1. ENTEROBACTERIACEAE

3. Morphology & Identification Gram-negative non-spore forming rods, catalase +ve, Oxidase - ve. Primarily normal flora of gastrointestinal tract. transient colonizers of skin, water streams. Facultative anaerobes: mixed acid fermentation. All ferment glucose; all reduce nitrates to nitrites. Lactose fermentation: usually normal flora positive and pathogens negative. Primary isolation media include eosin-methylene-blue (EMB) and MacConkey agar. Differential selective media for specific organisms include: Salmonella-Shigella (SS) medium, bismuth sulfite media, Hektoen enteric agar, XLD, deoxycholate-citrate agar.

8. Antigenic Structure – Most are motile by peritrichous flagella --H antigens. – Capsule – K antigen ( Vi for Salmonella). – LPS (endotoxin) – O antigen. – various outer membrane proteins. – Pili - various antigen types, some encoded by plasmids O (lipopolysaccharide) H (flagellar) K (capsular) – OKH (e.g., E. coli O55:K5:H21)

10. –septicemia, –pneumonia, –meningitis –urinary tract infections e.g., Citrobacter Enterobacter Escherichia Hafnia Morganella Providencia Serratia Opportunistic diseases -Enterobacteriaceae

11. Enterobacteriaceae: Foodborne infections – Escherichia coli – Salmonellae – Shigellae – Yersinia entercolitica

12. Associated with histocompatibility antigen (HLA) B27 Most common in young males. most common form of arthritis affecting men between 20-40 years old Symptoms Arthritis (pain, swelling, stiffness, redness) Conjunctivitis iritis/uveitis (rare but requires immediate treatment) Urinary tract infection (dysuria) may include reproductive organs Skin rash Heart problems in 10% of cases. Causes : – Not Enterobacteriaceae *Campylobacter *Chlamydia Reiter's syndrome – Enterobacteriaceae *Salmonella *Shigella *Yersinia

13. community acquired otherwise healthy people – Klebsiella pneumoniae * respiratory diseases * prominent capsule –urinary tract infection –fecal contamination *E. coli *Proteus – urease (degrades urea) – alkaline urine.. Cause stones

14. Escherichia coli

15. Toxins: two types of enterotoxin; Shiga-type toxin; ST toxin; Hemolysins; Endotoxin, LT toxin Adhesions: colonization factors; both pili or fimbriae ;non-fimbrial factors involved in attachment. There are at least 21 different types of adhesions. Virulence factors that protect the bacteria from host defenses: Capsule/Iron capturing ability Outer membrane proteins

16. E.coli urinary tract infection Is the leading cause of urinary tract infections which can lead to acute cystitis (bladder infection) and pyelonephritis (kidney infection). Polyuria, dysuria, hematouria, pyuria, flank pain..Can cause bacteremia. Typically uropathogenic E. coli produce hemolysins

17. E.coli-Meningitis and Sepsis Neonatal meningitis – is a leading cause of neonatal meningitis and septicemia with a high mortality rate. Usually caused by strains with the K1 capsular antigen.

18. Enteropathogenic E. coli infant diarrhea fever vomiting nausea non-bloody stools Destruction of surface microvilli This is a problem mainly in hospitalized infants and in day care centers.

19. Enterotoxigenic E. coli A watery diarrhea, nausea, abdominal cramps and low-grade fever for 1-5 days. Traveller’s diarrhea diarrhea in children in developing countries Transmission is via contaminated food or water.

20. Enterotoxigenic E. coli Causes diarrhea like cholera but milder caused by LT, ST, or LT/ST and colonization factors

21. Enterotoxigenic E. coli Heat labile toxin – Adenyl cyclase activation – cyclic AMP – secretion water/ions Heat stable toxin – Guanylate cyclase activation – cyclic GMP – uptake water/ions

22. E.coli-Enteroinvasive (EIEC) The organism attaches to the intestinal mucosa via pili 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, and watery diarrhea followed by bloody diarrhea (+ mucous, and pus). resembles shigellosis

23. Enteroinvasive E. coli (EIEC) Dysentery -resembles shigellosis -The EIEC are like Shigella: non-motile and non-lactose fermenter.

24. Enterohemorrhagic E. coli Also called Shiga toxin producing E. coli (STEC) Usually O157:H7 Transmission electron micrograph

25. E.coli Enterohemorrhagic (EHEC) Hemorrhagic colitis – bloody, copious diarrhea – few leukocytes – afebrile hemolytic-uremic syndrome – hemolytic anemia – thrombocytopenia (low platelets) – kidney failure

26. Enterohemorrhagic E. coli O157:H7 Diagnosis Usually sorbitol –ve MUG –ve Can be also identified by serology (for shiga toxin) Testing using Vero cells PCR

27. Enteroaggregative E. coli a cause of persistent, watery diarrhea with vomiting and dehydration in infants. the bacteria adheres to the intestinal mucosa and elaborates enterotoxins (heat-stable toxin (ST)). The result is mucosal damage, secretion of large amounts of mucus, and a secretory diarrhea.

28. Sanitary significance Total bacterial number: number of bacteria contained per ml or gm of the sample; the standard of drinking water is less than 100. Coliform bacteria index: the number of coliform bacteria detected out per 1000 ml sample; the standard of drinking water is less than 3

29. Escherichia coli Genetically E. coli and Shigella are genetically highly closely related. For practical reasons (primarily to avoid confusion) they are not placed in the same genus. Not surprisingly there is a lot of overlap between diseases caused by the two organisms. 1) Enteropathogenic E. coli (EPEC). Certain serotypes are commonly found associated with infant diarrhea. The use of gene probes has confirmed these strains as different from other groups listed below. There is a characteristic morphological lesion with destruction of microvilli without invasion of the organism that suggests adhesion is important. Clinically one observes fever, diarrhea, vomiting and nausea usually with non-bloody stools. 2) Enterotoxigenic E. coli (ETEC) produce diarrhea resembling cholera but much milder in degree. Also cause "traveler’s diarrhea". Two types of plasmid-encoded toxins are produced. a) Heat labile toxins which are similar to choleragen (see cholera section below). Adenyl cyclase is activated with production of cyclic AMP and increased secretion of water and ions. b) Heat stable toxins; guanylate cyclase is activated which inhibits ionic and water uptake from the gut lumen. Watery diarrhea, fever and nausea result in both cases. 3) Enteroinvasive E. coli (EIEC) produce dysentery (indistinguishable clinically from shigellosis, see bacillary dysentery below). 4) Enterohemorrhagic E. coli (EHEC). These are usually serotype O157: H7. These organisms can produce a hemorrhagic colitis (characterized by bloody and copious diarrhea with few leukocytes in afebrile patients). Outbreaks are often caused by contaminated hamburger meat. The organisms can disseminate into the bloodstream producing systemic hemolytic-uremic syndrome (hemolytic anemia, thrombocytopenia and kidney failure). Production of Vero toxin (biochemically similar to shiga toxin thus also known as "shiga-like") is highly associated with this group of organisms; encoded by a phage. Hemolysins (plasmid encoded) are also important in pathogenesis. As noted above, there are at least 4 etiologically distinct diseases. However, in the diagnostic laboratory generally the groups are not differentiated and treatment would be on symptomatology. Generally fluid replacement is the primary treatment. Antibiotics are generally not used except in severe disease or disease that has progressed to a systemic stage (e.g.hemolytic-uremia syndrome). Two major classes of pili are produced by E. coli : mannose sensitive and mannose resistant pili. The former bind to mannose containing glyocoproteins and the latter to cerebrosides on the host epithelium allowing attachment. This aids in colonization by E. coli.

30. Shigella

31. S. flexneri, S. boydii, S. sonnei, S. dysenteriae – bacillary dysentery – shigellosis bloody feces intestinal pain pus

32. Genral features Contain pili. Most strains can not ferment lactose; S. sonnei can slowly_ ferment lactose. According to O antigen, 4 serogroups. Easily acquiring drug-resistance.

33. Shigellosis within 2-3 days – epithelial cell damage

34. Shiga toxin enterotoxic cytotoxic inhibits protein synthesis – lysing 28S rRNA

35. Shigella attachment and penetration Within 2-3 days Epithelial cell damage

36. After the incubation period: 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. S. dysenteria is the most pathogenic followed by S. flexneri, S. sonnei and S. boydii.

37. Clinical significance man only "reservoir” Highly infectious 10 3 rather then 10 5 -10 8 for salmonellae and vibrios. Transmission via: – fecal to oral contact – Insects (flys..etc) transmitted by adult food handlers – unwashed hands

38. Diagnosis of Shigella infection Specimen: fresh stool, mucus flecks, rectal swabs / or serum to demonstrate rise in titer of agglutination antibodies. Primary media: MacConkey, EMB, Selective media: Hektoen enteric agar, SS agar, XLD, Deoxycholate-citrate agar Identification: TSI, Slide agglutination

39. Treating shigellosis manage dehydration patients respond to antibiotics, Problem of drug-resistance + dysentery vaccine.

40. Shigella Shigella (4 species; S. flexneri, S. boydii, S. sonnei, S. dysenteriae) all cause bacillary dysentery or shigellosis, (bloody feces associated with intestinal pain). The organism invades the epithelial lining layer, but does not penetrate. Usually, within 2-3 days, dysentery results from bacteria damaging the epithelium lining layers of the intestine often with release of mucus and blood (found in the feces) and attraction of leukocytes (also found in the feces as "pus"). Shiga toxin (chromosomally encoded) is neurotoxic, enterotoxic and cytotoxic plays a role. The toxin inhibits protein synthesis (acting on the 80S ribosome and lysing 28S rRNA). This is primarily a fecal-oral disease and it can be transmitted by infected food handlers, contaminating food. The source in each case is unwashed hands. Man is the only "reservoir". Patients with severe dysentery are usually be treated with antibiotics (e.g. ampicillin). In contrast to salmonellosis, patients respond to antibiotic therapy and disease duration is diminished.

41. Salmonella Salmonellosis may present as one of several syndromes including gastroenteritis, enteric (typhoid) fever or septicemia.

43. 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

45. Enteric or typhoid fever Enteric or typhoid fever occurs when the bacteria leave the intestine and multiply within cells of the reticuloendothelial system. The bacteria then re-enter the intestine, causing gastrointestinal symptoms. Typhoid fever has a 10-14 day incubation period and may last for several weeks. Salmonella typhi is the most common species isolated from this type salmonellosis. Human reservoir: carrier state common Contaminated food: water supply Poor sanitary conditions

46. Typhoid acute phase, gastroenteritis gall bladder –shedding, weeks Septicemia -occurs 10-14 days – lasts 7 days gastrointenteritis

47. Typhoid -Therapy Antibiotics – essential Vaccines Vi (capsular) antigen :protective

48. Salmonella gastroenteritis Salmonella gastroenteritis is the most common form of salmonellosis and generally requires an 8- 48 hour incubation period and may last from 2-5 days. Symptoms include nausea, vomiting and diarrhea (non-bloody stool). Salmonella enteritidis is the most common isolate. Poultry, eggs. no human reservoir self-limiting (2 - 5 days)

49. Salmonella septicemia Salmonella septicemia (bacteremia) may be caused by any species but S. choleraesuis is the most common. This disease resembles other Gram-negative septicemias and is characterized by a high, remittent fever with little gastrointestinal involvement.

50. Immunity (S. typhi) Vi (capsular) antigen – protective

51. Diagnosis – the samples A. Specimens a) Enteric fever: blood, bone marrow, stool, urine. b) gastroenteritis: stool, vomit, suspected food. c) Septicemia: blood.

52. Culture Differential media: MacConkey, EMB, Selective: Hektoen enteric agar, SS agar, XLD, Deoxycholate-citrate agar Enrichment: Selenite F or tetrathionate. Identifications: TSI, Bismuth sulfite media, Slide agglutination

55. Salmonella Using appropriate antibodies more than 2000 antigenic “types” have been recognized. There are, however, only a few types that are commonly associated with characteristic human diseases (most simply referred to as S. enteritidis, S. cholerae-suis and S. typhi). Salmonellosis, the common salmonella infection, is caused by a variety of serotypes (S. enteritidis) and is transmitted from contaminated food (such as poultry and eggs). It does not have a human reservoir and usually presents as gastroenteritis (nausea, vomiting and non-bloody stools). The disease is usually self-limiting (2-5 days). Like Shigella they invade the epithelium and do not produce systemic infection. In uncomplicated cases of salmonellosis, which are the vast majority, antibiotic therapy is not useful. S. cholerae- suis (seen much less commonly) causes septicemia after invasion. In this case, antibiotic therapy is required.. The severest form of salmonella infections "typhoid" (enteric fever), caused by Salmonella typhi. Although it is one of the historical causes of widespread epidemics and still is in the third world. The organism is transmitted from a human reservoir or in the water supply (if sanitary conditions are poor) or in contaminated food. It initially invades the intestinal epithelium and during this acute phase, gastrointestinal symptoms are noted. The organism penetrates, usually within the first week, and passes into the bloodstream where it is disseminated in macrophages. Typical features of a systemic bacterial infection are noted. The septicemia usually is temporary with the organism finally lodging in the gall bladder. Organisms are shed into the intestine for some weeks. At this time the gastroenteritis (including diarrhea) is noted again. The Vi (capsular) antigen plays a role in the pathogenesis of typhoid. A carrier state is common; thus one person e.g. a food handler can cause a lot of spread. Antibiotic therapy is essential. Vaccines are not widely effective and not generally used

56.  Typhoid fever is still a big health-problem in many countries, including Saudi Arabia.  The WHO estimates that there are worldwide about 16 million of clinical cases annually, of which about 600,000 result in death.  Foodborne infections account for 1.3 billion cases of acute diarrhea with 3 million deaths world-wide

57. Klebsiella – Normal flora of GI tract, but potential pathogen in other areas – 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

58. Klebsiella K. pneumoniae: may cause primary pneumonia, urinary tract and wound infections, bacteremia, meningitis, etc. K. rhinoscleromatis: pathogen of granumatous destruction of nose and pharynx. K. ozaenae: causes chronic atrophic rhinitis.

59. Proteus General characteristics: “swarming” phenomenon, urease positive. (P.vulgaris and P.mirabilis) P.vulgaris strains have common antigen with Rickettsia (Weil-Felix test). urinary tract infections; food poisoning. CLED (cystine-lactose-electrolyte-deficient medium)

60. End of Todays Lecture Thanks you Any questions Good luck