Mycopathologia

, Volume 174, Issue 4, pp 283–291

Cryptococcus neoformans/Cryptococcus gattii Species Complex in Southern Italy: An Overview on the Environmental Diffusion of Serotypes, Genotypes and Mating-Types

Authors

  • Orazio Romeo
    • Section of Microbiological, Genetic and Molecular Sciences, Department of Life SciencesUniversity of Messina
  • Fabio Scordino
    • Section of Microbiological, Genetic and Molecular Sciences, Department of Life SciencesUniversity of Messina
  • Valeria Chillemi
    • Section of Microbiological, Genetic and Molecular Sciences, Department of Life SciencesUniversity of Messina
    • Section of Microbiological, Genetic and Molecular Sciences, Department of Life SciencesUniversity of Messina
Article

DOI: 10.1007/s11046-012-9547-6

Cite this article as:
Romeo, O., Scordino, F., Chillemi, V. et al. Mycopathologia (2012) 174: 283. doi:10.1007/s11046-012-9547-6

Abstract

Given the lack of comprehensive molecular epidemiology studies in Reggio Calabria and Messina, Italy, we decided to perform an extensive environmental sampling to describe the current molecular epidemiology of C. neoformans/C. gattii species complex in southern Italy. In this study, we report the occurrence of serotypes, genotypes and mating-types of isolates of the C. neoformans/C. gattii species complex recovered from environmental sources. In addition, a number of environmental C. neoformans var. grubii strains, isolated in 1997 by our laboratory, were also retrospectively examined in order to compare their genotypes with those recently found and to infer the possible epidemiological changes in our country. One hundred and twenty-two isolates were identified as being C. neoformans, whereas only one was found to belong to C. gattii serotype B, genotype VGI and mating-type alpha. Our data revealed that all environmental isolates of C. neoformans recovered here as well as those previously isolated in 1997 belong to serotype A and genotype VNI and posses a mating-type alpha allele.

Keywords

Cryptococcus gattiiCryptococcus neoformansCryptococcosisMolecular typingEpidemiologySerotypesMating-types

Introduction

The genus Cryptococcus is comprised of at least 100 fungal species [1] of which only two in particular, Cryptococcus neoformans and Cryptococcus gattii, have been associated with life-threatening infections in humans and other animals.

However, in recent years, the taxonomy of C. neoformans has undergone important changes due to the rapid development of molecular biology techniques, which have shown that this species is, in reality, a ‘cryptic species complex’ [2, 3].

Currently, C. neoformans is classified into two varieties: C. neoformans var. grubii (serotype A), which has a worldwide distribution, and C. neoformans var. neoformans (serotype D), which is found mainly in temperate countries [4]. Cryptococcus neoformans var. gattii (serotypes B and C) is now recognized as separate species (C. gattii) on the basis of phenotypic and genetic differences [5, 6].

The increasing application of molecular epidemiology methods as well as phylogenetic and genotypic analysis of many clinical, environmental and hybrid isolates showed that the C. neoformans/C. gattii species complex is highly divergent and it can be subdivided into nine major molecular types: VNI/AFLP1, VNII/AFLP1B and VNB/AFLP1A (C. neoformans var. grubii, serotype A), VNIII/AFLP3 (AD hybrid, serotype AD), VNIV/AFLP2 (C. neoformans var. neoformans, serotype D), and VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5 and VGIV/AFLP7/AFLP10 (C. gattii serotypes B and C). This kind of classification has, significantly, improved our knowledge of the epidemiology of C. neoformans/C. gattii species complex [2, 3, 79] allowing a better understanding of the incidence and geographic distribution of particular hypervirulent strains [6, 10].

Moreover, recent epidemiological studies have confirmed that strains of serotype A are much more ubiquitous and prevalent and VNI genotype predominates worldwide causing the majority of cases of cryptococcosis [9]. The VNII genotype is also globally distributed but rarely encountered, whereas the VNB/AFLP1A genotype although, initially, found only in southern Africa, it has recently been recovered from clinical and environmental samples in Brazil and patients in Europe [7, 11, 12].

The occurrence of serotype D (VNIV/AFLP2 genotype) is scarcely reported in literature although, in past, it has frequently been found in several European countries [2, 11] and occasionally in Australia, New Zealand, Thailand, United States [2, 13] and India, where this serotype even outranks C. neoformans var. grubii in the pellets of different avifauna [14].

Cryptococcus gattii (genotypes VGI–IV) has traditionally been associated with tropical and subtropical climates where it is repeatedly found in Eucalyptus trees [15]. However, the recent emergence of hypervirulent genotypes (VGII genotype) in Canada and United States [16] as well as the publication of several clinical and environmental isolations of C. gattii in more temperate regions [1723] shows that it is able to occur in a wide range of geographical areas, thus stimulating an intensive research into this species. However, notwithstanding the efforts made so far, global population genetic structure of C. neoformans/C. gattii species complex still remains undetermined and needed more investigations [9].

At the end of nineties, in Italy, the epidemiology of C. neoformans serotype A in the southern regions mirrored the picture observed worldwide [24]; in fact, it has long been believed that there were only C. neoformans serotype A isolates but more recently strains of C. gattii serotype B have also been isolated [20, 23], indicating a probable change over time of the epidemiology and the occurrence of multiple genotypes in our natural environment. On the basis of these observations, we decided to perform an extensive environmental sampling to describe the current molecular epidemiology of C. neoformans/C. gattii species complex in southern Italy.

In this study, we report the occurrence of serotypes, genotypes and mating-types of isolates of the C. neoformans/C. gattii complex recovered from environmental sources in Italy.

Materials and Methods

Samples Collection and Processing

A total of 495 samples of bird droppings, soil, Ceratonia siliqua (carob tree), Eucalyptus camaldulensis and Prunus dulcis (almond tree) debris were collected from a variety of public and private places (city seafront, parks, squares, bird owners and pet shops) in Messina and Reggio Calabria, southern Italy (Table 1).
Table 1

Environmental samples examined and phenotypic and molecular results obtained in this study

Samples

City/sampling area

TS

PS (%)

Phenotypic analysis

Molecular analysis

No strains/species

Serotype

Genotype/mating-type

Bird droppings

Messina, University

9

4 (44)

7/C. neoformans

A

VNI/α

2/C. albidus

Bird droppings

Messina, Pet shop1

3

1 (33)

3/C. neoformans

A

VNI/α

Bird droppings

Messina, Pet shop2

5

2 (40)

2/C. neoformans

A

VNI/α

3/C. laurentii

Bird droppings

Messina, Bird owner

12

3 (25)

4/C. neoformans

A

VNI/α

Bird droppings

Reggio Calabria, Pet shop

5

Bird droppings

Reggio Calabria, Bird owner

8

2 (25)

5/C. neoformans

A

VNI/α

Pigeon Guano

Messina, Bird owner

5

1 (20)

1/C. neoformans

A

VNI/α

Pigeon Guano

Messina, City hall

7

3 (42.8)

3/C. neoformans

A

VNI/α

1/C.laurentii

Pigeon Guano

Messina, Tribunal

4

Pigeon Guano

Messina, University

9

3 (33.3)

3/C. neoformans

A

VNI/α

1/C. albidus

Pigeon Guano

Messina, Main square

8

3 (37,5)

7/C. neoformans

A

VNI/α

Pigeon Guano

Messina, Cathedral

5

Pigeon Guano

Messina, Train station

11

6 (54.5)

13/C. neoformans

A

VNI/α

4/C. terreus

3/C. laurentii

Pigeon Guano

Reggio Calabria, Bird owner1

6

4 (66.6)

5/C. neoformans

A

VNI/α

1/C. terreus

Pigeon Guano

Reggio Calabria, Bird owner2

7

2 (28.5)

5/C. neoformans

A

VNI/α

Pigeon Guano

Reggio Calabria, Bird owner3

6

3 (50)

3/C. neoformans

A

VNI/α

Pigeon Guano

Reggio Calabria, Cathedral

4

1 (25)

3/C. neoformans

A

VNI/α

Pigeon Guano

Reggio Calabria, Train station

5

1 (20)

1/C. neoformans

A

VNI/α

Pigeon Guano

Reggio Calabria, City seafront

13

5 (38.4)

3/C. neoformans

A

VNI/α

2/C. laurentii

1/C. albidus

Pigeon Guano

Reggio Calabria, City hall

4

E. camaldulensis

Messina, University

9

1 (11)

1/C. neoformans

A

VNI/α

E. camaldulensis

Messina, Ganzirri lake

12

E. camaldulensis

Messina, City centre

13

1 (7.7)

1/C. neoformans

A

VNI/α

E. camaldulensis

Messina, Public parks

8

E. camaldulensis

Reggio Calabria, City seafront

13

3 (23)

1/C. gattii

B

VGI/α

3/C. neoformans

A

VNI/α

1/C. laurentii

E. camaldulensis

Reggio Calabria, Train station

4

E. camaldulensis

Reggio Calabria, Public parks

9

2 (22.2)

2/C. neoformans

A

VNI/α

Rhizosphere Soil (E)a

Messina, University

9

Rhizosphere Soil (E)a

Messina, Ganzirri lake

12

Rhizosphere Soil (E)a

Messina, City centre

13

Rhizosphere Soil (E)a

Messina, Public parks

8

Rhizosphere Soil (E)a

Reggio Calabria, City seafront

13

Rhizosphere Soil (E)a

Reggio Calabria, Train station

4

Rhizosphere Soil (E)a

Reggio Calabria, Public parks

9

Ceratonia siliqua

Reggio Calabria, private tree

12

Ceratonia siliqua

Reggio Calabria, hilly area

7

1 (14.2)

1/C. neoformans

A

VNI/α

Ceratonia siliqua

Messina, University

4

Ceratonia siliqua

Messina, private tree

9

Rhizosphere Soil (C)b

Reggio Calabria, private tree

26

2 (7.7)

2/C. neoformans

A

VNI/α

1/C. laurentii

Rhizosphere Soil (C)b

Reggio Calabria, hilly area

13

Rhizosphere Soil (C)b

Messina, University

9

Rhizosphere Soil (C)b

Messina, private tree

16

Prunus dulcis

Reggio Calabria, private trees

15

Prunus dulcis

Reggio Calabria, hilly area

8

Prunus dulcis

Messina, private trees

9

1 (11)

1/C. neoformans

A

VNI/α

1/C. terreus

Prunus dulcis

Messina, hilly area

11

Rhizosphere Soil (A)c

Reggio Calabria, private trees

29

1 (3.4)

1/C. neoformans

A

VNI/α

Rhizosphere Soil (A)c

Reggio Calabria, hilly area

14

Rhizosphere Soil (A)c

Messina, private trees

18

2 (11)

2/C. neoformans

A

VNI/α

1/C. albidus

1/C. terreus

Rhizosphere Soil (A)c

Messina, hilly area

23

1 (4.3)

1/C. neoformans

A

VNI/α

Canary droppings

Yeast culture collectiond

27/C. neoformans

A

VNI/α

Pigeon dropping

Yeast culture collectiond

11/C. neoformans

A

VNI/α

Parrot droppings

Yeast culture collectiond

1/C. neoformans

A

VNI/α

Total

495

59

146

123e

123e

TS total samples, PS positive samples, a Eucalyptus tree, Carob tree, Almond tree, d Criseo et al. [24], One isolate of C. gattii ser. B-VGI/α

All environmental samplings were performed, intermittently, during the 3 months of spring 2011 (March–June).

Pigeon guano was primarily collected from the main squares and parks of the two cities where the pigeons were observed feeding or roosting for several weeks.

Samples of carob, almond and Eucalyptus trees were a mixture of shredded bark and leaves, including vegetable materials found where there were hollows in the tree for the pooling of water. Rhizosphere soil was also collected from each sampled tree and included in the study (Table 1).

The samples were collected in sterile plastic bags, stored at 4 °C and processed within 24–48 h after collection.

A 10-g (1 g for bird droppings) quantity of each environmental sample was suspended in 90 ml of sterile physiological solution (supplemented with 20 U/ml penicillin and 40 U/ml streptomycin) and homogenized using a Stomacher blender (Lab-blender 400, PBI International Milan, Italy) for 1 min. After allowing the suspension to settle for 15 min at room temperature, 1 ml of the supernatant was serially diluted up to 10−4 and 0.1 ml aliquots were plated, in duplicate, onto Staib agar containing penicillin (20 U/ml), streptomycin (40 U/ml) and biphenyl (0.1 %).

The plates were incubated at 26 °C and daily examined, up to 2 weeks for the presence of colonies showing the characteristic ‘brown colour effect’ (BCE).

In order to avoid any error possibly resulting from the presence of multiple species on primary cultures, yeasts colonies suspected of being Cryptococcus spp. were subcultured onto modified Sabouraud dextrose agar (SDA) supplemented with antibiotics (20 U penicillin; 40 μg/ml streptomycin) and identified by using both phenotypic and molecular methods. The reference strains used in this study are listed in Table 2.
Table 2

Reference strains used in this study

Strains

Species

Source/origin

Molecular types

Serotype

Mating-type

JEC21

C. neoformans

Derived from JEC20

VNIV/AFLP2

D

α

CBS132

C. neoformans

Fermenting fruit juice

VNIII/AFLP3

AD

αADa

WM626

C. neoformans

Human/Australia

VNII/AFLP1B

A

α

H99

C. neoformans

Human/USA

VNI/AFLP1

A

α

WM163

C. gattii

Eucalyptus/Australia

VGI/AFLP4

B

α

NIH444

C. gattii

Human/USA

VGII/AFLP6

B

α

NIH18

C. gattii

Human/USA

VGIII/AFLP5

C

α

WM779

C. gattii

Cheetah/South Africa

VGIV/AFLP7

C

α

Thirty-nine environmental strains of C. neoformans serotype A isolated, in 1997, in Reggio Calabria and Messina by Criseo and Gallo [24] (Table 1) were retrospectively examined in order to compare their genotypes with those recently found.

Phenotypic Identification

All yeast isolates with positive reaction for phenoloxidase (BCE) were, initially, microscopically examined using Indian ink preparations for detecting the presence of the polysaccharide capsule.

The strains were further characterized by studying urease hydrolysation on Christensen’s urea medium and assimilation of carbohydrates using the ID32 C system (bioMerieux, Marcy l’Étoile, France) with additional phenotypic tests such as Dalmau plate culture on corn meal agar, nitrate assimilation, starch production and thermotolerance at 37 °C [1].

The biovarieties were determined by culturing the isolates (48–72 h at 25 °C) on the selective medium l-canavanine glycine bromothymol blue (CGB) agar [25] and by the d-proline and the d-tryptophan assimilation tests [26, 27].

Molecular Typing of Fungal Isolates

DNA Isolation

Genomic DNA was extracted from yeast colonies, grown after 48 h at 30 °C in yeast extract peptone dextrose (YEPD) broth, using glass beads and phenol/chloroform method as described by Muller et al. [28].

PCR–Restriction Fragment Length Polymorphism (RFLP) Analysis of GEF1 Gene

The GEF1 gene, located within the MAT locus of C. neoformans, was selected for RFLP analysis because, compared to the PCR–RFLP of URA5 gene [3], it has the potential to identify both molecular and mating-types simultaneously [29].

For each strain, amplification of GEF1 gene was performed using DreamTaqTM PCR master mix (Fermentas, Italy), a ready-to-use pre-mixed solution containing Taq DNA Polymerase, specific buffer, MgCl2 and dNTPs to which 2 μl of DNA template and 0.5 μl of GEF1F-GGACCCATGCCTGAAATGTG and GEF1R-TACGCTTGC CCCGATCTG (10 mM) primer was added [29].

The expected 1,339 bp amplicon was digested, separately, with the restriction endonucleases EcoT14I and HapII (Fermentas) according to the manufacturer’s instructions. The resulting DNA fragments were separated and visualized on a 2 % (wt/vol) agarose gel stained with ethidium bromide.

The assignment of both molecular and mating-types was done by comparing, visually, the obtained RFLP patterns with those of reference strains used in this study (Table 2).

Molecular Determination of Serotypes by PCR/RFLP Analysis of CAP59 Gene

The CAP59 gene was amplified using the DreamTaq™ PCR master mix (Fermentas) with primers CH-Cap59F-CCTTGCCGAAGTTCGAAACG and CH-Cap59R-AATCGGTGGTTGGATTCA GTGT which yield an expected DNA fragment of ~400 bp [30]. Each amplicon was then digested, individually, with the enzymes BsmFI, HpaII and AgeI (Fermentas) according to the standard protocol of the supplier, and the resulting restriction fragments were separated on a 3 % agarose gel and stained with ethidium bromide.

Serotypes were manually assigned according to the PCR–RFLP patterns obtained from reference strains included in this study.

Results

The data obtained in this study are summarized in Table 1 and further illustrated in Fig. 1.
https://static-content.springer.com/image/art%3A10.1007%2Fs11046-012-9547-6/MediaObjects/11046_2012_9547_Fig1_HTML.gif
Fig. 1

Restriction analysis of the GEF1 gene showing the VNI/mating-type alpha genotype of some random selected C. neoformans isolates examined in this study. Lanes 5–7 DNA profiles obtained from isolates recovered in this study; Lanes 8–9 DNA profiles obtained from isolates recovered in 1997 by Criseo and Gallo [24]. Lanes 1–4 reference strains: JEC21 (VNIV), CBS132 (VNIII), WM626 (VNII) and H99 (VNI), respectively. Lane M molecular size marker. E profile obtained with EcoT14l enzyme, H profile obtained with HapII enzyme

Among 495 environmental samples examined, a total of 146 yeast isolates were recovered. Twenty-three isolates were phenotypically identified as Cryptococcus laurentii (n = 11), Cryptococcus terreus (n = 7) and Cryptococcus albidus (n = 5) (Table 1) by ID32C system and conventional methods [1].

Using phenotypic methods, 122 isolates were identified as being C. neoformans, whereas only one was found to belong to C. gattii (Table 1). All these yeast isolates were capsulated and showed urease, phenoloxidase activity and growth at 37°C. Subcultures of the yeasts onto CGB agar confirmed the presence of one C. gattii isolate recovered from E. camaldulensis tree in Reggio Calabria. It showed degradation of glycine resulting into alkalization of the medium and conversion of colonies from yellow into blue. In addition, this isolate assimilated d-proline and d-tryptophan but all other 122 yeast isolates tested did not; furthermore, most of these latter isolates (n = 109) did not grow on CGB agar or left the medium unchanged (n = 13) indicating thus that all of them belonged to C. neoformans.

These data show that C. gattii is present in our environment, and it is associated with E. camaldulensis in Reggio Calabria. In fact, this strain was recovered from an Eucalyptus tree close to those where it was already previously isolated [23]. Molecular analysis showed that this C. gattii strain belonged to serotype B and VGI genotype and posses a mating-type alpha allele (Table 1). No C. gattii isolates were recovered from carob, and almond trees examined in this study.

Molecular data obtained from all environmental isolates of C. neoformans recovered here revealed that they were all serotype A with a unique genotypic profile: VNI/mating-type alpha (Table 1; Fig. 1). Identical results have also been obtained by retrospective genetic analysis of 39 environmental C. neoformans strains recovered in 1997 from the same geographical areas re-considered in this study (Table 1) [24].

Discussion

The members of the C. neoformans/C. gattii species complex have always attracted considerable attention because of their intrinsic pathogenic nature, but, in recent years, even more because cryptococcosis has become a significant public global health problem afflicting not only immunocompromised individuals but also apparently immunocompetent subjects [31].

Over the past decade, the emergence of hypervirulent strains of C. gattii has resulted in a constant and intensive research in the biology of this microorganism, especially in the countries where it is currently considered as an endemic species [6, 32].

Numerous studies have focused on virulence, antifungal susceptibility, genotyping and epidemiology of this new species, thus providing invaluable data on its pathogenicity, role in human infections and impact on public health [10, 16].

Historically, C. gattii has been ecologically associated with Eucalyptus trees and geographically restricted to tropical and sub-tropical regions of the world [6]. However, more recent epidemiological data indicated that this species can adapt to new climatic conditions including those found in the Mediterranean area where this fungus is responsible for many human and animal infections [1722]. Recently, we demonstrated that C. gattii is present in the Italian environment [23] and the data reported in this study confirm that it can be consistently isolated. In addition, according to previous studies [18, 23, 33], the present results showed that the VGI/mating-type alpha is the only genotype of C. gattii known to occur in the Mediterranean environment and it can be found, with low rate of isolation, in Eucalyptus trees [23]. However, a high incidence of VGI strains has also been recently reported in carob tree samples from Spain [18] and an additional environmental association of C. gattii with almond trees has also described in Tunisia [22]. On the basis of these findings, we decided to examine both these species of plants but they were all negative for C. gattii. This indicates that the data obtained about on the ecology of this species in our geographical area need more investigations.

Another interesting evidence that emerges from this study is that during the years 1997–2011 the epidemiology of C. neoformans in southern Italy has almost remained unchanged. In fact, all previously serotype A strains isolated in 1997, as well as those recently recovered here, have similar molecular pattern (VNI/mating-type alpha). Other molecular types of C. neoformans such as VNII, VNIV or hybrids VNIII were not found among our isolates, and these data tend to confirm the assumption that different geographic and climatological conditions play an important role in serotype and genotype distribution [34, 35]. In fact, to our knowledge, in past years, C. neoformans var. neoformans isolates have never been found in clinical and environmental samples in southern Italy, whereas they are frequently isolated in northern regions [3638].

Taken together, these observations, including the data reported in this study, indicate that the occurrence and distribution of C. neoformans serotypes in southern Italy have largely remained unaltered during the past 15 years, although the presence of serotype B strains of C. gattii has recently been reported [23]. However, the occurrence of C. gattii in our country has been known since 1997 when it was reported that C. gattii occurs in Apulia (Italy) [39, 40]. More recently, in the same region, a new case of C. gattii (VGI/mating-type alpha) infection has been reported [20] concurrently with an environmental isolation of the same genotype in a close geographical area [23]. The patient was immunocompetent, and although he visited Toronto and Montreal (Canada) for a month in 2007, the presence of the VGI genotype suggested that the patient was probably infected in his living area, rather than during his visit to Canada where instead it is well known that the VGII genotype represents the most common infectious type encountered so far [6, 10, 20, 41]. These data highlight the need for additional environmental and clinical studies as C. gattii is present in our geographic area and it could represent a potential emerging fungus in the future as has happened in North America where the hypervirulent VGII genotype has been present in the environment for more than 30 years before causing the recent outbreaks [41].

Although serotype A/VNII (both AFLP1A/AFLP1B types) strains have been reported in the environments of several European countries [2, 11], they have never been isolated in southern Italy. However, based on concordance of different molecular typing methods used for C. neoformans isolates [42], it was surprising to find that the VNII genotype, reported as PCR-fingerprinting molecular type VN7 (strain IUM896897) by Viviani et al. [43], was responsible for human infection in the north Italian regions [43] where the environmental climatic conditions and abiotic factors are probably most favourable for survival and dissemination of serotype A/VNII and serotype D/VNIV strains. However, such an observation was already done in the past when it was demonstrated that C. neoformans var. neoformans strains are more susceptible to high temperatures [35] that are commonly found in southern regions of Europe.

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© Springer Science+Business Media B.V. 2012