产氢细菌Enterbacter sakazakii HP的分离及产氢特性

在自然环境中分离到一株具有高产氢活性的微生物菌株,经细菌鉴定仪及16S rRNA基因序列分析,鉴定该菌株为Enterbacter sakazakii HP。分析了起始pH值、反应温度、碳源、起始糖浓度、起始氧浓度及菌体密度等因素对菌株产氢活性的影响。研究表明,该菌株发酵产氢较适合的条件为:以葡萄糖为产氢底物,起始pH值8.0,菌体密度OD600=0.7,反应温度35℃,糖浓度为0.1mol/L,氧浓度为0%的条件下,此时产氢菌株的最高产氢活性为5.34μmolH2/h.mgdw,氢的得率为1.94molH2/mol葡萄糖。

Insights into Cronobacter sakazakii Biofilm Formation and Control Strategies in the Food Industry

Cronobacter sakazakii(C.sakazakii)is a foodborne opportunistic pathogen that can cause life-threatening invasive diseases,such as necrotizing enterocolitis,meningitis,and sepsis in infants.The potential risk of C.sakazakii contamination of powdered infant formula(PIF)is an issue that has attracted considerable attention from manufacturers,regulators,and consumers.C.sakazakii biofilms on the surfaces of equipment and in diverse food-production environments constitute a mode of cell growth that protects the pathogen from hostile environments,and are an important source of persistent contamination of food products.Bacterial biofilms are difficult to remove due to their resistant properties.Conventional cleaning and sterilizing procedures may be insufficient for biofilm control,and may lead to further biofilm development and dispersal.Consequently,novel control strategies are being developed,such as nanotechnology-based delivery systems,interspecies interactions,antimicrobial molecules of microbial origin,natural extracts,and phages.This review focuses on describing the mechanisms underlying the biofilm formation and behavior of C.sakazakii and discussing potential control strategies.

Examine the Correlation between Heat Shock Protein IbpA and Heat Tolerance in Cronobacter sakazakii

We used a proteomic approach to identify IbpA in Cronobacter sakazokii （C. sakazaki）, which is related to heat tolerance in this strain. The abundance of IbpA in C. sakazakii strains strongly increased after heat shock. C sakazakii CMCC 45402 ibpA deletion mutants were successfully constructed. The C. sakazakii CMCC 45402 AibpA and wild-type strains could not be distinguished based on colony morphology on LB agar plates or biochemical assays. The growth of the C. sakazakii CMCC 45402 AibpA mutant in heat shock conditions was indistinguishable from that of the isogenic wild-type, but showed greater heat resistance than E. coil O157：H7 strain CMCC 44828. This study suggests that the absence of a single ibpA gene has no obvious effect on the phenotype or heat resistance of the strain C. sakazakii CMCC 45402.

Detection and Molecular Characterization of <i>Cronobacter sakazakii</i>Isolated from Powdered Infant Formula (PIF) from North Central Region, Nigeria

Cronobacter sakazakii is an emerging ubiquitous and opportunistic pathogen that currently contaminates a wide spectrum of foods including powdered milk and poses a lethal threat to neonates, the elderly and persons with immune deficiencies. They cause life threatening neonatal meningitis, septicemia, and necrotizing enterocolitis. A total of 360 samples of powdered infant formula were collected from postnatal hospital attendees reconstituting the PIF for their children in the North Central region of Nigeria where cases of infant mortality are very high and presenting as enterocolitis and diarrhea. Pre-enriched samples were cultured in chromogenic Cronobacter broth and were then further sub-cultured into a chromogenic Cronobacter sakazakii agar. They were positive, exhibiting yellowish cultures typical of Cronobacter sakazakii. Biochemical tests of the isolates were also carried out and indicated the presence of Cronobacter sakazakii. The isolates were then characterized molecularly using specie specific PCR detection of Cronobacter sakazakii. The targeted genes of interest were ompA gene and CPA gene. The isolates tested showed bands for ompA gene on electrophoresis imager and were confirmed as Cronobacter sakazakii. In Nigeria, majority of infants are still fed with PIF. There is no existing data on the detection of Cronobacter sakazakii previously reported in the North central region of Nigeria hence the need to carry out the present study. The result of the study demonstrated the need for effective prevention and control measures as contamination of PIF with Cronobacter sakazakii constituted potential public health risk to neonates and infants.

Establishment of Real-Time Quantitative PCR Method for the Determination of Transposon Copy Number in Cronobacter sakazakii

[Objective] This study aimed to establish a Real-Time quantitative PCR method for the determination of transposon copy number in C. sakazakii. [ Method ] With single-copy housekeeping gene atpD as the reference gene, recombinant plasmid containing both single-copy housekeeping gene atpD and EZ-TN5 transposon was constructed; based on the established standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon, copy number of atpD gene and EZ-TN5 transpason in three C. sakazakii mutants was detected and the ratio was calculated. [ Result] Correlation coefficients of the standard curves for real-time quantitative detection of atpD gene and EZ-TN5 transposon were 0. 999 and 0.998, respectively ; the ratios of copy number of atpD gene and EZ-TN5 transposon in three C. sakazakii mutants were 0.98, 1.17 and 0.91, respectively, which indicates that EZ-TN5 transpeson in C. sakakii mutants is a single-copy. [ Conclusion] Real-time quantitative PCR method established in this study had high availability and could replace the Southern blot method to detect the copy num- ber of EZ-TN5 transposon in different bacteria.

Detection of <i>Enterobacter sakazakii</i>from Commercial Children Dry Milk

This study included isolation and identification of E. sakazakii from 51 different samples of powdered infant formula milk involved (Dialak 1 & 2, Celia 1 & 2, Primalac, Biomil 1 & 2, Similac, Nictalia 1 & 2, MAMi, Novolac (AD), Novolac (AR), Novolac (Allernova), S-26 AR, Nursoy, S-26 PDF gold. The results showed that one batch from three of the batches identified of Novolac and Dialak were contaminated and all the types of other infants were non-contaminated. The strains code given as (E1, E2, E3, E4);the bacteria showed resistance to antibiotics used was cephalosporin batch;the third generation showed sensitive to antibiotics life results through inhibition processes such as (Cefotaxime, Sifutetan and Siftadizim), where the diameters of inhibition zone for Siftadizim (18 mm), Sifutetan (22 mm) & Cefotaxime (25 mm) confirmed the bacteria by API and Vitek Compact- 2 (biomero).

Application of GFPuv Labeled Cronobacter sakazakii for Evaluation of Its Survival during Cornstarch Processing

Cronobacter sakazakii is an emerging pathogen that can cause diseases for several infant groups. These bacteria were contaminated in foods, clinical utensils, and environments. In Indonesia, C. sakazakii has been isolated from powdered infant formulas, weaning foods, and other dried foods such as cornstarch. The objective of this research is to trace survival of C. sakazakii during cornstarch production step using its mutant. Mutant was constructed by inserting Green Fluorescent Protein plasmid inside to the bacterial cell that appeared green fluorescent colonies under UV observation. The presence of C. sakazakii during processing was conducted by artificial contamination. This research consists of three steps, i.e. determination of the suitable enumeration method of C. sakazakii’s mutant, cornstarch production from yellow corn, and survival analysis of C. sakazakii during endosperm soaking and cornstarch drying. The suitable enumeration method was surface plating method on TSA-ampicillin medium combining with UV light application because of ineffectiveness of ampicillin inhibition for growth of yeasts and molds. The cornstarch produced in laboratory has the same properties with commercial cornstarch in parameters of moisture content, density, and starch granule structure. The yield of cornstarch final product was 48.90% (dry whole kernel-based). C. sakazakii cannot survive in 48 hours soaking process at 52?C and 24 hours drying process at 50?C that is applied during cornstarch production.

Occurrence of Enterobacter sakazakii(Cronobacter)and Other Enterobacteriaceae in Commercial Powdered Infant Formula in Abidjan,Ivory Coast

To determine the occurrence of Enterobacter sakazakii and other Enerobateriaceae in commercial powdered infant formula (PIF), 185 packages of PIF from different manufacturers, supermarkets and drug-stores in Abidjan were analyzed. Ten g of sample was homogenized in 90 ml of buffered peptone water (PBW, Biorad, Paris) for further studies. Enterobacteriaceae (coliforms) were enumerated according to French Association of Standardization methods. E. sakazakii was detected according to Kandhai’s method. Bacteria were identified using API20 system. Thirty-eight samples (20.5%) were positive for Enterobacteriaceae. Twenty-four samples (13%) yielded Enterobacter sakazakii. Other Enterobacteriaceae isolated included Pantoea spp. 21 (11.5%), Klebsiella pneumoniae subsp. Pneumonia 8 (4.3%), Citrobacter diversus 1 (0.5%), Citrobacter freundii 1 (0.5%), Enterobacter cloacae 1 (0.5%), Salmonella reading 1 (0.5%), Serratia ficara 1 (0.5%) Serratia odorifera 1 (0.5%). This study is the first report to describe the contamination of PIF from Abidjan with E. sakazakii and several other Enterobacteriaceae that could be opportunistic pathogens. Therefore, well-controlled studies need to be conducted to assess the extent of risk associated with contaminated PIF for infants in Abidjan.

上海市某区生菜中阪崎克罗诺杆菌的半定量风险评估

为了评估上海市某区居民通过生菜暴露阪崎克罗诺杆菌(Cronobacter sakazakii)的健康风险,本文按照微生物风险评估的程序,应用半定量风险评估工具Risk Ranger和sQMRA软件,结合居民膳食消费量数据进行评估。预计上海市某区健康成人每人每天因进食污染了阪崎克罗诺杆菌的生菜而患病的概率为1.66×10^(-9),每年发病7.63×10^(-3)例,风险系数为9,健康成人的发病概率很低。说明上海市某区即食生菜中阪崎克罗诺杆菌危害为低风险,可维持现有风险控制措施,但需严格执行生产过程风险监测,制定安全事件发生应急预案,并加强对消费者家庭食品安全的科普宣传。本研究为生菜中致病微生物风险评估补充数据,可供定量风险评估和食源性疾病的防控提供科学参考。

阪崎克罗诺杆菌双抗夹心酶联免疫吸附检测方法的建立

为建立阪崎克罗诺杆菌(Cronobacter sakazakii)的免疫学快速检测方法,分别制备了阪崎克罗诺杆菌的特异性单抗和多抗,建立阪崎克罗诺杆菌的双抗夹心酶联免疫吸附DAS-ELISA检测方法。结果:获得4株抗阪崎克罗诺杆菌单克隆抗体阳性细胞株,分泌抗体效价分别是1∶128000、1∶32000、1∶256000和1∶256000,抗阪崎克罗诺杆菌兔多克隆抗体效价是1∶128000。建立的阪崎克罗诺杆菌DAS-ELISA检测方法,分别以抗阪崎克罗诺杆菌兔多抗和单抗作为捕获抗体和检测抗体,兔多抗的最佳稀释度为1∶4000,单克隆抗体最佳稀释度为1∶1000,灵敏性可达1×106 CFU/mL;该检测方法具有良好的特异性,与常见的6种食源性病原菌都没有交叉反应,且能够同时检测6种克罗诺杆菌属细菌;重复性结果显示,该检测方法的批内批间变异系数均小于15%,具有良好的重复性。综上,利用抗阪崎克罗诺杆菌的单抗和兔多抗成功建立了DAS-ELISA方法,该方法具有良好的敏感性、特异性和重复性,可为阪崎克罗诺杆菌的快速检测提供技术支撑。

基于杂交链式反应扩增检测奶粉中阪崎肠杆菌的适配体磁珠荧光传感器

构建一种基于杂交链式反应(hybridization chain reaction,HCR)扩增的适配体磁珠荧光传感器。巧妙设计序列HP和发卡序列H1、H2,其中HP是由适配体序列与触发序列结合而成的,并且序列互补形成稳定的二级结构。然后采用戊二醇反应和亲和素-生物素反应进行适配体功能化磁珠的制备。将阪崎肠杆菌与适配体磁珠一起孵育,HP中的适配体序列识别靶标,引起HP构象变化,露出触发序列,通过HCR触发H1和H2的链状组装,产生长双链DNA。荧光指示剂SYBR Green I以插层和小槽结合的方式与HCR产物的长双链结合。最后加入氧化石墨烯(graphene oxide,GO)后,游离的H1、H2和SYBR Green I将通过π-π堆积紧密吸附在GO表面,荧光信号被猝灭。HCR产物不能被吸附在GO表面,因此与HCR产物结合的SYBR Green I发出依赖于靶浓度的强荧光信号,从而实现阪崎肠杆菌的定量检测。本方法在纯培养条件下的检出限为2CFU/mL,对奶粉的检出限为8CFU/g,对奶粉样品的检测结果与传统微生物培养法具有良好的一致性。该方法具有无需DNA提取,快速、稳定性高、高特异性和高灵敏度等优点,因此为阪崎肠杆菌的现场快速检测提供了一种很有潜力的方法。

鸡源阪崎克罗诺杆菌的分离鉴定和生物学特性分析

为确定新疆阿克苏地区某鸡场鸡只精神不振、采食量减少和死亡的原因,本试验对采集自该鸡场病死鸡的肝脏等器官进行病原的分离鉴定、形态学观察、生化鉴定和16S rRNA基因PCR鉴定,通过PCR检测毒力基因的携带情况,并进行动物回归试验和药物敏感性试验以分析其致病力和耐药性。结果显示,从病死鸡内脏器官中分离到1株革兰阴性短杆菌,鉴定该分离菌株为阪崎克罗诺杆菌(Cronobacter sakazakii);该分离菌株携带sip和ompX两种毒力基因;在3~7 h处于对数生长期;可感染雏鸡,并引起雏鸡严重脱毛和死亡,死亡雏鸡以肝脏肿大出血和肺脏有出血点为主要病理变化,与自然发病鸡症状一致;对替加环素、阿米卡星和多黏菌素B表现为敏感,对多西环素、链霉素和新霉素表现为中介,对其他18种抗生素表现为耐药。结果表明,分离菌株对动物具有潜在的致病性。本试验首次在病死鸡中分离出阪崎克罗诺杆菌,扩大了鸡体内致病微生物的种类,并对该菌的生物学特性进行了初步探究,以期对动物源阪崎克罗诺杆菌感染的临床用药和防控提供参考。

婴幼儿配方粉中阪崎肠杆菌分离株的药敏分析

目的研究中国婴幼儿配方粉中阪崎肠杆菌分离株的药物敏感性和耐药性。方法采用纸片扩散法测定了16株婴幼儿配方粉阪崎肠杆菌分离株对亚胺培南、环丙沙星、阿米卡星、头孢他啶等6大类28种抗生素的药敏结果。结果所有阪崎肠杆菌均对万古霉素、苯唑西林和青霉素G耐药,除1株阪崎肠杆菌之外,其他菌株同时也对头孢唑林和头孢泊肟耐药;所有菌株对13种以上的抗生素敏感。结论加强对预防用药和临床用药的重视,以减少阪崎肠杆菌多重耐药性的增加和经验用药的失误。

奶粉中阪崎肠杆菌的风险评估

阪崎肠杆菌能引起脑膜炎、NEC和菌血症等,约2.5%～14%婴儿配方奶粉含有阪崎肠杆菌,0%～12%的普通奶粉含有阪崎肠杆菌,含量从0.36～66.0cfu/100g。婴幼儿奶粉中的阪崎肠杆菌问题已受到了全世界的普遍关注。本文基于大量研究和调查数据,从奶粉中阪崎肠杆菌的危害识别、奶粉中阪崎肠杆菌的暴露评估、奶粉中阪崎肠杆菌危害特性以及阪崎肠杆菌的检测方法等方面客观地对奶粉中阪崎肠杆菌进行风险评估,并对降低我国婴儿配方奶粉中阪崎肠杆菌风险提出建议。

阪崎肠杆菌的生物学特性及其检测技术

阪崎肠杆菌(Enterobactersakazakii)是寄生于人和动物肠道的条件性肠道致病菌,能引起新生儿脑膜炎,致死性小肠结肠炎以及菌血症等,具有产生α-葡萄糖苷酶和吐温80脂酶、不发酵三梨醇以及无磷酸胺酶活性等生理生化特征,并且对干燥、渗透压和抗生素等有很强的抗性。该菌自然来源非常广泛,在水、土壤、植物根茎、动物肠道甚至加工食品都可存在,其中婴儿配方奶粉是婴儿感染阪崎肠杆菌的主要渠道。近年来基于α-葡萄糖苷酶反应的特异性生化检测和PCR扩增的分子检测技术已取得重要进展,但是目前还缺乏一种稳定检测该菌的分子分型检测技术。

阪崎克罗诺肠杆菌致病性机理研究进展

阪崎克罗诺肠杆菌是一种革兰氏阴性无芽孢肠道杆菌,可导致新生儿和免疫功能不全的婴幼儿严重脑膜炎、菌血症和坏死性结肠炎,是一种非常重要的食源性条件致病菌。目前,国内外对阪崎克罗诺肠杆菌的致病性及其致病机理的研究报道非常有限。本文通过阪崎克罗诺肠杆菌外膜蛋白A、脂多糖、生物膜、宿主细胞骨架、铁获得机制、海藻糖的存在、超广谱β-内酰胺酶的产生和细胞间相互作用等方面对阪崎克罗诺肠杆菌的致病性机理进行综述。

克罗诺杆菌检测方法研究进展

克罗诺杆菌(Cronobacter spp.)是一类重要的食源性致病菌,其引起的奶粉安全事件和新生儿致病问题受到了社会的高度重视。随着对该菌研究的深入和现代分类技术的不断发展,克罗诺杆菌的分类经历了从种到属的变化。但是截至目前,克罗诺杆菌的污染源和致病机制仍不清楚,为了防止易感人群的大规模疾病暴发,对于该菌的检测和预防尤为重要。本文对克罗诺杆菌的生化检测和分子检测的发展历程及存在问题进行综述,以期为该菌的研究提供参考。

阪崎肠杆菌脉冲场凝胶电泳分型及耐药研究

目的:研究北京、新疆、广东、辽宁等地和新西兰、美国、印度进口食品中分离的阪崎肠杆菌(ES)分子型别和耐药情况。方法:用限制性内切酶XbaⅠ酶切ES染色体DNA进行脉冲场凝胶电泳(PFGE)实验,用BioNumerics软件对不同地区分离的ES进行比对,分析菌株之间的相似度。用Vitek2进行药敏实验,分析ES耐药情况。结果:38株ES(其中ES12为标准菌株)分成37个PFGE型,相似度在25%～100%,未表现出优势带型和地区特异性,而部分食品被遗传紧密相关的ES克隆株污染。药敏实验结果显示37株ES共有9种耐药谱,部分菌株对呋喃类、头孢类、β-内酰胺类耐药。结论:建立的PFGE方法可应用于ES的分子分型和溯源。

利用环介导等温扩增技术对奶粉中阪崎肠杆菌进行检测

利用阪崎肠杆菌16S-23S rDNA间区(ITS)序列,在比较阪崎肠杆菌与其近源株ITS序列的基础上,设计了4条阪崎肠杆菌LAMP检测特异性引物,建立了奶粉中阪崎肠杆菌LAMP检测方法。用15株阪崎肠杆菌,61株近源菌验证试验表明,所建立的LAMP方法准确且灵敏度高。加菌试验表明,奶粉样品中阪崎肠杆菌检测低限为1.2CFU/100g。新建的LAMP方法与FDABAM方法比较试验表明,两种方法的检测结果完全符合。

傅里叶变换近红外技术在乳制品微生物鉴别中的应用研究

乳制品的质量安全问题受到广泛的关注。为快速、准确判定乳制品污染源,利用傅里叶变换近红外光谱技术采集被阪崎肠杆菌、金葡萄球菌、大肠杆菌三种致病菌污染的牛奶样品的近红外透射光谱(NP),使用一阶求导(FD),标准正态变量变换(SNV),多元散射校正(MSC)对光谱进行预处理,结合偏最小二乘判别分析(partial least squares-discriminant analysis,PLS-DA)对三种细菌判别的可行性进行探究。研究表明,利用FD与SNV预处理方法得到PLS-DA模型判别结果均比NP差,而利用MSC预处理后准确率与NP一致达到了100%,且预测集相关系数(Rp)比NP高,预测均方根误差(RMSEP)更小,表明模型在经过MSC预处理后预测性能更理想。阐明利用FT-NIR技术结合化学计量学方法经过合适预处理方法后能有效用于乳制品中微生物类别的鉴别。