VGII isolated from woody debris of divi-divi (Caesalpinia coriaria), Bonaire, Dutch Caribbean

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Molecular characterization of Cryptococcus gattii genotype AFLP6/VGII isolated from woody debris of divi-divi (Caesalpinia coriaria), Bonaire, Dutch Caribbean

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Ferry Hagen a,b,∗ , Anuradha Chowdhary c , Anupam Prakash c , Jan-Bart Yntema d , Jacques F. Meis a,b a

Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital, Nijmegen, The Netherlands Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands c Department of Medical Mycology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, India d Department of Pediatric Pulmonology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands b

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Article history: Received 23 July 2013 Accepted 1 October 2013 Available online xxx

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Keywords: Cryptococcus gattii Multi-locus sequence typing (MLST) Amplified fragment length polymorphism (AFLP) fingerprinting environmental sampling Caesalpinia coriaria

Background: The basidiomycetous yeast Cryptococcus gattii is an emerging and primary pathogen. There is a lack of information about its environmental spread outside outbreak regions in Mediterranean Europe, North and South America. Environmental sampling for C. gattii and molecular characterization of the obtained isolates will provide an insight into the global spread of the various genotypes. Methods: Woody debris of native divi-divi (Caesalpinia coriaria) trees were sampled across Bonaire, Dutch Caribbean. Colonies suspected for Cryptococcus species were subjected to standard mycology investigations and identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Isolates identified as C. gattii were subjected to amplified fragment length polymorphism genotyping, mating-type analysis and multi-locus sequence typing. Results: Ten colonies of C. gattii were cultured from different trunk hollows of the same divi-divi tree. Molecular characterization showed that all isolates were genotype AFLP6/VGII and mating-type ␣. Multilocus sequence typing revealed that all isolates were genetically indistinguishable from each other. Conclusions: C. gattii is present in the environment of Bonaire, which suggests that this yeast is likely to be present in the environment of other Caribbean islands. © 2014 Revista Iberoamericana de Micología. Published by Elsevier España, S.L. All rights reserved.

Caracterización molecular del genotipo AFLP6/VGII Cryptococcus gattii aislado de restos de madera de divi-divi (Caesalpinia coriaria), Bonaire, Caribe Holandés r e s u m e n 25 26 27 28 29 30 31 32 33

Palabras clave: Cryptococcus gattii tipado por secuenciación de múltiples loci (MLST) ˜ de fragmentos Polimorfismo de tamano amplificados (AFLP) muestreo medioambiental Caesalpinia coriaria

Antecedentes: La levadura Cryptococcus gattii es un basidiomiceto emergente y patógeno primario. Existe poca información acerca de su dispersión en medio ambiente fuera de las regiones con brotes descritos por esta levadura en la Europa mediterránea, Norte y Sur de América. Los muestreos del medio ambiente para la búsqueda de C. gattii y la caracterización molecular de los aislamientos obtenidos puede proveer de una visión global sobre la dispersión de varios de sus genotipos. Métodos: Se tomaron muestras de residuos de madera de árboles nativos divi-divi (Caesalpinia coriaria) en Bonaire, Caribe Holandés. Las colonias susceptibles de pertenecer a las especies de Cryptococcus se sometieron a un estudio micológico estándar e identificación por espectrometría de masas MALDI-TOF (Matrix-Assisted Laser Desorption Ionization-Time of Flight). Los aislamientos identificados como C. gattii se sometieron a genotipado mediante AFLP (Amplified Fragment Length Polymorphism), obtención del tipo sexual y MLST (Multi-locus Sequence Typing). Resultados: Se obtuvieron diez colonias de C. gattii en el cultivo de nuestras de diferentes agujeros de un mismo árbol divi-divi. La caracterización molecular mostró que todos los aislamientos eran genotipo AFLP6/VGII y tipo sexual ␣. El tipado mediante MLST reveló que todos los aislamientos eran genéticamente indistinguibles unos de otros.

∗ Corresponding author. E-mail addresses: [email protected], [email protected] (F. Hagen). 1130-1406/$ – see front matter © 2014 Revista Iberoamericana de Micología. Published by Elsevier España, S.L. All rights reserved. http://dx.doi.org/10.1016/j.riam.2013.10.007

Please cite this article in press as: Hagen F, et al. Molecular characterization of Cryptococcus gattii genotype AFLP6/VGII isolated from woody debris of divi-divi (Caesalpinia coriaria), Bonaire, Dutch Caribbean. Rev Iberoam Micol. 2014. http://dx.doi.org/10.1016/j.riam.2013.10.007

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Conclusiones: C. gattii está presente en el medio ambiente de Bonaire, lo que sugiere que esta levadura podría estar presente en el ambiente de otras islas del Caribe. © 2014 Revista Iberoamericana de Micología. Publicado por Elsevier España, S.L. Todos los derechos reservados.

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Cryptococcus gattii and its sibling Cryptococcus neoformans are the major basidiomycetous yeast pathogens within the genus Cryptococcus that comprises over one hundred recognized species.14 Annually, nearly an estimated one million individuals with HIV/AIDS develop cryptococcal meningitis with high estimated death rates of 6,25,000 subjects.19 C. neoformans, with its two varieties grubii and neoformans, has a global distribution and affects mainly immunocompromised individuals, such as those with HIV/AIDS or those under immunosuppressive therapy. Although C. gattii is mainly restricted to subtropical and tropical climate zones and is known to be a major source of cryptococcosis among immunocompetent subjects, it has also emerged as a significant pathogen in Canada and the Pacific Northwest of the USA.1,2,9,10,21 Molecular techniques, such as amplified fragment length polymorphism (AFLP) fingerprinting, PCR-fingerprinting, restriction fragment length polymorphism fingerprinting and multi-locus sequence typing (MLST), have provided a detailed insight into the genetic diversity of the C. neoformans/C. gattii species complex.1,18 Cryptococcus neoformans var. grubii (serotype A) can be divided into the three genotypes: AFLP1/VNI, AFLP1A/VNII/VNB and AFLP1B; C. neoformans var. neoformans (serotype D) is represented by genotype AFLP2/VNIV, and the intervarietal C. neoformans hybrid (serotype AD) corresponds with genotype AFLP3/VNIII.1,18 C. gattii consists of five haploid genotypes, with genotypes AFLP4/VGI, AFLP6/VGII and AFLP10/VGIV representing the serotype B isolates, and genotypes AFLP5/VGIII and AFLP7/VGIV corresponding with isolates exhibiting the C serotype.9 Interspecies hybrids have been described and thus far have only been isolated from clinical sources.1 During the past decade ongoing and expanding outbreaks of C. gattii occurred in temperate climate zones affecting previously healthy humans and animals.6,10,21 Besides the changing distribution pattern, it has been observed that certain C. gattii genotypes may be found more often in immunocompromised individuals than in immunocompetent subjects.9 Notably, environmental isolation of C. gattii was found to be a valuable tool to enhance the current knowledge of the geographical spread, genotypic diversity and the environmental niche of this pathogenic species.3,4,6,16,21 Within the Caribbean, C. gattii has rarely been reported as clinical or veterinary infection.9–11,13 Despite large-scale environmental sampling of a plethora of environmental sources that includes many tree and cacti species9,10,12,15 the fungus has been, up until

now, only recovered from the environment of Puerto Rico. Here we describe the environmental isolation and molecular characterization of C. gattii isolates from decayed woody debris of a native divi-divi tree (Caesalpinia coriaria) in Bonaire, Dutch Caribbean. Woody debris, collected from inside trunk hollows of living dividivi trees in April 2013, were cultured on simplified niger seed agar as described.4 The sampled divi-divi trees were located in Lagun Goto (N12◦ 14 3.1344 , E-68◦ 22 6.366 ), Rincon village (N12◦ 14 24.1116 , E-68◦ 19 32.5662 ) and neighboring surroundings of Hato village (N12◦ 10 11.8734 , E-68◦ 17 1.2366 ). Plates were incubated at 28 ◦ C and periodically observed for chocolate brown colonies of C. gattii and C. neoformans up to 7 days. Suspected colonies of Cryptococcus spp. were purified by dilution plating and identified by their morphological and biochemical profiles using VITEK2 and API 20C AUX (bioMérieux, Marcy I’Etoile, France). Cryptococcus spp. colonies were also inoculated on l-canavanineglycine bromothymol blue medium; a blue-color change suggestive of C. gattii was observed.1 Identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry was performed using the MALDI Biotyper, and spectra were compared to the reference spectra in the commercial database (Bruker Daltonics, Bremen, Germany).17 Ten colonies identified as C. gattii were obtained from decayed wood of different trunk hollows of the same divi-divi tree. The isolates have been deposited in the culture collection of the CBS-KNAW Fungal Biodiversity Centre under accession numbers CBS12864–CBS12873. All ten C. gattii isolates were subsequently subjected to molecular characterization as described previously.4,6,9 Analysis of the AFLP fingerprints, using UPGMA clustering and the Pearson correlation coefficient in BioNumerics version 6.6.8 (Applied Maths, Sint-Martens-Latem, Belgium) showed that all ten isolates clustered together with the reference strain representing genotype AFLP6/VGII (Fig. 1). Mating-type analysis by conventional PCR revealed that all ten isolates exhibit the STE12-allele with the ␣ mating-type.9 In-depth genetic analysis was initiated by amplification and sequencing of the ten nuclear loci CAP10, CAP59, GPD1, IGS1, LAC1, MPD1, PLB1, SOD1, TEF1 and URA5, as previously described.9 Multilocus sequence typing links clinical isolates to their geographical origin and demonstrates their relatedness to the isolates obtained from environmental and veterinary sources.4,5,7,9,11–13 Sequences

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Fig. 1. Amplified fragment length polymorphism fingerprint dendrogram of ten C. gattii isolates from Bonaire compared to the reference strains WM179 (AFLP4/VGI), WM161 (AFLP5/VGIII), WM178 (AFLP6/VGII), WM779 (AFLP7/VGIV) and IHEM14941 (AFLP10/VGIV) representing all known AFLP genotypes within C. gattii.

Please cite this article in press as: Hagen F, et al. Molecular characterization of Cryptococcus gattii genotype AFLP6/VGII isolated from woody debris of divi-divi (Caesalpinia coriaria), Bonaire, Dutch Caribbean. Rev Iberoam Micol. 2014. http://dx.doi.org/10.1016/j.riam.2013.10.007

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RB59FH ENV AFLP6A Canada RKI01/774 CLIN AFLP6A Germany A1M-F3016 CLIN AFLP6A Canada KB17821 VET AFLP6A Canada ENV152 ENV AFLP6A Canada CBS 10933 CLIN AFLP6A Canada A1M-R406 CLIN AFLP6A Canada A1M-R265 CLIN AFLP6A Canada 1070609048 VET AFLP6A Canada 0260201186 VET AFLP6A Canada 0260403732 VET AFLP6A Canada 1080211019 VET AFLP6A USA A1M-R269 CLIN AFLP6A Canada A1M-R409 CLIN AFLP6A Canada CBS 10934 CLIN AFLP6A Canada ICB 107 CLIN AFLP6A Brazil CBS7750 ENV AFLP6A USA KB9101 ENV AFLP6A Canada 0260201100 VET AFLP6A Canada 84 1070607935 VET AFLP6A Canada A1M-F2932 CLIN AFLP6A Canada A1M-R376 UNK AFLP6A Canada CBS10866 CLIN AFLP6A The Netherlands ENV131 ENV AFLP6A Canada KB916519 VET AFLP6A Canada CBS6956 CLIN AFLP6A USA RB50 ENV AFLP6A Canada 1070512906 VET AFLP6A Canada A1M-F2866 CLIN AFLP6A Canada A1M-R271 CLIN AFLP6A Canada CBS10485 CLIN AFLP6A Denmark CN308 ENV AFLP6B USA KB915181 VET AFLP6A Canada RB14 ENV AFLP6A Canada RB59JF ENV AFLP6A Canada RKI06/496 CLIN AFLP6A Switzerland 0170102053 VET AFLP6A Canada 0260209860 VET AFLP6A Canada 0260401664 VET AFLP6A Canada 0260609249 VET AFLP6A Canada 1080405336 VET AFLP6A Canada MMC2005000026 CLIN AFLP6A USA 97 AV55 CLIN AFLP6A Greece IUM00-5363 CLIN AFLP6A Greece RKI96/767 CLIN AFLP6A Brazil CBS 10602 CLIN AFLP6A Hong Kong CBS 10603 CLIN AFLP6A Hong Kong CBS 10604 CLIN AFLP6A Hong Kong 96 CBS 10605 CLIN AFLP6A Hong Kong WM178 CLIN AFLP6A Australia IP1998/1037-1 CLIN AFLP6B France IP1998/1037-2 CLIN AFLP6B France IP2003/125 CLIN AFLP6B France 96 CCA242A CLIN AFLP6B Spain CCA242G CLIN AFLP6B Spain CCA242O CLIN AFLP6B Spain CCA242X CLIN AFLP6B Spain WA861 VET AFLP6B Australia AV54S VET AFLP6B Greece AV54W CLIN AFLP6B Greece 98 IUM01-4731 CLIN AFLP6B Greece ICB97 CLIN AFLP6B Brazil CBS12864 ENV AFLP6B Bonaire CBS12865 ENV AFLP6B Bonaire CBS12866 ENV AFLP6B Bonaire CBS12867 ENV AFLP6B Bonaire CBS12868 ENV AFLP6B Bonaire CBS12869 ENV AFLP6B Bonaire CBS12870 ENV AFLP6B Bonaire CBS12871 ENV AFLP6B Bonaire CBS12872 ENV AFLP6B Bonaire CBS12873 ENV AFLP6B Bonaire CBS1930 VET AFLP6B Aruba HEC11102 CLIN AFLP6B Brazil ICB183 ENV AFLP6B Brazil IFM56776 CLIN AFLP6B Brazil IP2006/194 CLIN AFLP6B France ICB184 ENV AFLP6B Brazil 97 46A ENV AFLP6A Uruguay CBS8684 ENV AFLP6A Uruguay IP1993/980 CLIN AFLP6B France IP1997/170 CLIN AFLP6B French Guyana KB15506-2 ENV AFLP6B Canada KB16481 VET AFLP6B Canada 1070514755 VET AFLP6B Canada 0260313271 VET AFLP6B Canada A1M-R272 CLIN AFLP6B Canada CBS10601 CLIN AFLP6B Hong Kong KB1522-1 ENV AFLP6B Canada RAM2 ENV AFLP6B Australia CCA358 VET AFLP6B Spain 98 IHEM11489W CLIN AFLP6B Congo IHEM11489S CLIN AFLP6B Congo NT8 CLIN AFLP6B Australia MMRL1340 CLIN AFLP6B Australia 99 MMRL2647 CLIN AFLP6B Australia NT3 CLIN AFLP6B Australia 87 RDH2 CLIN AFLP6B Australia RDH7 CLIN AFLP6B Australia RDH9 CLIN AFLP6B Australia A6M-R38 CLIN AFLP6C USA A6M-R41 CLIN AFLP6C USA EJB12 CLIN AFLP6C USA EJB14 VET AFLP6C USA 80 EJB15 VET AFLP6C USA EJB18 CLIN AFLP6C USA MMC2005000132 CLIN AFLP6C USA CRY0146 ENV AFLP6B Brazil IHEM15022 CLIN AFLP6B Brazil IP1996/1120-1 CLIN AFLP6B France IP1996/1120-2 CLIN AFLP6B France IP1996/901-1 CLIN AFLP6B France 99 IP1996/901-2 CLIN AFLP6B France 78 IP2000/87 CLIN AFLP6B France CBS11998 CLIN AFLP6B French Guyana IP2001/935-1 CLIN AFLP6B Senegal ICB180 ENV AFLP6B Brazil LA362 VET AFLP6B Brazil WM779 VET AFLP7 Reference WM179 CLIN AFLP4 Reference IHEM14941S CLIN AFLP10 Reference WM161 ENV AFLP5 Reference

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Fig. 2. Bootstrapped maximum likelihood phylogenetic analysis of the multi-locus sequence typing data from a global set of C. gattii genotype AFLP6/VGII isolates (data from 9) compared with those isolated from a divi-divi tree on Bonaire.

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DQ861595 (=MPD1), DQ198344 (=PLB1), HM207449 (=SOD1), DQ861593 (=TEF1) and HQ606091 (=URA5). Phylogenetic analysis was performed as described before by using a bootstrapped maximum likelihood analysis (1000 bootstraps) in MEGA version 5.2 with the best fitting nucleotide substitution model being Hasegawa-Kishino-Yano plus gamma distributed with invariant sites and with C. gattii reference strains from the other genotypes as an outgroup.9 To compare the C. gattii isolates with those from other localities, the recently published MLST-dataset of C. gattii AFLP6/VGII isolates was included.9 As depicted in Fig. 2, all isolates from Bonaire clustered together with the genotype AFLP6/VGII isolate CBS1930 from Aruba. This isolate, obtained in July 1953, originated from clinical material of a child. This isolate was experimentally inoculated in a goat to see whether the atypical yeast isolate was the culprit of infection (original correspondence about this isolate in the CBS-KNAW archive). Other available isolates from Latin America were only distantly related, with Brazilian and French Guyana isolates as the closest siblings (Fig. 2). Interestingly, isolate CBS1930 was found to have mating-type a while the isolates from Bonaire were all mating-type ␣. Genetically, the isolates from Aruba and Bonaire differed slightly from each other suggesting that there is a clonal population of C. gattii AFLP6/VGII on these Caribbean islands. However, due to the presence of different mating-types within this set of isolates the conclusion of one clonal population needs to be taken with caution. Another explanation may be that although isolates from Dutch Caribbean islands are genetically closely related they may form different clonal populations. This scenario is identical to the one observed in Mediterranean population of C. gattii AFLP4/VGI, where Italian isolates with the mating-type a and Spanish isolates with the mating-type ␣ were genetically indistinguishable from each other using the same set of ten sequenced nuclear loci.9 Unfortunately, the only C. gattii AFLP6/VGII isolates available from the Caribbean, isolated from cacti on the island of Puerto Rico, could not be compared with our data since these isolates are not available to the research community.8,15 To conclude, C. gattii AFLP6/VGII is present in the environment of the Dutch Caribbean and Puerto Rico, suggesting that C. gattii might be present in other Caribbean localities. Little is known about the epidemiology of C. gattii in Central America and the Caribbean; therefore environmental sampling at other Caribbean islands, as well as investigating the occurrence of this pathogenic yeast among clinical samples from this region, should be considered.

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for each of the ten loci and for all ten C. gattii isolates revealed that they were genetically indistinguishable from each other. Genbank accession numbers for sequences identical to those obtained in the current study were DQ861598 (=CAP10), DQ096433 (=CAP59), DQ096382 (=GPD1), [in progress] (=IGS1), DQ096400 (=LAC1),

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The authors declare that there are no competing interests. Uncited reference 20

Acknowledgment The authors would like to thank Dr. Kika Colom (Alicante, Spain) for preparing the Spanish-language abstract. References

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Please cite this article in press as: Hagen F, et al. Molecular characterization of Cryptococcus gattii genotype AFLP6/VGII isolated from woody debris of divi-divi (Caesalpinia coriaria), Bonaire, Dutch Caribbean. Rev Iberoam Micol. 2014. http://dx.doi.org/10.1016/j.riam.2013.10.007

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