Tosrisawatkasem Orada, Thairat Sarut, Tonput Pairin, Tantivitayakul Pornpen
Department of Oral Microbiology, Faculty of Dentistry, Mahidol University, 6 Yothi Street, Rajthevi, Bangkok, 10400, Thailand.
Research office, Faculty of Dentistry, Mahidol University, Bangkok, Thailand.
BMC Oral Health. 2025 Jul 2;25(1):1029. doi: 10.1186/s12903-025-06334-2.
Infections caused by uncommon spp. (species other than ,,,, and ) have been continuously reported worldwide. However, virulence factors and antifungal susceptibility profiles of uncommon species remain limited. This study aimed to investigate the extracellular hydrolytic enzyme activities, antifungal susceptibility in planktonic and biofilm forms of cryptic and uncommon species as well as their biofilm structures and extracellular polymeric substance (EPS) compositions.
species was identified by sequence analysis of the internal transcribed spacer (ITS) regions of ribosomal RNA. The phospholipase and proteinase activities were evaluated by agar plate method. Biofilm-forming activity was analyzed using XTT-metabolic assay. Antifungal susceptibility tests for fluconazole, voriconazole, 5-flucytosine, and caspofungin were performed with broth microdilution following the CLSI guideline. biofilms were stained with fluorescent dyes for fungal cells (SYTO9), extracellular proteins (SYPRO Ruby), exopolysaccharides (Alexa Fluor 635-conjugated Concanavalin A) and analyzed using confocal laser scanning microscopy (CLSM).
A total of 25 uncommon isolates including , , (new nomenclature: ), (), (), (), and ( were identified, with 8% and 40% of the oral isolates producing phospholipase and proteinase, respectively. All isolates in this study were susceptible to fluconazole, voriconazole, and 5-flucytosine whereas 2 isolates (8% of all tested isolates) reduce susceptibility to caspofungin. The antifungal concentrations required to inhibit biofilm (sMIC) of and complex were higher than those of other species. In addition, the and biofilms had relatively high quantities of proteins, exopolysaccharides, and extracellular DNA (eDNA) compared to other species. Significant positive correlations between eDNA and exopolysaccharides ( = 0.77, < 0.01) and proteins ( = 0.60, < 0.01) in biofilms were observed.
Our results indicated variation in the expression of hydrolytic enzyme production and biofilm forming activities among uncommon spp. All isolates exhibited susceptibility to azole and 5-flucytosine. This study also presented the visualization of biofilm structures and distribution of EPS components in biofilms.
The online version contains supplementary material available at 10.1186/s12903-025-06334-2.
由罕见菌种（除白色念珠菌、热带念珠菌、近平滑念珠菌和光滑念珠菌之外的其他菌种）引起的感染在全球范围内不断被报道。然而，罕见念珠菌的毒力因子和抗真菌药敏谱仍然有限。本研究旨在调查隐匿性和罕见念珠菌种浮游态和生物膜态的细胞外水解酶活性、抗真菌药敏情况，以及它们的生物膜结构和细胞外聚合物（EPS）组成。
通过核糖体RNA内部转录间隔区（ITS）区域的序列分析鉴定念珠菌种。采用琼脂平板法评估磷脂酶和蛋白酶活性。使用XTT代谢分析法分析生物膜形成活性。按照CLSI指南，采用肉汤微量稀释法对氟康唑、伏立康唑、5-氟胞嘧啶和卡泊芬净进行抗真菌药敏试验。用荧光染料对念珠菌生物膜进行染色，分别标记真菌细胞（SYTO9）、细胞外蛋白质（SYPRO Ruby）、胞外多糖（Alexa Fluor 635标记的伴刀豆球蛋白A），并使用共聚焦激光扫描显微镜（CLSM）进行分析。
共鉴定出25株罕见念珠菌分离株，包括葡萄牙念珠菌、涎液念珠菌（新命名：罗伦隐球菌）、季也蒙念珠菌、法勒念珠菌、乳酒念珠菌、解脂念珠菌、吉列蒙念珠菌和无名念珠菌，其中8%的口腔念珠菌分离株产生磷脂酶，40%产生蛋白酶。本研究中的所有念珠菌分离株对氟康唑、伏立康唑和5-氟胞嘧啶敏感，而2株分离株（占所有测试分离株的8%）对卡泊芬净敏感性降低。抑制葡萄牙念珠菌和无名念珠菌复合物生物膜（sMIC）所需的抗真菌浓度高于其他菌种。此外，与其他菌种相比，葡萄牙念珠菌和无名念珠菌生物膜中的蛋白质、胞外多糖和细胞外DNA（eDNA）含量相对较高。在葡萄牙念珠菌生物膜中观察到eDNA与胞外多糖（r = 0.77，P < 0.01）和蛋白质（r = 0.60，P < 0.01）之间存在显著正相关。
我们的结果表明，罕见念珠菌种在水解酶产生和生物膜形成活性的表达上存在差异。所有念珠菌分离株对唑类药物和5-氟胞嘧啶均敏感。本研究还展示了念珠菌生物膜的结构可视化以及EPS成分在其中的分布。
在线版本包含可在10.1186/s12903-025-06334-2获取的补充材料。
