated molecular profiles and phospholipase pro-
duction was found. As an example, the isolates CL29
and CL30 which were 100% similar were low and
moderate producer of this enzyme. This fact was still
observed for the isolates CL2, CL7, and CL8 for
phospholipase and hemolysins production.
Although C. laurentii has been considered a
heterogeneous species, all isolates evaluated by the
ITS sequencing clustered within the phylogenetic
group I. Their similarity with other Brazilian envi-
ronmental isolates may suggest a clonality prevalence
of the phylogenetic group I in this country. However,
this needs to be confirmed through new studies that
should include representative number of strains from
different geographical areas. Interestingly, environ-
mental isolates clustered near the clinical ones
pointing out the relevance of C. laurentii as a
potential pathogen.
Thus, the C. laurentii evaluated isolates expressing
virulence factors, a decreased sensitivity antifungal
profile, and a wide genetic variability would suggest
that this species is a potential pathogen in the context
of the immunocompromised population. However,
other studies are required to confirm these results and
improve the knowledge about this specie.
Acknowledgments We thank Mrs. Angela Azor for her
technical assistance. DNA samples were sequenced at the
Laboratorio Multiusuario, UFTM. This work was supported by
Fundacao de Amparo a Pesquisa de Minas Gerais-FAPEMIG
Grant number (APQ-01735/2010). K.F-P, L.A-S, D.J.M. are
fellows from CAPES.
123
50
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