1. Identification of Enterobacteriaceae coliform organisms and related diseases
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2. Contents Characterisitics of Enterobacteriaceae Coliform/ Non-coliform
Identification
Antigenic Structure & Virulence Factor
E.coli – Pathogenic strains
– Clinical diseases
Other coliform

3. Family Enterobacteriaceae

4. Enterobacteriaceae Inhabit : Soil, water, decaying matter,
large bowel of humans and animals
Characteristics :
- Small and Regular & straight rods
- Gram (-)
- Non-spore-forming
- Glucose ferment
- Nitrate reduction
- Oxidase (-)
- Catalase (+)
- Most motile(except Shigella & Klebsiella & Yersinia )

5. Facultative → Grow best in air & ferment Carbohydrates
Diarrheal illnesses : two basic mechanism
1. Toxigenic infection – (a)
2. Invasive infection – (b)
Toxigenic infection : 미생물들이 상피세포 표면에 머무르면서 세포안쪽에 독소를 분비한다. 그 독소는 소장 표면의 receptor에 붙게 된다. 그러면 cell metabolism에 붕괴가 일어나고, 전해질과 수분을 잃게 된다. 이러한 증상은 E.coli 와 shigella 감염시 나타난다.
Invasive infection : 미생물이 상피세포를 직접 침입하여 파괴한 후, 궤양을 형성한다. 그렇게 되면 장 내벽이 손상되고 출혈이 일어난다. 증상으로는 혈변이 나타나게 된다.

6. Enterics : more than 50% of nosocomial infection
-E.coli, Klebsiella, Proteus, Enterobacter, Serratia, Citrobacter.
Isolated from soap dishes, sink, invasive devices
(fiberopic probes, trachea cannulas, indwelling catheters)
균은 뒤에서 설명합니다.

7. Coliform / Non-coliform

8. Family Enterobacteriaceae
Coliforms : Rapid lactose-fermenting
E.coli, Klebsiella, Enterobacter, Hafnia,Citrobacter,Serratia
Non-coliforms : Non-lactose-fermenting
Slow lactose-fermenting
Bacteria
- Opportunistic, normal gut flora
- Pathogenic enterics
- Pathogenic nonenterics

9. Identification

10. Identification Enrichment media : selenite or GN broth Selective media
- MacConkey agar - EMB agar - Hektoen enteric agar
TSI agar
IMViC(Indole test, MR test, VP test, Citrate test)
Urease test, motility test
Selective media (lactose-fermenters/ non lactose – fermenters)
MacConkey agar는 indicator로 Neutral red 사용 lactose+ 이면 pink lactose-면 Not colored
EMB agar는 indicator로 Eosin Y Methylene blue 사용 lactose+면 Metallic sheen blue, Dark nucleus lactost-면 Pale lavender
Hektoen enteric agar는 indicator로 Acid fuchsin Thymol blue사용 lactose+면 Salmon pink Orange lactose-면 Green, blue-green
또 Hektoen enteric agar는 indicator로 Ferric ammonium citrate 사용하여 색이 black이 되면 H2S production을 알 수 있다.
TSI agar: Test for it’s ability to ferment sugars and to produce hydrogen sulfide
Urease test양성이면 red 음성이면 yellow

11. MacConkey agar Bile salts 와 Crystal violet 을 함유한 배지
 그람양성균 성장 억제
Lactose와 Neutral red
Coliform : Pink
Non- coliform : 반투명

12. Eosin Methylene Blue (EMB agar)
Bile salt 와 Methylene blue
 그람양성균 성장억제
Coliform  검은색 핵을 만드는 것을 유발하며, 때때로 표면에 금속성 광택을 띄게 한다
Non – coliform  옅은 연보라색을 띄며 핵이 없다.

13. Hektoen enteric agar Bile salts  그람양성균 성장 억제 Non-coliform : 초록색과
BTB용액, acid fuchsin지시약
Coliform : 주황색이나,
주황색이 도는 핑크색
(orange,salmonpink)
Non-coliform : 초록색과
파란빛이 도는 초록색
(green, blue-green)
H2S 가스 생성여부도 진단할 수 있다.
H2S 가스가 생성되면 colony가 검은색으로 변한다.

14. TSIA(Triple Sugar Iron Agar) test
Triple agar : glucose 1g/L, lactose 10g/L, sucrose 10g/L
pH indicator : Phenol red - Acid : yellow (A)
- Alkali : red (K)
세 가지의 가능성 : K/A, A/A, K/K
- K/A : glucose 만을 분해하는 세균
처음에는 균이 glucose를 분해하여 산을 생성하여
A/A를 띠지만, 세균분해산물인 Amine 에 의해
중화되어 K/A 성상을 띠게 된다.
- A/A : glucose와 lactose 또는 glucose와
sucrose를 분해하 는 세균
- K/K : 어떠한 당도 이용하지 못하는 세균

15. IMViC test Indole test Methyl red test
- Tryptophan을 분해하여 indole을 생산하 는 능력
- Kovac’s reagent
- (+): 붉은색 (-): 황색 (변화X)
Methyl red test
- Glucose를 분해하여 생기는 mixed acid의 농도차이
- pH 4.4 이하이면 methyl red와 반응하여 적색, pH 6부근은 황색(변화X)

16. Vogues-Proskauer test (V-P test)
- 세균이 glucose를 발효시켜 유 기산 acetoin 형성여부
- (+): 붉은색
(-): 황색 (변화X)
Citrate test
- 세균이 탄소원으로 citratrate를 이용하는지
Glucose나 lactose가 없을때 탄소 원으로 citrate를 이용하는지
- (+): 청색
(-): 초록색 (변화X)

17. + - Genus Indole Methy red Voges-Proskauer Citrate TSIA Motility
Escherichia + -
A/A G
MOTILE
Klebsiella
SPECIES VARIABLE
NON-MOTILE
Enterobacter
Citrobacter
H2S
Serratia
K/A
Lf는 lactose fermentation Nlf는 non lactose fermentation
A/A G는 glucose and lactose and/or sucrose fermentation and gas!!
H2s는 H2S생성
K/A는 glucose fermentation only, peptone catabolized
Species variable 종마다 다양

18. Urea  ammonia (pH상승) Urease test Urea broth (Phenol red 함유)
(요소를 분해할 수 있는 세균을 동정)
Urea broth (Phenol red 함유)
 pH 상승 시 분홍
양성 : 진분홍 , 음성 : 변화 없음

19. Motility test Semi –solid agar test - SIM medium, MIO medium
- LB broth +0.4% agar  반고체 형태
(고체배지의 경우 1.2~ 1.5% agar)
Hanging drop wet mount for ability
Dark field microscope
- 배경 : 검은색
- 균체 : 흰색

20. Identification

21. Antigenic Structures and Virulence Factors

22. Antigenic Structures Complex surface antigens H : flagellar antigen
K : capsule and/or
fimbrial antigen
O : somatic or cell wall
antigen

23. Antigenic Structures Variety of subspecies or serotypes Serotyping
- Classifying some species
- Pinpoint the source of outbreaks
Epidemiological tool

24. Antigenic Structures Endotoxin (lipopolysaccharide,LPS) Exotoxin
Capacity to overcome host defenses
Multiply in the tissues and blood

25. Virulence Fator
Gram-negative bactera freely transfer ‘chromosome or plasmid’
- Drug resistance
- Toxigenicity
- Adaptive traits
- Alter their pathogenicity
Virulence genes : code for enterotoxins, capsules, hemolysins, fimbriae

27. E.coli -Pathogenic strains and Clinical diseases

28. E.coli Best known coliform Facultative anaerobic & non-fastidious
Colon bacillus – in the intestine of humans

29. Pathogenic strains of E.coli
(1) Enterotoxigenic E.coli (ETEC) -Several diarrheal illness(similar to cholera) - Two exotoxins - LT(heat-labile toxin) - ST(heat-stable toxin)  Heightened secretion, fluid loss -Fimbriae : adhesion to the small intestine

30. Pathogenic strains of E.coli
(2) Enteroinvasive E.coli (EIEC) -Inflammatory disease -Invasion and ulceration of the mucosa of the large intestine (3) Enteropathogenic E.coli (EPEC) -Infantile diarrhea

31. Pathogenic strains of E.coli
(4) Enterohemorrhagic E.coli (EHEC) -E.coli O157:H7 (verotoxin) -Hemorrhagic syndrome -Damage to kidney (5) Enteroaggregatory E.coli (EAEC) - Infantile diarrhea
-The true epidemiologic significance and impact of EAEC as a diarrheal pathogen cannot be assessed with the current diagnostic tools to detect its presence

32. Clinical diseases of E.coli
(1) Infantile diarrhea - Crowded tropical region, sanitary facilities, contaminated water -Immature·nonimmune neonatal intestine -Malnourished, loss of body fluids & electrolytes→ fatal
(2) Traveler’s diarrhea -70% exotic pathogen(ETEC) -Within 7-8days, profuse, watery diarrhea, low-grade fever, nausea, vomit

33. Clinical diseases of E.coli
(3) Urinary tract infections(UTI) -50~80% of UTI in healthy people -Invaded by its own endogenous bacterial colonists
(4)Other extraintestinal infections -Neonatal meningitis, pneumonia, septicemia, wound infection

34. Other coliforms

35. Other coliforms Klebsiella, Enterobacter, Citrobacter
Infection : sites other than the intestine, host defenses fail
Septicemia, endotoxemia, nosocomial infection

36. 1) Klebsiella pneumoniae
Respiratory tract → chronic lung infections
Large capsule : prevents phagocytosis
Nosocomial pneumonia, meningitis, bacteremia, wound infection, UTI

37. 2) Enterobacter & Hafnia spp.
Inhabit soil, sewage, diary products
Surgical wound, spinal fluid, sputum, blood
UTI, lethal when introduced into the bloodstream

38. 3) Citrobacter spp. Inhabit in soil, water, human feces
Opportunist (UTI & bacteremia)
Citrate test positive (blue)

39. 4) Serratia marcescens In soil, water, intestine
Produce intense ‘red pigment’
 red in sputum false impression of blood
Nosocomial infection, burn & wound infection fatal septicemia & meningitis in immunosuppressed patients.

40. Reference 임상 미생물학 실습/전국임상병리학과 교수 협의회/고려의학/159-169p
MICROBIOLOGY/Kathleen Park Talaro/McGraw Hill/ p
신 종합미생물학/미생물면역분과위원회/도서출판 신일북스/ p
밀레니엄미생물학/김관천 외 10명/ 정문각/ p
최신진단미생물학/정윤섭 외 6명/서흥출판사/ p
제12판 진단미생물학Ⅱ/Forbes Sahm Weissfeld/E.PUBLIC/ p
MEDICAL MICROBIOLOGY/김정엽,남혜봉,윤기주/청구문화사/ p
전북대학교 식품공학과 카페

41. What is not characteristic of Enterobacteriaceae?
Gram positive
Catalase positive
Oxidase negative
Non-spore-forming
Glucose ferment

42. What are two basic mechanisms of diarrheal illnesses?
Toxigenic infection
Invasive infection

43. E.Coli displays which antigens?
Capsular
Somatic
Flagellar
All of these

44. What is not coliforms?
1. Klebsiella 2. Enterobacter 3. Serratia 4. Proteus 5. Citrobacter 6. Morganella 7. Providencia