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 Acid-fast

Mycobacterium Wall-less

Mycoplasma

Unusual Obligate intracellular

Rickettsia, Chlamydia

G+ G- AF WL IC

Bacteria

Bacterial classification

Cell morphology Shapes

Rod Cocci Spiral

Associations Individual Diplo- Staphylo- Strepto-

G+ G- AF WL IC

Bacteria

Rod Cocci Rod Cocci Spiral

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

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 H2S

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

GGATCCCCTAGG

DNA hybridization

In situHybridization

Rate of increase2n

PCR RT-PCR

RAPD of P. aeruginosa

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

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) -hemolytic Sensitive to bacitracin

S. agalactiae (group B) -hemolytic Resistant to bacitracin

S. pneumoniae (pneumococcus) -hemolytic Sensitive to optochin

Viridans -hemolytic Resistant to optochin

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-

Citrate+ Citrate- H2S+ H2S-

Klebsiella E. coli Salmonella Shigella

Campy blood agar42oC+ 25oC-

Campylobacter

TCBS agarYellowOxidase+

Vibrio

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