Culture and identification of infectious agents, Lecture 25

1. Culture and identification of infectious agents, Lecture 25 Dr. Alvin Fox

2. Key Terms After culture • Biochemical (physiological) tests • Genetic tests • - Polymerase chain reaction (PCR) • - Sequencing • - DNA-DNA homology/arrays • - Restriction enzymes (digests) • Chemical • - fatty acid/protein profiling • Immunological • Direct detection (i.e. without culture) • PCR • Antigen detection • Staining (e.g. Gram stain) • Serology (antibody detection) • Isolation (culture) • Agar plate plate/colonies • Liquid media • Identification & taxonomy • Family • Genus • Species • Type • Strain

3. Taxonomy • Defines common traits among strains for a bacterial species • Usually genetic • Allows development of diagnostic kits

4. Species versus strains- selecting discriminating features

5. Classification • Strain: one single isolate or line • Type: sub-set of species • Species: related strains • Genus: related species • Family: related genera

6. Both terms define the species name Streptococcuspyogenes Genus part Species part So Streptococcus pyogenes and Streptococus agalactiae would be two species in the same genus In a report On first usage: Streptococcus pyogenes On second usage: S. pyogenes

7. Identification of infectious agents in the diagnostic laboratory • Aids treatment • Helps antibiotic selection • General hospital laboratory • physiological tests • More fully equipped laboratories • Genetic tests

8. Steps in isolation and identification • Step 1: Streaking culture plates • colonies on incubation (e.g 24 hr) • size, texture, color, hemolysis • oxygen requirement

9. Sheep blood agar plate culture Bacillus cereus. Bacillus anthracis CDC/Dr. James Feeley

10. Mixed colonies

11. Isolation and identification • Step 2: Colonies Gram stained • cells observed microscopically

12. Gram Stain Gram negative Gram positive Heat/Dry Crystal violet stain IodineFix Alcoholde-stain Safranin stain

13. Gram stain morphology • Shape • cocci (round) • bacilli (rods) • spiral or curved (e.g. spirochetes) • Single or multiple cells • clusters (e.g. staphylococci) • chains (e.g. streptococci) • Gram positive or negative

14. Step 3:Isolated bacteria are speciated • Generally using physiological tests

15. Clinical Microbiology Laboratory Bench

16. Step 4: Antibiotic susceptibility testing

17. Antibiotic susceptibility testing Susceptible Not susceptible Bacterial lawn Growth No growth Antibiotic disk

18. Molecular differentiation • Genomics • Gene characterization • PCR • Sequencing • Restriction digestion • Hybridization (probes, arrays) • % guanine-cytosine

19. 16S rRNA Sequencing • Differentiates bacterial species • Development of clinical tests based on sequence (e.g. PCR)

20. Real-time PCR ds DNA Cycle one Dye Cycle two Cycle 30 2 30

21. DNA-DNA hybridization Strain 1 Heat + Strain 2 0% Homology 100% Homology

22. Profiles • Long chain fatty acids • - structural (e.g. cell membrane) • Short chain • - metabolic • - volatiles • - Fatty acids/alcohols

23. Protein profiling: defining a species by characteristic (low molecular weight) proteins • Proteomics: defining all proteins expressed by a species under specific growth conditions

24. Bacterial DNA sequences can be amplified directly from human body fluids

25. Laboratory diagnosis without culture • In general, when speed is of essence • Of additional importance: • The organism grows poorly/slowly • The organism can’t be cultured

26. Microscopy • spinal fluids (meningitis) • sputum (tuberculosis) • sensitivity poor

27. Streptococcal Agglutination Test Streptococcal antigenic extract Antibody Latex beads

28. Serologic identification • antibody response to the infecting agent • several weeks after an infection has occurred

29. Epidemics • associated with particular strains/types • state or federal laboratory system