1. B.A.Fontanilla, MD Department of Microbiology and Parasitology
ENTEROBACTERIACEAE
B.A.Fontanilla, MD
Department of Microbiology and Parasitology

2. GENERAL CHARACTERISTICS
Taxonomy
Morphology
Physiology
Antigenic Structure
Determinants of Pathogenicity
Clinical Infection

3. ENTEROBACTERIACEAE Large number of closely related species
- found in soil, water, decaying matter
- found in large intestines of human, animals and
insects ----”enterics” or “enteric bacilli”
- includes causative agents of gastrointestinal
diseases
- causative agents of nosocomial infections

4. ENTEROBACTERIAECEAE TAXONOMY
Cedecea
Citrobacter
Edwardsiella
Enterobacter
Escherichia
Ewingella
Hafnia
Klebsiella
Kluyvera
Morganella
Proteus
Providencia
Rhanella
Salmonella
Serratia
Shigella
Enteric group

5. ENTEROBACTERIACEAE MORPHOLOGY
Small 0.5 x 3.0 µ
Gram-negative
non-spore-forming bacilli
May be motile or non-motile
If motile – peritrichous flagella
nonmotile – Shigella and Klebsiella

6. ENTEROBACTERIACEAE Biochemically diverse Facultative organisms
When grown in anaerobic or low O2
- ferment carbohydrates
When grown in sufficient O2
- utilize the TCA cycle and the electron transport system for energy production

7. ENTEROBACTERIACEAE PHYSIOLOGY
Ferment glucose
Reduce nitrates to nitrite
Do not liquify alginate
Oxidase negative
Basis for speciation within a family
- differences in carbohydrate they ferment
- variations in end-product production
- variation in substrate utilization

8. ENTEROBACTERIACEAE CULTURAL CHARACTERISTICS
On non differential or nonselective media
blood agar or infusion agar
- no species distinction
- appear as moist, smooth, gray colonies
Selective media -To isolate Shigella and salmonella from fecal matter
Differential media – selectively inhibit gram-positive organisms and to separate enterics in broad categories

9. ENTEROBACTERIACEAE ANTIGENIC STRUCTURE
In serologic typing, antigenic structures are used for classification and epidemiologic studies
- Capsular (K) antigens
- Flagellar (H) antigens
- Somatic (O) antigens

10. ENTEROBACTERIACEAE ANTIGENIC STRUCTURE
Capsular (K) antigens
- Klebsiella species has a well defined
polysaccharide capsule
- in other genera, amorphous slime layer
surrounding the bacterial cell
- in E.coli – proteins and form fimbriae not
capsules
- Vi antigen of Salmonella typhi

11. ENTEROBACTERIACEAE ANTIGENIC STRUCTURE
Flagellar antigens
- proteins
-antigenic variation is due fo differences in amino acid sequences
-antigenic typing of Salmonella is based on serologic typing of flagellar antigens

12. ENTEROBACTERIACEAE ANTIGENIC STRUCTURE
Somatic antigens
- O antigens is the most external part of the cell wall lipopolysaccharide (LPS)
- may enhance the establishment of the organism in the host.

13. ENTEROBACTERIACEAE ANTIGENIC STRUCTURE
Flagellar (H) antigens
- proteins
- antigenic variation of various flagellar
types due to differences in amino acid
sequences
- serologic typing of flagellar antigens –
basis for antigenic typing of Salmonella
typhi

14. ENTEROBACTERIACEAE DETERMINANTS OF PATHOGENICITY
1. ENDOTOXIN
- LPS (review structure p. 26 Jawetz)
- resides in the lipid A portion of the LPS
- produces, fever, fatal shock, leukocytic
alterations, regression of tumors, alteration
in host response to infection
- pooling of blood in the microcirculation - causing cellular hypoxia and metabolic failure due to inadequacy of blood in vital organs

15. ENTEROBACTERIACEAE DETERMINANT OF PATHOGENICITY
2. ENTEROTOXIN -Toxins that affect the small intestines - transduction of fluid in the lumen –diarrhea 3. SHIGA TOXINS AND SHIGALIKE TOXINS (Verotoxins) – actions on Vero (African green monkey) tissue culture cells - Shigella – toxin that interferes with protein synthesis of cells - E.coli – hemolytic diarrhea

16. ENTEROBACTERIACEAE DETERMINANTS OF PATHOGENICITY
4. COLONIZATION FACTORS - cellular surface factors: capsule – Klebsiella pneumoniae “Vi” antigen – S. typhi 5. Other Factors - ability to penetrate epithelial lining – E.coli - Shigella - Salmonella

17. ESCHERICHIA

18. ESCHERICHIA Includes 6 species 5 species associated with human disease
Escherichia blattae
Escherichia coli
Escherichia fergusonii
Escherichia hermanii
Escherichia vulneris

19. ESCHERICHIA COLI Grows well on commonly used media
On enteric isolation media
– lactose fermenting colonies
On blood agar – β-hemolytic (assoc with UTI)
Majority – non-pigmented, motile
Produce lysine decarboxylase, use acetate as carbon source, hydrolysis of tryptophan to indole

20. ESCHERICHIA COLI
Serologic typing is based on the determination of the O antigen type, the H antigen type and when applicable the K antigen type.
- 164 O antigens
- 100 K antigens
- 50 H antigens
Example: Serotype O157:H7 – hemorrhagic colitis
Serotype O124:H30 –enteroinvasive;
bacillary dysentery

21. ESCHERICHIA COLI DETERMINANTS OF PATHOGENICITY
Surface Factors
a. K1 capsule – E.coli with K1 capsule cause neonatal meningitis
b. O antigen
c. S fimbriae
2. Enterotoxins –produces watery diarrhea caused by the outpouring of fluids and electrolytes
- plasmid mediated

22. ESCHERICHIA COLI DETERMINANTS OF PATHOGENICITY
2. Enterotoxin – produces watery diarrhea - plasmid mediated a. LT enterotoxin - similar to enterotoxin of Vibrio cholera - stimulates adenylate cyclase in the epithelial cells of the small intestines, increasing perrmeability of the intestinal lining, resulting to loss of fluids and electrolytes

23. ESCHERICHIA COLI DETERMINANTS OF PATHOGENICITY
2. Enterotoxin
b. ST enterotoxin – ST producing E.coli do not cause diarrhea
Verotoxins (Shigalike Toxins)
- associated with 3 human syndrome:
- diarrhea, hemorrhagic colitis, hemolytic
uremic syndrome
- inhibit protein synthesis similar to Shigatoxin

24. ESCHERICHIA COLI DETERMINANT OF PATHOGENICITY
4. Other factors a. Enteroinvasiveness – strains have large plasmids that encode for O antigens b. Hemolytic – strains are nephropathogenic

25. ESCHERICHIA COLI CLINICAL MANIFESTATION
Pulmonary infections – nosocomial pneumonia
- most patients are 50 yrs or older
- with underlying chronic disease
- main source: endogenous aspiration of oral secretions containing E.coli
2. Neonatal meningitis – with subsequent neurologic or developmental abnormalities

26. ESCHERICHIA COLI CLINICAL MANIFESTATION
3. Wound infections – especially occurring in the abdomen 4. Sepsis – can invade the bloodstream from any of the primary infection sites

27. ESCHERICHIA COLI CLINICAL MANIFESTATIONS
5. Diarrheal disease a. Enteropathogenic E.coli (EPEC) - cause of infantile diarrhea - adhere to mucosal cells of small bowell - loss of microvilli - produce watery diarrhea

28. ESCHERICHIA COLI CLINICAL MANIFESTATION
5. Diarrheal disease b. Enterotoxigenic E.coli – common cause of travelers diarrhea - some strains produce LT, plasmid mediated - toxin activates adenylyl cyclase - intense and prolonged hypersecretion of water and chlorides and inhibits reabsorption of sodium

29. ESCHERICHIA COLI CLINICAL MANIFESTATION
5. Diarrheal disease c. Enterohemorrhagic E. coli (EHEC) - produces verotoxin - associated with hemorrhagic colitis and hemolytic uremic syndrome - serotype O157:H7 – most common

30. ESCHERICHIA COLI CLINICAL MANIFESTATION
5. Diarrheal diseases d. Enteroinvasive E. coli (EIEC) - cause bacillary dysentery in all age groups - disease is very similar to shigellosis - occurs most commonly in children - invade intestinal mucosal cells

31. ESCHERICHIA COLI CLINICAL MANIFESTATION
5. Diarrheal diseases f. Enteroaggregative E.coli (EAEC) - causes acute and chronic diarrhea in persons in developing countries - produce ST-like toxins

32. To be continued….

33. KLEBSIELLA

34. KLEBSIELLA TAXONOMY
Klebsiella pneumoniae (Friedlander’s bacillus) Klebsiella oxytoca – Klebsiella ozeana – Klebsiella rhinoscleromatis

35. KLEBSIELLA BIOCHEMICAL AND CULTURAL CHARACTERISTICS
Appear as lactose fermenting colonies on differential enteric media
Non-motile
Large capsule – colonies appear large, moist and mucoid

36. KLEBSIELLA ANTIGENIC STRUCTURE
Possess O and K antigens
K antigens are most useful in serologic typing

37. KLEBSIELLA DETERMINANTS OF PATHOGENICITY
Capsule – resist phagocytosis
- encapsulated strains are more virulent
2. Endotoxin
3. Enterotoxin – isolated in patients with tropical sprue
- similar to E.coli ST and LT
- plasmid mediated

38. KLEBSIELLA CLINICAL INFECTION
Klebsiella pneumoniae
- cause primary community-acquired
pneumonia
- typical patient: middle or older aged with underlying medical problems – alcoholism, chronic bronchopulmonary disease, diabetes mellitus
- most patients – thick, non-putrid bloody sputum – necrosis and abscess formation

39. KLEBSIELLA CLINICAL INFECTION
Klebsiella pneumonia
- can cause urinary tract infection
-wound infections, bacteremia, meningitis
2. Klebsiella oxytoca – causes chronic atrophic rhinitis – fetid odor
3. Klebsiella rhinoscleromatis – infects nose and pharynx; produces granulomatous inflammation

40. ENTEROBACTER

41. ENTEROBACTER
Enterobacter cloaca Enterobacter aerogenes

42. ENTEROBACTER BIOCHEMICAL AND CULTURAL CHARACTERISTICS
Motile
Grows on media used for the isolation of enterics
Rapid lactose fermenters and produce pigmented colonies

43. ENTEROBACTER ANTIGENIC STRUCTURE
- Antigenic subgrouping not as developed as E.coli and Klebsiella

44. ENTEROBACTER CLINICAL INFECTION
Isolated less frequently than E. coli and Klebsiella
Capable of infecting any tissue
Most frequently associated with urinary tract infection
Most infections occur in patients with underlying problems – nosocomial
Risks: long term hospitalization, plac ement of intravenous catheters, respiratory colonization; prior use of antibiotics,

45. SERRATIA

46. SERRATIA Serratia marcescens Serratia liquifaciens
Can be differentiated from other members of the Enterobacteriaceae by:
- ability to produce extracellular deoxyribonuclease (Dnase),
lipase and gelatinase
- resistance to colistin and cephalosporin

47. SERRATIA O and A antigens are important epidemiologic markers
All Serratia infections – associated with underlying disease, changing physiologic patterns, immunosuppressive therapy or mechanical manipulations
- 90% are hospital acquired: UTI, wound infections, pneumonia, septicemia

48. PROTEUS

49. PROTEUS
Proteus mirabilis
Proteus vulgaris

50. PROTEUS CULTURAL CHARACTERISTICS
Produce a translucent sheet of growth on non-selective media such as blood agar
- swarming

51. PROTEUS BIOCHEMICAL CHARACTERISTICS
- Distinguished from other enterics – produce phenylalanine deaminase
- All species produce urease:
urea = ammonia + CO2
Proteus mirabilis does not hydrolyze tryptophan to indole – basis of grouping into indole-positive and indole-negative

52. PROTEUS ANTIGENIC STRUCTURE
All members possess O, H and K antigens
certain P. vulgaris strains share antigens with Rickettsia – used as -antigens for the detection of rickettsial antibodies in Weil-Felix test
Strains OX-19, OX-K, OX-2

53. PROTEUS CLINICAL INFECTION
Proteus mirabilis -2nd leading cause of community acquired UTI - major cause of nosocomial infection - urease formation causes urine to become alkaline – stone formation - rapid motility – invasion of urinary tract

54. PROVIDENCIA

55. PROVIDENCIA Providencia rettgeri – previously Proteus
Providencia alcalifaciens
Providencia stuartii
Members of the normal intestinal flora
All cause urinary tract infections
Often resistant to antimicrobial therapy

56. CITROBACTER

57. CITROBACTER
Citrobacter freundii – isolated from patients with diarrhea Citrobacter diversus- neonatal meningitis and brain abscesses

58. TO BE CONTINUED…..

59. SHIGELLA

60. SHIGELLA TAXONOMY
Shigella – genetically indistinguishable from E.coli - divided into 4 serogroups given species name serogroup A – Shigella dysenteriae serogroup B – Shigella flexneri serogroup C – Shigella boydii serogroup D – Shigella sonnei

61. SHIGELLA BIOCHEMICAL PROPERTIES AND CULTURAL CHARACTERISTICS
Factors that distinguish from Salmonella
Appear as non-lactose fermenting colonies
Non-motile
Do not produce H2S
Do not produce gas from glucose (except S. flexneri)

62. SHIGELLA RESISTANCE TO PHYSICAL AND CHEMICAL AGENTS
Less resistant than most enterics to physical and chemical agents
Susceptible to most common disinfectants
Can tolerate low temperatures if adequate moisture is present
Can survive for more than 6 months in water and room temperature

63. SHIGELLA ANTIGENIC STRUCTURE
-Shigella are divided into 4 major O antigenic groups – A, B, C, D subgrouping – based on minor O antigens ex: 12 serologic types of group A 6 serologic types of group B 18 serologic types of group C - No H antigens - nonmotile

64. SHIGELLA DETERMINANTS OF PATHOGENESIS
1. Surface properties – due to O antigens
Survive the passage through upper GIT,
2. Invasiveness - attach to colonic cells and penetrate the epithelial cells by induced phagocytosis, escape from phagocytic vacuole, multiply and spread inside the cytoplasm and passage to adjacent cells.

65. SHIGELLA DETERMINANTS OF PATHOGENICITY
3. Toxins
-Shiga toxin interferes with protein synthesis
Subunit A - inactivates the 60S ribosomal unit
Subnunit B – serve as binding factor
bacillary dysentery is a 2-stage disease
early stage - Shigella multiply in a noninvasive manner in the jejunum- and produce the toxin which is taken up by small bowel receptors- result in an activated secretory process
second phase – involve the large intestines

66. SHIGELLA TOXINS Endotoxin - from the LPS
- causes irritation of the bowel wall
2. Shigella dysenteriae exotoxin
- neurotoxic –meningisimus, coma
- enterotoxic – produces diarrhea

67. SHIGELLA PATHOGENESIS
Spectrum of disease asymptomatic infection to severe bacillary dysentery with high fever, chills, convulsions, abdominal cramps, tenesmus and frequent bloody stools Organisms rarely penetrate the intestinal wall and spread to other parts of the body For children and elderly - dehydration

68. SHIGELLA DIAGNOSTIC LABORATORY TESTS
Specimen – rectal swab for culture
- feces – is not a good specimen,
- Shigella is sensitive to acids in feces
Culture – differential media- MacConkey or EMB
- selective media – SS agar

69. To be continued…..

70. SALMONELLA

71. SALMONELLA TAXONOMY Complicated nomenclature
CDC: Salmonella serotype typhimurium
taxonomically correct:
Salmonella enterica subspecies enterica serotype typhimurium
( read p 257 Jawetz)
4 serotypes that cause enteric fever
Salmonella paratyphi A/B
Salmonella cholerasuis
Salmonella typhi

72. SALMONELLA BIOCHEMICAL AND CULTURAL CHARACTERISTICS
- Do not ferment lactose
Most strains are motile
Produce H2S from thisulfate
Produce gas from glucose except serotype typhi

73. SALMONELLA RESISTANCE TO PHYSICAL AND CHEMICAL AGENTS
Capable of tolerating high concentrations of bile than most enterics
Serotype cholerasuis is used as standard test organism for determining the efficacy of disinfectants

74. SALMONELLA ANTIGENIC STRUCTURE
Antigens used to serotype – O and H antigens
O antigens - similar to O antigens of other Enterobacteriaceae – basis for serotyping
H antigens are different from the other Enterobcteriaceae
Capsular antigens – minor role in classification
- but with pathogenic significance
Vi (virulence) of serotype typhi prevent intracellular destruction

75. SALMONELLA DETERMINANT OF PATHOGENICITY
Surface antigens
a. O antigens – attach to host cells
- survive intracellularly
- resist killing by complement mediated
immune response
b. Vi antigens of serotype typhi – survival
inside the cell; prevents phagocytosis

76. SALMONELLA DETERMINANT OF PATHOGENICITY
2. Invasiveness – unlike Shigella, penetrate into subepithelial layers - serotypes causing gastroenteritis target cell – epithelial cells - serotype typhi – target cell - macrophage

77. SALMONELLA DETERMINANT OF PATHOGENICITY
3. Endotoxin - responsible for fever - cause activation of chemotactic factors of complement – cause localization of leukocytes in the lesions of typhoid fever 4. Enterotoxins – similar to LT and ST of E.coli - no clear role in disease production - affects small intestines but in salmonella infections large bowel is affected

78. SALMONELLA PATHOGENESIS AND CLINICAL INFECTION
Typhoid fever – Salmonella typhi
- uniquely adapted to humans
-sole source of infection: human carriers
convalescent carriers – short period
chronic carriers – longer than 1 year
- majority: older female with
gallbladder disease
- contaminate food or water

79. SALMONELLA PATHOGENESIS AND CLINICAL INFECTION
Typhoid fever – incubation period: days – lethargy, fever, malaise, bradycardia
- constipation – rule rather than diarrhea
- organisms penetrate intestinal wall
- infect regional lymphatic system
-some invade the bloodstream – other RES
like spleen (WBC ct normal or low)
- organisms are ingested by monocytes
- not killed: multiply

80. SALMONELLA PATHOGENESIS AND CLINICAL INFECTION
Typhoid fever
2nd week: reenter bloodstream
- prolonged bacteremia
- infection of biliary tree
- patient is severely ill, high fever and delirious
- tender abdomen: rose-colored spots
- diarrhea begins up to third week

81. SALMONELLA PATHOGENESIS AND CLINICAL INFECTION
Typhoid fever
3rd week: reinfection of intestinal tract from gallbladder
- may cause necrosis of Peyer’s patches
Complications: perforation
sever bleeding
abscess formation

82. SALMONELLA PATHOGENESIS AND CLINICAL INFECTION
2. Enteric fever - Typhoid fever – enteric fever - other enteric fevers caused by Salmonella serotypes paratyphi A paratyphi B - milder symptoms - lower mortality

83. SALMONELLA PATHOGENESIS AND CLINICAL INFECTION
3. Enterocolitis -due to Salmonella Typhimurium Salmonella Enteritides - actual infection of the colon hrs after ingestion of the organism - diarrhea, fever and abdominal pain, nausea - resolves 2-5 days - dehydration and electrolyte imbalance - inflammatory lesions of small and large int.

84. SALMONELLA PATHOGENESIS AND CLINICAL MANIFESTATION
4. Septicemia with focal lesions - commonly Salmonella cholerasuis - fever, chills, anorexia, anemia - focal lesions occur in any organ: secondary osteomyelitis pneumonia pulmonary abscess meningitis endocarditis

85. SAMONELLA DIAGNSOSIS SPECIMEN A. Feces –acute gastroenteritis
b. blood – septicemia and 1st week of illness
in enteric fever
typhoid fever: use feces in 3rd week of fever
Reading assignment: review methods of isolation of salmonella, culture and serologic methods

86. SALMONELLA SEROLOGIC METHODS
Agglutination test
known sera + unknown culture
clumping within few minutes (+)
rapid preliminary identification of cultures
2. Tube Dilution Agglutination test (Widal Test)
- test to detect antibodies formed vs O and H
antigen
- rise in antibody titers, serum obtained at intervals of 7-10 days

87. THE END….
THANK YOU