高灵敏假单胞菌铁载体的平板检测方法*

CAS蓝色检测平板是一种筛选、检测各类细菌铁载体的常用方法,而蔗糖-天冬酰氨培养基被用于假单胞菌产铁载体规律的研究。用天冬氨酸替代天冬酰氨,将CAS蓝色检测液与蔗糖-天冬氨酸培养基(MSA培养基)相结合,得到一种改进的MSA-CAS检测平板。通过对假单胞菌属7个种8个株进行荧光与非荧光铁载体检测方面的比较研究,结果表明MSA-CAS检测平板假单胞菌铁载体的检测灵敏度比通用CAS检测平板高,而且在检测荧光铁载体方面具有荧光背景低、荧光铁载体晕圈明显和晕圈与背景的对比度大的优点。     

假单胞菌荧光与非荧光铁载体对铁离子的应答差异

假单胞菌既能产荧光铁载体也能产非荧光铁载体.通过对假单胞菌在不同铁离子浓度下,在通用CAS(Chrome azroul S)检测平板、改进的蔗糖-天冬氨酸(SA)平板(MSA)上以及通用液体CAS培养基和MSA培养基内的铁载体产生情况的比较,发现在通用CAS的液体培养基上产生的主要为非荧光铁载体(pyochelin),而在改进的MSA培养基上产生的主要为荧光铁载体(pyoverdine);在铁离子的应答方面,pyoverdine较pyochelin灵敏,较低的铁离子浓度即可抑制荧光铁载体的产生,但是不能抑制非荧光铁载体.     

双层平板法检测嗜盐古菌铁载体

采用双层平板法应用于嗜盐古菌铁载体的原位检测。双层平板的上层为不添加铁离子的嗜盐古菌培养基, 嗜盐古菌可在其上生长, 在缺铁胁迫下可向外界分泌铁载体; 下层为含有CAS检测液用于铁载体检测的琼脂。当上层平板生长的嗜盐古菌分泌的铁载体透过培养基渗透到下层检测琼脂后, 即可在下层检测平板上产生明显的特征性的铁载体螯合晕圈, 表明双层平板法在嗜盐古菌的铁载体检测中确实可行, 且较原有的嗜盐古菌铁载体检测方法简便、直接。     

双层平板法检测嗜盐古菌铁载体

采用双层平板法应用于嗜盐古菌铁载体的原位检测.双层平板的上层为不添加铁离子的嗜盐古菌培养基,嗜盐古菌可在其上生长,在缺铁胁迫下可向外界分泌铁载体;下层为含有CAS检测液用于铁载体检测的琼脂.当上层平板生长的嗜盐古菌分泌的铁载体透过培养基渗透到下层检测琼脂后,即可在下层检测平板上产生明显的特征性的铁载体螯合晕圈,表明双层平板法在嗜盐古菌的铁载体检测中确实可行,且较原有的嗜盐古菌铁载体检测方法简便、直接.     

CAS平板覆盖法检测氢氧化细菌铁载体

【目的】用CAS平板覆盖法检测氢氧化细菌铁载体,解决通用CAS琼脂平板法中十六烷基三甲基溴化铵对真菌和某些细菌的生长抑制问题。【方法】将改良的CAS检测培养基覆盖在长满菌落的无铁培养基上,生长抑制问题因微生物未与十六烷基三甲基溴化铵直接接触而解决。【结果】3株氢氧化细菌SDW-5、SDW-9和AaP-13均能产生单菌落,加入CAS检测培养基1 h后,菌落周围产生明显的铁载体晕圈。【结论】本方法成功解决了生长抑制问题,可以作为检测微生物铁载体的通用方法。     

一株花生根际铁载体产生菌的分离鉴定及耐药性分析

目的：从花生根际筛选铁载体产生能力较强的菌株,对其进行鉴定及耐药性分析。方法：用梯度稀释法从花生根际中分离出细菌,在刃天青（CAS）检测平板上依显色圈的大小从中筛选出一株铁载体产生能力较强的菌,并对其进行生理生化鉴定和16S rDNA序列分析,用抗生素梯度平板检测其对9种常见抗生素的抗性。结果：筛选出一株产铁载体的菌株D15,13项生理生化指标除甲基红试验呈阳性外,其他均与恶臭假单胞菌相同;其16S rDNA与恶臭假单胞菌的同源性为100%;该菌对氨苄青霉素、氯霉素、利福平、红霉素、新霉素、链霉素、四环素都有不同程度的的抗性,对卡那霉素和庆大霉素表现较强的敏感性。结论：获得铁载体产生菌D15,经鉴定为恶臭假单胞菌,该菌耐药性符合用转座子诱变法研究铁载体合成的相关基因的条件。     

一株棉花根际铁载体产生菌E19的分离鉴定

目的：从棉花根际细菌中筛选出铁载体产生能力较强的菌株并对其进行鉴定。方法：用梯度稀释法从棉花根际中分离出细菌,再通过在CAS检测平板上显色圈的大小从中筛选出铁载体产生能力较强的菌株,并对其进行生理生化鉴定和16S rDNA序列分析。结果：筛选出一株铁载体产生能力较强的菌株E19,13项生理生化指标除甲基红试验呈阳性外,其它均与恶臭假单胞菌相同。其16S rDNA与恶臭假单胞菌（Pseudomonas putida）的同源性为100％。     

小麦根圈细菌铁载体的检测

本文报道微生物铁载体定性与定量测定的方法,以及小麦根圈细菌铁载体的室内检测结果。２２株小麦根圈耐寒细菌和２株固氮菌在定性检测平板上产生铁载体,另有３株固氮菌不产。定量测定结果表明：３６株水溶性色素产生菌合成铁载体的量在０．７１５－０．１０６（Ａ／Ａｒ）之间,其中属于荧光假单胞菌群的２５个菌株铁载体产量的测定值（Ａ／Ａｒ）在０．１８４－０．１０６之间,达极高量水平。     

高产铁载体荧光假单胞菌Pseudomonas fluorescens sp-f的筛选鉴定及其铁载体特性研究

采用改进的CAS检测平板从东湖中筛选得到了一株高产铁载体细菌sp-f,并用CAS检测液定量检测其分泌铁载体量,发现其As/Ar仅0.09(OD680),Su(Siderophore Unit)为90%,达到产铁载体菌最高级。用BIOLOG检测板,结合细菌生理生化反应、形态观察和16S rDNA序列比对分析等分类鉴定方法,确定sp-f为一株荧光假单胞菌。P.fluorescenssp-f生长过程中胞外铁载体的量在对数生长前期累积达到最高后有所减少,至稳定期时菌液中铁载体量达到稳定。在已知铁载体特异吸收峰波长下,用反向高效液相色谱检测无铁环境和高铁环境下培养液上清,比较发现sp-f上清含有3种含儿茶酚胺类基团铁载体,其中包括荧光和非荧光性的脓菌素,200μmol/L Fe2 可完全抑制荧光性质脓菌素的分泌,但非荧光脓菌素的分泌不受抑制,并且对非脓菌素的儿茶酚胺类铁载体的合成分泌反而具有一定的诱导作用。     

Pseudomonas fluorescens sp-f铁载体高效液相凝胶色谱分析

利用高效液相凝胶色谱法对荧光假单胞菌所产铁载体进行了分析,结果显示高产铁载体P.fluorescens sp-f与P.fluorescens AB92001均可分泌3种铁载体,其中具有荧光的pyoverdine容易被细胞外的铁抑制。高效液相凝胶色谱法适用于假单胞菌铁载体的分析检测。     

适合高产铁载体细菌筛选、检测体系的改进与探析

采用pH6．8的磷酸缓冲液代替通用CAS固体检测培养基中的MM9缓冲体系,不加哌嗪二乙醇磺酸,得到颜色稳定、检测效果明显、配制方便,适合高产铁载体细菌筛选与检测的新型检测平板。根据CAS检测液连续吸收光谱比较分析结果,将CAS液体定量检测体系中的检测波长由630nm改为680nm,可以克服OD630读数偏高、易受干扰的问题,而且OD680与铁载体浓度线性关系不变,但采用OD680检测不同细菌产铁载体能力的差异更为明显,因此提高了CAS定量检测的灵敏度,更适合高产铁载体细菌筛选与检测。     

CAS agar diffusion assay for the measurement of siderophores in biological fluids

We developed a simple and universal method, by modifying the universal CAS (Chrome azurol S) assay, measuring siderophores in various biological fluids. We named the assay as CAS agar diffusion (CASAD) assay. CAS plate devoid of nutrients was prepared by using Bacto-agar (1.5%, w/v) as a matrix. Holes with 5-mm-diameter were punched on the CAS agar plate. Each hole was added by 35 microl of the test fluids containing Desferal that was twofold serially diluted. After incubating at 37 degrees C or room temperature for 4-8 h, the size of orange haloes formed around the holes was measured. The size of orange haloes correlated well with the concentration of Desferal in all the biological fluids tested in this study. CASAD assay showed consistent results in wide pH range from 5 to 9. Addition of iron to the test fluids containing Desferal decreased the size of orange haloes in a dose-dependent manner, which suggests that the CASAD assay detects only iron non-bound siderophore. These results suggest that CASAD assay would serve as a simple, stable, and highly reproducible test for screening and quantitative siderophore analysis in biological fluids.     

Detection of siderophores in growing cultures ofPseudomonas spp.

Summary The siderophores produced byPseudomonas fluorescens andP. chlororaphis were detected from the culture supernatants in MM9 and modified King's medium by the universal CAS assay at wavelengths 620–690 nm. The CAS assay was applied to detectPseudomonas siderophores directly in situ, during their production phase, in modified King's medium. Optimum results were detected with a final CAS concentration of 0.025 mM and an iron concentration of 1.25 M. The problems of the method are discussed with respect to the absorbance spectrum, the toxicity of the HDTMA detergent, the influence of the iron concentration and the complexity of media for siderophore production.     

Use of CAS-agar plate modified to study the effect of different variables on the siderophore production by Aspergillus

AIMS: To evaluate the suitability of chrome azurol S (CAS) agar plate assay as a quantitative methodology for siderophore production. METHODS AND RESULTS: Aspergillus species (A. flavus, A. niger, A. tamarii) were inoculated in the CAS-agar plates and the siderophores production was determined and expressed as CAS-reaction rate (mm per day). All the species showed positive CAS reaction with different rates depending on culture conditions and A. flavus showed the highest CAS-reaction rate. The siderophore production in solid medium expressed as CAS-reaction rate was correlated with siderophore production in liquid medium. CONCLUSIONS: The use of CAS-agar plate assay was modified and the evaluation of CAS reaction in mm per day made it possible to study and quantify the effect of several variables on the siderophore production by Aspergillus fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: We describe the CAS-agar plate assay as a quantitative methodology, which make it possible to select and evaluate the siderophore production by several microorganisms (fungi and bacteria) according to different culture conditions.     

Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay.

A well-known and widely used method for detection of siderophore production by microorganisms in solid medium is the universal chrome azurol S (CAS)-agar plate assay. However, the high toxicity of CAS-blue agar medium caused by the presence of a detergent impedes its utilization with many varieties of fungi and Gram-positive bacteria. To solve this problem, a modification of the CAS-agar plate assay was made by incorporating the CAS-blue dye in a medium with no contact with the microorganisms tested. Half of each plate used in our experiments was filled with the most appropriate culture medium for each type of microorganism and the other half with CAS-blue agar. This modification allowed us to study several strains of fungi (basidiomycetes, deuteromycetes, ascomycetes and zygomycetes) and bacteria (Gram positive and negative), some of them appearing for the first time in the literature. All the microorganisms grew properly and reacted in different manners to the CAS assay. Some strains of wood-decaying basidiomycetes (mainly white-rot fungi) and Aspergillus species produced the fastest color-change reactions in the CAS-blue agar. This modified method could facilitate optimization of culture conditions, since both CAS-blue agar and growth medium were prepared and added in the Petri plate separately.     

Iron requirement and siderophore production in Rhizobium ciceri during growth on an iron-deficient medium

Under conditions of iron limitation many rhizospheric bacteria produce siderophores, ferric iron-specific ligands, which may enhance plant growth by increasing the availability of iron near the roots. Thirty-five strains of Rhizobium ciceri, specific to chickpea (Cicer arietinum L.), were screened for their ability to grow on iron-deficient medium and to produce siderophores. Maximal growth of all strains previously depleted in iron was obtained in medium containing 5 to 10 m of ferric iron. When iron limitation was achieved by the addition of 2,2-bipyridyl or EDDHA [ethylene diamine di(o-hydroxyphenyl) acetic acid] to the medium, only two strains were able to scavenge iron and grow. Siderophore production by these two strains was detected by the Chrome Azurol S assay (CAS), a universal test for siderophores. No hydroxamate-type siderophores were detected in the supernatants of Rhizobium ciceri cultures. However, some strains secreted salicylic acid and 2,3-dihydroxybenzoic acid as phenolate-type siderophores. Addition of ferric iron to the culture medium increased growth yield significantly but depressed the production of siderophores. Although these compounds are produced in response to iron deficiency, nutritive components of the culture medium significantly affected their production. It seems that CuII, MoVI and MnII ions bound competitively with iron to siderophores, resulting in a 34 to 100% increase in production.     

Siderophores of halophilic archaea and their chemical characterization

Nine halophilic archaea viz., Halobacterium salinarum, Halobacterium sp.1, Halobacterium sp.2, Halobaculum sp., Halococcus saccharolyticus, Halorubrum saccharovorum, Haloterrigena turkmenica, Halogeometricum sp. and Natrialba sp. isolated from marine salterns around Bhavnagar coast were screened for siderophore production. Five isolates viz., Halococcus saccharolyticus, Halorubrum saccharovorum, Haloterrigena turkmenica, Halogeometricum sp. and Natrialba sp. produced siderophores as evidenced by positive reaction in FeCl3 test, CAS assay and CAS agar plate test. Determination of chemical nature of siderophores by chemical assays and bioassays identified them as carboxylates. Quantification of siderophores indicated Halorubrum saccharovorum to be the maximum siderophore producer (2.62 RE mg/ml) and Halococcus saccharolyticus to be the least (1.33 RE mg/ml). The present study is the first report on siderophore production in Indian haloarchaeal strains. Mechanism of iron assimilation in four non-siderophore isolates still needs to be investigated further.