Candida Albicans의 Crude Extract와 Carcinogen의 투여에 의한 Mouse의 위점막 변화

정정명,이명수,김수찬,황문수,김동수,설상영,최하진 인제대학교 1990 仁濟醫學 Vol.11 No.1

저자들은 candida albicans와 cancinogen을 공동으로 투여받은 실험 mice에서 위암의 발생이 촉진되는 결과를 확인한 바 있는데 이 경우 candida albicans의 독소에 의한 영향인가 아닌가를 확인하기 위하여 candida albicans의 crude extract를 추출하고 이 물질이 실험 mice의 위점막에 미치는 영향을 관찰하였다. 본 실험의 결과로 candida albicans의 crude extract는 살아있는 candida albicans와 동일한 위점막 변화를 발생시킬 수 있으며 또 위암의 발생에 하나의 promotor로 작용할 수 있음이 확인되었다. 그리고 이러한 현상은 아마도 crude extract 내 포함된 candida albicans의 독소에 의한 것으로 사료되어 진다. MNNG(N-methyl-N'-nitro-N-nitrosoguanidine), having a potent mutagenic and carcinogenic effect, has been widely used as a carcinogen for production of gastric carcinoma in experimental animals. But when used alone, the carcinogenic effect of the MNNG is much interfered by the mucosal barter on the gastric mucosa of animal models, and some surfactants are employed to break down the mucosal barrier Nitrosamine, a carcinogen, can be synthesized by the action of pathogenic bacteria in the gut. As it was found that nitrosamine can be produced In the human gastric juice, much attention has been paid to the relationship between human stomach cancer and nitrosamine. Moreover infections by the pathogenic micro-organisms in the human stomach were confirmed and their long-standing infections are suggested to be a pathogenic factor on chronic gastritis and peptic ulcer. However, at present, it seems so hard to confirm the relationship between stomach cancer and the infection of pathogenic micro-organisms that far more information is needed to identify it accurately. Therefore we observed the effect of candida albicans, which is found in high frequency in the stomach cancer and peptic ulcer patients, on the change of gastric mucosa and pathogenesis of stomach cancer, and suggested that the concomittant administration of candida albicans and carcinogen to the experimental mouse may promote the development of stomach cancer. But in the study administrating live candida albicans we couldn't identify which of the factors of candida albicans induced those mucosal changes in experimental mouse. Therefore in this study we used the crude extract of candida albicans and investigated the effect of toxin in the crude extract of candida albicans. To find out which of factors of candida albicans made those carcinogenic effect in mice stomach, we have extracted the crude extract of candida albicans and investigated the effect of toxin in crude extract of candida albicans on the gastric mucosa of the experimental mouse. The results were as follows ; 1.When the crude extract of candida albicans was used alone ; Among 5 mice 3 showed local inflammatory change in the antrum and 4 showed In the body at the 3 months group. And an infammatory change in stomach antrum occured in all of 5 mice, however, among 5 mice 2 showed mucosal atophy in stomach body at 6 months group. 2.When MNNG was used alone ; Among 5 mice 2 showed grade A mucosal changes in 6 month group, and in the 9 months group among 6 mice 2 showed grade A and 3 showed grade B mucosal changes respectively. 3.When both the crude extract and carcinogen were used ; Among 4 mice 2 showed grade B mucosal change in the 6 months group. And among 6 mice 2 showed grade C mucosal change, 2 showed gastric antrum adenoma and 2 showed gastric cancer in the 9 months group. With these results, we can conclude that the application of crude extract of candida albicans may cause the same gastric mucosal change that was caused by live candida albicans, and it can act as a promoter in the pathogenesis of stomach cancer ; and it is suspected that a kind of toxin in candida albicans cause these muscosal changes in experimental mice stomach.

화상을 입힌 흰쥐에서 장관내 진균의 전위와 면역능의 변화

임풍,한기택,양성열,조성필 大韓成形外科學會 1993 Archives of Plastic Surgery Vol.20 No.5

Sepsis still remains the major cause of morbidity and mortality after initial survival from extensive burn. It has been confirmed that increased translocation of enteric microrganism to the mesenteric lymph nodes and blood flow in association with burn injuries had taken place in otherwise healthy animals. Candida albicans is one of the microflora responsible for sepsis, and its incidence in burned patients has steadily increased. The passage of viable C.albicans through the gastrointestinal mucosa into the host blood stream is believed to be an important mechanism leading to systemic candidiasis. Several lines of evidence suggest that the immunosuppression of cell mediated immuity(CMI) was due to decreased Th/Ts ratio in burned patients and CMI was specifically suppressed by C. albicans. The aim of this study is to investigate the relationship and serial changes between the translocation of C.albicans and burn injuries and to clarify the fact that whether the translocation of C.albicans suppress the immunocompetence by observing the changes of lymphocyte subpopulation in burned rats in which the translocation of C.albicans were found. Normal Wistar rats were challenged intragastrically with C.albicans prior to a 40% scald burn in the candida-burn experimental group. Tissues harvested from the mesenteric lymph node, liver, spleen, thymus, jejunum, cecum and colon on the lst, 3rd, 5th&7th postexperimental days and cultured on Sabouraud dextrose agar and then sitained with periodic-acid Schiff(PAS) to observe the transolcation of C.albicans. Also the lymphocytes were isolated from peripheral blood of the rats at the immediate before the experimentation, lst, 3rd, 5th and 7th postexperimental days and then the subpopulations were measured by flow-cytometry; All findings were compared with those of each control(gavage with phosphate buffered saline(PBS)+shame burn), candida experimental(gavage with C. albicans+shame burn) and burn experimental(gavage with PBS+40% scald burn) groups. The results were as follows; 1. In the candida experimental group, C.albicans were found to only the mesenteric lymphnode in the early experimental period. In the candida-burn experimental group, the translocation of C.albicans increased more significantly than that of the candida experimental group and the translocation was maintained up to the 7th post experimental day. 2. On microscopic examination, intestinal mucosal atrophy occured after burn injuries. C. albicans seem to have passed through the intestinal mucosal barrier and then they were phagocytized by macrophages in the lamina propria. Also most of C. albicans which had infiltrated into the other organs were phagocytized by macrophages, but some were free. In the candida-burn experimental group, C. albicans were found in the blood vessel of the liver and thymus on the lst postexperimental day and they had infiltrated into the organs at a greater rate than that of the candida experimental group. 3. In the candida-burn experimental group, the Th/Ts ratio was significantly decreased on the 3rd, 5th and 7th postexperimental days compared to that of the pre-experimental day. On the lst and 7th days, such diminution was significant as compared with the control group and on the 3rd, 5th days, the Th/Ts ratio had decreased more significantly than that of the control and candida experimental groups. But at all intervals, no statistical significance was found as compared with the burn experimental group. With these results it is inferred that burn stress promotes the translocation of C.albicans from the gastrointestinal tract to other organs and the translocation induced by burn injuries is maintained persistantly up to the 7th postexperimental day. And the translocation of C.albicans in burned rats seems not to enhance the depression of CMI through burn injury. But more study will be necessary to understant the immunologic alteration between the translocation of C.albicans and burn injuries.

화상을 입힌 흰쥐에서 장관내 Candida albicans 전위에 대한 Ketoconazole의 예방적 효과

정성한,조성필,한기택,오영환,임 풍 大韓成形外科學會 1994 Archives of Plastic Surgery Vol.21 No.4

Despite of many advances in both topical and systemic control of infections, candida sepsis continues to be a serious complication following major thermal injury. The translocation of enteric C. albicans may be occured to be associate with major burn injuries. Ketoconazole is an oral antifungal agent that has been effective in the treatment of many fungal infections. However, the results of prophylactic clinical and laboratory trials using ketoconazole have been inconsistent. The aim of this study is to determine the prophylactic effect of oral ketoconazole on the translocation of enteric C. albicans in major burn injuries. Normal rate recieved ketoconazole (10㎎/㎏/day) 24 hours prior to a 40% scald burn and were challenged intragastrically with C. albicans 1 hour prior to the burn in a ketoconazole - candida treated group. Tissues were harvested from the mesenteric lymph node, liver, spleen and cecum on the 1st and 4th postburn days and cultured on Sabouraud dextrose agar and then stained with periodic-acid Schiff (PAS) to observe the translocation of C. albicans. All findings were compared with those of each control(40% scald burn) and candida treated(gavage with C. albicans+40% scald burn) group. The results were as follows ; 1. There was no translocation of C. albicans to mesenteric lymph node or other organs in the control group. In the ketoconazolee-candida treated group, C. albicans were found in only the mesenteric lymph node on the l st postburn day, but they tanslocated to the liver and spleen on the 4th postburn day. In the candida treated group, C. albicans were observed in all examined organs on the 1st postburn day and the rate of translocation incresed on the 4th postburn day. The translocation of C. albicans in the candida treated group increased more significantly than that of the ketoconazole-candida treated group. 2. On microscopic examination, C. albicans seems to have passed through the intestinal mucosal barrier and then they were phagocytized by macrophages in the laminal propria. Also most C. albicans which had infiltrated into the other organs were phagocytized by macrophages. In the candida treated group, C. albicans had infiltrated into the organs at a greater rate than that of the ketoconazole-candida treated group. With these result, ketoconazole reduced the translocation rate of enteric C. albicans in major burn injures. But more study will be necessary to determine the relationship between the degree of translocation.

생체 내 상에서 Candida albicans로 유도한 염증인자에 미치는 스쿠알렌의 효과

이준행(Jun Haeng Lee) 대한방사선과학회(구 대한방사선기술학회) 2016 방사선기술과학 Vol.39 No.4

본 연구에서는 Candida albicans로 유도한 염증반응에 대해 스쿠알렌의 처치를 통해 염증반응을 경감시키는가에 대해 확인하였다. 실험동물은 (ICR계 생쥐) 각 실험군별 7마리씩을 사용하여 생체 내 실험을 하였다. Candida albicans -유도 염증인자 중 종양괴사인자-알파, 인터루킨-6, 산화질소는 ELISA kits 방법을 이용하여 관찰하였다. 실험을 통해 다음과 같은 결론을 얻었다. 1. Candida albicans를 감염시킨 군에서 1, 3일째 모두 신장조직 내에서의 산화질소 생성량을 감소시키는 것을 확인할 수 있었다 (p< 0.05). 2. 스쿠알렌 (80㎖/㎏)을 7일간 1일 1회 전 처치(복강투여)한 다음, Candida albicans를 감염시킨 군에서 3일째 군에서만 신장 조직 내에서의 종양괴사인자-알파 생성량을 감소시키는 것을 확인할 수 있었다(p< 0.05). 3. 스쿠알렌 (80㎖/㎏)을 7일간 1일 1회 전 처치(복강투여)한 다음. Candida albicans를 감염 시킨 군에서 3일째 군에서만 신장 조직 내에서의 인터루킨-6 생성량을 감소시키는 것을 확인할 수 있었다(p< 0.05). 결론적으로 스쿠알렌이 Candida albicans-유도 염증인자들의 억제를 위해서는 예방적 차원에서 스쿠알렌을 공급하는 것이 효과가 있음을 알 수 있었다. 따라서 스쿠알렌 처치가 Candida albicans-유도 면역억제에 대해 회복 효과를 기대할 수 있을 것으로 사료된다. In the present study, whether squalene treatment relives inflammatory reactions induced by Candida albicans was checked. The experiment was conducted in vivo using seven experimental animals (ICR mice) per experimental group. Among C. albicans-induced inflammatory factors, TNF-α, IL-6, and NO were observed using the ELISA kits method. Through the experiment, the following conclusions were obtained. 1. In the group infected with C. albicans, it could be identified that squalene treatment was inducing NO generation in renal tissues both on the 1st and 3rd days (p < 0.05). 2. In the group pre-treated(intraperitoneal administration) with SQ (80㎖/㎏) once per day for seven days and infected with C. albicans, it could be identified that squalene treatment was inducing TNF-α generation in renal tissues only on the 3rd day(p < 0.05). 3. In the group pre-treated(intraperitoneal administration) with SQ (80㎖/㎏) once per day for seven days and infected with C. albicans, it could be identified that squalene treatment was inducing IL-6 generation in renal tissues only on the 3rd day(p < 0.05). In conclusion, it could be seen that for squalene to suppress C. albicans-induced inflammatory factors, preemptively supplying SQ should be effective. Therefore, effects for recovery from C. albicans-induced immunodepression can be expected from SQ treatment.

단삼에 의한 Candida albicans 바이오필름 발달의 억제

이흥식 ( Heung-shick Lee ),김연희 ( Younhee Kim ) 한국미생물생명공학회(구 한국산업미생물학회) 2019 한국미생물·생명공학회지 Vol.47 No.3

Candida albicans는 기회감염을 유발하는 주요한 병원성 진균 중의 하나이다. 캔디다증 치료과정에서 항진균제에 대한 내성이 흔히 발견되는데, 그 이유는 Candida가 바이오필름을 형성할 수 있기 때문이다. 이전의 연구에서 우리는 단삼(Salvia miltiorriza)의 에탄올추출물이 세포막의 투과성을 변화시키고 세포벽 합성을 저해하여 항캔디다 활성을 나타냄을 밝혔다. 본 연구에서는 10개 C. albicans 임상균주가 형성한 초기단계의 바이오필름을 대상으로 XTT 환원분석법으로 대사활성을 측정하니, 78 μg/ml 단삼 에탄올추출물에 의해 바이오필름의 대사활성이 평균 51.3% 감소되었다. C. albicans 세포들이 폴리스티렌 표면에 부착하거나 germ tube를 형성하는 과정에서의 단삼 에탄올추출물의 영향을 현미경으로 분석하니, 39 μg/ml 단삼 에탄올추출물에 의해 부착된 세포의 밀도는 현저하게 감소하였으나 germ tube 형성은 거의 억제하지 못했다. 단삼 에탄올추출물이 C. albicans SC5314 세포의 균사에 특이적인 유전자 발현에 미치는 영향을 qPCR로 분석한 결과, EAP1은 34.7% (p < 0.001), ALS1은 45.0% (p < 0.001), ALS3는 48.1% (p < 0.001), ECE1은 21.3% (p = 0.006) 억제하였다. 결론적으로 단삼의 에탄올추출물은 초기단계의 C. albicans 바이오필름 발달을 효율적으로 저해하며, 이는 EAP1, ALS1, ALS3 유전자의 발현억제에 따른 세포부착 억제와 관련이 있다. 더불어 단삼 에탄올추출물의 C. albicans 세포막 기능저해와 세포벽 합성억제에 의한 구조변화 또한 세포부착단계에서의 바이오필름 발달억제에 기여할 것으로 추정된다. Candida albicans is an opportunistic human pathogen that causes infections. Candidiasis is often related to antifungal resistance because the pathogen has the ability to form biofilms. In a previous study, we found that the Salvia miltiorriza ethanol extract demonstrated anticandidal activity by altering membrane permeability and inhibiting the cell wall synthesis in C. albicans. Our results here demonstrate that 78 μg/ml of the S. miltiorriza extract significantly diminished the early stage biofilms formed by 10 clinical C. albicans isolates by 51.3%; this was analyzed by 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide salt (XTT) reduction assay. The effect of the S. miltiorrhiza extract on the adhesion of C. albicans cells to polystyrene plates and germ tube formation was examined via microscopic investigation. Although the density of the adhered cells was remarkably reduced up on incubation with 39 μg/ml S. miltiorrhiza extract, germ tube formation by C. albicans was rarely affected. Quantitative real-time PCR analysis showed that the S. miltiorrhiza extract downregulated the expression of C. albicans hypha-specific genes, EAP1 by 34.7% (p < 0.001), ALS1 by 45.0% (p < 0.001), ALS3 by 48.1% (p < 0.001), and ECE1 by 21.3% (p = 0.006), respectively. Our data suggest that the S. miltiorrhiza ethanol extract significantly inhibited the early stage of biofilm formation by C. albicans by interfering with cell adhesion, by downregulating EAP1, ALS1 and ALS3, and presumably by modifying the cell wall and membrane structure.

Candida albicans 의 단백질분해효소, 인지질분해효소 및 세포벽 단백분획으로 면역시킨 생쥐에서의 칸디다증 방어 효과

신운섭,최선주,박수정,박주영,김수기,이경호,양영철 대한의진균학회 2009 대한의진균학회지 Vol.14 No.1

Background: The opportunistic fungus Candida albicans is a major pathogen especially to immunocompromised patients. Objectives: We examined the protective effect of the active and passive immunizations to evaluate the applicability for the treatment of candidosis in Candida-infected mice model. Methods: Candida cell wall components were obtained by treatment of lyticase, proteinase K, and dithiothreitol. The proteinase was purified from the culture filtrates of C. albicans using a series of chromatographic steps consisting of DEAE-Sepharose FF, Sephacryl S-200 HR and size-exclusion high performance liquid chromatography. The phospholipase was purified from the culture supernatant of C. albicans with DEAE column chromatography, reverse phase column chromatography, revere phase HPLC and size-exclusion HPLC. Antibodies to cell wall protein components, proteinase and phospholipase were produced by immunization into mice of same strain. Results: The mean survival times of active and passive immunized mice groups were longer than those of non-immunized groups. Conclusion: These results showed that immunization with proteinase and its antibody were the most effective to prolong survival time in Candida-infected mice. Background: The opportunistic fungus Candida albicans is a major pathogen especially to immunocompromised patients. Objectives: We examined the protective effect of the active and passive immunizations to evaluate the applicability for the treatment of candidosis in Candida-infected mice model. Methods: Candida cell wall components were obtained by treatment of lyticase, proteinase K, and dithiothreitol. The proteinase was purified from the culture filtrates of C. albicans using a series of chromatographic steps consisting of DEAE-Sepharose FF, Sephacryl S-200 HR and size-exclusion high performance liquid chromatography. The phospholipase was purified from the culture supernatant of C. albicans with DEAE column chromatography, reverse phase column chromatography, revere phase HPLC and size-exclusion HPLC. Antibodies to cell wall protein components, proteinase and phospholipase were produced by immunization into mice of same strain. Results: The mean survival times of active and passive immunized mice groups were longer than those of non-immunized groups. Conclusion: These results showed that immunization with proteinase and its antibody were the most effective to prolong survival time in Candida-infected mice.

세포막손상 유발로 인한 황련의 캔디다 바이오필름 형성 억제

김연희,Kim, Younhee 한국미생물학회 2013 미생물학회지 Vol.49 No.1

Candida 바이오필름은 숙주조직이나 인체에 삽입된 의료기구의 표면에 자라는 진균의 군락으로 전통적인 항진균제에 대한 내성을 유발한다. 황련(Coptidis chinensis)의 뿌리는 극동지방에서 의료용 목적으로 널리 사용되어 왔다. 본 연구의 목적은 임상에서 분리한 C. albicans 바이오필름 형성 균주가 형성한 바이오필름에 대한 C. chinensis 수용성 추출물의 효과와 C. albicans 바이오필름 형성을 저해하는 데 기여하는 항진균활성을 평가하는 데 있다. 바이오필름에 대한 효과는 XTT [2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide)] 환원분석법을 사용하였으며, 조사된 모든 균주에 대한 대사활성은 $98{\mu}g/ml$의 C. chinensis 수용성 추출물에 의해 현저하게 감소($57.3{\pm}14.7%$)되어 유의성 있는 항바이오필름 활성을 나타내었다. Fluorescein diacetate와 propidium iodide로 이중 염색한 결과 C. chinensis 추출물은 C. albicans의 세포막을 손상시켰다. C. chinensis 수용성 추출물은 살진균 활성을 나타냈고, C. albicans 바이오필름의 폴리스티렌 표면으로의 부착을 억제하였으며 C. albicans를 $G_o/G_1$기에 머무르게 하여 바이오필름이나 출아법에 의한 증식을 억제시켰다. 본 연구의 결과는 C. chinensis 추출물이 목표가 되는 C. albicans에 복합적으로 유해한 효과를 내어 궁극적으로는 C. albicans 바이오필름 형성을 억제함을 나타낸다. 따라서 C. chinensis 추출물은 바이오필름과 관련된 캔디다의 감염을 치료하고 제거하기 위한 천연물 기반항진균제 개발에 대한 높은 가능성을 가진다. Candida biofilms are organized microbial communities growing on the surfaces of host tissues or indwelling medical devices, and the biofilms show enhanced resistance against the conventional antifungal agents. The roots of Coptidis chinensis have been widely used for medicinal purposes in East Asia. The present study was aimed to assess the effect of C. chinensis aqueous extract upon preformed biofilms of 10 clinical Candida albicans isolates and the antifungal activities which contribute to inhibit the C. albicans biofilm formation. Its effect on preformed biofilms was judged using XTT [2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide)] reduction assay, and metabolic activity of all tested strains was reduced significantly ($57.3{\pm}14.7%$) at $98{\mu}g/ml$ of the C. chinensis extract. The extract damaged the cell membrane of C. albicans which was analyzed by fluorescein diacetate and propidium iodide staining. The anticandidal activity was fungicidal, and the extract obstructed the adhesion of C. albicans biofilms to polystyrene surfaces, arrested C. albicans cells at $G_o/G_1$ as well, and reduced the growth of biofilms or budding yeasts finally. The data suggest that C. chinensis has multiple antifungal effects on target fungi resulting in preventing the formation of biofilms. Therefore, C. chinensis holds great promise for exploring antifungal agents from natural products in treating and eliminating biofilm-associated Candida infection.

Isolation Frequency Characteristics of Candida Species from Clinical Specimens

( Ga-yeon Kim ),( Jae-sik Jeon ),( Jae Kyung Kim ) 한국균학회 2016 Mycobiology Vol.44 No.2

Candida spp. is an invasive infectious fungus, a major risk factor that can increase morbidity and mortality in hospitalized patients. In this study, 2,508 Candida spp. were isolated from various clinical specimens collected from university hospitals from July 2011 to October 2014. They were identified in order to determine isolation frequencies and characteristics by specimen, gender, age group, year, season, and month. The strain-specific isolation rate of Candida spp. is in the order of Candida albicans (1,218 strains, 48.56%), Candida glabrata (416 strains, 16.59%), Candida utilis (305 strains, 12.16%), Candida tropicalis (304 strains, 12.12%), and Candida parapsilosis (116 strains, 4.63%) and these five species accounted for more than 94% of the total strains. Of the specimens, Candida spp. were most frequently isolated from urine-catheter, followed by urinevoided, blood, sputum, other, open pus, vaginal discharge, Tip, ear discharge, bronchial aspiration and bile, in that order. Looking at the age distribution, the detection rate of patients in their 60s and older was significantly higher at 75.8% (1,900/2,508). The detection rate of patients in their 20s and younger was shown to be very low at 2.55% (64/2,508). By year, the detection rate of non-albicans Candida spp. showed a tendency to gradually increase each year compared with C. albicans. As isolation of Candida spp. from clinical samples at the specie level can vary depending on characteristics of the patient, sample, season, etc., continual studies are required.

Candida albicans 추정 동정을 위한 발아관 형성 조건

박금보래,이미경 대한의진균학회 2008 대한의진균학회지 Vol.13 No.1

Background: Candida albicans is the most prevalent species found in human yeast infections. The germ tube test is still frequently used for its rapid presumptive identification. Recently Candida dubliniensis as well as C. albicans has been reported to form germ tubes. Objective: The purpose of this study was to evaluate the germ tube test at various conditions for rapid presumptive identification of C. albicans. Methods: C. albicans ATCC 14053, C. albicans ATCC 18804, C. dubliniensis ATCC MYA 646, and C. dubliniensis KCTC 17427 were tested. Human pooled serum (HPS), HBV, HCV infected patient serum, fetal bovine serum (FBS) and rabbit serum (RS) were used for germ tube test. The germ tube formation was evaluated at different keeping condition of various sera, after mixing with 5 different bacterial suspensions and at various incubation conditions. Results: The germ tube formation of C. albicans was more in the RS or FBS than in the HPS. For the various sera fresh sample was always the best expression of germ-tube forming ability. In the HCV infected patient serum and mixing with Pseudomonas aeruginosa germ tube formation was suppressed. C. dubliniensis did not form germ tube in the HPS, only formed in the FBS or RS. Conclusion: For rapid presumptive identification of C. albicans not C. dubliniensis the best selection of serum is the fresh HPS. We recommend the examination with isolated colony free from bacteria after incubation for 2 to 3 hours. Background: Candida albicans is the most prevalent species found in human yeast infections. The germ tube test is still frequently used for its rapid presumptive identification. Recently Candida dubliniensis as well as C. albicans has been reported to form germ tubes. Objective: The purpose of this study was to evaluate the germ tube test at various conditions for rapid presumptive identification of C. albicans. Methods: C. albicans ATCC 14053, C. albicans ATCC 18804, C. dubliniensis ATCC MYA 646, and C. dubliniensis KCTC 17427 were tested. Human pooled serum (HPS), HBV, HCV infected patient serum, fetal bovine serum (FBS) and rabbit serum (RS) were used for germ tube test. The germ tube formation was evaluated at different keeping condition of various sera, after mixing with 5 different bacterial suspensions and at various incubation conditions. Results: The germ tube formation of C. albicans was more in the RS or FBS than in the HPS. For the various sera fresh sample was always the best expression of germ-tube forming ability. In the HCV infected patient serum and mixing with Pseudomonas aeruginosa germ tube formation was suppressed. C. dubliniensis did not form germ tube in the HPS, only formed in the FBS or RS. Conclusion: For rapid presumptive identification of C. albicans not C. dubliniensis the best selection of serum is the fresh HPS. We recommend the examination with isolated colony free from bacteria after incubation for 2 to 3 hours.

막투과성 변화로 인한 대황의 Candida albicans에 대한 항진균 활성

이흥식,김연희,Lee, Heung-Shick,Kim, Younhee 한국미생물학회 2014 미생물학회지 Vol.50 No.4

Candida albicans는 면역력이 약화된 환자의 표재성에서 전신적 감염까지 다양한 부위에서 감염을 유발시킬 수 있는 기회 감염적 병원성 진균이다. C. albicans는 효모형에서 균사형으로 변환될 수 있으며 이 때 바이오필름을 형성할 수 있다. 바이오필름과 관련된 C. albicans의 감염은 일반적으로 통상적인 항진균제에 대해 내성을 보이므로 새로운 항진균제에 대한 개발이 절실하다. 대황(Rheum undulatum)은 전통적으로 한국과 중국에서 하제나 소염제로 사용되는 약용 식물이다. 본 연구의 목적은 R. undulatum이 캔디다증 환자로부터 분리한 C. albicans 바이오필름 형성 균주에 대한 바이오필름 형성 억제효과와 이에 대한 항진균 활성 기작을 알아보는 것이다. R. undulatum (0.098 mg/ml)은 12종의 바이오필름 형성 임상균주의 캔디다 바이오필름을 $49.4{\pm}6.0%$ 감소시켰고 C. albicans의 폴리스티렌 표면으로의 부착을 억제시켰다. CFDA, AM과 propidium iodide로 이중 염색한 결과 R. undulatum은 C. albicans의 세포막을 손상시켰으며 propidium iodide와 neutral red로 염색하여 공초점 레이저 현미경과 위상차 현미경으로 관찰한 결과 C. albicans의 세포용해를 야기함을 관찰할 수 있었다. Crystal violet 흡수율 실험으로 R. undulatum에 의한 세포막 투과성의 변화를 관찰하였다. 따라서 R. undulatum은 세포막의 손상과 세포막의 투과성 변화로 야기된 세포의 용해와 관련된 항진균 활성이 C. albicans의 바이오필름 형성을 억제하는 것으로 보여진다. 본 연구의 결과는 R. undulatum이 바이오필름과 관련된 캔디다의 감염을 치료하고 제거하기 위한 천연물 기반 항진균제 개발에 대한 좋은 후보물질임을 보여준다. Candida albicans is an opportunistic and the most prevalent fungal pathogen that can cause superficial and systemic infections in immunocompromised patients. C. albicans can promote the transition from budding yeast to filamentous form, generating biofilms. Infections associated with C. albicans biofilms are frequently resistant to conventional antifungal therapy. Therefore, the development of more effective antifungal drugs related with biofilm formation is required urgently. The roots of Rheum undulatum have been used for medicinal purposes in Korea and China traditionally. The aim of present study was to evaluate the effect of R. undulatum extract upon preformed biofilms of 12 clinical C. albicans isolates and the antifungal activities. Its effect on preformed biofilms was evaluated using XTT reduction assay, and metabolic activity of all tested strains was reduced significantly ($49.4{\pm}6.0%$) at 0.098 mg/ml R. undulatum. The R. undulatum extract blocked the adhesion of C. albicans biofilms to polystyrene surfaces, and damaged the cell membrane integrity of C. albicans which was analyzed by CFDA, AM, and propidium iodide double staining. It caused cell lysis which was observed by Confocal laser scanning and phase contrast microscope after propidium iodide and neutral red staining, respectively. Membrane permeability was changed as evidenced by crystal violet uptake. The data suggest that R. undulatum inhibits biofilm formation by C. albicans, which can be associated with the damage of the cell membrane integrity, the changes in the membrane permeability and the cell lysis of C. albicans.