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ENTEROBACTERIACEAE. Morphology & Identification Gram-negative non-spore forming rods. When motile, by peritrichous flagella. Primarily normal flora of.

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Presentation on theme: "ENTEROBACTERIACEAE. Morphology & Identification Gram-negative non-spore forming rods. When motile, by peritrichous flagella. Primarily normal flora of."— Presentation transcript:

1 ENTEROBACTERIACEAE

2 Morphology & Identification Gram-negative non-spore forming rods. When motile, by peritrichous flagella. Primarily normal flora of gastrointestinal tract. E. coli>Klebsiella>Proteus>Enterobacter Free living, also transient colonizers of skin. Facultative anaerobes: mixed acid fermentation All ferment glucose; all reduce nitrates to nitrites; all oxidase negative. Lactose fermentation: normal flora positive and pathogens negative. Primary isolation media include eosin-methylene-blue (EMB) and MacConkey agar. Differential selective media for specific organisms including dyes and bile salts. (Salmonella-Shigella (SS) medium, bismuth sulfite media.)

3 Classification ~29 genera, over 100 species. – Escherichia – Shigella – Edwardsiella – Salmonella – Citrobacter – Klebsiella – Enterobacter – Hafnia – Serratia – Proteus – Providencia – Morganella – Yersinia – Erwinia – Pectinobacterium

4 Antigenic Structure –Most are motile by peritrichous flagella --H antigens. –Capsule – K antigen ( Vi for Salmonella). –Cell envelope (wall) –LPS (endotoxin) – O antigen. –various outer membrane proteins. –Pili - various antigen types, some encoded by plasmids

5 鞭毛抗原( H ) 菌体抗原 (O) K 或 Vi 抗原

6 –septicemia, –pneumonia, –meningitis –urinary tract infections Citrobacter Enterobacter Escherichia Hafnia Morganella Providencia Serratia Opportunistic diseases -Enterobacteriaceae

7 Enterobacteriaceae: gastrointestinal diseases – Escherichia coli – Salmonella – Shigella – Yersinia entercolitica

8 Histocompatibility antigen (HLA) B27 Histocompatibility antigen (HLA) B27 – Enterobacteriaceae *Salmonella *Shigella *Yersinia – NotEnterobacteriaceae – Not Enterobacteriaceae *Campylobacter *Chlamydia Reiter's syndrome

9 community acquired otherwise healthy people – Klebsiella pneumoniae * respiratory diseases * prominent capsule –urinary tract infection –fecal contamination *E. coli *Proteus – urease (degrades urea) – alkaline urine Enterobacteriaceae

10 Enterobacteriaceae gram negative facultative anaerobic rodsgram negative facultative anaerobic rods – oxidase negative (no cytochrome oxidase)

11 E. coli – lactose positive – not usually identified – lactose positive sp. common, healthy intestine Shigella, Salmonella,Yersinia – lactose negative – identified Feces

12 other sites other sites identified biochemically – identified biochemically Enterobacteriaceae

13 Serotypes reference laboratory reference laboratory – antigens O (lipopolysaccharide) O (lipopolysaccharide) H (flagellar) H (flagellar) K (capsular) K (capsular)

14 Escherichia coli

15 Toxins: two types of enterotoxin; Shiga-type toxin; Enteroaggregative ST-like toxin; Hemolysins; Endotoxin Type III secretion system Adhesions – colonization factors ; both pili or fimbriae ;non-fimbrial factors involved in attachment. There are at least 21 different types of adhesions. Virulence factors that protect the bacteria from host defenses: Capsule/Iron capturing ability (enterochelin) Outer membrane proteins

16 E. coli fimbriae mannose Type 1 galactose galactose – glycolipids – glycoproteins P

17 E.coli-urinary tract infection Is the leading cause of urinary tract infections which can lead to acute cystitis (bladder infection) and pyelonephritis (kidney infection).

18 E.coli-Meningitis and Sepsis Neonatal meningitis – is the leading cause of neonatal meningitis and septicemia with a high mortality rate. Usually caused by strains with the K1 capsular antigen.

19 Enteropathogenic E. coli fever infant diarrhea vomiting nausea non-bloody stools Destruction of surface microvilli 1. loose attachment mediated by bundle forming pili (Bfp); 2. Stimulation of intracellular calcium level; 3. rearrangement of intracellular actin,

20 Enterotoxigenic E. coli A watery diarrhea, nausea, abdominal cramps and low-grade fever for 1-5 days. A watery diarrhea, nausea, abdominal cramps and low-grade fever for 1-5 days. Travellers diarrhea and diarrhea in children in developing countries Travellers diarrhea and diarrhea in children in developing countries Transmission is via contaminated food or water. Transmission is via contaminated food or water.

21 Enterotoxigenic E. coli diarrhea like cholera diarrhea like cholera milder milder nursery travellers diarrhea nursery travellers diarrhea caused by LT, ST, or LT/ST. caused by LT, ST, or LT/ST.

22 Enterotoxigenic E. coli Heat labile toxin Heat labile toxin –like choleragen –Adenyl cyclase activated –cyclic AMP –secretion water/ions Heat stable toxin Heat stable toxin – Guanylate cyclase activated – cyclic GMP – uptake water/ions – uptake water/ions

23 LT vs ST activity

24 E.coli-Enteroinvasive (EIEC) The organism attaches to the intestinal mucosa via pili Outer membrane proteins are involved in direct penetration, invasion of the intestinal cells, and destruction of the intestinal mucosa. There is lateral movement of the organism from one cell to adjacent cells. Symptoms include fever,severe abdominal cramps, malaise, and watery diarrhea followed by scanty stools containing blood, mucous, and pus. resembles shigellosis

25 Enteroinvasive E. coli (EIEC) Dysentery - resembles shigellosis - elder children and adult diarrhea

26 E.coli-c. Enteropathogenic (EPEC) Malaise and low grade fever diarrhea, vomiting, nausea, non-bloody stools Bundle forming pili are involved in attachment to the intestinal mucosa. This leads to changes in signal transduction in the cells, effacement of the microvilli, and to intimate attachment via a non-fimbrial adhesion called intimin. This is a problem mainly in hospitalized infants and in day care centers.

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28 E.coli-d. Enterohemorrhagic (EHEC) Hemorrhagic – bloody, copious diarrhea – few leukocytes – afebrile hemolytic-uremic syndrome – hemolytic anemia – thrombocytopenia (low platelets) – kidney failure

29 Enterohemorrhagic E. coli Usually O157:H7 Transmission electron micrograph

30 Enterohemorrhagic E. coli Vero toxin Vero toxin – “shiga-like” Hemolysins Hemolysins younger than 5 years old,causing hemorrhagic colitis

31 Enteroaggregative E. coli 肠集 聚型大肠杆菌 a cause of persistent, watery diarrhea with vomiting and dehydration in infants. That is autoagglutination in a ‘stacked brick’ arrangement. the bacteria adheres to the intestinal mucosa and elaborates enterotoxins (enteroaggregative heat-stable toxin, EAST). The result is mucosal damage, secretion of large amounts of mucus, and a secretory diarrhea.

32 E.coli-Enteroaggregative (EAggEC) Mucous associated autoagglutinins cause aggregation of the bacteria at the cell surface and result in the formation of a mucous biofilm. The organisms attach via pili and liberate a cytotoxin distinct from, but similar to the ST and LT enterotoxins liberated by ETEC. Symptoms incluse watery diarrhea, vomiting, dehydration and occasional abdominal pain.

33 Various Types of E. coli

34 Summary of E.coli strains that cause gastroenteritis.

35 Sanitary significance Totoal bacterial number: number of bacteria contained per ml or gm of the sample; the standard of drinking water is less than 100. Totoal bacterial number: number of bacteria contained per ml or gm of the sample; the standard of drinking water is less than 100. Coliform bacteria index: the number of coliform bacteria detected out per 1000 ml sample; the standard of drinking water is less than 3 Coliform bacteria index: the number of coliform bacteria detected out per 1000 ml sample; the standard of drinking water is less than 3

36 Escherichia coli Genetically E. coli and Shigella are genetically highly closely related. For practical reasons (primarily to avoid confusion) they are not placed in the same genus. Not surprisingly there is a lot of overlap between diseases caused by the two organisms. 1) Enteropathogenic E. coli (EPEC). Certain serotypes are commonly found associated with infant diarrhea. The use of gene probes has confirmed these strains as different from other groups listed below. There is a characteristic morphological lesion with destruction of microvilli without invasion of the organism that suggests adhesion is important. Clinically one observes fever, diarrhea, vomiting and nausea usually with non-bloody stools. 2) Enterotoxigenic E. coli (ETEC) produce diarrhea resembling cholera but much milder in degree. Also cause "traveler’s diarrhea". Two types of plasmid-encoded toxins are produced. a) Heat labile toxins which are similar to choleragen (see cholera section below). Adenyl cyclase is activated with production of cyclic AMP and increased secretion of water and ions. b) Heat stable toxins; guanylate cyclase is activated which inhibits ionic and water uptake from the gut lumen. Watery diarrhea, fever and nausea result in both cases. 3) Enteroinvasive E. coli (EIEC) produce dysentery (indistinguishable clinically from shigellosis, see bacillary dysentery below). 4) Enterohemorrhagic E. coli (EHEC). These are usually serotype O157: H7. These organisms can produce a hemorrhagic colitis (characterized by bloody and copious diarrhea with few leukocytes in afebrile patients). Outbreaks are often caused by contaminated hamburger meat. The organisms can disseminate into the bloodstream producing systemic hemolytic-uremic syndrome (hemolytic anemia, thrombocytopenia and kidney failure). Production of Vero toxin (biochemically similar to shiga toxin thus also known as "shiga-like") is highly associated with this group of organisms; encoded by a phage. Hemolysins (plasmid encoded) are also important in pathogenesis. As noted above, there are at least 4 etiologically distinct diseases. However, in the diagnostic laboratory generally the groups are not differentiated and treatment would be on symptomatology. Generally fluid replacement is the primary treatment. Antibiotics are generally not used except in severe disease or disease that has progressed to a systemic stage (e.g.hemolytic-uremia syndrome). Two major classes of pili are produced by E. coli : mannose sensitive and mannose resistant pili. The former bind to mannose containing glyocoproteins and the latter to cerebrosides on the host epithelium allowing attachment. This aids in colonization by E. coli.

37 Shigella

38 Shigella S. flexneri, S. boydii, S. sonnei, S. dysenteriae S. flexneri, S. boydii, S. sonnei, S. dysenteriae – bacillary dysentery – shigellosis bloody feces bloody feces intestinal pain intestinal pain pus pus

39 Genral features Pili. Pili. Most strains can not ferment lactose; S. sonnei can slowly_ ferment lactose. Most strains can not ferment lactose; S. sonnei can slowly_ ferment lactose. According to O antigen, 4 groups According to O antigen, 4 groups Easily causing drug-resistence. Easily causing drug-resistence.

40 Shigellosis within 2-3 days within 2-3 days – epithelial cell damage

41 Shiga toxin enterotoxic enterotoxic cytotoxic cytotoxic inhibits protein synthesis inhibits protein synthesis – lysing 28S rRNA

42 Shigella attachment and penetration Within 2-3 days Within 2-3 days Epithelial cell damage Epithelial cell damage

43 Clinical significance man only "reservoir" man only "reservoir" mostly young children mostly young children – fecal to oral contact – children to adults transmitted by adult food handlers transmitted by adult food handlers – unwashed hands

44 Clinical significance The infective dose required to cause infection is very low (10-200 organisms). There is an incubation of 1-7 days followed by fever, cramping, abdominal pain, and watery diarrhea (due to the toxin)for 1-3 days. This may be followed by frequent, scant stools with blood, mucous, and pus (due to invasion of intestinal mucosa). Is is rare for the organism to disseminate. The severity of the disease depends upon the species one is infected with. S. dysenteria is the most pathogenic followed by S. flexneri, S. sonnei and S. boydii.

45 Immunity SIgA.

46 Diagnosis of Shigella infection Specimen: stool. Culture and Identification Quick immunological methods: 1. Immunofluorescent “ball” test; 2. Coagglutination.

47 Prevention streptomycin dependent (SD) dysentery vaccine.

48 Treating shigellosis manage dehydration patients respond to antibiotics, Problem of drug-resistance – disease duration diminished

49 Shigella Shigella (4 species; S. flexneri, S. boydii, S. sonnei, S. dysenteriae) all cause bacillary dysentery or shigellosis, (bloody feces associated with intestinal pain). The organism invades the epithelial lining layer, but does not penetrate. Usually, within 2-3 days, dysentery results from bacteria damaging the epithelium lining layers of the intestine often with release of mucus and blood (found in the feces) and attraction of leukocytes (also found in the feces as "pus"). Shiga toxin (chromosomally encoded) is neurotoxic, enterotoxic and cytotoxic plays a role. The toxin inhibits protein synthesis (acting on the 80S ribosome and lysing 28S rRNA). This is primarily a disease of young children occurring by fecal-oral contact. Adults can catch this disease from children. However it can be transmitted by infected adult food handlers, contaminating food. The source in each case is unwashed hands. Man is the only "reservoir". Patients with severe dysentery are usually treated with antibiotics (e.g. ampicillin). In contrast to salmonellosis, patients respond to antibiotic therapy and disease duration is diminished.

50 Salmonella Salmonellosis may present as one of several syndromes including gastroenteritis, enteric (typhoid) fever or septicemia.

51 The antigenic structures of salmonellae used in serologic typing

52 Salmonella 2000 antigenic "types” 2000 antigenic "types” disease category disease category – S. enteritidis –many serotypes – S. cholerae-suis – S. typhi

53 Virulence factors Endotoxin – may play a role in intracellular survival Capsule (for S. typhi and some strains of S. paratyphi) Adhesions – both fimbrial and non-fimbrial Type III secretion systems and effector molecules – 2 different systems may be found: –One type is involved in promoting entry into intestinal epithelial cells –The other type is involved in the ability of Salmonella to survive inside macrophages Outer membrane proteins - involved in the ability of Salmonella to survive inside macrophages Flagella – help bacteria to move through intestinal mucous Enterotoxin - may be involved in gastroenteritis Iron capturing ability

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55 Enteric or typhoid fever Enteric or typhoid fever occurs when the bacteria leave the intestine and multiply within cells of the reticuloendothelial system. Enteric or typhoid fever occurs when the bacteria leave the intestine and multiply within cells of the reticuloendothelial system. The bacteria then re-enter the intestine, causing gastrointestinal symptoms. The bacteria then re-enter the intestine, causing gastrointestinal symptoms. Typhoid fever has a 10-14 day incubation period and may last for several weeks. Typhoid fever has a 10-14 day incubation period and may last for several weeks. Salmonella typhi is the most common species isolated from this salmonellosis. Salmonella typhi is the most common species isolated from this salmonellosis. Human reservoir: Human reservoir:carrier state common Contaminated food:water supply Contaminated food:water supply Poor sanitary conditions Poor sanitary conditions

56 Typhoid acute phase, gastroenteritisacute phase, gastroenteritis gall bladder –shedding, weeks Septicemia -occurs 10-14 days – lasts 7 days gastrointenteritis

57 胆囊 --- 肠道 --- 粪排菌 / 肠 壁淋巴组织 肾 ----- 尿 肝脾 ----- 肿大 骨髓 ------ 受抑制 皮肤 ---- 血栓出血 -- 玫瑰疹 伤寒和付伤寒的致病过程 伤寒和付伤寒沙门菌 小肠上部粘膜 肠系膜淋巴结 固有层淋巴结 进入血液再次进入血液 第一次菌血症 第二次菌血症

58 Typhoid -Therapy Antibiotics Antibiotics – essential Vaccines Vaccines Vi (capsular) antigen :protective Vi (capsular) antigen :protective

59 Salmonella gastroenteritis Salmonella gastroenteritis is the most common form of salmonellosis and generally requires an 8-48 hour incubation period and may last from 2-5 days. Salmonella gastroenteritis is the most common form of salmonellosis and generally requires an 8-48 hour incubation period and may last from 2-5 days. Symptoms include nausea, vomiting and diarrhea (non-bloody stool). Salmonella enteritidis is the most common isolate. Symptoms include nausea, vomiting and diarrhea (non-bloody stool). Salmonella enteritidis is the most common isolate. poultry, eggs. no human reservoir poultry 家禽, eggs. no human reservoir self-limiting (2 - 5 days) self-limiting (2 - 5 days)

60 Salmonella septicemia Salmonella septicemia (bacteremia) may be caused by any species but S. cholerae-suis is common. This disease resembles other Gram-negative septicemias and is characterized by a high, remittent fever with little gastrointestinal involvement.

61 Immunity (S. typhi) Vi (capsular) antigen Vi (capsular) antigen – protective

62 Diagnosis A. Specimens A. Specimens a) Enteric fever: blood, bone marrow, stool, urine. a) Enteric fever: blood, bone marrow, stool, urine. b) Food poisoning: stool, vomitus, suspected food. b) Food poisoning: stool, vomitus, suspected food. c) Septicemia: blood. c) Septicemia: blood. B. Culture and identification B. Culture and identification C. Widal test C. Widal test

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64 Salmonella Using appropriate antibodies more than 2000 antigenic “types” have been recognized. There are, however, only a few types that are commonly associated with characteristic human diseases (most simply referred to as S. enteritidis, S. cholerae-suis and S. typhi). Salmonellosis, the common salmonella infection, is caused by a variety of serotypes (S. enteritidis) and is transmitted from contaminated food (such as poultry and eggs). It does not have a human reservoir and usually presents as gastroenteritis (nausea, vomiting and non-bloody stools). The disease is usually self-limiting (2-5 days). Like Shigella they invade the epithelium and do not produce systemic infection. In uncomplicated cases of salmonellosis, which are the vast majority, antibiotic therapy is not useful. S. cholerae- suis (seen much less commonly) causes septicemia after invasion. In this case, antibiotic therapy is required.. The severest form of salmonella infections "typhoid" (enteric fever), caused by Salmonella typhi. Although it is one of the historical causes of widespread epidemics and still is in the third world. The organism is transmitted from a human reservoir or in the water supply (if sanitary conditions are poor) or in contaminated food. It initially invades the intestinal epithelium and during this acute phase, gastrointestinal symptoms are noted. The organism penetrates, usually within the first week, and passes into the bloodstream where it is disseminated in macrophages. Typical features of a systemic bacterial infection are noted. The septicemia usually is temporary with the organism finally lodging in the gall bladder. Organisms are shed into the intestine for some weeks. At this time the gastroenteritis (including diarrhea) is noted again. The Vi (capsular) antigen plays a role in the pathogenesis of typhoid. A carrier state is common; thus one person e.g. a food handler can cause a lot of spread. Antibiotic therapy is essential. Vaccines are not widely effective and not generally used

65 Klebsiella – NF of GI tract, but potential pathogen in other areas – Virulence factors Capsule Adhesions Iron capturing ability – Clinical significance Causes pneumonia, mostly in immunocompromised hosts. Permanent lung damage is a frequent occurrence (rare in other types of bacterial pneumonia) A major cause of nosocomial infections such as septicemia and meningitis

66 Klebsiella K. pneumoniae (Friedlander bacilli): may cause primary pneumonia, urinary tract and wound infections, bacteremia, meningitis, etc. K. rhinoscleromatis: pathogen of granumatous destruction of nose and pharynx. K. rhinoscleromatis: pathogen of granumatous destruction of nose and pharynx. K. ozaenae: causes chronic atrophic rhinitis. K. ozaenae: causes chronic atrophic rhinitis.

67 Proteus General characteristics: “swarming” phenomenon on nonselective agar (P.vulgaris; P.mirabilis and P.myxofaciens) General characteristics: “swarming” phenomenon on nonselective agar (P.vulgaris; P.mirabilis and P.myxofaciens) P.vulgaris strains (OX-19, OX-K, OX-2)have common antigen with Rickettsia (Weil-Felix test). P.vulgaris strains (OX-19, OX-K, OX-2)have common antigen with Rickettsia (Weil-Felix test). urinary tract infections; food poisoning. urinary tract infections; food poisoning.


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