ENTEROBACTERIACEAE

1. ENTEROBACTERIACEAE Dr. Rouchelle Dept of Microbiology

2. Characteristics of the Enterobacteriaceae • Gram-negative bacilli Fourmajor features: • Ferment glucose • Reduce nitrates to nitrites • Oxidase negative • All except Klebsiella, Shigella and Yersinia are motile by peritrichous flagella

3. Grow readily on • MacConkey (MAC) agar • Eosin methylene blue (EMB) agar • Grow readily at 35oC except Yersinia (25o-30oC) • Do not form spores • Natural Habitat: • Environment (soil, water, and plants) • Intestines of humans and animals

4. Classification of Enterobacteriaceae: • Very large number of organisms in Family Enterobacteriaceae, species are grouped into - TRIBES • Tribe 1 – Escherichieae • Tribe 2 – Klebsiellae • Tribe 3: Proteae • Tribe 4 : Yersiieae • Tribe 5 : Erwinieae • Within each Tribe, species are further subgrouped under genera

5. Major Genera of family Enterobacteriaceae: • Escherichia • Shigella • Salmonella • Yersinia • Klebsiella • Proteus

6. Antigenic Factors of Enterobactericeae: Ability to colonize, adhere, produce various toxins and invade tissues Some possess plasmids that may mediate resistance to antibiotics Imp antigens used to identify the organisms: O antigen – somatic, cell wall, heat-stable Ag H antigen – flagellar, heat labile Ag K antigen – capsular, heat-labile Ag

7. Clinical Significance of Enterics: Ubiquitous in nature Except for Salmonella, Shigella and Yersinia, most are present in the intestinal tract of animals and humans as commensal flora; “fecal coliforms” Some live in water, soil and sewage

8. Clinical Significance of Enterics: Based on clinical infections produced, enterics are divided into two categories: Opportunistic pathogens – normally part of the usual intestinal flora, that may produce infection outside the intestine Primary intestinal pathogens – Salmonella, Shigella, and Yersinia sp.

9. Enterobacteriaceae: Modes of Infection • Contaminated food and water (Salmonella spp., Shigella spp., Yersiniaenterocolitica, Escherichia coli O157:H7) • Endogenous infection (urinary tract infection, primary bacterial peritonitis, abdominal abscess) • Abnormal host colonization (nosocomial pneumonia) • Transfer between debilitated patients • Insect (flea) vector: unique for Yersiniapestis

10. Urinary tract infection: Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., and Proteus mirabilis • Pneumonia: Klebsiella pneumoniae; Enterobacter spp., • Wound Infection: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis • Bacteremia: Escherichia coli, Enterobacter spp., Klebsiella pneumoniae, and Proteus mirabilis

11. Enterobacteriaceae: Intestinal Infection • Escherichia coli • Shigella • Salmonella • Yersinia enterocolitica

12. Identification of Enterobacteriaceae: • IMViC Test • Indole, Methyl Red, Voges-Prosakaur, Citrate (IMViC) Tests: • The IMViC series of reactions allows for the differentiation of the various members of family Enterobacteriaceae.

13. A) Indole –Positive, B) MR –Positive C) VP- Positive, D) Citrate- Negative

14. Methyl Red-VogesProskauer (MR-VP) Tests Principle Glucose Acidic pathway Or Neutral pathway Acety methyl carbinol (ACETOIN) Mixed acids  pH less than 4.4 Barrit’s A Barrit;s B Methyl Red indicator VP positive Klebsiella MR positive E. coli Pink color Red color

15. MR/VP test Voges-Proskauer test Methyl Red test • Pink: Positive VP (Klebsiella) • Red: Positive MR (E. coli) • No pink: Negative VP (E. coli) • Yellow or orange: Negative MR (Klebsiella)

16. Result

17. TSI Reactions of the Enterobacteriaceae A/A + g = acid/acid plus gas (CO2) A/A = acid/acid A/A + g, H2S = acid/acid plus gas, H2S Alk/A = alkaline/acid Alk/A + g = alkaline/acid plus gas Alk/A + g, H2S = alkaline/acid plus gas, H2S Alk/A + g, H2S (w) = alkaline/acid plus gas, H2S (weak)

18. A/A + g Escherichia coli Klebsiella A/A + gas, H2S Citrobacterfreundii Proteus vulgaris Non-lactose, sucrose fermenter

19. Alk/A Shigella Providencia Alk/A + g Salmonella serotype Paratyphi A Alk/A + g, H2S Salmonella Proteus mirabilis Edwardsiella tarda

20. Lactose fermenting colonies Non lactose fermenting colonies

22. Escherichia coli Most significant species in the genus Important potential pathogen in humans Common commensal in the intestine Pink (lactose positive) colony with surrounding pink area on MacConkey

23. Ferments glucose, lactose, trehalose, & xylose Usually motile Positive indole and methyl red tests Does NOT produce H2S Simmons citrate negative Voges-Proskauer test negative

24. Infections caused by E.coli: Meningitis, gastrointestinal, urinary tract, wound, and bacteremia Gastrointestinal Infections : Enteropathogenic (EPEC) Primarily in infants and children; Outbreaks in hospital nurseries and day care centers Stool has mucous but not blood Identified by serotyping

25. EnteropathogenicE.coli • Destruction of surface microvilli • Fever • Diarrhea • Vomiting • Nausea • Non-bloody stools Gut lumen 25

26. Enterotoxigenic E. coli • Diarrhea resembling cholera • Travelers diarrhea 26

27. Enterotoxigenic E. coli Heat labile toxin Like cholera toxin Adenylcyclase activated Cyclic AMP Secretion water/ions Heat stable toxin Guanylatecyclase activated cyclic GMP uptake water/ions 27

28. Enteroinvasive E. coli (EIEC ) • Dysentery • - Resembles shigellosis

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

30. O157:H7transmitted- through meat products or sewage-contaminated vegetables Hemorrhagic diarrhea Bloody, copious diarrhea Few leukocytes Afebrile Hemolytic-uremic syndrome (HUS) Hemolytic anemia Thrombocytopenia (low platelets) Kidney failure 30

31. Enterohemorrhagic E. coli • Vero toxin- “shiga-like” • Hemolysins Enteroaggregative (EaggEC) Cause diarrhea by adhering to the mucosal surface of the intestine; watery diarrhea; symptoms may persist for over two weeks 31

32. Urinary Tract Infections E. coli is most common cause of UTI and pyelo-nephritis in humans Usually originate in the large intestine Able to adhere to epithelial cells in the urinary tract

33. Septicemia & Meningitis Most common causes of septicemia and meningitis among neonates Acquired in the birth canal before or during delivery E. coli also causes bacteremia in adults, secondary to genitourinary tract infection or a gastrointestinal source

34. Klebsiella, Enterobacter Usually found in intestinal tract Wide variety of infections, primarily pneumonia, wound, and UTI General characteristics: Non-motile Simmons citrate positive H2S negative, Weakly urease positive MR negative; VP positive

35. Usually found in Gastro-intestinal tract K. pneumoniae is most commonly isolated species Possesses a polysaccharide capsule - which protects against phagocytosis and antibiotics Makes the colonies moist and mucoid Frequent cause of nosocomial pneumonia

36. Significant biochemical reactions Lactose positive Most are urease positive Non-motile

37. Nosocomial pneumonia:Spread by health care personnel and equipment • Frequently caused by K. pneumoniae • Often seen in middle-aged males who abuse alcohol • Difficult to diagnose due to commensals in sputum

38. Proteus, Morganella & Providencia species All are normal intestinal flora Opportunistic pathogens Deaminate phenylalanine Non lactose fermenters

39. Proteus species P. mirabilis and P. vulgaris are widely recognized human pathogens Isolated from urine, wounds, and ear and bacteremic infections Both produce swarming colonies and have a distinctive “burned chocolate” odor Both are strongly urease positive Both are phenylalanine deaminase positive

40. Exhibits characteristic “swarming”

41. Laboratory Diagnosis of Enterics Specimen collection: Specimens collected and transported in Cary-Blair, Amies, or Stuart media Isolation and Identification Site of origin must be considered Enterics from sterile body sites are highly significant Routinely cultured from stool

42. Media for Isolation and Identification of Enterics: Blood agar and a selective/differential medium such as MacConkey On MacConkey, lactose positive are pink; lactose negative are clear and colorless

43. For stool, highly selective media, such as Hektoen Enteric (HE), XLD, or SS is used along with MacConkey agar Identification: All enterics are Oxidase negative Ferment glucose Reduce nitrates to nitrites

44. SHIGELLA & SHIGELLOSIS

45. Characteristics of Shigella: • Short gram negative bacilli • Non-motile • Do not produce gas from glucose • Fragile organisms • Non-lactose fermenting • Resistant to bile salts

46. CLASSIFICATION: • Shigellae are classified into 4 subgroups (A, B, C, D) based on biochemical and serological specificity • Subgroup A – Mannitol fermentation negative • Subgroup B, C, D – Mannitol positive

48. Clinical Infections • Dysentery (bloody stools, mucous, and numerous WBC) • S. sonnei and S. flexneri common • Humans are only known reservoir • Oral-fecal transmission • Low infectious dose (102-104 CFU)

49. Incubation period = 1-3 days • Watery diarrhea with fever; changing to dysentery • Outbreaks in daycare centers, nurseries, institutions

50. Pathogenesis of Shigellosis (2 stages) • Early stage: • Watery diarrhea - Enterotoxic activity of Shiga toxin • Following ingestion and noninvasive colonization, multiplication, and production of enterotoxin in the small intestine • Second stage: • Adherence and tissue invasion of large intestine - symptoms of dysentery • Cytotoxic activity of Shiga toxin increases severity of dysentry