Langsiri Nattapong, Banlunara Wijit, Klaychontee Oranong, Worasilchai Navaporn, Cognialli Regielly, Queiroz-Telles Flavio de, Spruijtenburg Bram, Groot Theun de, Meijer Eelco F J, Chindamporn Ariya
Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.
Department of Veterinary Pathology, Chulalongkorn University, Patumwan, Bangkok Thailand.
PLoS Negl Trop Dis. 2025 Jun 30;19(6):e0013253. doi: 10.1371/journal.pntd.0013253. eCollection 2025 Jun.
Sporothrix spp. are dimorphic fungi capable of undergoing morphological changes in response to temperature variations. The genus Sporothrix includes the species S. schenckii sensu stricto, S. brasiliensis, S. globosa, and S. luriei that cause sporotrichosis, which can range from local skin infections to systemic infections in immunocompromised individuals. As these species are morphologically similar, molecular techniques that utilize barcoding genes are required for accurate identification. While the Internal Transcribed Spacer (ITS) region is the universal fungal barcode, the calmodulin gene offers higher resolution for phylogenetic classification of Sporothrix using Sanger sequencing. This study evaluated the ability of long-read nanopore sequencing of calmodulin and ITS to identify species level and allow phylogenetic analysis of Sporothrix strains isolated from humans and felines in Thailand. We found that the calmodulin sequencing with Oxford Nanopore Technology (ONT) consistently classified all isolates as S. schenckii sensu stricto, whereas the ITS region showed a lower discriminatory power, complicating species identification in some isolates. The phylogenetic analysis of the calmodulin region indicated that all isolates localized in a specific S. schenckii sensu stricto subclade together with other isolates from Southeast Asia, while the three residual S. schenckii subclades were associated with other geographical locations. Antifungal susceptibility testing on all Sporothrix strains demonstrated elevated in vitro minimum inhibitory concentrations (MICs) to itraconazole for 8 out of 26 isolates. Altogether, this study demonstrated that ONT sequencing of calmodulin allows accurate species identification and phylogenetic analysis of S. schenckii sensu stricto isolates from Thailand, of which some also demonstrated elevated MIC values for itraconazole.
申克孢子丝菌属真菌是二态性真菌，能够根据温度变化发生形态改变。申克孢子丝菌属包括申克孢子丝菌狭义种、巴西孢子丝菌、球形孢子丝菌和鲁里孢子丝菌，这些菌种可引起孢子丝菌病，病情可从局部皮肤感染到免疫功能低下个体的全身感染。由于这些菌种在形态上相似，因此需要利用条形码基因的分子技术来进行准确鉴定。虽然内部转录间隔区（ITS）是通用的真菌条形码，但钙调蛋白基因通过桑格测序法对申克孢子丝菌属进行系统发育分类时具有更高的分辨率。本研究评估了利用纳米孔长读长测序法对钙调蛋白和ITS进行测序，以鉴定泰国人类和猫科动物分离出的申克孢子丝菌菌株的种水平并进行系统发育分析的能力。我们发现，采用牛津纳米孔技术（ONT）对钙调蛋白进行测序能始终如一地将所有分离株鉴定为申克孢子丝菌狭义种，而ITS区域的鉴别力较低，使得一些分离株的菌种鉴定变得复杂。对钙调蛋白区域的系统发育分析表明，所有分离株与东南亚的其他分离株一起定位于申克孢子丝菌狭义种的一个特定亚分支中，而申克孢子丝菌的另外三个亚分支与其他地理位置相关。对所有申克孢子丝菌菌株进行的抗真菌药敏试验表明，26株分离株中有8株对伊曲康唑的体外最低抑菌浓度（MIC）升高。总之，本研究表明，对钙调蛋白进行ONT测序能够准确鉴定泰国申克孢子丝菌狭义种分离株并进行系统发育分析，其中一些分离株对伊曲康唑的MIC值也升高。
