Microbiology

MicrobiologyCheng ZhangThurs 1 Dec 11MM Tutorial

Gram stainingFixed film (heat kill bacteria etc...)Methyl violet Lugols/grams iodineDecolourise with acetoneMethyl red STAIN 2 (counter stain): pink-red colour

STAIN 1: violet-blueGram staining G+ve keeps stain 1 VIOLET/BLUEG-ve recolourises with stain 2 PINK/RED

Organisms stain poorly with grams stain:Mycobacteria Acid fast ZN stain insteadSpiral bacteria Treponema, Leptospira, BorreliaMycoplasma Has no cell wallRickettsia, Coxiella, Chlamydia obligate intracellular

Nota beneBacterial pathogens can be...Extracellular Examples: Staphylococcus, StreptococcusFacultative intracellular = capable of living and reproducing inside and outside a cellExamples: Listeria, NeisseriaObligate intracellular = cannot reproduce outside host cellGram stainingPeptidoglycan which is stained most of cell wall in G+ve but only about 10% in G-veAcetone destroys outer lipopolysaccharide membrane of G-ve washing away STAIN 1Peptidoglycan 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; EnterococcusG+ve bacilli: Clostridium, Listeria only ones you need to know

G-ve spirals: Helicobacter, CampylobacterG-ve cocci (HAN): Haemophilius , Acinetobacter, NeisseriaFor 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 + EnterococcusG+ve bacilli... Clostridium + Listeria

G-ve cocci... NAH (What other N, A and H do you know at this stage?)G-ve spirals: Helicobacter, CampylobacterG-ve bacilli... Everything elseBoring stuffVirulence, Infective dose, Virulence determinants (genes), Pathogenicity islands (clusters of genes)Factors in virulence?TropismReplication (find nutrients)Immune evasionToxic (exotoxins, endotoxins)TransmissionRoutes of infection?What organism causes what?Use your common sense...Dont memorise the ridiculous listRoutes include: Respiratory e.g. TB, pneumoniaFaecal-oral e.g. cholera, shigellaDirect 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 itll stickMost are aptly named e.g. Strep pneumoniae, Neisseria meningitidis, Neisseria gonorrhoea, Mycobacterium TB/leprae, Vibrio cholerae, Salmonella typhiSome can still be worked out e.g. Campylobacter JEJUNI, Helicobacter PYLORI, Bacillus ANTHRACISSome are confusingly named e.g. Haemophilius influenzae, RickettsiaThe rest youll 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 negativeNeisseria (meningitidis and gonorrhoeae)

Haemophilus influenzae

E. coli (EPEC, EHEC, ETEC, UPEC)Salmonella spp.Vibrio choleraeShigella

Gram positiveStaph aureus (PVL)StreptococcusGroup A = S. pyogenesGroup B = S. agalactiae (newborn)Strep viridans = oral bacteriaPneumococcus = S. pneumoniaeClostridium (difficile, tetani, botulinum, perfringens)Listeria spp.

Opportunistic bacterial pathogensGram negativesPseudomonas aeruginosaUTIAcinetobacter baumaniiITU infections, pneumonia

Gram positivesStaphylococcus epidermidisCommensal

Enterococcus faecalisNot VRE, common15A little bit of detail...Vibrio cholerae (Genus species or G. species)Gram stain? Rod? Ball? You tell me...Extracellular, colonises small bowelProfuse watery diarrhoea (faecal-oral) fluid replace1A5B 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 channelsChloride and water leak out into the lumenA little bit of detail...Clostridium difficileGram stain? Rod? Ball? You tell me...Hospital exposure to sporesOpportunistic pathogen antibiotics clear normal floraDiarrhoea (symptomatic infection)If severe.. abdo pain, pseudomembranous colitis, perforated colon leading to faecal peritonitis..Rx: Stop other ABx, use metronidazole, vancomycinA little bit of detail...Neisseria meningitidisGram? Rod? Ball?Vaccine for menC not menBSubepithelial colonisation in nasopharynxSepticaemia (10% fatality); non-blanching rashCSF neck stiffness, photophobia, vomittingHAIsNosocomial = HAI >48h after admissionImmunocompromised; ImmunosuppressedWHY? Lines, catheters, intubation, chemo, prophylactic AB, prostheticsUK BIG FIVEMRSAVRE (Enterococcis faecium)E. coli/Klebsiella (NDM-1) (ESBL enterobacteraciae)P. aeruginosaAcinetobacter baumannii

Clostridium difficleVancomycin-insensitive S. aureus (VISA)Stenotrophomonas maltophilia (what the!??!)

AntibioticsThink of it in families:Inhibit cell wall synthesisInhibit protein synthesis Inhibit DNA synthesisMetabolic targetsInhibit RNA synthesis (rifampicin)MetronidazoleAntibioticsBacteriostatic/BacteriocidalThe 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. cariniiAminoglycosides (bind to 30S subunit)Gentamicin, streptomycin: many UWEsMacrolides (bind to 50S) e.g. erythromycinAntibiotic resistanceDecreased influxIncreased efflux e.g. TetracyclineDrug inactivation e.g. Penicillin/ESBLTarget modification e.g. Penicillin/QuinolonesTarget amplication e.g. SulphonamidesOther: biofilms, spores, intracellularTransfer of antibiotic resistancePlasmidsTransposons mobile genetic elements integrate to chromosomal DNAIntegrons gene cassettes in clusters, collect resistance genesVaccinationActive immunity host response to antigen vaccination induces thisPassive immunity acquiring protection from another immune individual through transfer of antibody or activated T cellsHerd immunity provides protection to unvaccinated individuals. Ring vaccination.

Vaccine formulationsAntigen(s) to stimulate an immune responseAdjuvant to enhance and modulate the immune responseDelivery systems e.g. slow release depotImmune potentiators stimulate immune system e.g. PAMPs such as TLRsExcipients e.g. buffer, salts, saccharides, proteins to maintain pH, osmolarity, stability

Vaccine antigensLive attenuated organisms e.g. BCG, Sabin (oral)Killed organisms e.g. Cholera, Salk (IM)Component vaccines e.g. TetanusDNA vaccines Conjugate vaccines saccharide linked to protein carrier e.g. MenC

Fungal infections3 major subclassesAllergies over-exuberant immune response to spores e.g. ABPAIgE blood testMycotoxicoses no immune component, mycotoxin ingestion e.g. Aflatoxin, magic mushroomsRx: gastric lavage, charcoal, organ transplant, supportiveFungal infectionsMycoses (fungal infections) result of impaired immunitySuperficial (cosmetic of skin or hair shaft) e.g. Black PiedraCutaneous e.g. T. capitis, T. PedisSubcutaneous e.g. Eumycetoma (often after traumatic implantation of agent)Systemic (deep)/invasive e.g. Candida, IPADx: Gold standard is microscopy of sample e.g. BAL, skin, sputum, vaginal smear, CSF...Also PCR, Ig/Ag-based assaysFungal pathogensTrue or primary pathogensEndemic in well-defined areasYou dont need to be able to name anyOpportunisticUbiquitousCryptococcus, Candida (1/4 carriers), AspergillusN.B. C. albicans is a yeast at low temp and pHNitrogen nutrient starvation: pseudohyphae (elongated cells looking for nutrients)Serum pH: hyphae (cells divide)

Antifungal targetsCell membrane (fungal ergosterol)Polyene antibiotics e.g. Amphotericin B, NystatinAzole antifungalsDNA/RNA synthesis e.g. FlucytosineFungal cell wall (glucans, chitin)Echinocandins e.g. Caspofungin acetateViruses20-450nm obligate intracellular parasitesNucleic acid (DNA or RNA) + protein (nucleocapsid helical or icosahedral) + sometimes lipid + sometimes CHOMany 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, HIVZoonosisVirusesBinding to host cell specificityHIV gp120 to CD4EBV gp340 to CD21Influenza HA to sialic acidPenetrationEnveloped viruses fuse e.g. HIV, measlesNon-enveloped disrupt host cell membrane genome crosses into cytosol e.g. polio, bacteriophage T4Eclipse phase (period of non-infectivity)Virus disassembled so no infectious particles presentExpression of viral proteins in highly regulated wayNucleic acid... Protein coat... Proteins for cell lysisAssembly of new particlesRelease cell lysis or budding (viruses with envelopes bleb)

Baltimore classificationBased 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. retrovirusesssDNA first copied to dsDNA (host machinery)Retroviruses reverse transcribed to DNA, integrated with our DNA, transcribed by our enzymesViral genome ssRNA +ve = same sense as mRNAdsRNA viruses have to provide enzymessRNA ve viruses must provide enzyme to form opposite strand polarity

Virus infection outcomesINFLUENCED BY... Virus dose, Route of entry (variolation), Age/sex/physiological state (VZV, EBV asymptomatic in child, HBV > in neonates)CELL DEATHPolio (paralysis), rotavirus (diarrhoea), HIV (immunodeficiency), HBV (hepatitis), rhabovirus (hydrophobia)PERSISTENTHBV (hepatitis), measles (subacute sclerosing panencephalitis)LATENTHSV-1 or 2 (cold sores, genital herpes), VZV (chickenpox, shingles)CELL TRANSFORMATION/CANCERHBV (hepatocellular carcinoma)HPV-6 and 11 (common warts)HPV-16 and 18 (cervical/penile cancer)EBV (Burkitts, nasopharyngeal carcinoma)

Viral routes of entryRespiratory: influenze, measles, mumps, variola, VZV, rhinovirusSkin: HPV, HSV-1 and 2, rhabovirus, yellow fever virus (mosquito)Blood products: HIV, HBV, HCVGenital tract: HIV, HSV-2, HPV-16 and 18GI: Polio, HAV, rotavirusRELEASEBlood, Skin, Gut, Respiratory, Saliva, Semen, Breast milk (HCMV), PlacentaViruses evade the immune system by...Antigenic variationHidingExpress inhibitory proteinsHIV (retrovirus env. +ve ssRNA)>95% AIDS in developing countrySexual, IVDU, mother-to-baby, blood productsGenome integrated into host DNA as provirusVirus gp120 binds to CD4 + CCR5 (macrophage) or CXCR4 (T cell)HA (Highly Active) ART

HIVAcute infection 2-3 months active virus replication, temporary reduction in CD4CD8 HIV-specific CTL produced virus titres decrease and CD4 recovers. Patient may become asymptomatic. Virus replication continues in LNs variation to escape immune systemVirus variants escape control by CTL, titres increase, CD4 drops and patient develops AIDSHIV drugsBinding/entry binds CXCR4, CCR5 or gp41 (T-20)Reverse transcription Nucleoside reverse-transcriptase inhibitors e.g. AZT and NNRTI (allosteric enzyme inhibition) e.g. EFVProtease inhibitors (prevent cleavage of polyprotein precursors) e.g. RitonavirIntegrase inhibitors prevent integration with host DNA e.g. RaltegravirLearn short names e.g. T-20 for Enfuvirtide, AZT for ZidovudineIf youre hell bent on getting a ridiculously high exam mark, learn the whole list.Influenza-ve sense ssRNA envelopedAntigenic 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) tamifluM2 ion channelVirus vaccinesSmallpox, diphtheria, tetanus, yellow fever, pertussis, MMR, poliomyelitis, HBsAgSmallpox eradicated because...No animal reservoirNo latent/persistent infectionEasily recognisable diseaseVaccine effective against all strains, low cost, abundant, potent (vaccinia vaccine and variola envelope highly conserved)WHO co-ordinated a global effortQuestions?