Dr.Babasaheb N. Kumbhar M.V.Sc bobbyvph11@gmail

1. ENTEROBACTERIACEAE Dr.Babasaheb N. Kumbhar M.V.Sc bobbyvph11@gmail.com

2. Enterobacteriaceae Habitat digestive tube(colon) of human and animals 150 Species Facultative anaerobes Diarrheal illnesses 3 million death/year 4 billion infectoions/ worldwide

3. Enterobacteriaceae • Classification – more than15 different genera • Escherichia • Shigella • Edwardsiella • Salmonella • Citrobacter • Klebsiella • Enterobacter • Hafnia • Serratia

4. Enterobacteriaceae • Proteus • Providencia • Morganella • Yersinia • Erwinia • Pectinobacterium

5. Family Enterobacteriaceae Certain E .coli strains can be considered true pathogens True pathogen (nonmotile) (nonmotile) True pathogen True pathogen

6. Morphology and General Characteristics • Are facultative anaerobes • Gram-negative, non-sporing, rod shaped bacteria • If motile, motility is by peritrichous flagella(Except Shigella & Klebsiella) • Many are normal inhabitants of the intestinal tract of man and other animals • Some are enteric pathogens and others are urinary or respiratory tract pathogens • Differentiation is based on biochemical reactions and differences in antigenic structure

7. Morphology and Physiology Klebsiella spp. have large capsule (form large and very mucoid colonies); those of Enterobacter have smaller capsule; the others produce diffusible slime layers (form circular, convex and smooth colonies). Short gram-negative rods. Facultative anaerobes. Grow readily and rapidly on simple media. K. pneumoniae

8. ENTEROBACTERIACEAE PHYSIOLOGY • Glucose is fermented with strong acid formation and often gas • 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

9. Enterobacteriaceae Meningitis Opportunistic pathogens Escherichia coli Klebsiella pneumoniae Enterobacter aerogenes Serratia marcescens Proteus spp. Providencia spp. Citrobacter spp. Pneumonia Sepsis Diarrhea UTI Obligate pathogens Salmonella spp. Shigella spp. Yersinia spp. Some E. coli strains

10. Incidence of Enterobacteriaceae Associated with Bacteremia

11. 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

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

13. Urinary Tract Infection, Pneumonia • Escherichia coli, Klebsiellapneumoniae, Enterobacter spp., and Proteus mirabilis • Pneumonia: Enterobacter spp., Klebsiellapneumoniae, Escherichiacoli, and Proteus mirabilis • Wound Infection: Escherichia coli, Enterobacterspp., Klebsiellapneumoniae, and Proteus mirabilis • Bacteremia: Escherichia coli, Enterobacter spp., Klebsiellapneumoniae, and Proteus mirabilis Intestinal Infection • Shigellasonnei (serogroup D) • Salmonella serotype enteritidis • Salmonella serotype typhimurium • Shigellaflexneri (serogroup B) • Escherichia coli O157:H7 • Yersiniaenterocolitica

14. MacConkey (MAC) Agar

15. Eosin Methylene Blue (EMB) Agar (Levine)

16. Antigenic Structure • Most are motile by peritrichous flagella --H antigens. • Capsule – Kantigen (Vifor Salmonella). • Cell envelope (wall) • LPS (endotoxin) – • O antigen. • various outer • membrane proteins. • Pili- various antigen • types, some encoded by plasmids

17. Pathogenesis and Immunity Common virulence factors Endotoxin (Lipid A of LPS) Capsule Antigenic phase variation Acquisition of growth factors (e.g. Fe) Resistance to serum killing Antimicrobial resistance Type III secretion systems: possessed by some Enterobacteriaceae pathogens, e.g., E. coli, Yersinia, Salmonella, and Shigella; facilitate transport of bacterial virulence factors directly into host cells.

18. HE Agar: Growth of Enteric Pathogens and Commensals

19. Escherichia coli • Toxins: two types of enterotoxin; Shiga-type toxin; Enteroaggregative ST-like toxin; Hemolysins; Endotoxin • Virulence factors that protect the bacteria from host defenses: Capsule/Iron capturing ability (enterochelin)

20. E. coli • May be hemolytic on CBA – more common in pathogenic strains • KEY tests for the normal strain: • TSI is A/A + gas • LIA K/K • Urea – • Indole + • Citrate – • Motility + • There is an inactive biotype that is anaerogenic, lactose –, and nonmotile.

21. EnteropathogenicE. coli • fever • infantdiarrhea • 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, EnterotoxigenicE. coli • 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.

22. E.coli-Enteroinvasive (EIEC) • The organism attaches to the intestinal mucosa via pili • Outermembrane 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- fever,severe abdominal cramps, malaise, and watery diarrhea followed by scanty stools containing blood, mucous,& pus. • resembles shigellosis E.coli-c. Enteropathogenic (EPEC) • Malaiseand low grade feverdiarrhea, 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. Enterohemorrhagic (EHEC) • Usually O157:H7 • Hemorrhagic • bloody, copious diarrhea • few leukocytes • afebrile • hemolytic-uremic syndrome • hemolytic anemia • thrombocytopenia (low platelets) • kidney failure Transmission electron micrograph

24. EnteroaggregativeE. coli • 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.

25. Shigella

26. Shigella species • Shigella • Contains four species that differ antigenically and, to a lesser extent, biochemically. • S. dysenteriae (Group A) • S. flexneri (Group B) • S. boydii (Group C) • S. sonnei (Group D) bacillary dysentery, shigellosis, bloody feces, intestinal pain, pus • Biochemistry • TSI K/A with NO gas • LIA K/A • Urea – • Motility - • All ferment mannitol except S. dysenteriae • S. sonnei may show delayed lactose fermentation

27. Shiga toxin 1. Chromosomally encoded 2. Neurotoxic 3. Enterotoxic 4. Cytotoxic Enterotoxicity can make the disease clinically appear as a diarrhea. The toxin inhibits protein synthesis (acting on the 60S ribosome and lysing 28S rRNA).

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

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

30. XLD Agar: Growth of Shigella and Proteus

31. Salmonella Only a few types that are commonly associated with characteristic human diseases - 1. S. enteritidis 2. S. cholerae-suis 3. S. typhi several syndromes including gastroenteritis, enteric (typhoid) fever or septicemia

32. Salmonella • Biochemistry • TSI K/A + gas and H2S: S. typhi produces only a small amount of H2S and no gas , and S. paratyphi A produces no H2S • LIA K/K with H2S with S. paratyphi A giving K/A results • Urea – • Motility + • Citrate +/- • Indole - • 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

33. Salmonella typhi • The organism is transmitted from: 1. a human reservoir 2. in the water supply (if poor sanitary conditions) 3. in contaminated food The antigenic structures of salmonellae used in serologic typing

34. 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 • 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 • 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

35. XLD Agar: Appearance of Salmonella

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

37. Klebsiella • NF of GI tract, but potential pathogen in other areas • TSI A/A + gas • LIA K/K • Urea + • Citrate + • MR-, VP+ • Motility - • Has both O and K antigens

38. Klebsiella K.Pneumoniae on BA and MAC • Virulence factors • Capsule • Adhesions • Iron capturing ability • Clinical significance • Causes pneumonia, mostly in immunocompromised hosts. • Permanent lung damage is a frequent occurrence (rare in other types of bacterial pneumonia) • A major cause of nosocomial infections such as septicemia and meningitis K.Pneumoniae On BA

39. Proteus • General characteristics: “swarming” phenomenon on nonselective agar(P.vulgaris; P.mirabilis and P.myxofaciens) • P.vulgaris strains (OX-19, OX-K, OX-2)have common antigen with Rickettsia (Weil-Felix test). • urinary tract infections; food poisoning.

40. Some enteric bacteria are motile. Klebsiella species are not motile, while Proteus species move very actively by means of peritrichous flagella, resulting in "swarming" on solid medium. Some strains of E. coli produce hemolysis on blood plates. Proteus spp.

41. CITROBACTER Resident of soil, water, stool • C.freundii • UTI and bacteremia

42. Serratia

43. Serratia • A free-living saprophyte • TSI A/A or K/A; +/- gas (does not ferment lactose) • LIA usually K/K • Citrate + • Motility + • Urea +/- • Has been found in RT and UT infections • Is resistant to many antimicrobics

44. Positive test e.g. E. coli Negative test e.g. Klebsiella Citrate Utilization Test Special Features: Used in the differentiation of genera and species. e.g. E. coli (+) from Klebsiella (-). Urease Test Indole test Negative test Positive test Positive Klebsiella, Enterobacter Negative E. coli

45. MR/VP test • Results 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)

46. Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae

47. Summary of morphology, cultural characteristics, and biochemical reactions of Enterobacteriaceae

48. Identification of Gram's -ve rods Oxidase Test Negative Positive Enterobacteriaceae Pseudomonas MacConkey’s agar & TSI • O/F test: O+/F- • Nitrate test: +ve further reduction to N2 Pink colonies on MacConkey & acidic butt and slant on TSI colorless colonies on MacConkey & acidic butt alkaline slant onTSI • Growth on cetrimide agar: Pale colonies with green pigmentation Lactose non-fermenter Lactose fermenter IMViC test & EMB No H2S production (no blacking in TSI) H2S production (blacking in TSI) Urease production IMViC ++ - - & black colonies with metalic shines on EMB IMViC - - ++ Shigella Motility -ve +ve SS agar Not motile Motile colorless colonies with black centers Proteus E.coli Salmonella Klebsiella Enterobacter

49. Thank you

50. References: • www.slideshare.net