Available via license: CC BY 4.0
Content may be subject to copyright.
Citation: Florek, M.;
Korzeniowska-Kowal, A.; Wzorek,
A.; Włodarczyk, K.; Marynowska, M.;
Pogorzelska, A.; Brodala, M.; Ploch,
S.; Buczek, D.; Balon, K.; et al.
Prevalence, Genetic Structure and
Antifungal Susceptibility of the
Cryptococcus neoformans/C. gattii
Species Complex Strains Collected
from the Arboreal Niche in Poland.
Pathogens 2022,11, 8. https://
doi.org/10.3390/pathogens11010008
Academic Editor: Lawrence S. Young
Received: 16 November 2021
Accepted: 19 December 2021
Published: 22 December 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
pathogens
Article
Prevalence, Genetic Structure and Antifungal Susceptibility of
the Cryptococcus neoformans/C. gattii Species Complex Strains
Collected from the Arboreal Niche in Poland
Magdalena Florek 1, * , Agnieszka Korzeniowska-Kowal 2, Anna Wzorek 2, Katarzyna Włodarczyk 3,
Maja Marynowska 1, Aleksandra Pogorzelska 1, Maria Brodala 1, †, Sebastian Ploch 4, Daniel Buczek 3 ,†,
Katarzyna Balon 3, 5, † and Urszula Nawrot 3
1
Department of Pathology, The Faculty of Veterinary Medicine, Wrocław University of Environmental and Life
Sciences, Norwida 31, 50-375 Wroclaw, Poland; maja.marynowska@upwr.edu.pl (M.M.);
aleksandra.pogorzelska@upwr.edu.pl (A.P.); 113077@student.upwr.edu.pl (M.B.)
2Department of Immunology of Infectious Diseases, Hirszfeld Institute of Immunology and Experimental
Therapy, Polish Academy of Sciences, St. Weigla 12, 53-114 Wroclaw, Poland;
agnieszka.korzeniowska-kowal@hirszfeld.pl (A.K.-K.); anna.wzorek@hirszfeld.pl (A.W.)
3
Department of Pharmaceutical Microbiology and Parasitology, Wrocław Medical University, Borowska 211a,
50-556 Wroclaw, Poland; katarzyna.wlodarczyk@umed.wroc.pl (K.W.); danielbuczek@onet.pl (D.B.);
katarzyna.balon@hirszfeld.pl (K.B.); urszula.nawrot@umw.edu.pl (U.N.)
4IT Lab, The Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences,
Norwida 31, 50-375 Wroclaw, Poland; sebastian.ploch@upwr.edu.pl
5Laboratory of Genomics & Bioinformatics, Hirszfeld Institute of Immunology and Experimental Therapy,
Polish Academy of Sciences, 53-114 Wroclaw, Poland
*Correspondence: magdalena.florek@upwr.edu.pl
† The following authors are students.
Abstract:
Fungi belonging to the Cryptococcus neoformans/C. gattii species complex (CNGSC) are
etiological agents of serious and not infrequently fatal infections in both humans and animals. Trees
are the main ecological niche and source of potential exposition concerning these pathogens. With
regard to epidemiology of cryptococcosis, various surveys were performed worldwide, enabling the
establishment of a map of distribution and genetic structure of the arboreal population of the CNGSC.
However, there are regions, among them Central and Eastern Europe, in which the data are lacking.
The present study shows the results of such an environmental study performed in Wrocław, Poland.
The CNGSC strains were detected in 2.2% of the tested trees belonging to four genera. The obtained
pathogen population consisted exclusively of C. neoformans, represented by both the major molecular
type VNI and VNIV. Within the tested group of isolates, resistance to commonly used antimycotics
was not found, except for 5-fluorocytosine, in which about 5% of the strains were classified as a
non-wild type.
Keywords:
Cryptococcus neoformans/C. gattii species complex; environmental; arboreal; RFLP; MALDI-
TOF MS; susceptibility
1. Introduction
Cryptococcosis is a serious, not infrequently fatal infection of humans and animals
caused by fungi belonging to the genus Cryptococcus. Though several species of the genus
were found responsible for the disease occurrence [1], those belonging to the Cryptococcus
neoformans/Cryptococcus gattii species complex (CNGSC) were the etiological agents of the
majority of cases. The worldwide impact of cryptococcosis in the human population only
is assumed to be more than 220,000 infections and up to 180,000 deaths annually [
2
]. The
disease induced by C. neoformans is usually observed in persons with impaired immune
status, while C. gattii is thought to be able to cause infection in immunocompetent individ-
uals; yet, there are reports suggesting genetically mediated differences both in host and
pathogen may affect this typical epidemiological pattern [3].
Pathogens 2022,11, 8. https://doi.org/10.3390/pathogens11010008 https://www.mdpi.com/journal/pathogens
Pathogens 2022,11, 8 2 of 15
The nomenclature concerning the CNGSC has been changing during the previous
and ongoing century. These pathogens can be described by their species, major molecular
type, or serotype. With respect to the species, presently, the two-species or seven-species
schemes relating to the complex are of use, as the discussion concerning classification of
the fungi did not reach a consensus among the scientific community [
3
,
4
]. According to
the consensus [
5
] based on various molecular methods, the described fungal complex has
been divided into eight major molecular types (MMT), and lately, a ninth one has been
discovered [
6
]. The fungi can be also divided into four serotypes, namely A, B, C, or D. In
the present paper, the nomenclature with regard to a two-species scheme as well as division
into nine MMT were adopted [
4
–
6
]: VNI, VNII, and VNB (all representing Cryptococcus
neoformans var. grubii, serotype A); VNIV (C. neoformans var. neoformans, serotype D); and
VNIII (the hybrid of these two varieties, serotype AD) as well as VGI, VGII, VGIII, VGIV,
and VGV (C. gattii, serotype B or C).
The discussed fungi are in possession of a bipolar mating system consisting of a single
mating-type locus (MAT) represented by two different mating types (MATa and MAT
α
).
Both bisexual and unisexual mating was observed within the CNGSC [7,8].
With respect to the geographical distribution of pathogens belonging to the described
complex, C. neoformans seems to be global, while C. gattii was regarded as associated rather
with tropical and subtropical zones [
9
]. For about the last twenty years, however, the latter
one has also been detected in regions with typically temperate climate [10,11].
It is assumed that infection with fungi of the CNGSC is a result of inhalation of
spores or dehydrated yeast cells and is acquired from environmental sources, such as plant
materials, soil, or bird excreta [
12
,
13
]. Arboreal sources are considered the main natural
niche [
13
] of the fungi, and according to a growing number of available publications, the
presence of CNGSC in certain parts of the world is often associated with specific species
of flora [
13
–
16
]. Interestingly, in some surveys comparing clinical and environmental
strains within the same area, it was impossible to recognise the environmental sources
of those clinical strains [
17
–
20
]. Moreover, when compared on a genetic level, these
two groups of strains often show discrepancies [
21
]. While some of those discrepancies
may be regarded as the result of dormant infections imported from other areas of the
world, another may indicate the presence of indigenous, as-yet-unrevealed sources of the
pathogen [
22
]. Importantly, it was also observed that genetic variability among the CNGSC
strains (e.g., among different MMTs) may influence their virulence and susceptibility to
antifungals [
23
,
24
]. Taking into consideration the results of the above-mentioned reports,
both extensive analysis of the prevalence of the pathogen within an environment as well as
analysis of the genetic structure of its local population may deepen our understanding of the
epidemiology of cryptococcosis. Nowadays, such investigations may grow in importance,
as epidemiological studies regarding SARS-CoV-2 infections show that insufficiency of the
respiratory system and immunological impairment observed in COVID-19 patients increase
the risk for opportunistic fungal infections. In the paper of Song et al. [
25
], Cryptococcus
was indicated among the top potential agents causing fungal co-infections in COVID-19
patients. Despite the seriousness of cryptococcosis, the data concerning the occurrence of
the agent and its population structure in Poland were not very well documented to date. In
our previous study, the population of non-clinical strains of animal origin isolated in Poland
was analysed [
26
]. With the present research, we would like to extend the knowledge of
the Polish community of the CNGSC, this time by analysing isolates obtained from the
arboreal specimen.
2. Results
There were 13 (2.16%) positive outcomes for the presence of the CNGSC strains out
of 602 tested samples. With reference to arboreal material, the fungi were detected within
specimens collected from 12 trees, among them 9 oaks, as well as one specimen each from
birch, pine, and Douglas fir. Statistically significant differences considering the isolation
frequency were not detected with regard to tree species (p= 0.227). The fungal strains were
Pathogens 2022,11, 8 3 of 15
also cultivated from one of the tested soil samples, which was collected around one of
the positive trees. The detailed information concerning the results of isolation of CNGSC
are given in Table 1and Figure 1. Eighteen strains of the CNGSC were isolated from the
examined trees. In two cases, multiple strains were detected simultaneously, while other
isolates were obtained alone. Since two of the trees were swabbed more than once, a few
strains had the same source, yet they were collected on different occasions. One isolate was
cultivated from the soil sample.
Table 1. The results of isolation of CNGSC strains from environmental sources.
Source of the Sample No. of Trees No. of Positive
Trees
Isolation
Percentage
(%)
No. of
Isolates Strains
Oak (Quercus L.) 309 9 2.91 14
1o*, 4’, 9a, D4, D2aˆ,
D2bˆ, K1aˆ, K1bˆ, Fw1,
Fw4*, Fw5, Fw6, Fw7*,
Fw8*
Black locust (Robinia
pseudoacacia L.) 23 0 0 0 -
Lime tree (Tilia L.) 11 0 0 0 -
Plane tree (Platanus L.) 36 0 0 0 -
Horse-chestnut (Aesculus
hippocastanum L.) 12 0 0 0 -
Hornbeam (Carpinus L.) 10 0 0 0 -
Birch (Betula L.) 10 1 10 1 Fw3
Pine (Pinus L.) 13 1 7.69 2 Fw2*, Fw9*
Fir (Abies Mill.) 6 0 0 0 -
Willow (Salix L.) 4 0 0 0 -
European beech (Fagus
sylvatica L.) 5 0 0 0 -
Common yew (Taxus
baccata L.) 2 0 0 0 -
Maple (Acer L.) 30 0 0 0 -
Swedish whitebeam
(Sorbus intermedia L.) 2 0 0 0 -
Sycamore maple (Acer
pseudoplatanus L.) 2 0 0 0 -
Sea buckthorn (Hippophaë
rhamnoides L.) 1 0 0 0 -
Thuja (Thuja L.) 10 0 0 0 -
Cottonwood (Populus L.) 10 0 0 0 -
Larch (Larix Mill.) 4 0 0 0 -
Spruce (Picea A. Dietr.) 8 0 0 0 -
Alder (Alnus Mill.) 9 0 0 0 -
Ash tree (Fraxinus L.) 9 0 0 0 -
Douglas fir (Pseudotsuga
menziesii) 66 1 1.51 1 9x
Pathogens 2022,11, 8 4 of 15
Table 1. Cont.
Source of the Sample No. of Trees No. of Positive
Trees
Isolation
Percentage
(%)
No. of
Isolates Strains
Trees in total: 592 12 2.20 18
Soil: 10 1 10 1 Fw10
In total: 602 13 2.16 19 -
* strains obtained from the same tree on several occasions; ˆ strains obtained from the same trees simultaneously.
Figure 1.
Locations of sample collection points and positive isolation sites (with number of isolates
and their MMT).
All the 19 obtained strains were identified as C. neoformans. According to the URA5-
RFLP method, the majority of them (11; 57.9%) represented the MMT VNIV, while the rest
(8; 42.1%) belonged to the MMT VNI. One of the MMT VNIV strains was the bearer of a
point mutation giving it an atypical URA5-RFLP banding profile [
27
]. Neither strains of
the MMT VNII nor AD hybrids were observed within the group of isolates. The obtained
serotyping results were in agreement with those obtained in the MMT identification, and
all the strains were assigned as representing mating type α.
Applying the MALDI-TOF MS method, all the tested strains achieved scores above
1.7 (regardless of whether the original base or its supplemented version was used), which
enabled reliable recognition. With respect to the manufacturers’ base alone, scores of
eight isolates made it possible to recognise a possible genus, and another 10 strains were
recognised on the level of secure genus and probable species. Only one isolate exceeded
the score of 2.3 and was identified as a highly probable species. The mean score secured
within the tested group was 2.051, with this value slightly higher among strains belonging
to the MMT VNI (2.154) when compared to the MMT VNIV (1.976). Using the best-match
strains’ MMTs for comparison, all the tested strains were assigned to MMTs that remained
in accordance with results of the other used methods. With the supplemented version of
the base, the score values obtained for seven strains (36.8%) increased, resulting in a shift of
four of them to the groups with the higher threshold, enabling more secure identification.
The mean score obtained for the whole tested group of strains using the updated tool was
2.120, whereas the value concerning the MMT VNI remained almost the same (2.157), and
for the MMT VNIV (2.093), it increased enough to exceed the threshold value of the more
secure recognition group (2.0–2.3). Considering identification of the MMTs, however, two
among the tested strains (10.5%) were misrecognised, as their best-match strains’ MMTs
Pathogens 2022,11, 8 5 of 15
were MMT VNIII instead of the MMT VNIV. The details concerning results of URA5-RFLP,
sero-, and mating type and MALDI-TOF MS analyses are given in the Table 2.
Table 2.
The results of examination of the MMT (using URA5-RFLP and MALDI-TOF MS techniques),
sero- and mating- types of the CNGSC strains.
The Strain
Name
PCM
Number
Sero- and
Mating Type
MMT
According to
URA5-RFLP
MALDI-TOF MS
Original Base
MALDI-TOF MS
Extended Base
Score MMT
Identification Score MMT
Identification
1o3145 DαVNIV 1.865 VNIV 2.553 VNIV
9a 3146 DαVNIV 1.968 VNIV 2.080 VNIV
9x 3147 DαVNIV 1.881 VNIV 1.953 VNIV
4’ 3150 DαVNIV 1.946 VNIV 1.946 VNIV
D2a 3148 AαVNI 2.234 VNI 2.260 VNI
D2b 3149 AαVNI 2.221 VNI 2.221 VNI
D4 3151 AαVNI 2.090 VNI 2.090 VNI
FW1 3153 AαVNI 2.112 VNI 2.112 VNI
FW2 3155 AαVNI 2.252 VNI 2.252 VNI
FW3 3158 DαVNIV 2.212 VNIV 2.212 VNIV
FW4 3160 DαVNIV 1.762 VNIV 1.762 VNIV
FW5 3161 AαVNI 2.045 VNI 2.045 VNI
FW6 3162 DαVNIV 1.963 VNIV 2.032 VNIV
FW7 3152 DαVNIV 2.108 VNIV 2.127 VNIII
FW8 3157 DαVNIV 2.002 VNIV 2.041 VNIII
FW9 3159 AαVNI 1.853 VNI 1.853 VNI
FW10 3154 AαVNI 2.426 VNI 2.426 VNI
K1a 3156 DαVNIV 1.965 VNIV 1.965 VNIV
K1b 3000 DαVNIV* 2.070 VNIV 2.070 VNIV
* strain presenting atypical banding pattern.
The result of drug-susceptibility tests of the investigated environmental CNGSC
isolates are presented in Table 3.
All the tested isolates were classified as susceptible to amphotericin B (AMB) with a
MIC
≤
1 mg/L [
28
]. The median value of MICs of AMB was 1 mg/L (range
0.125–1 mg/L
)
for the whole tested population of isolates as well as for the subgroups representing the
MMT VNI (range 0.5–1 mg/L) and the MMT VNIV (range 0.125–1 mg/L). In the case
of 5-fluorocytosine (5-FC), the MICs ranged from 1 to 64 mg/L, except of the MMT VNI,
for which it was in the range of 8–32 mg/L. The median value of MICs for 5-FC was
16 mg/L
, and all the isolates except one (18/19; 94.7%) were classified as belonging to the
WT population (MIC ≤32 mg/L).
The MIC values established for triazole derivatives ranged from 0.5 to 32 mg/L
for fluconazole (FLU), from 0.03 to 0.25 mg/L for voriconazole (VOR), from 0.015 to
0.25 mg/L
for isavuconazole (ISV), from 0.03 to 0.5 mg/L for itraconazole (ITR), and from
0.015 to 0.5 mg/L
for posaconazole (POS). The MIC50 for FLU, VOR, ISV, ITR, and POS
was 4.0, 0.06, 0.06, 0.125, and 0.125, respectively, whereas MIC90 amounted to 32, 0.25,
0.125, 0.25, and 0.25, respectively. According to adopted ECVs for triazoles, all but one
isolate were classified as belonging to the WT population. The only exception was strain
FW2 (3168), which showed an ISV MIC of 0.25 mg/L, which is one dilution above the
adopted ECV (0.125 mg/L). Regarding statistically significant differences concerning the
Pathogens 2022,11, 8 6 of 15
resistance to specific antimycotics, when compared, the MMT VNI and the MMT VNIV
were not detected.
Table 3.
The susceptibility of the investigated CNGSC isolates to antifungal agents in relation to
major molecular type (MMT).
The Strain
Name
MIC (mg/L)
AMB 5-FC FLU VOR ISV ITR POS
Major Molecular Type VNI
D2a 1 16 0.5 0.06 0.03 0.03 0.06
D2b 1 16 1 0.03 0.06 0.03 0.125
D4 1 16 1 0.03 0.06 0.03 0.125
FW1 0.5 32 32 0.25 0.125 0.25 0.25
FW 10 1 32 32 0.25 0.125 0.25 0.5
FW2 1 8 16 0.25 0.25 0.5 0.25
Fw9 1 32 32 0.125 0.015 0. 25 0.015
FW5 0.5 16 16 0.125 0.125 0.25 0.25
Median (range)
of VNI
1
(0.5–1)
16
(8–32)
16
(0.5–32)
0.125
(0.03–0.25)
0.0925
(0.015–0.25)
0.25
(0.03–0.5)
0.187
(0.015–0.5)
Major Molecular Type VNIV
1o1 16 0.5 0.125 0.125 0.03 0.06
9a 0.5 16 0.5 0.06 0.06 0.03 0.06
9x 1 32 0.5 0.06 0.06 0.03 0.06
4 1 16 0.5 0.03 0.06 0.03 0.25
Fw7 0.5 16 16 0.25 0.125 0.25 0.125
K1a 1 8 8 0.125 0.015 0.125 0.015
Fw8 1 16 4 0.125 0.015 0.03 0.015
FW3 0.5 8 4 0.06 0.015 0.03 0.015
FW4 1 8 8 0.06 0.015 0.125 0.015
FW6 0.125 1 4 0.06 0.06 0.5 0.25
K1b 1 64 1 0.03 0.03 0.125 0.125
Median (range)
of VNIV
1
(0.125–1)
16
(1–64)
4
(0.5–16)
0.06
(0.03–0.25)
0.06
(0.015–0.125)
0.03
(0.03–0.5)
0.06
(0.015–0.25)
Median (range)
of VNI and
VNIV
1
(0.125–1)
16
(1–64)
4
(0.5–32)
0.06
(0.03–0.25)
0.06
(0.015–0.25)
0.0775
(0.03–0.5)
0.0925
(0.015–0.5)
MIC50
(VNI and VNIV) 1 16 4 0.06 0.06 0.125 0.125
MIC90
(VNI and VNIV) 1 32 32 0.25 0.125 0.25 0.25
Abbreviations: AMB, amphotericin B; 5-FC, 5-fluorocytosine; FLU, fluconazole; ISV, isavuconazole; ITR, itracona-
zole; POS, posaconazole; VOR, voriconazole.
3. Discussion
Though pathogenesis of the cryptococcal infections has been extensively examined
over the last decades, knowledge considering the environmental origin of the agent re-
mains less well established. In the review presented by Cogliati [
29
], among an analysis
of 69 thousand globally reported strains of the CNGSC, less than 10% represented those
of environmental or veterinary origin. The detailed studies over the sources of this envi-
Pathogens 2022,11, 8 7 of 15
ronmentally distributed complex are essential in order to define its geographical extent,
population structure, and mode and risk of transmission. It was also proven that certain
environmental conditions, among them those associated with arboreal ancestry of these
fungi, may be responsible for the acquisition of virulence factors allowing them to establish
infection within human/animal hosts [
30
–
34
]. In Europe, several studies concerning the
arboreal source of the CNGSC were performed, the majority of which were related to west-
ern and southern regions of the continent as well as to Mediterranean Basin [
13
,
17
,
35
–
38
].
Contrastingly, in Central and Eastern Europe, investigations concerning the matter, to the
best of our knowledge, are almost absent [39].
In Poland, only a handful of examinations with reference to the environmental preva-
lence of the pathogenic complex were carried out. C. neoformans has been detected in 35.3%
of the tested sandpits and 19% of soil samples obtained from children’s recreational areas
in Łód´z, a city located in the central of Poland [
40
,
41
]. The fungus was also recognised in
the waters of Charzykowskie Lake and its influents as well as in Szczecin Lagoon, both
located in the north of the country [
42
,
43
]. In the present study, an analysis of arboreal
specimen collected in the southwestern part of Poland was performed. The CNGSC iso-
lates were obtained from 12 out of 592 (2.2%) trees tested as well as from one in ten soil
samples collected in the trees’ proximity. The positive trees represented genera Quercus,
Betula,Pinus, and Pseudotsuga; yet, none of these were associated with a significantly higher
frequency of isolation of the fungi. The prevalence of the CNGSC in the arboreal sources
in Europe varied substantially. In the ISHAM’s project [
13
], surveying samples collected
in nine European and three non-European countries, the CNGSC strains were recovered
from 5% of the trees. Similar results (4.8%) were reported by Montagna et al. [
17
] with
regard to Southern Italy. A slightly higher number was presented by Romeo et al. [
36
]
again in Southern Italy, as the authors reported nine positives among 143 trees tested. In
the Netherlands, depending on the study, the fungi were not recovered at all [
13
], or the
isolation frequency amounted to 3.8% (2 of 52 trees) [
11
]. A ratio far higher, reaching about
16%, has been noticed in Spain (1 of 6 trees) [
35
] and in Greece [
13
]. In Portugal, the CNGSC
was isolated from 3 out of 28 samples [
38
]. However, prevalence lower than that observed
in our study was noted in Croatia [
37
] (4 in 472 samples) and Central Italy [
44
] (1 in 265
trees). Negative results in the context of arboreal specimen were reported in Russia [
39
] and
Germany [
13
]. The observed discrepancies were probably related to the climate conditions
within the tested zones, species composition of the used tree population, or number of the
samples tested [
13
,
37
,
45
,
46
]. It was determined [
45
,
46
] that distribution range of certain
species belonging to the CNGSCN in the environment is strongly correlated with climatic
conditions, such as minimum/mean temperature in the coldest season/quarter, maximum
temperature, summer rainfall/precipitation in the driest month, water vapor pressure,
solar radiation, distance from the coast, and canopy closure [
45
–
48
]. With respect to Euro-
pean arboreal niche, C. neoformans has been isolated from trees belonging to the following
genera: Eucaliptus,Olea,Pinus,Creatonia,Pyrus,Prunus,Platanus,Aesculus,Carpinus,Juglans,
Juniperus,Gleditsia, and Quercus, representing both Mediterranean and temperate climate
regions [
17
,
35
,
36
,
45
]. Regarding C. gattii, the fungus has been obtained from trees of the
following genera: Eucaliptus,Olea,Creatonia,Pinus,Pseudotuga [
14
,
17
,
35
,
36
,
45
]. It was
suggested that C. gattii prefers trees with waxier cuticles [
49
]. In the matter of prevalence,
it was observed [
13
] that the percentage of isolation of C. gattii was statistically higher
pertaining to the genus Creatonia.C. neoformans var. grubii has been isolated statistically
more often from the Creatonia and Olea, while C. neoformans var. neoformans as well as
the hybrid of these two have been obtained mainly from the genus Platanus. None of the
36 tested trees of the genus Platanus swabbed in our study were positive with regard to
presence of the CNGSC.
In the present study, 57.9% of the isolated fungi represented the MMT VNIV, while
the rest (42.1%) belonged to the MMT VNI. Other molecular types, among them those
assigned to the species C. gattii, were not detected. Results of surveys concerning arboreal
sources of the fungi performed in Europe showed predominance of the MMT VNI. The
Pathogens 2022,11, 8 8 of 15
type constituted from 64.45% (330 of 512 total strains isolated) to 100% of the populations
analysed in various studies [
11
,
13
,
17
,
36
,
38
,
44
]. To the contrary, the MMT VNIV was often
absent [
11
,
17
,
36
,
38
,
44
] or was detected in a percentage ranging from 20.9 (107 of 512) to
25 [
13
,
37
]. The high frequency of isolation of the MMT VNIV in the present study remains,
however, in accordance with results of work reported by Cogliati et al. [
45
], predicting a
fundamental niche for this MMT as positioned in the sub-continental region of Europe and
not along the coast, which corresponds with the location of Wrocław. Of interest may be
the observation that, while comparing populations of non-clinical/environmental strains
isolated from animal and arboreal specimen within the same area, those of animal origin
were reported to show a higher frequency of isolation of the MMT VNIV. In northern Por-
tugal, [
38
] from arboreal specimens, the MMT VNI was obtained exclusively, whereas 15 in
23 isolates obtained from the samples originated from pigeons represented the MMT VNIV
(while the rest was equally distributed between the MMTs VNI and VNIII). Similarly, in our
previous study [
26
] among the non-clinical strains of animal origin, the MMTs VNIV, VNI,
and VNIII were represented by 74.36%, 15.38%, and 10.26% of isolates, respectively. It was
observed that in Europe, the MMT VNIII was detected more frequently compared to other
continents [
29
]. While among clinical strains, AD hybrids could comprise about 30% [
50
],
and it was not uncommon to isolate the type from specimen of animal origin
[26,38]
, to
the best of our knowledge, the only European arboreal isolation of the MMT VNIII was
reported in Greece [
13
]. Another rare type, the MMT VNII, has not been isolated from trees
in Europe to date. Cryptococcus gattii is the species that has been reported as occurring in
different genera of trees of southern Europe [
13
,
17
,
36
]. It was also isolated in the temperate
climate zone of northern Europe; the pathogen inhabited one Douglas fir tree found in the
Netherlands [
11
]. With respect to the MMTs, in arboreal specimens collected in Europe, C.
gattii was represented by the MMT VGI and the MMT VGIV [11,13,17,36].
With respect to mating type, within the population of the CNGSC, the MAT
α
locus is
regarded as the most prevalent both among clinical and environmental strains [
29
], whereas
MATa is rather rare, and it is more commonly observed within the MMT VNIV strains [
51
].
All the strains isolated in the present study, regardless of their serotype, represented mating
type
α
. Similar to our work, populations of the CNGSC isolated in Europe from tree
material consisting purely of mating type
α
were reported [
36
,
37
]. On the other hand, in the
study performed by Montagna et al. [
17
], the locus MATa was detected in one of 40 strains
of the serotype A, whereas in the survey presented by ISHAM [
13
], the locus was detected
in 7 of 330 isolates representing serotype A, 29 of 107 belonging of the serotype D, and in
3 out of 35 AD hybrids (
α
ADa). It is worth mentioning that within the population of the
strains isolated from animals in Poland, locus MATa was found in aD or aADa isolates [
26
].
Among the arboreal strains of C. gattii isolated in Europe, researchers observed sero- and
mating types αB, aB, and αC [11,13,17,36].
The population of the yeasts investigated in the present study showed a low rate of
resistance to the tested antimycotics. According to the criteria adopted, all the CNGSC
isolates were classified as susceptible to AMB or belonging to WT-population in regard
to the susceptibility to FLU, ITR, POS, and VOR. Similar results were presented in our
previous study [
26
] in which, among the CNGSC isolates obtained from asymptomatic
animals (mostly pigeons), triazole resistance was not detected. Comparison of the results
obtained by other authors with respect to the CNGSC susceptibility to triazole is difficult. In
those studies, in which we could find data concerning strains of arboreal origin, those were
rarely presented alone [
15
,
37
,
52
], and the overall data presenting the matter were sparse.
Often, mixed results for both plant- and pigeon-derived specimens [
53
] or soil contaminated
with human/animal material were presented [
54
]. Moreover, applied interpretation criteria
or different testing methods made it impossible to compare the results even by means of
the MIC values. As an example, authors analyzing the CNGSC strains in Croatia using the
ATB fungus test found all the population susceptible to FLU, ITR, and VOR, while in Brazil
(AFST-EUCAST), 78.9% of the obtained isolated were classified as non-WT with regard to
FLU [
15
,
37
]. Interestingly, in both the present and our previous study, high MIC values
Pathogens 2022,11, 8 9 of 15
were obtained for 5-FC. According to the applied ECV (32 mg/L;
Cordoba et al. [55]),
in the present study, 1 out of 19 isolates (5%) was classified as resistant (or rather as
non-WT) to this drug (Table 3). If applied to the epidemiological cut-off proposed by
Espinel-Ingroff [
56
], however, four other strains with the MIC amounting to 32 mg/L (three
MMT VNI and one MMT VNIV) could also be listed as non-WT. The number of non-WT
strains detected according to the former ECV in the present study was lower compared
to the results reported in our previous paper [
26
], in which 10% of the strains showed the
MIC values for 5-FC equal to 64 mg/L. The reason for the observed discrepancy between
arboreal and animal isolates was probably the size of both studied groups (19 vs. 39),
and statistically significant differences were not confirmed. It should be emphasized that
those resistant to 5-FC strains evaluated in our studies (four of animal and one of arboreal
origin) were collected in different locations and over subsequent four years; thus, they
rather were not related epidemiologically. Of importance may also be the fact that all
of these isolates represented the same MMT (VNIV). With regard to 5-FC, the reports of
other authors showed that the resistance among clinical and environmental Cryptococcus
isolates was rather low (1–2.5%) [
55
–
58
] or undetectable [
59
]. Interestingly, in their work,
Chowdhary et al. [
59
], while presenting low MIC values for 5-FC regarding both clinical
and environmental strains, estimated that environmental isolates (in this case arboreal) C.
neoformans var. grubii presented significantly reduced susceptibility compared to clinical
strains of the same variety. Although the primary resistant strains identified in our studies
were rare (5–10%), the phenomenon is of particular concern, as 5-FC is a drug recommended
for treatment of cryptococcal meningitis. Due to the severity of this type of infection and
the possibility of a fatal outcome, the therapy is usually introduced in an empirical or
preemptive manner before obtaining the final results of microbiological examination and
susceptibility tests. Therefore, knowledge with regard to the prevalence of resistant strains
within the local environment may enhance the selection of the most potent therapy.
The significant impact of factors, which may be present in the natural habitat of the
CNGSC, including nutrient limitation (among them nitrogen limitation), temperature,
ultraviolet radiation, enzymatic degradation on the susceptibility of the CNGSC to FLU,
and AMB, has been described [
34
,
52
,
60
–
63
]. The above-listed factors are probably able to
activate adaptive processes, favoring the survival of the microorganism in the presence
of the drugs; however, their role in the development of persistent, mutation-dependent
resistance cannot be excluded. Additionally, it has been documented that both antifungal
and non-antifungal agrochemicals may exert a similar effect on environmental strains of
the CNGSC [
64
,
65
]. To the best of our knowledge, however, neither use of agrochemicals
nor recognized mechanisms of the resistance [
66
–
68
] can explain environmental sources
of the CNGSC resistance to 5-FC. Nevertheless, detection of the primary resistant strains
may suggest existence of as yet not recognized environmental factors supporting the
development of their resistance.
It is possible that, for the sake of the small isolate number obtained in the present study,
it was impossible to detect statistically significant differences in the MIC distributions of
any of the tested drugs with regard to MMTs of the tested strains. On the contrary, our
previous work results proved that the average observed MIC value of amphotericin B
was significantly lower and of fluconazole was significantly higher for the MMT VNIV
compared to the MMT VNI among strains isolated from animals in Poland [
26
]. Similar
significant differences in the drug-susceptibility among particular MMTs of the CNGSC
were described by other authors [59,69].
Due to the effort of several groups of researchers documenting the presence of arboreal
CNGSC strains within various European regions, it was possible to predict the niche of
the particular species within the complex and assess potential areas of exposure [
45
,
46
].
According to estimations presented by Alaniz et al. [
46
], the total area of distribution of these
fungi in Europe covers 2.7 million km
2
. Since the ranges of particular species within the
complex differ slightly, the authors suggest that the number of people potentially exposed
to infection on this continent may reach about 360, 266 and 137 million with reference to C.
Pathogens 2022,11, 8 10 of 15
neoformans var. grubii,C. neoformans var. neoformans, and C. gattii, respectively. Yet, in order
to calculate the risk, environmental surveys must firstly be performed. Unfortunately, the
data covering Central, Eastern, and Northern parts of Europe, to the best of our knowledge,
remain unavailable. With respect to regions characterised by continental climate, to date,
the only available studies were performed in Russia (Saint Petersburg), Germany, and the
continental part of Croatia, where analysis of arboreal specimens gave negative isolation
results [
13
,
37
,
39
]. Therefore, we believe results of the present study may contribute to
knowledge concerning ecology of the CNGSC in Europe.
There is still need for ongoing surveillance concerning environmental presence of the
CNGSC and not only with regard to those regions that were not examined to date [
70
].
With phenomena such as global warming or increasing reduction in biodiversity, changes
in the ecology of these fungi [
71
,
72
] may be expected, subsequently influencing people
at risk.
4. Materials and Methods
4.1. Study Design and Sample Processing
The arboreal specimen as well as the soil samples were collected between June 2014
and April 2019 in parks situated on the territory of Wrocław (51
◦
6
0
36” N, 17
◦
1
0
20” E) as
well as in forests located within the radius of 60 km from the city (Kotowice, Sobótka).
The territory is classified Dfb (warm-summer humid continental climate), according to
Köppen–Geiger classification. The material was obtained by the swabbing of tree hollows
or by collecting samples of woody detritus/soil. Usually, one sample was collected from
one tree (with the exception of one of the oaks, which was swabbed multiple times, and
one pine swabbed twice) or from the soil of the tree surroundings. A total of 592 samples of
arboreal specimen and 10 soil samples were obtained. With respect to the arboreal sources,
most of the tested samples (309; 52.2%) were collected from oaks, while other genera of the
trees were represented by 1 to 66 specimens. The detailed information concerning sources
of specimen used in the present study is given in Table 1.
All the samples were vortexed with 20 mL of a sterile saline solution for 5 min and
then left in order to let the suspension settle. The obtained supernatants were diluted 1:10.
Two sets of plates containing Niger seed agar (NSA) were inoculated with (100
µ
L) the
supernatant or its dilution. Then, the plates were incubated at 30
◦
C for up to 14 days
although positive samples could usually be detected at 48–96 h. All colonies showing the
brown colour effect obtained from each sample were sub-cultured as single-colony isolates
on NSA in order to purify cultures and then assessed using India Ink staining. Strains
positive in morphological evaluation and able to produce melanin on NSA were classified
as the CNGSC. A selection of colonies cultured from the same sample for further tests was
performed by means of analysis of the colony morphology and melanisation pattern.
Statistical analyses of isolation frequency with regard to certain tree species were
performed using Fisher’s exact test and PQStat v.1.8.2.208 (PQStat Software 2021) software.
In each analysis, a significance level of 5% was adopted.
4.2. Molecular Examination/Genotyping
For the DNA isolation, the obtained CNGSC strains were cultured on Sabouraud
dextrose agar (SDA) for 48 h at 30
◦
C. Extraction of the DNA was obtained using a Mas-
terPure Yeast DNA Purification Kit (Epicentre Biotechnologies, Madison, WI, USA), in
accordance with the manufacturer’s instructions. All PCRs presented in this study were
carried out in an MJ Mini Personal Thermal Cycler (BIO-RAD, Hercules, CA, USA) utilising
25-
µ
L reaction volume (1
µ
L of the extracted DNA, 12.5
µ
L of master mix (PCR Mix, A&A
Biotechnology, Gdynia, Poland), 20 pM of each primer, and 11.1
µ
L of water). For each
reaction, both positive and negative (sterile water) controls were used.
Recognition of species and/or variety was performed by sequencing of the SOD1
gene [
73
]. Amplification of the gene in C. gattii and C. neoformans var. grubii (MMT VNI)
was executed by applying two separate sets of primers presented in the MLST consensus
Pathogens 2022,11, 8 11 of 15
scheme [
5
]. For C. neoformans var. neoformans (MMT VNIV), an alternative reverse primer
for the SOD1 gene described by Sanchini et al. [74] was employed.
Sero- and mating types of the tested strains were established using the PCR-based
method (amplification of the serotype-specific and mating-type-specific STE20 gene) de-
scribed by Li et al. [
75
]. The following strains were used as positive controls: CBS 10084
(Aα), CBS 132 (αADa), IUM 96-2828 (Aa), and CBS 10079 (Dα).
Restriction fragment-length polymorphism analysis of the orotidine monophosphate
pyrophosphorylase gene (URA5-RFLP) was conducted according to Meyer et al. [
76
]. The
obtained PCR products were double digested using Cfr13I (Sau96I) and HhaI enzymes
(Thermo Fisher Scientific, Waltham, MA, USA) for 16 h and then separated in 3% agarose
gel (100 V for 3 h). RFLP patterns of the tested strains were analysed visually by comparison
with banding characteristic for standard strains representing major molecular types (CBS
8710-VNI, CBS 10084-VNII, CBS 132-VNIII, and CBS 10079-VNIV).
4.3. Identification with the Use of MALDI-TOF MS Method
Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry(MALDI-
TOF MS) analysis was performed on ultrafleXtremer spectrometer (Bruker Daltonics GmbH,
Germany), as described in our previous study [
26
]. Biotyper 3.1 software (Bruker Daltonics
GmbH, Germany) and an in-house supplemented [
26
] manufacturer’s database (8469 en-
tries) was used for the isolates’ identification. Manufacturer given score values were used:
<1.7 (identification not reliable), 1.7–2.0 (probable genus identification), 2.0–2.3 (secure
genus identification and probable species identification), and >2.3 (highly probable species
identification). The highest scores among the series of repetitions were given as the result.
In order to define the major molecular type of the examined strains, the best match strains’
MMTs were analysed.
4.4. Susceptibility to Antifungal Drugs
The susceptibility of CNGSC isolates to amphotericin B (AMB), 5-fluorocytosine (5-
FC), fluconazole (FLU), isavuconazole (ISV), itraconazole (ITR), posaconazole (POS), and
voriconazole (VOR) was tested using the microdilution method, according to the European
Committee on Antimicrobial Susceptibility Testing (EUCAST) Definitive Document E.DEF
7.3.1. [
77
]. All applied reagents were purchased from Sigma-Aldrich Life Science. The
minimal inhibitory concentration (MIC) definition was the lowest drug concentration
resulting in 90% (AMB) or 50% (5-FC and triazole derivatives) reduction of the OD530
when compared to the drug-free control.
The clinical breakpoints as well as epidemiological cut-off values (ECVs) applied
in the present study were consistent with our previous work [
26
] and amounted to for
amphotericin B (1 mg/L), POS (0.5 mg/L), and VOR (0.5 mg/L), 32 mg/L for 5-FC and
FLU, 0.5 mg/L for ITR, and 0.125 mg/L for ISV [
28
,
55
,
78
,
79
]. According to the clinical
breakpoints, strains were identified as susceptible or resistant, and using ECV’s values,
it was possible to categorise the isolates into wild-type (WT; population of isolates in
a species-drug combination with no detectable acquired resistance mechanisms [
78
]) or
non-wild-type (non-WT; strains that may hold mutation).
In order to compare the distribution of MICs between particular MMTs, the Mann–
Whitney U test was applied using PAST for Mac OS X v.4.0 (Øyvind Hammer 1999–2021)
software. In each analysis, a significance level of 5% was adopted.
Author Contributions:
Conceptualization, M.F.; methodology, M.F.; validation, M.F., U.N. and A.K.-
K.; formal analysis, S.P.; investigation, M.F., A.W., K.W., M.M., A.P., M.B., D.B. and K.B.; resources,
M.F., U.N. and A.K.-K.; data curation, M.F., U.N. and A.K.-K.; writing—original draft preparation,
M.F. and U.N.; writing—review and editing, U.N., A.K.-K., M.M. and A.P.; visualization, M.F. and
U.N.; supervision, M.F.; project administration, M.F.; funding acquisition, M.F. All authors have read
and agreed to the published version of the manuscript.
Pathogens 2022,11, 8 12 of 15
Funding:
This research was funded by Project supported by the Wroclaw Centre of Biotechnology
programme, The Leading National Research Centre (KNOW) for the years 2014–2018 (Project No
1/PB/2016/KNOW). The APC The was financed/co-financed under the Leading Research Groups
support project from the subsidy increased for the period 2020–2025 in the amount of 2% of the
subsidy referred to Art. 387 (3) of the Law of 20 July 2018 on Higher Education and Science, obtained
in 2019.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
All data generated or analysed during this study are included in this
published article. The strains used in the present study were deposited into Polish Collection of
Microorganisms (PCM) at Hirszfeld Institute of Immunology and Experimental Therapy PAS. The
deposit numbers are given in Table 2.
Acknowledgments:
Authors would like to express their special gratitude to Scott Richards for
scientific English language correction.
Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
References
1.
Khawcharoenporn, T.; Apisarnthanarak, A.; Mundy, L.M. Non-neoformans cryptococcal infections: A systematic review. Infection
2007,35, 51–58. [CrossRef] [PubMed]
2.
Rajasingham, R.; Smith, R.M.; Park, B.J.; Jarvis, J.N.; Govender, N.P.; Chiller, T.M.; Denning, D.W.; Loyse, A.; Boulware, D.R.
Global burden of disease of HIV-associated cryptococcal meningitis: An updated analysis. Lancet Infect. Dis.
2017
,17, 873–881.
[CrossRef]
3.
Hagen, F.; Khayhan, K.; Theelen, B.; Kolecka, A.; Polacheck, I.; Sionov, E.; Falk, R.; Parnmen, S.; Lumbsch, H.T.; Boekhout, T.
Recognition of seven species in the Cryptococcus gattii/Cryptococcus neoformans species complex. Fungal Genet. Biol.
2015
,78, 16–48.
[CrossRef] [PubMed]
4.
Kwon-Chung, K.J.; Bennett, J.E.; Wickes, B.L.; Meyer, W.; Cuomo, C.A.; Wollenburg, K.R.; Bicanic, T.A.; Castañeda, E.; Chang,
Y.C.; Chen, J.; et al. The case for adopting the “species complex” nomenclature for the etiologic agents of cryptococcosis. mSphere
2017,2, e00357-16. [CrossRef] [PubMed]
5.
Meyer, W.; Aanensen, D.M.; Boekhout, T.; Cogliati, M.; Diaz, M.R.; Esposto, M.C.; Fisher, M.; Gilgado, F.; Hagen, F.;
Kaocharoen, S.; et al.
Consensus multi-locus sequence typing scheme for Cryptococcus neoformans and Cryptococcus gattii.Med.
Mycol. 2009,47, 561–570. [CrossRef]
6.
Farrer, R.A.; Chang, M.; Davis, M.J.; van Dorp, L.; Yang, D.H.; Shea, T.; Sewell, T.R.; Meyer, W.; Balloux, F.; Edwards, H.M.;
et al. A new lineage of Cryptococcus gattii (VGV) discovered in the central Zambezian Miombo woodlands. mBio
2019
,10,
e02306–e02319. [CrossRef]
7.
Kwon-Chung, K.J. Morphogenesis of Filobasidiella neoformans, the sexual state of Cryptococcus neoformans.Mycologia
1976
,68,
821–833. [CrossRef]
8.
Lin, X.; Hull, C.M.; Heitman, J. Sexual reproduction between partners of the same mating type in Cryptococcus neoformans.Nature
2005,434, 1017–1021. [CrossRef]
9.
Firacative, C.; Roe, C.C.; Malik, R.; Ferreira-Paim, K.; Escandón, P.; Sykes, J.E.; Castañón-Olivares, L.R.; Contreras-Peres, C.;
Samayoa, B.; Sorrell, T.C.; et al. MLST and whole-genome-based population analysis of Cryptococcus gattii VGIII links clinical,
veterinary and environmental strains, and reveals divergent serotype specific sub-populations and distant ancestors. PLoS Negl.
Trop. Dis. 2016,10, e0004861. [CrossRef]
10.
Kidd, S.E.; Hagen, F.; Tscharke, R.L.; Huynh, M.; Bartlett, K.H.; Fyfe, M.; Macdougall, L.; Boekhout, T.; Kwon-Chung, K.J.; Meyer,
W. A rare genotype of Cryptococcus gattii caused the cryptococcosis outbreak on Vancouver Island (British Columbia, Canada).
Proc. National. Acad. Sci. USA 2004,101, 17258–17263. [CrossRef]
11.
Chowdhary, A.; Randhawa, H.S.; Boekhout, T.; Hagen, F.; Klaassen, C.H.; Meis, J.F. Temperate climate niche for Cryptococcus gattii
in Northern Europe. Emerg. Infecti. Dis. 2012,18, 172–174. [CrossRef]
12.
Kwon-Chung, K.J.; Fraser, J.A.; Doering, T.L.; Wang, Z.A.; Janbon, G.; Idnurm, A.; Bahn, Y.S. Cryptococcus neoformans and
Cryptococcus gattii, the etiologic agents of cryptococcosis. Cold Spring Harb. Perspec. Med. 2014,4, a019760. [CrossRef] [PubMed]
13.
Cogliati, M.; D’Amicis, R.; Zani, A.; Montagna, M.T.; Caggiano, G.; De Giglio, O.; Balbino, S.; De Donno, A.; Serio, F.; Susever, S.;
et al. Environmental distribution of Cryptococcus neoformans and C. gattii around the Mediterranean Basin. FEMS Yeast Res.
2016
,
16, 045. [CrossRef] [PubMed]
14.
Chowdhary, A.; Randhawa, H.S.; Prakash, A.; Meis, J.F. Environmental prevalence of Cryptococcus neoformans and Cryptococcus
gattii in India: An update. Crit. Rev. Microbiol. 2012,38, 1–16. [CrossRef] [PubMed]
Pathogens 2022,11, 8 13 of 15
15.
Castro e Silva, D.M.; Santos, D.C.S.; Martins, M.A.; Oliveira, L.; Szeszs, M.W.; Melhem, M.S.C. First isolation of Cryptococcus
neoformans genotype VNI MAT-alpha from wood inside hollow trunks of Hymenaea courbaril.Med. Mycol.
2016
,54, 97–102.
[PubMed]
16.
Ellis, D.H.; Pfeiffer, T.J. Ecology, life cycle, and infectious propagule of Cryptococcus neoformans.Lancet
1990
,336, 923–925.
[CrossRef]
17.
Montagna, M.T.; De Donno, A.; Caggiano, G.; Serio, F.; De Giglio, O.; Bagordo, F.; D’Amicis, R.; Lockhart, S.R.; Cogliati, M.
Molecular characterization of Cryptococcus neoformans and Cryptococcus gattii from environmental sources and genetic comparison
with clinical isolates in Apulia, Italy. Environ. Res. 2018,160, 347–352. [CrossRef]
18.
Hurst, S.; Lysen, C.; Cooksey, G.; Vugia, D.J.; Litvintseva, A.P.; Lockhart, S.R. Molecular typing of clinical and environmental
isolates of Cryptococcus gattii species complex from southern California, United States. Mycoses 2019,62, 1029–1034. [CrossRef]
19.
Chen, Y.; Litvintseva, A.P.; Frazzitta, A.E.; Haverkamp, M.R.; Wang, L.; Fang, C.; Muthoga, C.; Mitchell, T.G.; Perfect, J.R.
Comparative analyses of clinical and environmental populations of Cryptococcus neoformans in Botswana. Mol. Ecol.
2015
,24,
3559–3571. [CrossRef] [PubMed]
20.
Litvintseva, A.P.; Kestenbaum, L.; Vilgalys, R.; Mitchell, T.G. Comparative analysis of environmental and clinical populations of
Cryptococcus neoformans.J. Clin. Microbiol. 2005,43, 556–564. [CrossRef]
21.
Cogliati, M.; Desnos-Ollivier, M.; McCormick-Smith, I.; Rickerts, V.; Ferreira-Paim, K.; Meyer, W.; Boekhout, T.; Hagen, F.; Theelen,
B.; Inácio, J.; et al. Genotypes and population genetics of Cryptococcus neoformans and Cryptococcus gattii species complexes in
Europe and the Mediterranean Area. Fungal Genet. Biol. 2019,129, 16–29. [CrossRef] [PubMed]
22.
Hagen, F.; Colom, M.F.; Swinne, D.; Tintelnot, K.; Iatta, R.; Montagna, M.T.; Torres-Rodriguez, J.M.; Cogliati, M.; Velegraki, A.;
Burggraaf, A.; et al. Autochthonous and dormant Cryptococcus gattii infections in Europe. Emerg. Infect. Dis.
2012
,18, 1618–1624.
[CrossRef] [PubMed]
23.
Thompson, G.R.; Albert, N.; Hodge, G.; Wilson, M.D.; Sykes, J.E.; Bays, D.J.; Firacative, C.; Meyer, W.; Kontoyiannis, D.P.
Phenotypic differences of Cryptococcus molecular types and their implications for virulence in a Drosophila model of infection.
Infect. Immun. 2014,82, 3058–3065. [CrossRef]
24.
Torres-Rodriguez, J.M.; Morera, Y.; Baro, T.; Corominas, J.M.; Castaneda, E. Pathogenicity of Cryptococcus neoformans var gattii in
an immunocompetent mouse model. Med. Mycol. 2003,41, 59–63.
25.
Song, G.; Liang, G.; Liu, W. Fungal Co-infections Associated with Global COVID-19 Pandemic: A Clinical and Diagnostic
Perspective from China. Mycopathologia 2020,185, 599–606. [CrossRef] [PubMed]
26.
Florek, M.; Nawrot, U.; Korzeniowska-Kowal, A.; Włodarczyk, K.; Wzorek, A.; Wo´zniak-Biel, A.; Brzozowska, M.; Galli, J.;
Bogucka, A.; Król, J. An Analysis of the Population of Cryptococcus Neoformans Strains Isolated From Animals in Poland, in the
Years 2015–2019. Sci. Rep. 2021,11, 6639. [CrossRef] [PubMed]
27.
Florek, M.; Król, J.; Wo´zniak-Biel, A. Atypical URA5 gene restriction fragment length polymorphism banding profile in Cryptococ-
cus neoformans strains. Folia Microbiol. 2019,64, 857–860. [CrossRef]
28.
The European Committee on Antimicrobial Susceptibility Testing—EUCAST. Available online: https://www.eucast.org/
astoffungi/clinicalbreakpointsforantifungals/ (accessed on 4 November 2021).
29.
Cogliati, M. Global molecular epidemiology of Cryptococcus neoformans and Cryptococcus gattii: An atlas of the molecular types.
Scientifica 2013,2013, 675213. [CrossRef]
30.
Maliehe, M.; Ntoi, M.A.; Lahiri, S.; Folorunso, O.S.; Ogundeji, A.O.; Pohl, C.H.; Sebolai, O.M. Environmental factors that
contribute to the maintenance of Cryptococcus neoformans pathogenesis. Microorganisms 2020,8, 180. [CrossRef] [PubMed]
31.
Pini, G.; Faggi, E.; Campisi, E. Enzymatic characterization of clinical and environmental Cryptococcus neoformans strains isolated
in Italy. Rev. Iberoam. De Micol. 2017,34, 77–82. [CrossRef]
32.
Rizzo, J.; Albuquerque, P.C.; Wolf, J.M.; Nascimento, R.; Pereira, M.D.; Nosanchuk, J.D.; Rodrigues, M.L. Analysis of multiple
components involved in the interaction between Cryptococcus neoformans and Acanthamoeba castellanii.Fungal Biol.
2017
,121,
602–614. [CrossRef] [PubMed]
33.
Fu, M.S.; Liporagi-Lopes, L.C.; dos Santos, S.R.; Tenor, J.L.; Perfect, J.R.; Cuomo, C.A.; Casadevall, A. Amoeba Predation of
Cryptococcus neoformans Results in Pleiotropic Changes to Traits Associated with Virulence. mBio
2021
,12, e00567-21. [CrossRef]
[PubMed]
34.
Bosch, C.; Toplis, B.; Vreulink, J.M.; Volschenk, H.; Botha, A. Nitrogen concentration affects amphotericin B and fluconazole
tolerance of pathogenic cryptococci. FEMS Yeast Res. 2020,20, foaa010. [CrossRef] [PubMed]
35.
Linares, C.; Colom, M.F.; Torreblanca, M.; Esteban, V.; Romera, Á.; Hagen, F. Environmental sampling of Ceratonia siliqua (carob)
trees in Spain reveals the presence of the rare Cryptococcus gattii genotype AFLP7/VGIV. Rev. Iberoam. Micol.
2015
,32, 269–272.
[CrossRef] [PubMed]
36.
Romeo, O.; Scordino, F.; Chillemi, V.; Criseo, G. Cryptococcus neoformans/Cryptococcus gattii species complex in Italy: An overview
on the environmental diffusion of serotypes, genotypes and mating-types. Mycopathologia 2012,174, 283–291. [CrossRef]
37.
Pllana-Hajdari, D.; Cogliati, M.; ˇ
Ciˇcmak, L.; Pleško, S.; Mlinari´c-Missoni, E.; Marekovi´c, I. First isolation, antifungal susceptibility,
and molecular characterization of Cryptococcus neoformans from the environment in Croatia. J. Fungi.
2019
,5, 99. [CrossRef]
[PubMed]
38.
Ferreira, A.S.; Sampaio, A.; Maduro, A.P.; Silva, I.; Teles, F.; Martins, M.D.L.; Inácio, J. Genotypic diversity of environmental
Cryptococcus neoformans isolates from Northern Portugal. Mycoses 2014,57, 98–104. [CrossRef]
Pathogens 2022,11, 8 14 of 15
39.
Vasilyeva, N.V.; Bosak, I.A.; Bogomolova, T.S.; Vybornova, I.V. Environmental isolates of Cryptococcus neoformans in Saint
Petersburg, Russia. Mycoses 2009,52, 54–55.
40.
Wójcik, A.; Błaszkowska, J.; Kurnatowski, P.; Góralska, K. Sandpits as a reservoir of potentially pathogenic fungi for children.
Ann. Agric. Environ. Med. 2016,23, 542–548. [CrossRef]
41.
Wójcik, A.; Kurnatowski, P.; Błaszkowska, J. Potentially pathogenic yeasts from soil of children’s recreational areas in the city of
Łód´z (Poland). Int. J. Occup. Med. Environ. Health 2013,26, 477–487. [CrossRef]
42.
Kurnatowski, P.; Rózga, A.; Rózga, B.; Babski, P.; Wójcik, A. Potentially pathogenic fungi in the waters of the Charzykowskie
Lake in Zaborski Landscape Park. Wiad. Parazytol. 2007,53, 109–115.
43.
D ˛abrowski, W.; Bogusławska-W˛as, E.; Daczkowska-Kozon, E. Analysis od the Szczecin Lagoon waters fungi. Acta Mycol.
1988
,
33, 101–108. [CrossRef]
44.
Campisi, E.; Mancianti, F.; Pini, G.; Faggi, E.; Gargani, G. Investigation in central Italy of the possible association between
Cryptococcus neoformans var Gattii and Eucalyptus camaldulensis.Eur. J. Epidemiol. 2003,18, 357–362. [CrossRef]
45.
Cogliati, M.; Puccianti, E.; Montagna, M.T.; De Donno, A.; Susever, S.; Ergin, C.; Velegraki, A.; Ellabib, M.S.; Nardoni, S.; Macci, C.;
et al. Fundamental niche prediction of the pathogenic yeasts Cryptococcus neoformans and Cryptococcus gattii in Europe. Environ.
Microbiol. 2017,19, 4318–4325. [CrossRef] [PubMed]
46.
Alaniz, A.J.; Carvajal, J.G.; Carvajal, M.A.; Cogliati, M.; Vergara, P.M. Spatial quantification of the population exposed to
Cryptococcus neoformans and Cryptococcus gattii species complexes in Europe: Estimating the immunocompetent and HIV/AIDS
patients under risk. Risk Anal. 2020,40, 524–533. [CrossRef] [PubMed]
47.
Timarán, D.A.V.; Melo, C.J.B.; Caicedo, M.I.M.; Ceballos, A.M.C.; Vallejo, D.; Velásquez, C.A.C. Aislamiento de Cryptococcus
neoformans en heces de palomas (Columba livia) en el casco urbano del municipio de Pasto, Colombia. Biosalud
2016
,15, 62–71.
[CrossRef]
48.
Granados, D.P.; Castañeda, E. Isolation and characterization of Cryptococcus neoformans varieties recovered from natural sources
in Bogotá, Colombia, and study of ecological conditions in the área. Microb. Ecol. 2005,49, 282–290. [CrossRef]
49.
May, R.C.; Stone, N.R.; Wiesner, D.L.; Bicanic, T.; Nielsen, K. Cryptococcus: From environmental saprophyte to global pathogen.
Nat. Rev. Microbiol. 2016,14, 106–117. [CrossRef]
50.
Viviani, M.A.; Cogliati, M.; Esposto, M.C.; Lemmer, K.; Tintelnot, K.; Colom-Valiente, M.F.; Swinne, D.; Velegraki, A.; Velho, R.;
European Confederation of Medical Mycology (ECMM) Cryptococcosis Working Group. Molecular analysis of 311 Cryptococcus
neoformans isolates from a 30-month ECMM survey of cryptococcosis in Europe. FEMS Yeast Res. 2006,6, 614–619. [CrossRef]
51.
Cogliati, M.; Zani, A.; Rickerts, V.; McCormick, I.; Desnos-Ollivier, M.; Velegraki, A.; Escandon, P.; Ichikawa, T.; Ikeda, R.;
Bienvenue, A.L.; et al. Multilocus sequence typing analysis reveals that Cryptococcus neoformans var neoformans is a recombinant
population. Fungal Genet. Biol. 2016,87, 22–29. [CrossRef] [PubMed]
52.
Khan, Z.U.; Randhawa, H.S.; Kowshik, T.; Chowdhary, A.; Chandy, R. Antifungal susceptibility of Cryptococcus neoformans and
Cryptococcus gattii isolates from decayed wood of trunk hollows of Ficus religiosa and Syzygium cumini trees in north-western
India. J. Antimicrob. Chemother. 2007,60, 312–316. [CrossRef]
53.
Movahed, E.; Munusamy, K.; Tan, G.M.Y.; Looi, C.Y.; Tay, S.T.; Wong, W.F. Genome-wide transcription study of Cryptococcus
neoformans H99 clinical strain versus environmental strains. PLoS ONE 2015,10, e0137457. [CrossRef] [PubMed]
54.
Gutch, R.S.; Nawange, S.R.; Singh, S.M.; Yadu, R.; Tiwari, A.; Gumasta, R.; Kavishwar, A. Antifungal susceptibility of clinical and
environmental Cryptococcus neoformans and Cryptococcus gattii isolates in Jabalpur, a city of Madhya Pradesh in Central India.
Braz. J. Microbiol. 2015,46, 1125–1133. [CrossRef]
55.
Córdoba, S.; Isla, M.G.; Szusz, W.; Vivot, W.; Altamirano, R.; Davel, G. Susceptibility profile and epidemiological cut-off values of
Cryptococcus neoformans species complex from Argentina. Mycoses 2016,59, 351–356. [CrossRef] [PubMed]
56. Espinel-Ingroff, A.; Aller, A.I.; Canton, E.; Castañón-Olivares, L.R.; Chowdhary, A.; Cordoba, S.; Cuenca-Estrella, M.; Fothergill,
A.; Fuller, J.; Govender, N.; et al. Cryptococcus neoformans-Cryptococcus gattii species complex: An international study of wild-type
susceptibility endpoint distributions and epidemiological cutoff values for amphotericin B and flucytosine. Antimicrob. Agents
Chemother. 2012,56, 3107–3113. [CrossRef] [PubMed]
57.
Selb, R.; Fuchs, V.; Graf, B.; Hamprecht, A.; Hogardt, M.; Sedlacek, L.; Schwarz, R.; Idelevich, E.A.; Becker, S.L.; Held, J.; et al.
Molecular typing and
in vitro
resistance of Cryptococcus neoformans clinical isolates obtained in Germany between 2011 and
2017. Int. J. Med. Microbiol. 2019,309, 151336. [CrossRef]
58.
Pfaller, M.A.; Messer, S.A.; Boyken, L.; Rice, C.; Tendolkar, S.; Hollis, R.J.; Doern, G.V.; Diekema, D.J. Global trends in the
antifungal susceptibility of Cryptococcus neoformans (1990 to 2004). J. Clin. Microbiol. 2005,43, 2163–2167. [CrossRef] [PubMed]
59.
Chowdhary, A.; Randhawa, H.S.; Sundar, G.; Kathuria, S.; Prakash, A.; Khan, Z.; Sun, S.; Xu, J.
In vitro
antifungal susceptibility
profiles and genotypes of 308 clinical and environmental isolates of Cryptococcus neoformans var grubii and Cryptococcus gattii
serotype B from north-western India. J. Med. Microbiol. 2011,60, 961–967. [CrossRef]
60.
Carlson, T.; Lupinacci, E.; Moseley, K.; Chandrasekaran, S. Effects of environmental factors on sensitivity of Cryptococcus
neoformans to fluconazole and amphotericin B. FEMS Microbiol. Lett. 2021,368, fnab040. [CrossRef]
61.
Wang, Y.; Casadevall, A. Decreased susceptibility of melanized Cryptococcus neoformans to UV light. Appl. Environ. Microbiol.
1994
,
60, 3864–3866. [CrossRef]
Pathogens 2022,11, 8 15 of 15
62.
Thompson III, G.R.; Wiederhold, N.P.; Fothergill, A.W.; Vallor, A.C.; Wickes, B.L.; Patterson, T.F. Antifungal susceptibilities among
different serotypes of Cryptococcus gattii and Cryptococcus neoformans.Antimicrob. Agents Chemother.
2009
,53, 309–311. [CrossRef]
[PubMed]
63.
O’Meara, T.R.; Alspaugh, J.A. The Cryptococcus neoformans capsule: A sword and a shield. Clin. Microbiol. Rev.
2012
,25, 387–408.
[CrossRef]
64.
Carneiro, H.C.S.; Ribeiro, N.Q.; Bastos, R.W.; Santos, D.A. Effect of non-antifungal agrochemicals on the pathogenic fungus
Cryptococcus gattii.Med. Mycol. 2020,58, 47–53. [CrossRef]
65.
Bastos, R.W.; Carneiro, H.C.S.; Oliveira, L.V.N.; Rocha, K.M.; Freitas, G.J.C.; Costa, M.C.; Magalhães, T.F.F.; Carvalho, V.S.D.;
Rocha, C.E.; Ferreira, G.F.; et al. Environmental triazole induces cross-resistance to clinical drugs and affects morphophysiology
and virulence of Cryptococcus gattii and C. neoformans.Antimicrob. Agents Chemother. 2018,62, e01179-17. [CrossRef] [PubMed]
66.
Gusa, A.; Williams, J.D.; Cho, J.E.; Averette, A.F.; Sun, S.; Shouse, E.M.; Heitman, J.; Alspaugh, J.A.; Jinks-Robertson, S. Transposon
mobilization in the human fungal pathogen Cryptococcus is mutagenic during infection and promotes drug resistance
in vitro
.
Proc. Natl. Acad. Sci. USA 2020,117, 9973–9980. [CrossRef] [PubMed]
67.
Billmyre, R.B.; Clancey, S.A.; Li, L.X.; Doering, T.L.; Heitman, J. 5-fluorocytosine resistance is associated with hypermutation and
alterations in capsule biosynthesis in Cryptococcus.Nat. Commun. 2020,11, 127. [CrossRef]
68.
Pais, P.; Pires, C.; Costa, C.; Okamoto, M.; Chibana, H.; Teixeira, M.C. Membrane proteomics analysis of the Candida glabrata
response to 5-flucytosine: Unveiling the role and regulation of the drug efflux transporters CgFlr1 and CgFlr2. Front. Microbiol.
2016,7, 2045. [CrossRef] [PubMed]
69.
Lee, G.H.A.; Arthur, I.; Merritt, A.; Leung, M. Molecular types of Cryptococcus neoformans and Cryptococcus gattii in Western
Australia and correlation with antifungal susceptibility. Med. Mycol. 2019,57, 1004–1010. [CrossRef] [PubMed]
70.
Edwards, H.M.; Cogliati, M.; Kwenda, G.; Fisher, M.C. The need for environmental surveillance to understand the ecology,
epidemiology and impact of Cryptococcus infection in Africa. FEMS Microbiol. Ecol. 2021,97, fiab093. [CrossRef] [PubMed]
71.
Cogliati, M. Global warming impact on the expansion of fundamental niche of Cryptococcus gattii VGI in Europe. Environ.
Microbiol. Rep. 2021,13, 375–383. [CrossRef]
72.
Keesing, F.; Belden, L.K.; Daszak, P.; Dobson, A.; Harvell, C.D.; Holt, R.D.; Hudson, P.; Jolles, A.; Jones, K.E.; Mitchell, C.E.; et al.
Impacts of biodiversity on the emergence and transmission of infectious diseases. Nature 2010,468, 647–652. [CrossRef]
73.
Chowdhary, A.; Hiremath, S.S.; Sun, S.; Kowshik, T.; Randhawa, H.S.; Xu, J. Genetic differentiation, recombination and clonal
expansion in environmental populations of Cryptococcus gattii in India. Environ. Microbiol. 2011,13, 1875. [CrossRef] [PubMed]
74.
Sanchini, A.; Smith, I.M.; Sedlacek, L.; Schwarz, R.; Tintelnot, K.; Rickerts, V. Molecular typing of clinical Cryptococcus neoformans
isolates collected in Germany from 2004 to 2010. Med. Microbiol. Immunol. 2014,203, 333–340. [CrossRef] [PubMed]
75.
Li, W.; Averette, A.F.; Desnos-Ollivier, M.; Ni, M.; Dromer, F.; Heitman, J. Genetic diversity and genomic plasticity of Cryptococcus
neoformans AD hybrid strains. G3: Genes|Genomes|Genet. 2012,2, 83–97. [CrossRef] [PubMed]
76.
Meyer, W.; Castañeda, A.; Jackson, S.; Huynh, M.; Castañeda, E.; IberoAmerican Cryptococcal Study Group. Molecular typing of
IberoAmerican Cryptococcus neoformans isolates. Emerg. Infect. Dis. 2003,9, 189. [CrossRef]
77.
European Committee on Antimicrobial Susceptibility Testing (EUCAST) Definitive Document E.DEF 7.3.1. Available on-
line: https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/EUCAST_E_Def_7_3_1_Yeast_testing_
_definitive.pdf (accessed on 4 November 2021).
78. Espinel-Ingroff, A.; Aller, A.I.; Canton, E.; Castanon-Olivares, L.R.; Chowdhary, A.; Cordoba, S.; Cuenca-Estrella, M.; Fothergill,
A.; Fuller, J.; Govender, N.; et al. Cryptococcus neoformans-Cryptococcus gattii species complex: An international study of wild-
type susceptibility endpoint distributions and epidemiological cutoff values for fluconazole, itraconazole, posaconazole, and
voriconazole. Antimicrob. Agents Chemother. 2012,56, 5898–5906. [CrossRef] [PubMed]
79.
Espinel-Ingroff, A.; Chowdhary, A.; Gonzalez, G.M.; Guinea, J.; Hagen, F.; Meis, J.F.; Thompson, G.R.; Turnidge, J. Multicenter
Study of Isavuconazole MIC Distributions and Epidemiological Cutoff Values for the Cryptococcus neoformans-Cryptococcus
gattii Species Complex Using the CLSI M27-A3 Broth Microdilution Method. Antimicrob. Agents Chemother.
2014
,59, 666–668.
[CrossRef] [PubMed]
