Vitek 2 Compact GN卡用于不同培养基生长类鼻疽伯克霍尔德菌鉴定结果分析

目的评估不同培养基生长类鼻疽伯克霍尔德菌在Vitek 2 Compact中的鉴定结果。方法收集2010年6月至2017年5月海南省人民医院分离的类鼻疽伯克霍尔德菌127株,分别接种于哥伦比亚羊血平板(CBA)、麦康凯平板(MAC)和流感嗜血巧克力平板(CHA)上,以多位点序列分型(MLST)为金标准,评估Vitek 2 Compact VT2.R7.01版GN卡对不同培养基上生长的类鼻疽伯克霍尔德菌的鉴定准确性。结果 CBA平板上生长的类鼻疽伯克霍尔德菌鉴定准确率最高,为98.4%,2株被错误鉴定为洋葱伯克霍尔德菌;MAC次之,准确率为94.5%,其中4株被错误鉴定为洋葱伯克霍尔德菌,2株为伯克霍尔德菌某种,1株被错误鉴定为铜绿假单胞菌;CHA鉴定准确率最低,为91.3%,其中4株被错误鉴定为洋葱伯克霍尔德菌,2株为伯克霍尔德菌某种,5株无鉴定结果。结论 Vitek 2 Compact GN卡对不同培养基上类鼻疽伯克霍尔德菌鉴定准确率不同,建议优先选择CBA平板上菌落。     

类鼻疽伯克霍尔德菌误鉴定1例

正伯克霍尔德菌(Burkholderia)是一种革兰阴性非发酵菌,其中与人类疾病相关的病原菌主要包括洋葱伯克霍尔德菌复合群(B.cepacia complex)、类鼻疽伯克霍尔德菌复合群(B.pseudomallei complex)等。而类鼻疽伯克霍尔德菌复合群可进一步分为类鼻疽伯克霍尔德菌、鼻疽伯克霍尔德菌(B.mallei)、泰国伯克霍尔德菌(B.thailandensis)、俄克拉何马伯克霍尔德菌(B.oklahomensis)和汉普蒂社伯克霍尔德菌(B.humptydooensis)5个种[1],其中除泰国伯克霍尔德菌毒力较     

类鼻疽伯克霍尔德菌致血流感染1例

正类鼻疽病(melioidosis)是由类鼻疽伯克霍尔德菌所导致的地方性传染病,主要流行于澳大利亚中部、东南亚等热带和亚热带地区。该菌常存在于疫区水、土壤、粪便及尸体中,可经破损皮肤接触含有致病菌的水或土壤而感染,也可经呼吸道感染[1]。2014年10月,本组从1例高烧患者血液标本中分离出类鼻疽伯克霍尔德菌,报道如下。1资料与方法1.1一般资料患者,男,39岁,因发热、右下肢肿痛入院。     

1例高致病性类鼻疽伯克霍尔德菌的鉴定及溯源性分析

正类鼻疽是一种由类鼻疽伯克霍尔德菌感染引起的热带传染病。该病原菌主要侵犯人体肺部,早期易出现发烧、寒战、咳嗽、恶心、呕吐、头痛、腹痛、肌肉痛等症状。类鼻疽伯克霍尔德菌侵入人体肺部后,容易形成难治性肺炎、肺部空洞,并可能短期内快速地发展为败血症而死亡。此外,类鼻疽伯克霍尔德菌还可能引起脑膜炎、前列腺炎、尿路感染及肝脾等内脏的     

抗类鼻疽伯克霍尔德菌多抗血清的制备及评价

目的制备抗类鼻疽伯克霍尔德菌(Burkholderia pseudoamllei,其抗原简写为BP)多抗血清,评价不同处理方式对抗原免疫原性的影响.方法 采用超声破碎、甲醛灭活、热灭活3种方式处理细菌抗原接种新西兰兔和BALB/C小鼠,检测不同抗原免疫动物抗血清的ELISA效价及凝集试验效价.结果 (1)ELISA方案最佳条件:二抗稀释度为1/8 000,抗原包被浓度为4 μg/mL或8 μg/mL;凝集反应最佳条件:细菌浓度为6×109 CFU/mL、凝集温度为37 ℃、观察时刻为4 h.(2)超声破碎抗原(U-BP)免疫新西兰兔和小鼠的抗血清ELISA效价高达1/64 000,最高血清凝集效价分别为1/32和1/64,甲醛灭活抗原(F-BP)抗血清ELISA效价高达1/16 000,凝集效价为1/128和1/64;热灭活抗原(H-BP)抗血清ELISA效价达1/8 000,凝集效价为1/16和1/64.(3)F-BP和H-BP具有较强的免疫原性,U-BP在小鼠中的免疫原性较弱(P<0.05).结论 建立了抗类鼻疽伯克霍尔德杆菌多克隆抗体的检测方法,制备了高效价的抗类鼻疽伯克霍尔德杆菌多抗血清;甲醛灭活和热灭活细菌具有较好的免疫原性,为下一步类鼻疽伯克霍尔德杆菌的致病机制研究和疫苗研发打下了基础.     

类鼻疽伯克霍尔德菌重症肺炎并发脓毒血症1例

患者,男,35岁。因口干、多饮、多食、夜尿多1年余,伴呼吸困难、发热1周急诊于2011年7月16日入院。入院体检:T38℃,P90次/min,R26次/min,BP122/78mmHg(1mmHg=0.133kPa),APACHIII评分11分,指脉氧饱和度96%,神志     

泰国伯克霍尔德菌错误鉴定1例

目的鉴定1例曾被错误鉴定为类鼻疽伯克霍尔德菌的泰国伯克霍尔德菌。方法对1例肺脓肿合并败血症患者的痰液和血液标本进行细菌分离培养、生化鉴定、分子生物学鉴定和药敏试验鉴定。结果培养出革兰阴性短小杆菌;生化鉴定特征符合泰国伯克霍尔德菌;测序结果显示与泰国伯克霍尔德菌相似度达100%,与类鼻疽伯克霍尔德菌相似度为99%;全基因组测序结果显示该菌与泰国伯克霍尔德菌E444具有最近的亲缘关系。药敏试验结果显示该菌对亚胺培南、左氧氟沙星、哌拉西林/他唑巴坦、头孢他啶、甲氧苄啶/磺胺甲噁唑敏感。结论成功鉴定了1例泰国伯克霍尔德菌。     

哌拉西林他唑巴坦对类鼻疽伯克霍尔德菌的体外抗菌活性评估

目的 评估哌拉西林他唑巴坦体外抗类鼻疽伯克霍尔德菌的活性,为临床用药提供理论依据.方法 采用E-test法,对临床分离的109株类鼻疽伯克霍尔德菌进行哌拉西林他唑巴坦最低抑菌浓度(MIC)的检测,计算MIC50和MIC90,与有CLSI敏感性判断标准的亚胺培南、强力霉素、复方磺胺甲噁唑、头孢他啶、四环素、阿莫西林克拉维...     

类鼻疽伯克霍尔德菌在青瓜和生菜幼苗中的定植情况研究

  目的  研究类鼻疽伯克霍尔德菌在青瓜、生菜幼苗中的定植情况。  方法  用海南省三亚市人民医院保存的类鼻疽伯克霍尔德菌BP47、BP56、BP66分别感染青瓜、生菜无菌幼苗根部和叶片,从被感染幼苗根部与叶片的研磨液中分离该菌。 采用菌落平板计数检测被感染第1、3、5、7天的幼苗叶片中类鼻疽伯克霍尔德菌的增殖情况,绘制增殖曲线。 通过透射电镜对幼苗叶片中的类鼻疽伯克霍尔德菌进行定位。  结果  从被感染的青瓜、生菜幼苗中分离到了类鼻疽伯克霍尔德菌,而且该菌可以在幼苗中增殖,菌量随着时间推移而增加。 通过透射电镜在青瓜、生菜幼苗叶片中也观察到了该菌定植。  结论  类鼻疽伯克霍尔德菌可以在一些生食类蔬菜（青瓜、生菜）幼苗中定植,为探究其新的感染方式提供了实验依据。     

类鼻疽实验室诊断的研究进展

类鼻疽是由革兰阴性菌类鼻疽伯克霍尔德菌引起的人畜共患传染病,已成为一个新的全球公共卫生问题。传统的类鼻疽实验室诊断需要特殊的仪器设备,且操作繁琐,许多热带发展中国家缺乏实验室诊断工具和相关临床经验,延误了类鼻疽病的诊断和治疗。优化的血清学检测及分子生物学检测方法具有操作简便、快速、高灵敏度、高特异度等特点,对于类鼻疽的早期诊断具有重要意义。现就类鼻疽实验室诊断技术和研究进展进行综述,以便为该病制订最佳防控方案提供参考。     

DNA microarray-based detection and identification of Burkholderia mallei,Burkholderia pseudomallei and Burkholderia spp.

We developed a rapid oligonucleotide microarray assay based on genetic markers for the accurate identification and differentiation of Burkholderia (B.) mallei and Burkholderia pseudomallei, the agents of glanders and melioidosis, respectively. These two agents were clearly identified using at least 4 independent genetic markers including 16S rRNA gene, fliC, motB and also by novel species-specific target genes, identified by in silico sequence analysis. Specific hybridization signal profiles allowed the detection and differentiation of up to 10 further Burkholderia spp., including the closely related species Burkholderia thailandensis and Burkholderia-like agents, such as Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia vietnamiensis, Burkholderia ambifaria, and Burkholderia gladioli, which are often associated with cystic fibrosis (CF) lung disease. The assay was developed using the easy-to-handle and economical ArrayTube? (AT) platform. A representative strain panel comprising 44 B. mallei, 32 B. pseudomallei isolates, and various Burkholderia type strains were examined to validate the test. Assay specificity was determined by examination of 40 non-Burkholderia strains.     

PCR-RFLP analysis of the flagellin sequences for identification of Burkholderia pseudomallei and Burkholderia cepacia from clinical isolates

The flagellin genes of four Burkholderia pseudomallei and two Burkholderia cepacia clinical isolates were studied by a polymerase chain reaction (PCR)-based isolation method using the same pair of primers. The PCR-amplification products of the isolates showed a single band of about 1.1 kb, which is similar to a type II B. cepacia flagellin reported previously. In order to distinguish these two species based on the flagellin PCR-amplified products, Pst I and Xho I restriction endonuclease analysis was performed. The results suggest that there is sufficient diversity within the flagellin sequences of the closely related Burkholderia species, B. pseudomallei and B. cepacia, to enable flagellin-type identification on the basis of the pattern of restriction fragments. In addition, the flagellin gene should be of considerable use as a genetic marker for clinical identification of these organisms.     

Single gene target bacterial identification. groEL gene sequencing for discriminating clinical isolates of Burkholderia pseudomallei and Burkholderia thailandensis

Proper identification of Burkholderia pseudomallei and Burkholderia thailandensis is crucial in guiding clinical management of patients with suspected melioidosis, as more than 99% of cases of melioidosis are caused by B. pseudomallei, whereas B. thailandensis is only responsible for causing less than 1% of the cases. However, the difference between the 16S ribosomal RNA gene sequences of B. pseudomallei and that of B. thailandensis is only 1%, and is therefore not discriminative enough for distinguishing the 2 species confidently. In this study, we amplified and sequenced the groEL genes of 7 strains of B. thailandensis and 6 strains of B. pseudomallei, and compared the sequences with 7 other groEL gene sequences of Burkholderia species. BLAST analysis revealed that the putative protein encoded by the groEL gene of B. thailandensis has 99.6%, 99.5%, 98.4%, 98.5%, and 96.5% amino acid identity with the groEL of B. pseudomallei, B. mallei, B. cepacia, B. vietnamiensis, and B. fungorum respectively. The amino acid sequences of GroEL of the strains of B. thailandensis and B. pseudomallei all showed >99.5% amino acid identity with each other. The nucleotide sequence of the groEL gene of any of the strains of B. thailandensis showed >99.8% nucleotide identity with that of any of the other strains of B. thailandensis, and the nucleotide sequence of the groEL gene of any of the strains of B. pseudomallei showed >99.5% nucleotide identity with that of any of the other strains of B. pseudomallei. However, the nucleotide sequence of any of the strains of B. thailandensis showed <97.6% nucleotide identity with any of the strains of B. pseudomallei. The amino acid sequences of GroEL of the 20 strains of Burkholderia species all showed >96% amino acid identity with each other. Furthermore, the nucleotide sequence of the groEL genes of the 2 strains of B. cepacia showed >99.5% nucleotide identity with each other, and the nucleotide sequence of the groEL gene of B. mallei showed >99.5% nucleotide identity with any of the strains of B. pseudomallei. The groEL gene sequence is therefore good for distinguishing between B. thailandensis and B. pseudomallei, and the GroEL amino acid and groEL nucleotide sequences of this single gene locus may potentially be useful for a 2-tier hierarchical identification of medically important Burkholderia at the genus and species levels respectively.     

Polysaccharide microarray technology for the detection of Burkholderia pseudomallei and Burkholderia mallei antibodies

A polysaccharide microarray platform was prepared by immobilizing Burkholderia pseudomallei and Burkholderia mallei polysaccharides. This polysaccharide array was tested with success for detecting B. pseudomallei and B. mallei serum (human and animal) antibodies. The advantages of this microarray technology over the current serodiagnosis of the above bacterial infections were discussed.     

Rapid presumptive identification of Burkholderia pseudomallei with real-time PCR assays using fluorescent hybridization probes

Burkholderia pseudomallei (the etiologic agent of melioidosis) can cause pyogenic or granulomatous lesions in almost any organ. Septicemia has a case fatality rate of >40%. Early diagnosis and appropriate antibiotic therapy are crucial for survival, but cultivation, biochemical identification, and conventional PCR of B. pseudomallei are time consuming. We established real-time PCR assays using fluorescent hybridization probes targeting the 16S rDNA, the flagellin C (fliC) and the ribosomal protein subunit S21 (rpsU) genes. The test sensitivity and specificity were assessed with a representative panel of 39 B. pseudomallei, 9 B. mallei, 126 other Burkholderia strains of 29 species, and 45 clinically relevant non-Burkholderia organisms. The detection limit for the 16S rDNA, fliC, and rpsU assay was 40, 40, and 400 genome equivalents per reaction, however, in spiked blood samples it was 300, 300, and 3000, respectively. Specificity, positive and negative predictive value of the assays was 100%. In conclusion, we recommend the use of the 16S rDNA and/or fliC real-time PCR assays for the rapid identification of B. mallei and B. pseudomallei in positive blood cultures or from suspicious bacterial colonies.     

Development of a polymerase chain reaction assay for the specific identification of Burkholderia mallei and differentiation from Burkholderia pseudomallei and other closely related Burkholderiaceae

Burkholderia mallei and Burkholderia pseudomallei, the etiologic agents responsible for glanders and melioidosis, respectively, are genetically and phenotypically similar and are category B biothreat agents. We used an in silico approach to compare the B. mallei ATCC 23344 and B. pseudomallei K96243 genomes to identify nucleotide sequences unique to B. mallei. Five distinct B. mallei DNA sequences and/or genes were identified and evaluated for polymerase chain reaction (PCR) assay development. Genomic DNAs from a collection of 31 B. mallei and 34 B. pseudomallei isolates, obtained from various geographic, clinical, and environmental sources over a 70-year period, were tested with PCR primers targeted for each of the B. mallei ATCC 23344-specific nucleotide sequences. Of the 5 chromosomal targets analyzed, only PCR primers designed to bimA(Bm) were specific for B. mallei. These primers were used to develop a rapid PCR assay for the definitive identification of B. mallei and differentiation from all other bacteria.     

Characterization of Burkholderia pseudomallei isolated in Thailand and Malaysia

A total of 35 Burkholderia pseudomallei isolates from Thailand (16 clinical and eight soil isolates) and Malaysia (seven animal, two isolate each from clinical and soil) were investigated by their antimicrobial resistance, plasmid profiles and were typed by randomly amplified polymorphic DNA analysis. All isolates were found to be resistant to six or more of the 12 antimicrobial agents tested. Only two small plasmids of 1.8 and 2.4 megadalton were detected in two clinical isolates from Thailand. RAPD analysis with primer GEN2-60-09 resulted in the identification of 35 RAPD-types among the 35 isolates. The constructed dendrogram differentiated the 35 isolates into two main clusters and a single isolate. The wide genetic biodiversity among the 35 isolates indicate that RAPD-PCR can be a useful method to differentiate unrelated B. pseudomallei in epidemiological investigation.