Molecular identification of Sporothrix clinical isolates in China

Molecular identification of Sporothrix clinical isolates in China 中国地区孢子丝菌临床分离株的分子鉴定 1 first-author 2 1 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhouxun123@sina.com †E-mail:zhouxun123@sina.com zhouxun123@sina.com Department of Dermatology, Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China Department of Dermatology, Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China In this study, we investigated the molecular phylogeny of 64 clinical isolates which were identified as Sporothrix schenckii sensu lato by morphological identification. All of the strains were isolates from patients from several provinces in China. The phylogeny was inferred by DNA sequence analyses based on datasets of the ribosomal internal transcribed spacer (ITS) and a combined ITS and partial β-tubulin region. Reference sequences were retrieved from GenBank. Results showed that all of the isolates were clustered in a distinct clade with a type of Sporothrix globosa . Our analysis showed that S. globosa is the causal agent of the tested sporotrichosis in China, rather than S. schenckii that was generally believed to be the case. The existence of S. schenckii in China remains to be confirmed. This study improved our understanding of the distribution of the species in S. schenckii complex. 研究目的 2 明确中国地区孢子丝菌病的致病菌种。 创新方法 2 构建中国地区临床孢子丝菌系统进化树，用分子生物学方法对致病菌种进行重新鉴定。 研究手段 2 多基因测序和系统发育分析。 重要结论 2 球形孢子丝菌可能为中国地区孢子丝菌病的主要病原菌，申克孢子丝菌在中国地区是否存在尚需进一步验证。 Internal transcribed spacer (ITS) Diagnosis Sporothrix globosa Taxonomy Phylogeny 内含子转录间隔区 球形孢子丝菌 分类学 系统发育 Introduction 1 Sporotrichosis is an acute or chronic mycosis caused by infection of the Sporothrix schenckii , and recently is demonstrated to be a species complex (López-Romero et al., 2011 2011 ). Humans and animals acquire it from inoculation of the fungus through thorns, traumas and scratches or bite wounds (de Lima Barros et al., 2004 2004 ; Schubach et al., 2008 2008 ), even the spores can enter through the respiratory tract. Sporotrichosis may develop as a disseminated disease in immunocompromised individuals particularly in acquired immunodeficiency syndrome (AIDS) patients (Yap, 2011 2011 ). Sporotrichosis has a global distribution. In China, it is more frequently encountered in the northeast provinces including Jilin, Heilongjiang, and Liaoning (Zhang and Lin, 2008 2008 ; Mei et al., 2011 2011 ; Song et al., 2011a 2011a ; 2011b 2011b ). S. schenckii had long been believed to be the unique causal agent of sporotrichosis. Identifications were made primarily based on morphological characters (Lopes-Bezerra et al., 2006 2006 ). Phylogenetic analysis based on the sequences of the internal transcribed spacer (ITS) region suggested that more than one species existed in this morphologically defined group, indicating this is a species complex (de Beer et al., 2003 2003 ). Recently, isolates received as S. schenckii were regrouped into at least six cryptic species by multilocus phylogenetic analysis, and exhibited a degree of geographical specificity (Marimon et al., 2006 2006 ; 2007 2007 ; Madrid et al., 2010 2010 ). Different clinical forms of sporotrichosis are related to the host condition, the route of infection, and the virulence of pathogen, while the virulence was suggested to be associated with different species of Sporothrix (Arrillaga-Moncrieff et al., 2009 2009 ; Fernandes et al., 2013 2013 ). Therefore, it is important to recognize what are the real causal agents of the sporotrichosis in various regions, because of the significant differences in the guiding clinical management among these species. Clinical Sporothrix species have previously been classified based on the partial calmodulin sequences (Marimon et al., 2007 2007 ; 2008b 2008b ; Tan et al., 2013 2013 ), but calmodulin regions have been reported to have a lower amplification efficiency as compared to more commonly used regions such as the ribosomal ITS and β-tubulin. ITS is widely used in fungal identification, and recommended as the general barcode for fungi, although additional markers might be needed to distinguish species in certain complexes (Schoch et al., 2012 2012 ). Zhou et al. ( 2013 2013 ) concluded that all the Sporothrix complex species of clinical isolates could readily be recognized by ITS phylogeny. The aims of this study were to (1) collect a wide range representative strains of pathogens causing sporotrichosis in China, and (2) characterize these strains and confirm their identity through analyses of the ITS sequences and β-tubulin of these strains with relevant taxa. Materials and methods 1 Fungal isolates 2 Sixty-four clinical isolates identified as S. schenckii sensu lato were included in this study (Table 1 1 ). All these isolates were collected from the Dermatology Departments of three hospitals in China, mostly from farmers, housewives, or children. The breakdown is as follows: 26 from the First Hospital of Jilin University (Northeast China), 13 from the First Hospital of Beijing University (North China), and 25 from the Southwest Hospital of the Third Military Medical University (Southwest China). These isolates were subcultured on 2% potato-dextrose agar plates at 25 °C for 14 d. Isolates were then stored in sterilized water at 4 °C and on potato dextrose agar (PDA) slant at room temperature. Indicates the type strain CBS: culture collection of the Centraalbureau voor Schimmelcultures, Utrecht, the Netherlands; CMW: culture collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Pretoria, South Africa; ATCC: American Type Culture Collection, Virginia, USA; IPEC: Instituto de Pesquisa Clínica Evandro Chagas, Fiocruz, Brazil; FMR: Facultate de Medicina i Ciències de la Sault, Reus, Spain; KMU: Kanazawa Medical University, Ishikawa, Japan 2 Fungal species 2 Strain No. 2 Origin 2 GenBank No. 2 Reference ITS β-tubulin S. globosa LC2401 Clinical, Jilin, China JX997723 KC407844 This study S. globosa LC2402 Clinical, Jilin, China JX997718 KC407845 This study S. globosa LC2403 Clinical, Jilin, China JX997741 KC407846 This study S. globosa LC2404 Clinical, Jilin, China JX997737 KC407847 This study S. globosa LC2405 Clinical, Jilin, China JX997702 KC407848 This study S. globosa LC2406 Clinical, Jilin, China JX997734 KC407911 This study S. globosa LC2407 Clinical, Jilin, China JX997738 KC407849 This study S. globosa LC2408 Clinical, Jilin, China JX997715 KC407850 This study S. globosa LC2409 Clinical, Jilin, China JX997740 KC407851 This study S. globosa LC2410 Clinical, Jilin, China JX997711 KC407852 This study S. globosa LC2411 Clinical, Jilin, China JX997731 KC407853 This study S. globosa LC2412 Clinical, Jilin, China JX997733 KC407854 This study S. globosa LC2413 Clinical, Jilin, China JX997713 KC407855 This study S. globosa LC2414 Clinical, Jilin, China JX997712 KC407856 This study S. globosa LC2415 Clinical, Jilin, China JX997730 KC407857 This study S. globosa LC2416 Clinical, Jilin, China JX997709 KC407858 This study S. globosa LC2417 Clinical, Jilin, China JX997710 KC407859 This study S. globosa LC2418 Clinical, Jilin, China JX997724 KC407860 This study S. globosa LC2419 Clinical, Jilin, China JX997719 KC407861 This study S. globosa LC2420 Clinical, Jilin, China JX997720 KC407862 This study S. globosa LC2421 Clinical, Jilin, China JX997728 KC407863 This study S. globosa LC2422 Clinical, Jilin, China JX997698 KC407864 This study S. globosa LC2423 Clinical, Jilin, China JX997739 KC407865 This study S. globosa LC2424 Clinical, Jilin, China JX997729 KC407866 This study S. globosa LC2425 Clinical, Jilin, China JX997735 KC407867 This study S. globosa LC2426 Clinical, Jilin, China JX997700 KC407868 This study S. globosa LC2427 Clinical, Chongqing, China JX997722 KC407869 This study S. globosa LC2428 Clinical, Chongqing, China JX997692 KC407870 This study S. globosa LC2429 Clinical, Chongqing, China JX997725 KC407871 This study S. globosa LC2430 Clinical, Chongqing, China JX997717 KC407872 This study S. globosa LC2431 Clinical, Chongqing, China JX997732 KC407873 This study S. globosa LC2432 Clinical, Chongqing, China JX997704 KC407874 This study S. globosa LC2433 Clinical, Chongqing, China JX997701 KC407875 This study S. globosa LC2434 Clinical, Chongqing, China JX997705 KC407876 This study S. globosa LC2435 Clinical, Chongqing, China JX997707 KC407877 This study S. globosa LC2436 Clinical, Chongqing, China JX997742 KC407878 This study S. globosa LC2437 Clinical, Chongqing, China JX997708 KC407879 This study S. globosa LC2438 Clinical, Chongqing, China JX997721 KC407880 This study S. globosa LC2439 Clinical, Chongqing, China JX997703 KC407881 This study S. globosa LC2440 Clinical, Chongqing, China JX997695 KC407882 This study S. globosa LC2442 Clinical, Chongqing, China JX997697 KC407884 This study S. globosa LC2443 Clinical, Chongqing, China JX997693 KC407885 This study S. globosa LC2444 Clinical, Chongqing, China JX997694 KC407886 This study S. globosa LC2445 Clinical, Chongqing, China JX997726 KC407887 This study S. globosa LC2446 Clinical, Chongqing, China JX997727 KC407888 This study S. globosa LC2447 Clinical, Chongqing, China JX997716 KC407889 This study S. globosa LC2448 Clinical, Chongqing, China JX997696 KC407890 This study S. globosa LC2449 Clinical, Chongqing, China JX997699 KC407891 This study S. globosa LC2450 Clinical, Chongqing, China JX997714 KC407892 This study S. globosa LC2451 Clinical, Chongqing, China JX997736 KC407893 This study S. globosa LC2452 Clinical, Chongqing, China JX997706 KC407894 This study S. globosa LC2453 Clinical, Beijing, China KC407830 KC407895 This study S. globosa LC2454 Clinical, Beijing, China KC407831 KC407896 This study S. globosa LC2455 Clinical, Beijing, China KC407832 KC407897 This study S. globosa LC2456 Clinical, Beijing, China KC407833 KC407898 This study S. globosa LC2459 Clinical, Beijing, China KC407834 KC407900 This study S. globosa LC2460 Clinical, Beijing, China KC407835 KC407901 This study S. globosa LC2462 Clinical, Beijing, China KC407837 KC407903 This study S. globosa LC2463 Clinical, Beijing, China KC407838 KC407904 This study S. globosa LC2465 Clinical, Beijing, China KC407839 KC407906 This study S. globosa LC2466 Clinical, Beijing, China KC407840 KC407907 This study S. globosa LC2467 Clinical, Beijing, China KC407841 KC407908 This study S. globosa LC2468 Clinical, Beijing, China KC407842 KC407909 This study S. globosa LC2469 Clinical, Beijing, China KC407843 KC407910 This study S. schenckii CBS 117842 (CMW 7614) Clinical, South Africa AY280495 AY280477 Aghayeva et al. ( 2004 2004 ) S. schenckii CMW 7615 Clinical, South Africa AY280496 AY280478 Aghayeva et al. ( 2004 2004 ) S. schenckii CMW 7612 Clinical, South Africa AY280494 AY280476 Aghayeva et al. ( 2004 2004 ) S. schenckii ATCC 26331 (CBS 359.36 * * ) Clinical, USA FJ545232 AM116911 Marimon et al. ( 2006 2006 ) S. schenckii CMW 5681 Clinical, South Africa EF127886 EF139107 de Meyer et al. (2008) S. brasiliensis IPEC 17943 Clinical, Brazil FN549902 AM116935 Madrid et al. ( 2010 2010 ) S. brasiliensis IPEC 15572 Clinical, Brazil FN549904 AM116955 Madrid et al. ( 2010 2010 ) S. globosa FMR 8597 Clinical, Spain FN549904 AM116964 Madrid et al. ( 2010 2010 ) S. globosa CBS 120340 * * (FMR 8600) Clinical, Spain FN549905 AM116966 Madrid et al. ( 2010 2010 ) S. luriei KMU 2787 (CBS 937.72 * * ) Clinical, South Africa AB128012 AM747289 Kawasaki et al. (2003) S. mexicana CBS 120341 * * Environmental, Mexico FN549906 AM498344 Madrid et al. ( 2010 2010 ) S. pallida CBS 150.87 * * (CMW 17168) Environmental, Germany EF127879 EF139109 de Meyer et al. (2008) Ophiostoma nigrocarpum CMW 650 Abies sp. AY280489 AY280479 Aghayeva et al. ( 2004 2004 ) DNA extraction, amplification, and sequencing 2 Sporothrix spp. strains were inoculated on 2% PDA plates and incubated at 25 °C for 10 d. About 200 mg of fresh filamentous mycelia were scraped off for DNA extraction. The total genomic DNA was extracted using the cetyltrimethyl ammonium bromide (CTAB) method (Porebski et al., 1997 1997 ). The quality and quantity of genomic DNA were detected by using 1% agarose gel electrophoresis imaging. Products were stored at −20 °C. The BT2a / BT2b primers were used to amplify the partial β-tubulin gene (Glass and Donaldson, 1995 1995 ). The ITS region was amplified using the primer pair ITS1 / ITS4 (White et al., 1990 1990 ). The polymerase chain reaction (PCR) mixture consisted of 2.5 μl of 10 mmol/L PCR buffer, 2 μl of 2.5 mmol/L dNTP mix, 1 μl of each primer (10 μmol/L), 1 μl of DNA template, 0.2 μl Taq DNA polymerase, and 17.3 μl double-distilled water. Amplification was performed through the following steps: initial denaturation at 95 °C for 5 min, followed by 35 cycling consisting of denaturing at 95 °C for 30 s, annealing at 52 °C ( Bt2 ) or 54 °C (ITS) for 30 s, and extension at 72 °C for 1 min, followed by 10 min at 72 °C for extension. The PCR products were quantified by 1% agarose gel electrophoresis, and purified products were sequenced at the Beijing Genomics Institute (BGI; China) with the ITS1 / ITS4 and BT2a / BT2b primers mentioned above. Sequence alignment and phylogenetic analysis 2 Sequences generated from the forward and reverse primers were assembled using a Contig Express component to obtain consensus sequence. The sequence identified for all our isolates was verified by a BLAST search ( http://blast.ncbi.nlm.nih.gov http://blast.ncbi.nlm.nih.gov ). Several ITS regions and β-tubulin sequences published in GenBank were retrieved as reference sequences, together with sequences of new isolates (Table 1 1 ). Alignments of ITS and combined ITS and β-tubulin were made using an MAFFT program ( http://www.ebi.ac.uk/Tools/msa/mafft/ http://www.ebi.ac.uk/Tools/msa/mafft/ ) (Katoh and Toh, 2010 2010 ), and followed by manual adjustments in BioEdit V7.0.5 (Hall, 1999 1999 ). Both alignments were used for phylogenetic analyses. The maximum parsimony (MP) analysis was performed using PAUP V.4.0b10 (Swofford, 2003 2003 ). Ambiguously aligned regions were excluded from all analysis. Trees were inferred using the heuristic search by the 1000 random sequence addition and the tree-bisection-reconnection (TBR) branch-swapping algorithm. Branches of zero length were collapsed and all minimal-length trees were saved. Gaps were treated as missing data. Clade stability was assessed in a bootstrap test with 1000 replications. Tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for the generated parsimony trees. A Kishino-Hasegawa (KH) test was performed to determine whether the trees were significantly different. The trees were visualized in TreeView (Page, 1996 1996 ). The model of evolution was estimated by using MrModeltest 2.3 (Nylander, 2004 2004 ). The newly generated sequences in this study were deposited in the GenBank, with their accession numbers listed in Table 1 1 . Results 1 Full length ITS regions and partial β-tubulin genes were amplified in this study. The amplified DNA fragments of the ITS regions and β-tubulin genes were approximatively 450 and 600 bp, respectively. The combined dataset of the ITS regions and β-tubulin genes contained 1052 characters, of which 742 characters were constant, 191 variable characters were parsimony-uninformative, and 119 were parsimony-informative. One of the most parsimonious trees with the shortest tree length (TL=465, CI=0.856, RI=0.835, RC=0.714, and HI=0.144) is shown in Fig. 1 1 . In our combined phylogenetic tree, all the 64 clinical strains collected from different regions of China clustered in a well supported (99%) phylogenetic clade together with the ex-type strain of S. globosa (CBS 120340). This clade shows sufficient distance to the type of S. schenckii (CBS 359.36) (Fig. 1 1 ). The 64 isolates from China in the S. globosa clade were distributed among 18 different haplotypes. Bootstrap support values above 50% are shown above the branches, and the scale bar indicates 10 changes. The tree is rooted with Ophiostoma nigrocarpum . Reference sequences are shown in bold. “C” and “E” indicate clinical and environmental strains, respectively. * * Indicates the type strain Discussion 1 S. schenckii was attributed to a unique species as the pathogen of sporotrichosis since it was first isolated from a patient (Lopes-Bezerra et al., 2006 2006 ). In past years, various molecular approaches have demonstrated that isolates of S. schenckii which were identified through morphology displayed diverse genetic characteristics and phylogenetic groupings (Lin et al., 2000 2000 ; Mesa-Arango et al., 2002 2002 ). Genotyping by analysis of restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA) and ribosomal DNA (rDNA) demonstrated the existence of intraspecific genetic variability, and all of the four rDNA types showed different geographic distributions (Watanabe et al., 2004 2004 ). With molecular phylogenetic analysis, S. schenckii was discovered to represent more than one species (de Beer et al., 2003 2003 ). Marimon et al. ( 2007 2007 ) revealed that S. schenckii was a complex of at least six phylogenetic species, comprising currently four clinical relevant species, i.e., S. schenckii s. str ., S. brasiliensis , S. globosa (Marimon et al., 2007 2007 ), and S. luriei (Marimon et al., 2008b 2008b ). Marimon et al. ( 2007 2007 ) and Zhou et al. ( 2013 2013 ) both revealed the existence of the correlation between geographical distribution and genotypes among species of Sporothrix . S. brasiliensis was confirmed to be restricted to Brazil, while S. schenckii was mainly prevalent in the Americas, Africa, and Asia (Marimon et al., 2007 2007 ; Zhou et al., 2013 2013 ). S. globosa had been reported with widespread geographical distributions, such as in Chile, India, Italy, Japan, Spain, UK, and USA (Madrid et al., 2009 2009 ). Previously, based on calmodulin (CAL) sequence analysis, five Chinese environmental isolates received as S. schenckii had been clustered into the clade of S. globosa (Marimon et al., 2007 2007 ), together with six clinical isolates of Sporothrix using ITS region analysis (Zhou et al., 2013 2013 ), and a total of 11 (100%) Chinese isolates were confirmed to be S. globosa . In this study, we sampled 64 clinical strains from a wider geographic regions of China, and our combined analysis of ITS and β-tubulin regions further confirmed that the causal agent of sporotrichosis is S. globosa in China. However, none of these isolates exhibited the same haplotype as the type of S. globosa CBS 120340, which is a clinical strain from Spain. We did not find a significant correlation between geographic origins of these strains and their genetic variations based on the haplotype data. More research is needed to indicate whether S. schenckii or other Sporothrix species exist in China. Sporotrichosis is characterized by a wide range of cutaneous and extracutaneous clinical manifestations. Kong et al. ( 2006 2006 ) revealed that different randomly amplified polymorphic DNA (RAPD) genotypes of strains led to different clinical forms. There have been reports about cutaneous dissemination and invasive sporotrichosis caused by S. schenckii and S. brasiliensis since Marimon et al. ( 2007 2007 ) discovered the three new species of Sporothrix (Galhardo et al., 2010 2010 ; Silva-Vergara et al., 2012 2012 ). However, no case of such clinical classification attributed to S. globosa has been reported. Except of the absence of sporotrichosis forms caused by S. globosa in some studies (Zhou et al., 2013 2013 ), all of the clinical patterns in our study were characterized as fixed and lymphocutaneous, as well as five cases caused by S. globosa described in Brazil (de Oliveira et al., 2010 2010 ; Rodrigues et al., 2012 2012 ) and Chile (Cruz et al., 2012 2012 ). We considered that this observation could be explained by the different virulence levels of members in S. schenckii complex. Although a study on the murine model has shown that the most virulent species was S. brasiliensis , followed by S. schenckii and S. globosa (Arrillaga-Moncrieff et al., 2009 2009 ), further studies are needed to investigate the possible pathogenesis of different clinical types of sporotrichosis. Some in vitro studies of S. schenckii sensu lato showed variable antifungal susceptibility among different isolates (Brandsberg and French, 1972 1972 ; McGinnis et al., 2001 2001 ), which indicates the existence of cryptic species with variations in antifungal susceptibility. Similar conclusions were presented by the studies of other fungal complexes such as the Pseudallescheria boydii (Gilgado et al., 2006 2006 ) and the Coccidioides immitis complex (Koufopanou et al., 2001 2001 ). In a recent in vitro antifungal susceptibility test, S. brasiliensis showed the best response to antifungals in general, followed by S. schenckii , S. albicans , S. globosa , and S. mexicana (Marimon et al., 2008a 2008a ). Therefore, species identification of clinical isolates is essential to determine the effective treatment, and it is significant to study their epidemiology, geographic distribution, and the relevance among taxa, virulence, and clinical patterns. Conclusions 1 The data in our study showed that the sporotrichosis cases in China were caused by S. globosa rather than S. schenckii , and it is demonstrated that ITS and β-tubulin are also efficient to distinguish species in S. schenckii species complex. With the increasing multilocus sequence data in this group, efficient molecular diagnosis protocols using specific primers should be developed to enable a rapid and accurate identification of clinical Sporothrix species. 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We also thank the Dermatology Department of the First Hospital of Jilin University, the Beijing University First Hospital, and the Southwest Hospital of the Third Military Medical University for supplying the clinical strains used in this study. Compliance with ethics guidelines Ting-ting LIU, Ke ZHANG, and Xun ZHOU declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1300136 Project supported by the National Natural Science Foundation of China (No. 31270062) and the Chongqing Science and Technology Commission (No. cstc2011jjA 10089), China 2013-05-08 2013-12-27 2013-07-12 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2014 2014 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 01 15