多重PCR方法快速检测4种主要致腹泻性大肠埃希菌

肠毒素性大肠埃希菌(ETEC)、肠致病性大肠埃希菌(EPEC)、肠出血性大肠埃希菌(EHEC)和侵袭性大肠埃希菌(EIEC)是引起腹泻的主要大肠埃希菌,威胁着食品安全和人类健康,建立同时检测4种致腹泻性大肠埃希菌的方法具有重要意义。基于ETEC LT肠毒素基因、EPEC bfpA基因、EHECO抗原基因和EIEC侵袭性质粒特异性基因,设计了4对特异性引物,通过对单一PCR反应条件的优化建立了快速检测4种主要致腹泻性大肠埃希菌的多重PCR方法,彼此之间无交叉反应。该多重PCR方法具有良好的特异性和灵敏性,对24株致病菌进行检测,所试4株致腹泻性大肠埃希菌均为PCR阳性,其他菌株则为阴性。实践证明,利用所建立的多重PCR方法对124份肉类、奶类制品及人工污染样品等进行检测,检出15份阳性,与国标(GB4789.6-1994)检测结果相同。结果表明本文建立的多重PCR方法可用于ETEC、EPEC、EHEC和EIEC的单一或混合感染的鉴别诊断及食品安全风险评估,具有良好的实用性。     

大肠埃希菌O157及其检验

以往,对食品中的大肠埃希菌检验主要是从食品卫生指标角度进行。通过对大肠埃希菌的检验,以确定食品受粪便污染的程度,而O157：H7大肠埃希菌系临床腹泻病原菌,是致病性大肠埃希菌的重要成员。近年,由该菌引起的食物中毒来势颇为凶猛,深受世界各国政府所重视。本文试对该菌的有关情况做一简述。1腹泻原性大肠埃希菌的分类和引起的疾病（见表1）引起腹泻的大肠埃希菌,已知有肠致病性大肠埃希菌（EPEC）、肠侵袭性大肠埃希菌（EIEC）、产肠毒素大肠埃希菌（EIEC与肠出血性大肠埃希菌（EHEC）或称产Vero毒素大肠埃希菌（VTEC）…     

致病性大肠埃希菌和沙门菌毒力岛基因的双重PCR检测方法的建立

目的实现对致病性大肠埃希菌（E．coli）、沙门菌（Salmonella）的同时检测,建立快速灵敏的双重PCR检测方法。方法以致病性大肠埃希菌和沙门菌毒力岛基因为研究对象,根据GenBank发表的大肠埃希菌和沙门菌毒力岛基因序列,分别设计合成了大肠埃希菌毒力岛irpl、irl）2和fyuA,沙门菌毒力岛mgtC、sseL和sopB等6对引物,以禽致病性大肠埃希菌（CVCC1565）菌株和沙门菌（ATCC9150）菌株的核酸混合物为模板,经引物特异性试验,引物组合,成功建立了快速鉴别检测致病性大肠埃希菌和沙门菌的双重PCR方法。结果特异性试验结果显示,引物irpl、irp2和fyuA仅能扩增出大肠埃希菌（CVCC1565）的特异性片段,大小分别是799、414和948bp;引物mgtC、sseL和sopB仅能扩增出沙门菌（ATCC9150）的特异性片段,大小分别是500、269和1000bp。敏感性试验结果表明大肠埃希菌和沙门菌的最低检测限分别为2．2×101CFU／mL和2．0×101CFU／mL。结论本研究建立的双重PCR方法具有特异性强、敏感性高、快速简便等特点,可用于致病性大肠埃希菌和沙门菌的联合检测与鉴别诊断。     

2株乳酸菌对人工诱发鸡大肠埃希菌病的预防性实验

目的观察植物乳杆菌和粪链球菌2株乳酸菌预防鸡大肠埃希菌病的效果。方法把2株乳酸菌添加到肉鸡的饲料中饲喂,至14日龄时用鸡源致病性大肠埃希菌人工诱发鸡大肠埃希菌病,10 d后统计发病率、死亡率和有效预防率。结果成功诱发出鸡大肠埃希菌病,植物乳杆菌和粪链球菌预防鸡大肠埃希菌病的有效率非常高。结论植物乳杆菌和粪链球菌可以用于预防鸡大肠埃希菌病。     

合肥地区动物源性大肠埃希菌的血清型分布与耐药性分析

目的了解安徽省合肥地区动物源性大肠埃希菌的血清型分布和耐药状况,以期筛选出菌苗株和指导临床合理用药。方法对46份疑似大肠埃希菌病病料进行细菌分离培养、生化编码鉴定和致病性测定。采用玻片凝集试验对分离到的46株致病性大肠埃希菌进行血清型鉴定。同时分别采用K-B纸片琼脂扩散法和双纸片增效法检测致病性大肠埃希菌的耐药性和ESBLs阳性菌株。结果46株致病性大肠埃希菌中,除7株细菌未能定型外,其余39株细菌分布于10个血清型,O127：K63血清型为优势血清型,占定型菌株的33．33％。46株致病性大肠埃希菌对21种抗菌药物均呈现不同程度的耐药性,15个ESBLs阳性菌株表现为多重耐药,对各种抗菌药物的耐药率均高于ESBLs阴性菌株。结论O127：K63血清型为优势血清型,可作为菌苗株。合肥地区动物源性大肠埃希菌耐药性较为严重,尤其是产ESBLs大肠埃希菌多重耐药更为突出。     

妇科肿瘤患者尿液主要致病菌分布及大肠埃希菌耐药性的分析

目的探讨妇科肿瘤患者尿路感染情况。方法对688份尿液标本中主要致病菌分布及主要菌大肠埃希菌的药敏进行了总结和分析。结果尿液中主要菌为大肠埃希菌(59．3％),粪肠球菌(11．1％),铜绿假单胞菌(6．3％)。大肠埃希菌中产ESNLs菌株占51．8％,大肠埃希菌对青霉素类、头孢类耐药率均大于50％,对喹诺酮类耐药率大于70％,产ESBLs大肠埃希菌对氨苄西林。舒巴坦、阿莫西林-棒酸、替卡西林-棒酸耐药率很高,不适合用于临床治疗,对哌拉西林-他唑巴坦和头孢哌酮-舒巴坦敏感率均在90％左右。结论     

婴儿泌尿道感染的病原菌分布及耐药性分析

目的探讨婴儿泌尿道感染的病原菌构成及其耐药情况,以指导临床合理用药。方法对2012年5月至2013年7月某院门诊及住院婴儿送检的尿标本进行分离培养,用全自动微生物鉴定／药敏系统进行菌种鉴定和药敏试验,同时对大肠埃希菌和肺炎克雷伯菌进行超广谱β-内酰胺酶检测。结果婴儿泌尿道感染以大肠埃希菌检出率最高（46．24％）,其余依次是粪肠球菌（22．04％）、肺炎克雷伯菌（7．80％）、屎肠球菌（5．37％）、铜绿假单胞菌（4．30％）。大肠埃希菌和肺炎克雷伯菌超广谱β-内酰胺酶检出率分别为46．51％和58．62％。革兰阴性菌对亚胺培南、美罗培南的耐药率为0,对氨苄西林耐药率最高（〉80％）。革兰阳性菌对万古霉素耐药率为0,对苯唑西林、妥布霉素的耐药率100％。结论婴儿泌尿道感染病原菌以大肠埃希菌为主;大肠埃希菌、肺炎克雷伯菌超广谱β-内酰胺酶检出率高,应引起重视;临床医师要根据药敏结果指导用药,对危重病例和存在泌尿道畸形反复发作病例,革兰阴性菌可选用亚胺培南、美罗培南,革兰阳性菌可选用万古霉素。     

457株尿路感染的病原菌及其耐药性研究

目的了解尿路感染的病原菌分布及耐药情况,为临床合理用药提供依据。方法用VITEK-AMS微生物自动分析仪鉴定菌种和药敏试验,同时对大肠埃希菌和肺炎克雷伯菌进行ESBLs检测,对肠球菌进行氨基糖苷类高水平耐药（HLAR）的筛选。结果尿路感染以大肠埃希菌检出率最高（50．5％）其余依次为肠球菌属（10．5％）,克雷伯菌属（8．6％）,真菌（7．9％）等,大肠埃希菌和肺炎克雷伯菌ESBLs检出率分别为24．2％和17．9％,HLAR肠球菌检出率为60．4％．尿路感染病原菌对喹诺酮类等常用药物的耐药性较高。结论明确尿路感染的病原菌及其耐药性对合理使用抗生素具有重要意义。     

大肠埃希菌和肺炎克雷伯菌ESBLs检测及耐药性分析

由细菌超广谱β-内酰胺酶（ESBLs）引起的细菌耐药性一直是临床相关感染性疾病治疗中的棘手问题。从不同病区患者标本中分离了96株大肠埃希菌和80株肺炎克雷伯菌,分剐采用双纸片协同试验和药物敏感试验检测了上述菌株产生ESBLs情况及对17种抗生素的耐药性。结果发现,27．1％（26／96）的大肠埃希菌株和22．5％（18／80）肺炎克雷伯菌株产ESBLs。ICU病房分离的大肠埃希菌和肺炎克雷伯菌株ESBLs总阳性率（46．0％）与介入科病房和烧伤科病房分离菌株ESBLs总阳性率（28．6％和25．0％）无显著性差异（P〉0．05）,但明显高于呼吸科、骨科、其他病房及门诊部分离菌株ESBLs总阳性率（6．3％～14．3％,P〈0．01）。不产ESBLs大肠埃希菌株和肺炎克雷伯菌株对17种抗生素耐药率明显低于产ESBLs菌株。产ESBLs大肠埃希菌和肺炎克雷伯菌对氨曲南均敏感,对氨苄西林／舒巴坦、阿莫西林／棒酸、阿米卡星耐药率仅为15．8％-23．4％。上述实验结果提示,大肠埃希菌和肺炎克雷伯菌临床菌株中有较高的ESBLs阳性率,不同病区患者感染的大肠埃希菌和肺炎克雷伯菌ESBLs阳性率有很大差异,氨曲南、氨苄西林／舒巴坦、阿莫西林／棒酸、阿米卡星可作为治疗产ESBLs大肠埃希菌和肺炎克雷伯菌感染性疾病的首选药物。     

乳杆菌DM8909菌株抑制小鼠阴道感染的研究

目的 :探讨乳杆菌 DM880 9菌株对小鼠阴道内其他菌群的抑制及调整阴道菌群的作用。方法 :通过用阿莫西林钠溶液对小鼠阴道冲洗处理后 ,接种致病性大肠埃希菌 EPEC10 4 ,建立起大肠埃希菌在小鼠阴道内的定植 ;用乳杆菌 DM890 9菌液冲洗治疗来去除大肠埃希菌 EPEC10 4在小鼠阴道的定植。结果 :通过各组小鼠阴道冲洗液检出的平均对数值比较 ,乳杆菌治疗组的小鼠阴道内大肠埃希菌的数量 ( 3.82±0 .6 2 )较自然恢复组小鼠阴道内大肠埃希菌的数量 ( 6 .72± 1.16 )减少明显 ( P<0 .0 5 ) ,而且治疗组的症状好转率 ( 8/10 )显著好于自然恢复组的症状好转率 ( 2 /10 ) ( P<0 .0 5 )。结论 :乳杆菌 DM890 9菌株能有效去除大肠埃希菌 EPEC10 4在小鼠阴道的定植和改善小鼠外阴的炎症症状     

IgG antibodies to secretory component in normal human serum

While isolating free secretory component (FSC) by monoclonal antibody affinity chromatography, we demonstrated FSC-IgG complexes in human milk. We hypothesized that IgG antibody to secretory component (SC) might be transported into the milk from the serum. We therefore examined sera from 10 normal adults and 10 infants for IgG capable of binding to FSC in an enzyme-linked immunosorbent assay. Eight of 10 normal adult sera and nine of 10 infant sera demonstrated IgG binding to FSC with titers ranging from 1:54 to 1:4096. Quantitation of the IgG bound to FSC was hampered in adult sera by the binding of IgM and polymeric IgA to the FSC. Quantitation in five infant sera ranged from 0.5 to 6.4 micrograms/ml. A pepsin digest of an IgG fraction of serum demonstrated binding of the F(ab')2 fragments to the FSC. The specificity of the antibodies in human serum was evaluated by examining the binding to secretory IgA (sIgA) and FSC isolated from pooled human milk and polymeric IgA isolated from the ascitic fluid of a patient with an IgA myeloma. Eight of the 10 adults had antibody specific for FSC. Three of the eight, all female, also had antibody specific for sIgA. Two of the eight had antibody either to FSC and sIgA or to FSC plus an antibody that could bind to an epitope shared by sIgA and FSC. Competition experiments with monoclonal antibodies to human secretory component and sIgA were used to confirm and further define these specificities. The results of this study indicate that antibody to SC is common in normal adult and infant sera. The majority of antibodies seem to be directed against epitopes present on FSC but not on sIgA, which suggests sensitization to circulating or membrane-bound SC. The significance of these antibodies in normal human sera remains to be elucidated.     

Levels of cytokines (IL-1beta, IL-2, IL-6, IL-8, TNF-alpha) and trace elements (Zn, Cu) in breast milk from mothers of preterm and term infants

It has been well documented that human milk contains several immunomodulator components which are important during infant period when the newborn's immune system is still under development. In this study, we aim at examining levels of cytokines, zinc (Zn), and copper (Cu) in milk from mothers of premature and mature infants, and comparing changes during lactation periods consequently. Milk was collected from total of 40 mothers (group M: mothers of mature infants, n = 20; group PM: mothers of premature infants, n = 20) from four lactation stages: colostrum (0-7 days), transitional (7-14 days), mature milk (21 days), and mature milk (2nd month). Levels of cytokines (interleukin [IL]-lbeta, IL-2, IL-6, IL-8, tumor necrosis factor-alpha [TNF-alpha]) were determined by chemiluminesence method, whereas atomic absorption spectrophotometer was used for the determination of Zn and Cu levels. Cytokine levels were determined to be high in colostrum and transient milk from mothers of full-term infants, whereas their levels were reduced drastically in the 21st day and the 2nd month milk (P < .01, P < .001). Similar trends were observed in milk from mothers of premature infants, but cytokine levels were significantly lower in colostrum compared to colostrum from mothers of mature infants (P < .01). The differences in cytokine levels were continuous in transient milk (P < .05) and mature milk (21 days) (P < .05), whereas there was no statistically significant differences between milk from both groups of mothers in the 2nd month (P > .05). Zn levels in milk from mothers of premature infants were significantly lower compared to the ones from mothers of mature infants (P < .01) and these differences continued through the 2nd month. Although Cu levels were lower in milk from mothers of premature infants, there was no statistically significant difference except colostrum (P > .05). Our results clearly demonstrate that the level of immunomodulating agents such as cytokines and trace elements in milk from mothers of premature infants is less than the level of the same agents in milk from mothers of full-term infants. Although there are commercially available products for infant feeding, human milk is still the best natural nutrient for newborns. Therefore, when premature infants are breastfed, necessary precautions such as supplemantary diets must be considered for possible infections and risks related with immune system deficiency.     

Seric and secretory antibodies reactive to α, β and γ intimins of Escherichia coli in healthy Brazilian adults

Intimin is essential for attaching and effacing lesions by pathogens such as enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC), and the antigenic polymorphism of intimin determines distinct subtypes. Our aim was to investigate the presence of immunoglobulin G (IgG) and IgA antibodies reactive to α, β and γ intimins in serum and colostrum from healthy Brazilian adults. We found seric IgG and secretory IgA antibodies reactive to conserved and variable regions of α, β and γ intimins and a positive correlation between the concentrations of these antibodies in both serum and colostrum that suggested cross reactivity among anti-intimin antibodies, as was confirmed by immunoblotting and absorption. The concentrations of anti-conserved region antibodies were higher than those of variable region antibodies. The presence of antibodies reactive to EHEC antigens could result from contact with EPEC or with other bacteria of the environment even though this bacterium is not frequent in Brazil, and suggests possible protection against EHEC.     

Exploration of bovine milk proteome in colostral and mature whey using an ion-exchange approach

In addition to milk's nutritional role, it contains immunoglobulins (antibodies) and immunoregulatory proteins that are active in the digestive tract of newborns. However, knowledge of the repertoire of milk proteins remains meager. In this work, we report an ion-exchange-based protein fractionation method that allows in-depth exploration of the whey proteome in bovine milk; 293 unique gene products were identified, of which 176 were newly identified in whey. This work also demonstrated qualitatively for the first time the consistency, albeit differing in protein levels, in milk proteome between colostrum and mature milk (3 mo. post calving). Semiquantitative analysis showed a number of up-regulated proteins in colostrum that may provide extra natural defenses for the neonate. Increased understanding of the composition and functions of bovine milk proteins and their potential health benefits may, in the future, play an important role in nutritional and biomedical applications as properly processed cow's milk proteins could potentially confer the same bioactivity as their human counterparts.     

A sensitive solid phase radioimmunoassay for secretory IgA: its application for the determination of urinary levels of secretory IgA in children and adults

A sensitive solid phase radioimmunoassay method was established for the specific quantitative determination of secretory IgA (sIgA) by taking advantage of the dual antigenicities of sIgA, one specific for alpha-chain and the other for secretory component (SC). The sIgA and IgA in the sample were first bound by anti-IgA antibodies coated on the polystyrene tube, then the amount of bound sIgA was quantified by the use of 125I-labeled anti-SC antibodies. This method is quite sensitive and allows us to distinguish sIgA from IgA and free SC which usually coexist in exocrine secretions. Linear relationship was observed between the bound radioactivity of radioiodinated anti-SC and the amount of sIgA in the range of 5 to 60 ng of sIgA. With this method, the urinary sIgA levels in normal children and adults were measured. Urinary sIgA was detected in half of infants within 7 days after birth, while it could be demonstrated in almost all of the infants by 14th day. Then, it gradually increased and reached about a half of the adult level by the age of puberty. This method will be useful for the sensitive and specific measurement of sIgA in various exocrine secretions.     

Gel electrophoretic analysis of proteins in human milk and colostrum

Protein patterns of skim milk and fat globule phases were relatively unchanging throughout 9 months lactation, the only notable differences being a rise in lysozyme and a decline up to 6 months with increase thereafter in lactoferrin. Profound changes in protein patterns of the breast secretion were observed during the first week postpartum. True colostrum, in which peptides of sIgA dominate the protein patterns, exists for at most the 2 initial days of secretion. Proteins of mature milk are not coordinated to appear in the colostral secretion simultaneously. A band corresponding to alpha-lactalbumin is present from the initial secretion; that for beta-casein emerges approximately 2 days layer.     

双歧杆菌对SD大鼠空肠黏膜分泌型免疫球蛋白A的影响

目的研究双歧杆菌的免疫佐剂效应。方法将SD大鼠随机分成3组,即卵清白蛋白（OVA）组,双歧杆菌＋OVA组和PBS空白对照组。各组给予免疫后,收集长约3cm的空肠,通过免疫组织化学技术和Qwin图像处理系统,对空肠分泌型免疫球蛋白A（sIgA）的定位进行系统分析,研究同时注射OVA和双歧杆菌后对SD大鼠在不同时期空肠中sIgA阳性细胞分泌的影响。结果在免疫后的SD大鼠的空肠内均产生了特异性的分泌型抗体sIgA,其中双歧杆菌＋OVA组显著高于其他2组（P〈0．05）。结论双歧杆菌经胃肠道黏膜免疫可诱导有效的免疫佐剂效应。     

Detection and characterization of IgG-and sIgA-abzymes capable of hydrolyzing histone H1

Immunoglobulins IgG and sIgA actively hydrolyzing histone H1 have been detected on analyzing proteolytic activity of antibodies isolated by chromatography on Protein A-agarose from blood serum of patients with multiple sclerosis and from colostrum of healthy mothers. These antibodies hydrolyze other histones less actively and virtually failed to cleave lysozyme of chicken egg. By gel filtration at acidic pH and subsequent analysis of protease activity of chromatographic fractions, it was shown that IgG and sIgA molecules were responsible for hydrolysis of histone H1. Anti-histone H1 antibodies of IgG and sIgA classes were purified by affinity chromatography on histone H1-Sepharose from catalytically active antibody preparations. The protease activity of anti-histone H1 IgG antibodies was inhibited by serine proteinase inhibitors, whereas anti-histone H1 sIgA antibodies were insensitive to inhibitors of serine, asparagine, and cysteine proteases.     

Lipid kinase activity of antibodies from milk of clinically healthy human mothers

We have shown recently that polyclonal human milk sIgA contains a subfraction of antibodies (Abs) tightly bound to unusual minor milk lipids containing sialic acid. Here, we show that a small subfraction of milk IgG is tightly bound to the similar or the same minor lipids. The ability of small fractions of sIgA and IgG from human milk to phosphorylate selectively two minor lipids in the presence of [gamma-(32)P]nucleoside triphosphates was shown here for the first time to be an intrinsic property of these antibodies. In contrast to known kinases, antibodies with lipid kinase activity can transfer phosphoryl group to lipids not only from ATP but also from other different nucleotides (dATP, GTP, dGTP, UTP, TTP) with comparable efficiencies (30-100%). To our knowledge, there are no examples of enzymes using orthophosphate as a substrate of phosphorylation reactions. An extremely unusual property of lipid kinase Abs is their high affinity for orthophosphate (K(m)=1.6-5.6 microM) and capability to phosphorylate minor lipids using [(32)P]orthophosphate as donor of phosphate group. The relative specific activity and affinity of abzymes for orthophosphate and ATP depend significantly on donor milk. However, the levels of Ab-dependent phosphorylation of lipids for all Abs in the case of ATP (100%) and orthophosphate (60-80%) as substrates are comparable. The first example of natural abzymes with synthetic activity was milk sIgA with protein kinase activity. Most probably, lipid kinase sIgA and IgG of human milk are the second example of Abs with synthetic activity.     

Lipid composition and vitamin E content in human colostrum and mature milk

Lipidic components, as well as fatty acid composition and vitamin E content were determined in colostrum (days 3-5 of postpartum) and mature milk (day 21) in 8 women from Murcia (Spain). Triglycerides concentration was higher and cholesterol and esterified cholesterol were lower in mature milk than in colostrum, whereas phospholipid content was similar. These differences indicate that the diameter of milk fat globules increases in mature milk. The percentage of medium-chain fatty acids (12:0 and 14:0) increased in mature milk as compared to colostrum, reflecting de novo synthesis of fatty acids. With the only exception of stearic acid which was lower in mature milk than in colostrum, the remaining long-chain fatty acids was similar. The proportion of both linoleic (18:2) and eicosapentaenoic (20:5) acids found in mature milk and colostrum is higher than in studies from other countries and may reflect the intake of high proportions of polyunsaturated fat from vegetable oils and fish in the studied women. Both vitamin E content and vitamin E/linoleic acid ratio in mature milk are lower than in colostrum, evidencing the efficient mechanism of mammary gland vitamin E uptake around parturition.