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Molecular epidemiology of environmental Cryptococcus species isolates based on amplified fragment length polymorphism
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Research Paper
Molecular epidemiology of environmental Cryptococcus species isolates based on amplified fragment length polymorphism K. Pakshir a, H. Fakhim b,c, A. Vaezi d, J.F. Meis e,f, M. Mahmoodi a, K. Zomorodian a, J. Javidnia d,g, S. Ansari h, F. Hagen e,i, H. Badali d,j,* a
Basic Sciences in Infectious Diseases Research Center, Department of Medical Parasitology and Mycology, Shiraz University of Medical Sciences, Shiraz, Iran Department of Medical Mycology and Parasitology, Urmia University of Medical Sciences, Urmia, Iran Cellular and Molecular Research Center, Urmia University of Medical Sciences, Urmia, Iran d Department of Medical Mycology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran e Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital (CWZ), Nijmegen, The Netherlands f Centre of Expertise in Mycology Radboudumc/CWZ, Nijmegen, The Netherlands g Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran h Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran i Department of Medical Mycology, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands j Invasive Fungi Research Center, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 June 2018 Received in revised form 15 September 2018 Accepted 25 September 2018 Available online xxx
Objective. – Cryptococcosis is a major opportunistic fungal infection caused by members of the genus Cryptococcus, mainly those belonging to the Cryptococcus neoformans/Cryptococcus gattii species complexes. Here, we report a comprehensive molecular epidemiological study of the environmental distribution of Cryptococcus isolates in Shiraz, Iran with review of litreature. Method. – A total of 406 samples were obtained from Eucalyptus trees and 139 samples from pigeon droppings. Cryptococcus species identification and genotyping were performed by amplified fragment length polymorphism (AFLP) fingerprinting sequencing and sequencing of the ITS rDNA region. Results. – Majority of the isolates belonged to the Naganishia taxon (n = 69) including N. albida (formerly C. albidus, n = 62), N. globosa (formerly C. saitoi, n = 4), N. adeliensis (formerly C. adeliensis, n = 2), N. diffluens (formerly C. diffluens, n = 1), and the identified C. neoformans isolates (n = 25) belonged to genotype AFLP1/VNI (n = 22) and AFLP1B/VNII (n = 3). Conclusion. – More research efforts should be employed to isolate C. gattii species complex from environmental niches in Iran and provide additional evidence related to novel molecular types.
C 2018 Elsevier Masson SAS. All rights reserved.
Keywords: Cryptococcus species Amplified Fragment Length Polymorphism (AFLP) fingerprinting Environmental sampling Iran
1. Introduction Cryptococcosis is a life-threatening fungal infections and is caused by members of the basidiomycetous yeast genus Cryptococcus that belongs to the class of Tremellomycetes [1]. Majority of cases are diagnosed in immunocompromised and also immunocompetent hosts with high mortality despite the application of antifungal therapy [2–4]. Recently, a new taxonomy was proposed for the pathogenic Cryptococcus neoformans/Cryptococcus gattii species complexes, i.e., C. neoformans (genotype AFLP1/VNI,
* Corresponding author. Department of Medical Mycology/Invasive Fungi Research Center, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. E-mail address: [email protected] (H. Badali).
AFLP1A/VNB/VNII and AFLP1B/VNII), followed by C. deneoformans (genotype AFLP2/VNIV) and Cryptococcus gattii into five species (C. gattii sensu stricto, genotype AFLP4/VGI; C. bacillisporus genotype AFLP5/VGIII; C. deuterogattii genotype AFLP6/VGII; C. tetragattii genotype AFLP7/VGIV and C. decagattii genotype AFLP10) [5]. Interspecies hybrids between C. neoformans and C. deneoformans (genotype AFLP3/VNIII) and hybrids with other cryptococcal species have been described [5]. Also the genus Cryptococcus has been taxonomically revised, reducing the number of species from over a hundred to only ten [1,6]. Despite the evidence for the taxonomic revision there has been some ongoing discussion [5,7]. C. neoformans s.s. has a worldwide geographical distribution, and is generally present in pigeon droppings [8–12], while C. gattii s.l. was traditionally considered to have a restricted geographic distribution in tropical and subtropical climates
https://doi.org/10.1016/j.mycmed.2018.09.005 C 2018 Elsevier Masson SAS. All rights reserved. 1156-5233/
Please cite this article in press as: Pakshir K, et al. Molecular epidemiology of environmental Cryptococcus species isolates based on amplified fragment length polymorphism. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.09.005
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associated with trees such as Eucalyptus, but through the years it has been found in other, more temperate climate, areas in the World [13–16]. Currently, molecular taxonomy based on PCR fingerprinting, Restriction Fragment Length Polymorphism (RFLP) analysis, Amplified Fragment Length Polymorphism (AFLP) fingerprinting, microsatellite typing and Multilocus Sequence Typing (MLST), have been regarded as more reliable and universally applicable than conventional techniques in unravelling the genotypic diversity [5]. AFLP fingerprinting is a highly discriminating genotyping tool based on the detection of genomic restriction fragments by PCR amplification. Although cryptococcosis is known for a long time, limited data are available from Iran [17]. Therefore, in the present investigation we compiled the first comprehensive study of environmental distribution of Cryptococcus isolates based on conventional and AFLP fingerprinting to gain insights into their molecular epidemiology in Shiraz, Iran. In addition, there is no comprehensive review of Cryptococcus species isolates in Iran describing the geographic distribution in the eastern portion of the Middle East with both arid and semi-arid climates. Therefore, we reviewed the English and Persian published papers on this topic.
university of Shiraz, and pet shops (Fig. 1). Samples were placed in sterile plastic bags, packaged and transferred to the mycological laboratory. Briefly, trunk and hollows samples were collected by rubbing the sterile cotton-tipped swab moistened in a solution of sterile distilled water, 5–10 g of flowers and leaves, fragmented with a pestle and 2 g of pigeon dropping were suspended in 50 mL of sterile distilled water followed by the addition of antibiotics (penicillin 1000 U and streptomycin 2000 U) with subsequent vigorous shaking for 3 minutes and allowed to settle for 15 minutes. Subsequently, 0.1 mL of the supernatant was spread onto the bird seed agar medium supplemented with biphenyl (5% Guizotia abyssinica seeds, 0.1% glucose, 0.1% KH2PO4, 0.1% creatinine, 0.1% biphenyl, 1.5% agar) and penicillin/streptomycin as described above. In order to observe brown pigmented colony formation, cultures were incubated at 28 8C for 10 days and were monitored daily after the first 3 days. Colonies were selected from positive plates and subsequently streaked to single colonies onto Sabouraud’s dextrose agar supplemented with 50 mg/L chloramphenicol (SDA; Difco, Franklin Lakes, NJ, USA) plates. Isolates producing brown pigmented colonies were identified as Cryptococcus species.
2. Materials and methods
2.2. Conventional identification, coding and storage
2.1. Sampling and Isolation
The grown isolates on bird seed agar medium were preliminarily identified based on India ink test and urease production. All identified isolates were suspended in tryptic soy broth medium (TSB, Scharlau, Spain) containing 2% glucose, 2% peptone and 20% glycerol at 70 8C for further investigation and deposited at the reference culture collections of Department of Medical Mycology, Shiraz University of Medical Sciences and Invasive Fungi Research Center (IFRC), Sari, Iran.
A total of 406 samples were obtained from Eucalyptus trees comprising trunk and hollows (n = 200), flowers (n = 100), and leaves (n = 106), and 139 samples from pigeon droppings were collected from ten different locations in Shiraz, Iran. Samples were collected during different seasons for the period 2012–2014 from houses, mainly from roofs, holly places, hospitals, medical
Fig. 1. Graphical summary of this study on molecular epidemiology of environmental Cryptococcus species isolates in Iran based on AFLP.
Please cite this article in press as: Pakshir K, et al. Molecular epidemiology of environmental Cryptococcus species isolates based on amplified fragment length polymorphism. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.09.005
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Fig. 2. Location of collected samples. Numbers indicate correlatively the follows: first total number of environmental samples from that region of Shiraz; second number of Cryptococcus species isolates.
3. Molecular characterization of the isolates
Animal Mycology ITS Database (ISHAM-ITS database) and submitted to GenBank.
3.1. DNA extraction and sequencing 3.2. AFLP genotyping Genomic DNA of cryptococcal isolate was extracted from 2-day-old culture as described by Makimura et al. and stored at 20 8C prior to use [18]. The ITS rDNA region was amplified and sequenced using internal primers (ITS1 and ITS4) as previously described [18]. Sequence data were aligned manually using MEGA 5.05 and BioEdit version 7.0.9 (Alignment, BioEdit Sequence 2011) software packages and obtained DNA sequences were compared with the International Society for Human and
AFLP genotyping was performed as previously described [19,20]. Fragments were analyzed on an ABI3500xL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA); for this purpose, the amplicons were 10 diluted with ddH2O, and 1 mL of this dilution was added to 0.1 mL LIZ600 internal size marker (Promega, Leiden, The Netherlands) and 8.9 mL ddH2O. Raw data was further processed by using Bionumerics v7.5 (Applied Maths,
Please cite this article in press as: Pakshir K, et al. Molecular epidemiology of environmental Cryptococcus species isolates based on amplified fragment length polymorphism. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.09.005
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Figure. 3. Amplified fragment length polymorphism fingerprint (AFLP) dendrogram of hundred Cryptococcus species of environmental isolates from Shiraz, Iran.
Please cite this article in press as: Pakshir K, et al. Molecular epidemiology of environmental Cryptococcus species isolates based on amplified fragment length polymorphism. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.09.005
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Sint-Martens Latem, Belgium) and a dendrogram was generated based on the UPGMA algorithm. Determination of the genotypes for the isolates was performed using a set of reference strains representing all C. neoformans s.l. and C. gattii s.l. haploid and hybrid genotypes.
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3.3. Data analysis Statistical analysis was performed using the SPSS software package version 18.0.0 (IBM, New York, NJ, USA). P values < 0.05 was considered statistically significant.
Table 1. Overview of published studies on environmental Cryptococcus species in Iran (1997–2018). Reference/year
Location
Sources
Total Identification methods samples
No. of positive isolates(%)
Species (no.)
Khosravi et al. (1997) [24] Nasr Isfahani et al. (2001) [25] Mikaeili et al. (2001) [26] Bineshian et al. (2002) [27]
Rasht, Ramsar, Babol, Sari,Gorgan
Pigeon droppings
983
Culture (NSA, CGB)
175 (17.8%)
C. deneoformans (175)
Isfahan
Pigeon dropping
136
Kermanshah
Pigeon droppings
1000
11 (8.1%) Culture (NSA, CGB), India ink, Ureas test, Assimilation test Culture (NSA), India ink 8 (0.8%)
Garmsar, Gonbad, Sadd-eGhaboos
Eucalyptus camaldulensis
1140
Culture (NSA, CGB), India ink, Urease test, API 20c Aux
138 (36.3%)
Shahrekoord
Pigeon droppings
800
50 (6.25%)
Ahwaz
Pigeon droppings
65
22 (34%)
C. neoformans (22)
Sari, Qaemshahr Hedayati et al. (2011) [30] Afshari et al. (2012) Mazandaran provience [31]
Swallow droppings
97
Culture (NSA), India ink, Urease test, Assimilation test Culture (NSA), India ink, Urease test, Assimilation test Culture(NSA, CGB), Urease test
C. gattii (2), Naganishia albida (43), Papiliotrema laurentii (36), Cryptococcus species (57) C. neoformans(50)
5 (5.2%)
C. neoformans (5)
Pigeon droppings
400
20 (5%)
C. neoformans (18), C. deneoformans (2)
Soltani et al. (2013) Isfahan [32]
Pigeon droppings
120
3 (2.5%.)
C. neoformans (3)
Beglari et al. (2013) West of Tehran Tehran Hashemi et al. (2014) [33]
Pigeon droppings Fecal sample (Lovebirds, Mynah, Parrot, Kasku, Finches, Bridal Dutch, Canaries) Eucalyptus trees, Soil
100 262
Culture (NSA), India ink, Urease test, Sequencing D1D2 and IGS regions), Mating type India ink, Urease test, Culture (NSA, CGB), RapID yeast plus system (RYP) Sequencing ITS regions Culture (NSA), India ink, Urease test, Assimilation test
11 (11%) 0 (0%)
C. neoformans (11) –
0 (0%)
–
13 (2.6%)
C. gattii (13)
Ebrahimi et al. (2004) [28] Zarrin et al. (2010) [29]
Salehei et al. (2015) [35] Nowrozi et al. (2015) [34]
Ahvaz Mazandaran provience, Golestan, Tehran, Isfahan, Fars, Khuzestan, Hormozgan Mazandaran provience
Amirrajab et al. (2016) [36] Mokhtarnejad et al. West and East Azarbaijan (2016) provinces [37]
Dehghan-Niri et al. (2017) [38] Kamari et al. (2017) [39]
156
Leaf, Soil, Flower, Air 495 around the Eucalyptus trees
Wild migratory birds
700
National Park of Urmia Lake 112 soil
Isfahan, Qom, Yazd, Canker-affected stone fruit 42 Chaharmahal and Bakhtiari trees Province Isfahan Pigeon nests 186
Eucalyptus trees
88
Culture (NSA), India ink, Urease test Culture (NSA,CGB), India ink, Urease test
4 (0.6%) Culture (NSA, CGB), India ink, Urease test, Sequencing ITS regions Sequencing the D1/D2 and 53 (47.3%) ITS 1 & 2 regions
Sucrose Nutrient Agar, Sequencing ITS regions
42 (100%)
Culture (NSA), Sequencing ITS regions
7 (3.7%)
Culture (NSA), Sequencing ITS regions
2 (2.2%)
C. deneoformans (11)
C. neoformans (8)
C. neoformans (4)
N. adeliensis (13), N. albidosimilis (8), N. albida (1), N. bhutanensis (2), N. diffluens (1), N. friedmannii (1), N. globosa (1), N. uzbekistanensis (26) Cryptococcus magnus (42)
N. albida (5), N. uzbekistanensis (2) N. adeliensis (1), C. neoformans var. grubii (1) Filobasidium uniguttulatum (1) N. adeliensis (2)
CGB: Canavanine Glycine–Bromothymol blue medium agar; NSA: Niger Seed Agar.
Please cite this article in press as: Pakshir K, et al. Molecular epidemiology of environmental Cryptococcus species isolates based on amplified fragment length polymorphism. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.09.005
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4. Results Graphical summary of molecular epidemiology were shown in Fig. 1. According to the conventional tests (urease production and India ink test) performed on the brown colonies that appeared onto bird seed agar, 47 out of 406 (11.6%) Eucalyptus trees samples and 47 of 139 (33.8%) pigeon droppings were identified as potential Cryptococcus species. Initial identification and distribution results of all samples are shown in Fig. 2. Based on sequencing of the ITS rDNA regions, a variety of species were identified, i.e., Naganishia albida, formerly Cryptococcus albidus (n = 62) consisting of samples from Eucalyptus trees (n = 42) and pigeon dropping (n = 20), and Cryptococcus neoformans s.s. from pigeon droppings (n = 25), Naganishia globosa (formerly Cryptococcus saitoi) from Eucalyptus trees (n = 3) and pigeon dropping (n = 1), Naganishia adeliensis (formerly Cryptococcus adeliensis) from Eucalyptus tree (n = 1) and pigeon dropping (n = 1), and N. diffluens (formerly C. diffluens) from Eucalyptus tree (n = 1). Nucleotide sequence accession numbers for determined isolates were deposited in GenBank under accession numbers KY216167–KY216188 and KY238135–KY238199. The genotype as determined by AFLP fingerprinting revealed that 22 isolates belonged to C. neoformans s.s. genotype AFLP1/VNI, 3 isolates were found to be AFLP1/VNB/VNII and 69 isolates fell in the Naganishia taxon including, N. albida (n = 62), N. globosa (n = 4), N. adeliensis (n = 2), N. diffluens (n = 1) (Fig. 3). Sampling during different seasons did not greatly influence the recovery of Cryptococcus species isolates. Overall, internal transcribed spacer (ITS) regions were not a suitable marker for species differentiation within this genus.
all the sampled areas, as well as the failure to isolate this pathogen from Eucalyptus trees, does not mean that the fungus is not present in these areas. Besides, C. gattii species complex has been reported from Asia, Africa, and Europe [14,42,43]. Further sampling in a larger area over an extended period of time would increase the recovery C. gattii species complex, given the fact that the C. gattii species complex in nearby countries has been reported [9]. Therefore, more research efforts should be employed to isolate C. gattii species complex from environmental niches in Iran and provide additional evidence related to novel molecular types. Funding This research was financially supported by Shiraz University of Medical Sciences (grants no. 6763 and 6764), Shiraz, Iran and Mazandaran University of Medical Sciences (grant no. 2550), school of Medicine, Sari, Iran. Disclosure of interest The authors declare that they have no competing interest. The authors alone are responsible for the content and writing of the paper. Acknowledgements F.H. and J.F.M thankfully acknowledge international collaboration for AFLP typing from Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital (CWZ), Nijmegen, The Netherlands and Department of Medical Mycology, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands.
5. Discussion References The current study is the first epidemiological and molecular characterization of environmental Cryptococcus species isolated in Shiraz, which is located in the eastern region of the Middle East and South of Iran. This first environmental survey generated data, which are currently accessible for detailed follow-up investigation in future studies. Only few studies are available from Jordan, Egypt and Turkey the in Middle East [21–23]. Although in Asian countries genotypic diversity studies have been carried out [13], at present, no epidemiological data have been reported in Iran. So far, there are only few studies, notably from a different city in Iran that reported Cryptococcus species from pigeon dropping and Eucalyptus trees with C. neoformans being more prevalent than other Cryptococcus species (Table 1) [24–39]. It appears that in climatically divergent geographical regions of Iran, the prevalence of C. neoformans is different and varies from 0.6% to 36% (Table 1). This variation could be due to the methodology used, sample size studied, climate of the region and culture techniques. Although the number of cryptococcal infections is low in Iran, in the period 1969 to 2015, the majority of the cases was caused by C. neoformans [17]. Our findings suggest that trees may represent an important environmental niche and a stable reservoir for N. albida, formerly C. albidus, and that bird excreta could represent a secondary niche. The majority of cryptococcal isolates from pigeon dropping represented C. neoformans, of which 22 isolates belonged to the major genotype AFLP1/VNI and a small proportion (n = 3) belonged to the genotype AFLP1B/VNII. The majority of isolates in the present study belong to the Naganishia taxon which suggests that the local geographic differences of Cryptococcus species prevalence could be the result of differences in the local environment and climate [40]. According to Simwami et al. C. neoformans was genetically less diverse than those occurring in Africa and North America in comparison with Asian countries [41]. The failure to isolate C. gattii species complex from
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[28] Ebrahimi A, Halajian A, Nikookhah F. Isolation of Cryptococcus neoformans from pigeon droppings in shahrekord, Iran. Indian Vet J 2004;81:1293–4. [29] Zarrin M, Jorfi M, Amirrajab N, Rostami M. Isolation of Cryptococcus neoformans from pigeon droppings in Ahwaz, Iran. Turk J Med Sci 2010;40:313–6. [30] Hedayati MT, Mayahi S, Fakhar M, Shokohi T, Majidi M. Cryptococcus neoformans isolation from swallow (Hirundo rustica) excreta in Iran. Rev Inst Med Trop Sao Paul 2011;53:125–7. [31] Afshari SAK, Shokohi T, Aghili R, Badali H. Epidemiology and molecular characterization of Cryptococcus neoformans isolated from pigeon excreta in Mazandaran province, northern Iran. J Mycol Med 2012;22:160–6. [32] Soltani M, Bayat M, Hashemi SJ, Zia M, Pestechian N. Isolation of Cryptococcus neoformans and other opportunistic fungi from pigeon droppings. J Res Med Sci 2013;18(1):56. [33] Hashemi SJ, Jabbari AG, Bayat M, Rafiei SM. Prevalence of Cryptococcus neoformans in domestic birds referred to veterinary clinics in Tehran. Eur J Exp Biol 2014;4:482–6. [34] Nowrozi H, Kazemi A, Mohammad ganji Nik B, Sabokbar A. Isolation of Cryptococcus neoformans var. gatti from Around of Eucalyptus trees in Different Regions of Iran. Sci J Ilam Uni Med Sci 2016;23:1–8. [35] Salehei Z, Mahmoudabadi AZ, Zarrin M. Lack of Cryptococcus gattii from Eucalyptus in Ahvaz. Curr Med Mycol 2015;1:1. [36] Amirrajab N, Haghani I, Rasuli M, Shokohi T. MigratoryBirds as a Potential Reservoirs of Cryptococcus neoformans. Int J Environ Res 2016;10:459–64. [37] Mokhtarnejad L, Arzanlou M, Babai-Ahari A, Di Mauro S, Onofri A, Buzzini P, et al. Characterization of basidiomycetous yeasts in hypersaline soils of the Urmia Lake National Park, Iran. Extremophiles 2016;20:915–28. [38] Dehghan-Niri M, Rahimian H, Babaeizad V. Cryptococcus magnus as the causative agent of stem and branch canker of stone fruit trees in some central provinces of Iran. J App Entomol Phytopathol 2017;84:239–50. [39] Kamari A, Sepahvand A, Mohammadi R. Isolation and molecular characterization of Cryptococcus species isolated from pigeon nests and Eucalyptus trees. Curr Med Mycol 2017;3:20. [40] Jain N, Wickes B, Keller S, Fu J, Casadevall A, Jain P, et al. Molecular epidemiology of clinical Cryptococcus neoformans strains from India. J Clin Microbiol 2005;43:5733–42. [41] Simwami SP, Khayhan K, Henk DA, Aanensen DM, Boekhout T, Hagen F, et al. Low diversity Cryptococcus neoformans variety grubii multilocus sequence types from Thailand are consistent with an ancestral African origin. PLoS Pathog 2011;7:e1001343. [42] Hagen F, Colom MF, Swinne D, Tintelnot K, Iatta R, Montagna MT, et al. Autochthonous and dormant Cryptococcus gattii infections in Europe. Emerg Infect Dis 2012;18:1618. [43] Springer DJ, Chaturvedi V. Projecting global occurrence of Cryptococcus gattii. Emerg Infect Dis 2010;16:14.
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Available online at
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Research Paper
Molecular epidemiology of environmental Cryptococcus species isolates based on amplified fragment length polymorphism K. Pakshir a, H. Fakhim b,c, A. Vaezi d, J.F. Meis e,f, M. Mahmoodi a, K. Zomorodian a, J. Javidnia d,g, S. Ansari h, F. Hagen e,i, H. Badali d,j,* a
Basic Sciences in Infectious Diseases Research Center, Department of Medical Parasitology and Mycology, Shiraz University of Medical Sciences, Shiraz, Iran Department of Medical Mycology and Parasitology, Urmia University of Medical Sciences, Urmia, Iran Cellular and Molecular Research Center, Urmia University of Medical Sciences, Urmia, Iran d Department of Medical Mycology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran e Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital (CWZ), Nijmegen, The Netherlands f Centre of Expertise in Mycology Radboudumc/CWZ, Nijmegen, The Netherlands g Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran h Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran i Department of Medical Mycology, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands j Invasive Fungi Research Center, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 June 2018 Received in revised form 15 September 2018 Accepted 25 September 2018 Available online xxx
Objective. – Cryptococcosis is a major opportunistic fungal infection caused by members of the genus Cryptococcus, mainly those belonging to the Cryptococcus neoformans/Cryptococcus gattii species complexes. Here, we report a comprehensive molecular epidemiological study of the environmental distribution of Cryptococcus isolates in Shiraz, Iran with review of litreature. Method. – A total of 406 samples were obtained from Eucalyptus trees and 139 samples from pigeon droppings. Cryptococcus species identification and genotyping were performed by amplified fragment length polymorphism (AFLP) fingerprinting sequencing and sequencing of the ITS rDNA region. Results. – Majority of the isolates belonged to the Naganishia taxon (n = 69) including N. albida (formerly C. albidus, n = 62), N. globosa (formerly C. saitoi, n = 4), N. adeliensis (formerly C. adeliensis, n = 2), N. diffluens (formerly C. diffluens, n = 1), and the identified C. neoformans isolates (n = 25) belonged to genotype AFLP1/VNI (n = 22) and AFLP1B/VNII (n = 3). Conclusion. – More research efforts should be employed to isolate C. gattii species complex from environmental niches in Iran and provide additional evidence related to novel molecular types.
C 2018 Elsevier Masson SAS. All rights reserved.
Keywords: Cryptococcus species Amplified Fragment Length Polymorphism (AFLP) fingerprinting Environmental sampling Iran
1. Introduction Cryptococcosis is a life-threatening fungal infections and is caused by members of the basidiomycetous yeast genus Cryptococcus that belongs to the class of Tremellomycetes [1]. Majority of cases are diagnosed in immunocompromised and also immunocompetent hosts with high mortality despite the application of antifungal therapy [2–4]. Recently, a new taxonomy was proposed for the pathogenic Cryptococcus neoformans/Cryptococcus gattii species complexes, i.e., C. neoformans (genotype AFLP1/VNI,
* Corresponding author. Department of Medical Mycology/Invasive Fungi Research Center, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran. E-mail address: [email protected] (H. Badali).
AFLP1A/VNB/VNII and AFLP1B/VNII), followed by C. deneoformans (genotype AFLP2/VNIV) and Cryptococcus gattii into five species (C. gattii sensu stricto, genotype AFLP4/VGI; C. bacillisporus genotype AFLP5/VGIII; C. deuterogattii genotype AFLP6/VGII; C. tetragattii genotype AFLP7/VGIV and C. decagattii genotype AFLP10) [5]. Interspecies hybrids between C. neoformans and C. deneoformans (genotype AFLP3/VNIII) and hybrids with other cryptococcal species have been described [5]. Also the genus Cryptococcus has been taxonomically revised, reducing the number of species from over a hundred to only ten [1,6]. Despite the evidence for the taxonomic revision there has been some ongoing discussion [5,7]. C. neoformans s.s. has a worldwide geographical distribution, and is generally present in pigeon droppings [8–12], while C. gattii s.l. was traditionally considered to have a restricted geographic distribution in tropical and subtropical climates
https://doi.org/10.1016/j.mycmed.2018.09.005 C 2018 Elsevier Masson SAS. All rights reserved. 1156-5233/
Please cite this article in press as: Pakshir K, et al. Molecular epidemiology of environmental Cryptococcus species isolates based on amplified fragment length polymorphism. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.09.005
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associated with trees such as Eucalyptus, but through the years it has been found in other, more temperate climate, areas in the World [13–16]. Currently, molecular taxonomy based on PCR fingerprinting, Restriction Fragment Length Polymorphism (RFLP) analysis, Amplified Fragment Length Polymorphism (AFLP) fingerprinting, microsatellite typing and Multilocus Sequence Typing (MLST), have been regarded as more reliable and universally applicable than conventional techniques in unravelling the genotypic diversity [5]. AFLP fingerprinting is a highly discriminating genotyping tool based on the detection of genomic restriction fragments by PCR amplification. Although cryptococcosis is known for a long time, limited data are available from Iran [17]. Therefore, in the present investigation we compiled the first comprehensive study of environmental distribution of Cryptococcus isolates based on conventional and AFLP fingerprinting to gain insights into their molecular epidemiology in Shiraz, Iran. In addition, there is no comprehensive review of Cryptococcus species isolates in Iran describing the geographic distribution in the eastern portion of the Middle East with both arid and semi-arid climates. Therefore, we reviewed the English and Persian published papers on this topic.
university of Shiraz, and pet shops (Fig. 1). Samples were placed in sterile plastic bags, packaged and transferred to the mycological laboratory. Briefly, trunk and hollows samples were collected by rubbing the sterile cotton-tipped swab moistened in a solution of sterile distilled water, 5–10 g of flowers and leaves, fragmented with a pestle and 2 g of pigeon dropping were suspended in 50 mL of sterile distilled water followed by the addition of antibiotics (penicillin 1000 U and streptomycin 2000 U) with subsequent vigorous shaking for 3 minutes and allowed to settle for 15 minutes. Subsequently, 0.1 mL of the supernatant was spread onto the bird seed agar medium supplemented with biphenyl (5% Guizotia abyssinica seeds, 0.1% glucose, 0.1% KH2PO4, 0.1% creatinine, 0.1% biphenyl, 1.5% agar) and penicillin/streptomycin as described above. In order to observe brown pigmented colony formation, cultures were incubated at 28 8C for 10 days and were monitored daily after the first 3 days. Colonies were selected from positive plates and subsequently streaked to single colonies onto Sabouraud’s dextrose agar supplemented with 50 mg/L chloramphenicol (SDA; Difco, Franklin Lakes, NJ, USA) plates. Isolates producing brown pigmented colonies were identified as Cryptococcus species.
2. Materials and methods
2.2. Conventional identification, coding and storage
2.1. Sampling and Isolation
The grown isolates on bird seed agar medium were preliminarily identified based on India ink test and urease production. All identified isolates were suspended in tryptic soy broth medium (TSB, Scharlau, Spain) containing 2% glucose, 2% peptone and 20% glycerol at 70 8C for further investigation and deposited at the reference culture collections of Department of Medical Mycology, Shiraz University of Medical Sciences and Invasive Fungi Research Center (IFRC), Sari, Iran.
A total of 406 samples were obtained from Eucalyptus trees comprising trunk and hollows (n = 200), flowers (n = 100), and leaves (n = 106), and 139 samples from pigeon droppings were collected from ten different locations in Shiraz, Iran. Samples were collected during different seasons for the period 2012–2014 from houses, mainly from roofs, holly places, hospitals, medical
Fig. 1. Graphical summary of this study on molecular epidemiology of environmental Cryptococcus species isolates in Iran based on AFLP.
Please cite this article in press as: Pakshir K, et al. Molecular epidemiology of environmental Cryptococcus species isolates based on amplified fragment length polymorphism. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.09.005
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Fig. 2. Location of collected samples. Numbers indicate correlatively the follows: first total number of environmental samples from that region of Shiraz; second number of Cryptococcus species isolates.
3. Molecular characterization of the isolates
Animal Mycology ITS Database (ISHAM-ITS database) and submitted to GenBank.
3.1. DNA extraction and sequencing 3.2. AFLP genotyping Genomic DNA of cryptococcal isolate was extracted from 2-day-old culture as described by Makimura et al. and stored at 20 8C prior to use [18]. The ITS rDNA region was amplified and sequenced using internal primers (ITS1 and ITS4) as previously described [18]. Sequence data were aligned manually using MEGA 5.05 and BioEdit version 7.0.9 (Alignment, BioEdit Sequence 2011) software packages and obtained DNA sequences were compared with the International Society for Human and
AFLP genotyping was performed as previously described [19,20]. Fragments were analyzed on an ABI3500xL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA); for this purpose, the amplicons were 10 diluted with ddH2O, and 1 mL of this dilution was added to 0.1 mL LIZ600 internal size marker (Promega, Leiden, The Netherlands) and 8.9 mL ddH2O. Raw data was further processed by using Bionumerics v7.5 (Applied Maths,
Please cite this article in press as: Pakshir K, et al. Molecular epidemiology of environmental Cryptococcus species isolates based on amplified fragment length polymorphism. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.09.005
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Figure. 3. Amplified fragment length polymorphism fingerprint (AFLP) dendrogram of hundred Cryptococcus species of environmental isolates from Shiraz, Iran.
Please cite this article in press as: Pakshir K, et al. Molecular epidemiology of environmental Cryptococcus species isolates based on amplified fragment length polymorphism. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.09.005
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Sint-Martens Latem, Belgium) and a dendrogram was generated based on the UPGMA algorithm. Determination of the genotypes for the isolates was performed using a set of reference strains representing all C. neoformans s.l. and C. gattii s.l. haploid and hybrid genotypes.
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3.3. Data analysis Statistical analysis was performed using the SPSS software package version 18.0.0 (IBM, New York, NJ, USA). P values < 0.05 was considered statistically significant.
Table 1. Overview of published studies on environmental Cryptococcus species in Iran (1997–2018). Reference/year
Location
Sources
Total Identification methods samples
No. of positive isolates(%)
Species (no.)
Khosravi et al. (1997) [24] Nasr Isfahani et al. (2001) [25] Mikaeili et al. (2001) [26] Bineshian et al. (2002) [27]
Rasht, Ramsar, Babol, Sari,Gorgan
Pigeon droppings
983
Culture (NSA, CGB)
175 (17.8%)
C. deneoformans (175)
Isfahan
Pigeon dropping
136
Kermanshah
Pigeon droppings
1000
11 (8.1%) Culture (NSA, CGB), India ink, Ureas test, Assimilation test Culture (NSA), India ink 8 (0.8%)
Garmsar, Gonbad, Sadd-eGhaboos
Eucalyptus camaldulensis
1140
Culture (NSA, CGB), India ink, Urease test, API 20c Aux
138 (36.3%)
Shahrekoord
Pigeon droppings
800
50 (6.25%)
Ahwaz
Pigeon droppings
65
22 (34%)
C. neoformans (22)
Sari, Qaemshahr Hedayati et al. (2011) [30] Afshari et al. (2012) Mazandaran provience [31]
Swallow droppings
97
Culture (NSA), India ink, Urease test, Assimilation test Culture (NSA), India ink, Urease test, Assimilation test Culture(NSA, CGB), Urease test
C. gattii (2), Naganishia albida (43), Papiliotrema laurentii (36), Cryptococcus species (57) C. neoformans(50)
5 (5.2%)
C. neoformans (5)
Pigeon droppings
400
20 (5%)
C. neoformans (18), C. deneoformans (2)
Soltani et al. (2013) Isfahan [32]
Pigeon droppings
120
3 (2.5%.)
C. neoformans (3)
Beglari et al. (2013) West of Tehran Tehran Hashemi et al. (2014) [33]
Pigeon droppings Fecal sample (Lovebirds, Mynah, Parrot, Kasku, Finches, Bridal Dutch, Canaries) Eucalyptus trees, Soil
100 262
Culture (NSA), India ink, Urease test, Sequencing D1D2 and IGS regions), Mating type India ink, Urease test, Culture (NSA, CGB), RapID yeast plus system (RYP) Sequencing ITS regions Culture (NSA), India ink, Urease test, Assimilation test
11 (11%) 0 (0%)
C. neoformans (11) –
0 (0%)
–
13 (2.6%)
C. gattii (13)
Ebrahimi et al. (2004) [28] Zarrin et al. (2010) [29]
Salehei et al. (2015) [35] Nowrozi et al. (2015) [34]
Ahvaz Mazandaran provience, Golestan, Tehran, Isfahan, Fars, Khuzestan, Hormozgan Mazandaran provience
Amirrajab et al. (2016) [36] Mokhtarnejad et al. West and East Azarbaijan (2016) provinces [37]
Dehghan-Niri et al. (2017) [38] Kamari et al. (2017) [39]
156
Leaf, Soil, Flower, Air 495 around the Eucalyptus trees
Wild migratory birds
700
National Park of Urmia Lake 112 soil
Isfahan, Qom, Yazd, Canker-affected stone fruit 42 Chaharmahal and Bakhtiari trees Province Isfahan Pigeon nests 186
Eucalyptus trees
88
Culture (NSA), India ink, Urease test Culture (NSA,CGB), India ink, Urease test
4 (0.6%) Culture (NSA, CGB), India ink, Urease test, Sequencing ITS regions Sequencing the D1/D2 and 53 (47.3%) ITS 1 & 2 regions
Sucrose Nutrient Agar, Sequencing ITS regions
42 (100%)
Culture (NSA), Sequencing ITS regions
7 (3.7%)
Culture (NSA), Sequencing ITS regions
2 (2.2%)
C. deneoformans (11)
C. neoformans (8)
C. neoformans (4)
N. adeliensis (13), N. albidosimilis (8), N. albida (1), N. bhutanensis (2), N. diffluens (1), N. friedmannii (1), N. globosa (1), N. uzbekistanensis (26) Cryptococcus magnus (42)
N. albida (5), N. uzbekistanensis (2) N. adeliensis (1), C. neoformans var. grubii (1) Filobasidium uniguttulatum (1) N. adeliensis (2)
CGB: Canavanine Glycine–Bromothymol blue medium agar; NSA: Niger Seed Agar.
Please cite this article in press as: Pakshir K, et al. Molecular epidemiology of environmental Cryptococcus species isolates based on amplified fragment length polymorphism. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.09.005
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4. Results Graphical summary of molecular epidemiology were shown in Fig. 1. According to the conventional tests (urease production and India ink test) performed on the brown colonies that appeared onto bird seed agar, 47 out of 406 (11.6%) Eucalyptus trees samples and 47 of 139 (33.8%) pigeon droppings were identified as potential Cryptococcus species. Initial identification and distribution results of all samples are shown in Fig. 2. Based on sequencing of the ITS rDNA regions, a variety of species were identified, i.e., Naganishia albida, formerly Cryptococcus albidus (n = 62) consisting of samples from Eucalyptus trees (n = 42) and pigeon dropping (n = 20), and Cryptococcus neoformans s.s. from pigeon droppings (n = 25), Naganishia globosa (formerly Cryptococcus saitoi) from Eucalyptus trees (n = 3) and pigeon dropping (n = 1), Naganishia adeliensis (formerly Cryptococcus adeliensis) from Eucalyptus tree (n = 1) and pigeon dropping (n = 1), and N. diffluens (formerly C. diffluens) from Eucalyptus tree (n = 1). Nucleotide sequence accession numbers for determined isolates were deposited in GenBank under accession numbers KY216167–KY216188 and KY238135–KY238199. The genotype as determined by AFLP fingerprinting revealed that 22 isolates belonged to C. neoformans s.s. genotype AFLP1/VNI, 3 isolates were found to be AFLP1/VNB/VNII and 69 isolates fell in the Naganishia taxon including, N. albida (n = 62), N. globosa (n = 4), N. adeliensis (n = 2), N. diffluens (n = 1) (Fig. 3). Sampling during different seasons did not greatly influence the recovery of Cryptococcus species isolates. Overall, internal transcribed spacer (ITS) regions were not a suitable marker for species differentiation within this genus.
all the sampled areas, as well as the failure to isolate this pathogen from Eucalyptus trees, does not mean that the fungus is not present in these areas. Besides, C. gattii species complex has been reported from Asia, Africa, and Europe [14,42,43]. Further sampling in a larger area over an extended period of time would increase the recovery C. gattii species complex, given the fact that the C. gattii species complex in nearby countries has been reported [9]. Therefore, more research efforts should be employed to isolate C. gattii species complex from environmental niches in Iran and provide additional evidence related to novel molecular types. Funding This research was financially supported by Shiraz University of Medical Sciences (grants no. 6763 and 6764), Shiraz, Iran and Mazandaran University of Medical Sciences (grant no. 2550), school of Medicine, Sari, Iran. Disclosure of interest The authors declare that they have no competing interest. The authors alone are responsible for the content and writing of the paper. Acknowledgements F.H. and J.F.M thankfully acknowledge international collaboration for AFLP typing from Department of Medical Microbiology and Infectious Diseases, Canisius-Wilhelmina Hospital (CWZ), Nijmegen, The Netherlands and Department of Medical Mycology, Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands.
5. Discussion References The current study is the first epidemiological and molecular characterization of environmental Cryptococcus species isolated in Shiraz, which is located in the eastern region of the Middle East and South of Iran. This first environmental survey generated data, which are currently accessible for detailed follow-up investigation in future studies. Only few studies are available from Jordan, Egypt and Turkey the in Middle East [21–23]. Although in Asian countries genotypic diversity studies have been carried out [13], at present, no epidemiological data have been reported in Iran. So far, there are only few studies, notably from a different city in Iran that reported Cryptococcus species from pigeon dropping and Eucalyptus trees with C. neoformans being more prevalent than other Cryptococcus species (Table 1) [24–39]. It appears that in climatically divergent geographical regions of Iran, the prevalence of C. neoformans is different and varies from 0.6% to 36% (Table 1). This variation could be due to the methodology used, sample size studied, climate of the region and culture techniques. Although the number of cryptococcal infections is low in Iran, in the period 1969 to 2015, the majority of the cases was caused by C. neoformans [17]. Our findings suggest that trees may represent an important environmental niche and a stable reservoir for N. albida, formerly C. albidus, and that bird excreta could represent a secondary niche. The majority of cryptococcal isolates from pigeon dropping represented C. neoformans, of which 22 isolates belonged to the major genotype AFLP1/VNI and a small proportion (n = 3) belonged to the genotype AFLP1B/VNII. The majority of isolates in the present study belong to the Naganishia taxon which suggests that the local geographic differences of Cryptococcus species prevalence could be the result of differences in the local environment and climate [40]. According to Simwami et al. C. neoformans was genetically less diverse than those occurring in Africa and North America in comparison with Asian countries [41]. The failure to isolate C. gattii species complex from
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Please cite this article in press as: Pakshir K, et al. Molecular epidemiology of environmental Cryptococcus species isolates based on amplified fragment length polymorphism. Journal De Mycologie Me´dicale (2018), https://doi.org/10.1016/j.mycmed.2018.09.005