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Published in final edited form as: Fungal Genet Biol. 2016 Jan 6;87:22–29. doi: 10.1016/j.fgb.2016.01.003

Multilocus sequence typing analysis reveals that Cryptococcus neoformans var. neoformans is a recombinant population

Massimo Cogliati a,*, Alberto Zani a, Volker Rickerts b, Ilka McCormick b, Marie Desnos-Ollivier c, Aristea Velegraki d, Patricia Escandon e, Tomoe Ichikawa f, Reiko Ikeda f, Anne-Lise Bienvenue g,h, Kathrin Tintelnot b, Okan Tore j, Sevim Akcaglar j, Shawn Lockhart k, Anna Maria Tortorano a, Ashok Varma i
PMCID: PMC4754669  NIHMSID: NIHMS753767  PMID: 26768709

Abstract

Cryptococcus neoformans var. neoformans (serotype D) represents about 30% of the clinical isolates in Europe and is present less frequently in the other continents. It is the prevalent etiological agent in primary cutaneous cryptococcosis as well as in cryptococcal skin lesions of disseminated cryptococcosis. Very little is known about the genotypic diversity of this Cryptococcus subtype. The aim of this study was to investigate the genotypic diversity among a set of clinical and environmental C. neoformans var. neoformans isolates and to evaluate the relationship between genotypes, geographical origin and clinical manifestations. A total of 83 globally collected C. neoformans var. neoformans isolates from Italy, Germany, France, Belgium, Denmark, Greece, Turkey, Thailand, Japan, Colombia, and the USA, recovered from different sources (primary and secondary cutaneous cryptococcosis, disseminated cryptococcosis, the environment, and animals), were included in the study. All isolates were confirmed to belong to genotype VNIV by molecular typing and they were further investigated by MLST analysis. Maximum likelihood phylogenetic as well as network analysis strongly suggested the existence of a recombinant rather than a clonal population structure. Geographical origin and source of isolation were not correlated with a specific MLST genotype. The comparison with a set of outgroup C. neoformans var. grubii isolates provided clear evidence that the two varieties have different population structures.

Keywords: Cryptococcus, C. neoformans var. neoformans, C. neoformans var. grubii, MLST, Recombination

1. Introduction

Cryptococcus neoformans and C. gattii are two sibling yeast species responsible for cryptococcosis. This life-threatening disease is mainly associated with AIDS patients in the countries where the HIV infection burden is still high such as in sub-Saharan Africa and in South East Asia (Assogba et al., 2015; Park et al., 2009). In developed countries, however, the incidence of cryptococcosis in HIV-infected population is decreasing due to the introduction of high active antiretroviral therapy (HAART). In contrast, the disease is increasingly found in non-AIDS patients such as those with hematological neoplasms, recipients of organ transplantation, and victims of autoimmune diseases (Bratton et al., 2012; Henao-Martínez and Beckham, 2015; Sanchini et al., 2014).

C. neoformans is classified into two varieties, three serotypes and five molecular types. C. neoformans var. grubii, serotype A, is identified as the molecular types VNI, VNII and VNB, whereas C. neoformans var. neoformans, serotype D, belongs to molecular type VNIV. In addition, diploid or aneuploid intervarietal AD hybrids are identified as molecular type VNIII. Cryptococcus gattii has two serotypes, B and C, and four molecular types VGI, VGII, VGII, and VGIV (Heitman et al., 2010).

The recent C. gattii emergence from Vancouver Island (Canada) south to the Pacific Northwest of the United States, has contributed to the interest in C. gattii in different parts of the world and, at the same time, to highlight the differences from C. neoformans in both ecological distribution and clinical manifestations (Espinel-Ingroff and Kidd, 2015; Chen et al., 2014).

C. neoformans var. grubii is the prevalent agent of cryptococcosis and it is globally distributed. It is commonly isolated from pigeon and other bird excreta and soil as well as many species of trees (Cogliati, 2013). The main clinical manifestation and the cause of death is meningoencephalitis especially in immunocompromised patients (Kwon-Chung et al., 2014).

C. neoformans var. neoformans has not been extensively investigated and little is known about its ecology, distribution and clinical manifestations. The majority of clinical isolates were reported from Europe where it has a prevalence of 30% (Viviani et al., 2006), but it has also been found in North (Yan et al., 2002; Litvintseva et al., 2005) and South America (Pérez et al., 2008; Cortés et al., 2011; Meyer et al., 2003; Trilles et al., 2003) as well as in Asia (Sukroongreung et al., 1996; Feng et al., 2008; Capoor et al., 2008; Ikeda and Shinoda, 2000). Very few isolates have been recovered from the environment mainly from pigeon droppings. A recent large environmental survey carried out in Europe identified the association of this variety with different tree species (Cogliati et al., 2014). At present, little information about specific clinical manifestations is available although a skin tropism of this yeast has been shown. A study carried out on 108 Cryptococcus isolates recovered from patients with skin lesions clearly indicated that infection with serotype D isolates was one of the risk factors in cutaneous manifestations (Neuville et al., 2003).

The present study aims to investigate a large number of C. neoformans var. neoformans strains isolated from different geographical areas and from different sources in order to elucidate the genetic population structure of this variety.

2. Materials and methods

2.1. Isolates

Eighty-three C. neoformans var. neoformans isolates were investigated (Table 1). Twenty isolates were from Germany, 19 from Italy, 13 from Greece, 8 from Japan, 7 from France, 4 from Belgium, 4 from Denmark, 4 from Colombia, and 1 each from Australia, Thailand, Turkey and the USA. Twenty-six isolates were recovered from the environment (soil, trees, pigeon droppings and dust), 22 from cases of disseminated cryptococcosis, 17 from cases of documented primary cutaneous cryptococcosis, 4 from cases of probable primary cutaneous cryptococcosis, 9 from cases of secondary cutaneous cryptococcosis, and 5 from veterinary cases. All clinical cases were independent cases and no multiple isolates from the same patient were included in the study. In addition, data obtained from previous studies of 30 C. neoformans var. grubii isolates (11 VNB, 10 VNI, and 9 VNII) were included in the analysis as outgroup strains (Khayhan et al., 2013; Litvintseva et al., 2006; Sanchini et al., 2014; Cogliati et al., 2013; Meyer et al., 2009; Kaocharoen et al., 2013; Choi et al., 2010; Umeyama et al., 2013; Wiesner et al., 2012) (Table S1).

Table 1.

Clinical and molecular information of the 83 C. neoformans var. neoformans isolates investigated in the present study.

Strain code Category Origin Source Date Underlying disease Molecular
type		 Mating
type		 Sequence
type		 CAP59 GPD1 IGS1 LAC1 PLB1 SOD1 URA5 Reference
WM629 DC Australia Blood 1987 AIDS VNIV αD 117 16 21 30 19 13 1 19 Meyer
IUM 01-4729 ENV Belgium Pigeon droppings 2001 – VNIV αD 510 16 21 24 18 13 20 32 Cogliati
IUM 01-4730 ENV Belgium Dust 2001 – VNIV αD 514 16 40 24 20 13 20 32 Cogliati
IUM 97-4899 PPCC Belgium Skin 1997 No risk factors VNIV αD 507 16 3 26 39 14 20 34 Cogliati
IUM 98-5036 PPCC Belgium Skin 1998 Diabete VNIV αD 506 16 3 24 20 13 20 32 Cogliati
H0058-I-1406 DC Colombia CSF 2002 AIDS VNIV αD 335 27 28 30 19 14 17 41 Escandon
H0058-I-2250 DC Colombia CSF 2004 AIDS VNIV αD 336 16 22 32 14 14 18 17 Escandon
H0058-I-2291 DC Colombia CSF 2004 AIDS VNIV αD 160 16 21 30 19 13 17 19 Escandon
H0058-I-2880 DC Colombia CSF 2007 AIDS VNIV αD 336 16 22 32 14 14 18 17 Escandon
NIH-424 ENV Denmark Pigeon nest 1970 – VNIV αD 180 20 21 26 21 19 17 21 Kwon-Chung
NIH-429 ENV Denmark Pigeon nest 1970 – VNIV αD 512 16 21 31 19 13 19 16 Kwon-Chung
NIH-430 ENV Denmark Pigeon nest 1970 – VNIV aD 509 16 20 24 16 14 20 16 Kwon-Chung
NIH-433 ENV Denmark Pigeon nest 1970 – VNIV aD 515 17 21 28 13 14 17 16 Kwon-Chung
CNRMA00.330 PCC France Skin 2000 No risk factor VNIV αD 135 27 22 43 24 13 17 20 Dromer
CNRMA00.840 SCC France Skin 2000 Hematological malignancy VNIV αD 125 16 21 45 21 13 21 22 Dromer
CNRMA07.1501 SCC France CSF 2007 AIDS VNIV αD 180 20 21 26 21 19 17 21 Dromer
CNRMA97.697 DC France CSF 1997 AIDS VNIV αD 121 16 21 32 19 13 17 20 Dromer
CNRMA98.480 DC France CSF 1998 AIDS VNIV αD 511 16 21 29 16 14 17 24 Dromer
CNRMA99.1037 PCC France Skin 1999 No risk factor VNIV αD 122 16 21 32 24 13 17 20 Dromer
MKT6301 PCC France Skin 2011 No risk factor VNIV αD 121 16 21 32 19 13 17 20 Bienvenu
RKI 04-0061 DC Germany – 2004 Liver disorder VNIV αD 110 15 21 24 21 13 20 22 Rickerts
RKI 04-0089 PCC Germany Skin, hand 2004 Chronical asthma, corticosteroids VNIV αD 486 16 22 32 19 13 17 22 Rickerts
RKI 05-0151 DC Germany Blood 2005 Diabetes VNIV αD 531 16 29 24 20 13 20 18 Rickerts
RKI 07-0173 DC Germany – 2007 Liver disorder VNIV αD 530 31 21 83 13 37 59 53 Rickerts
RKI 08-0429 DC Germany CSF 2008 AIDS VNIV αD 487 16 21 32 24 13 17 32 Rickerts
RKI 08-0572 DC Germany – 2008 No risk factors VNIV αD 116 16 21 29 13 14 17 24 Rickerts
RKI 08-0591 DC Germany – 2008 No risk factors VNIV αD 116 16 21 29 13 14 17 24 Rickerts
RKI 09-0102 DC Germany – 2009 Sarcoidosis VNIV αD 116 16 21 29 13 14 17 24 Rickerts
RKI 09-0103 DC Germany – 2009 Sarcoidosis VNIV αD 116 16 21 29 13 14 17 24 Rickerts
RKI 09-0388 PCC Germany Skin 2009 No risk factor VNIV αD 519 26 22 32 24 13 17 20 Rickerts
RKI 09-0393 PCC Germany Skin 2009 No risk factor VNIV αD 505 15 21 31 15 13 19 18 Rickerts
RKI 09-0515 DC Germany BAL 2009 Solid organ Tx VNIV αD 168 22 21 30 22 14 17 18 Rickerts
RKI 09-0545 DC Germany CSF 2009 AIDS VNIV αD 160 16 21 30 19 13 17 19 Rickerts
RKI 11-0047 DC Germany CSF 2010 AIDS VNIV αD 116 16 21 29 13 14 17 24 Rickerts
RKI 11-0048 DC Germany CSF 2010 AIDS VNIV αD 116 16 21 29 13 14 17 24 Rickerts
RKI 12-0155 PCC Germany Skin 2012 No risk factor VNIV αD 116 16 21 29 13 14 17 24 Rickerts
RKI 12-0559 PCC Germany Skin 2012 No risk factor VNIV αD 513 16 21 43 21 13 20 18 Rickerts
RKI 13-0490 ENV Germany Pigeon droppings 2013 – VNIV aD 522 27 21 53 19 14 19 16 Rickerts
RKI 13-0491 ENV Germany Pigeon droppings 2013 – VNIV aD 523 29 21 83 21 14 58 53 Rickerts
RKI 13-0492 ENV Germany Pigeon droppings 2013 – VNIV aD 524 29 21 83 40 14 58 53 Rickerts
GRACA18BK1-3 ENV Greece Eucalyptus tree 2013 – VNIV aD 502 26 38 30 22 13 19 18 Velegraki
GRACP14BK1-1 ENV Greece Pine tree 2013 – VNIV aD 499 26 21 30 22 13 19 18 Velegraki
GRACP15SO1-1 ENV Greece Pine tree 2013 – VNIV aD 499 26 21 30 22 13 19 18 Velegraki
GRACP15SO1-2 ENV Greece Pine tree 2013 – VNIV aD 499 26 21 30 22 13 19 18 Velegraki
GRACP16HO1-1 ENV Greece Pine tree 2013 – VNIV aD 499 26 21 30 22 13 19 18 Velegraki
GRACP30BK1-1 ENV Greece Plane tree 2013 – VNIV aD 498 26 20 30 17 13 19 18 Velegraki
GRAKI10SO1-1 ENV Greece Olive tree 2013 – VNIV aD 499 26 21 30 22 13 19 18 Velegraki
GRAKI11HO1-1 ENV Greece Olive tree 2013 – VNIV aD 499 26 21 30 22 13 19 18 Velegraki
GRAKI12SO1-1 ENV Greece Plane tree 2013 – VNIV aD 503 26 39 30 22 13 19 18 Velegraki
GRAKI13HO1-1 ENV Greece Plane tree 2013 – VNIV aD 503 26 39 30 22 13 19 18 Velegraki
GRAKI28HO1-1 ENV Greece Olive tree 2013 – VNIV aD 500 26 22 44 17 14 23 23 Velegraki
GRLMM26HO1-2 ENV Greece Olive tree 2013 – VNIV aD 489 1 1 1 1 1 27 1 Velegraki
GRSAB18HO1-1 ENV Greece Pine tree 2013 – VNIV aD 501 26 37 30 38 13 19 18 Velegraki
ITMPV22BK7-1 ENV Italy Oak tree 2014 – VNIV αD 496 16 3 30 19 13 17 19 Cogliati
IUM 01-0956 SCC Italy Skin, head 2001 AIDS VNIV αD 521 27 13 12 6 9 8 13 Cogliati
IUM 02-0826 VET Italy Cat 2002 – VNIV aD 517 22 21 30 24 21 17 20 Cogliati
IUM 02-4295 PPCC Italy Skin 2002 No risk factors VNIV aD 516 22 3 31 24 14 17 20 Cogliati
IUM 73-0017 DC Italy CSF 1973 No risk factors VNIV αD 508 16 3 31 24 14 17 16 Cogliati
IUM 77-0033 SCC Italy Skin, right ear 1977 No risk factors VNIV αD 135 27 22 43 24 13 17 20 Cogliati
IUM 79-0801 PCC Italy Skin, left leg 1979 Common variable immunodeficiency VNIV αD 520 27 3 43 24 13 17 20 Cogliati
IUM 91-2588 SCC Italy Skin, hand 1991 AIDS VNIV αD 135 27 22 43 24 13 17 20 Cogliati
IUM 92-0701 DC Italy CSF 1992 AIDS VNIV αD 508 16 3 31 24 14 17 16 Cogliati
IUM 93-1543 SCC Italy Skin 1993 AIDS VNIV αD 135 27 22 43 24 13 17 20 Cogliati
IUM 93-1656 DC Italy CSF 1993 AIDS VNIV αD 279 22 22 31 22 14 17 34 Cogliati
IUM 97-4851 SCC Italy Skin 1997 AIDS VNIV αD 135 27 22 43 24 13 17 20 Cogliati
IUM 98-0824 SCC Italy Skin, right hand and arm 1998 Solid tumor prostate VNIV αD 135 27 22 43 24 13 17 20 Cogliati
IUM 98-2742 SCC Italy Skin, left hand 1998 AIDS VNIV αD 112 16 22 31 24 14 17 16 Cogliati
IUM 98-4987 PPCC Italy Skin 1998 Solid tumor breast VNIV αD 518 26 21 31 15 21 19 15 Cogliati
NIH-530 VET Italy Cow 1972 – VNIV αD 112 16 22 31 24 14 17 16 Kwon-Chung
PD1596 VET Italy Cat 2010 – VNIV αD 252 22 22 30 19 14 23 41 Danesi
PD2270 VET Italy Cat 2011 – VNIV αD 135 27 22 43 24 13 17 20 Danesi
PD32 VET Italy Cat 2009 – VNIV αD 294 16 21 24 20 13 22 32 Danesi
M9112 PCC Japan Skin 1985 No risk factors VNIV αD 168 22 21 30 22 14 17 18 Ikeda
M9117 PCC Japan Skin 1988 Severe cellular immunity deficiency VNIV αD 168 22 21 30 22 14 17 18 Ikeda
M9118 PCC Japan Skin 1988 No risk factors VNIV αD 168 22 21 30 22 14 17 18 Ikeda
M9119 PCC Japan Skin 1988 No risk factors VNIV αD 168 22 21 30 22 14 17 18 Ikeda
M9120 PCC Japan Skin 1983 No risk factors VNIV αD 168 22 21 30 22 14 17 18 Ikeda
M9196 PCC Japan CSF 1989 Malignant lymphoma VNIV αD 168 22 21 30 22 14 17 18 Ikeda
M9214 PCC Japan Skin 1989 No risk factors VNIV αD 168 22 21 30 22 14 17 18 Ikeda
M9215 PCC Japan Skin 1993 Systemic lupus erythematosus VNIV αD 168 22 21 30 22 14 17 18 Ikeda
CBS7816 ENV Thailand Cuckoo droppings 1997 – VNIV αD 126 17 21 28 19 14 1 20 Kaochaoen
TRNCGB1H01-1 ENV Turkey Pine tree 2013 – VNIV αD 497 16 21 24 20 13 1 32 Tore O
NIH-116 ENV USA, Virginia Pigeon nest 1960 – VNIV αD 135 27 22 43 24 13 17 20 Kwon-Chung
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PCC = primary cutaneous cyrptococcosis; PPCC = probable primary cutaneous cryptococcosis; SCC = secondary cutaneous cryptococccosis; DC = disseminated cryptococcosis; ENV = environment; VET = veterinary; CSF = cerebrospinal fluid; BAL = Bronchoalveolar lavage.

2.2. Clinical case definitions

The cases of primary cutaneous cryptococcosis presented a single cutaneous lesion often on the arms or the legs which are primarily due to a traumatic injury. No Cryptococcus antigens from serum were detected and no isolates from other body sites were recovered. The cases that presented isolated cutaneous lesions but were not supported by the other clinical evidences were defined as probable.

Secondary cutaneous cryptococcosis patients presented multiple skin lesions with no specific body sites, positive Cryptococcus antigens or positive cultures from other clinical samples (blood, CSF, urine).

Disseminated cryptococcosis cases presented positive cultures from multiple body sites, positive Cryptococcus antigens, but no skin lesions.

2.3. Molecular analysis

Molecular type and mating type were determined by multiplex PCRs as previously reported (Cogliati et al., 2000; Esposto et al., 2004). Multilocus sequence typing was performed according to the ISHAM consensus scheme (Meyer et al., 2009) and all sequences were deposited in the Cryptococcus MLST database (www.mycologylab.org).

The data of strains WM629, CBS7816, PD32, PD2270, PD1596, RKI 08-0429, RKI 04-0089, RKI 09-0388, RKI 09-0515, RKI 07-0173, RKI 05-0151, RKI 04-0061, RKI 09-0393, RKI 09-0545, RKI 09-0102, RKI 09-0103, RKI 11-0048, RKI 08-0591, RKI 08-0572, RKI 11-0047) were obtained from previous studies (Sanchini et al., 2014; Kaocharoen et al., 2013; Meyer et al., 2009; Danesi et al., 2014).

The concatenated sequences of the seven MLST genes (CAP59, GPD1, IGS1, LAC1, PLB1, SOD1, URA5) of the 83 C. neoformans var. neoformans and 30 C. neoformans var. grubii isolates were aligned by ClustalW algorithm (www.ebi.ac.uk) and the resulting file was converted in a Roehl data file by DnaSP software (Universitat de Barcelona, www.ub.edu/dnasp). Network analysis was performed using the median joining method included in the software Network v4.6 (Fluxus Technology Ltd., www.fluxus-engeneering.com).

Genetic population parameters and population comparisons were performed by DnaSP software whereas maximum likelihood phylogenetic analysis and average evolutionary divergence were calculated with the software Mega v6.0 (www.megasoftware.net).

The degree of recombination inside the population was also calculated using both the linkage disequilibrium test and the Watterson estimator (theta) method (DnaSP software). The linkage disequilibrium test is an extension of Fisher's exact probability test on contingency tables. The test consists in obtaining the probability of finding a table with the same marginal totals and which has a probability equal or less than the observed table. The null-hypothesis of non-random association between the two tested loci was confirmed if the probability was less than 0.05. The Watterson estimator (theta) method extrapolates two values that correspond to the expected theta value for a non-recombinant population and to the expected theta value for a free-recombinant population, and then it calculates the observed theta value in the investigated population.

3. Results

Molecular identification confirmed that all the C. neoformans var. neoformans isolates belonged to molecular type VNIV and that 63 were mating type α and 20 mating type a (Table 1).

The alignment of the 4092-bp sequences resulting by concatenating the seven MLST loci showed the presence of 425 polymorphic sites that identified 49 sequence types with a haplotype diversity (Hd) value of 0.965. LAC1 and URA5 were the most polymorphic loci discriminating 16 and 15 haplotypes, respectively. In contrast, the IGS1 locus was the least discriminatory locus with an Hd value of 0.241 (Table 2).

Table 2.

Genetic population parameters defining the population structure of the 83 var. neoformans isolates investigated.

Locus Sequence length (bp) Polymorphic sites Haplotypes Haplotype diversity (Hd) Recombining events Linkage disequilibrium Theta (no recombination; free recombination)
CAP59 560 41 9 0.761 4 No –
GPD1 558 58 5 0.241 1 No –
IGS1 781 57 11 0.772 1 No –
LAC1 481 149 15 0.866 20 No –
PLB1 534 40 7 0.584 0 No –
SOD1 537 73 11 0.628 2 No –
URA5 639 55 16 0.865 3 No –
All loci 4092 425 49 0.965 31 No 87.1 (484.0; 17.4)
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Parameters were calculated by DnaSP v.5 software.

Maximum likelihood phylogenetic reconstruction showed that most of C. neoformans var. neoformans isolates were grouped in a unique cluster with an average evolutionary divergence of 0.008, twofold lower than that observed among C. neoformans var. grubii isolates. Four isolates (WM629, CBS7816, TRNCGB1HO1-1, IUM 01-0956) were situated between the two varieties with an ambiguous topology and one isolate (GRLMM26HO1-2) was unexpectedly more related to the VNI group (Fig. 1). A network analysis showed the same topology with a core region grouping the majority of C. neoformans var. neoformans isolates all linked by star-like branches. The topology of C. neoformans var. grubii population was very different with clear clusters and higher genetic distances. The two populations were linked by a long branch, along which were the five ambiguous isolates (Fig. 2A).

Fig. 1.

Fig. 1

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Maximun likelihood phylogenetic reconstruction including 83 C. neoformans var. neoformans (VNIV) and 30 C. neoformans var. grubii (VNI, VNII, VNB) isolates. Black dots indicate VNIV isolates with an ambiguous position. Numbers near the nodes represent the bootstrap values obtained for 1000 replications.

Fig. 2.

Fig. 2

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Network analysis performed by median joining algorithm. Panel A shows the different genotypes, panel B shows the different geographical origin, and panel C the different source of isolation. Double slash means that the branch has been shortened to fit the image.

The investigated isolates were therefore divided in three groups, C. neoformans var. neoformans, C. neoformans var. grubii and putative intervarietal recombinants, and average evolutionary divergence between the populations was calculated. The results showed that C. neoformans var. neoformans and C. neoformans var. grubii diverged 0.09 and both diverged from the putative recombinants around 0.05. The five putative recombinants were then checked to exclude mixed cultures and five single colonies from each of the original strains were collected and processed for molecular typing and ploidy determination by flow cytometry. The results confirmed that all strains from single colonies were VNIV aD or αD and that they were haploids (Fig. S1).

The linkage disequilibrium analysis of C. neoformans var. neoformans populations was also calculated for both the concatenated sequences and the single loci alignments. The results confirmed the absence of linkage disequilibrium in all the cases confirming that recombination in this population could not be excluded (Table 2). The Watterson estimator (theta) method results were in perfect agreement with that obtained in the linkage disequilibrium test strengthening for the hypothesis of a recombinant population (Table 2). In addition, the estimation of recombination events among the whole C. neoformans var. neoformans population reveals that at least 31 recombination events had occurred and that the most recombinant locus was LAC1 (20 events) (Table 2).

Fig. 2B displays geographical origin of each isolate on the network tree. No correlation was observed between the country of isolation and the MLST profile. Only the eight Japanese isolates belonged all to the same genotype (ST168). Similarly, comparison between MLST profiles and source of isolation did not provide evidence of any specific correlation. ST135 and ST180 grouped together isolates from the environment with isolates from veterinary cases, from primary cutaneous cryptococcosis and from secondary cutaneous cryptococcosis whereas none of the environmental isolates shared the same ST with isolates from disseminated cryptococcosis (Fig. 2C).

4. Discussion

The present study investigated, for the first time by MLST, the genetic population structure of a large number of C. neoformans var. neoformans isolates from different geographical origin and from different sources. The results strongly support the hypothesis that this population is recombinant and are in agreement with the recent study carried out by other authors (Desnos-Ollivier et al., 2015) in a population of French clinical C. neoformans isolates. The high haplotype diversity as well as the low evolutionary divergence among C. neoformans var. neoformans population confirm that isolates are strictly correlated each to the other but they are characterized by high variability due to recombination. Statistical tests such as linkage disequilibrium analysis, Watterson estimator calculation, and recombining events evaluation also corroborate this hypothesis. Furthermore, the C. neoformans var. neoformans population investigated in the present study included 20 mating type a isolates recovered from both patients and the environment. This suggests that isolates of C. neoformans var. neoformans mating type a are more prevalent than those of C. neoformans var. grubii and therefore sexual reproduction may occur more frequently.

The high polymorphism observed for the LAC1 locus suggests that the laccase enzyme plays a crucial role for both the host infection and the environmental survival of this yeast. The survival in a particular ecological niche, such as a tree, could depend on the capacity of this yeast to degrade a wide range of phenolic compounds present in lignin of the tree trunk. In addition, since laccase is a key enzyme for melanin production this variety could gain an advantage for growth on surfaces exposed to light, for example the bark of the tree.

Both maximum likelihood phylogenetic analysis and network analysis identified a group of five isolates with an ambiguous position on the tree. The average evolutionary divergence between C. neoformans var. grubii and C. neoformans var. neoformans groups was twice than that observed between the two groups and the ambiguous isolates suggesting that the five isolates could represent haploid strains generated by intervarietal recombination (Xu et al., 2000; Kavanaugh et al., 2006). Laboratory isolated haploid intervarietal recombinant clones have been reported by crossing between H99, a strain of C. neoformans var. grubii VNI, and JEC20, a strain of C. neoformans var. neoformans (Kwon-Chung and Varma, 2006). This is in contrast with the recent published proposal (Hagen et al., 2015), advocating that C. neoformans var. neoformans and C. neoformans var. grubii are separate species. Further investigations and a larger number of C. neoformans var. neoformans isolates are needed to clarify this important issue.

The comparison between MLST profiles and geographical origin showed that isolates recovered from different countries and from different continents were grouped in the same cluster confirming the role of recombination in shortening the genetic divergence. However, clonal expansion of some genotypes could occur in geographical areas where physical barriers exist that prevent recombination. This could be true in Japan where all the isolates investigated belonged to the same ST even though they were isolated from different patients, at different time points and from different regions of Japan.

A similar analysis to this one, which compared the source of isolates, showed that the environmental genotypes could be the source of infections for animal, primary cutaneous cryptococcosis and secondary cutaneous cryptococcosis. In contrast, this was not observed for the isolates from disseminated cryptococcosis cases, which shared an identical ST but only with those from primary and secondary cutaneous cryptococcosis cases. Further studies are required to confirm this discrepancy.

In conclusion, our study showed that C. neoformans var. neoformans population is not evolving primarily by clonal expansion, as observed for C. neoformans var. grubii (Khayhan et al., 2013), and that intravarietal recombination is largely occurring and intervarietal recombination could not be excluded.

Supplementary Material

Figure S1
Table S1

Acknowledgments

We thank Prof. F. Dromer and Prof. J. Kwon-Chung for providing some of the isolates and for her suggestions to improve this study, and Dr. T. Boekhout and Dr. B. Theelen for sequencing support.

Footnotes

Disclosures: The findings and conclusions of this article are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Appendix A. Supplementary material: Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fgb.2016.01.003.

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Associated Data

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Supplementary Materials

Figure S1
NIHMS753767-supplement-Figure_S1.pdf (373.7KB, pdf)
Table S1
NIHMS753767-supplement-Table_S1.pdf (52.5KB, pdf)

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