I&I 5: The Biology of Pathogenic Micro-organisms

- These are single-celled organisms which are characterised by these features:
• small (1-10 μm, cf 10 - 100 μm for eukaryotes);
• the cell is enclosed by a rigid cell wall = protective and to prevent cell lysing at low osmolarity
• plasma membrane = semipermeable barrier
• no membrane about their DNA (“nucleoid”= where the main cell DNA is);
• most of the cell’s DNA is in a single circular molecule, but there may be additional much smaller circular DNA molecules present termed plasmids (note that the nucleoid and plasmids are attached to the cell membrane)
• they do not have membrane-bound compartments (e.g. mitochondria (!!)).
• some have flagella which differ from cilia of eukaryotes and allow motility
• some have polysaccharide “capsule” to protect the cell e.g. from antibodies
• some have short fibres projecting from the cell surface - pili (s: pilum) = join cells to other cells and allow transmission of DNA, especially plasmids, from cell to cell; or fimbriae (s: fimbria)= attach cells to surfaces so that they are not washed away
- On the whole, bacteria have similar metabolism (e.g. tricarboxylic acid cycle) although may differ in detail e.g. protein synthesis.

1. Morphology
- cocci (s: coccus) – more-or-less spherical;
- bacilli (s: bacillus) – more-or-less rods;
- in-betweens (cocco-bacilli);
- others e.g. spirals (e.g. T. pallidim)
- may be in clumps or chains or pairs (diplo-)

2. Cell wall chemistry
- Bacteria fall into two great groups which can be distinguished by a simple staining procedure = the Gram stain → the most important single technique in medical microbiology
- The Gram stain depends on the on the composition of the cell wall which is EITHER:
• mostly all PEPTIDOGLYCAN;
OR
• mostly LIPOPOLYSACCHARIDE (LPS aka endotoxin) with a thin layer of peptidoglycan inside the LPS layer.
- The principle of the Gram stain is that peptidoglycan binds the stain crystal violet/iodine very tightly:
• cells with thick outer peptidoglycan layers appear purple/black GRAM POSITIVE
• cells with thin inner peptidoglycan layers covered with LPS do not stain with crystal violet/iodine: they are made visible by counterstaining with one of several pink-ish red dyes and are GRAM NEGATIVE.

3. Other features e.g specific biochemical attributes = some are anaerobic, or produce substances which lyse red blood cells, or have characteristic metabolic patterns.
(4. Ultimately DNA sequence.)

Hence, bacteria are divided into:
- Gram positive cocci:
e.g. Staphylococcus spp.; Streptococcus spp.
- Gram negative cocci: are rare e.g. Neisseiria spp are G-ve diplococci (pairs).
- Gram positive bacilli:
e.g. Bacillus anthracis; Clostridium spp.
- Gram negative bacilli:
e.g. Shigella spp.; Bacteroides spp. = coliforms e.g. E. coli, Salmonella

- HOWEVER: there are some exceptions
• A few bacteria do not Gram stain at all either because either they have lost the cell wall e.g. Mycoplasma spp. or the cell wall is very specialised & excludes both Gram’s stain and counterstain e.g. Mycobacteria spp.
• These are sometimes called Gram-null and must be stained with acid-fast stains
• Interestingly both these are intracellular parasites.

- Bacteria being cells reproduce by binary fission.
- However some do this v. fast = 30’ per division.
- Many bacteria have very basic growth requirements = water, salts, a carbon source; but grow much better in rich media e.g. beef broth, blood.
- Hence our bodies (inside & out) are ideal for bacterial growth
- One problem for bacterial growth in tissues is availability of Fe: free Fe is at very low concentrations and so may limit bacterial growth.

- The vast majority of bacterial species are “free living” in the natural environment = soil & water.
- A few inhabit the external surfaces of the body, GI tract etc and do no harm.
- These are described as “commensal”.
- A very few inhabit us and do harm = Pathogenic bacteria.
- This is because they invade regions where they should not be
e.g. lungs, blood, soft tissues such as skin & muscle, body cavities such as bowel, bladder and do damage there.

- The bacterial factors which confer pathogenicity on bacteria are referred to as pathogenicity factors (aka virulence factors)
- These are sometimes encoded by plasmids which may spread from pathogenic to non-pathogenic bacterial strains.

1. Invasion of the host directly causes damage
2. Resistance to the pathogen causes inflammatory response which may also cause damage

- Infection with pathogenic bacteria does not necessarily result in disease/damage e.g. less than 30% of those drinking water contaminated with Vibrio cholerae develop cholera
- Hence
Host + pathogen = disease
- Obviously it is the immune system (innate and adaptive) that determines whether disease develops.
- It follows that host factors related to immunosuppression, e.g. malnutrition, influence whether disease develops.

1. Adherence to body surfaces (adhesins);
2, Invasion of tissues (invasins);
3. Toxins
• endotoxins (LPS) which induce inflammation;
• exotoxins, secreted bacterial proteins which cause tissue damage & so inflammation;
4. The ability to evade immune response.
- Within a bacterial species, e.g. Escherichia coli, not all strains (aka types) will be equally pathogenic
e.g. E. coli O157 causes severe disease through secreting an exotoxin unlike the lab strain K12.

- Allow binding of bacteria to human cells
- E.g. fimbriae of uropathogenic Echerichia coli; adhesins HMW1 & 2 of Haemophilus influenzae; pili of Neisseria meningitidis; fibronectin-binding proteins of Staphylococcus aureus.
- Note that adherence necessarily precedes invasion and so adhesins overlap with invasins.

- Allow the bacteria to invade tissues
- e.g. collagenase of Clostridium tetani; streptokinase of Streptococcus pyogenase which dissolves blood clots; opaCEA of Neisseria meningitidis which by binding to proteins on the apical surface of epithelial cells promotes transcytosis (transport of the bacteria across the epithelium).

- AKA lipopolysaccharides of baterial capsule
- Since this is expressed by all Gram negative bacteria, in situations where these come into contact with cells of the innate immune system (i.e. after invasion of tissues) there will be inflammation.
- It is this inflammation that causes the symptoms of disease (in part).

- Broadly, these kill host cells, so maybe promoting invasion (through destroying tissues)
- e.g Shiga toxin from Shigella dysenteriae and related toxins from some E. coli strains (e.g. O157) which enter cells and block protein synthesis; haemolysins (so-called because assayed through lysis of red cells) from Staphylococcus aureus which lyses cell by pore formation.
- It is the tissue damage caused by these exotoxins which (in part) cause the symptoms of disease.

- May have exotoxins which kill phagocytes
- c5a peptidase of Streptococcus pyogenes which inactivates complement
- Polysaccharide capsules of S. pneumonia which prevent antibody binding
- Inhibition of intracellular bacterial killing mechanisms e.g. catalase to inactivate H2O2 in Mycobacterium tuberculosis.

Powerful Fe-chelating agents to grab Fe e.g. from transferrin (remember, Fe may be growth limiting): e.g. enterobactin from E. coli → allows easier growth

- As effector mechanisms certainly the most important of these are
• complement;
• phagocytosis, particularly by neutrophils;
• antibody (especially those activating complement and acting as opsonins, increasing phagocytes ability to endocytose targets)
• cytokines (especially those activating phagocytosis).

1. Classic pathogens e.g. Mycobacterium tuberculosis
2. Commensal bacteria in the wrong place e.g. Escherichia coli in the bladder
3. Opportunistic pathogens infecting individuals with imparied immunity e.g. Pneumocystis jirovecii (was carinii)

- In the third world today diseases caused by parasites are of enormous importance = malaria leads the way caused by Plasmodium spp.
- In the UK today very few eukaryotic pathogens are of significance:
• yeasts especially Candida albicans;
• the fungus Pneumocystis jiroveci ex P. carinii (in AIDS);
• threadworms (aka pinworms) Enterobius vermicularis.
• Athlete’s foot & suchlike minor things;
• Aspergillosis due to Aspergillus fumigatus = can be fatal, especially in the immunosuppressed.
• Imported infections e.g. malaria which if undiagnosed can be v. bad news.

- Candida albicans:
• the causative organism of thrush
• oral, vaginal
• can be a problem with babies.
• C. albicans is commensal in about 20% & only becomes a problem if there is some disturbance in its environment which allows it to grow
→ destruction of normal flora through antibiotic use;
→ immunosuppression (AIDS);
→ in vagina, rise of pH (vaginal secretions are usually acid) due e.g. to pregnancy.

- Pneumocystis jiroveci:
• a unicellular fungus with a complex life cycle;
→ widespread in the environment.
• Infects immunosuppressed people (mostly AIDS) ONLY and is described as an opportunistic pathogen (i.e. only when it gets the chance).

- Enterobius vermicularis:
• little worms that inhabit the rectum particularly of children & cause anal pruritis;
• avidly spread from child to child (itching – scratching …);
• common infection, easily treated.

- World-wide, viruses are a major cause of mortality
• HIV, hepatitis B
- Especially for children.
- And there is the ever present risk of new viral pathogens emerging
• Ebola, SARS;
- Or familiar viruses developing greater virulence
• influenza
- or a pandemic.
- Within the UK viruses are rarely cause of death except in “at risk” individuals
- But are a major cause of morbidity and GP consultations - 35% of GP consultations will be for infections, many of these viral.

- Packages of nucleic acids wrapped in a protein coat (capsid) which invade cells and use the cells’ biosynthetic machinery to make more viruses
- The viral nucleic acid is its genome which encodes essential functions not supplied by the cell.

- Viruses can only “grow” (“replicate”) within cells = “obligate intracellular parasites”
- But need to spread from cell to cell.
- Hence there are 2 phases to the virus life cycle: intracellular replication and extracellular transmission.

- The intracellular phase involves replication of the genome and synthesis of proteins
• 2 kinds: “non-structural” and “structural”
• Non-structural = produced early in the growth cycle. Have intracellular functions = often involved in genome replications etc
• Structural = produced late in the growth cycle and are important in the extracellular phase
- This is largely dependent on host cell synthetic systems.
1. Virion attaches to the cell via specific receptors. The term virion refers to the extracellular form in which the viral genome is wrapped in the protein capsid
2. Virion is taken up by the cell. The uptake of the cell (penetration) usually involves endosomal mechanisms
3. The viral nucleic acid is released and 'early' genes are expressed. Early genes encode functions essential for viral genome replication and for 'late' gene expression. Viral gene transcription may occur either in the cytoplasm, or on the nucleus, depending on the virus
4. Genome replication occurs, then 'late' gene products are produced. Late genes encode (mostly) proteins which are incorporated into the virion e.g. capsid
5. The new genome and structural proteins assemble into progeny virus. Assembly often occurs at the cell membrane
6. New virion is released into the extracellular space. Release often occurs by a type of budding mechanism resulting in the virion being wrapped in part of the cell membrane - the envelope.
7. Virus replication often causes so much damage to the cell that it lyses - then the virions are released without being enveloped

- Replication of viruses within cells usually (but not always) results in some level of cell damage which usually results in cell death = cytopathic effect (cpe) which may be characteristic and useful in diagnosis.

- The extracellular phase involves transmission by the virion of the viral genome from the previous host cell to the next.
- The role of virion is to protect the genome in the extracellular environment and to allow attachment to the next host cell.

- The virion is comprised of structural proteins (the “capsid”) together with genomic nucleic acid
• These are often regular structures e.g. adenovirus;
• or irregular (“pleomorphic”) surrounded by an “envelope” (derived from the cell membrane, containing viral proteins) e.g. influenza.
- Virion shape may be useful in diagnosis.
- Viral genomes may be DNA, RNA, single or double stranded, linear or circular in not quite all permutations.
- Knowledge of genome structure (sequence) provides diagnostic technology
• PCR, RT-PCR.

- The viral proteins are targets for the immune system and can be used to develop vaccines
• vaccines are a means of inducing artificial immunity.
- Can also be used in immunological techniques to identify viral proteins are important in diagnosis = seorlogy → use of antibodies to detect the presence of antigen
- The enzymic functions of some of these proteins can be targets for drugs
• only where the enzymic function is specific to the virus.

4 key steps
1. Transmission from an infected host to a new host
2. Entry into the host;
3. Replication within the host;
4. Release form the host and transmission to the next.

- 2 main modes of transmission
• horizontal i.e. not vertical e.g. rhinovirus;
• vertical: mother-to-child either in utero e.g. rubella or perinatal e.g. CMV.
- Horizontal transmission may be:
• direct, person-to-person = airborne, faecal-oral, via “fomites” (infected materials e.g. bedding), contact (more-or-less intimate, including sexual transmission), exchange of blood.
• Or indirect, involving a vector
usually biting arthropods hence arboviruses (arthropod bourne) which may involve a reservoir.
- Understanding virus transmission is an important step in controlling disease e.g.
• sewage & enteric fevers (John Snow mid 19th C);
• mosquitos & yellow fever (Walter Reed, late 19th C);
• sexual hygiene & HIV (late 20th C);
• bats? & Ebola (early 21st ).

- For entry into the host the virus must pass physical barriers
• chiefly keratinised layer of squamous epithelia or “mucociliary escalator” of other epithelia
• vectors bite through skin and inject virus directly into blood stream e.g. 'warts' papilloma virus won't infect skin unless there is a cut

- Replication within the host may occur
• locally e.g. HPV;
• systemically e.g. measles;
• or in particular organs/tissues (tropism) e.g. polio, hepatitis;
- Dissemination occurs via circulation: virus gains entry from local site of replication either as free virus or carried by migrating leukocytes
• inflammation aids dissemination.
- Release from host depends on site of replication
• gut, respiratory tract, buccal cavity, blood, liver.

- Symptoms of disease are caused by direct damage caused by the virus OR host response – particularly inflammation
- Infection does not necessarily mean disease = many viruses cause no or trivial disease e.g. EBV in young children has no symptoms
- Infection may be
1. Acute: virus either kills the patient quite rapidly
2. “Cleared” by immune system, usually with immunity to re-infection e.g. mumps
3. May be persistent: chronic or latent despite immune responses e.g. HIV. In both cases causing more-or less severe disease
- Persistent infections may result in
• persistent disease (e.g. hepatitis);
• recurrence of same or similar disease (e.g. herpes);
• development of a quite different disease (e.g. HPV 16 = benign initially but can be serious later)

- Replication in tissues causes direct cellular damage & loss of function;
- Inflammatory and immune mechanisms may cause “collateral damage”.

2 kinds of symptoms
1. Non-specific symptoms which are largely immune mediated : headache, fever, lymphadenopathy, malaise, myalgia, rash, arthritis (not all at once!!)
2. Specific symptoms due to damage to target tissues/organs.
- The specific symptoms which occur lead to the definition of syndromes
• gastroenteritis, hepatitis, meningitis etc.
- Note that syndromes can have many different causes: not all respiratory tract infections are due to influenza virus!

- The severity of disease varies with strain of virus and with condition of host
• malnutrition, pregnancy, age, immunosuppression, other underlying disease.
- Compare bacterial diseases: the reasons for varied pathogenicity of viruses are not clear but may relate to target tissue e.g. polio.
- Malnutrition is a major cause of enhanced susceptibility to infectious disease through reduced cellular immunity
• Observed by Jewish physicians in Warsaw 1942 that starving people were anergic to PPD (Heaf test); had little fever or rash when infected e.g. with measles, but invariably died; had lowered blood white cell counts; and allergies disappeared (Heaf test disappeared → cellular immune system disappears).
- This accounts for high mortality to e.g. measles in 3rd world today – a case fatality rate up to 20% cf UK today 0.2%.

- Broadly antiviral mechanisms are
1. Cytotoxic cells to kill virus infected cells prior to production of virus progeny;
2. Antibodies to inactivate extracellular viruses;
3. Cytokines to render cells resistant to infection.
- These mechanisms may be:
• Non-specific i.e effective against all viruses & immediately available
= “innate” immunity: NK cells and some cytokines;
• Specific for particular viruses and requiring time to develop but having “memory” = “adaptive” immunity: CTL and antibody and some cytokines.
- Cytokines produced either by infected cells non-specifically activated by viral components (especially dsRNA) or specifically activated T lymphocytes have three functions:
1. Block virus replication e.g. interferon;
2. Activate inflammation e.g. interleukin 1;
3. Control the immune response e.g. interleukin 2.
- NK cells recognise non-specific changes in cell-surface features due to infection;
- CTL recognise fragments of viral proteins transported to the cell surface by specialised proteins “class I MHC antigens”;
• both kill the infected cell before it can release progeny virus.
- Antibodies bind to virions and prevent their infecting cells
• either by preventing attachment or by preventing the intracellular release of the viral genome from the virion.
- Broadly speaking “cellular” mechanisms clear the acute infection (or not)
• NK, CTL & cytokines;
- Humoral mechanisms” (long-lived antibodies) prevent re-infection (or not)
• IgA in mucosal secretions, IgG in circulation.