Page 1
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
Page 2
Antibiotic sensitivity
Page 3
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
Page 4
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
G+ G- AF WL IC
Bacteria
Page 5
Bacterial classification
Cell morphology Shapes
Rod Cocci Spiral
Associations Individual Diplo- Staphylo- Strepto-
G+ G- AF WL IC
Bacteria
Rod Cocci Rod Cocci Spiral
Page 6
Bacterial classification
Growth characteristics Oxygen requirement
Aerobic Anaerobic
Microaerophilic, aerotolerant
Facultative Spore formation Intracellular/extracellular Fastidious/non-fastidious
G+ G- AF WL IC
Bacteria
Rod Cocci Rod Cocci Spiral
+ spore - + +/- -O2
Page 7
Classification & Diagnosis
Type of colonies Appearance
Color, shape, size and smoothness On differential media
Blood, MacConkey, EMB On selective media
MacConkey, Thayer-Martin
Page 8
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 H2S
Page 9
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
Page 10
Immunological detection
Page 11
Conventional diagnosis methods
Page 12
Conventional method
Depend on ability to culture Treponema pallidum
Slow, esp. for fastidious species Mycobacterium spp.
Not always definitive
Page 13
Molecular diagnosis
Ribotyping Restriction fragment length polymorphism
(RFLP) DNA hybridization PCR, RT-PCR and RAPD Nucleic acid sequence analysis Phage-GFP (TB)
Page 14
RFLP
GGATCCCCTAGG
Page 15
DNA hybridization
Page 16
In situHybridization
Page 17
Rate of increase2n
PCR RT-PCR
Page 18
RAPD of P. aeruginosa
Page 19
Molecular diagnosis
Reduce reliance on culture
Faster More 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
Page 20
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 )
Page 21
Staphylococci
S. aureus: coagulase + S. epidermidis: coagulase -
Page 22
Streptococci
Streptococci On blood agar Growth inhibition disc
S. pyogenes (group A) -hemolytic Sensitive to bacitracin
S. agalactiae (group B) -hemolytic Resistant to bacitracin
S. pneumoniae (pneumococcus) -hemolytic Sensitive to optochin
Viridans -hemolytic Resistant to optochin
Page 23
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
Page 24
Gram negative
Straight rods Curved rods
Lactose+ Lactose-
Citrate+ Citrate- H2S+ H2S-
Klebsiella E. coli Salmonella Shigella
Campy blood agar42oC+ 25oC-
Campylobacter
TCBS agarYellowOxidase+
Vibrio
Page 25
BacteriaGram+ Gram- Acid Fast
IntraCellular
WallLess
Cocci Rod CocciRodSpiral
Staph. Strep. Non-spore Spore
StraightCurve
+O2 -O2+/-O2 Other
S. a.S. e.S. s.
ABPnVir
Fil Rod
A.i. C.d.L. m.
M.t.M.l.N.c.
+O2 -O2
B.a.B.c.
C.b.C.t.C.p.C.d.
TreponemaBorreliaLeptospira
NeisseriaMoraxella
P.a. Enteric Bact.
Resp. Zoo GU
Bordetella.H. influenzaeLegionella
YersiniaPasteurellaBrucellaFrancisellaStreptobacillus
H. ducreyiGardnerellaCalymmatobacterium
RickettsiaCoxiellaErlichiaChlamydia
Mycoplasma
VibrioCampylobacterHelicobacter