Amino acid- or protein-dependent growth of Trichophyton mentagrophytes and Trichophyton rubrum

Culture conditions were examined for Trichophyton mentagrophytes and Trichophyton rubrum, which are major pathogens involved in dermatophytosis. They grew well in Sabouraud's dextrose broth or RPMI 1640. Growth in phosphate-buffered yeast nitrogen base supplemented with glucose was very slow, although growth improved significantly with the addition of amino acids or proteins to the medium. The fungi could also grow using human nail fragments as the only source of nutrition. Examination of proteases by substrate gel electrophoresis indicated that distinct sets of proteases are secreted from the dermatophytes in two different media, Sabouraud's dextrose broth and nail fragments. A protease inhibitor, phenylmethanesulfonyl fluoride, inhibited the growth of the fungi on nail fragments, but it did not inhibit their growth in Sabouraud's dextrose broth.     

须癣毛癣菌的形态学及分子生物学鉴定

目的对临床上分离自人(36株)及狐狸(5株)的须癣毛癣菌菌株进行新的分类系统鉴定,并检测传统的分类方法是否能满足临床鉴定需要。方法①观察原鉴定为须癣毛癣菌菌株在沙氏培养基、1%蛋白胨培养基、溴甲酚紫乳固体葡萄糖琼脂培养基(BCP-MSG)和显微镜下形态学及尿素酶、毛发穿孔等生理学试验表现。②通过ITS区段和LSU区段分子生物学序列分析进行新的分类系统的菌种分型,并对传统形态学和生理学鉴定方法进行检测。结果①41株须癣毛癣菌形态学及生理学试验符合须癣毛癣菌(38株)和红色毛癣菌(3株)菌落的特点。②ITS区段序列分析发现ITS区段能将须癣毛癣菌和红色毛癣菌准确的鉴定到种,但无法明确其种内分型;而LSU区段序列分析可对36株(36/38)须癣毛癣菌有性型做出明确的鉴定。结论传统实验室鉴定方法仍具有其有效性及可靠性。通过分子生物学鉴定,临床分离的须癣毛癣菌皆为指(趾)间毛癣菌(38/38),而LSU区段的序列分析鉴定狐狸源性菌株皆属于本海姆节皮菌,有别于大多数人源性菌株有性型为万博节皮菌,对于须癣毛癣菌的菌种鉴定更优于ITS区段,但分子生物学试验还需结合形态学的观察,才能够对菌种做出正确的鉴定。     

Characterization of the cyclophilin of Trichophyton mentagrophytes

A genetic approach to cyclophilins in a dermatophyte, Trichophyton mentagrophytes, was carried out. The nucleotide and deduced amino acid sequences of the cyclophilin of T. mentagrophytes shared about 70% sequence similarity with those of Schizosaccharomyces pombe, Saccharomyces cerevisiae and Candida albicans. However, the first 21 amino acid and the C-terminal amino acid regions of 188 to 226 of the T. mentagrophytes cyclophilin were distinct from those of the other fungal cyclophilins. The recombinant glutathione S-transferase (GST)-T. mentagrophytes cyclophilin fusion protein produced by Escherichia coli was purified. The protease digest of the fusion protein had a molecular weight of about 13 kDa and peptidyl-prolyl cis-trans isomerase (PPI) activity. This digest protein from T. mentagrophytes was confirmed to be cyclophilin by proving PPI activity.     

联合应用抗真菌药物对不同地区来源红色毛癣菌的体外敏感性实验

目的调查联合应用抗真菌药物对不同地区来源红色毛癣菌的体外抗真菌作用,探讨药物的体外相互作用及地区因素对红色毛癣菌药物敏感性的影响。方法以M38-A方案测定酮康唑、萘替芬、联合使用酮康唑萘替芬以及单用特比萘芬对红色毛癣菌的最小抑菌浓度(MIC),并计算了酮康唑与萘替芬的药物间抑菌浓度指数(FICI)。结果联合使用酮康唑萘替芬组的最小抑菌浓度显著低于单用酮康唑、萘替芬组,和单用特比萘芬组的疗效相似。北京的红色毛癣菌株对上述药物的敏感度均低于上海和南京的菌株。结论联合应用抗真菌药物优于单用,地区差异可能会影响红色毛癣菌对药物的敏感性。     

须癣毛癣菌所致脓癣病发的扫描和透射电镜观察及菌体外酶活性分析：2例报告

目的报道2例由须癣毛癣菌引起的脓癣,并对病发做扫描和透射电镜观察及菌体外分泌酶活性测定。方法例1为6岁女孩,表现为头顶部炎性肿块、脓血伴脱发;例2为1岁8个月男孩,表现为头部红斑,无脓肿及脱发。家中分别饲养宠物狗和兔。病发真菌镜检均阳性后做真菌培养、生化鉴定及分子生物学鉴定。对2例患儿病发用扫描和透射电镜进行超微结构观察。对2株分离的菌株分别进行细胞外分泌酶测定。结果2株菌均鉴定为万博节皮菌（须癣毛癣菌的有性期）。扫描电镜见例1的病发受累毛干表面大量炎性组织碎屑、红细胞、脓细胞及上皮细胞;透射电镜在毛皮质内发现菌体。扫描电镜见例2的受累毛干表面真菌孢子及炎性渗出物;透射电镜见毛发内外菌体。2株菌的外分泌酶主要为碱性磷酸酶、白氨酸芳胺酶、β-葡萄糖甙酶、N-乙酰-葡萄糖胺酶、α-甘露糖甙酶。经口服伊曲康唑胶囊等综合治疗后2例均痊愈,未见不良反应。结论确诊2例须癣毛癣菌所致脓癣,伊曲康唑胶囊等药物联合治疗有效。     

Experimental penetration of Trichophyton mentagrophytes into human stratum corneum

We present confirmation of the experimental penetration of Trichophyton mentagrophytes into human stratum corneum under designated conditions of temperature and humidity. When stratum corneum, obtained from healthy human heel region, was incubated at 100% humidity, mycelium was observed in the corneum layer on day 2 at 35 °C and 27 °C, and on day 4 at 15 °C. At 90% humidity, the hyphae penetrated into the stratum corneum on day 4 at 35 °C, and on day 6 at 27 °C. Whereas, at 80% humidity, no fungal elements were observed in the stratum corneum at both 27 °C and 35 °C for up to 7 day. These data suggested that humidity was a more important environmental factor for penetration than temperature, and that at least 90% humidity is necessary for dermatophytes to penetrate into the stratum corneum within a few days. Mean humidity in the interdigital space between the fourth and fifth toes was found to be approximately 98%. This revised version was published online in June 2006 with corrections to the Cover Date.     

须癣毛癣菌致人兔共患体癣3例

报道须癣毛癣菌引起的人兔共患体癣3例.患者皮损均为环状红斑丘疹,上覆鳞屑,炎症明显,伴瘙痒,均与兔子有密切接触史,且兔子均有明显皮损.取患者及兔子的皮屑真菌镜检及培养,鉴定为须癣毛癣菌,经口服及外用抗真菌药物后治愈.     

A Study on Stability of Phenotype and Genotype of Trichophyton rubrum

Objective: To explore the stability of phenotype and genotype in Trichophyton rubrum. Methods: All the strains were cultured on Sabouraud’s dextrose agar slopes, and identified to species level. Strains isolated recently were subcultured on Sabouraud agar slopes four times at an interval of 4 weeks. DNA was extracted with CTAB method. A probe consisting of 3′ end of 18S rDNA, adjacent ITS1, 5.8S rDNA and ITS2 regions was amplified from template DNA of the T. rubrum standard strain using fungal universal primers NS5 and ITS4, labelled by P³² and hybridized with EcoR I-digested T. rubrum genomic DNA. Results: (1) Four phenotypes were isolated from 207 T. rubrum strains, with downy type (45.4%) in the first place, and granular type not found. After 1 year of conservation, 54 strains showed morphological variations with the total variation rate of 26.1%. (2) Eleven strains showed variations in colony morphology or pigment upon subculture. (3) AP-PCR analysis of 10 T. rubrum isolates and one T. rubrum standard strain showed similar DNA patterns with main bands at 2.2, 1.7, 1.3, 0.9 and 0.7 kb. No changes in DNA pattern were found upon subculture. (4) Hybridization analysis revealed that all the 11 T. rubrum strains presented three bands and were identified into two types (2.4, 3.9, 5.9 kb and 2.4, 4.4, 6.5 kb). No changes in band pattern were found upon subculture. Conclusions: Phenotype of T. rubrum was instable and the colonial morphology and pigment easily changed during conservation or subculture, while its genotype was relatively stable.     

Nucleotide sequence of a Trichophyton mentagrophytes HindIII mitochondrial DNA fragment containing at RNA gene cluster

Abstract A 0.85-kb Hin dIII mitochondrial DNA fragment of the dermatophytic fungus Trichophyton mentagrophytes has been sequenced. The fragment contains eight complete genes which corresponds to a tRNA gene cluster. From 5' to 3', the sequenced genes code for tRNA^thr, tRNA^glu, tRNA^val, tRNA^met1, tRNA^met3, tRNA^leu, tRNA^ala, and tRNA^phe. This tRNA gene cluster is located downstream of the larger ribosomal RNA gene. The particularities ofthe sequenced genes and their comparison with other fungal tRNA mitochondrial genes are reported.     

红色毛癣菌分泌性蛋白酶的分析

分泌性蛋白酶是红色毛癣菌致病的潜在毒力因子。在构建红色毛癣菌6个不同时间段cDNA文库的基础上,共获得了9683条uniqueESTs,通过生物信息学分析从中得到了18个可能的分泌性蛋白酶的EST序列,包括4个分泌性肽酶、1个分泌性金属蛋白酶、2个细胞外丝氨酸蛋白酶、1个分泌性天冬氨酸蛋白酶、9个分泌性枯草杆菌蛋白酶样丝氨酸蛋白酶、1个空泡丝氨酸蛋白酶。这些分泌性蛋白酶在红色毛癣菌感染过程中可能分别与其获得营养、扩大侵袭范围及激起宿主免疫应答有关,这些结果为进一步研究红色毛癣菌感染和发病机制提供了重要的分子基础和线索。     

Effective photodynamic treatment of Trichophyton species with Rose Bengal

Photodynamic therapy (PDT) with Rose Bengal has previously achieved eradication of Trichophyton rubrum infections causing toenail onychomycosis; however, its antifungal activity against other clinically relevant dermatophytes has yet to be studied. Here, we test the efficacy of PDT using Rose Bengal (140 μM) and 532 nm irradiation (101 J/cm²) against Trichophyton mentagrophytes and Trichophyton interdigitale spores, in comparison to T. rubrum. A significant reduction (>99%) of T. mentagrophytes and T. interdigitale was observed, while actual eradication of viable T. rubrum was achieved (99.99%). Laser irradiation alone inhibited growth of T. rubrum (55.2%) and T. mentagrophytes (45.2%) significantly more than T. interdigitale (25.5%) (P = .0086), which may indicate an increased presence of fungal pigments, xanthomegnin and melanin. The findings suggest that Rose Bengal-PDT can act against a broader spectrum of fungal pathogens, and with continued development may be employed in a wider range of clinical antifungal applications.     

In vitro susceptibility of dermatomycoses agents to six antifungal drugs and evaluation by fractional inhibitory concentration index of combined effects of amorolfine and itraconazole in dermatophytes

To investigate the antifungal drug susceptibility of fungi responsible for dermatomycoses, minimum inhibition concentration (MIC) tests were performed in 44 strains of dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum, with six antifungal drugs (amorolfine, terbinafine, butenafine, ketoconazole, itraconazole and bifonazole) by broth microdilution assay according to Clinical Laboratory Standard Institute protocols. Six possible dermatomycosis‐causing non‐dermatophytic fungi were also tested. The two major causes of tinea, T. rubrum and T. mentagrophytes, showed significantly different sensitivities to ketoconazole and bifonazole. Clinically derived dermatophytes were sensitive to the six antifungal drugs tested. However, non‐dermatophytes, especially Fusarium spp., tended to be resistant to these antifungal drugs. In Trichophyton spp., the MICs of non‐azole drugs had narrower distributions than those of azoles. To evaluate the effects of antifungal drug combinations, the fractional inhibitory concentration index was calculated for the combination of amorolfine and itraconazole as representative external and internal drugs for dermatophytes. It was found that this combination had synergistic or additive effects on most dermatophytes, and had no antagonistic effects. The variation in susceptibility of clinically derived fungal isolates indicates that identification of causative fungi is indispensable for appropriately choosing effective antifungal drugs in the early stages of infection. The results of combination assay suggest that multiple drugs with different antifungal mechanisms against growth of dermatophytes should be used to treat refractory dermatomycoses, especially onychomycosis.     

麻类织物对须癣毛癣菌生长抑制作用的研究

目的通过分析汉麻、亚麻和苎麻对须癣毛癣菌生长的影响,探讨麻类织物对浅部真菌的抑制作用。方法实验采用振荡法。将3种麻类织物分别与须癣毛癣菌混合培养,对照组为棉布组,3d后分别取培养液稀释5倍后涂布培养皿,计算各自的菌落数和抑菌率并进行统计分析。结果麻类织物组菌落数明显少于对照组棉布组(P<0.01),3种麻类织物对须癣毛癣菌的抑菌率均高于60%,但3种麻类织物的抑菌率之间无明显差异(P<0.01)。结论3种麻类织物均可显著抑制须癣毛癣菌的生长,麻类织物有可能用来预防由须癣毛癣菌引起的足癣、股癣等浅部真菌病。     

家庭内红色毛癣菌病患者间菌株差异性分析

目的用PCR技术比较分离自同一家庭红色毛癣菌病患者的菌株差异性,分析家庭内多发的红色毛癣菌病的致病菌株是家内相互感染,还是家外感染。方法以家庭内多发的皮肤癣菌病患者为研究对象,分离致病菌株并以传统方法鉴定菌种。再分别用随机扩增多态性DNA(RAPD)和巢式PCR特异扩增红色毛癣菌的串联重复亚元件(TRSS:TRS-1/TRS-2)产生的指纹图谱分析种内株间有无差异性。结果纳入实验的16株菌分离自8个家庭,用形态学等方法及种特异引物均鉴定为红色毛癣菌。RAPD显示4个家庭内的菌株间有差异性,TRS-1区PCR指纹图谱显示5个家庭内菌株有株间差异,TRS-2区能鉴定出2个家庭内菌株间有差异。综合各方法共区分出6个家庭内的菌株间有带型差异。结论该研究提示家庭内多发红色毛癣菌病从家外途径感染率高于家内感染。TRS-1区PCR指纹图谱对红色毛癣菌的菌株区分度高于RAPD,更适于红色毛癣菌株间分型。结合多种分子分型方法可最大限度发现不同菌株间的差异。     

红色毛癣菌的生物学特性研究

目的 观察红色毛癣菌在不同温度、不同培养基上的生长和产孢情况,并对其进行分子生物学鉴定.方法 ①大培养:采用沙堡葡萄糖琼脂(SDA)和马铃薯葡萄糖琼脂(PDA)平皿,27℃、35℃黑暗培养,测量菌落直径,绘成生长曲线.②小培养(钢圈法):采用SDA、PDA、溴甲酚紫乳固体葡萄糖琼脂(BCP-MSG)、乳蜜琼脂(M)和复合维生素B(VitB)培养基,27℃、30℃黑暗培养,观察镜下菌丝生长、孢子产生情况.③进行rDNA 18S和ITS序列测定.结果 在SDA,PDA上,27℃条件下菌落生长速度较35℃快;在5种培养基上,SDA、PDA产孢较快较多,复合维生素B培养基产孢较慢,但产生大分生孢子较多.30℃产孢更丰富.对部分菌株rDNA ITS、18S PCR扩增产物纯化后直接测序,结果在GenBank中比对、分析,相似度为98%～100%,均鉴定为红色毛癣菌.结论 SDA、PDA均为鉴定和分离红色毛癣菌的合适培养基.5种培养基均可用来刺激红色毛癣菌产孢,其中SDA、PDA产孢较早、较丰富.红色毛癣菌rDNA 18S和ITS序列测定是一种快速准确的红色毛癣菌分子生物学鉴定方法.     

红色毛癣菌蛋白酶MEP和SUB的表达及临床意义

目的探讨红色毛癣菌蛋白酶MEP和SUB的表达及临床意义。方法抽提红色毛癣菌总RNA,采用半定量RTPCR法检测红色毛癣菌金属蛋白酶(Metalloproteinases,MEP)、枯草菌素蛋白酶(subtilisins,SUB)基因表达量的变化。结果不同病例的红色毛癣菌SUB的表达水平与临床症状的严重程度密切相关,而与患者的年龄、性别、病程等无明显相关性;MEP的表达水平在不同年龄、性别、病程和临床分型等方面存在一定差异,但无显著意义。结论红色毛癣菌致病力的大小可能与SUB的不同表达有关。     

红色毛癣菌临床分离株的菌落形态和镜下结构特征再分析

目的探讨红色毛癣菌的菌落形态和镜下结构特征以及与感染部位的相关性。方法采用传统培养方法对192株红色毛癣菌进行表型分型,选取其中39株在28℃、30℃、35℃3种温度孵育6d、10d、14d时观察菌落形态和生长速度。结果192株红色毛癣菌共分离出3种表型：绒毛型、沟纹型、粉末型（或颗粒型）。在相同培养基上,28℃、30℃时菌落生长速度无显著差异（P〈0.05）,均快于35℃（P〈0.05）。在相同温度时,菌落在SDA上的生长速度快于PDA培养基。在28℃、30℃时菌落形态比较稳定,在35℃时变异较大。菌落在PDA培养基上产孢丰富,镜下显示有较多的大、小分生孢子,而在SDA培养基上只有少量的小分生孢子,几乎见不到大分生孢子。各种浅部感染均以绒毛型为主,绒毛型在手足癣中占比例最高,沟纹型在体股癣中所占比例较高,粉末型在甲癣中占的比例较高。结论红色毛癣菌的菌落形态与培养基和培养温度有关,其表型和镜下结构与感染部位均有一定的相关性。     

蒺藜TTS-12对须癣毛癣菌的体内外抗菌活性研究

为考察天然产物蒺藜TTS-12对须癣毛癣菌的体外和体内抗菌活性,本研究采用CLSI的M38-A2方案对须癣毛癣菌进行最小抑菌和杀菌浓度的检测,选取多重指标考察TTS-12对须癣毛癣菌的抑菌作用;建立豚鼠感染须癣毛癣菌豚鼠体癣模型,分组给药观察低、中、高剂量蒺藜TTS-12凝胶剂对豚鼠体癣模型在背部病变程度评分,病灶皮肤真菌培养阴性率,病灶皮肤病理变化的影响。结果发现,TTS-12对须癣毛癣菌的标株菌株的MIC值为1μg/mL,MFC为8μg/mL,其还能够显著抑制须癣毛癣菌的菌丝生长,浓度为4μg/mL的TTS-12作用15天时,菌丝生长抑制率已经高于60%,且对须癣毛癣菌的孢子萌发抑制作用呈时间和浓度依赖性。动物实验中,低、中、高剂量蒺藜TTS-12凝胶剂均能显著降低背部病变程度评分(P<0.01),中、高剂量能增高病灶皮肤真菌培养阴性率(P<0.05),病灶皮肤HE染色切片表明高剂量组棘层肥厚降低,背部炎症减弱,使角质层恢复正常。以上结果表明蒺藜TTS-12在体外抗须癣毛癣菌和豚鼠体癣模型中均有较好的活性。