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 CocciRod 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 CocciRod Cocci Spiral + spore - + +/- -O 2

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  H 2 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

Rate of increase 2 n PCRRT-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

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

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-H 2 S+ H 2 S- Klebsiella E. coli Salmonella Shigella Campy blood agar 42 o C+ 25 o C- Campylobacter TCBS agar Yellow Oxidase+ Vibrio

Bacteria Gram+Gram-Acid Fast Intra Cellular Wall Less Cocci RodCocciRodSpiral Staph.Strep.Non-sporeSpore Straight Curve +O 2 -O 2 +/-O 2 Other S. a. S. e. S. s. A B Pn Vir FilRod A.i.C.d. L. m. M.t. M.l. N.c. +O 2 -O 2 B.a. B.c. C.b. C.t. C.p. C.d. Treponema Borrelia Leptospira Neisseria Moraxella P.a.EntericBact. Resp.ZooGU Bordetella. H. influenzae Legionella Yersinia Pasteurella Brucella Francisella Streptobacillus H. ducreyi Gardnerella Calymmatobacterium Rickettsia Coxiella Erlichia Chlamydia Mycoplasma Vibrio Campylobacter Helicobacter