Enterobacteriaceae

1. Enterobacteriaceae

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

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

4. Enterobacteriaceae • Morphology and General Characteristics • Gram-negative, non-sporing, rod shaped bacteria • Oxidase – • Ferment glucose and may or may not produce gas in the process (aerogenic vs anaerogenic) • Reduce nitrate to nitrite (there are a few exceptions)

5. Enterobacteriaceae • Are facultative anaerobes • If motile, motility is by peritrichous flagella • 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

6. Enterobacteriaceae • Most grow well on a variety of lab media including a lot of selective and differential media originally developed for the the selective isolation of enteric pathogens. • Most of this media is selective by incorporation of dyes and bile salts that inhibit G+ organisms and may suppress the growth of nonpathogenic species of Enterobacteriaceae. • Many are differential on the basis of whether or not the organisms ferment lactose and/or produce H2S.

7. Enterobacteriaceae • On CBA they all produce similar colonies that are relatively large and dull gray. They may or may not be hemolytic. • The three most useful media for screening stool cultures for potential pathogens are TSI, LIA, and urea or phenylalanine agar. • The antigenic structure is used to differentiate organisms within a genus or species. • Three major classes of antigens are found:

8. Enterobacteriaceae • Somatic O antigens – these are the heat stable polysaccharide part of the LPS. • Variation from smooth to rough colonial forms is accompanied by progressive loss of smooth O Antigen. • Flagellar H antigens – are heat labile • Envelope or capsule K antigens – overlay the surface O antigen and may block agglutination by O specific antisera. • Boiling for 15 minutes will destroy the K antigen and unmask O antigens. • The K antigen is called the Vi (virulence) antigen in Salmonella typhi.

9. Antigenic Structure of Enterobacteriaceae

10. Enterobacteriaceae • Escherichia coli • Normal inhabitant of the G.I. tract. • Some strains cause various forms of gastroenteritis. • Is a major cause of urinary tract infection and neonatal meningitis and septicemia. • May have a capsule. • Biochemistry • Most are motile.

11. 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.

12. E. coli • Antigenic structure - has O, H, and K antigens. K1 has a strong association with virulence, particularly meningitis in neonates. • Virulence factors • Toxins • Enterotoxins – produced by enterotoxigenic strains of E. coli (ETEC). Causes a movement of water and ions from the tissues to the bowel resulting in watery diarrhea. There are two types of enterotoxin: • LT – is heat labile and binds to specific Gm1 gangliosides on the epithelial cells of the small intestine where it ADP-ribosylates Gs which stimulates adenylate cyclase to increase production of cAMP. • Increased cAMP alters the activity of sodium and chloride transporters producing an ion imbalance that results in fluid transport into the bowel.

14. E. coli toxins • ST – is heat stable and binds to specific receptors to stimulate the production of cGMP with the same results as with LT.

15. LT vs ST activity

16. E. coli toxins • Both enterotoxins are composed of five beta subunits (for binding) and 1 alpha subunit (has the toxic enzymatic activity).

17. Composition of subunits of enterotoxins

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

19. E. coli toxins • Enteroaggregative ST-like toxin – produced by enteroaggregative strains of E. coli (EAEC) – causes watery diarrhea. • Hemolysins – two different types may be found: cell bound and secreted. • They lyse RBCs and leukocytes and may help to inhibit phagocytosis when cell bound. • Endotoxin • Type III secretion system to deliver effector molecules directly into the host cells. • Involved in inducing uptake of EIEC into intestinal cells. • Involved in development of an attachment and effacing lesion in EPEC characterized by microvilli destruction and pedestal formation.

20. Type III secretion system

21. Pedestal formation

22. E. coli • Adhesions – are also called colonization factors and include both pili or fimbriae and non-fimbrial factors involved in attachment (e.g. intimin). • There are at least 21 different types of adhesions. • Antibodies to these may protect one from colonization. • Virulence factors that protect the bacteria from host defenses • Capsule • Iron capturing ability (enterochelin) • Outer membrane proteins - are involved in helping the organism to invade by helping in attachment (acting as adhesion) and in initiating endocytosis.

23. Types of adhesions (intimin)

24. E. coli • Clinical significance • Is the leading cause of urinary tract infections which can lead to acute cystitis (bladder infection) and pyelonephritis (kidney infection).

25. Ascending urinary tract infection

26. Urinary tract infections (UTI) • New evidence in women who suffer from recurrent UTIs suggests that this is due to the formation of pod-like E. coli biofilms inside bladder epithelial cells. • Bacteria living on the edges of the biofilms nay break off leading to a round of infection.

27. Pod-like biofilm

28. E. coli infections • Neonatal meningitis – is the leading cause of neonatal meningitis and septicemia with a high mortality rate. • Usually caused by strains with the K1 capsular antigen. • 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), and • enterohemorrhagic E. coli (EHEC).

29. Various types of E. coli

30. E. coli gastroenteritis • ETEC – is a common cause of traveler’s diarrhea and diarrhea in children in developing countries. • The organism attaches to the intestinal mucosa via colonization factors and then liberates enterotoxin. • The disease is characterized by a watery diarrhea, nausea, abdominal cramps and low-grade fever for 1-5 days. • Transmission is via contaminated food or water. • EPEC – Bundle forming pili are involved in attachment to the intestinal mucosa. • The type III secretion system inserts the tir (translocated intimin receptor) into target cells, and intimate attachment of the non-fimbrial adhesion called intimin to tir occurs. • Host cell kinases activated to phosphorylate tir which then causes a reorganization of host cytoskeletal elements resulting in pedestal formation and development of an attaching and effacing lesion • The exact mode of pathogenesis is unclear, but it is probably due to the attachment and effacement. • Diarrhea with large amounts of mucous without blood or pus occurs along with vomiting, malaise and low grade fever. • This is a problem mainly in hospitalized infants and in day care centers.

31. BFP EPEC EPEC EPEC

32. Pedestal formation

33. EPEC Tir injected

34. E. coli gastroenteritis • EIEC – The organism attaches to the intestinal mucosa via pili and outer membrane 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 include fever,severe abdominal cramps, malaise, and watery diarrhea followed by scanty stools containing blood, mucous, and pus. • EAEC – Mucous associated autoagglutinins cause aggregation of the bacteria at the cell surface and result in the formation of a mucousbiofilm. • The organisms attach via pili and liberate a cytotoxin distinct from, but similar to the ST and LT enterotoxins liberated by ETEC. • Symptoms include watery diarrhea, vomiting, dehydration and occasional abdominal pain.

35. E. coli gastroenteritis • EHEC – The organism attaches via pili to the intestinal mucosa and liberates the shiga-like toxin. • The symptoms start with a watery diarrhea that progresses to bloody diarrhea without pus and crampy abdominal pain with no fever or a low-grade fever. • This may progress to hemolytic-uremic syndrome that is characterized by low platlet count, hemolytic anemia, and kidney failure. • This is most often caused by serotypes O157:H7. • This strain of E. coli can be differentiated from other strains of E. coli by the fact that it does not ferment sorbitol in 48 hours (other strains do). • A sorbitol-Mac (SMAC) plate (contains sorbitol instead of lactose) is used to selectively isolate this organism. • One must confirm that the isolate is E. coli O1547:H7 using serological testing and confirm production of the shiga-like toxin before reporting out results. • Serotypes of E. coli other than O157H7 have now been found to cause this disease

36. Summary of E.coli strains that cause gastroenteritis.

37. E.coli • Antimicrobic therapy- E. coli is usually susceptible to a variety of chemotherapeutic agents, though drug resistant strains are increasingly prevalent. • It is essential to do susceptibility testing. • Treatment of patients with EHEC infections is not recommended because it can increase the release of shiga-like toxins and actually trigger HUS

38. 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) • 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

39. Shigella species • Antigenic structure • Differentiation into groups (A, B, C, and D) is based on O antigen serotyping; K antigens may interfere with serotyping, but are heat labile. • O antigen is similar to E. coli, so it is important to ID as Shigellabefore doing serotyping. • Virulence factors • Shiga toxin – is produced by S. dysenteriae and in smaller amounts by S. flexneri and S. sonnei. • Acts to inhibit protein synthesis by inactivating the 60S ribosomal subunit by cleaving a glycosidic bond in the 28S rRNA constituents. • This plays a role in the ulceration of the intestinal mucosa.

40. Shigella species • Outer membrane and secreted proteins • These proteins are expressed at body temperature and upon contact with M cells in the intestinal mucosa they induce phagocytosis of the bacteria into vacuoles. • Shigella destroy the vacuoles to escape into the cytoplasm. • From there they spread laterally (Polymerization of actin filaments propels them through the cytoplasm.) to epithelial cells where they multiply but do not usually disseminate beyond the epithelium.

42. Shigella attachment and penetration

43. Shigella attachment

44. Shigella penetration

45. Shigella invasion continued

46. Shigella • Clinical significance • Causes shigellosis or bacillary dysentery. • Transmission is via the fecal-oral route. • The infective dose required to cause infection 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 one is infected with. • S. dysenteria is the most pathogenic followed by S. flexneri, S. sonnei and S. boydii.

47. Shigella • Antimicrobial therapy • Sulfonamides are commonly used as are streptomycin, tetracycline, ampicillin, and chloramphenicol. • Resistant strains are becoming increasingly common, so sensitivity testing is required.

48. Salmonella • Salmonella • 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 infections • Clinically Salmonella isolates are often still reported out as serogroups or serotypes based on the Kauffman-White scheme of classification. • Based on O and H (flagella) antigens • The H antigens occur in two phases; 1 and 2 and only 1 phase is expressed at a given time. • Polyvalent antisera is used followed by group specific antisera (A, B, C1, C2, D, and E) • Salmonella typhi also has a Vi antigen which is a capsular antigen.

49. Phase variation of Salmonella

50. 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