Determination of biofilm production by Candida tropicalis isolated from hospitalized patients and its relation to cellular surface hydrophobicity,plastic adherence and filamentation ability

Candida tropicalis is an emerging virulent species. The aim of this study is to determine the biofilm‐forming ability of 29 strains of C. tropicalis isolated from inpatients, and to examine its relation with other virulence factors such as cellular surface hydrophobicity (CSH), immediate (15 min, IA) and late (24 h, LA) plastic adherence and filamentation ability. The study was performed in parallel using two incubation temperatures – 37 and 22 °C – to determine the effect of growth temperature variations on these pathogenic attributes of C. tropicalis. Biofilm formation (BF) was measured by optical density (OD) and by XTT reduction (XTT); Slime index (SI), which includes growth as a correction factor in BF, was calculated in both methods. All strains were hydrophobic and adherent – at 15 min and 24 h – at both temperatures, with higher values for 22 °C; the adhered basal yeast layer appears to be necessary to achieve subsequent development of biofilm. Filamentation ability varied from 76.2% of strains at 37 °C to 26.6% at 22 °C. All C. tropicalis strains were biofilm producers, with similar results obtained using OD determination and XTT measurement to evaluation methods; SI is useful when good growth is not presented. BF at 37 °C was similar at 24 h and 96 h incubation; conversely, at 22 °C, the highest number of biofilm‐producing strains was detected at 96 h. CSH is an important pathogenic factor which is involved in adherence, is influenced by the filamentation of yeast, and plays a critical role in BF. Copyright © 2013 John Wiley & Sons, Ltd.     

Assignment of most genes encoding major peroxisomal polypeptides to chromosomal band V of the asporogenic yeast Candida tropicalis

The peroxisomes of the asporogenic yeast Candida tropicalis contain about 20 major polypeptides (PXPs). We have isolated a number of genes encoding them; 11 POX genes encoded independent PXPs and three POY genes were likely to encode three other PXPs. To locate these genes on the chromosomes, chromosomes of C. tropicalis were separated by pulsed-field gel electrophoresis. Eight chromosomal bands were observed over the range of 1.0 Mbp (band 1) to 2.8 Mbp (band VIII); the genome size was estimated to be about 20 Mbp. Southern blot analysis showed that ten genes were on band V, three genes were on band IV, and the other gene was on band VI. Three genes gave hybridization signals of nearly equal intensity on two different chromosomal bands: POX6A and POX8B, on bands V and VII; and POX8A, on bands IV and VI. Ribosomal RNA genes also hybridized to two bands, VI and VII. Most genes assigned to only one band hybridized to two restriction fragments produced by either NotI or SfiI endonuclease. The results suggested that C. tropicalis was diploid and that restriction sites were conserved little between homologues. The three POX genes that were found on two chromosomal bands hybridized to not more than two restriction fragments, implying that the allelic genes were present on different chromosomal bands.     

响应面法优化不同型态热带假丝酵母发酵生产木糖醇的工艺

利用Design Expert软件对菌丝型和酵母型热带假丝酵母发酵生产木糖醇实验进行设计及结果分析,建立木糖和木糖醇浓度与4个关键因子(菌型、发酵温度、pH、初始木糖浓度)的二次多项式回归模型,并对模型进行解析。结果表明:菌丝型热带假丝酵母转化木糖为木糖醇的能力高于酵母型;升高发酵温度,有利于木糖转化为木糖醇,而pH升高对转化过程并没有明显促进;发酵液中初始木糖浓度与木糖转化率呈正相关关系;获得最佳发酵工艺条件为菌种采用菌丝型酵母,发酵温度37℃,pH8,初始木糖浓度60mg/mL,此时木糖醇浓度达到17.21mg/mL。     

富核糖核酸热带假丝酵母发酵培养基优化

以热带假丝酵母（Candida tropicalis）AY91009为试验菌株,以其核糖核酸（RNA）含量为目标,选取糖蜜为碳源,采用响应面法对其发酵培养基进行优化,建立酵母浸粉、NH4Cl和ZnSO4·7H2O的二次回归模型,确定培养基最佳配方为：糖蜜（30%含糖量）140 mL/L、酵母浸粉2.90%、NH4Cl 1.37%、NaH2PO4·2H2O 0.10%、MgSO4·7H2O 0.20%、FeSO4·7H2O 0.05%、ZnSO4·7H2O 0.10%。在此优化培养基中发酵培养12 h,RNA含量达到11.58%,比优化前提高了35.6%。20 L罐分批发酵试验结果表明,细胞干质量达到32.38 g/L,RNA含量为6.69%,总RNA含量为2.17 g/L,为后续的高密度发酵研究奠定了良好的基础。     

热带假丝酵母木糖还原酶的酶学性质研究

研究了从热带假丝酵母(Candida tropicalis)菌体中获得的木糖还原酶(XR)的酶学性质。实验结果证实,C.tropicalis的细胞浆粗提液经盐析、透析及阴离子交换柱层析后得到的酶液中木糖还原酶比酶活为9·3U/mg、最适酶反应pH为6·0、最适反应温度为35℃;以木糖为底物时,Km·Xyl为64·8mmol/L、Km·N·X为0·0622mmol/L;以阿拉伯糖为底物时,Km·Ara为172mmol/L、Km·N·A为0·0375mmol/L。Zn2+是木糖还原酶的激活剂,Fe3+为抑制剂。固定木糖为反应底物,分别以NADPH及NADH为辅酶测定酶活,实验结果显示该菌体中木糖还原酶的活性主要依赖于辅酶NADPH。     

高甾醇含量热带假丝酵母细胞的培养条件研究

以大豆油脱臭馏出物为唯一碳源,研究了高产甾醇的热带假丝酵母（Candida tropicalis）1253细胞的培养条件。研究发现,无机氮源培养使得菌体甾醇含量显著高于有机氮源培养。当尿素与牛肉膏复合作为氮源时,菌体甾醇含量最高（7.55%）。最佳培养基配方为：脱臭馏出物50.0 g/L、尿素1.0 g/L、牛肉膏1.0 g/L、甘氨酸0.02 g/L、十二烷基苯磺酸钠0.5 g/L、MgSO4 0.1 g/L、K2HPO4 0.2 g/L、KH2PO4 0.2 g/L。最适培养条件为：初始pH 7.0、摇床转速200 r/min。采用上述最适的发酵条件,按10%（V/V）接种量接入热带假丝酵母1253种子液,30 ℃摇床振荡培养96 h。发酵结束后,经过测定,酵母细胞中甾醇含量为8.83%。     

Effect of NaCl on the biofilm formation by foodborne pathogens

This study was designed to evaluate the effect of NaCl on the biofilm formation of Listeria monocytogenes, Staphylococcus aureus, Shigella boydii, and Salmonella Typhimurium. The biofilm cells were cultured in media containing different NaCl concentrations (0% to 10%) for 10 d of incubation at 37 °C using a 24-well polystyrene microtiter plate, collected by swabbing methods, and enumerated using plate count method. The attachment and detachment kinetic patterns were estimated according to the modified Gompertz model. The cell surface hydrophobicity and auto-aggregation were observed at different NaCl concentrations. Most strains showed 2 distinctive phases at lower than 6% NaCl, while the numbers of adhered cells gradually increased throughout the incubation period at 4% to 10% NaCl. At 0% NaCl, the numbers of adhered L. monocytogenes, S. aureus, S. boydii, and S. Typhimurium cells rapidly increased up to 7.04, 6.47, 6.39, and 7.27 log CFU/cm(2), respectively, within 4 d of incubation. The maximum growth rate (k(A)) and specific growth rate (μ(A)) of adherent pathogenic cells were decreased with increasing NaCl concentration. Noticeable decline in the numbers of adherent cells was observed at low concentration levels of NaCl (<2%). The adherence abilities of foodborne pathogens were influenced by the physicochemical surface properties. The hydrophobicity and auto-aggregation enhanced the biofilm formation during the incubation periods. Therefore, this study could provide useful information to better understand the adhesion and detachment capability of foodborne pathogens on food contact surfaces.     

Molecular cloning of the RPS0 gene from Candida tropicalis.

The Saccharomyces cerevisiae RPS0 A and B genes encode proteins essential for maturation of the 40S ribosomal subunit precursors. We have isolated a homologue of the RPS0 gene from Candida tropicalis, which we named CtRPS0. The C. tropicalis RPS0 encodes a protein of 261 amino acid residues with a predicted molecular weight of 28.65 kDa and an isoelectric point of 4.79. CtRps0p displays significant amino acid sequence homology with Rps0p from C. albicans, S. cerevisiae, Neurospora crassa, Schizosaccharomyces pombe, Pneumocystis carinii and higher organisms, such as human, mouse and rat. CtRPS0 on a high copy number vector can complement the lethal phenotype linked to the disruption of both RPS0 genes in S. cerevisiae. Southern blot analysis suggests that CtRPS0 is present at a single locus within the C. tropicalis genome.     

Cloning and heterologous expression of the NADPH cytochrome P450 oxidoreductase genes from an industrial dicarboxylic acid-producing Candida tropicalis

NADPH cytochrome P450 oxidoreductase (CPR) catalyses the transfer of electrons during P450-mediated oxidation, which plays an important role in the omega-oxidation pathway of Candida tropicalis. Two putative allelic genes, CPR-a and CPR-b, were cloned from the long chain dicarboxylic acid-producing Candida tropicalis 1230, using cassette PCR methods. Both the identified open reading frames predict the gene products of 679 amino acid residues. The deduced amino acid sequences of CPR-a and CPR-b are highly homologous to CPR genes from C. tropicalis ATCC 750 and Candida maltosa. Both genes were individually expressed in a cpr mutant of Saccharomyces cerevisiae with high CPR activities, in which only a small distinction was observed between recombinant CPR-a and CPR-b. Both CPR-a and CPR-b contain one CTG codon, which codes for serine (amino acid 50) in C. tropicalis rather than universal leucine. A mutated cDNA of CPR-a with a TCG codon instead of CTG codon was constructed and expressed, resulting in little increase in CPR activity. This indicates that the alteration of Ser-50 has little effect on functional expression of CPR. Furthermore, high ketoconazole sensitivity for the cpr mutant was complemented by heterologous expression of the cloned CPR-a or CPR-b.     

生长因子和乳化剂对热带假丝酵母产十二碳二元酸发酵的影响

通过液体摇瓶分批发酵,研究生长因子和乳化剂对热带假丝酵母(Candida tropicalis)利用正十二烷发酵产十二碳二元酸的影响,生长因子为多种氨基酸和维生素,乳化剂为吐温60和吐温80.研究结果表明,对产酸的促进作用依次为维生素B2,丝氨酸,维生素B1和脯氨酸.添加60μg/L维生素B2可维持较高产酸水平,添加1.0g/L丝氨酸比不添加丝氨酸的产酸量约提高30%.与不添加乳化剂相比,添加吐温的菌体产酸量明显提高,添加10g/L吐温60和吐温80的最大产酸量分别提高51.84%和48.14%.添加吐温60还能发酵缩短周期,添加20g/L吐温60的菌体产酸量最高可达81.63g/L.     

食品中金黄色葡萄球菌生物被膜形成基因分析及影响因素研究

分析金黄色葡萄球菌食品分离菌株生物被膜形成相关基因,研究茶多酚和乳酸链球菌素(Nisin)对不同菌株生长及生物被膜形成的影响。以实验室保存的16株金黄色葡萄球菌食品分离菌株为研究对象,采用聚合酶链式反应(PCR)检测14种生物被膜形成相关基因;通过微孔板法研究不同浓度茶多酚和Nisin对不同菌株生长的抑制作用,确定最小抑菌浓度(MIC);在此基础上进一步研究茶多酚和Nisin对生物被膜形成能力影响。结果表明:14种生物被膜形成相关基因中有10种被检出,其中cna、ebp S、fib和ica AD基因检出率为100%,sas C为87.5%、fnb A为68.75%、sas G为68.75%、clf B为68.75%、eno为62.5%,ica BC为56.25%;16株菌株中均不携带bbp、bap、clf A和fnb B基因。16株菌株均能形成生物被膜,但形成能力有差异,其中F23、F44、F58、F64、F71、F86、F107和F109等8株菌株为被膜强形成菌株,F7、F19、F40、F46、F60、F99、F101、F106等8株菌株为被膜弱形成菌株。茶多酚和Nisin对16株金黄色葡萄球菌食品分离菌株的MIC范围分别为0.1~0.2 g/L和0.125~1 g/L,茶多酚和Nisin在1/2MIC和1/4MIC浓度下对生物被膜形成抑制作用明显(p<0.05),且茶多酚抑制效果强于Nisin。本研究结果为食品中金黄色葡萄球菌生物被膜形成控制具有参考意义。      

产碱性脂肪酶热带假丝酵母LYSC-3的筛选、鉴定及发酵条件的优化

以橄榄油为唯一碳源,以溴钾酚紫为显色剂,采用琼脂平板法从富含油脂的土壤中筛选到一株产碱性脂肪酶野生型菌株LYSC-3。经形态学、生理生化和分子生物学的鉴定,确定菌株LYSC-3为热带假丝酵母(Candida tropicalis)。经发酵条件优化,菌株LYSC-3的最佳产碱性脂肪酶的发酵条件为:橄榄油10g·L-1、蛋白胨5g·L-1、蔗糖20g·L-1、(NH4)2SO40.5g·L-1、MgSO4·7H2O0.2g·L-1,K2HPO40.2g·L-1,pH8.0,35℃,150r·min-1摇床振荡培养3d,获得碱性脂肪酶最高酶活力达38.6U·mL-1。     

食用菌发酵液体外对白色念珠菌生长和生物被膜形成影响

该文研究食用菌液体发酵液体外对白色念珠菌（Candida albicans）生长及生物被膜形成的影响。采用纸片扩散法测定食用菌液体发酵液中白色念珠菌的抑菌活性;甲基四氮盐（XTT）减低法测定其对白色念珠菌生物被膜形成的影响。结果表明,各种食用菌发酵液对白色念珠菌均有不同程度的抑制作用,其中对发酵液抑菌作用从强到弱依次为香菇发酵液、鸡腿菇发酵液,抑菌圈直径分别为（2.967±0.160） mm、（2.433±0.214） mm。对白色念珠菌生物被膜形成的抑制作用强弱依次为鸡腿菇发酵液和香菇发酵液,其相对抑菌率分别为（39.25±1.87）%、（28.72±2.59）%。鸡腿菇和香菇均对白色念珠菌在培养12 h时形成的生物被膜的抑制作用最强,相对抑菌率分别达到（46.37±3.14）%和（42.86±3.38）%。香菇及鸡腿菇发酵液对白色念珠菌生长及其生物被膜的形成有一定的抑制作用,有进一步的开发价值。     

高产核酸的热带假丝酵母诱变选育及其发酵条件优化

以热带假丝酵母（Candida tropicalis）HY4-17为出发菌株,采用电子束辐射技术诱变,经过初筛、复筛及遗传稳定性试验选育核酸高产菌株,通过Plackett-Burman试验、最陡爬坡试验及响应面法优化培养基,并优化接种量和发酵时间以提高核酸产量。结果表明,获得一株生长快速,遗传稳定的突变菌株EBI-21,其核酸含量较出发菌株HY4-17提高了25%。最佳培养基组成为：糖蜜185 g/L,硫酸铵3 g/L,硫酸锌0.05 g/L,硫酸镁0.5 g/L,硫酸亚铁0.05 g/L,磷酸1 mL/L,pH值为4.5。在优化培养基及接种量8%,发酵时间12 h条件下,核酸产量达到1.73 g/L,较未优化前提高了80.21%,培养时间缩短了10 h。     

热带假丝酵母ctfat1p基因在吸收脂肪族化合物中的功能研究

以热带假丝酵母(Candida tropicalis)1798中的ctfat1p基因为研究对象,利用重叠PCR将ctfat1p基因内约500 bp片段与G418抗性基因(kanr)相连接,经末端单酶切后电转化至C. tropicalis 1798感受态细胞中,通过一次同源单交换,将抗性基因kanr插入至ctfat1p基因内部,实现目的基因的敲除,并通过发酵实验分析Ctfat1p在热带假丝酵母脂肪酸跨膜转运过程中的功能。结果表明,经过G418抗性筛选和基因组PCR鉴定,成功获得ctfat1p基因缺失菌株C. tropicalis 1798 Δctfat1p;分析发现ctfat1p基因敲除对C. tropicalis 1798以油脂为底物培养12 h后重组菌OD600 nm值仅为野生型菌株的47.9%,表明ctfat1p基因敲除后影响C. tropicalis 1798对油脂吸收利用。通过基因敲除手段构建ctfat1p基因缺失菌株,可以减弱细胞对长链脂肪酸的摄取,验证了ctfat1p基因为热带假丝酵母油脂吸收和利用的关键基因。     

Inactivation of Cronobacter malonaticus cells and inhibition of its biofilm formation exposed to hydrogen peroxide stress

Presence of Cronobacter malonaticus in powdered infant formula (PIF) poses a high risk to infant and public health. Cronobacter malonaticus has been widely distributed in food and food processing environments, and the true origin of C. malonaticus in PIF is poorly understood. Control and prevention of C. malonaticus is necessary for achieving microbial safety of PIF. However, little information about decontamination of C. malonaticus is available. In this study, effects of hydrogen peroxide on inactivation and morphological changes of C. malonaticus cells were determined. Furthermore, inhibitory effects of H₂O₂ on biofilm formation in C. malonaticus were also performed. Results indicated that H₂O₂ could completely inactivate C. malonaticus in sterile water with 0.06% H₂O₂ for 25 min, 0.08% H₂O₂ for 15 min, and 0.10% for 10 min, respectively, whereas the survival rates of C. malonaticus in tryptic soy broth medium significantly increased with the same treatment time and concentration of H₂O₂. In addition, morphological changes of C. malonaticus cells, including cell shrinkage, disruption of cells, cell intercession, and leakage of intercellular material in sterile water after H₂O₂ treatment, were more predominant than those in tryptic soy broth. Finally, significant reduction in biofilm formation by H₂O₂ was found using crystal violet staining, scanning electron microscopy, and confocal laser scanning microscopy detection compared with control samples. This is the first report to determine the effects of H₂O₂ on C. malonaticus cells and biofilm formation. The findings provided valuable information for practical application of H₂O₂ for decontamination of C. malonaticus in dairy processing.     

A novel assay of biofilm antifungal activity reveals that amphotericin B and caspofungin lyse Candida albicans cells in biofilms

The ability of Candida albicans to form drug-resistant biofilms is an important factor in its contribution to human disease. Assays to identify and characterize molecules with activity against fungal biofilms are crucial for the development of drugs with improved anti-biofilm activity. Here we report the application of an adenylate kinase (AK)-based cytotoxicity assay of fungal cell lysis to the characterization of agents active against C. albicans biofilms. We have developed three protocols for the AK assay. The first measures AK activity in the supernatants of biofilms treated with antifungal drugs and can be performed in parallel with a standard 2,3-bis-(2-methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-caboxanilide-based biofilm susceptibility assay; a second, more sensitive protocol measures the AK activity present within the biofilm matrix; and a third procedure allows the direct visualization of lytic activity toward biofilms formed on catheter material. Amphotericin B and caspofungin, the two most effective anti-biofilm drugs currently used to treat fungal infections, both directly lyse planktonic C. albicans cells in vitro, leading to the release of AK into the culture medium. These studies serve to validate the AK-based lysis assay as a useful addition to the methods for the characterization of antifungal agents active toward biofilms and provide insights into the mode of action of amphotericin B and caspofungin against C. albicans biofilms.     

Predominant localization of non-specific lipid-transfer protein of the yeast Candida tropicalis in the matrix of peroxisomes

PXP-18 is a 14-kDa major peroxisomal protein of the yeast Candida tropicalis and a homologue of the non-specific lipid-transfer protein (nsLTP) of mammals. Mammalian nsLTP is thought to facilitate the contact of membranes, to stimulate lipid-transfer between them. If PXP-18 functions like nsLTP, it must be present on organelle membranes. Immunoelectron microscopy of C. tropicalis cells indicated that gold particles, which visualized PXP-18, localized exclusively in the matrix of peroxisomes. Subcellular fractionation followed by Western blotting revealed the association of PXP-18 with peroxisomes in C. tropicalis cells. An enzyme-linked immunosorbent assay revealed that almost all the PXP-18 associated with peroxisomes was detectable after the solubilization of the organelle but not before, implying the predominance of PXP-18 inside peroxisomes. This differential assay was applied to the intracellular import of the intact and truncated PXP-18s expressed in Saccharomyces cerevisiae cells. Most of the intact PXP-18 was shown to be imported into the matrix of host-cell peroxisomes, whereas the truncated PXP-18, which lacked the C-terminal tripeptide Pro-Lys-Leu, no longer targeted peroxisomes. These results are consistent with the view that PXP-18 is the matrix protein of peroxisomes and must function in a system other than that of lipid transfer.     

Effects of tolC on tolerance to bile salts and biofilm formation in Cronobacter malonaticus

Bile salts is one of essential components of bile secreted into the intestine to confer antibacterial protection. Cronobacter species are associated with necrotizing enterocolitis in newborns and show a strong tolerance to bile salts. However, little attempt has been made to focus on the molecular basis of the tolerance to bile salts. In this study, we investigated the roles of tolC on growth, cell morphology, motility, and biofilm formation ability in Cronobacter malonaticus under bile salt stress. The results indicated that the absence of tolC significantly affected the colony morphology and outer membrane structure in a normal situation, compared with those of the wild type strain. The deletion of tolC caused the decline in resistance to bile salt stress, inhibition of growth, and observable reduction in relative growth rate and motility. Moreover, the bacterial stress response promoted the biofilm formation ability of the mutant strain. The expression of the AcrAB-TolC system (acrA, acrB, and tolC) was effectively upregulated compared with the control sample when exposed to different bile salt concentrations. The findings provide valuable information for deeply understanding molecular mechanisms about the roles of tolC under bile salt stress and the prevention and control of C. malonaticus.     

Emodin,a natural inhibitor of protein kinase CK2, suppresses growth,hyphal development,and biofilm formation of Candida albicans

Emodin (1,3,8‐trihydroxy‐6‐methyl‐anthraquinone) is a natural secondary plant product, originally isolated from the rhizomes of Rheum palmatum. Many reports show its diuretic, vasorelaxant, antibacterial, antiviral, anti‐ulcerogenic, immunosuppressive, hepatoprotective, anti‐inflammatory and anticancer potential. Emodin is a pleiotropic molecule capable of interacting with several major molecular targets, e.g. NF‐κB, AKT/mTOR and STAT3. The compound can also act as an inhibitor of some protein kinases, with special affinity to protein kinase CK2. The aim of the presented report was to evaluate antifungal properties of emodin and its activity towards CK2 isolated from Candida cells. Our studies revealed that the compound suppressed growth of the cells of reference strains as well as clinical Candida strains, with minimal inhibitory concentration and minimal fungicidal concentration values between 12.5 and 200 μg/mL. Moreover, at a low concentration, the compound was able to effectively stop hyphal formation, thus showing a distinct antivirulent potential. Interestingly, we showed that emodin added to Candida culture inhibited the phosphorylation of many cellular proteins, presumably owing to the inhibition of protein kinase CK2. Notably, the enzyme isolated from the Candida cells was susceptible to emodin with IC₅₀ of 2.8 μg/mL. Indeed, our computational modelling revealed that emodin was able to occupy the ATP‐binding pocket of CK2. Copyright © 2017 John Wiley & Sons, Ltd.