BACTERIAL CLASSIFICATION AND DIAGNOSIS OF BACTERIAL DISEASE

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BACTERIAL CLASSIFICATION AND DIAGNOSIS OF
BACTERIAL DISEASE

• Disease causing agent
• Bacterial, fungal, viral or other?
• Treatment
• Antibiotic sensitivity
• Source of infection
• Food, air or contact?
• Epidemiology
• Cholera, Diphtheria
• Prevention
• E.coli O157H7 outbreak, Shigella, Listeria

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Antibiotic sensitivity
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Site of sampling

• Sterile sites
• Blood
• Celebrospinal fluid (CSF)
• Body fluids (Peritoneal and pleural)
• Non-sterile (normal flora)
• Respiratory tract
• Ear, eye and mouth
• Skin (wound and abscess)
• Urine (mid-stream)
• Feces

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Bacterial classification

• Wall structure
• Gram
• Staphylococcus, Streptococcus, Clostridium,
  Bacillus
• Gram -
• Enteric, respiratory and others
• Acid-fast
• Mycobacterium
• Wall-less
• Mycoplasma
• Unusual
• Obligate intracellular
• Rickettsia, Chlamydia, Treponema

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Bacterial classification

• Cell morphology
• Shapes
• Rod
• Cocci
• Spiral
• Associations
• Individual
• Diplo-
• Staphylo-
• Strepto-
• Filaments

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Gram stains
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Gram Stains of Mixtures
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Cell shapes
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Bacterial classification

• Growth characteristics
• Oxygen requirement
• Aerobic
• Anaerobic
• Microaerophilic, aerotolerant
• Facultative
• Spore formation
• Intracelular/extracellular
• Fastidious/non-fastidious

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Endospore
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Classification Diagnosis

• Type of colonies
• Appearance
• Color, shape, size and smoothness
• On differential media
• Blood, MacConkey, EMB, Manitol salt agar
• On selective media
• MacConkey, EMB, Manitol salt agar

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Colony morphology/color
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Bacterial colony morphology
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Capsule
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MacConkey Agar Plate
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Blood Agar Plates and Hemolysis
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Mannitol salt agar
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Classification Diagnosis

• Metabolism
• Utilization of specific substrates
• Lactose (Sal/Shi/Yer/)-
• Citrate (E.coli-/Klebsiela)
• Production of certain end products
• Fermentation end products
• Acid (acetate, propionic acid, butyric acid etc.)
• Aceton
• Alcohol
• Amine
• H2S

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Triple Sugar Iron Agar Slants

• TSI
• Fermentation of glucose, lactose, and/or sucrose
• Reduction of sulfur to hydrogen sulfide
• Gas formation
• Used for Enterobacteriaceae

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Classification Diagnosis

• Specialized tests
• Immunological
• O-, H- K-Ag (serotype)
• Precipitation, agglutination
• Specialized enzymes
• Catalase--- Staph. vs. Strep-.
• Coagulase---S. aureus vs. S. epidermidis-
• Oxidase---Neisseria gonorrhoea
• Urease---Proteus, Helicobacter
• Antibiogram pattern
• Phage typing
• Fatty acid profile

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Urease Test
H2O
Urea
CO2 NH3
NH4 OH-
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Indole Test
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API test strip
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Bacteriophage Plaques
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Conventional method

• Depend on ability to culture
• Treponema pallidum
• Slow, esp. for fastidious species
• Mycobacterium spp.
• Not always definitive

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Immunological detection
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Instant View tests
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Molecular diagnosis

• Ribotyping
• Restriction fragment length polymorphism (RFLP)
• DNA hybridization
• PCR, RT-PCR and RAPD
• Nucleic acid sequence analysis
• Phage-GFP (TB)

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RFLP
GGATCC CCTAGG
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DNA hybridization
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In situHybridization
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PCR
Rate of increase 2n
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Real time PCR
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RAPD of P. aeruginosa
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DNA and protein chips
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Microarray array mediated pathogen detection
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Ab chip mediated pathogen detection
?-O157H7
E.coli O157H7
?-Salmonella
?-O157H7
Salmonella typhimurium
?-Salmonella
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Molecular diagnosis

• Reduce reliance on culture
• Faster
• High sensitive
• More definitive
• More discriminating
• Techniques adaptable to all pathogens

• Technically demanding
• Relatively expensive
• Can be too sensitive
• Provides no information if results are negative

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Bioterrorism

• Pathogen detection
• Fast and accurate
• Mobile
• Inexpensive
• Source investigation

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Biosensor Mediated Pathogen Detection
Step 1 Attach Capture Antibody to Fiber Optic
Waveguide
Step 2 Apply Sample. Target Organism Binds to
Waveguide
Step 3 Wash Away Other Cells and Debris
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Biosensor Mediated Pathogen Detection
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Step 1 Attach Capture Antibody to Fiber Optic
Waveguide
Step 2 Apply Sample. Target Organism Binds to
Waveguide
Step 3 Wash Away Other Cells and Debris
Step 4 Add Specific Detection Antibody
Step 5 Turn on Laser. Detection Antibody
Fluoresces, Indicating a Positive Sample.
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Biosensors
Analyte 2000TM for laboratory research and
development
RAPTORTM automated version for field analysis
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Differentiating Staphylococci from Streptococci

• Gram stain and morphology
• Both Gram
• Staphylococci bunched cocci
• Streptococci chained cocci
• S. pneumoniae diplococcus
• Enzyme tests
• Staphylococci catalase
• Streptococci catalase -
• Growth
• Staph. large colonies (non-fastidious), some
  hemolytic
• Strep. small colonies (fastidious), many
  hemolytic (? or ?)

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Staphylococci

• S. aureus coagulase
• S. epidermidis coagulase -

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Streptococci
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Differentiating the Gram- bacteria

• Cocci
• Neisseria
• Rods
• Type of disease they cause
• Enteric Gram- rods
• API test
• Curved
• Vibrio, Campylobacter, Helicobacter
• Spiral Gram- organisms
• Spirochetes

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Gram negative
Curved rods
Straight rods
Lactose Lactose-
TCBS agar Yellow Oxidase Vibrio
Campy blood agar 42oC 25oC- Campylobacter
Citrate Citrate-
H2S H2S-
Klebsiella E.coli Salmonella
Shigella
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Bacteria