革兰阴性菌引起的血培养阳性标本直接细菌鉴定和药敏的应用

目的 评估血培养阳性标本直接细菌鉴定和药敏可行性.方法 将血培养瓶放入Bact/Alert 3D 60血培养系统进行培养筛选.选取78份含革兰阴性杆菌的阳性血培养瓶进行试验.抽取培养液,用BD真空分离管离心血细胞.在收集到足量菌液后,用VITEK-32革兰阴性菌鉴定药敏卡做直接鉴定药敏试验.用标准方法及亚培养后的鉴定药敏试验对直接鉴定药敏试验进行评估.结果 VITEK-32直接鉴定试验,78株中的74株(94.9％)准确鉴定,直接药敏试验标准符合率95.6％.KB法血标本直接药敏试验标准符合率96.2％,但微小错误率高于VITEK-32直接药敏法.结论 Bact/Alert血培养阳性标本直接VITEK-32细菌鉴定和药敏对革兰阴性菌是切实可行的,可大幅度缩短时间,为临床及时修正用药提供依据,具有较高的应用价值.     

血培养三级报告的临床应用价值

目的探讨血培养三级报告对临床诊疗菌血症的指导和应用价值。方法对我院2015年1月至9月237份阳性血液标本进行三级报告,分析一级报告时间和准确性,比较直接药敏(二级报告)与仪器法(三级报告)药敏结果的二者符合率。结果 237份血培养阳性标本,共检出革兰阳性菌105例,革兰阴性菌126列,真菌6例。一级报告时间平均为29.5h,准确率为97.89%(232/237)。革兰阳性葡萄球菌直接药敏与仪器法药敏的平均符合率为93.45%,严重错误率为0.11%,重大错误率为1.48%,一般错误率为4.97%,革兰阴性菌直接药敏与仪器法药敏的平均符合率为90.87%,严重错误率为0.32%,重大错误率为2.38%,一般错误率为6.43%,总体符合率为91.92%。结论血培养阳性标本三级报告的建立,为临床诊疗提供了准确和快速的结果,具有较大的临床应用价值。     

239株脑脊液标本非重复分离株病原学分布与药物敏感试验结果分析

目的通过回顾性分析脑脊液培养结果的病原菌分布与药物敏感试验结果,分析中枢神经系统感染相关细菌的耐药性及其变化趋势。方法选取西安交通大学医学院第一附属医院2012年1月至2014年9月期间从临床患者脑脊液标本中分离培养出的细菌,所有菌株均采用全自动微生物分析仪进行鉴定,仪器法与纸片法检测其对抗生素的敏感性。用WHONET 5.6软件进行数据统计分析。结果本研究共包含非重复分离菌株239株,其中革兰阳性菌175株(74%),革兰阴性菌59株(24%),真菌5株(2%)。革兰阳性菌主要为葡萄球菌属、肠球菌属与链球菌属细菌,其中表皮葡萄球菌最为常见。革兰阴性菌主要为肠杆菌科细菌与非发酵革兰阴性杆菌,其中鲍曼不动杆菌、肺炎克雷伯杆菌、大肠埃希菌最为常见。真菌均为新型隐球菌。分离菌株中革兰阳性菌与革兰阴性菌均有不同程度的耐药现象,革兰阳性菌仅对利奈唑胺、替考拉宁、万古霉素敏感率为100%,革兰阴性菌中肠杆菌仅对阿米卡星、美洛培南、哌拉西林/他唑巴坦、亚胺培南敏感率为90%以上,而鲍曼不动杆菌对各类抗生素耐药率均在80%以上。结论中枢神经系统感染以革兰阳性菌为主,表皮葡萄球菌为最常见的分离菌株。多重耐药菌的出现,使中枢神经系统感染的治疗面临巨大挑战,提示临床合理使用抗生素进行抗感染治疗至关重要。     

成人血流感染病原菌的分布及耐药性分析

目的了解成人血流感染病原菌的分布及耐药情况,为临床合理使用抗菌药物提供科学依据。方法采用Bact/Alert 3D血培养仪和VITEK-2 Compact鉴定仪,进行病原菌分离培养、鉴定和药敏试验。结果3 595份血培养送检标本中分离出230株病原菌,其中革兰阴性菌147株占63.91%,革兰阳性菌78株占33.91%,真菌5株占2.17%。革兰阴性菌以大肠埃希菌和肺炎克雷伯杆菌为主,对阿米卡星、碳青霉烯类药物敏感率高。革兰阳性菌以金黄色葡萄球菌和表皮葡萄球菌为主,对万古霉素、利奈唑胺保持高敏感率。结论本院血培养病原菌以革兰阴性菌为主,菌种复杂多样,不同菌种耐药性差异较大,应该定期对病原菌分布和耐药情况进行监测。     

新生儿血培养阳性病原菌分布及耐药状况分析

目的了解本院新生儿血培养阳性标本病原菌分布情况及主要病原菌的耐药特点,为临床合理治疗提供依据。方法对我院新生儿科4 750例患儿入院时行血培养,并以VITEK32全自动微生物分析仪进行菌株鉴定,K-B法进行药敏试验。结果共分离出病原菌617株,其中革兰阳性菌382株(61.91%),前三位为凝固酶阴性葡萄球菌(41.49%)、金黄色葡萄球菌(7.94%)和肠球菌属(6.00%),革兰阴性菌216株(35.01%),前四位为大肠埃希菌(12.97%)、肺炎克雷伯菌(9.08%)、粘质沙雷菌(5.51%)和阴沟肠杆菌(4.86%),真菌19株(3.08%)。药敏结果显示主要革兰阳性菌对青霉素G耐药率90%,对利福平和呋喃妥因的耐药率较低,未检出耐利奈唑烷和万古霉素菌株。主要革兰阴性菌对阿米卡星、左氧氟沙星、哌拉西林/他唑巴坦和头孢哌酮/舒巴坦耐药率较低,未检出耐亚胺培南、美罗培南和替加环素菌株。结论本院新生儿血培养病原菌以革兰阳性菌为主,凝固酶阴性葡萄球菌是主要病原菌,并且耐药率较高。     

237例新生儿败血症的病原分布及耐药性分析

目的了解近年本地区新生儿败血症病原学及细菌耐药状况,用以指导临床治疗。方法对2006年5月至2010年4月收治的237例病例的血液进行培养,并对分离的菌株进行鉴定和药敏试验。结果革兰阳性菌为主要病原菌,排名前3位的病原菌分别是:表皮葡萄球菌(48.52%)、人葡萄球菌(16.03%)、凝固酶阴性葡萄球菌(10.13%)。对培养出的革兰阳性菌最敏感的抗生素是万古毒素、阿米卡星,对培养出的革兰阴性菌最敏感的抗生素是丁胺卡那霉素、亚胺培南。结论本地区新生儿血培养病原菌以革兰阳性菌为主,耐药菌株较多,临床医师应根据细菌鉴定及药敏试验选择敏感药物治疗。     

新生儿血培养病原菌分布及与超敏C-反应蛋白的关系

目的分析新生儿血培养阳性标本中各病原菌分布情况及与hs-CRP的相关性。方法统计分析2015年1月至2016年1月大连市妇幼保健院新生儿ICU血培养临床资料并采用CRP检测试剂盒对血培养阳性的患者进行hs-CRP的检测。结果血培养阳性结果中,凝固酶阴性葡萄球菌46株,占65.7%,肺炎克雷伯菌8株,占11.4%,大肠埃希菌5株,占7.1%,肠杆菌、鲍曼不动杆菌、洛非不动杆菌各2株,分别占2.9%,摩根摩根菌、B群链球菌、金黄色葡萄球菌、肠球菌、微球菌各1株,分别占1.4%;hs-CRP的检出量,革兰阴性菌感染组为(20.60±3.40)mg/L,革兰阳性菌感染组为(15.80±2.30)mg/L,正常对照组为(4.10±0.95)mg/L。结论新生儿血培养阳性的主要致病菌为凝固酶阴性葡萄球菌、肺炎克雷伯菌和大肠埃希菌;有细菌感染的新生儿血中hs-CRP值升高,革兰阴性菌感染hs-CRP值高于革兰阳性菌。     

深圳地区新生儿血培养病原菌分布及耐药性分析

目的了解深圳地区新生儿败血症病原菌分布及其常见病原菌的耐药情况,为临床合理选择抗生素提供依据.方法用Bact/Alert全自动血培养系统进行血培养,VITEK-32全自动微生物鉴定仪对2003～2005年深圳市儿童医院794例新生儿血培养阳性标本进行细菌鉴定和药敏试验.结果794例新生儿血培养标本共检出细菌177株,总阳性率为22.29%,其中革兰阳性菌134株,占75.71%,革兰阳性菌以凝固酶阴性葡萄球菌为主,占阳性菌的85.07%,占总分离率的64.41%.革兰阴性菌43株,占24.92%,革兰阴性菌以肺炎克雷伯菌居多,占阴性菌的41.03%,占总分离率的9.04%.革兰阳性菌对抗生素耐药率最高的为青霉素,其次为头孢唑啉、苯唑西林、红霉素和氨苄西林/舒巴坦.在常见的革兰阴性菌中大肠埃希菌和肺炎克雷伯菌的产ESBLs均在50%左右,这些菌株对亚胺培南、头孢替坦、环丙沙星、左旋氧氟沙星表现了较低的耐药率.结论凝固酶阴性葡萄球菌是该地区新生儿败血症最常见的病原菌,其次为肺炎克雷伯菌.对常用抗菌药物有不同程度的耐药.了解该地区病原菌的流行分布和抗生素的耐药情况对临床合理选用抗生素具有重要意义.     

235例泌尿系统感染的病原菌分布及药敏分析

目的:了解本地区泌尿系统感染的病原菌分布情况,指导临床合理使用抗生素.方法:细菌鉴定及药敏试验采用VITEK-60全自动细菌鉴定仪.结果:革兰阴性菌是本地区泌尿系统感染的主要致病菌70.64%(166/235),其中大肠埃希菌占革兰阴性菌的66.87%(111/166),其次为克雷伯菌属10.84%(18/166).主要致病革兰阳性菌以葡萄球菌为主38.7%(24/62).革兰阴性菌对环丙沙星、左氧氟沙星和甲氧苄啶/磺胺甲恶唑耐药率高,革兰阳性菌对氨苄西林/舒巴坦、环丙沙星和甲氧苄啶/磺胺甲恶唑等 8种抗生素耐药率均偏高.治疗首选万古霉素、呋哺类、利福平等抗生素.结论:大肠埃希菌是本地区泌尿系统感染的主要病原菌.细菌对多种抗生素耐药,建议临床根据药敏结果使用抗生素.     

Vitek-AMS鉴定临床细菌的应用研究

目的探讨Vitek-AMS对临床细菌鉴定的应用价值。方法对玉溪市人民医院1999年至2008年临床分离11 537株细菌(临床株)和省、部级临床检验中心下发微生物学室间质量评价鉴定菌种(参考株)的Vitek-AMS鉴定结果作对比分析。结果 11 537株临床分离菌中,不能鉴定细菌8株(0.07%);除外传染病因子,鉴定到属细菌114株(1.62%),鉴定到种(含亚种、物生型)细菌6 908株(98.38%)。共有64属193种。细菌类型的分布革兰阴性杆菌革兰阳性球菌酵母菌革兰阳性杆菌厌氧菌革兰阴性球菌。用参考菌种作比较,Vitek鉴定种的符合率为83.08%(54/65),属的符合率为98.46%(64/65);其中革兰阴性杆菌符合率最高(100%,24/24),酵母菌类符合率最低(66.67%,14/21)。7种鉴定卡菌种的阳性检出率平均为56.80%(234/412),其中GPI卡最高(100%),NHI卡最低(13.33%);但应用机会最多是GNI+卡。Vitek-AMS检测葡萄球菌产β-内酰胺酶阳性率为88.89%,大肠埃希菌和肺炎克雷伯菌产ESBLs阳性率分别为59.74%和32.20%。结论 Vitek-AMS的应用为临床细菌学检验提供了一种高效、快速、可靠的实验方法;但对于个别菌种、细菌酶的检测必要时应以参考方法确认。     

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry for Identification of Microorganisms in Clinical Urine Specimens after Two Pretreatments

Rapid identification of microorganisms in urine is essential for patients with urinary tract infections (UTIs). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed as a method for the direct identification of urinary pathogens. Our purpose was to compare centrifugation-based MALDI-TOF MS and short-term culture combined with MALDI-TOF MS for the direct identification of pathogens in urine specimens. We collected 965 urine specimens from patients with suspected UTIs, 211/965 isolates were identified as positive by conventional urine culture. Compared with the conventional method, the results of centrifugation-based MALDI-TOF MS were consistent in 159/211 cases (75.4%), of which 135/159 (84.9%) had scores ≥ 2.00; 182/211 cases (86.3%) were detected using short-term culture combined with MALDI-TOF MS, of which 153/182 (84.1%) had scores ≥ 2.00. There were no apparent differences among the three methods (p = 0.135). MALDI-TOF MS appears to accelerate the microbial identification speed in urine and saves at least 24 to 48 hours compared with the routine urine culture. Centrifugation-based MALDI-TOF MS is characterized by faster identification speed; however, it is substantially affected by the number of bacterial colonies. In contrast, short-term culture combined with MALDI-TOF MS has a higher detection rate but a relatively slow identification speed. Combining these characteristics, the two methods may be effective and reliable alternatives to traditional urine culture.     

Comparison of the Bruker MALDI-TOF Mass Spectrometry System and Conventional Phenotypic Methods for Identification of Gram-Positive Rods

In recent years, MALDI-TOF Mass Spectrometry (MS) method has emerged as a promising and a reliable tool for bacteria identification. In this study we compared Bruker MALDI-TOF MS and conventional phenotypic methods to identify a collection of 333 Gram-positive clinical isolates comprising 22 genera and 60 species. 16S rRNA sequencing was the reference molecular technique, and rpoB gene sequecing was used as a secondary gene target when 16Sr RNA did not allow species identification of Corynebacterium spp. We also investigate if score cut-offs values of ≥1,5 and ≥1,7 were accurate for genus and species-level identification using the Bruker system. Identification at species level was obtained for 92,49% of Gram-positive rods by MALDI-TOF MS compared to 85,89% by phenotypic method. Our data validates the score ≥1,5 for genus level and ≥1,7 for species-level identification in a large and diverse collection of Gram-positive rods. The present study has proved the accuracy of MALDI-TOF MS as an identification method in Gram-positive rods compared to currently used methods in routine laboratories.     

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.     

A simple and sensitive method to extract bacterial, yeast and fungal DNA from blood culture material

This study investigated the various commercially available kits and 'in-house' methods to extract DNA from Gram-negative and Gram-positive bacteria, yeast and fungal agents in commonly employed blood culture material. The main methods investigated were as follows; Qiagen QIAmp Blood kit, Roche high PCR template preparation kit, Puregene DNA extraction kit, boiling, glass beads/sonication and wash/alkali/heat lysis. The results indicated that a simple wash/alkali/heat lysis method was the most sensitive, reproducible, simple and cost-effective extraction method. This was the only method which removed any PCR inhibitors and inherent DNA which existed in virgin BacT/Alert aerobic, anaerobic and paediatric blood culture material. Contaminating microbial DNA from Lactococcus lactis or Bacillus coagulans was identified in all batches of BacT/Alert FAN aerobic blood culture material examined.     

胆总管结石患者胆汁病原菌分布特点及耐药性分析

目的:探讨胆总管结石患者胆汁病原菌的分布特点以及耐药性的分析。方法:选择2016年6月-2017年6月期间我院收治的胆总管结石合并胆道感染患者160例为研究对象,所有患者均进行逆行内镜胰胆管造影(ERCP)并抽取胆汁标本,进行细菌培养和耐药性实验,评价分析胆汁病原菌的分布特点及耐药性情况。结果:160例患者中有117例(73.13%)检出病原菌,共培养出病原菌130株,其中有13例患者为两种病原菌同时感染。革兰阴性菌有95株(73.08%)、革兰阳性菌有31株(23.85%)、真菌有4株(3.08%)。比例由高到低的前六位病原菌依次为:大肠埃希菌、肺炎克雷伯菌、阴沟肠杆菌、屎肠球菌、铜绿假单胞菌、粪肠球菌。革兰阴性菌对亚胺培南、阿米卡星、美罗培南、他唑巴坦、头孢吡肟等三四代头孢菌素耐药率较低,对头孢曲松、环丙沙星、左氧氟沙星、哌拉西林、氨苄西林等耐药率较高。革兰阳性菌对替拉考宁、万古霉素、利奈唑胺等耐药率较低,对四环素、环丙沙星、左氧氟沙星、克林霉素、氨苄西林等耐药率较高。真菌对酮康唑、伊曲康唑、氟康唑等耐药率较低,对两性霉素B耐药率较高。结论:胆总管结石患者胆汁病原菌主要为革兰阴性菌、其次为革兰阳性菌,各病原菌对各种抗菌药物表现出不同的耐药性,因此在临床治疗时应参考药敏试验结果进行合理选择治疗药物。     

Distinction of Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of water-soluble tetrazolium salts with a selection medium

Bacteria are fundamentally divided into two groups: Gram-positive and Gram-negative. Although the Gram stain and other techniques can be used to differentiate these groups, some issues exist with traditional approaches. In this study, we developed a method for differentiating Gram-positive and -negative bacteria using a colorimetric microbial viability assay based on the reduction of the tetrazolium salt {2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt} (WST-8) via 2-methyl-1,4-napthoquinone with a selection medium. We optimized the composition of the selection medium to allow the growth of Gram-negative bacteria while inhibiting the growth of Gram-positive bacteria. When the colorimetric viability assay was carried out in a selection medium containing 0.5μg/ml crystal violet, 5.0 μg/ml daptomycin, and 5.0μg/ml vancomycin, the reduction in WST-8 by Gram-positive bacteria was inhibited. On the other hand, Gram-negative bacteria produced WST-8-formazan in the selection medium. The proposed method was also applied to determine the Gram staining characteristics of bacteria isolated from various foodstuffs. There was good agreement between the results obtained using the present method and those obtained using a conventional staining method. These results suggest that the WST-8 colorimetric assay with selection medium is a useful technique for accurately differentiating Gram-positive and -negative bacteria.     

Comparison of ribosomes from gram-positive and gram-negative bacteria with respect to the presence of protein S1

Ribosomes from Gram-positive and Gram-negative bacteria have been analysed for the presence of ribosomal protein S1 by three methods, sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoreaction with E. coli anti-S1 and chromatography on poly (U)-Sepharose. We observed that protein S1 is predominantly present in Gram-negative bacteria in comparison with Gram-positive bacteria. Exceptions are noted in both species.     

Gram-positive merA gene in gram-negative oral and urine bacteria

Clinical mercury resistant (Hg(r)) Gram-negative bacteria carrying Gram-positive mercury reductase (merA)-like genes were characterized using DNA-DNA hybridization, PCR and sequencing. A PCR assay was developed which discriminated between the merA genes related to Staphylococcus and those related to the Bacillus/Streptococcus merA genes by the difference in size of the PCR product. DNA sequence analysis correlated with the PCR assay. The merA genes from Acinetobacter junii, Enterobacter cloacae and Escherichia coli were sequenced and shared 98-99% identical nucleotide (nt) and 99.6-100% amino acid identity with the Staphylococcus aureus MerA protein. A fourth merA gene, from Pantoeae agglomerans, was partially sequenced (60%) and had 99% identical nt and 100% amino acid identity with the Streptococcus oralis MerA protein. All the Hg(r) Gram-negative bacteria transferred their Gram-positive merA genes to a Gram-positive Enterococcus faecalis recipient with the resulting transconjugants expressing mercury resistance. These Gram-positive merA genes join Gram-positive tetracycline resistance and Gram-positive macrolide resistance genes in their association with mobile elements which are able to transfer and express in Gram-negative bacteria.     

Gram-positive and Gram-negative bacteria elicit different patterns of pro-inflammatory cytokines in human monocytes

Pro-inflammatory cytokines secreted by tissue macrophages recruit polymorphonuclear leukocytes and evoke fever, cachexia and production of acute phase proteins. This study investigates whether Gram-positive and Gram-negative bacteria equally and efficiently trigger production of the pro-inflammatory cytokines IL-1 beta, IL-6, IL-8 and TNF-alpha in human monocytes. A range of aerobic and anaerobic Gram-positive and Gram-negative bacteria were killed by UV-light and added in different concentrations to human monocytes. Cytokines were measured in 24 h supernatants by ELISA. Gram-positive and Gram-negative bacteria were equally efficient inducers of IL-1 beta, but Gram-positive bacteria generated twice as much TNF-alpha as did Gram-negative bacteria (p<0.001 for 25 and 250 bacteria/cell). In contrast, Gram-negative bacteria induced at least twice as much IL-6 and IL-8 as did Gram-positive bacteria (p<0.001 for 2.5, 25 and 250 bacteria/cell). While the cytokine responses to LPS were similar to those induced by the corresponding amount of Gram-negative bacteria, the strong IL-1 beta and TNF-alpha responses to Gram-positive bacteria could not be induced by soluble peptidoglycan or lipotheicoic acid. The particular nature of the bacteria, thus seem to modify the response to Gram-positive bacterial components. The different cytokine profiles evoked by Gram-positive and Gram-negative bacteria might optimize clearance of bacteria that differ in cell wall structure.     

Proteomic analysis of acrylamide gel separated proteins immobilized on polyvinylidene difluoride membranes following proteolytic digestion in the presence of 80% acetonitrile

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) combined with mass spectrometry (MS) is a highly accurate and sensitive means of identifying proteins. We have developed a novel method for digesting proteins on polyvinylidene difluoride (PVDF) membranes for subsequent matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis. After Tricine sodium dodecyl sulfate (SDS)-PAGE, separated proteins were electroblotted onto PVDF membranes in a semidry discontinuous buffer system, visualized by staining with Coomassie Blue, excised, digested with trypsin or lysC in 80% acetonitrile, and then analyzed by MALDI-TOF MS. This method has several advantages over in-gel digestion in terms of sample handling, sensitivity, and time. We identified 105 fmol of Bacillus subtilis SecA and 100 approximately 500 fmol of standard proteins. We also analyzed the submembrane protein fraction solubilized by 1% n-dodecyl-beta-D-maltoside from B. subtilis membranes after separation by 2-D PAGE, and identified 116 protein spots. This method can detect proteins at the 10 approximately 50 fmol level by pooling more than ten identical electroblotted protein spots.