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  1. FAMILY ENTEROBACTERIACEAE Tsegaye Alemayehu (Assistant Professor) 1
  2. Outlines Family Enterobacteriaceae • Genus Escherichia • Genus Salmonella • Genus Shigella • Genus Klebsiella • Genus Citrobacter • Genus Serratia • Genus Enterobacter • Genus Yersinia • Genus Proteus Family Vibrionaceae • Genus Vibrio 2
  3. Enterobacteriaceae General Characteristics • Gram negative rods, found as normal flora in intestinal tract of human & animals. • It is named as well coliforms or enterobacilli • Non-spore forming, aerobic & facultative anaerobic bacteria • Most are motile 3
  4.  All ferment glucose with acid production.  These bacteria grow well on most simple media such as nutrient agar.  Release endotoxin from their cell wall.  They are catalase positive (with one exception) (Shigella dysentery type 1),  Oxidase negative & reduce nitrates to nitrites. Cont’d… 4
  5. Cont’d…  Antigens: possess a wide variety of antigens which are used in serotyping, particularly Salmonellae, Shigellae & E. coli.  Cross-reactions however, can occur due to a sharing of antigens.  Most of them have possessed three type of antigens H-antigen - Flagellar protein Found in flagella Possessed by motile enterobacteriaceae Heat labile K-antigen - Capsular polysaccharides Surrounds the cell wall Heat labile O-antigen - Outer membrane lipopolysaccharide (Somatic Antigen) It is found in the cell wall of enterobacteriaceae  Heat stable 5
  6. Cont’d… Note • K-antigens can interfere with the testing of underlying O-antigens • K- antigens can be heat-inactivated, enabling O- antigens to be detected. • The K-antigens of some Salmonellae, e.g. S. Typhi, are called Vi-antigens. 6
  7. Reading assignment • About source of naming for O - antigen, H – antigen, K- antigen, and Vi-antigen which language they were derived? 7
  8. 1. Antiphagocytic surface properties  Capsules  K-antigens &  LPS 2. Adhesins  Fimbriae / pili  Intimin (non-fimbrial adhesin)  Invasins: haemolysin, siderophores &  Shigella-like "invasins" for intracellular invasion & spread 3. Toxins  Heat labile(LT) toxin  Heat stable (ST) toxin  Shiga -like toxin  Cytotoxins &  Endotoxin (LPS) Virulence factors 8
  9. Members of Enterobacteriaceae 1. Lactose fermenting  Escherichia 1  Enterobacter1,2  Klebsiella1  Citrobacter1,2 2. Non-lactose fermenting  Salmonella  Providencia2  Shigella  Serratia2  Proteus  Yersinia  Morganella 2  Edwardsiella  Hafnia2 1 Often referred to as coliforms. 2 Less common human pathogen, often opportunistic or hospital-acquired. 9 MacConkey agar plate : lactose fermentation (A) vs non-fermentation of lactose (B)
  10. Lactose Fermenter’s (LF) Enterobacteriaceae 10
  11. Genus Escherichia General characteristics  Gram negative bacilli consisting of five species,  E. albetii, E. coli, E. fergusonii, E. hermanii & E. vulneris  E. coli is the most common & clinically important.  It is the most abundantly found bacteria in colon & faeces.  Found in soil, water & vegetation.  Most are motile; some are capsulated. Virulence factors: • Toxin, Pilli 11
  12. E.coli on Mac 12 Colonies of Escherichia coli on MacConkey agar plate are pink to dark pink, dry and donut-shaped, surrounded by a dark pink area of precipitated bile salts
  13. E. coli causes:  Urinary tract infections  It is the commonest pathogen isolated from patients with cystitis.  Recurring infections are common in women.  Infections of wounds, peritonitis, sepsis & endotoxin induced shock. Meningitis & bacteraemia in neonates. E. coli capsular type K1 is associated with neonatal meningitis. Pathogenesis & clinical manifestations 13
  14. Cont’d… Diarrhoeal disease: • Infantile gastroenteritis • Traveller’s diarrhoea • Dysentery, & • Haemorrhagic diarrhoea leads to haemolytic uraemic syndrome. 14
  15. E. coli strains associated with diarrhoeal disease 1. Enterotoxigenic E. coli (ETEC) • Causes watery (secretory) diarrhoea with vomiting, cramps, nausea, & low-grade fever • Due to the production of plasmid mediated toxins (LT, ST) in infants & adults. • The diarrhea is also called “Traveller's diarrhea”. • Pathogenic serogroups includes O6, O8, O15, O25, O27 15
  16. 2. Enteropathogenic E. coli (EPEC) • Causes vomiting, fever & prolonged diarrhoea mainly in infants (less than 2 year). • Due to bacteria adhering to epithelial cells, multiplying & causing lesions. • Pathogenic serogroups includes O26, O55, O86, O111, O114, O125-O128 & O142. 16
  17. • Causes dysentery (similar to shigellosis), fever and colitis, with blood, mucus, and many pus cells in faecal specimens. • Due to bacteria invading and multiplying in epithelial cells. • Pathogenic serogroups includes O78, O115, O148, O153, O159 & O167. 3. Entero-invasive E. Coli (EIEC) 17
  18. 4. Enterohaemorrhagic E. coli (EHEC) • Causes life-threatening haemorrhagic diarrhoea (colitis) in all ages, without pus cells, & often without fever. • It can progress to haemolytic uraemic syndrome with renal failure. • EHEC is due to cytotoxins damaging vascular endothelial cells, & is mainly associated with the serogroup 0157:H7. • It is sometimes referred to as VTEC (vero cytotoxin-producing E. coli, because it is toxic to vero monkey cells in culture). • Infection occurs by ingesting contaminated meat products, un pasteurized milk & dairy products. 18
  19. • Causes chronic watery diarrhoea & vomiting, mainly in children. • There are more than 50 pathogenic serogroups responsible for the infection. 5. Enteroaggregative E. coli (Eagg EC) 19
  20. Laboratory diagnosis Specimen: Urine, pus, blood, stool, body fluid Smear: Gram-negative rods Culture: Lactose-fermenting mucoid colonies on macConkey agar & some strains are haemolytic on blood agar . Biochemical reaction 20 Biochemical Tests Reaction Lactose Fermenters Lysine decarboxylase (LDC) + Beta-glucuronidase (PGUA) +, O157 “_” Hydrogen sulphide _ IMViC ++_ _ Indole + Methyl Red + Voges-Proskauer _ Citrate Utilization Test _
  21. Treatment Trimethoprim-sulphamethoxazole, ampicillin, cephalosporins, aminoglycosides & cefotaxime. Prevention & control  Reducing rise of nosocomial infections such as restricting use of antibiotics &  avoiding use of urinary catheters Maintenance of hygienic standards to reduce gastroenteritis. Proper cooking of beef reduces risk of EHEC infections. Cont’d… 21
  22. Genus Klebsiella General Characteristics  Gram-negative rods  Non-motile  Lactose-fermenting  Capsulated  Main species of medical importance: • K. pneumoniae • K. rhinoscleromatis • K. ozenae • K. oxytoca • Klebsiella aerogenes Virulence factors  Pili - adherence to respiratory & urinary epithelium.  Capsule - prevent phagocytosis.  About 80 capsular (K) antigens are presently recognized.  Type K1, K2, K3 & K21 are of particular significance in human disease.22
  23. Klebsiella Colony on MAC 23 MacConkey agar plate : Klebsiella colonies are often mucoid, large (4-6 mm) & dark to pale pink
  24. Pathogenesis & clinical manifestations Klebsiella pneumoniae • Causes chest infections & occasionally severe bronchopneumonia with lung abscesses. • Infections are often opportunistic, occurring in those with existing chest disease or D. mellitus, or in malnourished persons. • It also causes UTI, septicaemia, meningitis (especially in neonates), wound infection & peritonitis. 24
  25. Klebsiella aerogenes • Is associated with HAI of wounds & UTI. • It is also found in the respiratory tract where it may cause infection, particularly in immunocompromised patients. Klebsiella rhinoscleromatis • Causes rhinoscleromatis (granulomatous disease) of the nose & pharynx. • Chronic inflammatory growths can lead to deformity of the nose or distortion of the respiratory passages. Klebsiella ozaenae • It causes ozenae manifesting with foul smelling nasal discharge leading to chronic atrophic rhinitis. Cont’d… 25
  26. Laboratory diagnosis  Specimen: Sputum, urine, pus, CSF, body fluid  Smear: Gram-negative rods  Culture:  Large mucoid  Lactose-fermenting colonies on macConkey agar  Shows stringy type growth when cultured in broth medium. Serology: • Capsular polysaccharide serotyping. • More than 80 serotypes of K. pneumoniae recognized 26
  27. Biochemical tests Key: VP = Voges-Proskauer, Lact = Lactose fermentation, Ure = Urease, Cit = Citrate, Mal = Malonate utilization, LDC = Lysine decarboxylase • Klebsiella are indole negative (K. oxytoca is indole positive), • Ornithine decarboxylase negative & • do not produce H2S Klebsiella Species Biochemicals Vp Lact Ure Cit Mal LDC K. pneumoniae - + + + + + K. aerogenes + + + + + + K. ozaenae - ± - ± - ± K. rhinoscleromatis - - - - + - 27
  28. Cont’d…. 28
  29. Cont’d… Treatment • Since isolates from HAI are frequently resistant to multiple antibiotics. • The choice of drugs depends on results of sensitivity testing. • However, gentamycin & cefotaxime can be used. Prevention & control • Removing of urinary catheters when they are no longer needed. • Taking proper care of respiratory therapy devices. 29
  30. Genus Enterobacter • It is gram-negative • Lactose fermenting • Motile rods, & • Found as a commensal in the intestinal tract of humans & animals • Also found in moist environments.  Medical important species  Enterobacter aerogenes  Enterobacter agglomerrans  Enterobacter cloacae  It produces mucoid colony resembling Klebsiella on MacConkey agar. 30
  31. Enterobacter on MAC 31 Enterobacter spp., On the left, are often late lactose fermenters, & so colonies may appear colourless to light pink
  32. Enterobacter aerogenes Can cause • Urinary tract infection • Wound infection & • Septicaemia in immuno-compromised & chronically debilitated patients. 32
  33. Genus Citrobacter • It is gram-negative • Lactose fermenting • Motile rods & • Opportunistic pathogen  Medical important species is  Citrobacter freundii  Citrobacter diversus  Citrobacter koseri 33
  34. Citrobacter freundii Is associated with • Urinary tract infections • Wound infection & • Septicaemia in immuno-compromised & chronically debilitated patients. • It is H2S positive while C. diversus is negative 34
  35. Citrobacter on MAC 35
  36. Non-Lactose Enterobacteriaceae 36
  37. Genus Salmonellae General characteristics  Gram-negative, facultative rod-shaped bacteria  Motile, non-spore forming  Live in the intestinal tracts of warm and cold blooded animals.  Some species are ubiquitous.  Other species are specifically adapted to a particular host. 37
  38.  Classifies in different O groups, or O sero groups which contain a number of sero - types possessing a common O antigen.  The O groups first defined were designated by capital letters A to Z and those discovered later by the number.  Group A , for example, is characterized by O antigen 2, group B by O antigen 4 and group D by O antigen 9 (Refer Mackie and McCartney). Kauffmann- White Classification 38
  39. Group, Serogroup, Serotype O Antigens H antigens: Phase 1 Phase 2 Group A, serogroup 2 S. Paratyphi A 1,2,12 a - Group B, serogroup 4 S. Paratyphi B S. Derby S. Typhimurium S. Heidelberg 1,4,5,12 1,4,5,12 1,4,5,12 (1)*,4,5,(12)* f,9 (1,2) * i 1,2 r 1,2 c 1,5 Group C, serogroup 7 S. Cholerae-suis S. Paratyphi C S. Oranienburg S. Garoli S. Thompson S. Bareilly 6,7 6,7,(Vi)* 6,7 6,7 6,7 6,7 c 1,5 m,t - i 1, 6 k 1, 5 y 1,5 d - Group D, serogroup 9 S. Typhi S. Enteritidis S. Pullorum Gallinarum 9,12,(Vi)* 1,9,12 1,9,12 g,m - (Non motile) - r z6 Group E1, serogroup 3,10 S. Weltevreden S. Anatum Group G, serogroup 13,22 S. Poona S. Worthington S. Cubana 3,10 3,10 13,22 1,13,23 1,13,23 e,h 1,6 z 1,6 z 1,w z29 - Table: Antigenic structure and grouping of some salmonellae according to the Kauffmann- White classification system. 39
  40. Antigenic Structure 1. Somatic (O) or cell wall antigens  are heat stable & alcohol resistant  Are lipopolysaccharide layer on surface of bacterial cell wall.  Used for serological tests  Not always antigenically identical & over 60 different O antigens are recognized 40
  41. 2. Surface (Envelope) Antigens  Includes the capsular ( K) antigens including the Vi antigen ; the slime (mucus) or M antigen ; and the fimbrial, or F antigen.  Such antigens may mask O antigens, and the bacteria will not be agglutinated with O antisera.  One specific surface antigen is well known: the Vi antigen which occurs in only three Salmonella Serovars (out of about 2,200); S.Typhi, S. Paratyphi C, & S. Dublin. 41
  42. 3. Flagellar (H) Antigens Are located on flagella & are denatured or removed by heat or alcohol. In many but not all salmonellae, the production of flagellar antigen is diphasic ( phase I and phase II). Anti flagellar antibodies can immobilize bacteria with corresponding H antigens. 42
  43. Medically important serovar  Salmonella typhi  Salmonella paratyphi  Salmonella choleriasis  Salmonella typhimurium and  Salmonella enteritidis  Salmonella strain produce a thermo labile enterotoxin that bears a limited relatedness to cholera toxin both structurally & antigenically 43
  44. Classification of Salmonella 44
  45. Virulence factors 1. Lipopolysaccharide (endotoxin) - released into the bloodstream resulting in septicemia. 2. Invasins - proteins that mediate adherence to & penetration of intestinal epithelial cells. 3. Factors involved in resistance to phagocytosis A. Catalase & super oxide dismutase - protect the bacteria from intracellular killing by neutralizing oxygen radicals. B. Defensins - small cationic proteins that facilitate killing of bacteria by phagolysosomes. 45
  46. Cont’d… 4. Factors involved in resistance to acid pH Salmonellae are protected from stomach acid & acid pH Phagosome by acid tolerance response (ATR) genes of chromosome. 5. Vi (virulence ) antigen - this surface antigen of Salmonella typhi has anti-phagocytic properties. However, its exact role as a virulent factor is not clear. 46
  47. Pathogenesis & clinical manifestations  The bacteria enter the human digestive tract, penetrate the intestinal mucosa (causing no lesion), and are stopped in the mesenteric lymph nodes.  Bacterial multiplication occurs and part of the bacterial population lyses.  From the mesenteric lymph nodes, viable bacteria and LPS (endotoxin) may be released into the bloodstream resulting in septicemia.  Release of endotoxin is responsible for cardiovascular problems. 47
  48. Diseases are caused by Salmonella 1. Salmonellosis (enteric fever) (typhoid) - resulting from bacterial invasion of the bloodstream. 2. Acute gastroenteritis  Resulting from a food borne infection/intoxication.  Produce a thermo labile enterotoxin that bears a limited relatedness to cholera toxin. 3. Septicemia -is feature of enteric fever caused by Salmonella typhi & Salmonella paratyphi 48
  49. Laboratory diagnosis Specimen: Blood, Bone marrow, stool, urine , serum, left over food , & duodenal aspirates.  Blood - 80% positive in the first week.  Stool (gastroenteritis) - 70-80% positive in the second & third week.  Urine - 20% positive in the third & fourth week.  Serum - for widal test- positive after the second week of illness.  Gram reaction - Gram-negative rods 49
  50. Culture 1. Differential medium-for rapid isolation of lactose fermenter from non-fermenter. • Eg: EMB agar, MacConkey agar & Deoxycholate Citrate agar. 2. Selective medium-favour growth of salmonella & shigella over other enterobacteriaceae. • Eg: Salmonella-Shigella (SS) agar, Hekton Enteric agar, XLD agar, Deoxycholate-Citrate agar. 3. Enrichment cultures: Inhibit replication of normal intestinal flora and permit replication of salmonella. • Eg: Selenite F broth and Tetrathionate broth Salmonellae are non-lactose fermenting & some produce H2S. 50
  51. Cont’d… 51
  52. Biochemical reaction Generally Salmonealeae produce gas & acid from carbohydrate; except Salmonella typhi which does not produce gas. 52
  53. Biochemical Test 53 LDC test results – negative (left) & positive (right)
  54. Triple sugar agar 54
  55. Serotyping  Based on their O and H antigen composition, more than 2300 Salmonella serovars are described in the Kauffmann-White scheme.  Salmonellae are placed in groups by their O antigens (A, B, C, etc) and subdivided by their H (phase 1 and 2) antigens.  Polyclonal anti-sera containing antibodies to the major groups can be used to identify an isolate bio-chemically suspected of being Salmonella.  Full sero-typing (for epidemiological purposes) requires the use of polyvalent and monovalent O and H anti-sera and is usually carried out in a specialist Public Health Laboratory. 55
  56. Widal test The diagnostic value of the Widal test remains controversial.  Most agree that the test is not sufficiently sensitive or specific to be clinically useful when only a single acute-phase serum sample is tested (common practice). It measures agglutinating antibody levels against O(somatic) and H (flagellar) antibodies. In acute typhoid fever, O agglutinins can usually be detected 6-8 days after the onset of fever & H agglutinins after 10-12 days. 56
  57. Interpretation of results 1. Higher or rising titer of O ( > 1: 60) suggests active infection. 2. Higher titer of H ( > 1: 60) suggests past immunization or past infection. 3. Higher titer of antibody to Vi antigen occur in some carriers. Treatment  Ampicillin, Cephalosporin, Chloramphenicol  Plasmids mediated drug resistance is a problem of concern currently. 57
  58. RDT • The Salmonella Rapid Detection is a qualitative test for a broad spectrum of Salmonella serotypes. • Results can be recorded in 20-25 minutes 58
  59. Prevention & Control  Personal hygiene  Proper storage of food  Use of pasteurized milk and milk products.  Proper cooking of Vegetables and fruits  Health education 59
  60. Genus Shigella General characteristics  Shigellosis is an infectious disease caused by various species of Shigella.  Natural habitat: Intestinal tracts of humans & other primates.  are slender gram-negative rods; coco bacillary forms occur in young cultures.  Non-motile, non-spore forming • Based on antigenic structure and biochemical reactions, Shigella organisms are divided into four subgroups corresponding to the following species: 60
  61. Shigella on different Agars 61 SS agar
  62. Cont’d… Subgroup A: Shigella dysenteriae • Contains 13 distinct serotypes • Serotype 1 was formerly called S. shiga • Serotype 2 was formerly called S. schmitzii Subgroup B: Shigella flexneri • Contains 6 related serotypes and 4 serotypes • Divided into subsero-types Subgroup C: Shigella boydii: • Contains 18 distinct serotypes Subgroup D: Shigella sonnei: • Contains one serotype 62
  63. Virulence factors 1. Endotoxin: irritate the bowel wall 2. Exotoxin: Enterotoxin and neurotoxin 3. S. dysenritiae type 1 (shiga bacillus) produce heat labile exotoxin mediated diarrhea. 4. Long chain LPS - preventing the effect of serum complement. 63
  64. Pathogenesis & clinical manifestations  It is almost always limited to the gastrointestinal tract, bloodstream invasion is quite rare.  It is highly communicable.  It invade the mucosal epithelial cells (eg, M cells) by induced phagocytosis, escape from the phagocytic vacuole, multiplication and spread within the epithelial cell cytoplasm, and passage to adjacent cells. 64
  65. Cont’d…  Micro-abscesses in the wall of the large intestine & terminal ileum lead to necrosis of the mucous membrane, superficial ulceration, bleeding, and formation of a "pseudo membrane" on the ulcerated area.  This consists of fibrin, leukocytes, cell debris, a necrotic mucous membrane, & bacteria.  As the process subsides, granulation tissue fills the ulcers and scar tissue forms. 65
  66. Contd’…  Plasmid encoded proteins are required for shigella to break free from cellular endo-somes and for the migration between epithelial cells.  Long chain LPS plays a role in virulence by preventing the effect of serum complement. 66
  67. Laboratory diagnosis  Specimen: Fresh stool or rectal swabs  Gram reaction: Gram-negative non-motile rods.  Culture The following media can be used:  MacConkey  Eosin - methylene Blue agar  Hekton enteric agar or Salmonella Shigella agar 67
  68. 68 Species Man Cat Ind LDC ODC S.dysenteriae 1 (Sd 1) 2 3–12 - - - - + + - + d - - - - - - S. flexneri 1–5 d 6 + + + + d - - - - - S. sonnei + + - - + S. boydii 1–18 + + d - - Man = Mannitol, Cat = Catalase, Ind = Indole, LDC = Lysine decarboxylase, ODC = Ornithine decarboxylase. Notes *Most strains positive. d Different strains give different reactions. Biochemical reactions which help to differentiate shigellae
  69. Sero grouping  Are sero-grouped by their O-antigens using polyvalent group anti-sera & when indicated,  mono-specific (monovalent) antiserum.  e.g. monovalent S. dysenteriae 1 antiserum is required to identify S. dysenteriae 1. Non-agglutinating Shigella  Some Shigella strains (mostly S. dysenteriae & S. sonnei) possess surface (K) antigens that can ‘hide’ the O-antigens being tested & so prevent agglutination. 69
  70. Treatment • Ciprofloxacin • Ampicillin • Tetracycline • Trimethoprim – sulphamethoxazole & chloramphenicol Prevention & control  Sanitary control of water, food, & milk.  Proper sewage disposal.  Disinfection of excreta.  Early detection & treatment of carriers. e.g : Food handlers 70
  71. Genus Proteus General characteristics • Gram negative rods & it differ from other members of Enterobacteriaceae by : 1.Their ability to produce the enzyme urease & phenylalanine diaminase. 2. Certain species are very motile & produce a striking swarming colonies on blood agar plate 3. Cell wall (O antigens) of certain strains of Proteus (such as OX-2, OX-19 & OX-K) cross react with antigens of several species of rickettsia. 71
  72. Cont’d… 4. The Proteus antigens can be used in tests ( Weil Felix test) to detect the presence of antibodies against certain rickettsia in patient sera.  Proteus species are found in the intestinal tract of humans, animals, soil, sewage & water.  They are motile, non-capsulated & pleomorphic rods. Species of medical importance are:  P. mirabilis  P. Vulgaris Other species  P. penneri  P. myxofaciens &  P. hauseri 72
  73. Swarming on BAP but not MAC 73
  74. Virulence factors 74 1. These bacteria are characteristically highly motile & chemotaxis may play a part in pathogenesis. 2. Strains of Proteus species may also express calcium - dependent & calcium- independent haemolysins in addition to a range of proteases such as an IgAase. 3. Proteus species & others urease producing organisms tested alkaline conditions in the urine & may provoke the formation of calculi (stones) in the urinary tract.
  75. Pathogenesis & clinical manifestations 75 Proteus mirabilis causes: 1. Urinary Tract infections • Proteus infected urine has an alkaline reaction. • It is a common cause of UTI in the elderly & young males often following catheterization or cystoscopy. • Infections are also associated with the presence of renal stones. 2. Abdominal & wound infections • It is often a secondary invader of ulcers, pressure sores, burns & damaged tissues. 3. Septicaemia, occasionally meningitis & chest infections. 4. Secondary invader of ulcer, burn, pressure sores & chronic discharging ear.
  76. Proteus vulgaris 76 • Important nosocomial pathogen. • Isolated from wound infection & urinary tract infection. • Proteus mirabilis infections usually respond better to antimicrobial therapy than those caused by P. vulgaris & other related organisms.
  77. Laboratory diagnosis 77  Specimen: Urine, pus, blood, ear discharge  Smear: Gram-negative rods  Culture: Produce characteristic swarming colonies over the surface of blood agar.  Are non-lactose fermenting colonies on MacConkey agar.  Proteus species have a characteristic smell, rotten egg/pungent smell.
  78. Ether shaking technique to kill Proteus for isolation of other bacteria 78 • When a mixed culture (Proteus & other bacteria) observed on blood agar plate, take a loop-full of mixed culture & mix it in ether solution. • Ether specifically kills Proteus species which allows to isolate the other bacteria.
  79. Biochemical tests 79 Proteus species:  Do not ferment lactose.  Rapidly hydrolyze urea within 4 hours  Phenylalanine deaminase (PDA), positive.  Beta - galactosidase (ortho-Nitrophenyl-β- galactoside)(ONPG) --------- negative.  Proteus species………….Urease positive  Proteus vulgaris………... Indole positive  Proteus mirabilis……….. Indole negative
  80. Proteus on Urea broth & Agar 80
  81. Biochemical reactions that differentiate Proteus species, M. morganii, & Providencia species 81
  82. Serology • Some of the antigens of Proteus strains OX19, OXK & OX2, agglutinate with sera from patients with rickettsial diseases. • These reactions form the basis of the Weil-Felix test. Treatment  Cephalosporins, aminoglycosides & ampicillin.  Some strains of Proteus mirabilis are beta-lactamase producing and therefore resistant to ampicillin.  Proteus species are resistant to polymyxin & nitrofurantoin. 82
  83. Prevention & control • No specific preventive measure is indicated, but many hospital acquired UTIs can be prevented by prompt removal of urinary catheters. 83
  84. Genus Yersinia General characteristics  Animals are natural hosts of Yersinia & humans are accidental hosts.  They are short, pleomorphic, microaerophilic or facultative anaerobic  Gram negative rods  Oxidase negative  Exhibiting bipolar staining with special stains  The genus consist of 10 species . 84
  85. Medical important Species 1. Yersinia pestis, the cause of Bubonic plague 2. Yersinia pseudotuberculosis & 3. Yersinia enterocolitica, important causes of human diarrheal diseases; & others. 85
  86. Virulence factors 1. ST lipopolysaccharide that have endotoxic activity and contribute to the toxaemia of plague. 2. LT Fraction 1 (F1) protein capsular antigen helps the organism to resist phagocytosis & is a protective immunogen. 3. The V-antigen, part of the type III secretion system, is an important protective antigen. 86
  87. Cont’d… 4. Yersinia pestis also produces a plasminogen activator & fibrinolysin, which may play a critical stage of infection. 5. Other proteins associated with virulence includes adhesion & iron acquisition factors which are common to Yersinia enterocolitica & Yersinia pseudo tuberculosis. 87
  88. Pathogenesis & clinical manifestations  Y. pestis causes plague, a zoonotic disease which is transmitted from rats & other rodents to humans by infected fleas (main vectors: Xenopsylla cheopis & X. brasiliensis).  Occasionally, infection occurs by inhaling the organisms in airborne droplets.  or by handling infected rodents or domestic animals (e.g. cats, dogs) that harbour infected fleas. 88
  89. Cont’d… • Rat flea (Xenopsylla cheopis) gets infected by biting an infected rodent • Infected rat flea bites human (accidental host) • Organism migrate to regional lymphnodes from the site of bite (bubonic plague) & • Gets into the blood via lymphatics (septicemic plague). • or Primary pneumonic plague results from inhalation of infective droplets, usually from an infected coughing person. 89
  90. Oriental rat flea 90
  91. There are three main forms of the disease 1. Bubonic plague • Characterized by high fever & acute lymphadenitis with painful haemorrhagic swellings called buboes, usually in the groin area. • Occasionally lymph nodes in the neck or armpits are involved, depending on the site of the flea bite. • There is a markedly raised white cell count with neutrophilia. 91
  92. 2. Pneumonic plague  Inhalation of the organism or its spread to the lungs via the blood stream.  Pulmonary infection causes severe bronchopneumonia with haemorrhaging.  It is rapidly fatal unless treated at an early stage.  Highly infectious & can spread quickly in conditions of poverty & overcrowding.  The sputum contains large numbers of plague bacilli & is often blood stained. 92
  93. 3. Septicaemic plague  Serious haemorrhagic condition in large numbers of Y. pestis are present in the blood.  The organisms can often be seen in peripheral blood smears.  Buboes are usually absent.  There is a haemorrhagic rash.  Septicaemic plague is rapidly fatal. 93
  94. Plague 94 Plague patient with an axillary lymphadenopathy Plague that included gangrene of the hand causing necrosis of the fingers
  95. Laboratory diagnosis Specimen: Lymphnode aspirate, CSF, & blood Smears: Wright’s stain, immuno-fluorescence stain, methylene blue stains, basic fuchsine stain , & Wayson’s stain to demonstrate bipolar granules. Culture: Grow on blood agar or MacConkey agar. NB: All cultures are highly infectious & must be handled with extreme caution Biochemical tests Y. pestis is catalase positive & oxidase negative. A microbiology specialist laboratory is required to identify Y. pestis. 95
  96. • Some of the Y. pestis organisms have bipolar staining, • Which gives them a hairpin- like appearance. 96 Dark stained bipolar ends of Yersinia pestis can clearly be seen in this Wright's stain of blood from a plague victim
  97. Y. pestis 97 Y. pestis on sheep blood agar, 72 hours. grows well on most standard laboratory media. After 48 to 72 hours, it shows gray-white to slightly yellow opaque raised, irregular "fried egg" morphology; alternatively, colonies may have a "hammered copper" shiny surface
  98. Serology  Fluorescent antibody technique using Y. pestis antisera  Prognosis: Mortality rate is 50% (100%) for pneumonic plaque) Treatment  Streptomycin  Tetracycline  Streptomycin plus tetracycline or chloramphenicol 98
  99. Prevention & control  Chemoprophylaxis for contacts of patients.  Formalin-killed vaccine for travellers to hyper endemic areas and high risk persons.  Plague is controlled by reduction of the rodent population.  Other Yersinia infections are controlled by proper preparation of food and food products. 99
  100. Other Gram Negative Enteric Pathogens 100
  101. It includes • Genus Vibrio • Genus Campylobacter • Genus Helicobacter 101
  102. Genus Vibrio General characteristics  Gram-negative straight or curved rods  Motile by means of a single polar flagellum  Oxidase-positive  Species of medical importance Vibrio cholera 01  Found in fresh water, shell fish & other sea food  Man is the major reservoir of V. cholera 01, which causes epidemic cholera.  Readily may survive in clean stagnant water 102
  103. Medical Important species • V. cholerae • V. parahaemolyticus • V. vulnificus 103
  104. Cont’d…  Readily killed by heat and drying  dies in polluted water but may survive in clean stagnant water, esp. if alkaline, or sea water for 1-2 weeks. 104
  105. Vibrio cholerae  More than 130 different O serogroups have been described.  The classical cause of epidemic cholera possess the O1 antigen, and it is known Vibrio cholera 01. 105
  106. Virulence factors V. cholera requires two major pathogenic mechanisms to cause disease. 1.The ability to produce cholera toxin. 2.Expression of toxin -co- regulated pili. 106
  107. Pathogenesis & clinical manifestations  Route of infection is fecal-oral route.  After ingestion of the V. cholerae 01, the bacteria adheres to the intestinal wall with out invasion  Then produces an exotoxin causing excessive fluid secretion & diminished fluid absorption resulting in diarrhea (rice water stool)  which is characterized by passage of voluminous watery diarrhea containing vibrio's, epithelial cells and mucus and result in severe dehydration. 107
  108. Non-01 V. cholerae  Cause mild, some times bloody, diarrhoea often accompanied by abdominal cramp.  Also cause wound infection in patients exposed to aquatic environments, and bacteraemia and meningitis.  May elaborate a wide range of virulence factors including enterotoxin, cytotoxin, haemolysins and colonizing factors.  A few strains produce cholera toxin. 108
  109. Laboratory diagnosis  Specimen: Stool  Smear: Gram-negative motile curved rods  Motility of vibrio is best seen using dark-field microscopy.  Presumptive diagnosis: Inactivation of vibrio in a wet preparation after adding vibrio antiserum. 109
  110. Culture 1. Thiosulphate citrate bile salt sucrose agar (TCBS) • selective media for primary isolation of V. cholerae. • Observe for large yellow sucrose fermenting colonies after 18-24 hrs of incubation. 2. Alkaline peptone water: • Enrichment media for V. cholerae 01 growth on and just below the surface of peptone water with in 4-6 hours at room temperature as well as 37 oc. 110
  111. Vibrio on TCBS 111
  112. Biochemical Reaction  Oxidase-positive  Ferment sucrose and maltose (acid; no gas)  Do not ferment L-arabinose Treatment  Fluid and electrolyte replacement.  Occasionally short-course antibiotic therapy, e.g. with tetracycline (but resistance is common) or doxycycline. 112
  113. Prevention & control  Prevention mainly achieved by clean water & food supply.  Use of tetracycline for prevention is effective during close contact with infected patients. 113