Microbiology Cheng Zhang Thurs 1 Dec 11 MM Tutorial Gram staining 1. 2. 3. 4. 5. Fixed film (heat kill bacteria etc...) Methyl violet STAIN 1: violet-blue Lugol’s/gram’s iodine Decolourise with acetone Methyl red STAIN 2 (counter stain): pink-red colour Gram staining G+ve keeps stain 1 VIOLET/BLUE G-ve recolourises 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 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 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 For your stage... • Bacteria are either: • G+ve or G-ve or other (as mentioned) • Cocci (balls) or bacilli (rods) [or spirals] • G+ve cocci: Streptococcus; Staphylococcus; Enterococcus • G+ve bacilli: Clostridium, Listeria only ones you need to know • G-ve spirals: Helicobacter, Campylobacter • G-ve cocci (HAN): Haemophilius , Acinetobacter, Neisseria 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... For your stage... • In summary... • G+ve cocci... Staph + Strep + Enterococcus • G+ve bacilli... Clostridium + Listeria • G-ve cocci... NAH (What other N, A and H do you know at this stage?) • G-ve spirals: Helicobacter, Campylobacter • G-ve bacilli... Everything else 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 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: Erythema migrans Familiarise yourself with names! • The more you hear it, the more it’ll stick • Most are aptly named e.g. Strep pneumoniae, Neisseria meningitidis, Neisseria gonorrhoea, Mycobacterium TB/leprae, Vibrio cholerae, Salmonella typhi • Some can still be worked out e.g. Campylobacter JEJUNI, Helicobacter PYLORI, Bacillus ANTHRACIS • Some are confusingly named e.g. Haemophilius influenzae, Rickettsia • The rest you’ll have to learn... Enjoy! • Large and small bowel: Gram negs, anaerobes, candida! 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) “Pathogens to know....” Gram negative • Neisseria (meningitidis and gonorrhoeae) • Haemophilus influenzae • E. coli (EPEC, EHEC, ETEC, UPEC) • Salmonella spp. • Vibrio cholerae • Shigella 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. “Opportunistic bacterial pathogens” Gram negatives • Pseudomonas aeruginosa UTI • Acinetobacter baumanii ITU infections, pneumonia Gram positives • Staphylococcus epidermidis Commensal • Enterococcus faecalis Not VRE, common A little bit of detail... Vibrio cholerae (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 adenylate cyclase to increase cAMP to open apical ion channels • Chloride and water leak out into the lumen 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 A little bit of detail... Neisseria meningitidis • Gram? Rod? Ball? • Vaccine for menC not menB • Subepithelial colonisation in nasopharynx • Septicaemia (10% fatality); non-blanching rash • CSF neck stiffness, photophobia, vomitting HAIs Nosocomial = HAI >48h after admission Immunocompromised; Immunosuppressed WHY? Lines, catheters, intubation, chemo, prophylactic AB, prosthetics UK BIG FIVE • MRSA • VRE (Enterococcis faecium) • E. coli/Klebsiella (NDM-1) (ESBL enterobacteraciae) • P. aeruginosa • Acinetobacter baumannii • Clostridium difficle • Vancomycin-insensitive S. aureus (VISA) • Stenotrophomonas maltophilia (what the!??!) Antibiotics Think of it in families: • Inhibit cell wall synthesis • Inhibit protein synthesis • Inhibit DNA synthesis • Metabolic targets • Inhibit RNA synthesis (rifampicin) • Metronidazole 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 Antibiotic resistance 1. 2. 3. 4. 5. 6. 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 Transfer of antibiotic resistance 1. Plasmids 2. Transposons – mobile genetic elements integrate to chromosomal DNA 3. Integrons – gene cassettes in clusters, collect resistance genes 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. Vaccine formulations • Antigen(s) to stimulate an immune response • Adjuvant to 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 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 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 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 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) Antifungal targets 1. Cell membrane (fungal ergosterol) • • Polyene antibiotics e.g. Amphotericin B, Nystatin Azole antifungals 2. DNA/RNA synthesis e.g. Flucytosine 3. Fungal cell wall (glucans, chitin) • Echinocandins e.g. Caspofungin acetate 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 Viruses 1. Binding to host cell – specificity – – – 2. Penetration – – 3. 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) – – – 4. 5. HIV gp120 to CD4 EBV gp340 to CD21 Influenza HA to sialic acid 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) 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 • 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 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 (subacute sclerosing panencephalitis) • 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) 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 Viruses evade the immune system by... 1. Antigenic variation 2. Hiding 3. Express inhibitory proteins HIV (retrovirus env. +ve ssRNA) • • • • >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 HIV 1. Acute infection 2-3 months – active virus replication, temporary reduction in CD4 2. 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 3. Virus variants escape control by CTL, titres increase, CD4 drops and patient develops AIDS HIV drugs 1. Binding/entry binds CXCR4, CCR5 or gp41 (T-20) 2. Reverse transcription Nucleoside reversetranscriptase inhibitors e.g. AZT and NNRTI (allosteric enzyme inhibition) e.g. EFV 3. Protease inhibitors (prevent cleavage of polyprotein precursors) e.g. Ritonavir 4. 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. 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 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 Questions?