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Microbiology

Microbiology. Cheng Zhang Thurs 1 Dec 11 MM Tutorial. Gram staining. Fixed film (heat kill bacteria etc...) Methyl violet Lugol’s /gram’s iodine Decolourise with acetone Methyl red  STAIN 2 (counter stain): pink-red colour. STAIN 1: violet-blue. Gram staining.

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Microbiology

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  1. Microbiology Cheng Zhang Thurs 1 Dec 11 MM Tutorial

  2. Gram staining • Fixed film (heat kill bacteria etc...) • Methyl violet • Lugol’s/gram’s iodine • Decolourise with acetone • Methyl red  STAIN 2 (counter stain): pink-red colour STAIN 1: violet-blue

  3. Gram staining G+ve keeps stain 1  VIOLET/BLUE G-verecolourises with stain 2  PINK/RED Organisms stain poorly with gram’s stain: • Mycobacteria  ‘Acid fast’  ZN stain instead • Spiral bacteria  Treponema, Leptospira, Borrelia • Mycoplasma  Has no cell wall • Rickettsia, Coxiella, Chlamydia  obligate intracellular

  4. Nota bene Bacterial pathogens can be... • Extracellular • Examples: Staphylococcus, Streptococcus • Facultative intracellular = capable of living and reproducing inside and outside a cell • Examples: Listeria, Neisseria • Obligate intracellular = cannot reproduce outside host cell

  5. Gram staining • Peptidoglycan which is stained – most of cell wall in G+ve but only about 10% in G-ve • Acetone destroys outer lipopolysaccharide membrane of G-ve washing away STAIN 1 • Peptidoglycan matrix retains STAIN 1

  6. For your stage... • Bacteria are either: • G+ve or G-veor other (as mentioned) • Cocci (balls) or bacilli (rods) [or spirals] • G+vecocci: Streptococcus; Staphylococcus; Enterococcus • G+ve bacilli: Clostridium, Listeria only ones you need to know • G-ve spirals: Helicobacter, Campylobacter • G-vecocci (HAN): Haemophilius , Acinetobacter, Neisseria

  7. For your stage... • G-ve bacilli: Literally everything else you are likely to be asked at this stage... you name it! • Salmonella, Shigella, Proteus, ESBL i.e. E. Coli and Klebsiella, Pseudomonas, Vibrio sp., Legionella...

  8. For your stage... • In summary... • G+vecocci... Staph + Strep + Enterococcus • G+ve bacilli... Clostridium + Listeria • G-vecocci... NAH (What other N, A and H do you know at this stage?) • G-ve spirals: Helicobacter, Campylobacter • G-ve bacilli... Everything else

  9. Boring stuff • Virulence, Infective dose, Virulence determinants (genes), Pathogenicity islands (clusters of genes) • Factors in virulence? • Tropism • Replication (find nutrients) • Immune evasion • Toxic (exotoxins, endotoxins) • Transmission

  10. Routes of infection?What organism causes what? • Use your common sense... • Don’t memorise the ridiculous list Routes include: • Respiratory e.g. TB, pneumonia • Faecal-oral e.g. cholera, shigella • Direct contact e.g. UG: name any STI • or skin e.g. Staph • Vector borne (tick-borne)  Lyme disease (Borrelia) • Note: Erythemamigrans

  11. Familiarise yourself with names! • The more you hear it, the more it’ll stick • Most are aptly named e.g. Strep pneumoniae, Neisseriameningitidis, Neisseria gonorrhoea, Mycobacterium TB/leprae, Vibriocholerae, Salmonella typhi • Some can still be worked out e.g. Campylobacter JEJUNI, Helicobacter PYLORI, Bacillus ANTHRACIS • Some are confusingly named e.g. Haemophiliusinfluenzae, Rickettsia • The rest you’ll have to learn... Enjoy! • Large and small bowel: Gram negs, anaerobes, candida!

  12. So, get to know what causes what! • What are the bacterial causes of pneumonia? • Diarrhoea? • ETC... • N.B. Single gram positive cause of pneumonia is Streptococcus pneumoniae (pneumococcus)

  13. “Pathogens to know....” Gram negative Gram positive Staph aureus (PVL) Streptococcus Group A = S. pyogenes Group B = S. agalactiae (newborn) Strep viridans = oral bacteria Pneumococcus = S. pneumoniae Clostridium (difficile, tetani, botulinum, perfringens) Listeria spp. • Neisseria (meningitidis and gonorrhoeae) • Haemophilusinfluenzae • E. coli (EPEC, EHEC, ETEC, UPEC) • Salmonella spp. • Vibriocholerae • Shigella

  14. “Opportunistic bacterial pathogens” Gram negatives Gram positives Staphylococcus epidermidis Commensal Enterococcusfaecalis Not VRE, common • Pseudomonas aeruginosa UTI • Acinetobacterbaumanii ITU infections, pneumonia

  15. A little bit of detail... Vibriocholerae(Genus species or G. species) • Gram stain? Rod? Ball? You tell me... • Extracellular, colonises small bowel • Profuse watery diarrhoea (faecal-oral) – fluid replace • 1A5B toxin co-regulated pilus (similar Shigella, E. coli) • A = active, B = binding. Hence... 1A injected intracellularly and ADP-ribosylates G-proteins • The constantly active G protein stimulates adenylatecyclase to increase cAMP to open apical ion channels • Chloride and water leak out into the lumen

  16. A little bit of detail... Clostridium difficile • Gram stain? Rod? Ball? You tell me... • Hospital exposure to spores • Opportunistic pathogen – antibiotics clear normal flora • Diarrhoea (symptomatic infection) • If severe.. abdo pain, pseudomembranous colitis, perforated colon leading to faecal peritonitis.. • Rx: Stop other ABx, use metronidazole, vancomycin

  17. A little bit of detail... Neisseriameningitidis • Gram? Rod? Ball? • Vaccine for menC not menB • Subepithelial colonisation in nasopharynx • Septicaemia (10% fatality); non-blanching rash • CSF  neck stiffness, photophobia, vomitting

  18. HAIs Nosocomial = HAI >48h after admission Immunocompromised; Immunosuppressed WHY? Lines, catheters, intubation, chemo, prophylactic AB, prosthetics UK BIG FIVE • MRSA • VRE (Enterococcisfaecium) • E. coli/Klebsiella(NDM-1)  (ESBL enterobacteraciae) • P. aeruginosa • Acinetobacterbaumannii • Clostridium difficle • Vancomycin-insensitive S. aureus(VISA) • Stenotrophomonasmaltophilia (what the!??!)

  19. Antibiotics Think of it in families: • Inhibit cell wall synthesis • Inhibit protein synthesis • Inhibit DNA synthesis • Metabolic targets • Inhibit RNA synthesis (rifampicin) • Metronidazole

  20. Antibiotics • Bacteriostatic/Bacteriocidal • The ones in your slides: • Beta-lactams (inhibits transpeptidation enzyme) • Tetracycline (competes with tRNA for A site) • Chloramphenicol (bind to 50S subunit) • Quinolones(inhibits DNA synthesis) • Sulphonamides (competes for dihydropteroate) • Co-trimoxazole for p. carinii • Aminoglycosides(bind to 30S subunit) • Gentamicin, streptomycin: many UWEs • Macrolides (bind to 50S) e.g. erythromycin

  21. Antibiotic resistance • Decreased influx • Increased efflux e.g. Tetracycline • Drug inactivation e.g. Penicillin/ESBL • Target modification e.g. Penicillin/Quinolones • Target amplication e.g. Sulphonamides • Other: biofilms, spores, intracellular

  22. Transfer of antibiotic resistance • Plasmids • Transposons – mobile genetic elements integrate to chromosomal DNA • Integrons – gene cassettes in clusters, collect resistance genes

  23. Vaccination • Active immunity – host response to antigen – vaccination induces this • Passive immunity – acquiring protection from another immune individual through transfer of antibody or activated T cells • Herd immunity provides protection to unvaccinated individuals. Ring vaccination.

  24. Vaccine formulations • Antigen(s)to stimulate an immune response • Adjuvantto enhance and modulate the immune response • Delivery systems e.g. slow release depot • Immune potentiators stimulate immune system e.g. PAMPs such as TLRs • Excipients e.g. buffer, salts, saccharides, proteins to maintain pH, osmolarity, stability

  25. Vaccine antigens • Live attenuated organisms e.g. BCG, Sabin (oral) • Killed organisms e.g. Cholera, Salk (IM) • Component vaccines e.g. Tetanus • DNA vaccines • Conjugate vaccines – saccharide linked to protein carrier e.g. MenC

  26. Fungal infections • 3 major subclasses • Allergies – over-exuberant immune response to spores e.g. ABPA • IgE blood test • Mycotoxicoses – no immune component, mycotoxin ingestion e.g. Aflatoxin, magic mushrooms • Rx: gastric lavage, charcoal, organ transplant, supportive

  27. Fungal infections • Mycoses (fungal infections) – result of impaired immunity • Superficial (cosmetic of skin or hair shaft) e.g. Black Piedra • Cutaneous e.g. T. capitis, T. Pedis • Subcutaneous e.g. Eumycetoma (often after traumatic implantation of agent) • Systemic (deep)/invasive e.g. Candida, IPA • Dx: Gold standard is microscopy of sample e.g. BAL, skin, sputum, vaginal smear, CSF... • Also PCR, Ig/Ag-based assays

  28. Fungal pathogens • True or primary pathogens • Endemic in well-defined areas • You don’t need to be able to name any • Opportunistic • Ubiquitous • Cryptococcus, Candida (1/4 carriers), Aspergillus • N.B. • C. albicans is a yeast at low temp and pH • Nitrogen nutrient starvation: pseudohyphae (elongated cells looking for nutrients) • Serum pH: hyphae (cells divide)

  29. Antifungal targets • Cell membrane (fungal ergosterol) • Polyene antibiotics e.g. Amphotericin B, Nystatin • Azoleantifungals • DNA/RNA synthesis e.g. Flucytosine • Fungal cell wall (glucans, chitin) • Echinocandins e.g. Caspofungin acetate

  30. Viruses • 20-450nm obligate intracellular parasites • Nucleic acid (DNA or RNA) + protein (nucleocapsid – helical or icosahedral) + sometimes lipid + sometimes CHO • Many asymptomatic. Cause epidemics/pandemics when viruses jump from native species to unnatural host e.g. H1N1 (Spanish Flu) 1918/19 killed 40 million, SARS-CoV, HIV • Zoonosis

  31. Viruses • Binding to host cell – specificity • HIV gp120 to CD4 • EBV gp340 to CD21 • Influenza HA to sialic acid • Penetration • Enveloped viruses fuse e.g. HIV, measles • Non-enveloped disrupt host cell membrane – genome crosses into cytosol e.g. polio, bacteriophage T4 • Eclipse phase (period of non-infectivity) • Virus disassembled so no infectious particles present • Expression of viral proteins in highly regulated way • Nucleic acid... Protein coat... Proteins for cell lysis • Assembly of new particles • Release – cell lysis or budding (viruses with envelopes bleb)

  32. Baltimore classification • Based on how +ve sense mRNA is made! All viruses must make mRNA. • Single strand? double strand? • +ve sense? –ve sense? • Some degree of common sense can be applied e.g. retroviruses

  33. ssDNA first copied to dsDNA (host machinery) • Retroviruses reverse transcribed to DNA, integrated with our DNA, transcribed by our enzymes • Viral genome ssRNA +ve = same sense as mRNA • dsRNA viruses have to provide enzyme • ssRNA –ve viruses must provide enzyme to form opposite strand polarity

  34. Virus infection outcomes • INFLUENCED BY... • Virus dose, Route of entry (variolation), Age/sex/physiological state (VZV, EBV asymptomatic in child, HBV > in neonates) • CELL DEATH • Polio (paralysis), rotavirus (diarrhoea), HIV (immunodeficiency), HBV (hepatitis), rhabovirus (hydrophobia) • PERSISTENT • HBV (hepatitis), measles (subacutesclerosingpanencephalitis) • LATENT • HSV-1 or 2 (cold sores, genital herpes), VZV (chickenpox, shingles) • CELL TRANSFORMATION/CANCER • HBV (hepatocellular carcinoma) • HPV-6 and 11 (common warts) • HPV-16 and 18 (cervical/penile cancer) • EBV (Burkitt’s, nasopharyngeal carcinoma)

  35. Viral routes of entry • Respiratory: influenze, measles, mumps, variola, VZV, rhinovirus • Skin: HPV, HSV-1 and 2, rhabovirus, yellow fever virus (mosquito) • Blood products: HIV, HBV, HCV • Genital tract: HIV, HSV-2, HPV-16 and 18 • GI: Polio, HAV, rotavirus RELEASE • Blood, Skin, Gut, Respiratory, Saliva, Semen, Breast milk (HCMV), Placenta

  36. Viruses evade the immune system by... • Antigenic variation • Hiding • Express inhibitory proteins

  37. HIV (retrovirus env. +vessRNA) • >95% AIDS in developing country • Sexual, IVDU, mother-to-baby, blood products • Genome integrated into host DNA as provirus • Virus gp120 binds to CD4 + CCR5 (macrophage) or CXCR4 (T cell) • HA (Highly Active) ART

  38. HIV • Acute infection 2-3 months – active virus replication, temporary reduction in CD4 • CD8 HIV-specific CTL produced – virus titres decrease and CD4 recovers. Patient may become asymptomatic. Virus replication continues in LNs – variation to escape immune system • Virus variants escape control by CTL, titres increase, CD4 drops and patient develops AIDS

  39. HIV drugs • Binding/entry  binds CXCR4, CCR5 or gp41 (T-20) • Reverse transcription  Nucleoside reverse-transcriptase inhibitors e.g. AZT and NNRTI (allosteric enzyme inhibition) e.g. EFV • Protease inhibitors (prevent cleavage of polyprotein precursors) e.g. Ritonavir • Integrase inhibitors prevent integration with host DNA e.g. Raltegravir • Learn short names e.g. T-20 for Enfuvirtide, AZT for Zidovudine • If you’re hell bent on getting a ridiculously high exam mark, learn the whole list.

  40. Influenza • -ve sense ssRNA enveloped • Antigenic drift (AA mutations) and shift (e.g. zoonosis e.g. human + avian co-infection) • 100nm: • HA (glycoprotein, binds sialic acid) • NA (removes sialic acid to allow new viruses to escape) – tamiflu • M2 ion channel

  41. Virus vaccines • Smallpox, diphtheria, tetanus, yellow fever, pertussis, MMR, poliomyelitis, HBsAg • Smallpox eradicated because... • No animal reservoir • No latent/persistent infection • Easily recognisable disease • Vaccine effective against all strains, low cost, abundant, potent (vaccinia vaccine and variola envelope highly conserved) • WHO co-ordinated a global effort

  42. Questions?

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