Antifungal Susceptibility, Enzymatic Activity, PCR-Fingerprinting and ITS Sequencing of...

Antifungal Susceptibility, Enzymatic Activity,PCR-Fingerprinting and ITS Sequencing of EnvironmentalCryptococcus 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

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

Introduction

Cryptococcus genus includes several species world-

wide distributed and found in different environments

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: [email protected]

M. L. Silva-Vergara

e-mail: [email protected]

A. L. Pedrosa

Department of Molecular Biology, Triangulo Mineiro

Federal University, Uberaba, MG, Brazil

P. R. da Silva � A. A. Andrade

Department of Microbiology, Triangulo Mineiro Federal

University, Uberaba, MG, Brazil

123

Mycopathologia (2012) 174:41–52

DOI 10.1007/s11046-011-9500-0

[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 [5–10].

Cryptococcus laurentii has been recovered from

pigeons and captive bird droppings and from trees

hollows with decaying wood [11–13]. 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 [14–21].

Virulence factors such as the capsular polysaccha-

ride, ability to growth at 37�C and the melanin,

phospholipase, proteases, DNase, collagenase, and

urease production were previously characterized to

C. neoformans and C. gattii [22–24]. 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

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 [34–36]. 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 37�C 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 -20�C. 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

42 Mycopathologia (2012) 174:41–52

123

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 32�C. 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 (5–10 volumes for three

times each). The cell volume was quantified and stored

at –4�C until dry. Then, a 15% veronal buffered

solution (v/v) was prepared and added on the dried

cells, autoclaved at 120�C for 20 min, and then

centrifuged for 30 min at 2,000 rpm. The supernatant

containing the antigen was stored at -4�C.

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 30�C. 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 30�C 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 120�C 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 30�C for 7 days

and after this, the readings were taken [41].

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 = 0.7–0.99, low enzymatic

activity, Pz = 0.5–0.69, 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 30�C. 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 30�C 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 33�C 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

Mycopathologia (2012) 174:41–52 43

123

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 Tris–HCl 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

94�C, followed by 36 cycles of 1 min at 94�C, 1 min at

50�C, 1 min at 72�C, with a final extension of 10 min at

72�C. 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, Tris–HCl 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 94�C for 2 min initial

denaturation, followed by 1 min of denaturation at

94�C, 1 min of annealing at 52�C and 1 min of

extension at 72�C, and a final extension cycle of

15 min at 72�C. 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 -20�C 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 -20�C for sequencing reactions.


123

Fig. 1 Agarose gel

electrophoresis and

phenogram of polymerase

chain reaction fingerprinting

profiles obtained from 38

environmental

Cryptococcus laurentiiisolates 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

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 37�C, although the optimal

growth was obtained at 35�C. 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

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 Resistant Range GM MIC50 MIC90

Amphotericin B 33 (86.9%) – 5 (13.1%) 0.12–4.0 0.50 0.5 2.0

Ketoconazole 35 (92.1%) – 3 (7.9%) 0.06–4.0 0.41 0.5 1.0

Itraconazole 8 (21.0%) 21 (55.3%) 9 (23.7%) 0.12–2.0 0.34 0.25 1.0

Voriconazole 36 (94.7%) 1 (2.65%) 1 (2.65%) 0.12–4.0 0.44 0.50 1.0

Fluconazole 11 (28.9%) 27 (71.1%) 0 8–32 14.87 16.0 32.0

GM geometric mean, SDD susceptible dose dependent

Fig. 2 Evolutionary relationship of C. laurentii environmental

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

c


123


123

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

non-HIV patients who presented diverse underlying

immunodeficiency status. Curiously, most of these

individuals developed fungemia [52–54]. Different

from C. neoformans and C. gattii, the tropism of this

species for the central nervous system (SNC) seems to

be uncommon [55–57].

The commercial kits to detect capsular antigen of

C. neoformans in serum and CSF have been used for

C. neoformans cryptococcosis diagnosis [58–60].

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


123

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

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

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