1. Enterobacteriaceae are a family of Gram-negative, non-spore forming rods that are normal flora of the gastrointestinal tract. They include many important pathogens like Escherichia, Salmonella, Shigella. 2. They are facultative anaerobes that ferment glucose and reduce nitrates. Identification is based on lactose fermentation and reactions on selective media. 3. Shigella causes bacillary dysentery. It is non-motile and invades the colonic epithelium, causing severe diarrhea, fever and tenesmus. Virulence factors include invasion plasmid antigens.

1. 杆菌科肠
ENTEROBACTERIACEAEENTEROBACTERIACEAE

2. ※ G-, non-spore forming rods. Most of them have
peritrichous flagella ( 周身鞭毛 ).
※ Primarily normal flora of gastrointestinal tract.
※ Free living, also transient colonizers of skin.
※ Facultative anaerobes: mixed acid fermentation
※ All ferment glucose; all reduce nitrates ( 硝酸盐 ) to nitrites
（亚硝酸盐） ; all are oxidase （ - ） .
Morphology & Identification

3. ※ Lactose fermentation: normal flora （ + ） and
pathogens
（ - ） .
※ Primary isolation media include eosin ['i əs n]-methylene-ː ɪ
blue (EMB) and MacConkey agar.
※ Differential selective media for specific organisms
including dyes and bile salts. (Salmonella-Shigella (SS)
medium, bismuth ['b zməθ]sulfite media.)ɪ

4.  Escherichia （埃希菌属）
 Shigella （志贺菌属）
 Edwardsiella （爱德华氏菌
属）
 Salmonella （沙门菌属）
 Citrobacter （柠檬酸细菌
属）
 Klebsiella （克雷伯氏菌属）
 Enterobacter （肠杆菌属）
 Hafnia （二氧化铪属）
 Serratia （沙雷氏菌属）
 Proteus （变形杆菌
属）
 Providencia
（普罗威登斯菌属）
 Morganella
（摩根菌属）
 Yersinia
（耶尔森氏菌属）
 Erwinia （欧文氏菌
属）
 Pectinobacterium
ClassificationClassification
29 genera, over 100 species29 genera, over 100 species

5. 1 、 Similar appearance and dyeability
§1 Common biological characteristics

6. §1 Common biological characteristics
1 、 Similar appearance and dyeability
2 、 Simple cultural conditions
※ Aerobic or facultative anaerobic —— can grow in
the presence or absence of O2

7. 1. Similar appearance and dyeability
2. Simple cultural conditions
3. Vivid in biochemical reaction
※ Typically oxidase （ - ）
※ Most strains ferment lactose & glucose with
the
production of acid and/or gas
§1 Common biological characteristics ( )★

8. Preliminary identification of enteric pathogens and intestinalPreliminary identification of enteric pathogens and intestinal
opportunisticopportunistic pathogenspathogens
Enteropathogenic bacteria
Intestinal opportunistic pathogens
do not ferment lactose
ferment lactose
lactose-fermentation ( 乳糖发酵试验）
Reactions on TSI （ TRIPLE
SUGAR IRON ） agar slants

9. ※ （ + ） in Nitrate Reduction Test ( 硝酸盐还原试验 )
对氨基 苯磺酸
萘胺
百浪
多息

10. ※ IMViC reaction :
Distinguish!

11. 4. Complicated Antigenic Structure4. Complicated Antigenic Structure
※※ Most are motile by peritrichous flagella --H antigens.Most are motile by peritrichous flagella --H antigens.
※※ Capsule – K antigen ( Vi for Salmonella).Capsule – K antigen ( Vi for Salmonella).
※※ LPS (endotoxin) – O antigen.LPS (endotoxin) – O antigen.
※※ various outer membrane proteins.various outer membrane proteins.
※※ Pili - various antigen types, some encoded by plasmidsPili - various antigen types, some encoded by plasmids

12. Antigenic structure

13. Over 700 antigenic types (serotypes) are
recognized based on O, H, and K antigens in E. coliE. coli (K
antigens be also called capsular antigen).

14. 5 、 Resistance
These organisms have no spores, so the resistance are
not strong. The resistance of pathogenic strains to bile salts
（胆盐） and brilliant green （煌绿） is much higher
than that of opportunisticopportunistic strains, so these two substances
are usually used as the inhibitors in enterobacteriaceae
selective medium.

15. 6 、 Liable to variation
drug resistance variation
Virulence variation
Biochemical reaction variation
Conjugation
Transduction
lysogenic conversion

16. 7 ． Production of bacteriocins
8 ． Some of enteric bacilli can produce
exotoxins.

17. 9. Opportunistic pathogen -9. Opportunistic pathogen -EnterobacteriaceaeEnterobacteriaceae
—— septicemia(septicemia( 败血症）败血症）
—— pneumoniapneumonia （肺炎）（肺炎）
—— meningitismeningitis （脑炎）（脑炎）
—— urinary tract infectionsurinary tract infections
（泌尿系统感染）（泌尿系统感染）
Citrobacter
Enterobacter
Escherichia
Hafnia
Morganella
Providencia
Serratia

18. Enterobacteriaceae:Enterobacteriaceae: gastrointestinal diseasesgastrointestinal diseases
—— some serological type of Escherichia colisome serological type of Escherichia coli
—— SalmonellaSalmonella
—— ShigellaShigella
—— Yersinia entercoliticaYersinia entercolitica

19. §2 Escherichia coliEscherichia coli （（ E. coliE. coli ））
Coliform enterobacteria which ferment lactose

20. Characteristics
Morphology
※ usually motile, produce peritrichous
flagella
※ some produce polysaccharide capsules
※ smooth, translucent （半透明） , colourless
colonies, 2-3 mm in diameter in 18h on nutrient
agar
※ temperature (15-45℃)

21. Characteristics
Antigenic structureAntigenic structure
Over 700 antigenic types (serotypes) are recognized
based on O, H, and K antigens.
▲ Lipopolysaccharide LPS (heat-stable somatic
antigens ) — O antigens
▲ flagellar antigens — H antigens
▲ "capsular" antigens — K antigens

22. Diseases caused byDiseases caused by E. coliE. coli
E. coli is responsible for three types of infections
in humans:
※ urinary tract infections (UTI),
※ neonatal meningitis,
※ intestinal diseases (gastroenteritis ，肠胃
炎 ).

23. 1. pathogenic material
Pathogenicity
⑴ Adhesins
Include:
— Colonization Factor Antigen Ⅰ 、Ⅱ、Ⅲ
— aggregative adherence fimbriae Ⅰ 、 II
— bundle-forming pili
—Intimin
— P pili
— invasive plasmid antigen proteins (Ipa),
— Dr pili

24. (2) Exotoxins
△ Shiga-type toxin:Ⅰ 、Ⅱ
△ Heat labile enterotoxin:Ⅰ 、Ⅱ
△ Heat stable enterotoxin: a 、 b
△ Hemolysin A(HlyA)

25. △ Shiga-type toxin:Ⅰ 、Ⅱ
△ Heat labile enterotoxin:Ⅰ 、Ⅱ
△ Heat stable enterotoxin:a 、 b
△ Hemolysin A(HlyA)

26. Shiga-type toxins( 志贺毒素 )
Shiga-type toxins – also called verotoxin -produced by
enterohemorrhagic strains of E. coli (EHEC) – is cytotoxic,
enterotoxic, neurotoxic, and may cause diarrhea and ulceration
of the G.I. tract.
Two types: shiga-like toxin 1 and 2.
Inhibit protein synthesis by
cleaving a 28S rRNA that’s part of the
60S subunit

27. Stxlysogenic phage
intestinal villi( ['v li ]ɪ ː 绒毛）
kindey epithelial cells
Gb3(glycolipid receptor)
Split 28S rRNA in 60S subunit
Termination of host protein synthesis
Upregulation gene
expression of
inflammatory
factor
The pathogenic mechanisms of shiga(-type) toxins
Cell death
villus

28. △ Shiga-type toxin:Ⅰ 、Ⅱ
△ Heat labile enterotoxin:Ⅰ 、Ⅱ
△ Heat stable enterotoxin:a 、 b
△ Hemolysin A(HlyA)

29. The chemcial constitution and pathogenesis of
heat labile enterotoxin （不耐热肠毒素 )
Share 70% homology with Cholera toxin
Subunit ASubunit B
cell membrane
Adenyl Cyclase
ATP cAMP 腹泻腹泻diarrhea
GM1 receptor
H2O CI-
Na+
are Secreted greatly

30. △ Shiga-type toxin:Ⅰ 、Ⅱ
△ Heat labile enterotoxin:Ⅰ 、Ⅱ
△ Heat stable enterotoxin: a 、 b
△ Hemolysin A(HlyA)

31. The chemcial constitution and pathogenesis ofand pathogenesis of Heat
stable enterotoxin ( 耐 毒素热肠 )
ST
ST
a
STb
guanylate cyclase
cGMP↑ 腹泻腹泻diarrhea
Disturbance in Water-Electrolyte Balance
⊕
No homology with Cholera toxin

32. ST – is heat stable and binds to specific receptors to stimulate
the production of cGMP with the same results as with LT.

33. Both enterotoxins are composed of five beta subunits (for
binding) and 1 alpha subunit (has the toxic enzymatic
activity).

34. LT vs ST activityLT vs ST activity

35. △ Shiga-type toxin:Ⅰ 、Ⅱ
△ Heat labile enterotoxin:Ⅰ 、Ⅱ
△ Heat stable enterotoxin:a 、 b
△ Hemolysin A(HlyA)

36. Hemolysin A( 溶血素 A)
This toxin is considered play an important role in the
pathogenesis of diseases caused by uropathogenic E.coli,
but the mechanisms are not known nowadays.

37. Clinical finding
(1) Extraintestinal infection: Opportunities pathogenic
① urinary tract infections ： infected by UPEC
(uropathogenic E.coli ）
② septicaemia, bacteremia
③ neonatal meningitis
Results of ectopic parasitism ['pærəsa t zəm](ɪ ˌɪ 异常寄生）

38. (2) intestinal diseases (Gastroenteritis)
Gastroenteritis – there are several distinct types of
E. coli that are involved in different types of
gastroenteritis:
enterotoxigenic E. coli (ETEC)
enteroinvasive E. coli (EIEC)
enteropathogenic E. coli (EPEC)
enteroaggregative E. coli (EAEC)
enterohemorrhagic E. coli (EHEC)
caused by Pathogenic E. coli

39. EnterotoxigenicEnterotoxigenic E. coliE. coli (ETEC):(ETEC): cause diarrhea in infants andcause diarrhea in infants and
travelerstravelers
Pathogenic E. coli — ETEC

40. Involves two steps: Intestinal colonization, Diarrheagenic
enterotoxin(s)
Transmission is via contaminated food or water.
Pathogenesis of ETECPathogenesis of ETEC
ETEC – is a common cause of traveler’s diarrhea and
diarrhea in children in developing countries.
The disease is characterized by a watery diarrhea, nausea,
abdominal cramps and low-grade fever for 1-5 days.
Pathogenic E. coli — ETEC

41. EnterotoxinsEnterotoxins
LT (heat-labile) toxin
ST (heat-stable) toxin
Pathogenic E. coli — ETEC
固有层

42. Pathogenic E. coli — EIEC (enteroinvasive E. coli )
▲ Shigella-like E coli
▲ invasive (penetrate and multiply within epithelial
cells)
▲ does not produce shiga toxin
▲ EIEC – The organism attaches to the intestinal
mucosa via pili and outer embrane 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.

43. Pathogenic E. coli — EIEC (enteroinvasive E. coli )
▲ Symptoms include fever, severe abdominal cramps, malaise
（不适） , and watery diarrhea followed by scanty stools
containing blood, mucous, and pus.

44. Pathogenic E. coli — EPEC
(enteropathogenic E. coli )
▲ bundle forming pili (intimin)
▲ moderately invasive "attaching and effacing"
▲ does not produce LT or ST; some reports of shiga-like toxin
▲ usually infantile diarrhea; watery diarrhea similar to
ETEC, some inflammation, no fever; symptoms probably
result mainly from invasion rather than toxigenesis

45. Pathogenic E. coli — EAEC (enteroaggregative E. coli )
EAEC –aggregation of the bacteria at the cell surface and result
in the formation of a mucous biofilm.
▲ The organisms attach via AAFI/II and Bfp and produce a
cytotoxin .
▲ Symptoms include watery diarrhea, vomiting, dehydration
and occasional abdominal pain.

46. Pathogenic E. coli — EHEC
EnterohemorrhagicEnterohemorrhagic E. coliE. coli (EHEC)(EHEC)
▲ represented by a single strain (serotype O157:H7)
▲ adhesins (probably mediated by fimbriae)
▲ moderately invasive
▲ produces shiga toxin but not LT or ST
▲ copious bloody discharge intense inflammatory response, may
be complicated by hemolytic uremia ( 出血性尿毒症）
▲ pediatric diarrhea caused by this strain can be fatal due to
acute kidney failure.

47. Pathogenic E. coli
TreatmentTreatment
▲ Treatment is based on symptomatology （症候学） .
▲ Fluid replacement is the primary treatment
▲ Antibiotics are generally not used except in severe diseases or
diseases that has progressed to a systemic stage
(e.g.hemolytic-uremia syndrome).

48. §2 ShigellaShigella （（志 菌属）贺
agents of bacillary dysentery

49. Shigella
Morphology and StructureMorphology and Structure
※ G-,
※ PiliPili, nonmotile,
※ facultatively anaerobic,
※ non-spore-forming rods
※ Most strainsMost strains failure to ferment lactose; S. sonnei (ferment lactose; S. sonnei ( 宋内宋内
志贺菌志贺菌） can slowly ferment lactose.can slowly ferment lactose.
※ closely related with Escherichia coli
※ Easily causing drug-resistence.Easily causing drug-resistence.

50. Antigenic structure and classification
species group type subtype
S. dysenteriae A 1 ～ 10 8a,8b,8c
S.flexneri
B 1 ～ 6 ， X ， Y
Variant
1a,1b,2a,2b,3a,3b,3c ， 4a,4
b
S.boydii C 1 ～ 18
According to the specific O antigen, Shigella are classified
into 4 groups, more than 40 serotypes(include subtypes).
O antigen
K antigen: non-specificity
Group-specific
Type-specific
Can be use for typing

51. Shigella
里急后重
结肠炎
[t 'nezməs]ɪ

52. Shigella
VirulenceVirulence
1. invasin
▲ encoded by plasmids — Invasion Plasmid Antigens (Ipa) A-D.
▲ induces the endocytic uptake of shigellae by M cells, epithelial cells,
and macrophages.
▲ deform the plasma membrane of contiguous cells.
▲ IcsB plasmid-encoded protein lyses the plasma membranes,
resulting in intercellular bacterial spread.

53. Shigella
2. Endotoxin
cause fever, shock, bloody, mucoid stools, and abdominal
pain (cramps and tenesmus) .
VirulenceVirulence

54. Shigella
3. exotoxin-- Shiga toxin(vero toxin)
▲ chromosomally-encoded
▲ neurotoxic, enterotoxic and cytotoxic
▲ The toxin inhibits protein synthesis (acting on the 60S
ribosome and lysing 28S rRNA). Its enterotoxicity can make
the disease clinically appear as a diarrhea.
VirulenceVirulence

55. ShigellaShigella attachmentattachment
and penetrationand penetration

56. ShigellaShigella
— Clinical significance
△ Causes shigellosis ( 志贺氏菌病 )or bacillary dysentery.
○○ bloody fecesbloody feces
○○ intestinal painintestinal pain
○○ puspus
△ Transmission is via the fecal-oral route.
△ The infective dose 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).
△ It is rare for the organism to disseminate.
△ The severity of the disease depends upon the species .
— S. dysenteria is the most pathogenic followed by S. flexneri, S.
sonnei and S. boydii.

57. Shigella
▲ occurring by fecal-oral contact
▲ Man is the only “reservoir”, mostly young childrenmostly young children
children to adultschildren to adults
transmitted by adult food handler (unwashed hands)transmitted by adult food handler (unwashed hands)
Epidemiology(Epidemiology( 流行病学 )

58. ImmunityImmunity
SIgA

59. Shigella
Treatment and ControlTreatment and Control
▲ Managing of dehydration is of primary concern.
▲ Patients with severe dysentery are usually treated with
antibiotics (e.g. ampicillin).
▲ prevention: attenuated live vaccine

60. slide agglutination tests
Sample
Macc 、 SS
plate
Biochemical reaction
Serological identificationSugar fermentation
※ procedure of laboratory diagnosis
DiagnosisDiagnosisShigella

61. Shigella
Diagnosis — SamplingDiagnosis — Sampling
▲ Positive cultures are most often obtained from blood-tinged
plugs of mucus in freshly passed stool specimens obtained
during the acute phase of disease
▲ Rectal swabs may also be used if the specimen is deposited in
a buffered glycerol saline holding solution

62. Shigella
Diagnosis — IsolationDiagnosis — Isolation
primary differential/selective media
SS agar

63. Shigella
Diagnosis — IsolationDiagnosis — Isolation
▲ colorless, non-lactose-fermenting colonies
▲ Secondly tubed slants ( 斜面 ) of Kligler's
Iron Agar or Triple Sugar Iron
Agar.
▲ Shigella species produce an
alkaline slant and an acid butt
with no bubbles of gas in the agar.

64. Shigella
Diagnosis — IdentificationDiagnosis — Identification
▲ slide agglutination tests with antisera
▲ polymerase chain reaction (PCR).
▲ Enzyme-linked immunosorbent assay (ELISA)

65. Salmonella (Salmonella ( 沙 菌属门 )

66. Salmonella
Structure & Antigenic TypesStructure & Antigenic Types
▲ G-, flagellated, facultatively anaerobic bacilli
▲ three major antigens:
H or flagellar antigen;
O or somatic antigen;
Vi antigen (only a few serovars)

67. SalmonellaSalmonella
▲ Classification has been changing in the last few years.
There is now 1 species: S. enteritica , and 7 subspecies: 1, 2 ,3a ,
3b ,4 ,5, and 6.
▲ Subgroup 1 causes most human infection
△ Based on O and H (flagella) antigens
There are 58 O antigens in salmonella, put the
salmonella that have the same O antigen together and
make a group. There are 42 groups of salmonella (A,
B,C and so on).

68. △ The H antigens occur in two phases; 1 and 2 and only 1
phase is expressed at a given time.
phase 1: high specific, the basis of subtype
phase 2: non-specific.
△ Salmonella typhi also has a Vi antigen which is a capsular
antigen, it is also non-specific.

69. Salmonella
Virulence FactorsVirulence Factors
▲ the ability to replicate intracellularly
▲ the ability to invade cells
invasin: Vi (capsular) antigen
▲ Adhesions – both fimbrial and non-fimbrial
▲ a complete lipopolysaccharide coat-LPS (endotoxin), also may
play
a role in intracellular survival
▲ 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

70. Salmonella

71. Salmonella — Clinical Manifestations
1) Gastroenteritis—food poisoning
▲ Symptoms usually begin 6 to 48 hours after ingestion of
contaminated food or water
▲ the cardinal manifestation is diarrhea.
▲ nausea, vomiting, abdominal cramps, myalgia, headache,
fever and chills are common
▲ The duration of fever and diarrhea is usually 2 to 7 days

72. Salmonella — Clinical Manifestations
2) Septicemia
▲ an intermediate stage of infection – no intestinal symptoms
and the bacteria cannot be isolated from fecal specimens.
▲ it may remain localized in the intestine or disseminate to the
bloodstream

73. Salmonella — Clinical Manifestations
▲ severe systemic form, may be fatal
▲ The best studied enteric fever is typhoid fever
▲ may be preceded by gastroenteritis
▲ an incubation period of 10 to 14 days
▲ symptoms of enteric fevers are nonspecific:fever,
anorexia[ ænə'reksiə] (ˌ 厌食症） , headache, myalgias, and
constipation （便秘）
3) Enteric fevers — typhoid3) Enteric fevers — typhoid

74. Salmonella — Clinical Manifestations
3) Enteric fevers — typhoid3) Enteric fevers — typhoid
★ primary bacteraemic phase:
(7- 10 days of the incubation period )
▲ invade the epithelium
▲ spread to mesenteric [mesən'ter k]ɪ （肠系膜
的） lymph
nodes & throughout the body
▲ be taken up by the reticuloendothelial cells （网状内皮细胞）
△ infect the liver, spleen, gallbladder, bones, meninges
[mə'n nd i z]ɪ ʒ ː

75. Salmonella — Clinical Manifestations
3) Enteric fevers — typhoid3) Enteric fevers — typhoid
★ second and heavier bacteraemic phase(2-3 weeks)
▲ pass into the blood( the onset of the fever and other signs of
clinical illness)
▲ From the gallbladder a further invasion of the intestine
results.
▲ Peyer patches （淋巴集结） & other gut lymphoid tissues
become involved in an inflammatory reaction
▲ followed by necrosis, sloughing （蜕皮） and the
formation of typhoid ulcers

76. Salmonella — Clinical Manifestations
3) Enteric fevers — typhoid3) Enteric fevers — typhoid
▲ Onset: 2 weeks/ insidious/early symptoms
▲ Progression :
the temperature shows a stepladder rise over the 1st week of the
illness, remains high for 7-10 days and then falls by lysis during
the 3rd or 4th week.
physical signs include a relative bradycardia [bræd 'k d ə]ɪ ɑː ɪ
（心搏徐缓 ) , hepatomegaly, splenomegaly ( 肝脾肿
大） and often a rash of rose spots.
▲ Relapse: shorter and milder.
▲ Complications: severe intestinal haemorrhage and perforation

77. Salmonella — Clinical Manifestations
4) The prolonged carrier state4) The prolonged carrier state
▲ continue to excrete the salmonellae for a year or
more
▲ The bacilli are most commonly present in the
gallbladder

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

79. Salmonella — laboratory diagnosis
Isolation & identificationIsolation & identification
▲ Blood culture
the definitive diagnosis of enteric fever
most commonly found during the first 7-10 days
and during relapses
▲ Stool and urine culture
In typhoid fever, positive form the 2nd week and
urine cultures from the 3rd week of the infection.

80. ▲ Specimens should be plated on several EMB media.
▲ Laboratory identification of the genus Salmonella is done by
biochemical tests.
▲ Biochemical reactions of suspicious colonies are then determined
on triple sugar iron agar and lysine-iron agar.
▲ serologic type is confirmed by serologic testing.
▲ It can be confirmed by antigenic analysis of O and H antigens
using polyvalent and specific antisera.
Salmonella — laboratory diagnosis
Isolation & identificationIsolation & identification

81. Salmonella-laboratory diagnosis
Widal testWidal test
▲ a tube test for determining the quantity of
agglutinating antibodies, or agglutinins, in the
serum of a patient with typhoid fever

82. Salmonella-laboratory diagnosis-widal test
▲ Generally, in typhoid cases, it is valuable that the
titre of specific
O antibodies is ≥1:80 or the titre of specific H
antibodies is ≥1:160.
▲ In paratyphoid cases, if the titre of specific H
antibodies is ≥1:80, the result is （ + ） .

83. Salmonella
Control and TreatmentControl and Treatment
▲ Vaccines are available for typhoid fever and are
partially effective.
▲ Typhoid fever and enteric fevers should be treated
with antibiotics.

84. Exercises:
IMViC test ST LT ETEC EIEC
EPEC EHEC EAEC Enteric fevers
1.Please describe the Common biological
characteristics of enterobacteriaceae briefly.
2.Please describe the pathogenic mechanism of
salmonella.