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Microbiology Cheng Zhang Thurs 1 Dec 11 MM Tutorial.

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1 Microbiology Cheng Zhang Thurs 1 Dec 11 MM Tutorial

2 Gram staining 1.Fixed film (heat kill bacteria etc...) 2.Methyl violet 3.Lugol’s/gram’s iodine 4.Decolourise with acetone 5.Methyl red  STAIN 2 (counter stain): pink-red colour STAIN 1: violet-blue

3 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

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 -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

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 +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

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: Erythema migrans

11 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!

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 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.


15 “Opportunistic bacterial pathogens” Gram negatives Pseudomonas aeruginosa UTI Acinetobacter baumanii ITU infections, pneumonia Gram positives Staphylococcus epidermidis Commensal Enterococcus faecalis Not VRE, common

16 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

17 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

18 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

19 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!??!)

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

21 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

22 Antibiotic resistance 1.Decreased influx 2.Increased efflux e.g. Tetracycline 3.Drug inactivation e.g. Penicillin/ESBL 4.Target modification e.g. Penicillin/Quinolones 5.Target amplication e.g. Sulphonamides 6.Other: biofilms, spores, intracellular

23 Transfer of antibiotic resistance 1.Plasmids 2.Transposons – mobile genetic elements integrate to chromosomal DNA 3.Integrons – gene cassettes in clusters, collect resistance genes

24 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.

25 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

26 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

27 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

28 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

29 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)

30 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

31 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

32 Viruses 1.Binding to host cell – specificity – HIV gp120 to CD4 – EBV gp340 to CD21 – Influenza HA to sialic acid 2.Penetration – Enveloped viruses fuse e.g. HIV, measles – Non-enveloped disrupt host cell membrane – genome crosses into cytosol e.g. polio, bacteriophage T4 3.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 4.Assembly of new particles 5.Release – cell lysis or budding (viruses with envelopes bleb)

33 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

34 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

35 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)

36 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

37 Viruses evade the immune system by... 1.Antigenic variation 2.Hiding 3.Express inhibitory proteins

38 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


40 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

41 HIV drugs 1.Binding/entry  binds CXCR4, CCR5 or gp41 (T-20) 2.Reverse transcription  Nucleoside reverse- transcriptase 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.

42 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

43 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

44 Questions?

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