Ebac 2

• 2 Broad Categories of Enterobacteriaceae

  • Opportunistic pathogens
  • Primary pathogens

  
• Opportunistic pathogens


  Bacteria that are generally harmless but may cause harm to individuals if they have become immunocompromised or if that particular microbe reaches a part of the body to which they are not sterile
• Primary pathogens


  Species of bacteria that cause infections virtually to all individuals regardless of their age and immune status
• Shigella


  • Closely related to Escherichia – both belong to the same tribe, Escherichieae
  • Non-lactose fermenting bacteria
  • All species causes dysentery (bloody diarrhea with mucus, blood, and leukocytes)

  
• Shigella species are not members of the GI microbiota, and are considered as "primary pathogens"

  
• If you happen to isolate Shigella species from a stool sample of an individual, then that individual is likely infected with Shigella species

  
• Biochemical Characteristics of Shigella

  • Non-lactose fermenters
  • "Biochemically Inert"
  • Non-motile
  • Anaerogenic, except Shigella flexneri
  • Do not hydrolyse urea
  • Do not produce H2S gas from thiosulfate/sodium thiosulfate
  • Do not decarboxylate lysine
  • Do not use acetate or mucate as carbon source

  
• Shigella sonnei


  • Unique member of the genus
  • Able to ferment lactose slowly (LLF) producing pink colonies on MAC agar only after 48 hours of incubation
  • ONPG positive
• Shigella species are fragile and susceptible to various effects of physical and chemical agents

  
• Shigella are susceptible to the acid pH of the stool. Feces suspected of containing Shigella spp., must be immediately inoculated to primary culture media to increase recovery of the organisms

  
• All Shigella species possess O antigens, and certain strains possess K antigens

  
• Serotypes of Shigella based on O Antigen


  • Subgroup A - S. dysenteriae (Dysentery bacillus)
  • Subgroup B - S. flexneri (Strong's bacillus)
  • Subgroup C - S. boydii (S. ambigua)
  • Subgroup D - S. sonnei

  
• Biochemical and Serologic Differentiation of Shigella Species

  • Mannitol fermentation
  • ONPG
  • Ornithine decarboxylase
  • Serogroup

  
• Mode of Transmission

  • Humans and other large primates are the only known reservoir
  • Direct person-to-person contact, fecal-oral-route with carriers as the source
  • May also be transmitted by flies, fingers, and food and water contaminated by infected persons
• Disease Association

  • Shigella species cause all types of bacillary dysentery – painful type of diarrhea characterized by watery feces w/ mucus, blood streaks & pus cells
  • Infective dose: as low as 100 bacilli are needed to initiate the disease in some healthy individuals

  
• Predominant Shigella Isolates

  • S.sonnei – predominant in the U.S and other industrialized countries
  • S.flexneri – affects mostly young children, leading isolate of gastroenteritis among men who have sex with men
  • S.dysenteriae type 1 and S.boydii – most common isolates in developing countries
  • S.dysenteriae type 1 - most virulent

  
• Pathophysiology of Shigella

  1. Attach to and invade the M cells located in Peyer patches

  2. Secrete four invasion-plasmid antigen (Ipa) proteins into epithelial cells and macrophages that induce membrane ruffling

  3. Lyse the phagocytic vacuole and replicate in the cytoplasm

  4. Rearrangement of actin filaments in the host cells propel the bacteria through the cytoplasm through the adjacent cells where cell-to-cell passage occurs

  5. Proliferations of macrophages and other phagocytes destabilize the intestinal wall's integrity

  6. Infected epithelial cells undergo rearrangement of actin filaments, resulting in the polymerization of actin to propel the bacteria

  7. Invasion and damage of the intestine's epithelial cells, resulting to mucus secretion and presence of blood

  8. Shigella dysenteriae produces shiga toxins
• Ocytes

  • Ability to squeeze through the tight junctions of the intestinal wall
  • Destabilization of the intestinal wall's integrity
  • Allowing bacteria to further reach the deeper epithelial tissues

  
• Organism

  • Invasive
  • Able to get through the cells - cell-to-cell passage
• Invasion and damage of the intestine's epithelial cells

  1. Rearrangement of actin filaments

  2. Polymerization of actin

  3. Propel the bacteria

  
• Invasion results in mucus secretion and presence of blood
• Characteristic findings of a stool with dysentery: Mucus, Blood, Pus (PMNs)
• Shiga toxins


  Composed of an A and B subunit
• Shiga toxin mechanism

  1. B subunit binds to the host cell glycolipid Gb3 receptor

  2. Facilitates the transfer of the A subunit into the cell

  3. A subunit cleaves the 28S rRNA in the 60S ribosomal subunit

  4. Disruption of protein synthesis resulting in cell damage and death
• High concentrations of Gb3 are found on intestinal villi and renal endothelial cells. This results in hemorrhagic diarrhea (hemorrhagic colitis) and hemolytic uremic syndrome (HUS)
• Stool sample diagnosis of Shigella spp.

  1. Undergo enrichment first

  2. Selenite broth used as enrichment medium

  3. Incubated at 37°C for 18 to 24 hours

  4. After incubation, subcultured to primary media

  
• Primary culture media

  • EMB
  • MAC
  • XLD
  • SSA
  • HEA
• Biochemical testing is done when there is growth in the primary media
• Biochemical characteristics of Shigella

  • TSI: Alkaline slant over acid deep (K/A)
  • H2S: Negative
  • Gas in fermentation: Negative
  • MR: Positive
  • Motility: Negative
  • Citrate: Negative
  • ADH: Negative
  • LDC: Negative
  • ODC: Negative
  • Deaminase (phenylalanine): Negative
  • Urease: Negative
  • MacConkey: Clear colonies (lactose negative)
  • Hektoen enteric: Green colonies (lactose negative)
  • Adonitol: Negative
  • Dulcitol: Negative
  • D-mannose: Positive
  • Sucrose: Negative
  • Salicin: Negative

  
• Shigella spp. are non-lactose fermenters; only glucose has been fermented
• Shigella spp. most of the time produce negative results - biochemically inert

  
• Salmonella spp. on SSA medium - colorless colonies with black center

  
• Salmonella


  • Many serotypes found in cold-blooded animals, rodents, and birds
  • Gram-negative, facultative anaerobic bacilli
  • On selective and differential media - produce clear, colorless, non-lactose fermenting colonies with black centers

  
• Biochemical characteristics of Salmonella

  • TSI: K/Ⓐ H2S - Positive
  • Motility: Positive
  • MR: Positive
  • VP: Negative
  • Indole: Negative
  • Lysine: Positive
  • Urease: Negative
  • Deaminase (phenylalanine): Negative
  • D-mannitol: Positive
  • myo-Inositol: Negative
  • Sucrose: Negative
  • MacConkey: Clear (lactose negative)
  • Hektoen enteric: Green with black centers (lactose negative, H2S positive)

  
• Salmonella spp. are aerogenic
• Salmonella are motile, except S. pullorum and S. gallinarum Salmonella are NLF except Salmonella Arizona is LLF

  Salmonella are aerogenic except Salmonella Typhi is non-aerogenic

  Salmonella areH2S positive except Salmonella Paratyphi is H2S negative

  
• O Antigen


  Stable to boiling and acid alcohol
• H (flagger) Antigen


  More stable
• Vi Antigen


  Virulence Antigen (K antigen); occurs in Salmonella serotype Typhi and few strains of Salmonella serotype Cholerasius; plays a role in preventing phagocytosis
• Salmonella surface antigens

  • Invasiveness (enables them to traverse the intestinal mucosa)
  • Endotoxin, enterotoxins
  • Intracellular capability (survive without being harmed inside macrophages and other phagocytic cells)