Microbiology

Cheng ZhangThurs 1 Dec 11

MM Tutorial

Gram staining

1. Fixed film (heat kill bacteria etc...)2. Methyl violet 3. Lugol’s/gram’s iodine4. Decolourise with acetone5. Methyl red STAIN 2 (counter stain): pink-red colour

STAIN 1: violet-blue

Gram staining

G+ve keeps stain 1 VIOLET/BLUEG-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 listRoutes 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 aeruginosaUTI

• Acinetobacter baumaniiITU infections, pneumonia

Gram positives

• Staphylococcus epidermidisCommensal

• Enterococcus faecalisNot 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

HAIsNosocomial = HAI >48h after admissionImmunocompromised; ImmunosuppressedWHY? Lines, catheters, intubation, chemo, prophylactic AB, prostheticsUK 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. Decreased influx2. Increased efflux e.g. Tetracycline3. Drug inactivation e.g. Penicillin/ESBL4. Target modification e.g. Penicillin/Quinolones5. Target amplication e.g. Sulphonamides6. Other: biofilms, spores, intracellular

Transfer of antibiotic resistance

1. Plasmids2. Transposons – mobile genetic elements

integrate to chromosomal DNA3. 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. Flucytosine3. 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

Viruses1. 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 particles5. 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, rotavirusRELEASE– Blood, Skin, Gut, Respiratory, Saliva, Semen, Breast milk

(HCMV), Placenta

Viruses evade the immune system by...

1. Antigenic variation2. Hiding3. 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

HIV1. Acute infection 2-3 months – active virus

replication, temporary reduction in CD42. 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 drugs1. 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.

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?