1. Enterobacteriaceae

2. Transmitted by the fecal-oral route
Primary pathogens Organisms capable of causing disease in anyone
Opportunistic pathogens Organisms that can only cause disease under certain conditions or in certain hosts
Shigella
Salmonella
Yersinia
Klebsiella pneumoniae
E. coli
Senatia
Morganella
Providencia
Enterobacter
Proteus

3. Characteristics shared by all members of family Enterobacteriaceae

4. G-, non-spore forming rods

5. Peritrichous flagella
-shigella: nonmotile
Facultative anaerobe
Not fastidious

6. Active metabolism - All ferment glucose;
- All reduce nitrates to nitrites;
- All oxidase negative
- Lactose fermentation: the key measure to isolate
and identify the Enterobacteriaceae
+: nonpathogenic
-: pathogenic

7. EMB plate
伊红美兰琼脂：弱选择性培养基，用于肠道菌的选择性分离。蛋白胨提供碳源和氮源；乳糖是大肠菌群可发酵的糖类；磷酸氢二钾是缓冲剂；琼脂是培养基凝固剂；伊红和美蓝是抑菌剂和pH指示剂，可抑制革兰氏阳性菌，在酸性条件下产生沉淀，形成紫黑色菌落或具黑色中心的外围无色透明的菌落。

8. MacConkey agar
麦康凯琼脂培养基：用于肠道致病菌的选择性分离培养。胰酪蛋白胨、蛋白胨提供氮源、维生素和矿物质；乳糖是大肠菌群可发酵的糖类； 牛胆盐抑制非肠杆菌科细菌；氯化钠维持均衡的渗透压；结晶紫抑制革兰氏阳性菌；中性红是指示剂；琼脂是培养基凝固剂。

9. Triple sugar iron agar (TSI)
用于肠杆菌科细菌的生化反应筛选。蛋白胨、牛肉粉提供氮源、维生素、矿物质；乳糖、葡萄糖、蔗糖为可发酵糖类，其产酸时通过酚红指示剂测出，酸性呈黄色，碱性呈红色；硫代硫酸钠可被某些细菌还原为硫化氢，与硫酸亚铁中的铁盐生成黑色硫化铁；氯化钠维持均衡的渗透压；琼脂是培养基的凝固剂。

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

11. Enterobacteriaceae: gastrointestinal diseases
Escherichia coli
Salmonella
Shigella
Yersinia entercolitica

12. Serotypes reference laboratory antigens O (lipopolysaccharide)
H (flagellar)
K (capsular)
O6:K15:H6

13. (I) Escherichia coli

14. Extraintestinal infection Intestinal tract infection
Medical significance
Normal flora
E. coli is the majority of GI normal flora
Opportunistic pathogen
Extraintestinal infection
Urinary tract infection
Septicemia
Neonatal meningitis
pathogen
Intestinal tract infection
ETEC
EIEC
EPEC
EHEC
EAggEC

15. Escherichia coli
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. What is the pathogenesis of these five groups of pathogenic E. coli?
Gastroenteritis caused by E. coli

17. Enterotoxigenic E. coli (ETEC)
A watery diarrhea, nausea, abdominal cramps and low-grade fever for 1-5 days.
Travellers diarrhea and diarrhea in children in developing countries
Transmission is via contaminated food or water.
diarrhea like cholera
milder
nursery travellers diarrhea
caused by LT, ST, or LT/ST.

18. Enterotoxigenic E. coli (ETEC)
Heat labile toxin
like choleragen
Adenyl cyclase activated
cyclic AMP
secretion water/ions
Heat stable toxin
Guanylate cyclase activated
cyclic GMP
uptake water/ions

19. LT vs ST activity

20. Enteroinvasive E. coli (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

21. Enteroinvasive E. coli (EIEC)
Dysentery
rearrangement of intracellular actin
resembles shigellosis
elder children and adult diarrhea

22. Enteropathogenic E. coli (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.

23. Enteropathogenic E. coli (EPEC)
fever
infant diarrhea
vomiting
nausea
non-bloody stools
Destruction of surface microvilli
- loose attachment mediated by bundle forming pili (Bfp);
- Stimulation of intracellular calcium level;
- Rearrangement of intracellular actin

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

25. Enterohemorrhagic E. coli (EHEC)
Vero toxin
“shiga-like”
Hemolysins
younger than 5 years old,causing hemorrhagic colitis

26. Enterohemorrhagic E. coli (EHEC)
Usually O157:H7
Transmission electron
micrograph

27. Enteroaggregative E. coli (EaggEC)
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.

28. Enteroaggregative E. coli (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.

29. Summary of E. coli strains that cause gastroenteritis.

30. Organism Virulence factors
Adherence
Toxins
invasion
serovar
ETEC
Colonization factors of adherence (CFAs)
Type 1 pili
Endotoxin
Heat-labile enterotoxin (LT)
Heat-stable enterotoxin (ST)
Noninvasive
O6:K15:H6
EIEC
Afimbrial adhesins
Very invasive
Type III secretion system 28
EPEC
Bundle-forming pili (BFP)
Intimin
Poorly invasive
2, 55
EHEC
Shiga toxin
Probably poorly invasive
O157:H7
EaggEC
Mucus-associated autoagglutination
Cytotoxin (enteroaggregative ST-like toxin)
O42

31. Enterotoxigenic E. coli (ETEC) Small intestine
organism
Site
Disease
pathogenesis
Enterotoxigenic E. coli (ETEC)
Small intestine
Traveler’s diarrhea, infant diarrhea in under developed countries, watery diarrhea, cramps, nausea, low-grade fever
Heat-stable and/or heat-labile enterotoxins, stimulate guanylate or adenylate cyclase activity with fluid and electrolyte loss
Enteroinvasive E. coli (EIEC)
Large intestine
Fever, cramping, watery diarrhea followed by development of dysentery with scant, bloody stools
Plasmid-mediated invasion and destruction of epithelial cells lining colon
Enteropathogenic E. coli (EPEC)
Infant diarrhea with fever, nausea, vomiting, nonbloody stools
plasmid-mediated adherence and destruction of epithelial cells
Enterohemorrhagic E. coli (EHEC)
Hemorrhagic colitis with severe abdominal cramps, watery diarrhea initially, followed by grossly bloody diarrhea, little or no fever, hemolytic uremic syndrome (HUS)
Mediated by cytotoxic “vero-toxin”
Enteroaggregative E. coli (EAggEC)
Persistent infant diarrhea, sometimes with gross blood, low-grade fever
Aggregative adherence mediated by plasmid

32. Various Types of E. coli

33. “Nonintimate” association:
bacteria attach to host cell by bundle-forming pili
Bacterial attachment:
signal transduction event stimulated;
host cell tyrosine kinase activated;
Ca2+ levels increase
“Intimate” contact:
pedestallike structure (composed of actin fibers) forms in host cell under bacteria (intimin)

34. Escherichia coli
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.

35. Sanitary significance
Total 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

36. (II) Shigella dysentery bacterium

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

38. Classification Serogroup Species Number of serotypes A S. dysenteriae10 B
S. flexneri 13 C
S. boydii 18 D
S. sonnei 1
The classification is based on O antigens

39. What disease is caused by Shigella species?
Bacillary dysentery
shigellosis

40. Bacillary dysentery Source - humans are the only reservoir
Transmission
- the fecal-oral route
Clinical findings
-watery diarrhea
-abdominal cramps
-tenesmus
-bloody stool with mucus and pus
-fever
Clinical types
- acute dysentery
acute toxic dysentery
-chronic dysentery
Tenesmus: Spasmodic contraction of anal or vesical sphincter with pain and persistent desire to empty the bowel with involuntary ineffectual straining efforts

41. Shigellosis within 2-3 days epithelial cell damage bloody feces
intestinal pain
pus

42. Shigellosis Invasiveness Endotoxin Exotoxin: Shiga toxin
- S. dysenteriae
- neurotoxic, enterotoxic and cytotoxic

43. Shiga toxin enterotoxic cytotoxic inhibits protein synthesis
lysing 28S rRNA

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

45. Clinical significance
The infective dose required to cause infection is very low ( 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.

46. immunity Primary immunity defense - SIgA Immunity intensity - Limited
- reasons
surface infection
various types

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

48. Prevention & Treatment
streptomycin dependent (SD) dysentery vaccine.
manage dehydration
patients respond to antibiotics , Problem of drug-resistance
disease duration diminished

49. Summary 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").

50. Summary Shigella
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.

51. (III) Salmonella

52. Salmonella More than 2000 serotypes
Transmitted by the fecal-oral routed
Salmonellosis may present as one of several syndromes including gastroenteritis, enteric (typhoid) fever or septicemia.
Zoonosis

53. Antigenic composition of Salmonella
O antigen
H antigen
Vi antigen
- S. typhi and S. hirschfeldii
- Pathogenicity
- Screening of carriers (titer≥1:10)
- Inhibition of the agglutination of O Ags and the O Abs

54. The antigenic structures of salmonellae used in serologic typing

55. Pathogenicity 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
Enterotoxin - may be involved in gastroenteritis
Outer membrane proteins - involved in the ability of Salmonella to survive inside macrophages
Flagella – help bacteria to move through intestinal mucous

56. Pathogenicity Virulence factors
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

58. Disease caused by Salmonella
pathogens
Gastroenteritis
S. enteritidis, S. typhimurium,
S. choleraesuis
Septicemia
S. choleraesuis, S. typhimurium,
S. enteritidis, S. hirschfeldii
Enteric fever
S. typhi, S. paratyphi A,
S. schottmuelleri, S. hirschfeldii

59. Gastroenteritis
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.
poultry, eggs. no human reservoir
self-limiting (2 - 5 days)

60. Gastroenteritis The most common form of salmonellosis (70%)
Self-limited (2-5 d)
Not enter into blood
Culture
- blood (-)
- stool (+)

61. Septicemia
Salmonella septicemia (bacteremia) may be caused by any species but S. choleraesuis is common.
This disease resembles other G- septicemias and is characterized by a high, remittent fever with little gastrointestinal involvement.

62. Septicemia 5-10% of salmonellosis
Intestinal manifestations: often absent
Culture
- blood (+)
- stool (-)

63. The pathogenesis of typhoid
Enteric fever
The pathogenesis of typhoid

64. Enteric or typhoid fever
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.
Typhoid fever has a day incubation period and may last for several weeks.
Salmonella typhi is the most common species isolated from this salmonellosis.
Human reservoir: carrier state common
Contaminated food: water supply
Poor sanitary conditions

66. Typhoid -Therapy Antibiotics essential Vaccines
Vi (capsular) antigen :protective

67. What does the pathogenesis imply in terms of collection of clinical samples?

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

70. Widal test
A quantitative agglutination test for typhoid and paratyphoid, in which detects a patient’s antibodies to the specific O antigen of S. typhi and H antigens of S. typhi, S. paratyphi A, S. schottmuelleri and S. hirschfeldii

71. How to interpret the results of Widal test?
Consider the manifestaton, course, history, and local epidemiological conditions

72. How to interpret the results of Widal test?
TO<1:80,TH<1:160, PH<1:80
Normal value
TO≥1:80 & TH≥1:160 or
TO≥1:80 & PH≥1:80
Typhoid fever
Paratyphoid fever
TO≥1:80 & TH <1:160 or
TO≥1:80 & PH <1:80
Early stage of infection or cross-reaction of O antigen with other salmonellae
TO < 1:80 & TH ≥1:160 or
TO < 1:80 & PH ≥ 1:80
Vaccination or nonspecific memory reaction

73. How to interpret the results of Widal test?
Dynamic observation
-antibody titer give a rise gradually
-titer of convalescence serum≥4 times than that of early specimen
False negative
-pre-antobiotic
-immunosuppressed

74. immunity Primary immunity defense Immunity intensity - CMI
- strong and permanent

75. 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. choleraesuis (seen much less commonly) causes septicemia after invasion. In this case, antibiotic therapy is required. .

76. Salmonella
The severest form of salmonella infections "typhoid" (enteric fever), caused by Salmonella typhi.
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

77. Summary Common members of Enterobacteriaceae to cause human diseases
Common properties of Enterobaceriaceae
The medical significance of E. coli
The opposite traits of Shigella and Salmonella in terms of pathogenesis and immunity
Widal test: definition, result determination