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 O157:H7 outbreak, Shigella, Listeria
Antibiotic sensitivity
 Site of sampling
 Sterile sites
 Blood
 Cerebrospinal 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
 Bacterial classification
 Wall structure
 Gram +
 Staphylococcus, Streptococcus, Clostridium,
Bacillus
 Gram -
 Enteric, respiratory and others
Bacteria
 Acid-fast
 Mycobacterium
 Wall-less
 Mycoplasma
G+ G- AF WL IC
 Unusual
 Obligate intracellular
 Rickettsia, Chlamydia
 Bacterial classification
 Cell morphology
 Shapes Bacteria

 Rod

 Cocci

 Spiral
G+ G- AF WL IC
 Associations
 Individual Rod Cocci Rod Cocci Spiral

 Diplo-

 Staphylo-

 Strepto-
 Bacterial classification
 Growth characteristics Bacteria
 Oxygen requirement
 Aerobic

 Anaerobic
G+ G- AF WL IC
 Microaerophilic, aerotolerant
 Facultative
Rod Cocci Rod Cocci Spiral
 Spore formation
 Intracellular/extracellular + spore - + +/- -O2

 Fastidious/non-fastidious
Classification & Diagnosis

 Type of colonies
 Appearance
 Color, shape, size and smoothness
 On differential media
 Blood, MacConkey, EMB
 On selective media
 MacConkey, Thayer-Martin
Classification & Diagnosis
 Metabolism
 Utilization of specific substrates
 Lactose (Sal/Shi/Yer/)-

 Citrate (E. coli-/Klebsiella+)

 Production of certain end products

 Fermentation end products
 Acid (acetate, propionic acid, butyric acid etc.)
 Acetoin
 Alcohol
 Amine
 H2 S
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
Immunological detection
Conventional diagnosis methods
Conventional method

 Depend on ability to culture

 Treponema pallidum
 Slow, esp. for fastidious species
 Mycobacterium spp.
 Not always definitive
 Molecular diagnosis

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

GGATCC
CCTAGG
DNA hybridization
 In situ
Hybridization
 PCR RT-PCR

Rate of increase
2n
RAPD of P. aeruginosa
 Molecular diagnosis
 Reduce reliance on  Technically
culture demanding
 Faster  Relatively expensive
 More sensitive  Can be too sensitive
 More definitive  Provides no
 More discriminating information if results
are negative
 Techniques adaptable
to all pathogens
 Bioterrorism

 Pathogen detection
 Fast and accurate
 Mobile
 Inexpensive
 Source investigation
Differentiating Staphylococci from Streptococci

 Gram stain and morphology

 Both Gram +
 Staphylococci: bunched cocci
 Streptococci: chained cocci (S. pneumoniae form diplococcus)
 Enzyme tests
 Staphylococci: catalase +
 Streptococci: catalase -
 Growth
 Staph.: large colonies (non-fastidious), some hemolytic
 Strep.: small colonies (fastidious), many hemolytic ( or )
 Staphylococci

 S. aureus: coagulase +
 S. epidermidis: coagulase -
 Streptococci

Streptococci On blood agar Growth inhibition disc

S. pyogenes (group A) β-hem olytic Sensiti
ve o t bacitr
acin
S.agalacti
ae(groupB) β-hem olytic Resistant otbacitr
acin
S.pneumoniae(pneum ococcus) α-hem olytic Sensiti
ve o
t optochin
V iri
dans α-hem olytic Resistant otoptochin
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
 Gram negative

Straight rods Curved rods

Lactose+ Lactose- Campy blood agar TCBS agar

42oC+ 25oC- Yellow
Oxidase+

Citrate+ Citrate- H2S+ H2S-

Campylobacter Vibrio
Klebsiella E. coli Salmonella Shigella
 Bacteria
Intra Wall
Gram+ Gram- Acid Fast Cellular Less

Cocci Rod Spiral Rod Cocci M.t. Rickettsia Mycoplasma

M.l. Coxiella
N.c. Erlichia
Chlamydia
Staph. Strep. Non-spore Spore Treponema Neisseria
Borrelia Moraxella
Leptospira
Fil Rod +O2 -O2

B.a. C.b. Curve

S. a. A A.i. C.d. Straight
S. e. B L. m. B.c. C.t.
S. s. Pn C.p.
C.d. Vibrio
Vir
Campylobacter
+O2 +/-O2 -O2 Other Helicobacter

P.a. Enteric Bact.

Resp. Zoo GU
Bordetella. Yersinia H. ducreyi
H. influenzae Pasteurella Gardnerella
Legionella Brucella Calymmatobacterium
Francisella
Streptobacillus