Bacterial Infections II Gramnegatives

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Bacterial Infections IIGram-negatives

• Mark Pallen

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The Gram Stain
Gram's
Crystal
iodine
violet
Decolorise with
acetone
Gram-positives
appear purple
Counterstain with
e.g. methyl red
Gram-negatives
appear pink
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Gram-positive rods
Gram-positive cocci
Gram-negative rods
Gram-negative cocci
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Anaerobic
Anaerobic
Gram-positive rods
Gram-positive cocci
Gram-positive
cocci
Anaerobic
Anaerobic
Gram-negative rods
Gram-negative cocci
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Enteric Gram-negative rods

• Lactose-fermenters (LFs)
• Escherichia coli
• Klebsiella pneumoniae
• Non-lactose fermenters (NLFs)
• Salmonella enterica
• Shigella spp.
• Proteus mirabilis
• Pseudomonas aeruginosa

Pink on Macconkey Agar
Pale on Macconkey Agar
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Escherichia coli

• Gut commensal
• Model lab organism
• E. coli K12 isolated in 1922 from stool of a
  convalescent diphtheria patient in California
• Pathogen
• Causes UTI, bloodstream infections meningitis,
  diarrhoea
• E. coli O157 causes haemorrhagic colitis and
  haemolytic uraemic syndrome

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E. coli in the News
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E. coli in the News
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Salmonella enterica

• one species, 2000 serovars
• non-standard nomenclature common
• S. enterica serovar Typhimurium
• or S. typhimurium
• Appears as a non-lactose fermenter
• on MacConkey agar or similar selective agar

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Salmonella Antigenic Structure

• Kauffmann-White antigenic scheme
• agglutination reactions with specific antisera
  against Salmonella antigens
• O antigens
• characteristic sequence of repeating
  polysaccharide units in LPS.
• H antigens
• flagellar antigens (protein) and may occur in one
  of two phase variations.

flagellated
(motile)
H antigen
(flagellin)
can undergo phase variation
O antigen
lipopolysaccharide, part of cell wall
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Salmonella Laboratory Diagnosis

• Biochemical tests and serological tests must be
  done in parallel
• Some other bacteria, e.g. Citrobacter, may have
  similar serological profiles
• Commercial kits commonly used, e.g. API20

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Shigellathe cause of bacillary dysentery

• 4 species increasing severity of disease
• Shigella sonnei
• gt90 of cases in E W
• Shigella boydii
• 1 of cases in E W
• Shigella flexneri
• 6 of cases in E W
• Shigella dysenteriae
• lt1 of cases in E W
• Shigella sonnei is commonly acquired in
  UK,whilst the others tend to be imported,
  especially by holidaymakers

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Shigella

• Non-lactose-fermenting Gram-negative rod
• O but no H antigens
• non-motile
• Identification scheme similar to Salmonella
• Biochemistry and serology done in parallel
• Colicin typing possible

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Proteus mirabilis

• Non-lactose-fermenting Gram-negative rod
• Causes
• UTI
• Bloodstream infection
• Swarming motility
• Urease-positive

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Pseudomonas aeruginosa

• Opportunistic pathogen
• Causes UTI, blood-stream infections, infections
  of burns, pneumonia
• Targets
• immunocompromised patients
• burns patients
• cystic fibrosis patients
• grows aerobically on most media
• greenish pigment production

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Fastidious Gram-negative rods

• Bordetella pertussis
• Haemophilus influenzae
• Legionella pneumophila

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Bordetella pertussis

• causes whooping cough
• diagnosis by pernasal swab
• after 5 days colonies like mercury droplets on
  selective agar

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Legionella pneumophila

• causes Legionnaires disease, an atypical
  pneumonia
• associated with hot water systems and air
  conditioning
• grows on selective agar after several days

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Curved Gram-negative rods

• Campylobacter jejuni
• Helicobacter pylori
• Vibrio cholerae

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Campylobacter jejuni

• curved bacilli with spiral, S-shaped
  seagull-like forms
• highly motile
• single polar or bipolar unsheathed flagella

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Campylobacter jejuni

• Microaerophilic
• need high concentration CO2, low concentration O2
• Thermophilic
• optimal growth 42C
• Fastidious
• blood or serum normally required
• needs antibiotic-containing selective medium
• grows in colonies resembling water droplets

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Helicobacter Pylori

• spiral motile Gram-negative bacillus
• causally linked to
• gastritis (Kochs postulates proven by
  volunteer!)
• peptic ulceration
• gastric carcinoma
• produces huge amount of the enzyme urease
• diagnosis
• Biopsy microscopy, culture, rapid urease test
• Serological techniques
• C-14 urea breath test

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Vibrio choleraethe cause of cholera

• Vibrio cholerae O1
• Curved Gram-negative rod
• Traditionally two biotypes
• "classical" and "El Tor
• now also "Bengal O137
• Clinical features
• Profuse watery diarrhoea
• Diagnosis
• culture on selective medium, TCBS

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Anaerobic Gram-negatives

• Bacteroides fragilis
• Fusobacterium
• spindle-shaped
• cause polymicrobial abscesses
• often mixed in with aerobes
• F. necrophorum causes necrobacillosis

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Gram-negative cocci

• Neisseria meningitidis
• The meningococcus
• Neisseria gonorrhoeae
• The gonococcus

False-colour EM
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Oxidase positive Identified by sugar fermentation
reactions MeninGos do Maltose Glucose
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Neisseria gonorrhoeae

• fragile organism
• cannot survive in environment
• no animal reservoir
• Gram-negative diplococcus
• kidney bean shaped
• often intracellular in clinical samples
• Causes gonorrhoea

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Unusual Gram-negatives

• Chlamydias
• obligate intracellular pathogens with complex
  life cycle
• cause trachoma, pneumonia, STDs
• Rickettsias
• obligate intracelllar pathogens
• cause typhus, Rocky Mountain spotted fever etc.
• Coxiella burnetti
• obligate intracelllar pathogen
• causes Q fever

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Unusual Gram-negatives

• Spirochaetes
• thin spiral bacteria with endoflagellum
• visualised with silver staining or dark-ground
  microscopy
• Treponema pallidum
• causes syphilis
• Leptospira icterohaemorrhagica
• causes Weil's disease
• Borrelia burgdorferi
• causes Lyme arthritis

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Summary

• Enteric Gram-negative rods
• LFs versus NLFs
• E. coli, Salmonella, Shigella, Proteus,
  Pseudomonas
• Curved Gram-negative rods
• Campylobacter, Helicobacter, Vibrio
• Fastidious Gram-negative rods
• Bordetella, Haemophilus, Legionella

• Anaerobic Gram-negative rods
• Gram-negative cocci
• Meningococcus
• Gonococcus
• Unusual Gram-negatives
• Chlamydia, Rickettsia, Coxiella, Spirochaetes

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Further Reading

• http//medweb.bham.ac.uk/http/depts/inf/teach/BDS/
  bdslect.htm