肠杆菌科ppt课件

Synopsis of important definitions and characteristics of diarrhoeal diseases, based on recommendations put forth by the World Health Organization (WHO) and the Infectious Diseases Society of America (IDSA),肠杆菌科 (Enterobacteriaceae),第一节 埃希菌属 第二节 志贺菌属 第三节 沙门菌属 第四节 肠道杆菌科的其他菌属,肠杆菌科（Enterobacteriaceae）细菌：是一大类生物学性状相似的革兰阴性杆菌 ，常寄居在人和动物的肠道内，主要通过粪口途径进行传播。 分类学位置： Domain （界） : Bacteria Phylum（门）: Proteobacteria Class （纲） : Gamma Proteobacteria Order （目） : Enterobacteriales Family(科): Enterobacteriaceae Genus （属） : Escherichia Species （种） : E. coli,肠杆菌科中与医学有关的细菌,肠杆菌科细菌共同的生物学特征,形态结构 G 小杆菌，钝圆杆菌，无芽孢 多数有鞭毛及菌毛 培养特性 需氧或兼性厌氧 营养要求不高，普通培养基可生长，菌落相似，形成灰白色中等大小S型菌落。 肠道选择培养基,生化反应：活跃，一般非致病菌能分解乳糖，常用于鉴别肠道杆菌。 抵抗力不强，60 30min 即可灭活。 抗原结构复杂： 菌体（O）抗原 鞭毛（H）抗原 表面（K、Vi）抗原,细菌素：产生受质粒控制。大肠菌素、粘质沙雷菌素。 毒素与酶：LPS和外毒素。,Special selective and differential agar medium:,EMB agar plate SS agar plate KI agar medium MacCONKEY Agar,EMB agar plate:,Ingredients: Nutrient agar (PH7.6) 100 ml Lactose 1.00g 2Eosin- y 2ml 0.5methylene blue 1ml,E.coli on EMB,Principle: EMB is one of the usual media that is used to separate the pathogen and non-pathogen of the Enterobacteria. In EMB plate, the eosin and the methylene blue are the indicator. In acid condition, eosin and methylene blue combine into mauve (紫红色)or black production. The nonpathogen colony (e.g.E.coli) that can ferment lactose takes on mauve or black with metallic luster. The pathogen colony that cannot ferment lactose displays translucence.,EMB agar,Eosin methylene blue agar, as the name suggests, contains the dyes eosin and methylene blue. EMB agar is selective because the aniline dyes in this purple media inhibit growth of Gram-positive organisms. Lactose fermenters metabolize the lactose in the media and produce acid byproducts, causing a color change in the colony. Thus, EMB is also a differential medium. Strong acid production by organisms such as E. coli results in a metallic green sheen. Weaker fermentation of lactose results in colonies with a pinkish-purple color. Colonies of nonlactose fermenters remain colorless, or at least are no darker than the color of the media.,MacConkey Agar,MacConkey agar contains lactose, bile salts, neutral red and crystal violet. It is a selective media because Gram-positive organisms are inhibited by the bile salts and the crystal violet. When bacteria ferment lactose and produce enough acid products to reduce the pH below 6.8, the neutral red turns from colorless to red. Thus, MacConkey is a differential media on which lactose fermenting colonies appear red (or pink). Nonlactose fermenters are colorless.,SS(almonella Shigella Agar) agar plate:,Ingredients: Nutrient agar 100ml Lactose 1.00g Bile salts 0.85g inhibitor Sodium citrate 0.85g inhibitor Sodium thiosulfate 0.85g 10% ferric citrate 1ml 1% Neutral red 0.25ml indicator 1% brilliant green 0.033ml inhibitor,SS培养基（沙门志贺培养基） 乳糖、中性红指示剂、胆盐、煌绿等抑制剂,Principle: SS agar is often used to help isolate salmonellae and shigellae from other enteric gram-negative bacteria. Neutral red is the indicator. In acid condition it turns red, and it turns yellow in base condition.,E.coli that ferment lactose can produce acid so the colonies turns red . while other enteropathogen cannot ferment lactose, but can use peptone to produce base, so the colonies turns yellow.,KI (Kligler Iron Agar) agar medium:,Ingredients: Peptones 2.00 g NaCl 0.50g Glucose 0.10g Lactose 1.00 g ferrous sulfate 0.05g Sodium thiosulfate 0.075g Agar 0.50g 0.2 phenol red 1.25ml Distilled water 100ml,The medium can be used to obtain pure culture and primarily identify the suspect colonies of enteric bacteria. Phenol red is the indicator. When the colonies produce lots of acid from the fermentation of lactose, the media appear yellow completely. If the colonies only produce few of acid form the fermentation of glucose, only the bottom of the media appear yellow .For the combination of ferrous sulfate with H2S (sulfureted hydrogen) produced by the H2S-produced enterobacteria, the color becomes black. And if the colonies produce gas, there is bubble in the bottom of the tube.,MacCONKEY Agar,Typical Composition (g/litre) Peptone from casein 17.0; peptone from meat 3.0; sodium chloride 5.0; lactose 10.0; bile salt mixture 1.5; neutral red 0.03; crystal violet 0.001; agar-agar 13.5; 4-methylumbelliferyl-b-D- glucuronide 0.1. Experimental Procedure and Evaluation Inoculate plates in the usual way and incubate for 18 to 24 hours at 35 C aerobically. Lactose-negative colonies are colourless. Lactose-positive colonies are red and often surrounded by a turbid zone due to the precipitation of bile acids. Inspect the culture with UV light: light blue fluorescence denotes E. coli.,第一节 埃希菌属（Escherichia）,一、生物学性状 形态染色: G, 杆菌, 无芽胞 多数有周身鞭毛, 有菌毛,生物学性状,培养特性 符合肠杆菌特点 EMB 紫黑色有金属光泽 S-S 桃红色 生化反应 IMViC（+ + - - ） I 靛基质试验(吲哚实验) M 甲基红试验 Vi VP反应 C 枸橼酸盐利用试验,乳糖发酵菌落,E.coli 在EMB 紫黑色有金属光泽,抗原 O 抗原: 细胞壁脂多糖最外层的特异性多糖 （ O1-O173） H 抗原: 位于鞭毛上（H1-H56） 荚膜抗原: 多糖，位于O抗原外围，如大肠埃希菌的K抗原，伤寒沙门菌的Vi抗原。 K抗原（K1-K100） 血清型表示法：O：K：H 如肠出血性大肠杆菌O157:H7,The enterobacterial common antigen (ECA) occurs in three different forms: a phosphatidyl-linked linear polysaccharide anchored on the cell surface (ECAPG), a cyclic form built of 4-6 repeating units localized in the periplasm (ECACYC) a linear polysaccharide covalently linked to LPS core oligosaccharide (ECALPS). Postepy Hig Med Dosw (Online). 2015 Sep 8;69:1003-12. The structure and significance of enterobacterial common antigen (ECA). Article in Polish Godziewicz TK1, ukasiewicz J1, ugowski C2. Abstract The enterobacterial common antigen (ECA) is a carbohydrate-derived cell surface antigen present in all Gram-negative bacteria belonging toEnterobacteriaceae family. Biosynthetic pathways shared by ECA and LPS (endotoxin) suggest close connections between these antigens. ECA occurs in three different forms: a phosphatidyl-linked linear polysaccharide anchored on the cell surface (ECAPG), a cyclic form built of 4-6 repeating units localized in the periplasm (ECACYC) and as a linear polysaccharide covalently linked to LPS core oligosaccharide (ECALPS). Regardless of ECA form, poly- and oligosaccharides of ECA consist of the biological trisaccharide repeating units: 3)-d-Fucp4NAc-(14)-d-ManpNAcA-(14)-d-GlcpNAc-(1, where Fucp4NAc refers to 4-acetamido-2,4-dideoxygalactose, ManpNAcA to N-acetyl-mannosaminuronic acid and GlcpNAc to N-acetylglucosamine. ECAPG and ECALPS consisting of one unit with Fucp4NAc as a terminal sugar were also identified. The number of the studies shows its occurrence in all members of enteric bacteria with a few exceptions such as Erwinia chrysanthemi. The presence of ECA was also shown for such genera as Plesiomonas 4 and Yersinia 36, previously belonging to the Vibrionaceae and Pasteurellaceae families, respectively. It was one of the reasons to include these two taxa in the Enterobacteriaceae family. The function of ECA is not fully understood, but it was reported that its occurrence is important in resistance of bacterial cells to environmental conditions, such as bile salts in the human digestive tract. The immunogenicity of ECA seems very interesting in the fact that only sparse rough Gram-negative strains, such as Shigella sonnei phase II, Escherichia coli R1, R2, R4, K-12, and Yersinia enterocolitica O:3 are able to induce the production of specific anti-ECA antibodies. It is the effect of the ECALPS, and the evidence for the existence of such covalent linkage was provided by structural analysis of S. sonnei surface antigens.,致病性,致病物质 黏附素具有高度特异性, 吸附、定植、致病 定植因子抗原I, II, III 集聚黏附菌毛 束形成菌毛 紧密黏附素 P 菌毛等,外毒素 （1）不耐热肠毒素（heat labile enterotoxin, LT）: 蛋白质，分LT-I和LT-两型； 对热不稳定，与霍乱肠毒素相似。,ATP cAMP,小肠液过度分泌,肠腔积液、腹泻,机制,A亚单位毒素活性成分,B亚单位与肠上皮细胞受体（GM1神经节苷脂） 结合,B单位与受体结合导致A进入细胞内激活腺苷酸环化酶,LT的作用示意,（2）耐热肠毒素（heat stable enterotoxin, ST），分STa、STb两型 为低分子多肽，对热稳定 机制为激活细胞鸟苷酸环化酶，使cGMP导致腹泻 LT和ST同时存在，致病性要强 （3）志贺毒素和 （Shiga toxin，Stx-1，Stx-2） （4） 溶血素A,所致疾病 肠道外感染（内源性感染） 化脓性感染定位转移 腹膜炎、阑尾炎、创伤感染、手术刀口感染、肺炎、 败血症、新生儿脑膜炎等 泌尿道感染病原菌来自病人肠道（上行性） 占泌尿道感染首位、女性多见（尿道炎、膀胱炎、肾盂肾炎等） 尿路致病性大肠埃希菌(uropathogenic E. coli, UPEC),胃肠炎（腹泻） （外源性感染） 肠产毒性大肠埃希菌（ETEC）： 婴幼儿、旅游者腹泻，与霍乱功能相似 肠侵袭性大肠埃希菌（EIEC）： 成人腹泻，与菌痢相似 肠致病性大肠埃希菌（EPEC）： 婴儿腹泻（水样腹泻） 肠出血性大肠埃希菌（ EHEC，O157）： 出血性结肠炎， 溶血性尿毒综合征（HUS） 肠集聚性大肠埃希菌 （EAEC）： 婴儿腹泻（持续性）,引起胃肠炎的大肠埃希菌,微生物学检查与防治,肠道外感染 标本涂片、染色分离培养鉴定 尿路感染时每ml尿含菌10万，有诊断价值 肠道内感染 用弱选择性培养基 分离培养+生化反应+血清分型+毒素测定+核酸检测等 卫生细菌学检查 大肠菌群指数:每1000ml(g)样品中的大肠菌群数 我国卫生标准规定: 每1000ml水中大肠杆菌3个， 每100 ml 瓶装汽水、果汁中，大肠杆菌5个,大肠菌群指数（coli-index）指1升水中含有的大肠菌群数。 大肠菌群是一群以大肠埃希氏菌为主的需氧及兼性厌氧，革兰氏阴性，无芽孢杆菌，能在48小时内发酵乳糖并产酸产气。包括埃希菌属、枸橼酸杆菌属、肠杆菌属及克雷伯菌属等4种肠道细菌。 检测中规定生活饮用水的水质标准为1升水中大肠菌群不得超过3个，即大肠菌群指数不得大于3。也可用“大肠菌群值(colititre)”来表示。即以水样中可检出1个大肠菌群数的最小毫升数表示，两者的关系是：大肠菌群值=1000/大肠菌群指数。,1.大肠菌群值和大肠菌群指数 大肠菌群值(colititre)是指能检出大肠菌群的最小样品量(ml)。 大肠菌群指数（coli-index）是指1000ml样品的大肠菌群数。 卫生部规定的饮水卫生标准是大肠菌群值不小于333ml，大肠菌群指数不得超过3。 2.细菌总数测定 检测每毫升或每克样品中所含细菌数。 卫生部标准是饮用水、饮料中细菌总数100CFU/ml。,防治原则,食物彻底加热； 口服疫苗； 抗生素治疗：氨苄青霉素、庆大霉素、头孢菌素等。,第二节 志贺菌属（Shigella）,一、生物学性状 形态染色 无芽胞，无荚膜，无鞭毛，无动力； 多数有菌毛； 革兰氏阴性杆菌。,Kiyoshi Shiga (志賀 潔 Shiga Kiyoshi?, February 7, 1871 - January 25, 1957) was a Japanese physician and bacteriologist.,培养特性 肠道鉴别培养基上的无色透明、乳糖不发酵菌落 生化反应 葡萄糖（+）、乳糖（-）（宋内志贺菌迟缓发酵乳糖）、H2S-、尿素（-）、动力（-）,2志贺氏菌 3沙门氏菌 4大肠杆菌,抗原分类: 根据O抗原分4群，40余型 抵抗力: 比其它肠杆菌弱，尤其对酸敏感,志贺菌属的分类,二、致病性与免疫性,致病物质 侵袭力 菌毛：黏附肠黏膜上皮细胞 型分泌系统：诱导细胞膜凹陷,导致细菌内吞 内毒素强烈内毒素，作用于局部并可吸收入血引起全 身症状 外毒素A群（、型）产生志贺毒素（shiga toxin, ST）有3种生物学活性: （1）肠毒素：类似ETEC毒素、霍乱肠毒素作用水样泻 （2）细胞毒性：对人肝细胞、肠黏膜细胞有毒性，引起变性坏死 （3）神经毒性：损伤动物神经系统（麻痹、死亡）,Shiga toxin : The toxins are named for Kiyoshi Shiga, who first described the bacterial origin of dysentery caused by Shigella dysenteriae. Heat Labile Enterotoxins (LT): These are produced by some Enterotoxigenic E. coli (ETEC). Pertussis toxin (PT): is a protein-based AB5-type exotoxin produced by the bacterium Bordetella pertussis. PT is involved in the colonization of the respiratory tract and the establishment of infection.,Histopathology of acute colitis following peroral infection with shigellae,所致疾病细菌性痢疾 传染源：病人、带菌者 传播途径：经口 临床类型 急性菌痢 中毒性菌痢 慢性菌痢 免疫力 不牢固，易再感染,三、微生物学检查法,微生物学检查 分离鉴定 毒力实验: Sereny试验, 9X109/ml菌悬液, 接种于豚鼠眼结膜囊, 角膜结膜炎, Sereny试验阳性 快速诊断,四、防治原则,注意饮食卫生 易出现多重耐药菌株 链霉素依赖株(streptomycin dependent strain, Sd)活疫苗,第三节 沙门菌属 (salmonella),是肠杆菌科中最复杂的菌属，有2500种以上血清型。 对人类致病 肠热症伤寒沙门菌，甲型副伤寒沙门菌、肖氏沙门菌、希氏沙门菌； 食物中毒鼠伤寒沙门菌、猪霍乱沙门菌、肠炎沙门菌； 败血症猪霍乱沙门菌最常见。,Genus：Salmonella Species：S. bongori S. enterica,Subspecies Salmonella enterica enterica Salmonella enterica salamae Salmonella enterica arizonae Salmonella enterica diarizonae Salmonella enterica houtenae Salmonella enterica indica,Salmonella choleraesuis Salmonella Dublin Salmonella Enteritidis Salmonella Heidelberg Salmonella Paratyphi Salmonella Typhi Salmonella Typhimurium,主要沙门氏菌的生化特性,一、生物学性状,形态染色 符合肠杆菌特点，多为周身鞭毛菌，有菌毛,伤寒杆菌的周身鞭毛,培养特性 EMB、SS上呈无色透明乳糖不发酵菌落,麦康凯培养基上的伤寒杆菌菌落,麦康凯培养基，上为E.coli，下为伤寒杆菌,生化反应 乳糖（-），葡萄糖（+）/+，多数H2S+，动力+，尿素（-） 抗原结构 O抗原特异性低、稳定、分群（组） H抗原特异性高，不稳定，分型 Vi抗原（毒力抗原）不稳定，可阻止“O”凝集 抵抗力 不强,二、致病性与免疫性,致病物质 侵袭力 菌毛吸附 型分泌系统穿入细胞，扩散