申克孢子丝菌复合体在形态学和生理生化的研究

Morphological and physiological comparison of taxa comprising the Sporothrix schenckii complex 申克孢子丝菌复合体在形态学和生理生化的研究 1 first-author 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:zhouxun123@sina.com †E-mail:zhouxun123@sina.com zhouxun123@sina.com 1 3 Department of Dermatology, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China Department of Dermatology, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China Department of Dermatology and Cosmetology, Chongqing Hospital of Traditional Chinese Medicine, Chongqing 400021, China Department of Dermatology and Cosmetology, Chongqing Hospital of Traditional Chinese Medicine, Chongqing 400021, China The Laboratory of Pathogen Biology and Immunology, Basic Medical Experiment Teaching Center, Chongqing Medical University, Chongqing 400016, China The Laboratory of Pathogen Biology and Immunology, Basic Medical Experiment Teaching Center, Chongqing Medical University, Chongqing 400016, China Based on recent molecular data, it has been suggested that Sporothrix globosa is the main causal agent of sporotrichosis in China. The objective of this study was to compare the morphology, growth characteristics, patterns of carbon source usage, and susceptibility to antifungal agents among Sporothrix strains. A total of 15 clinical strains confirmed to be S. globosa , from three different regions of China, and 11 ex-type strains from the CBS-KNAW biodiversity center were obtained. The elongated conidia of S. pallida , S. variecibatus , S. schenckii , and S. schenckii luriei were clearly different from the subglobose and globose conidia of S. globosa strains. S. schenckii is able to assimilate sucrose, raffinose, and ribitol. Susceptibility profiles of these Sporothrix species were evaluated by measuring minimum inhibitory concentrations (MICs). Fluconazole, itraconazole, terbinafine, and amphotericin B showed good activity against most S. globosa clinical isolates from China. Potassium iodide also showed a low MIC against S. pallida , while fluconazole showed a high MIC for S. mexicana , S. humicola , S. globosa , S. schenckii , and S. inflata ; these strains might be considered tolerant. The species showed differences in susceptibility to antifungal drugs and should therefore be properly identified during diagnosis prior to designing therapeutic strategies. 目的 2 研究申克孢子丝菌复合体在形态学、糖同化和抗真菌药物敏感性的差异，探讨不同种类孢子丝菌的形态、生理生化及抗真菌药物敏感的特性。 创新点 2 首次对我国球形孢子丝菌与其它孢子丝菌模式菌株在形态学、生理生化和抗真菌药敏方面进行比较，从而找出我国孢子丝菌与模式菌株的差异和相同点。 方法 2 实验选用15株从我国三个不同地区分离的球形孢子丝菌和11株购买的孢子丝菌模式菌株 （表1），将这26株孢子丝菌菌株分别接种到2%的马铃薯葡萄糖琼脂平板培养基上，通过在不同时间段观察菌落生长特征，测量菌落直径，镜下观察菌丝、孢子形态，测定最小抑制浓度（MIC）并进行评价。 结论 2 实验显示：根据顶端分生孢子的形态（图2）、不同时期菌种的菌落直径（图1）、糖同化实验（表2和表3）可进行简单鉴定我国球形孢子丝菌与孢子丝菌复合体；特比萘芬有较好的体外抑菌活性，而碘化钾、氟康唑、伊曲康唑、两性霉素B对不同的菌株的抗菌敏感性不同（表4）。综上所述，我国孢子丝菌临床菌株与孢子丝菌复合体在表型、生理生化及体外抗真菌药物敏感性上均有不同程度的差异。 Sporothrix globosa Phenotypic characters Antifungal susceptibility 球形孢子丝菌 表型特点 抗真菌药物敏感性 Introduction 1 Sporotrichosis is a chronic subcutaneous mycosis that can be acquired by contact with contaminated objects or organic material (O'Reilly and Altman, 2006 2006 ). It also can be acquired through zoonotic transmission (Oliveira et al., 2011 2011 ; Silva-Vergara et al., 2012 2012 ). The incidence of sporotrichosis is increasing in China, particularly in the northeastern region of the country (Zhang and Lin, 2008 2008 ). Sporothrix globosa was identified as the causal agent of sporotrichosis in many of these cases (Tan et al., 2013 2013 ; Yu et al., 2013 2013 ; Liu et al., 2014 2014 ; Zhou et al., 2014 2014 ). Four species of the Sporothrix complex have been reported as causal agents of sporotrichosis: S. brasiliensis , S. globosa , S. schenckii s. str ., and S. schenckii luriei (Rodrigues et al., 2014 2014 ). S. pallida and S. mexicana are mainly environmental but may also be potentially pathogenic to humans and other mammals. It is not clear if these species have different physiological features that determine their pathogenicity or if they differ in their susceptibility to antifungals. Species in the S. schenckii complex have been shown to differ in their calmodulin (CAL), internal transcribed spacer (ITS), and a fragment of the β-tubulin gene sequences (Marimon et al., 2007 2007 ). Few studies have focused on the morphological and physiological variation among strains that comprise the S. schenckii complex. Recent studies have shown that morphologically similar species in this group could differ in their physiological features (Marimon et al., 2007 2007 ; Rodrigues et al., 2014 2014 ). Colony characteristics, growth rates, and carbon assimilation tests have been shown to be useful for distinguishing species of Sporothrix (Marimon et al., 2007 2007 ; de Meyer et al., 2008 2008 ). Moreover, the distribution of sessile conidia and their morphological variation have been suggested to be the key features distinguishing species within the Sporothrix complex. In this study, using type strains of different species of Sporothrix , we compared colony and microscopic morphologies, assimilation of carbon sources, and antifungal susceptibilities. The variation in these characteristics among different Sporothrix species, as reported here, provides new insights into their pathogenicity mechanisms and suggests different susceptibilities to antifungals, thereby providing new avenues for clinical therapeutic strategies. Materials and methods 1 Strains 2 Fifteen strains of Sporothrix from clinical sources in China were included in the study. Isolates consisted of 15 strains that were morphologically and genetically identified as S. globosa (Liu et al., 2014 2014 ) and 11 ex-type strains, including S. brunneoviolacea ( n =1), S. dimorphospora ( n =1), S. globosa ( n =1), S. humicola ( n =1), S. inflata ( n =1), S. lignivora ( n =1), S. mexicana ( n =1), S. pallida ( n =1), S. variecibatus ( n =1), S. schenckii ( n =1), and S. schenckii luriei ( n =1) (Table 1 1 ). Isolates were stored at room temperature on slant cultures containing 2% (0.02 g/ml) potato dextrose agar. Cultures were stored at 4 °C in distilled water for long-term preservation. Strain Name Source Country of origin  LC2454 S . globosa Clincal strain Beijing, China  LC2460 S . globosa Clincal strain Beijing, China  LC2466 S . globosa Clincal strain Beijing, China  LC2467 S . globosa Clincal strain Beijing, China  LC2469 S . globosa Clincal strain Beijing, China  LC2432 S . globosa Clincal strain Chongqing, China  LC2433 S . globosa Clincal strain Chongqing, China  LC2445 S . globosa Clincal strain Chongqing, China  LC2447 S . globosa Clincal strain Chongqing, China  LC2480 S . globosa Clincal strain Chongqing, China  LC2404 S . globosa Clincal strain Jilin, China  LC2405 S . globosa Clincal strain Jilin, China  LC2411 S . globosa Clincal strain Jilin, China  LC2413 S . globosa Clincal strain Jilin, China  LC2419 S . globosa Clincal strain Jilin, China  CBS 120341 S. mexicana Environmental Mexcio  CBS 124561 S. brunneoviolacea Soil Spain  CBS 118129 S. humicola Soil South Africa  CBS 239.68 S. inflata Wheat-field soil Germany  CBS 131.56 S. pallida Stemonitis fusca Japan  CBS 121961 S. variecibatus Trichopoda sp. from Protea repens South Africa  CBS 119148 S. lignivora Utility poles South Africa  CBS 120340 S. globosa Clincal strain Spain  CBS 553.74 S. dimorphospora Soil Canada  CBS 359.36 S. schenckii Clincal strain Unknown  CBS 937.72 S. schenckii luriei Clincal strain South Africa Morphological characteristics 2 Mycelial discs (2 mm diameter) of each strain were transferred to three replicate plates containing 2% (0.02 g/ml) potato dextrose agar, and the plates were incubated at 25 °C. Colony size, shape, and pigment production were recorded after 7, 14, 21, and 28 d of incubation. Colony diameter was measured daily for 7 d and growth rate was calculated as the 7-d average of mean weekly growth (mm/week). Growth rates were subjected to an analysis of variance ( P < 0.05) with Duncan’s multiple range test by using SPSS software (Version 16.0). After 7 d, the shape and size of the appressoria formed across the underside of the cover slip were studied (Marimon et al., 2007 2007 ). Physiological studies 2 Carbon assimilation was tested in liquid medium according to Marimon et al. ( 2007 2007 ). Variation in the ability to grow on sucrose, raffinose, and maltose was tested in all strains. The tests were conducted in 96-well microplates, containing either no carbon source (negative control) or glucose (positive control). The inocula were adjusted to a final concentration of from 2×10 5 to 2×10 6 colony-forming unit (CFU)/ml. Microplates were read (530 nm) after 5 d of incubation at 25 °C, as previously described (Marimon et al., 2007 2007 ). Antifungal susceptibility testing 2 The following antifungals were tested for their activity against Sporothrix strains: fluconazole (FLC), itraconazole (ITC), terbinafine (TRB), amphotericin B (AMB), and potassium iodide (KI). Antifungal susceptibility tests were performed on the isolates using the methods recommended by the Clinical Laboratory and Standards Institute standard document M38-A2 (Kohler et al., 2004 2004 ; Trilles et al., 2005 2005 ; Marimon et al., 2008 2008 ; Oliveira et al., 2011 2011 ). Ninety-six-well microplates containing 0.01 ml of RPMI-1640 nutrient broth in each well were incubated at 25 °C. Nutrient broth RPMI-1640 was amended with antifungal drug concentrations to final concentrations that spanned 0.50 to 500.00 μg/ml (KI), 0.125 to 64.00 μg/ml (FLC), and 0.03 to 16.00 μg/ml (ITC, TRB, and AMB). The concentration of cells used to inoculate assays was adjusted to a final concentration that ranged from 1×10 4 to 5×10 4 CFU/ml. Candida parapsilosis (ATCC 22019) was included as a control for each antifungal test (Marimon et al., 2008 2008 ). Growth was monitored spectrophotometrically at 530 nm during the growth period and growth in the presence of antifungals was compared with growth in the absence of antifungals to establish minimum inhibitory concentrations (MICs) (Espinel-Ingroff et al., 2002 2002 ). The MICs for AMB, ITC, and KI were defined as the lowest concentration showing 100% growth inhibition. MICs for TRB were defined as the lowest concentrations that showed 80% inhibition of growth, and for FLC as the lowest concentration that produced 50% growth inhibition (Trilles et al., 2005 2005 ; Marimon et al., 2008 2008 ; Oliveira et al., 2011 2011 ; Rodrigues et al., 2014 2014 ). The tests were carried out three times and an MIC based on these replicates was calculated. Carbon assimilation was tested in liquid medium according to Marimon et al. ( 2007 2007 ). Variation in the ability to grow on sucrose, raffinose, and maltose was tested in all strains. The tests were conducted in 96-well microplates, containing either no carbon source (negative control) or glucose (positive control). The inocula were adjusted to a final concentration of from 2×10 5 to 2×10 6 CFU/ml. Microplates were read (530 nm) after 5 d of incubation at 25 °C, as previously described (Marimon et al., 2007 2007 ). Results 1 Morphological characteristics 2 3.1.1 Colony characters Images of Sporothrix isolate colonies are depicted in Fig. 1 1 . All strains grew well on potato dextrose agar at 25 °C. Colonies of clinical isolates from China produced cream-colored, smooth or verrucous, moist colonies with occasional aerial mycelia. Over time, these colonies matured into black leathery colonies with a wrinkled and folded surface. Colonies of S. schenckii , S. schenckii luriei , S. pallida , and S. humicola produced white aerial mycelium, with white colonies in the center. These colonies remained white to cream-colored over time. (a) S. globosa (Beijing, China); (b) S. globosa (Jilin, China); (c) S. globosa (Chongqing, China); (d) S. mexicana (CBS 120341); (e) S. brunneoviolacea (CBS 124561); (f) S. humicola (CBS 118129); (g) S . inflata (CBS 239.68); (h) S. pallida (CBS 31.56); (i) S. variecibatus (CBS 121961); (j) S. lignivora (CBS 119148); (k) S. globosa (CBS 120340); (l) S. dimorphospora (CBS 553.74); (m) S. schenckii (CBS 359.36); (n) S. schenckii luriei (CBS 937.72) 3.1.2 Conidial morphology The morphology of conidia in S. schenckii isolates differed from that of the S. globosa isolates examined. The conidia of S. globosa isolates were septate hyphae with terminal clavate obovoid structures. The conidia of S. schenckii isolates were ovate, hyaline, and thin-walled conidia in sympodial conidiophores, with oval to oblong phialides. The type strain of S. inflata produced globose, sessile, brown conidia. The type strains of S. mexicana , S. variecibatus , S. lignivora , and S. pallida produced oval conidia and hyphae with terminal obovoid conidia. The type strain of S. brunneoviolacea showed hyaline, globose to smooth and thin-walled guttulate conidia.The conidia of ex-type S. humicola were solitary, straight, and hyaline. The type strain of S. dimorphosporas showed subglobose to obovoid sessile conidia in a conidiogenous terminal structure. The type strain of S. schenckii luriei produced septate, oblong, and hyaline conidia that were slightly irregular to irregular in shape (Fig. 2 2 ). (a) S . mexicana (CBS 120341); (b) S . brunneoviolacea (CBS 124561); (c) S. humicola (CBS 118129); (d) S. inflata (CBS 239.68); (e) S. pallida (CBS 131.56); (f) S. variecibatus (CBS 121961); (g) S. lignivora (CBS 119148); (h) S. globosa (CBS 120340); (i) S. dimorphospora (CBS 553.74); (j) S. schenckii (CBS 359.36); (k) S. schenckii luriei (CBS 937.72) 3.1.3 Growth rate Duncan’s multiple range test showed no significant differences in the growth rates among 15 S. globosa clinical strains from China ( P =0.45). However, there was a statistical difference in growth rates among the different species that comprise the complex. Moreover, statistical analysis revealed a faster growth rate of strains isolated from the natural environment compared with those obtained from clinical specimens ( P < 0.05). The maximum growth rate of S. globosa strains was (12.33±5.29) mm/week and the minimum growth rate was (8.41±3.02) mm/week; these were not significantly different ( P > 0.05). S. mexicana type strains grew the fastest with a rate of (19.93±2.15) mm/week. S. schenckii and S. globosa type strains had growth rates of (8.68±1.56) mm/week and (9.77±2.86) mm/week, respectively. The colony diameters for different isolates at the same temperature are summarized in Table 2 2 . 2 Fungus isolate 4 Diameter (mm) 2 Growth rate (mm/week) 7 d 14 d 21 d 28 d LC2454 10.15±4.83 21.33±1.36 26.83±1.72 38.33±3.20 8.41±3.02 LC2460 10.65±4.84 20.50±1.51 32.00±3.03 39.83±3.31 9.17±4.05 LC2466 11.66±1.21 21.16±1.47 30.33±1.36 40.66±2.94 9.05±1.84 LC2467 11.66±1.03 21.00±1.54 33.16±2.02 42.66±3.66 10.44±3.68 LC2469 12.16±1.16 21.16±1.72 32.66±2.33 44.33±3.93 10.72±2.63 LC2432 9.98±4.48 21.16±2.31 31.83±2.99 46.10±1.16 11.83±4.34 LC2433 8.16±6.26 22.63±2.16 34.33±3.20 44.33±1.50 11.11±3.06 LC2445 8.28±5.76 22.66±3.93 35.50±2.07 43.00±3.57 11.38±4.52 LC2447 9.98±4.57 21.50±1.37 30.33±2.58 44.66±2.16 11.11±3.37 LC2480 9.63±4.35 21.50±2.73 31.66±2.92 44.50±2.25 11.22±4.09 LC2404 9.96±4.63 20.16±1.16 31.00±3.09 39.50±3.01 9.55±2.70 LC2405 10.15±4.74 20.50±1.08 31.50±2.66 39.33±2.42 9.50±4.24 LC2411 9.80±4.86 20.33±2.33 31.33±3.38 47.66±1.36 12.33±5.29 LC2415 9.96±4.55 20.00±2.28 31.50±2.25 46.00±3.46 11.05±2.97 LC2419 9.46±6.67 21.00±0.89 32.16±2.31 47.83±3.18 11.88±4.01 S. mexicana 25.70±2.98 46.00±2.13 63.63±2.13 85.50±0.83 19.93±2.15 S. brunneoviolacea 16.83±2.50 33.16±1.72 52.66±2.25 64.50±0.57 15.85±3.65 S. humicola 27.25±4.52 47.25±3.19 59.66±2.73 71.33±1.21 14.74±4.38 S. inflata 13.75±1.28 24.66±2.50 39.00±1.78 45.66±3.72 10.63±6.77 S. pallida 28.62±3.50 51.00±4.14 62.33±2.58 71.50±1.51 14.29±6.46 S. variecibatus 29.33±5.20 48.22±5.08 65.16±1.16 74.50±0.54 15.05±5.16 S. lignivora 29.66±1.62 58.50±6.41 76.83±7.47 77.50±0.54 18.03±10.3 S. globosa 17.01±4.69 24.50±1.97 33.83±1.47 46.33±1.50 9.77±2.86 S. dimorphospora 15.50±2.07 30.66±1.86 42.66±3.01 55.00±1.09 13.20±2.05 S. schenckii 17.20±3.35 27.35±2.25 34.16±1.83 43.00±1.78 8.68±1.56 S. schenckii luriei 14.25±3.84 24.83±5.07 35.33±1.03 55.66±1.63 13.78±5.73 Physiological studies 2 The type strain of S. lignivora (CBS 119148) was unable to assimilate sucrose, unlike all of the other strains. All isolates were able to assimilate dextrose and ribitol with the exception of S. dimorphospora , S. inflata , S. lignivora , and S. mexicana . With respect to carbon source utilization, the clinical isolates from China were similar to the type strain of S. globosa (Table 3 3 ). Number of tests showing positive (+)/negative (−) as carbon source (+: able to grow sufficiently; −: unable to grow sufficiently) Strain Sucrose Raffinose Ribitol S. globosa ( n =15) + − + S. mexicana + + − S. brunneoviolacea + + + S. humicola + − + S. inflata + − − S. pallida + − + S. variecibatus + − + S. lignivora − − − S. globosa + − + S. dimorphospora + − − S. schenckii + + + S. schenckii luriei + + + Antifungal susceptibility test 2 Table 4 4 summarizes the MIC ranges for the Sporothrix isolates. Fifteen clinical isolates of S. globosa were subjected to antifungal susceptibility testing. These isolates showed a low MIC to KI (MIC range, 31.25–250.00 μg/ml), FLC (MIC range, 0.50– 4.00 μg/ml), ITC (MIC range, 0.25–1.00 μg/ml), TRB (MIC range, 0.06–1.00 μg/ml), and AMB (MIC range, 0.13–0.50 μg/ml) (Table 4 4 ). ITC, TRB, and AMB had the lowest MICs against S. schenckii with values of 2.00, 1.00, and 1.00 μg/ml, respectively, while FLC showed no activity (MIC > 64.00 μg/ml). We detected moderate activity for ITC and TRB against the S. globosa type strain, with MICs of 1.00 μg/ml, while KI and FLC showed a low activity indicating that they are not effective against this strain. FLC was not effective against S. mexicana , S. humicola , S. inflata , or S. schenckii luriei (MIC > 64.00 μg/ml). KI: potassium iodide; FLC: fluconazole; ITC: itraconazole; TRB: terbinafine; AMB: amphotericin B 2 Strain 2 Name 5 MIC (μg/ml) KI FLC ITC TRB AMB LC2454, LC2460, LC2466, LC2467, LC2469 > S. globosa 31.25–62.50 0.50–2.00 0.50–1.00 0.06–1.00 0.13–0.50 LC2404, LC2405, LC2411, LC2413, LC2419 > S. globosa 62.50–125.00 2.00–4.00 0.50–1.00 0.25–1.00 0.13–0.50 LC2432, LC2433, LC2445, LC2447, LC2480 > S. globosa 62.50–250.00 1.00–4.00 0.25–1.00 0.13–1.00 0.25–0.50 CBS 120341 > S. mexicana 62.50 >64.00 4.00 0.25 8.00 CBS 124561 > S. brunneoviolacea 62.50 4.00 1.00 2.00 1.00 CBS 118129 > S. humicola 31.25 >64.00 0.06 2.00 8.00 CBS 239.68 > S. inflata 31.25 >64.00 1.00 0.06 0.25 CBS 131.56 > S. pallida 7.80 4.00 0.13 0.25 >16.00 CBS 121961 > S. variecibatus 31.25 4.00 1.00 0.06 1.00 CBS 119148 > S. lignivora 62.50 1.00 >16.00 1.00 8.00 CBS 120340 > S. globosa >500.00 >64.00 1.00 1.00 4.00 CBS 553.74 > S. dimorphospora 25.65 1.00 4.00 1.00 4.00 CBS 359.36 > S. schenckii 250.00 >64.00 2.00 1.00 1.00 CBS 937.72 > S. schenckii luriei 31.25 >64.00 1.00 1.00 4.00 KI was the most effective antifungal agent against S. pallida (MIC=7.80 μg/ml). The MICs of FLU showed moderate activity against clinical isolates of S. globose from China compared with S. schenckii , S. globosa , and S. schenckii luriei . TRB was the most active drug for all the strains tested. AMB was slightly less effective against S. schenckii luriei and S. globosa compared with S. globosa clinical isolates from China. For ITC, a strain with an MIC of > 4.00 μg/ml was considered tolerant, while a strain with an MIC in the range of 1.00–2.00 μg/ml was considered susceptible. Our data showed that ITC was effective against S. globosa clinical isolates from China, but was ineffective for S. schenckii , S. globosa , and S. schenckii luriei . Discussion 1 Different strains comprising the S. schenckii complex exhibited variation in colony characteristics. The type strain of S. globosa showed terminal clavate obovoid conidia. However, we were unable to observe any dark colony characters, and we presume that the isolate had degenerated, thereby losing its ability to produce these dark mycelia (Marimon et al., 2007 2007 ). Moreover, pathogenic Sporothrix species differed in growth rates and physiological characteristics when compared with environmental isolates, suggesting that these strains have adapted specific, albeit yet to be defined, traits that enable their pathogenicity. ITC, when administered at a dose of 200 mg orally daily for 3–6 months, is the first antifungal treatment for cutaneous and lymphocutaneous sporotrichosis recommended by the Clinical Practice Guidelines for the Management of Sporotrichosis (de Lima Barros et al., 2004 2004 ; Silva-Vergara et al., 2012 2012 ; Rodrigues et al., 2014 2014 ). In this study, ITC showed good activity against all species, except for S. lignivora , consistent with the results of Kohler et al. ( 2004 2004 ). Moreover, TRB exhibited high activity against all the strains tested, as demonstrated by other authors (Marimon et al., 2008 2008 ). In this study, we found that S. globosa isolates from China were more susceptible to antifungals than the type strain of S. globosa . The study included strains isolated from the three different regions of China. Even in this limited sample we found variation in morphological characteristics and susceptibilities to antifungal agents. Therefore, our findings reinforce the importance of identifying S. schenckii sensu stricto and of evaluating its antifungal susceptibilities to determine the optimum therapeutic option for each case of sporotrichosis. Thus, it is imperative that species causing infection be identified during diagnosis and that treatment be prescribed accordingly. The results of this study provide fundamental data to assist in the selection of antifungal agents with enhanced activities against selected strains. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1500055 Project supported by the National Natural Science Foundation of China (No. 31270062) 2015-03-08 2015-10-21 2015-09-14 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2015 2015 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 11 16