口腔黏膜真菌鉴定方法的比较

比较常见用于黏膜真菌菌种鉴别的多种方法,探寻最佳的鉴别方法。采集230例普通人群口腔黏膜样本,分别用玉米吐温-80培养观察厚膜孢子法、糖发酵生化反应法、CHROMagar假丝酵母菌显色培养基法、ITS基因的PCR-RFLP(聚合酶链反应-限制性片段长度多态性)法、ITS测序菌种鉴定法,鉴别真菌各菌株。结果显示:有56例菌株至少通过1种方法检出真菌;玉米吐温-80分离培养假丝酵母菌37株;50例菌株ITS基因测序共鉴定出8个菌种,白假丝酵母菌(C.albicans)29株,近平滑假丝酵母菌(C.parapsilosis)10株,热带假丝酵母菌(C.tropicalis)5株,Candida metapsilosis 1株,Lodderomyces elongisporus 1株,克柔假丝酵母菌(Candida krusei)1株,乙醇假丝酵母菌(C.ethanolica)1株,季也蒙毕赤酵母菌(Pichia guilliermondii)2株;CHROMagar假丝酵母菌显色培养基法鉴定出3种菌株,分别是白假丝酵母菌、热带假丝酵母菌、近平滑假丝酵母菌;PCR-RFLP法检出5种菌株,分别是白假丝酵母菌、热带假丝酵母菌、近平滑假丝酵母菌、季也蒙毕赤酵母菌、克柔假丝酵母菌,与基因的测序鉴定一致率为91%;糖发酵生化反应法阳性标本占被检出真菌例数的46.4%(26/56)。结果表明:ITS基因的测序法可以准确鉴定真菌各个菌种;PCR-RFLP法能鉴定常见的菌种,但操作繁琐;CHROMagar假丝酵母菌显色培养基法能快速准确鉴别3种常见假丝酵母菌菌种;玉米吐温-80可以准确培养鉴别白假丝酵母菌;糖发酵生化反应法,缺乏足够的敏感度和特异性,难以准确鉴别各个菌种。     

PCR-SSCP技术在假丝酵母属菌种鉴别中的应用

选取中国工业微生物菌种保藏管理中心(CICC)保藏的假丝酵母属的7个种30株菌, 对其rDNA的ITS1区及ITS2区进行了PCR-SSCP指纹图谱分析, 结果表明在假丝酵母属种水平的区分鉴定中, ITS1区与ITS2区的PCR-SSCP图谱均能对本研究所选7个种的菌株进行显著区分, 比较两个区段的PCR-SSCP图谱及鉴别效果, 发现ITS2区的应用效果要优于ITS1区。     

The rate of isolation of fungi in the genus Candida from the nasopharyngeal mucosa of the those in contact with the products from microbial protein manufacture]

In the mycological study of the air in working rooms at enterprises for the production of paprin and in the airspace of residential areas, as well as the study of the fungal contamination of the upper respiratory ways in workers and residents of the development zone, the isolation rate of the production fungal strains of the genus Candida from the nasopharynx was shown to depend on their content in the air. In the absence of producer microorganisms in the atmospheric air they were not detected among the population. The detection of yeast-like fungi of the genus Candida on the pharyngeal mucosa is of sanitary demonstrative importance for the evaluation of the specific microbial contamination of the air in working rooms and the air space of the development zone. When the stable work of gas purification systems was ensured at modern enterprises for the production of protein vitamin concentrate no production strains were detected in the atmospheric air of the development zone and on the nasopharyngeal mucosa of the residents, which was indicative of the absence of any influence of gas and air discharges from these enterprises on the microbial contamination of the airspace.     

胃病患者胃黏膜真菌感染的检测

通过检测胃炎和胃溃疡、胃癌患者胃黏膜寄居的真菌,了解胃黏膜真菌的菌种多样性及其与胃溃疡的关系。采集消化科就诊患者胃镜钳取的胃黏膜标本63例,采用念珠菌显色培养基(CHROMagar)进行真菌分离培养鉴定。用玉米吐温80培养基进行真菌孢子形态学检查,用ITS(internal transcribed spacer region)序列限制性片段长度多态性(Restriction fragment length polymorphism,RFLP)检测分析真菌菌种多样性。分离培养真菌32(32/63,50.8%)株,经ITS序列RFLP法鉴定为白假丝酵母菌31株,光滑假丝酵母菌1株。真菌阳性率与病理诊断成正相关(r=0.263,P=0.027),与性别、年龄、吸烟、饮酒、学历的相关性均无统计学意义(P0.05)。结果表明,胃黏膜寄居的真菌存在多样性,且真菌阳性率与病理损害程度存在相关性。     

Characterization of a new xylitol-producer Candida tropicalis strain

A xylitol-producer yeast isolated from corn silage and designated as ASM III was selected based on its outstanding biotechnological potential. When cultivated in batch culture mode and keeping the dissolved oxygen at 40% saturation, xylitol production was as high as 130 g l(-1) with a yield of 0.93 g xylitol g(-1) xylose consumed. A preliminary identification of the yeast was performed according to conventional fermentation and assimilation physiological tests. These studies were complemented by using molecular approaches based on PCR amplification, restriction-fragment length polymorphism analysis and sequencing of the rDNA segments: intergenic transcribed spacer (ITS) 1-5.8S rDNA-ITS 2, and D1/D2 domain of the 26S rRNA gene. Results from both the conventional protocols and the molecular characterization, and proper comparisons with the reference strains Candida tropicalis ATCC 20311 and NRRL Y-1367, led to the identification of the isolate as a new strain of C. tropicalis.     

Cyberlindnera fabianii: primer aislamiento clínico en Chile

BackgroundAlthough considered an unusual etiological agent, Cyberlindnera (Candida) fabianii has been related to septicemia in several reports in recent years. Its doubtful or uncertain identification when using tests such as CHROMagar Candida, API® Candida, API® ID32C or VITEK® MS, leads to an underestimation of the cases produced by this yeast.AimsTo report the first isolation of C. fabianii in Chile and its identification.MethodsThe sequencing of the internal transcribed spacer region (ITS) was performed. Antifungal susceptibility profiles were obtained by means of the broth microdilution technique.ResultsThe identification was only reached by sequencing the ITS regions, which shows the limited usefulness of the conventional techniques in the identification of some yeast species. A dendrogram shows the phylogenetic relationship of the isolated strain with some other yeast species.ConclusionIn the identification of fastidious microorganisms or microorganisms whose identification is not completely reliable when using classical or even advanced methodologies, such as mass spectrometry, sequencing techniques are essential.     

M-PCR： a powerful method for rapid molecular identification of Trichogramma wasps （Hymenoptera： Trichogrammatidae）

Two species-specific primers were designed depending on ITS2 sequence variation of 37 Trichogramma wasps, and these primers were applied to establish an assay,multiplex PCR (M-PCR), for molecular diagnosis of two important Trichogramma wasps,T. confusum and T. dendrolimi, in China. Multiplex-PCR results showed that only target species produced two PCR products, one product of ITS2 region species-specific amplification and one product of its ITS 1 region universal amplification, but other species produced only one ITS1 universal PCR product. Using this method, the target Trichogramma species can be distinguished from other Trichogramma species. Molecular identification based on M-PCR has particular value over morphological technology and other approaches, such as normal molecular and biochemical methods. Furthermore, because M-PCR assay can avoid false negative results, which frequently happen in PCR reaction, this method will be much more accurate and useful for Trichogramma identification, and can be developed as an easy and rapid diagnostic kit applied in the identification and quality monitoring of Trichogramma mass products both in the factory and in the field. Such an easy and rapid diagnostic kit will be valuable in the application of Trichogramma species as a biological control.     

Genotypes of Candida dubliniensis in clinical isolates

Amplification of specific sequences of the ITS1 and ITS2 regions and the intervening 5.8S rRNA gene has lead to the identification of four separate genotypes in Candida dubliniensis. Using primers specific for each genotype, we have studied the prevalence of these genotypes among 68 clinical isolates, mostly from Spanish patients infected by HIV. The majority of the isolates tested belonged to genotype 1 (97%), while only one isolate each from genotypes 2 (1.5%) and 3 (1.5%) were detected in the oral cavity of two patients with HIV infection.     

Identification of yeast species from orange fruit and juice by RFLP and sequence analysis of the 5.8S rRNA gene and the two internal transcribed spacers

Yeast isolates from orange fruit and juice in a spontaneous fermentation were identified and classified by two molecular techniques. The first was analysis of the restriction pattern generated from the polymerase chain reaction (PCR)-amplified 5.8S rRNA gene and the two internal transcribed spacers (ITS) using specific primers. The second technique was sequence analysis of the ITS regions using the same two primers. Nine different restriction profiles were obtained from the size of the PCR products and the restriction analyses with three endonucleases (CfoI, HaeIII and HinfI). These groups were identified as Candida tropicalis, Clavispora lusitaniae, Hanseniaspora uvarum, Pichia anomala, Pichia fermentans, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Saccharomyces unisporus, and Trichosporon asahii. Checking against identification according to morphological, physiological and biochemical traits corroborated this molecular identification. A total concordance was found in the identification with PCR-restriction fragment length polymorphism of the ITS region after analysing certified yeast strains from two different culture collections. Consequently, a rapid and reliable identification of the yeast populations was achieved by using molecular techniques.     

Molecular Identification of <Emphasis Type="Italic">Candida orthopsilosis</Emphasis> Isolated from Blood Culture

The incidence of candidemia and invasive candidiasis have increased markedly due to the increasing number of immunocompromised patients. There are five major medically important species of Candida with their frequency of isolation in the diminishing order namely Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata and Candida krusei. In addition, there are numerous other species of Candida which differ in their genetic makeup, virulence properties, drug susceptibilities and sugar assimilation capabilities. In this report, an unusual Candida species was isolated from the blood of two leukaemic patients. Conventional culture and biochemical tests identified the Candida species as C. parapsilosis. Using fungal-specific oligonucleotide primers ITS1 and ITS4, we managed to amplify the ribosomal RNA gene and its internal transcribed spacer region from the genomic DNA of these isolates. The PCR products were then purified and subjected to automated DNA sequencing using BLAST and CLUSTAL sequence analysis identified these isolates to be Candida orthopsilosis. Candida orthopsilosis is a new species recently identified in 2005, being morphologically indistinguishable from C. parapsilosis and was previously classified as a subspecies of C. parapsilosis. This report highlights the importance of complementing traditional culture and biochemical-based identification methods with DNA-based molecular assays such as PCR as the latter is more superior in terms of its discriminatory power and speed.     

Fluconazole-resistant pathogens Candida inconspicua and C. norvegensis: DNA sequence diversity of the rRNA intergenic spacer region, antifungal drug susceptibility, and extracellular enzyme production

The opportunistic fungal pathogens Candida inconspicua and C. norvegensis are very rarely isolated from patients and are resistant to fluconazole. We collected 38 strains of the two microorganisms isolated from Europe and Japan, and compared the polymorphism of the rRNA intergenic spacer (IGS) and internal transcribed spacer (ITS) regions, antifungal drug susceptibility, and extracellular enzyme production as a potential virulence factor. While the IGS sequences of C. norvegensis were not very divergent (more than 96.7% sequence similarity among the strains), those of C. inconspicua showed remarkable diversity, and were divided into four genotypes with three subtypes. In the ITS region, no variation was found in either species. Since the sequence similarity of the two species is approximately 70% at the ITS region, they are closely related phylogenetically. Fluconazole resistance was reconfirmed for the two microorganisms but they were susceptible to micafungin and amphotericin B. No strain of either species secreted aspartyl proteinase or phospholipase B. These results provide basal information for accurate identification, which is of benefit to global molecular epidemiological studies and facilitates our understanding of the medical mycological characteristics of C. inconspicua and C. norvegensis.     

Use of molecular methods in identification of Candida species and evaluation of fluconazole resistance

The aim of this study was to evaluate the use of one of the molecular typing methods such as PCR (polymerase chain reaction) following by RFLP (restriction fragment length polymorphism) analysis in the identification of Candida species and then to differentiate the identified azole susceptible and resistant Candida albicans strains by using AP-PCR (arbitrarily primed-polymerase chain reaction). The identification of Candida species by PCR and RFLP analysis was based on the size and primary structural variation of rDNA intergenic spacer regions (ITS). Forty-four clinical Candida isolates comprising 5 species were included to the study. The amplification products were digested individually with 3 different restriction enzymes: HaeIII, DdeI, and BfaI. All the isolates tested yielded the expected band patterns by PCR and RFLP analysis. The results obtained from this study demonstrate that Candida species can be differentiated as C. albicans and non-C. albicans strains only by using HaeIII restriction enzyme and BfaI maintains the differentiation of these non-C. albicans species. After identification Candida species with RFLP analysis, C. albicans strains were included to the AP-PCR test. By using AP-PCR, fluconazole susceptible and resistant strains were differentiated. Nine fluconazole susceptible and 24 fluconazole resistant C. albicans were included to the study. Fluconazole resistant strains had more bands when evaluating with the agarose gel electrophoresis but there were no specific discriminatory band patterns to warrant the differentiation of the resistance. The identification of Candida species with the amplification of intergenic spacer region and RFLP analysis is a practical, short, and a reliable method when comparing to the conventional time-consuming Candida species identification methods. The fluconazole susceptibility testing with AP-PCR seems to be a promising method but further studies must be performed for more specific results.     

Evaluation of Medium Components by Plackett-Burman Statistical Design for Lipase Production by Candida rugosa and Kinetic Modeling

Lipase production by Candida rugosa was carried out in submerged fermentation. Plackett-Burman statistical experimental design was applied to evaluate the fermentation medium components. The effect of twelve medium components was studied in sixteen experimental trials. Glucose, olive oil, peptone and FeCl3?6H2O were found to have more significance on lipase production by Candida rugosa. Maximum lipase activity of 3.8 u mL-1 was obtained at 50 h of fermentation period. The fermentation was carried out at optimized temperature of 30oC, initial pH of 6.8 and shaking speed of 120 r/min. Unstructured kinetic models were used to simulate the experimental data. Logistic model, Luedeking-Piret model and modified Luedeking-Piret model were found suitable to efficiently predict the cell mass, lipase production and glucose consumption respectively with high determination coefficient(R2). From the estimated values of the Luedeking-Piret kinetic model parameters, α and β, it was found that the lipase production by Candida rugosa is growth associated.     

金黄色葡萄球菌的检测方法优化及其在舟山市水产品加工厂中的污染状况调查

金黄色葡萄球菌(Staphylococcus aureus)是引起食物中毒的重要致病菌。为简化金黄色葡萄球菌复杂的检测方法,并了解舟山市水产品中该致病菌的污染状况,通过比较已公开的3套针对耐热核酸酶编码基因的PCR鉴定体系,根据特异性和灵敏性实验筛选了1套最优的针对金黄色葡萄球菌的PCR鉴定体系。基于这一体系和国家规定的标准方法,对舟山市水产品加工厂各个环节采集的120份样品中金黄色葡萄球菌的污染状况进行了调查。结果显示,120份样品中金黄色葡萄球菌的检出率为8.3%,PCR检测方法与国标法检出阳性率比较无显著差异,而且集中在原料和环境这两个环节检出金黄色葡萄球菌,在半成品和成品中却未检测到。由此,可以认为舟山市水产品加工厂的原料和环境中存在着一定的安全隐患,而成品相对较为安全。     

Development and validation of a molecular method for the diagnosis of medically important fungal infections

The increasing incidence of severe fungal infections highlights the need for rapid and precise identification methods in clinical mycology. The aim of this study was to develop and validate a culture-indipendent molecular approach that could allow the detection of fungal pathogens in clinical samples, with particular attention to the identification of drug-resistant Candida and Aspergillus species. A real-time multiplex PCR assay was developed using TaqMan probes specific for highly discriminating ITS sequences. In its multiplex format the assay showed a high specificity, clearly discriminating among different species, as well as a high sensitivity (20 CFU/1 mL sample), making it a potentially useful starting point for the development of a more complete molecular diagnostic assay.     

Kinetic modeling of lipase catalyzed hydrolysis of (R/S)-1-methoxy-2-propyl-acetate as a model reaction for production of chiral secondary alcohols

The Candida antarctica lipase B catalyzed kinetic resolution of (R/S)-1-methoxy-2-propyl-acetate was studied as a model system for the biocatalytic production of chiral secondary alcohols. For this purpose, a kinetic model is proposed involving both enantiomers of this reaction using model discrimination and parameter identification. Starting from a ping-pong bi-bi mechanism, a simplified model with sensitive parameters was derived for the R- and S-enantiomer, respectively. It was validated at pH 7.0, using time-course measurements at varying temperatures (30-60 degrees C) and initial substrate conditions (0.05-1.5 M). This model was then used for mechanistic interpretation of the kinetic resolution on a biochemical level. The effect of temperature on kinetic parameters and enantiomeric ratio was investigated and compared to findings from the field of molecular modeling to obtain a better understanding of the reaction system for process design. Values of 21.2 and 9.7 kJmol-1 were determined for the enthalpic (DeltaR-S DeltaH ++ degrees) and the entropic (-T x DeltaR-S DeltaS ++ degrees) contribution of the difference in transition state energy of both enantiomers at 30 degrees C. High enantiomeric ratio's (E of 47-110) especially at lower temperatures, in addition to enzyme activity at a wide pH range, indicate this biotransformation is a promising example for the industrial production of chiral secondary alcohols.     

Isolation of aCandida utilis variant by using a nitrogen-limited continuous culture

During continuous culture ofCandida utilis the appearance of a morphologic variant yeast was detected. The new microorganism developed systematically whenever it was changed from normal to stressed propagation conditions. A simple system was used for the isolation of the yeast variant, which was defective in cellular division and showed improved kinetic parameters and oxygen uptake rate. An asynchronic nitrogen-limited continuous culture ofCandida utilis allowed us to enrich the population in the chemostat with the modified yeast and isolate it in a defined medium. Assimilation and fermentation tests indicated it to be a variant ofCandida utilis that showed stable morphologic and physiologic differences with the parental yeast.Candida utilis growing in this nitrogen-limited continuous culture also showed a high mutation rate.     

罗布麻及其易混品的DNA分子鉴定

为从分子水平更准确地鉴别罗布麻及其易混品,本研究利用PCR产物直接测序法对罗布麻、大叶白麻和Apoacynum cannabinum的核基因组（rDNA）ITS区与叶绿体基因组（cpDNA）trnL内含子及trnL-F间隔区序列进行测序与比较。结果显示,罗布麻和大叶白麻ITS序列完全一致,与A. cannabinum在ITS1区有13个位点、在ITS2区有10个位点不同;在trnL内含子区及trnL-F间隔区,罗布麻和大叶白麻共有3个位点不同,罗布麻和A. cannabinum间共有23个位点、大叶白麻和A. cannabinum间共有20个位点不同。研究表明,依据rDNA ITS区序列可鉴别A. cannabinum和国产“罗布麻”（罗布麻与大叶白麻）;利用cpDNA trnL内含子及trnL-F间隔区序列可鉴别罗布麻及其相似种。     

一株甲基对硫磷降解菌-布朗克假丝酵母JMUPMD-1的分离与鉴定

对一株新分离的、能以甲基对硫磷为唯一磷源生长的菌株JMUPMD-1,利用形态特征观察和生理生化特性,及结合rDNA ITS分子序列分析对JMUPMD-1进行鉴定;采用气相色谱法检测培养过程中甲基对硫磷浓度的变化,确定甲基对硫磷降解速率。该菌株的rDNA ITS序列与布朗克假丝酵母（Candida blankii）的同源性为99％。形态特征和生理生化特性与布朗克假丝酵母相符合,因此鉴定为布朗克假丝酵母。以350μg／L甲基对硫磷为唯一磷源和350μg／L甲基对硫磷及1g／L K2HPO4组成的混合磷源培养该菌株,测得该菌株的降解率为48．6％。该菌株的胞内提取液具有明显的甲基对硫磷降解酶活性。     

Determination of the nucleotide sequence of the 23S ribosomal RNA and flanking spacers of an Enterococcus faecium strain, reveals insertion-deletion events in the ribosomal spacer 1 of enterococci

The usefulness of 16S-23S (ITS1) and 23S-5S (ITS2) ribosomal spacer nucleotide sequence determination, as a complementary approach to the biochemical tests traditionally used for enterococcal species identification, is shown by its application to the identification of a strain, E27, isolated from a natural bacteria mixture used for cheese production. Using combined approaches we showed, unambiguously, that strain E27 belongs to the Enterococcus faecium species. However, its ITS1 region has an interesting peculiarity. In our previous study of ITS1s from various enterococcal species (NAIMI et al., 1997, Microbiology 143, 823-834), the ITS1s of the two E. faecium strains studied, were found to contain an additional 115-nt long stem-loop structure as compared to the ITS1s of other enterococci, only one out of the 3 ITS1s of E. hirae ATCC 9790, was found to contain a similar 107-nt long stem-loop structure. The ITS1 of strain E27 is 100% identical to that of E. faecium ATCC 19434T, except that the 115-nt additional fragment is absent. This strongly suggests the existence of lateral DNA transfer or DNA recombination events at a hot spot position of the ITS1s from E. faecium and E. hirae. Small and large ITS1 nucleotide sequence determination for strain E27 generalized the notion of two kinds of ITSs in enterococci: one with a tRNA(Ala) gene, one without tRNA gene. To complete strain E27 characterization, its 23S rRNA sequence was established. This is the first complete 23S rRNA nucleotide sequence determined for an enterococcal species.