Identification of Streptococcus species and Haemophilus influenzae by direct sequencing of PCR products from 16S-23SrDNA intergenic spacer regions

目的：利用16S-23SrRNA间区的特征建立一种简单、快速的细菌分类方法。方法：以化脓性链球菌16SrRNA的3'端和23SrRNA的5'端的保守区中合成3对引物,PCR扩增16S-23SrDNAISRs,纯化后直接测序,在GenBank上查找对应细菌的ISRs序列。用DNAMAN软件进行系统进货分析。结果：链球菌属为单拷贝16S-23SrRNAISRs片段,编码1个tRNA^Ala基因。流感嗜血杆菌各生物型出现2个大小相似的ISRs片段。与GenBank上的资料比较,7株链球菌归属5个种,流感嗜血杆菌均为b型。结论：ISRs作为细菌分类新的目标基因具有属、种、型和株特异性与灵敏性。     

不可分型流感嗜血杆菌对气管黏膜上皮细胞的作用

目的:探讨不可分型流感嗜血杆菌(NTHi)与气液界面无血清培养原代兔气管黏膜上皮细胞的相互作用.方法:利用低温酶消化法分离兔气管黏膜上皮细胞,无血清培养液及胶原覆盖膜形成的气液界面培养,使上皮细胞分化成假复层黏膜纤毛上皮细胞,然后加入NTHi感染上皮细胞,通过扫描电镜(SEM)和透射电镜(TEM)观察其形态结构变化.结果:NTHi感染24 h后,SEM示气管黏膜上皮细胞表面结构破坏,90%细胞凋亡或死亡,纤毛倒伏、断裂,细菌与非纤毛上皮细胞连接;TEM示细菌黏附在细胞表面,细胞表面有较多微绒毛,伴微绒毛延伸或伪足包绕细菌,将其吞噬胞内.结论:NTHi黏附在上皮细胞表面,上皮细胞通过伪足形成和微绒毛延伸包绕细菌,将细菌吞噬在胞内;NTHi对上皮细胞有毒性作用,致细胞变性坏死或凋亡.     

儿童流感嗜血杆菌氨苄青霉素耐药基因的研究

目的研究儿童流感嗜血杆菌（HI）对氨苄青霉素的耐药状况，并从分子生物学的角度研究其耐药机制。方法对儿童痰标本及咽拭子中分离的55株HI菌株进行β-内酰胺酶检测，PCR扩增β-内酰胺酶基因TEM-1、ROB-1的编码基因，并进行核酸序列测定与分析。结果分离的55株HI有17株产生β-内酰胺酶，并对氨苄青霉素耐药，耐药率30．9％。未发现β内酰胺酶阴性而对氨苄青霉素耐药的菌株。PCR法检测产酶HI中β-内酰胺酶基因TEM-1阳性率为100％，不产酶HI中无TEM-1阳性者。在32株菌株（17株产酶菌和15株不产酶菌）中未检测到ROB-1基因。结论流感嗜血杆菌是急性下呼吸道感染的主要病原体之一，对氨苄青霉素的耐药率较高。流感嗜血杆菌对氨苄青霉素的耐药主要由质粒介导的TEM-1型β-内酰胺酶所致。     

东莞地区健康学龄前儿童流感嗜血杆菌携带株耐药性研究

目的了解东莞地区学龄前健康儿童鼻咽部携带流感嗜血杆菌(Hi)状况及对常用抗生素的耐药情况。方法对546名学龄前健康儿童鼻咽部双份标本培养出的Hi107株做药敏试验。结果Hi携带率为19.96%,β-内酰胺酶阳性占9.3%,对氨苄西林不敏感率为11.2%。结论东莞地区学龄前健康儿童鼻咽部Hi携带率与国内各大城市无差异,耐药情况不甚严重。     

2005年至2006年南京地区儿童流感嗜血杆菌耐药性分析

目的:研究我院2005年至2006年儿童感染流感嗜血杆菌(Hi)对常用抗生素的敏感性、产β-内酰氨酶情况,以有效指导临床合理用药和预防。方法:临床标本进行流感嗜血杆菌分离培养,用Kirbry-Bauer(K-B)法和Etest法测定203株流感嗜血杆菌对8种抗生素的耐药率,以Nitrocefin纸片法测定β-内酰氨酶。结果:流感嗜血杆菌β-内酰氨酶总产生率为44.83%;流感嗜血杆菌对头孢他啶、头孢噻肟、头孢曲松、美洛培南、氧氟沙星、阿奇霉素耐药率分别为7.87%、10.34%、7.39%、7.87%、2.96%和7.39%;对氨苄西林、复方磺胺甲基异噁唑的耐药率分别为49.75%和51.23%。结论:南京地区儿童呼吸道感染流感嗜血杆菌耐药形势严峻,与该地区前2年资料比较,流感嗜血杆菌对氨苄西林和复方新诺明耐药率明显升高,产β-内酰氨酶是流感嗜血杆菌对氨苄西林耐药的重要机制。对头孢他啶、头孢噻肟、头孢曲松、美洛培南、阿奇霉素有较高的敏感性。     

流感嗜血杆菌抗生素敏感性检测及方法比较

目的 :检测北京儿童医院流感嗜血杆菌的抗生素敏感性 ,比较纸片扩散与E -test两种检测方法的结果。材料和方法 :选择 2 0 0 0年 4至 6月间从北京儿童医院门诊就诊的 1月～ 5岁的上呼吸道感染儿童鼻咽部分离培养出的流感嗜血杆菌 10 5株。检测抗生素有 :氨苄青霉素、阿莫西林 /克拉维酸、头孢克洛、头孢呋辛及头孢曲松。同时使用纸片扩散法和E -test方法。结果 :有 5株 (4.8% )流感嗜血杆菌产 β -内酰胺酶 ,对氨苄青霉素耐药。头孢克洛敏感率 98.1% ;所有菌株对阿莫西林 /克拉维酸、头孢呋辛及头孢曲松敏感。两种方法检测结果一致率除头孢曲松为 91.4 %外 ,均大于 99%。结论 :北京儿童医院流感嗜血杆菌的抗生素耐药率处于相对较低的水平。在严格按照规程操作的情况下 ,纸片法与E—test方法的结果有很好的一致性。     

老年人流感嗜血杆菌相关呼吸道疾病研究进展

老年人咽部携带的流感嗜血杆菌(Hi)与Hi疾病密切相关,在机体免疫力下降等情况下可下移引起各种呼吸道感染。本文对老年人咽部Hi携带、Hi相关呼吸道疾病及疫苗研究情况作一概述。     

不可分型流感嗜血杆菌外膜蛋白P6基因真核质粒的构建及表达

目的:构建不可分型流感嗜血杆菌(nontypeableHaemophilus influenzae,NTHi)外膜蛋白P6(outermembrane protein P6,P6)真核表达的重组质粒,并在HeLa细胞中表达,为核酸疫苗的开发奠定基础。方法:以NTHi基因组为模板,扩增编码P6蛋白的基因片段,酶切、纯化P6基因与真核载体pcDNA3.1/HisA后进行连接,之后转化并筛选含有目的基因的重组质粒pcDNA3.1/HisA-P6;将重组子转染至HeLa细胞,荧光蛋白法检测其表达产物。结果:经质粒PCR、酶切、测序证实插入的基因片段为NTHi-P6蛋白编码基因;荧光显微镜下显示,该重组质粒能够在HeLa细胞中表达目的蛋白。结论:成功地构建了真核重组质粒pcD-NA3.1/HisA-P6,并在HeLa细胞中表达。     

Effect of pneumococcal conjugate vaccine on prevalence of otitis media with effusion among children in Vietnam

PurposeOtitis media with effusion (OME) is common in young children and is associated with Streptococcus pneumoniae infection. We aimed to determine the impact of pneumococcal conjugate vaccine (PCV) introduction on the prevalence of OME and OME associated with vaccine-type (VT) or non-VT.MethodsPopulation-based cross-sectional surveys were conducted in pre- (2016) and post-PCV periods (2017, 2018, and 2019) at selected communes in Nha Trang, Vietnam. For each survey, we randomly selected 60 children aged 4–11 months and 60 aged 14–23 months from each commune. Nasopharyngeal sample collection and tympanic membrane examination by digital otoscope were performed. S. pneumoniae was detected and serotyped by lytA qPCR and microarray. Odds ratios (OR) and 95% confidence intervals (CIs) were calculated using Firth’s logistic regression, stratified by age group.ResultsOver the four surveys, 2089 children had a bilateral ear examination. Compared to pre-PCV, the prevalence of OME reduced in 2018 (OR 0.51, 95 %CI 0.28–0.93) and in 2019 (OR 0.53, 95 %CI 0.29–0.97) among the <12-month-olds, but no significant reduction among the 12–23-month-olds. The prevalence of OME associated with VT pneumococcus decreased in 2018 and 2019 (2018: OR 0.14, 95 %CI 0.03–0.55; 2019: OR 0.20, 95 %CI 0.05–0.69 in the <12-months-olds, 2018: OR 0.05, 95 %CI 0.00–0.44, 2019: OR 0.41, 95 %CI 0.10–1.61 in the 12–23-months-olds). The prevalence of OME associated with non-VT pneumococcus increased in the 12–23-month-olds in 2017 (OR 3.09, 95 %CI 1.47–7.45) and returned to the pre-PCV level of prevalence in 2018 and 2019 (OR 0.94, 95 %CI 0.40–2.43 and 1.40, 95 %CI 0.63–3.49).ConclusionPCV10 introduction was associated with a reduction of OME prevalence in infants but not in older children.     

A detailed analysis of possible efficacy signals of NTHi-Mcat vaccine against severe COPD exacerbations in a previously reported randomised phase 2b trial

BackgroundAn investigational vaccine containing non-typeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat) surface proteins did not show vaccine efficacy (VE) against combined moderate and severe (moderate/severe) exacerbations in a randomised, observer-blinded, placebo-controlled phase 2b trial of patients with chronic obstructive pulmonary disease (COPD). Nevertheless, observations on rates of severe exacerbations and hospitalisations encouraged further evaluation.MethodsPatients with stable COPD (moderate to very severe airflow limitation, Global Initiative for Chronic Obstructive Lung Disease [GOLD] stage 2–4), 40–80 years and at least one moderate/severe exacerbation in the last year received two doses of NTHi-Mcat vaccine or placebo plus standard care. Secondary analyses were conducted on VE against exacerbations according to severity. Potential predictive factors at baseline for VE against severe exacerbations were explored in post-hoc analyses.ResultsOf 606 patients enrolled, 571 were included in the efficacy analysis (279 in NTHi-Mcat vaccine group, 292 in placebo group). VE against severe acute exacerbations of COPD (AECOPD) in various subgroups was 52.11 % (p = 0.015; frequent exacerbators), 65.43 % (p = 0.015; baseline GOLD grade 4), 38.24 % (p = 0.034; previous pneumococcal and/or influenza vaccination). VE was 52.49 % (p = 0.044) for the 6–12 months period after 1 month post-dose 2. Multivariable analysis identified two factors (frequent exacerbator status plus inhaled corticosteroid use at baseline) associated with significant VE against severe AECOPD; in this subpopulation, VE was 74.99 % (p < 0.001).ConclusionResults suggest potential efficacy with the NTHi-Mcat vaccine against severe exacerbations in certain patients with COPD, in particular those who have frequent exacerbations and use inhaled corticosteroids. This potential signal requires confirmation in an appropriately designed prospective clinical trial.Trial registrationClinicalTrials.gov, NCT03281876.