Biolog细菌自动鉴定系统应用初探

利用BiloogMicrostation细菌自动鉴定系统（3.50版）对已知的9个属23株菌进行了鉴定。24hBiolog系统鉴定结果：12林革兰氏阴性菌中，9株可准确鉴定到种的水平（75％），3株未达到属的水平。11株革兰氏阳性菌均属于芽孢杆菌属，全部鉴定到属的水平，9株准确鉴定到种的水平（81.8％）。总计属的水平准确率86.9％（20／23），种的水平准确率78.2％（18／23）。     

真菌还原Cr（VI）的研究

从不同来源的样品中分离筛选出几株抗Ｃｒ（ＶＩ）的真菌,他们能在含３００￣５００ｍｇ／ＬＫ２Ｃｒ２Ｏ７的蔗糖合成培养基中生长,其中ＢＳ－１菌株抗Ｋ２Ｃｒ２Ｏ７达９００ｍｇ／Ｌ．ＢＳ－１等４株真菌在含２００ｍｇ／Ｌ Ｋ２Ｃｒ２Ｏ７的培养基中生长４￣６ｄ后,培养液中的Ｃｒ（ＶＩ）已全部消失。这些真菌经鉴定为青霉菌ＢＳ－１和ＢＳ－３,黑曲霉ＢＲ－４和黄曲霉ＢＸ－１。经紫外可见光扫描及化学分析证实,高毒的Ｃ     

多孔微球固定化CHO工程细胞生产rt-PA的研究

以 Ｃｙｔｏｐｏｒｅ 多孔微球固定产重组组织型纤溶酶原激活剂（ｒｔ Ｐ Ａ） Ｃ Ｈ Ｏ 工程细胞株４ Ｂ３ ,在２ Ｌ 搅拌式生物反应器用无血清培养基 Ｄ Ｆ５ Ｓ 连续灌流培养。４ Ｂ３ 细胞的最大活细胞密度和ｒｔ Ｐ Ａ 生产水平分别达到８８３ ×１０６／ ｍ Ｌ 和１２４７３ Ｉ Ｕ／ ｍ Ｌ。含ｒｔ Ｐ Ａ 的４ Ｂ３ 细胞培养上清经 Ｍ Ｐ Ｇ 吸附层析和 Ｌｙｓｉｎｅｓｅｐｈａｒｏｓｅ ４ Ｂ 亲和层析两步纯化,ｒｔ Ｐ Ａ 的纯度达到９８ ％ 。     

一株产虾青素的黄杆菌CF-60的研究

从土壤中分离到一株黄杆菌（Ｆｌａｖｏｂａｃｔｅｒｉｕｍｓｐｐ）Ｃ_Ｆ－６０,该菌的生长需Ｍｇ^２＋存在,ＭｇＳＯ_４·７Ｈ_２Ｏ的最适浓度为０．２％;蛋白胨是该菌株生长的最好氮源,它不能利用无机氮。种龄超过９６ｈ的菌体不能在新鲜培养基中生长。经５４ｈ的２Ｌ恒化器发酵,生物量达６．８ｇ／Ｌ,色素产量为１０．６ｍｇ／Ｌ。该菌产生的类胡萝卜素成分简单,主要成分的含量为９０．３％,该成分经初步鉴定是分子结构中含有羰基和羟     

细菌还原Au3+制备金催化剂的研究

从不同来源的细菌菌株筛选获得一株吸附还原Ａｕ３＋较强的菌株Ｄ０１,经鉴定为巨大芽孢杆菌（Ｂａｃｉｌｕｓｍｅｇａｔｈｅｒｉｕｍ）Ｄ０１。菌株Ｄ０１在Ａｕ^３＋浓度６００ｍｇ／Ｌ下仍能较好生长。从电化学反应表明,该菌具有较强的还原力,它能将金催化剂的前驱体Ａｕ^３＋／αＦｅ２Ｏ３还原成具有催化ＣＯ＋Ｏ２→ＣＯ２的高分散度的Ａｕ０／αＦｅ２Ｏ３催化剂     

BIOLOG系统鉴定黄瓜根围促生菌的初步研究

对筛选出的８株具有明显促生防病作用的黄瓜根围促生菌ＣＮ１１,ＣＮ３１,ＣＮ４５,ＣＮ１ １６,ＣＮ１２９,ＸＢ１２０,ＸＢ５,ＸＢ４１通过ＢＩＯＬＯＧ法进行了分类鉴定,分别为：铜绿假单胞菌（Ｐｓｅｎｄｏｍｏｎａｓ ａｅｒｕｇｉｎｏｓａ）,波纹假单胞菌（Ｐ． Ｃｏｒｒｕｇａｔａ）,短芽孢杆菌（Ｂａｃｉｌｌｕｓ ｂｒｅｖｉｓ）,荧光假单胞菌Ｂ型（Ｐ． Ｆｌｕｏｒｅｓｃｅｎｓ ｔｙｐｅ Ｂ）,铜绿假单胞菌（Ｐ． Ａｅｒｕｇｉｎｏｓａ）,荧光假     

黑山晋枣芽培养及值株再生研究

以珍稀、优质黑山晋枣液芽为外植体建立了原种无性繁殖系。继代繁殖培养基（ｍｇ／Ｌ）：ＭＳ＋ＢＡ０．１＋ＩＡＡ０．１和ＭＳ＋ＢＡ０．１＋ＩＢＡ０．５,培养３０ｄ平均苗高３．０－３．３ｃｍ,芽繁殖系数３．３－３．５;生根培养基为ＭＳ＋ＩＡＡ０．１＋ＩＢＡ０．２及ＭＳ＋ＩＡＡ０．２＋ＩＢＡ０．２两种,生根率８３．３％,培养４０ｄ平均具根苗高达４．９９ｃｍ,一次成苗,简化程序,缩短成苗期。移载成活率８３．０     

高密度培养大肠杆菌YK537/pSBHL-11生产重组人细胞白介素

在 Ｂ． Ｂｒａｕｎ Ｅ Ｓ１０ 型１５ Ｌ 和 Ｎ Ｂ Ｓ Ｂｉｏ Ｆｌｏ ３０００ 型５ Ｌ 发酵罐中,利用补料分批培养技术高密度表达培养含重组质粒ｐ Ｓ Ｂ Ｈ Ｌ１１ 的大肠杆菌 Ｙ Ｋ５３７ ,生产重组人白细胞介素３（ Ｉ Ｌ３） ,发现在发酵过程中,限制性流加甘油,控制溶解氧在３０ ％ ～４０ ％ 左右、３０ ℃生长１１ｈ ,４２ ℃诱导培养４ｈ ,能将发酵液中最终菌体密度从 Ｏ Ｄ１６６００ 提高到 Ｏ Ｄ５３６００（ 相当于每升发酵液含１０６ 克湿菌体） ,并且保持了白细胞介素３ 的表达量,占菌体总蛋白的３０ ％ 左右,含量超过３３ ％ ｇ／ Ｌ,使 Ｉ Ｌ３ 包涵体产量从湿重２２ｇ／ Ｌ 提高到８５ ｇ／ Ｌ,纯化步骤比较简单,超声破菌后经两次洗涤纯度就达到７０ ％ 以上。     

芽孢杆菌M_(50)产生β甘露聚糖酶的条件研究

从土壤中分离到９ 株产生β甘露聚糖酶的芽孢杆菌（ Ｂａｃｉｌｌｕｓ ｓｐ ．） 。Ｂａｃｉｌｌｕｓｓｐ ． Ｍ５０２５０ｍ Ｌ三角瓶摇瓶培养试验,以４ ％ 的魔芋粉为碳源,１－０ ％ （ ＮＨ４）２ＳＯ４ 为氮源,０－３５ ％Ｎａ２ＣＯ３ ,３０ ～３４ ℃培养６０ｈ 产酶达到高峰。酶活力为１８０ ～２００ｕ／ｍ Ｌ。１００Ｌ 罐发酵,在３０ ～３２ ℃,１∶０ ．７５ｖｖｍ 通气量,２００ｒ／ｍｉｎ 条件下,发酵液酶活力高达３３０ｕ／ｍ Ｌ。酶的最适反应温度和ｐＨ 分别为５０ ℃和６－０ ,低于５０ ℃,ｐＨ５ ．０ ～７ ．０ 酶稳定。Ｆｅ^３＋ 、Ａｌ^３＋ 、ＥＤＴＡ、Ｈｇ^２＋ 对酶有抑制作用,而Ｂａ^２＋ 、Ｍｎ^２＋ 对酶有激活作用。发酵粗酶液对苎麻精干麻精练,显示对精干麻的半纤维素残胶具有降解作用。     

脂质体IFNr对DBA／2小鼠P388白血病细胞转移的影响

给ＤＢＡ／２小鼠双前肢足垫皮下注射２×１０Ｐ３８８细胞后,腹腔注入Ｌ－ＩＦＮｒ（脂质体包裹ＩＦＮｒ）或ＩＦＮｒ４,２０,１００×１０４Ｕ／（ｋｇ·ｄ）,连续１０ｄ,结果小鼠生存期分别延长１８．３、２９．３、３３．９、２３．９、３１;９、３３．６ｄ。在第１５天循环血中的单核细胞分别相应降低２５．３％、５４．９％,８９．７％和５５．８％、８５．２％、９０．２％;移植瘤引流区腋窝淋巴结肿大呈剂量依赖性缩小。结果表明两种ＩＦＮｒ制剂均有显著的抗白血病细胞Ｐ３８８转移的作用,Ｌ－ＩＦＮｒ的效果优于ＩＦＮｒ。     

Evaluation of the Biolog automated microbial identification system

Biolog's identification system was used to identify 39 American Type Culture Collection reference taxa and 45 gram-negative isolates from water samples. Of the reference strains, 98% were identified to genus level and 76% to species level within 4 to 24 h. Identification of some authentic strains of Enterobacter, Klebsiella, and Serratia was unreliable. A total of 93% of the water isolates were identified.     

Evaluation of the Biolog automated microbial identification system.

Biolog's identification system was used to identify 39 American Type Culture Collection reference taxa and 45 gram-negative isolates from water samples. Of the reference strains, 98% were identified to genus level and 76% to species level within 4 to 24 h. Identification of some authentic strains of Enterobacter, Klebsiella, and Serratia was unreliable. A total of 93% of the water isolates were identified.     

A Side by Side Comparison of Bruker Biotyper and VITEK MS: Utility of MALDI-TOF MS Technology for Microorganism Identification in a Public Health Reference Laboratory

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, highly accurate, and cost-effective method for routine identification of a wide range of microorganisms. We carried out a side by side comparative evaluation of the performance of Bruker Biotyper versus VITEK MS for identification of a large and diverse collection of microorganisms. Most difficult and/or unusual microorganisms, as well as commonly encountered microorganisms were selected, including Gram-positive and negative bacteria, mycobacteria, actinomycetes, yeasts and filamentous fungi. Six hundred forty two strains representing 159 genera and 441 species from clinical specimens previously identified at the Laboratoire de santé publique du Québec (LSPQ) by reference methods were retrospectively chosen for the study. They included 254 Gram-positive bacteria, 167 Gram-negative bacteria, 109 mycobacteria and aerobic actinomycetes and 112 yeasts and moulds. MALDI-TOF MS analyses were performed on both systems according to the manufacturer’s instructions. Of the 642 strains tested, the name of the genus and / or species of 572 strains were referenced in the Bruker database while 406 were present in the VITEK MS IVD database. The Biotyper correctly identified 494 (86.4%) of the strains, while the VITEK MS correctly identified 362 (92.3%) of the strains (excluding 14 mycobacteria that were not tested). Of the 70 strains not present in the Bruker database at the species level, the Biotyper correctly identified 10 (14.3%) to the genus level and 2 (2.9%) to the complex/group level. For 52 (74.2%) strains, we obtained no identification, and an incorrect identification was given for 6 (8.6%) strains. Of the 178 strains not present in the VITEK MS IVD database at the species level (excluding 71 untested mycobacteria and actinomycetes), the VITEK MS correctly identified 12 (6.8%) of the strains each to the genus and to the complex/group level. For 97 (54.5%) strains, no identification was given and for 69 (38.7%) strains, an incorrect identification was obtained. Our study demonstrates that both systems gave a high level (above 85%) of correct identification for a wide range of microorganisms. However, VITEK MS gave more misidentification when the microorganism analysed was not present in the database, compared to Bruker Biotyper. This should be taken into account when this technology is used alone for microorganism identification in a public health laboratory, where isolates received are often difficult to identify and/or unusual microorganisms.     

Evaluation of a biochemical test scheme for identifying clinical isolates of Enterococcus faecalis and Enterococcus faecium

AIMS: To evaluate the full test scheme of Facklam and Sahm (1995) for the identification of clinical enterococcal isolates to genus and species level. METHODS AND RESULTS: Fifty-nine clinical isolates, previously provisionally classed as enterococci on the basis of just four biochemical tests of Facklam and Sahm and one other test, were subjected to genus and species identification using the full identification scheme of Facklam and Sahm; 98% of these strains were confirmed to be enterococci and of these, 69% were identified as Enterococcus faecalis and 31% as Enterococcus faecium. Six tests in the scheme (out of 24) gave anomalous or unreliable results for some strains, and two gave unexpected results for the majority of strains presumptively identified as Ent. faecium. CONCLUSIONS: Nine (out of 12) genus tests and nine (out of 12) species tests from the Facklam and Sahm scheme were reliable. Testing for the presence of the Lancefield antigen D was also useful. The majority of presumptive Ent. faecium strains gave different results for the sorbitol and raffinose tests from that expected. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates the level of reliability for each of the tests in a current enterococcal identification scheme for differentiating clinical isolates, and showed that two tests gave consistently different test results from those expected for Ent. faecium.     

16S rRNA sequencing in routine bacterial identification: a 30-month experiment

Accurate identification of bacterial isolates is an essential task in clinical microbiology. Phenotypic methods are time-consuming and either fail to identify some bacteria such as Gram-positive rods entirely or at least fail to do so in some clinical situations. 16S rDNA sequencing is a recent method of identification which offers a useful alternative. In this study, we investigate the usefulness of this method for identifying a range of bacteria in a clinical laboratory under routine conditions. Over a period of 30 months, 683 isolates were obtained from clinical specimens, sequenced and analysed. For 568 of these isolates (83.1%), the sequence provided species level identification. For 108 isolates (15.8%), the identification was limited to the genus level, and for 7 isolates (1%), the sequence remained unidentifiable by 16S rDNA sequence analysis. For the isolates identified only to the genus level, the 16S rDNA approach failed to identify bacteria to the taxonomic level for 3 reasons: failure to differentiate between species in 72 isolates (66%), the lack of any closely related sequence in the database for 15 isolates (13.8%) and the presence of more than 1% of undetermined position in the sequence for 13 isolates (12%).     

Comparison of 16S rRNA sequencing with conventional and commercial phenotypic techniques for identification of enterococci from the marine environment

AIMS: To compare accuracy of genus and species level identification of presumptive enterococci isolates from the marine environment using conventional biochemical testing, four commercial identification systems and 16S rRNA sequence analysis. METHODS AND RESULTS: Ninety-seven environmental bacterial isolates identified as presumptive enterococci on mEI media were tested using conventional and Enterococcus genus screen biochemical tests, four commercial testing systems and 16S rRNA sequencing. Conventional and Enterococcus genus screen biochemical testing, 16S rRNA sequencing and two commercial test systems achieved an accuracy of > or = 94% for Enterococcus genus confirmation. Conventional biochemical testing and 16S rRNA sequencing achieved an accuracy of > or = 90% for species level identification. CONCLUSIONS: For confirmation of Enterococcus genus from mEI media, conventional or genus screen biochemical testing, 16S rRNA sequencing and the four commercial systems were correct 79-100% of the time. For speciation to an accuracy of 90% or better, either conventional biochemical testing or 16S rRNA sequencing is required. SIGNIFICANCE AND IMPACT OF THE STUDY: Accurate identification of presumptive environmental Enterococcus isolates to genus and species level is an integral part of laboratory quality assurance and further characterization of Enterococcus species from pollution incidents. This investigation determines the ability of six different methods to correctly identify environmental isolates.     

Identification and Antimicrobial Susceptibility Testing of <Emphasis Type="Italic">Staphylococcus vitulinus</Emphasis> by the BD Phoenix Automated Microbiology System

This study evaluated the performance of the BD Phoenix system for the identification (ID) and antimicrobial susceptibility testing (AST) of Staphylococcus vitulinus. Of the 10 S. vitulinus isolates included in the study, 2 were obtained from the Czech Collection of Microorganisms, 5 from the environment, 2 from human clinical samples, and 1 from an animal source. The results of conventional biochemical and molecular tests were used for the reference method for ID, while antimicrobial susceptibility testing performed in accordance with Clinical and Laboratory Standards Institute recommendations and PCR for the mecA gene were the reference for AST. Three isolates were incorrectly identified by the BD Phoenix system; one of these was incorrectly identified to the genus level, and two to the species level. The results of AST by the BD Phoenix system were in agreement with those by the reference method used. While the results of susceptibility testing compared favorably, the 70% accuracy of the Phoenix system for identification of this unusual staphylococcal species was not fully satisfactory.     

Identification of Nocardia species using matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry

Background

The MALDI (matrix-assisted laser desorption/ionization) Biotyper system for bacterial identification has already been utilized in clinical microbiology laboratories as a successful clinical application of protoemics. However, in cases of Nocardia, mass spectra suitable for MALDI Biotyper identification are often not obtained if such specimens are processed like general bacteria. This problem is related to the insufficiencies in bacterial spectrum databases that preclude accurate specimen identification. Here, we developed a bacterial processing method to improve mass spectra from specimens of the genus Nocardia. In addition, with the new processing method, we constructed a novel in-house bacterial database that combines a commercial database and mass spectra of Nocardia strains from the Department of Clinical Laboratory at Chiba University Hospital (DCLC) and the Medical Mycology Research Center at Chiba University (MMRC).

Results

The newly developed method (Nocardia Extraction Method at DCLC [NECLC]) based on ethanol-formic acid extraction (EFAE) improved mass spectra obtained from Nocardia specimens. The Nocardia in-house database at Chiba University Hospital (NDCUH) was then successfully validated. In brief, prior to introduction of the NECLC and NDCUH, 10 of 64 (15.6%) clinical isolates were identified at the species level and 16 isolates (25.0%) could only be identified at the genus level. In contrast, after the introduction, 58 isolates (90.6%) were identified at the species level and 6 isolates (9.4%) were identified at the genus level.

Conclusions

The results of this study suggest that MALDI-TOF (time-of-flight) Biotyper system can identify Nocardia accurately in a short time in combination with a simple processing method and an in-house database.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9078-5) contains supplementary material, which is available to authorized users.     

A Universal Method for the Identification of Bacteria Based on General PCR Primers

The Universal Method (UM) described here will allow the detection of any bacterial rDNA leading to the identification of that bacterium. The method should allow prompt and accurate identification of bacteria. The principle of the method is simple; when a pure PCR product of the 16S gene is obtained, sequenced, and aligned against bacterial DNA data base, then the bacterium can be identified. Confirmation of identity may follow. In this work, several general 16S primers were designed, mixed and applied successfully against 101 different bacterial isolates. One mixture, the Golden mixture7 (G7) detected all tested isolates (67/67). Other golden mixtures; G11, G10, G12, and G5 were useful as well. The overall sensitivity of the UM was 100% since all 101 isolates were detected yielding intended PCR amplicons. A selected PCR band from each of 40 isolates was sequenced and the bacterium identified to species or genus level using BLAST. The results of the UM were consistent with bacterial identities as validated with other identification methods; cultural, API 20E, API 20NE, or genera and species specific PCR primers. Bacteria identified in the study, covered 34 species distributed among 24 genera. The UM should allow the identification of species, genus, novel species or genera, variations within species, and detection of bacterial DNA in otherwise sterile samples such as blood, cerebrospinal fluid, manufactured products, medical supplies, cosmetics, and other samples. Applicability of the method to identifying members of bacterial communities is discussed. The approach itself can be applied to other taxa such as protists and nematodes.