Gram negative

• Gram-negative bacteria


  Bacteria with a thin peptidoglycan layer in their cell wall
• Gram-negative bacteria


  • Coccus
  • Curved rods
  • Rods
• Identification of Gram-negative bacteria

  1. Aerobic

  2. Oxidase test positive

  3. Pseudomonas, Stenotrophomonas, Burkholderia

  4. Oxidase test negative

  5. Enterobacteriaceae

  6. Lactose fermenter

  7. Klebsiella, Escherichia, Enterobacter

  8. Non-lactose fermenter

  9. Salmonella, Shigella

  10. Anaerobic

  11. Fastidious
• Enterobacteriaceae


  Found in animal and human gut - 'enteric bacteria'; some are always pathogenic in humans while others are opportunists
• Enterobacteriaceae


  • Coccobacilli or bacilli, facultative anaerobes, motile members have peritrichous flagella; some have capsules, others a loose slime layer
  • All are oxidase negative and reduce nitrate to nitrite; ferment glucose anaerobically
  • Lactose fermenters (LF) or non-fermenters (NLF)
• Identification of Enterobacteriaceae


  Conventional biochemical tests (e.g. TSI, IMViC) or commercial test kits (e.g. API 20E)

  
• Triple Sugar Iron agar slant (TSI slant)


  • Used for the differentiation of members of the Enterobacteriaceae based on their fermentation of lactose, sucrose and glucose and the production of H2S
• Gram-negative bacteria


  • Thick peptidoglycan layer
  • Thin peptidoglycan layer
• Antigens of Enterobacteriaceae


  • Outer LPS membrane (O polysaccharide or O Ag, Core polysaccharide, Lipid A)
  • Flagellar (H) Ag
  • Capsular K or Vi Ag
• Virulence factors of Enterobacteriaceae

  • Lipid A
  • Capsules (protein and polysaccharide)
  • Fimbriae (adhesin)
  • Exotoxins, siderophores, haemolysins
  • Type III secretion system (T3SS)
• The presence of enteric bacteria in urine, blood, or CSF is always diagnostic of infection or disease
• Culture of Enterobacteriaceae


  Selective and differential media used (e.g. EMB, MacConkey agar)
• Treatment of diarrhoea caused by Enterobacteriaceae

  Fluid and electrolyte replacement; antibiotics are not usually used
• Antibiotics used for internal infections caused by Enterobacteriaceae

  Ampicillin, amoxycillin, cephalosporins, aminoglycosides, chloramphenicol, ciprofloxacin; resistance develops frequently because of transfer of R-plasmids (conjugation)
• Escherichia coli


  Most important coliform - rods, motile, sometimes capsulated, facultative anaerobe
• Escherichia coli


  • Numerous O, H, and K Ags; 0157, 0111, H8 and H7 are associated with virulence
  • Virulent factors: pili, capsule, endotoxins, exotoxins (enterotoxins)
  • Bile tolerant, oxidase negative, IMViC ++--, TSI A/AG H2S-ve
  • Grows well on BA, LF (pink on MacConkey; yellow on CLED)
  • Commercial identification kit e.g. API 20E
• Transmission of Escherichia coli

  Endogenous or exogenous; commensals of human intestinal tract
• Diseases caused by Escherichia coli


  • Sepsis in newborns
  • Meningitis in infants
  • UTI
  • Diarrhoeal diseases (gastroenteritis) - mild to severe leading to excessive fluid loss and dehydration
• Enteropathogenic E. coli (EPEC)


  Causes diarrhoea outbreaks in nurseries: fever, diarrhoea, vomiting and nausea usually with non-bloody stools
• Enterotoxigenic E. coli (ETEC)


  Common cause of 'traveler's diarrhoea'; LT (heat labile toxin) and ST (heat stable toxin) plasmid-coded enterotoxins
• Enteroinvasive E. coli (EIEC)


  Invade intestinal epithelial cells, cause inflammation and dysentery indistinguishable clinically from shigellosis
• Enterohaemorrhagic E. coli (EHEC e.g. O157:H7)

  Causes diarrhoea, (bloody and copious, haemorrhagic colitis); Vero toxin (similar to Shiga toxin) encoded by a phage, haemolysins (plasmid encoded) and T3SS are virulence factors
• Enteroaggregative E. coli (EAEC)

  Causes acute and chronic diarrhoea and food-borne illness; adhere to mucosae (pili); they clump spontaneously
• Shigella infection
• Klebsiella pneumoniae


  • Human intestine and respiratory tract; opportunistic pathogen
  • Mucoid growth (large polysaccharide capsule protects from phagocytosis) and is non-motile, LF, and with IMViC reaction of - -++
  • Can cause pneumonia, UTI, meningitis, bacteremia with focal lesions in debilitated patients and nosocomial infections (VAD-ventricular assist device, catheters, etc)

  
• Enterobacter aerogenes


  May be found free-living, in the intestinal tract and may cause UTI and sepsis
• Serratia marcescens


  Occasionally cause fatal disease in neonates, nosocomial infections and is a common opportunistic pathogen in the immunocompromised
• Gram-negative, capsulated Klebsiella pneumoniae

  • From a pneumonia lung abscess
• EHEC (E. coli 0157:H7)


  • Enterohemorrhagic verotoxin producing, potentially fatal to humans (especially infants and children), contracted when contaminated meat is cooked inadequately
• Salmonella


  • The genus Salmonella has >2,000 serotypes and found in man, mammals, birds, reptiles, insects
  • S. typhi, & S. paratyphi are pathogens for humans only; most other serotypes potentially pathogenic for man and animals
• Salmonella


  • Gram-negative, motile and non-spore forming rods
  • Facultative anaerobe, NLF, IMViC - + - +, TSI K/AG(+/-) H2S+ve
  • Have O and H Ags with S. typhi having Vi capsular (virulence) Ag; T3SS leading to endocytosis
• Serotyping of Salmonella


  Kauffmann-White classification system
• Diseases caused by Salmonella

  • Enteric fever (Typhoid and Paratyphoid)
  • Gastroenteritis
  • Septicaemia
• Enteric fever (Typhoid and Paratyphoid)

  • Caused by S. typhi (with Vi Ag) and S. paratyphi A, B, C
  • Transmitted from human reservoir (carriers esp. food handlers) or in water supply (if sanitary conditions are poor) or in contaminated food
  • Initially invades the intestinal epithelium (acute phase) with gastrointestinal symptoms
  • Penetration within the 1st week → bloodstream and disseminated in macrophages
  • 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 diarrhoea) is noted again
• Gastroenteritis caused by Salmonella


  • Commonest form, may be caused by any of the pathogenic serotypes with symptoms of nausea, vomiting, abdominal cramps, headache and diarrhoea (e.g. S. enteritidis, S. typhimurium)
  • Transmitted from contaminated food (such as poultry and eggs)
  • The disease is usually self-limiting (2-5 days)
  • Like Shigella these strains of salmonella invade the epithelium and do not produce systemic infection
• Septicaemia caused by Salmonella

  Fulminant and fatal sometimes, with pneumonia, meningitis and osteomyelitis resulting from haematogenous spread (e.g. S. enteritidis, S. dublin, S. choleraesuis)
• Treatment of Salmonella infections


  • For enteric salmonellosis fluid and electrolyte replacement; however in neonates antimicrobials are important
  • For enteric fevers and septicaemias – ampicillin, ciprofloxacin, trimethoprim-sulfamethoxazole
  • For carriers with chronic gallbladder infections– ampicillin or surgical removal of gallbladder
• Prevention of Salmonella infections

  • Sanitary measures for the prevention of contamination of food and water
  • Carriers should not be permitted to work as food handlers
  • Vaccines for typhoid fever: single i/m dose of capsular polysaccharide vaccine (TY2 strain of S. typhi) OR 3 capsules taken every other day of an oral live attenuated vaccine (S. typhi Ty21a strain)
• Shigella


  • 4 spp: S. dysenteriae, S. sonnei, S. flexneri, S. boydii
  • Gram-negative, non-motile, non-capsulated, NLF (except S. sonnei), IMViC + + - - (S. sonnei indole –ve)

  
• Pathogenicity of Bacillary dysentery (Shigellosis)

  • No systemic disease; produces endotoxin and an exotoxin (Shiga toxin) chromosomally encoded. It is neurotoxic, enterotoxic and cytotoxic. The toxin inhibits protein synthesis
  • Initially colonizes small intestine cells causing an enterotoxin-mediated diarrhoea
  • Later invasion of colon epithelial M cells by endocytosis → multiplies in the cytosol → polymerizes host's actin fibres and gets into adjacent cells → inflammation → bloody mucopurulent diarrhoea