多重耐药不动杆菌16S rRNA甲基化酶和同源性研究

目的 调查2007年7月-2008年5月山西医科大学第二附属医院临床分离的61株多重耐药不动杆菌中介导氨基糖苷类抗生素高水平耐药的16S rRNA甲基化酶基因和Ⅰ类整合子携带耐药基因的分布.方法 利用blaOXA-51基因及16S rRNA-23S rRNA序列进行菌株鉴定,琼脂稀释法测定12种抗菌药物对61株不动杆菌的MIC,PCR筛选6种16S rRNA甲基化酶基因以及整合子基因盒,脉冲场凝胶电泳(PFGE)分析菌株同源性.结果 61株临床分离不动杆菌中55株为鲍曼不动杆菌、3株为3TU不动杆菌、l株13TU不动杆菌、1株醋酸钙不动杆菌、l株溶血不动杆菌.48株不动杆菌对阿米卡星、庆大霉素、妥布霉素均耐药,其中有47株检出armA基因;未检出rmtA、rmtB、rmtC、mad和npmA基因.armA基因阳性的菌株中Ⅰ类整合子阳性27株,分别携带arr-3、accA4、aacCl、catB8、aadA1和dfrA12基因.PFGE条带分析发现47株armA阳性菌株分为5个克隆,其中A、B为主要克隆,分布在我院多个科室中.结论 16S rRNA甲基化酶基因armA在多重耐药不动杆菌中广泛存在,armA基因不位于Ⅰ类整合子中,不动杆菌Ⅰ类整合子携带耐药基因主要介导对氨基糖苷类及氯霉素的耐药性.PFGE结果显示armA基因阳性菌株在我院呈克隆播散,必须采取有效的措施来控制耐药菌的传播.     

4株威克汉姆无绿藻的鉴定及基因特征分析

目的对临床分离的4株无绿藻进行菌种鉴定和基因特征分析。方法对临床分离的4株酵母样微生物进行培养特性和形态学特征分析,商品化Vitek 2细菌鉴定系统(配套YST卡)和MALDI-TOF MS微生物鉴定系统鉴定;PCR扩增其16S rRNA基因和28S rRNA基因,并进行系统发育分析,以确定其分类学地位。结果该4株微生物在沙保氏葡萄糖琼脂培养基上培养3 d可形成灰白色、光滑、湿润的"酵母样真菌"菌落;光镜下观察可见大量圆形、卵圆形或椭圆形的孢子囊,且其内含内孢子,形似桑葚状或草莓状,与酵母菌的菌体形态差异显著;Vitek YST和MALDI-TOF MS系统,均鉴定为威克汉姆无绿藻。该4株微生物同时存在代表原核生物的16S rRNA基因以及代表真核生物的28S rRNA基因,其中,16S rRNA基因序列与威克汉姆无绿藻相似度最高,在99.7%以上;28S rRNA基因经克隆测序发现其存在多拷贝,且同一菌株不同克隆序列之间相似度在91.9%～100%。16S rRNA基因和28S rRNA基因系统发育分析均显示,该4株微生物与威克汉姆无绿藻聚类在同一个分枝。结论该4株微生物可鉴定为威克汉姆无绿藻,且单拷贝的小亚基16S rRNA更适合作为其菌种鉴定的靶基因。     

大肠埃希菌16S rRNA甲基化酶基因的接合传递及其与整合子的相关性

目的 对临床分离的多重耐药（MDR）大肠埃希菌株的16S rRNA甲基化酶基因特征与接合传递效率进行研究,探讨其与整合子的相关性。 方法 136株MDR大肠埃希菌经PCR筛检16S rRNA甲基化酶基因armA、rmtA、rmtB、rmtC、rmtD;对阳性菌株作整合酶基因intI1、intI2和intI3检测,并扩增Ⅰ类整合子可变区插入片段,对扩增产物进行测序与鉴定所含耐药基因盒;以阳性菌株为供体菌,耐叠氮化钠大肠埃希菌J53为受体菌进行接合试验,并结合质粒图谱对16S rRNA甲基化酶基因进行初步定位。 结果 在136株多重耐药大肠埃希菌中,共检出16S rRNA甲基化酶阳性菌12株（8.8%）,其中,armA阳性3株（2.2%）,rmtB阳性10株（7.4%）,未检出rmtA、rmtC、rmtD基因。阳性菌株均只含Ⅰ类整合子,对其可变区扩增片段（1 000～2 300 bp）的测序结果显示,该区域含有多种耐药基因盒,但不含16S rRNA甲基化酶基因。接合试验与质粒图谱结果初步表明armA和rmtB编码基因位于约23 000 bp的质粒上,接合试验的耐药质粒传递率高达83.3%(10/12)。 结论 在MDR大肠埃希菌中,armA和rmtB编码基因位于约23 000 bp质粒上,其中,rmtB为优势基因,接合试验和质粒图谱证明该类耐药质粒很容易在同种菌间传播。Ⅰ类整合子与16S rRNA甲基化酶基因虽然存在于同一菌体内和/或同处于一个质粒上,但整合子基因盒对该类基因的捕获率很低或根本不捕获。     

嗜麦芽寡氧单胞菌临床株与环境株的16S rRNA基因序列及系统发育分析

目的:比较嗜麦芽寡氧单胞菌临床株与环境株16S rRNA基因序列,构建系统发育树,分析其进化关系.方法:对选取的3株嗜麦芽寡养单胞菌临床株和1株环境株的16S rRNA基因进行PCR扩增并测序.将上述及从GenBank中挑选出的其他32株不同来源的嗜麦芽寡养单胞菌的16S rRNA基因序列进行对比分析.并绘制系统发育树.结果:系统发育分析表明大部分菌株可根据来源分为3个簇,序列分析显示某些高度可变区可能存在可区分临床株与环境株的关键序列.结论:嗜麦芽寡养单胞菌基因型及表现型具有多样性:大部分嗜麦芽寡氧单胞菌临床株与环境株可根据16S rRNA基因序列进行鉴别.     

Genetic diversity of Flavobacterium columnare examined by restriction fragment length polymorphism and sequencing of the 16S ribosomal RNA gene and the 16S-23S rDNA spacer

Genetic variability among strains of Flavobacterium columnare, isolated in the United States, was characterized by restriction fragment length polymorphism (RFLP) and phylogenetic analysis based on the sequence of the 16S rRNA gene. Twenty-seven isolates of F. columnare were differentiated into three genotypes. The isolates within the genotypes were further grouped based on RFLP of the 16S-23S rDNA spacer. The first genotype had five strains that were further divided into group A (4 strains) and B (1 strain) while the second genotype had 10 strains that were also further divided into group A (4 strains) and B (6 stains). The third genotype had 12 isolates with no differences in the RFLP patterns of the 16S-23S rDNA spacers. The 16S rRNA gene sequences representing the three identified genotypes were compared to the different published sequences by phylogenetic analysis and the results showed the American genotypes 1, 2 and 3 corresponding to genomovar 1, 2, and 3, respectively, reported by Triyanto and Wakabayashi [Triyanto, Wakabayashi H. Genotyping of strains of Flavobacterium columnare from diseased fishes. Fish Pathol 1999; 34: 65-71]. The study demonstrates a method for RFLP and sequencing of the 16S rRNA gene and the 16-23S rDNA spacer as a useful tool in epidemiological studies of F. columnare.     

Rapid molecular diagnosis of lactobacillus bacteremia by terminal restriction fragment length polymorphism analysis of the 16S rRNA gene

OBJECTIVE: Bacteremia due to lactobacilli is uncommon, yet it is increasing in frequency, especially among immunosuppressed patients. In the clinical laboratory, lactobacilli must be subcultured from positive blood cultures before identification by traditional biochemical methods. Delays in diagnosis are significant because the organisms are inherently resistant to vancomycin, a drug frequently prescribed for empiric therapy for gram-positive bacteremia. Recently, we developed a rapid terminal-restriction fragment length polymorphism (T-RFLP) diagnostic assay based on species-specific variations in the bacterial 16S rRNA gene. We sought to apply this technique to the identification of Lactobacillus spp. from three cases of bacteremia. DESIGN: The results of the T-RFLP analysis are compared with two standard biochemical identification methods. METHODS: Lactobacillus strains were isolated from positive clinical blood cultures. Initial suspect cultures were subcultured and characterized using an automated substrate hydrolysis system and Lactobacillus carbohydrate fermentation profiles. Further biochemical and molecular analyses were performed from isolates propagated in Lactobacillus MRS broth. DNA was extracted and the 16S rRNA gene sequenced. Two sets of fluorescent labeled primers targeting the 16S rRNA gene were used for polymerase chain reaction (PCR) with chromosomal preparations from reference strains and blood isolates. The PCR products were digested with restriction enzymes and terminal-restriction fragment profile analysis performed. RESULTS: T-RFLP analysis correctly identified the Lactobacillus species in each case. T-RFLP analysis could be completed within 8 hours of obtaining a positive blood culture as compared to more than the 24 to 48 hours required for traditional culturing and biochemical characterizations. CONCLUSION: T-RFLP analysis allows for rapid identification of Lactobacillus directly from positive blood cultures and circumvents the requirement for subculture. Reduced diagnostic time has implications for duration of infection, the cost of patient care, length of hospitalization, development of broad-spectrum antibiotic resistance, and mortality due to bacteremia. T-RFLP profiling represents a highly reproducible and predictive source for identification of many organisms associated with bacteremia.     

不同分子生物学方法快速鉴别非结核分枝杆菌的应用评价

目的 评价3种分子生物学方法快速鉴定非结核分枝杆菌的优缺点.方法 收集41株临床分离的非结核分枝杆菌,以16S rRNA基因测序方法为标准,同时以hsp65基因测序方法及PCR-RFLP方法鉴定菌株,与16S rRNA基因测序结果进行比较.结果 41株非结核分枝杆菌16SrRNA基因测序结果:9株龟分枝杆菌复合群,7株偶发分枝杆菌,7株胞内分枝杆菌,3株鸟分枝杆菌,3株堪萨斯分枝杆菌复合群,3株耻垢分枝杆菌,3株土分枝杆菌,2株草分枝杆菌,2株无色分枝杆菌,1株瘰疬分枝杆菌,1株M.arupense.与16S rRNA基因测序相比较,hs65 PCR-RFLP能鉴定9株龟分枝杆菌复合群至亚种脓肿分枝杆菌,3株堪萨斯分枝杆菌复合群鉴定至亚种堪萨斯分枝杆菌;1株偶发分枝杆菌及1株无色分枝杆菌与其不符;其余菌株鉴定结果一致,符合率为95.1%(39/41).hsp65基因测序结果显示,1株爱尔兰分枝杆菌与16S rRNA测序结果不符,其余菌株鉴定结果与其一致,符合率为97.6%(40/41),并且能进一步将9株龟分枝杆菌复合群鉴定至亚种脓肿分枝杆菌,3株堪萨斯分枝杆菌复合群鉴定至亚种堪萨斯分枝杆菌.结论 3种方法均能快速鉴定非结核分枝杆菌.与16S rRNA基因测序相比,hsp65基因测序及hsp65 PCR-RFLP更容易鉴定临床最常见非结核分枝杆菌(如堪萨斯分枝杆菌和脓肿分枝杆菌),可在临床推广使用.     

Use of DNA restriction endonuclease digest and ribosomal RNA gene probe patterns to fingerprint Helicobacter pylori and Helicobacter mustelae isolated from human and animal hosts.

Variation amongst strains of Helicobacter pylori and Helicobacter mustelae was examined by DNA restriction endonuclease digestion and rRNA gene patterns generated using a non-radioactive probe. Chromosomal DNA was extracted from 30 cultures of H. pylori from human, Rhesus monkey and pig gastric mucosa, and from three H. mustelae isolates from ferret gastric mucosa. DNA fingerprinting with Hae III and Hind III showed H. mustelae was relatively homogeneous but revealed genomic heterogeneity within H. pylori with at least 18 different DNA patterns identifiable amongst the 30 isolates. Five sets of strains other than duplicates with matching DNA fingerprints were identified within H. pylori. The Peruvian isolates were the largest identical set and comprised eight isolates from four different patients with five consecutive isolates from one patient. The Rhesus monkey strains were a relatively homogeneous set as were several Australian human isolates. The study demonstrates that rRNA gene restriction patterns provide a simple but highly discriminatory electrophoretic fingerprint for H. pylori with potential for use as a novel epidemiological marker in addition to total DNA digest analysis.     

Comparison between two PCR-based bacterial identification methods through artificial neural network data analysis

The 16S ribosomal ribonucleic acid (rRNA) and 16S-23S rRNA spacer region genes are commonly used as taxonomic and phylogenetic tools. In this study, two pairs of fluorescent-labeled primers for 16S rRNA genes and one pair of primers for 16S-23S rRNA spacer region genes were selected to amplify target sequences of 317 isolates from positive blood cultures. The polymerase chain reaction (PCR) products of both were then subjected to restriction fragment length polymorphism (RFLP) analysis by capillary electrophoresis after incomplete digestion by Hae III. For products of 16S rRNA genes, single-strand conformation polymorphism (SSCP) analysis was also performed directly. When the data were processed by artificial neural network (ANN), the accuracy of prediction based on 16S-23S rRNA spacer region gene RFLP data was much higher than that of prediction based on 16S rRNA gene SSCP analysis data (98.0% vs. 79.6%). This study proved that the utilization of ANN as a pattern recognition method was a valuable strategy to simplify bacterial identification when relatively complex data were encountered.     

Multiplex PCR for detection of Enterobacteriaceae in blood

BACKGROUND: Rapid and sensitive methods are needed to detect the small numbers of bacteria that may sometimes contaminate units of blood during collection. A multiplex 5'-nuclease TaqMan PCR assay (PE Applied Biosystems) was used to detect several bacterial species that may contaminate blood. STUDY DESIGN AND METHODS: Oligonucleotide primers were made for regions of the 16S rRNA gene conserved in four different bacterial species: Yersinia enterocolitica and Serratia, Klebsiella, and Enterobacter species. Two probes were designed: SL-1 detected Serratia, Klebsiella, and Enterobacter species, and YE-3 detected Y. enterocolitica. RESULTS: When TaqMan PCR was performed with chromosomal DNA isolated from pure cultures of Serratia liquefaciens, Klebsiella oxytoca, Klebsiella pneumoniae, Enterobacter cloacae, and Enterobacter agglomerans, the limit of detection with probe SL-1 was 1 to 2 CFUs. For S. marcescens, the sensitivity was 8 CFUs. The limit of detection for Y. enterocolitica with probe YE-3 was 2 CFUs. When total chromosomal DNA was extracted from whole-blood samples spiked with different numbers of Y. enterocolitica, S. liquefaciens, E. cloacae, or K. pneumoniae bacteria, the TaqMan PCR detected 12 to 16 organisms in 1 mL of blood. CONCLUSION: The 5'-nuclease TaqMan PCR assay takes only 3 hours to perform and has the potential to detect very small numbers of bacteria.     

16SrRNA甲基化酶基因在产ESBLs肠杆菌科细菌中的分布

目的了解临床分离的产超广谱β-内酰胺酶（ESBLs）的革兰阴性肠杆菌科细菌中16SrRNA甲基化酶基因的分布情况。方法对临床分离的70株革兰阴性肠杆菌科细菌用VITEK．32型全自动微生物分析系统进行细菌鉴定,用纸片扩散法检测ESBLs,并用聚合酶链反应（PCR）检测armA、rmtA、rmtB、rmtC、rmtD和npmA6种16SrRNA甲基化酶基因,对检测的阳性产物进行测序,并通过GenBank比对DNA序列。结果70株产ESBLs革兰阴性肠杆菌中,9株16SrRNA甲基化酶基因阳性,其中5株检出armA基因,4株检出rmtB基因,2株同时检出armA和rmtB基因,rmtA、rmtC、rmtD、npmA4种基因扩增均为阴性。结论不同地区医院16SrRNA甲基化酶基因的分布情况各不相同。     

小儿常见感染菌16S—23S rRNA基因区间的分子生物学研究

目的：探讨聚合酶链反应（PCR）加限制性内切酶片段长度多态性（RFLP）分析在细菌rDNA区间检测中的应用。方法：以16S－23S RNA基因区间为靶序列,设计引物,选择合适的内切酶,采用PCR法加RFLP法检测标准菌株及临床菌株的rDNA区间。结果：26株不同的标准菌株经PCR扩增后,分别出现一条带,两条带,三条带及多条带的不同DNA图谱,敏感性试验可检出2．5CFU的细菌,与人类基因组DNA,真菌及病毒无交叉反应,其中14种菌经一步PCR扩增即可区分,另12种菌除肺炎克雷伯氏菌与坚韧肠球菌经Hinf I或Alu I酶切后仍不能区分外,其余经其中一内切酶酶切后均能区分,32例血培养阳性标本均扩增出与相应标准菌株相一致的图谱。结论：16S－22S rRNA区间基因PCR．扩增加RFLP技术检测细菌rDNA区间,具有特异,敏感,快速,准确的特点,为细菌感染的病原诊断提供新的科学依据。     

Citrobacter koseri and Citrobacter amalonaticus isolates carry highly divergent beta-lactamase genes despite having high levels of biochemical similarity and 16S rRNA sequence homology

OBJECTIVES: Isolates previously identified as Citrobacter diversus are now known as Citrobacter koseri. We measured sequence variation at the beta-lactamase structural gene among a group of clinical isolates originally identified as C. diversus by API 20E profiling. METHODS: beta-Lactamase and 16S rRNA genes were amplified by PCR and sequenced by standard methods. beta-Lactamase induction was attempted in liquid-grown cultures using cefoxitin. Nitrocefin hydrolysis assays were performed using a spectrophotometer. RESULTS: Analysis of 16S rRNA gene sequences showed that Citrobacter spp. isolates with an inducible beta-lactamase gene, cdiA, closely related to 'C. koseri ' NF85 and ULA27 are actually Citrobacter amalonaticus. C. koseri isolates, whose identities were confirmed by 16S rRNA sequencing, produce a class A beta-lactamase, Cko, constitutively at low levels. The cko and cdiA beta-lactamase genes share <45% identity. CONCLUSIONS: We have confirmed that cko is a beta-lactamase gene carried by C. koseri, and that isolates previously identified as 'C. koseri ', but carrying the cdiA beta-lactamase gene are C. amalonaticus. Thus, beta-lactamase-gene-specific PCR may provide a valuable tool to differentiate these biochemically homogeneous Citrobacter species.     

Emergence of RmtB methylase-producing Escherichia coli and Enterobacter cloacae isolates from pigs in China

OBJECTIVES: To investigate the occurrence of 16S rRNA methylases conferring high-level resistance to aminoglycosides in Enterobacteriaceae isolated from two pig farms in China. METHODS: Enterobacteriaceae isolated from 151 pig rectal swab samples and 9 environmental samples were screened for the presence of the rmtA, rmtB, armA and rmtC genes by PCR and sequencing. Conjugation experiments were carried out to study the transferability of the 16S rRNA methylase genes. All isolates and their transconjugants were tested for susceptibility to antimicrobial agents. The clonal relatedness of RmtB-producing Escherichia coli was assessed by PFGE with XbaI. RESULTS: Of 152 Enterobacteriaceae isolates recovered from pigs, 49 (32%) were positive for the rmtB gene, including 48 E. coli and a single isolate of Enterobacter cloacae. Of the nine Enterobacteriaceae isolates from environmental samples, no 16S rRNA methylase gene was identified. The 49 rmtB-positive isolates showed resistance to ampicillin, tetracycline and trimethoprim and also carried a bla(TEM) gene. Transfer of the rmtB and bla(TEM) genes by conjugation experiments of all 49 isolates was successful, suggesting that the rmtB-containing plasmids in the E. coli and E. cloacae isolates were self-transmissible. Conjugative transfer frequencies varied from 2.2 x 10(-10) to 1.3 x 10(-6) transconjugants per recipient. The transfer of non-aminoglycoside antimicrobial resistance traits was also observed in most cases. Forty-four rmtB-positive E. coli showed 30 different PFGE types. CONCLUSIONS: The rmtB gene was detected on conjugative plasmids of porcine E. coli and E. cloacae isolates. Both horizontal gene transfer and clonal spread were responsible for the dissemination of the rmtB gene. The emergence of 16S rRNA methylases in Enterobacteriaceae isolates is described for the first time in China. This is also the first report of rmtB-positive Enterobacteriaceae among healthy food-producing animals.     

Mutations in the cyp51A gene and susceptibility to itraconazole in Aspergillus fumigatus serially isolated from a patient with lung aspergilloma

OBJECTIVES: To monitor changes in itraconazole susceptibility of isolates from a patient undergoing treatment for pulmonary Aspergillus infection and relate these changes to genotypic/phenotypic alterations. METHODS: Six Aspergillus fumigatus isolates were serially recovered from the patient. Itraconazole MICs were determined by Etest and NCCLS methodology. Growth characteristics and phenotype were monitored. Molecular analysis included random amplified polymorphic DNA (RAPD) assay and sequencing of the cyp51A gene. RESULTS: The MIC of itraconazole against the first isolate before treatment was 0.25 mg/L; the MIC against the second isolate, recovered after 6 months of itraconazole therapy, was >16 mg/L; and that against the third isolate, obtained 2 months after discontinuation of the therapy, was 0.5 mg/L. The MIC against the last three isolates, acquired after restoration of itraconazole therapy for 4-7 months, was >16 mg/L. The six isolates shared identical band patterns of RAPD assay using four primers and the same sequence in intertranscribed spacers (ITS). Therefore, the six isolates were likely to be the same strain of A. fumigatus, and mutations involving itraconazole resistance possibly occurred in these isolates after prolonged itraconazole therapy. Sequencing of the cyp51A gene in the coding region revealed a mutation of M220I in cytochrome P450 sterol 14-alpha-demethylase in the second resistant isolate and a mutation of G54R in the last three resistant isolates. Expression changes of some pump genes, such as MDR3, may also, in part, be related to the resistance to itraconazole. CONCLUSIONS: We conclude that resistance of A. fumigatus to itraconazole occurred in a patient treated with the drug, and the resistance may result from mutations in the cyp51A gene-the gene encoding the target enzyme for itraconazole.     

Detection of Chlamydia pneumoniae antigenin PBMNCs of healthy blood donors

BACKGROUND: Because it has been increasingly recognized that Chlamydia pneumoniae may be linked to some chronic inflammatory diseases, including atherosclerosis, detection of this pathogen in blood from patients may be valuable in the diagnosis of such diseases. However, the prevalence of chlamydia in the blood of healthy donors has not yet been extensively studied. STUDY DESIGN AND METHODS: The presence of C. pneumoniae in PBMNCs obtained from healthy persons who donated blood for blood transfusion was assessed by a PCR that was specific for the C. pneumoniae 16S rRNA gene and by the use of staining with FITC-conjugated chlamydia MoAb. RESULTS: Twenty-one (8.9%) of 237 blood samples tested showed the presence of C. pneumoniae DNA and antigen in the PBMNCs. There was no significant difference in the presence of chlamydia in blood according to sex or to age between 20 and 59 years of age. However, a possible seasonal variation in the presence of chlamydia in blood from healthy donors was suggested by the results obtained. CONCLUSION: A significant percentage of healthy donors carry C. pneumoniae, which may be a risk factor for some chronic diseases.     

Cloning and sequencing an unknown gene of Tropheryma whipplei and development of two LightCycler PCR assays

Whipple's disease is a rare infectious illness that can affect any organ system in the body. It is caused by Tropheryma whipplei, a Gram-positive rod-shaped bacterium with a high G + C content, classified within the actinobacteria. For decades, laboratory detection has been based on microscopy and the periodic acid-Schiff (PAS) staining of biopsies. Recently, PCR has become a useful tool to detect T. whipplei DNA in various clinical specimens. However, a positive PCR result does not confirm Whipple's disease as it has been shown that asymptomatic persons can harbor T. whipplei DNA. Since there is not yet much known about the genome of T. whipplei, genome-walking represents a convenient method to determine unknown gene sequences. Starting from a RAPD fragment we have sequenced and cloned an open reading frame (ORF) of 843 bp. Two real-time PCR assays targeting the ORF fragment and the 16S rRNA gene, respectively, were developed. Compared to a conventional 16S rRNA PCR system the ORF LightCycler assay proved to be very specific (100%) but not sufficiently sensitive (62.4%). In contrast, the 16S rRNA LightCycler assay showed a sensitivity of 95.7% and a specificity of 97.8%. Thus, the 16S rRNA gene assay but not that targeting the new ORF is a suitable alternative to conventional PCR methods.     

Evaluation of an automated culture system for detecting bacterial contamination of platelets: an analysis with 15 contaminating organisms

BACKGROUND: Approximately 1 in 2000 platelet components are bacterially contaminated. The time to detection of 15 seeded organisms in platelets recovered from an automated culture system was studied. STUDY DESIGN AND METHODS: Isolates of Bacillus cereus, Bacillus subtilis, Candida albicans, Clostridium perfringens, Corynebacterium species, Enterobacter cloacae, Escherichia coli, Klebsiella oxytoca, Propionibacterium acnes, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Serratia marcescens, Streptococcus pyogenes, and Streptococcus viridans were inoculated into Day 2 apheresis platelet components to obtain a final concentration of approximately 10 and 100 CFU per mL (2 units/organism). Each bag was sampled 10 times (20 mL/sample). Four mL of each sample was inoculated into standard aerobic and anaerobic bottles and into aerobic and anaerobic bottles containing charcoal; 2 mL was inoculated into pediatric aerobic bottles (so as to maintain a 1:10 ratio of sample to media) and 1 mL into thioglycollate broth. RESULTS: With the exception of P. acnes, all organisms were detected in a mean of 9.2 to 25.6 hours. A range of 10 serial dilutions in inoculating concentrations was associated with an overall 10.1-percent difference in detection time. A mean of 74.4 and 86.2 hours (100 and 10 CFU/mL inocula, respectively) was required for the detection of P. acnes in anaerobic bottles. CONCLUSION: Bacteria thought to be clinically significant platelet contaminants can be detected in 9.2 to 25.6 hours when the starting concentration is approximately 10 to 100 CFU per mL. P. acnes required considerably longer incubation times for detection (in either aerobic or anaerobic bottles). However, P. acnes is of questionable clinical significance. Such a detection system could be used in either a blood collection center or a transfusion service to screen platelet concentrates for bacterial contamination. Such testing (with sterile sampling performed so as to maintain a closed-bag system) would be expected to save lives and might allow an extension of platelet storage.