Microbiology Flashcards
Describe the Phylogenetic Tree of Life
Bacteria:
Thermotoga,
Green nonsulfur bacteria,
Bacteroides,
Cyanobacteria,
Gram positives
Purple bacteria.
Archaea:
Thermoproteus,
Pyrodictium,
Thermococcales,
Methanococcales,
Methanobacteriales,
Methanomicrobiales
Extreme Halophiles.
Eucarya:
Microsporidia,
Flagellates,
Animalia
Fungi,
Ciliates,
Plantae.
What organisms does the human microbiome involve?
Stomach 10^2: Lactobacillus, Candida, Streptococcus, Heliobacter pylori, Peptostreptococcus
Duodenum 10^2: Streptococcus, Lactobacillus
Jejunm 10^2: Streptococcus, Lactobacillus
Proximal ileum 10^2: Streptococcus, Lactobacillus
Distal ileum 10^8: Clostridium, Streptococcus, Bacteroides, Actinomycinae, Corneybacteria
Colon 10^12: Bacteroides, Clostridium, Bifidobacterium, Enterobateriacae
What are factors affecting development of the microbiome?
Maternal factors: gut microbiome, vaginal infection, periodontitis
Birth: vaginal (lactobacillus) vs caesarean (staphylococcus, propionibacterium) delivery
Postnatal factors: antibiotics (microbiota depletion), breast-feeding (bifidobacterium, lactobacillus), host-genetics (christensenellacae - associated with lower BMI), environment (familial transmission, environmental exposure)
Infant (<1 year): milk consumption (bifidobacterium, lactobacillus, veillonella), solid food introduction (bacteroides, clostridiales)
Toddler (1-3 years): full adult diet (adult-like microbiota)
How does breast milk affect the microbiota?
Rich in human milk oligosaccharides (HMO), but babies cannot digest them.
Bifidobacterium longum infantis contains all the enzymes required,
Releases short chain fatty acids (SFCA), which then provide energy for intestinal cells,
Promotes production of anti-inflammatory molecules.
B. infantis also produces sialic acid required for brain development.
What affects the adult microbiome?
Share microbiota with your household, environment, pets, food;
Altered by antibiotics;
Reduces in variety as we age.
What is the function of the microbiome?
• Energy biogenesis
• Protection from pathogenic bacteria
• Immune system education
• Vitamin production
• Host cell proliferation
• Brain function
• Bile salt metabolism
• Drug metabolism and activity
What is energy biogenesis?
Resistant starch broken down by the microbiome to short chain fatty-acid (SCFAs) by fermentation,
These include butyrate, pyruvate, acetate;
Butyrate main energy source for enterocyte (intestinal lining cell).
SFCAs have anti-inflammatory and anti-tumour properties,
SFCAs stimulate production of protein YY (PYY), which induces satiety.
Resistant starch as a pre-biotic also promotes weight loss.
How does the microbiota provide protection from pathogenic bacteria?
Niche competition and nutrient depletion from invading bacteria:
Complex inter-related niches,
Mucin layer of the gut mucosa heavily colonized and competes for cell surface receptors,
Promotes development of health epithelium.
SFCAs inhibit virulence gene expression and lower pH to below optimal growth conditions.
Microbiota produce bacteriocins. Directly kill Salmonella, Listeria, Clostridium.
What is the gut-brain axis?
Neural connections involving central, autonomic and enteral nervous system;
Strong links between GI and brain function - depression, anxiety and GI symptoms; irritable bowel syndrome;
Potential involvement in CNS disorders - Parkinson’s and Alzheimer disease, Multiple sclerosis.
Adrenergic nerve causes noradrenaline release in gut mucosa, can alter microbiota composition;
This effects:
Afferent nerve cell of vagus nerve/spinal cord carries signals back to brain,
Cytokines released from transmembrane dendritic cells (DC),
5-HT release from entero endocrine cells,
Bacterial molecules (fatty acids, GABA, 5-HT precursors);
Circulating molecules detected by area posterma, or afferent goes to brain directly;
This brings about an effect in the Limbic system, responsible for emotions and stress.
Beneficial molecules produced by bacteria: Oxytocin increase, GABA (gamma amino butyric acid) increase, BDNF (brain-derived neurotrophic factor) decrease, 4-EPS (4-ethylphenylsulfate) decrease, SCFA increase
What are some examples of gut microorganisms and their effect on the gut-brain axis?
Lactobacillus reuteri: increases Oxytocin, affects Vagus nerve - Regulates neuronal plasticity, increases social behaviour, increases oxytocin levels, increases oxytocin neurones.
Lactobacillus rhamnosus: increases GABA, affects Vagus nerve - decreases stress responsiveness, decreases anxiety and depressive-like behaviour, increases Vagal mesenteric nerve firing;
GABA-Aa2 and GABA-B1b altered mRNA expression in mesolimblic brain structures.
Bifidobacterium longum NCC3001: increases BDNF, affects Vagus nerve - decreases Anxiety-like and depressive behaviour, decreases Excitability ENS neurons
Bacteroides fragilis: decreases 4-EPS circulating - decreases anxiety-like behaviour, decreases repetitive behaviour, increases communication
SCFA-producing bacteria: increases SCFA circulating - decreases stress response, decreases anxiety and depressive like behaviour;
Resting microglia undergoes neuroinflammation to activate
GABA = gamma amino butyric acid
BDNF = brain-derived neurotrophic factor
4-EPS = 4-ethylphenylsulfate
What are Microbiome produced secondary bile acids?
Primary bile acids produced in liver from cholesterol metabolism, involved in emulsification of fat. Intestinal bacteria (microbiome) biotransform some of the primary bile acids into secondary bile acids.
Activate cell surface and nuclear hormone receptors on Hepatocytes, Intestinal cells, Inflammatory cells.
Important in maintaining normal health: Reduce gut inflammation, Regulate synthesis of bile acids, Activate vitamin D receptor.
Stimulates glucagon like peptide-1: increases insulin secretion, reduces glucagon secretion.
What is the microbiome role in disease?
Microbiome has important physiological and homeostatic roles. A number of studies has identified associations between altered microbiome and disease:
Obesity and type 2 diabetes mellitus,
Inflammatory bowel disease,
Colon cancer,
Asthma,
Neurodegenerative disease.
What is the effect of the microbiome on obesity?
Many reasons to consider the microbiome to be important in obesity:
Bacteria involved in energy production,
Stimulate production of mediators that alter insulin and glucagon production,
Involved in satiety,
Regulate intestinal integrity and inflammation.
In experiment, lean subjects had more bacteroidetes and weight loss was associated with increased bacteroidetes.
Clear associations, plausible mechanisms and possible treatments. Probiotics shown to lower cholesterol and fasting blood glucose in patients with type 2 diabetes mellitus.
In mouse experiments, mice with microbiome lay down more fat with less food consumption than germ-free mice. Also, germ-free mice inoculated with microbiota from obese or lean human twins take on the microbiota characteristics of the donor.
What is inflammatory bowel disease?
Two major conditions:
Ulcerative colitis - affects the colon,
Crohn’s disease - can affect any part of gut from mouth to anus.
Both have chronic inflammation, relapsing and remitting, usually treated by suppressing the immune system.
Incidence and prevalence appear to be increasing - increasing as countries have increasing wealth, more common in north than south of the globe.
Most common presentation is with abdominal pain, diarrhoea and weight loss.
Perturbations of intestinal microbiome implicated:
Decreased microbial diversity,
Some bacteria are decreased (Firmicutes, some Clostridium species),
Some bacteria are increased (Enterobacteriaceae - including E.Coli, Facultative anaerobes).
Alterations in gut microbiome composition during disease include reduced microbial diversity and expansion of facultative anaerobes due to increased nitrosative and oxidative stress in the gut.
What is treatment of inflammatory bowel disease?
Anti-inflammatory treatment:
Aminosalicylic acid, glucocorticoids;
Anti-tumour necrosis factor.
Enteral nutrition-used in Crohn disease, which will alter microbiome.
Surgery often required.
No routine place for microbiome based therapy such as probiotics.
What are risk factors for colon cancer?
Increased risk with obesity, insulin resistance, increased red meat intake;
Epidemiological studies show increased fibre intake probably protective,
Physical activity is protective.
Possible common mechanisms involving the microbiome or just associations - Microbiome differs in patients in colon cancer:
Seven bacterial species consistently elevated (e.g. Bacteroides fragilis-produces a tumorigenic toxin),
Large number of bacteria are reduced,
Also changes in the virome.
How is the microbiome related to colon cancer?
Microbiome differs in patients in colon cancer:
Seven bacterial species consistently elevated (e.g. Bacteroides fragilis-produces a tumorigenic toxin),
Large number of bacteria are reduced,
Also changes in the virome.
Microbiome protective effects: Production of SFCAs, Phytochemicals metabolized in colon. Both have anti-inflammatory effects.
Microbiome harmful effects:
Fermentation of diet derived protein to phenols, indoles,
N-nitroso compounds also produced which can be carcinogenic,
Ammonia also produced which can damage colonic epithelia,
Secondary bile acids can promote DNA damage.
Metabolic output of the microbiome likely to alter risk of development and progression of colorectal cancer.
What is does microbiome treatment involve?
Microbiome is a complex ecosystem with multiple bacteria, archae, viruses and fungi; Many genes and metabolic pathways; Therapy is at an early stage.
Role of probiotics:
Helicobacter pylori in peptic ulceration;
Clostridioides difficile infection.
Probiotics most commonly contain Lactobacillus and Bifidobacterium - derived from cultured milk sources;
Evidence of health benefit is limited:
Potential benefits in type 2 diabetes control;
Infectious diarrhoea - no evidence of affect on duration of diarrhoea or hospitalisation;
Ulcerative colitis - no evidence that helps treat acute disease or maintain remission;
Neonatal necrotizing enterocolitis (ischaemic damage to intestinal mucosa that occurs in preterm infants) - studies may show a benefit but flawed design, not routinely used.
What is helicobacter pylori infection?
Helicobacter pylori can be found in the gastric mucosa. Has the enzyme urease which breaks down urea to produce ammonia which neutralizes gastric acidity. Attaches to the gastric mucosa.
Most common chronic bacterial infection in humans, Present in early humans, Estimates 50% of human population infected, Most commonly acquired in childhood.
Pathophysiology:
Disrupts gastric mucus layer leading to exposure of mucosa to acidic environment,
Promotes inflammatory immune response,
Causes chronic gastritis (may be asymptomatic, but can lead to peptic ulceration),
Increases the risk of stomach cancer.
What is peptic ulcer disease?
Often asymptomatic but may cause bleeding leading to anaemia.
Upper abdominal pain, indigestion, heartburn. Occasionally they perforate causing severe pain.
What is investigation and treatment for H. Pylori?
Investigation:
Urea breath test - Give carbon-14 labeled urea and detect labelled CO2 in breath,
Stool antigen test,
Endoscopy and biopsy.
Treatment:
Proton pump inhibitor (e.g. lansoprazole) - suppress acid secretion,
Antibiotics 7 day course - Amoxicillin and clarithromycin or metronidazole.
What is Clostridioides difficile Infection?
How do you treat and prevent it?
Causes antibiotic associated colitis. During antibiotic usage C. Diff is resistant and has selective advantage.
Prevention: Antibiotic stewardship - some antibiotics are more likely to cause C. Diff infection (e.g. ciprofloxacin, clindamycin, cephalosporins); Infection control measures.
Treatment:
Most cases respond to antibiotic treatment (e.g. vancomycin or metronidazole),
But some are resistant and/or recurrent;
Faecal microbiota transplantation (FMT) - instillation of processed stool from a healthy donor into the intestinal tract - reserved for patients with recurrent disease, delivering a “healthy microbiome”, can be delivered by oral capsule/nasojejunal or nasoduodenal tube/Enema/Colonoscopy, 91% effective after 8 weeks with repeat FMT.
What are Infections, Pathogens, Pathogenicity, Colonizations, Carriers, Virulence factors, and Virulence?
Infections: present at a body site, causing disease, can be localised or systemic;
Pathogens: can cause disease, primary pathogen can cause disease in healthy individuals, opportunistic pathogen causes disease in certain hosts (e.g. immunocompsomised);
Colonizations: present at a body site, doing no harm, no symptoms, Patients can be ‘carriers’;
Carriers: have colonisation;
Virulence factors: Genes, molecules, or structures that contribute to virulence (may be cell membrane associated, Cytosolic, Excreted);
Virulence: The relative ability of a pathogenic organism to cause disease;
Pathogenicity: the ability to inflict disease on the host.
What is the ‘Iceberg concept of infection’?
Biological response gradient - can’t see everyone infected (no/mild symptoms), only those with severe/moderate symptoms are identified.
When does disease occur?
10 hours unrestricted growth > 1 billion organisms
Speed matters:
Early defence response= no disease,
Late response = disease
What is Staphylococcus aureus?
Causes Superficial ( e.g. skin and soft tissue) infections and Deep seated infections (bacteraemia, endocarditis, osteomyelitis).
Community and hospital acquired infection.
Leading cause of Surgical site infections,
Leads to other hospital acquired infections e.g. bacteraemias,
MRSA through acquisition of resistance genes.
Virulence factors:
Staphylococcal protein A - binds to immunoglobulins and reduces phagocytosis,
Coagulase - activates host prothrombin to convert prothrombin to thrombin and promote clotting of host plasma/blood,
Panton Valentin leukocidin (PVL) (some only) - skin abscesses and nectrotizing pneumonia,
Biofilm - extracellular polysaccharide network allows persistence on prosthetic material,
Clumping factor - mediates clumping and binding to fibrinogen,
Toxic Shock Syndrome Toxin (TSST1/2) (some only) - superactivation of T-cells,
Capsule - resists phagocytosis.
What is Streptococcus pneumoniae?
Colonisation in throat and nasopharynx ->
Mucosal infection (Sinusitis, Otitis Media, Pneumonia) ->
Invasive disease (Bacteraemia, Meningitis)
Virulence factors:
Capsule - Resists phagocytosis,
Pneumolysin - damages epithelium and inhibits mucous-ciliary beating,
Pili - binds to host epithelium,
Hydrogen peroxide - cell damage and inhibits other bacteria,
Neuraminidase - cleaves mucin,
Cytokine production - promotes inflammation.
What is Uropathogenic Escherichia coli?
Ascending bacterial infection of the urinary system.
Virulence factors:
Fimbriae (type 1 and P) - Adhesion to host cell, Biofilm formation, Cytokine induction;
Flagellum - motility;
Lipopolysaccharide (LPS) - colonize bladder and induces cytokines;
Outer membrane vesicles - bud off cell surface to deliver toxins to host;
Capsule - inhibits phagocytosis;
Siderophores - iron uptake system;
Alpha-haemolysin - induces cell lysis.
What is Neisseria meningitidis?
Bacteria: Gram negative cocci,
Carriage: nasopharynx of 5-10% population (Higher carriage rates in school-aged children and young adults),
Transmission: Respiratory droplets,
Diseases: Meningitis, Septicaemia (sepsis),
Virulence factors:
Pili - colonization of nasopharynx,
Lipooligosaccharide (LOS) - mediates toxic effects,
Capsule - inhibits phagocytosis.
Mechanism:
LOS > binds to cells in innate immune system > cytokine release > endothelial damage and capillary leakage
What are hospital acquired infections?
Hospitalized patients at great risk for new infections since greater opportunity for ‘opportunistic pathogens’. Host factors: breach in physical defences, loss of cellular immunity, prosthetic material, loss of microbiome.
Pneumonia - S. pneumoniae initially but also Gram negative bacteria;
Surgical site infections - S. aureus including MRSA but also other pathogens;
Urinary tract infections - E. coli but also other pathogens (e.g. proteus…);
C. difficile infections - loss of microbiome in bowel.
How have microbes establish relationships with
humans?
Humans evolved on a planet dominated by microbes. In the environment, microorganisms live in complex communities, with competitive and non-
competitive interactions, dependencies, and adaptation to the habitat.
Human commensals live in complex communities too: the human microbiome.
Some areas of the body microbial organisms are
not expected to occur under normal circumstances.
Sterile sites: Major organs – Brain, heart, liver; Bone/bone marrow; Joints.
What is the difference between a commensal and a pathogen?
Commensals live within us but do not cause us disease (can be carriers), pathogens cause disease.
In clinical practice distinguishing a commensal from a pathogen is complex.
Any microorganism capable of causing disease is a pathogen; for many commensals disease causation is an accident because it is not required for their evolutionary survival.
Obligate pathogens depend on disease causation for transmission and their evolutionary survival: Mycobacterium tuberculosis.
What are the types of pathogens?
Commensals: live in humans but don’t cause harm,
Obligate: depend on disease causation for transmission and their evolutionary survival (Mycobacterium tuberculosis),
Zoonotic: microorganism that is a colonizer of pathogen in animals, transmitted to humans via direct vector or with direct contact with the animal or its products (Chlamydophila psittaci, Borrelia burgdorferi, Bacillus antracis),
Environmental: microorganism able to cause disease transmitted to humans from an environmental source such as water or soil (Clostridium tetani, Clostridium botulinum)
What are the stages of microbial transmission?
Escape from the host or reservoir,
Transport to the new host,
Entry,
Escape.
What is involved in microbial pathogenesis?
Entry, Niche establishment, Multiply, Spread, Exit the host.
Entry via Adhesins Pili; leads to capsule formation; Detect changes in temp, O2, pH, metal concentrations; Quorum sensing; Small regulatory RNAs; Necrotic death means production of exudates that allows exit and transmission into a new host.
Can spread through the body as blood, lymph + blood, nerves, cerebrospinal fluid.
Portal of exit is the path used by the pathogen to leave the host. Usually corresponds to the site where the pathogen is located.
What are incubation times?
Time to infection with a microorganism to symptom development;
Symptom onset reflects pathogen growth and invasion, excretion of toxins, initiation of host-defense mechanisms;
Range between few hours (food poisoning) to decades (Tuberculosis),
The infected host can be infectious.
S. aureus food poisoning: 8-24 hours,
Bacillus cereus food poisoning: within 24 hours,
Salmonella: 6-48 hours,
SARS-associated coronavirus: 3-10 days,
Varicella-zoster: 10-21 days,
Legionella pneumophila: 7-21 days,
Treponema pallidum: 10-90 days,
Hepatitis A virus: 14-50 days,
HIV: <1 - >15 years
What are strategies for evading host defences?
Concealment of antigens
Intracellular persistence,
Colonizing privileged sites,
Concealment by taking up host membranes or molecules
What are diseases that allow infection more easily?
Diabetes,
Chronic renal disease,
Chronic liver disease,
Chronic obstructive pulmonary disease,
Malignancy,
Immunosuppression: Primary - congenital, Iatrogenic - Chemotherapy, Transplants (solid organ); Acquired - HIV.
What causes UTIs?
E. Coli is most common cause of urinary tract infections:
Lower urinary tract infection (bladder and urethra);
Upper UTI (ascending to ureter/kidneys) - pyelonethritis, renal abscess;
Prostatitis (form ascending or haematogenous infection).
Other Enterobacteriaceae:
Proteus mirabilis associated with renal calculi (urease protuction);
Citrobacter spp., Enterobacter spp., Serratia spp. = associated with antibiotic resistance.
What are the modes of transmission of pathogens?
Direct: Direct contact, Droplet spread;
Indirect: Airborne, Vehicle borne, Vector borne (mechanical or biologic).
What are principles of interventions to prevent transmission of infections?
Directed against the area in the infection chain most susceptible to intervention.
Controlling or eliminating the agent at the source:
Antimicrobial treatment,
Isolating the infected patient,
Soil might be covered;
Mode of transmission:
Water treatment,
Hand washing,
Decontamination of the environment,
Modification of air pressure/quality – filters,
Controlling vectors – mosquitoes;
Portals of entry:
Use of Personal protective equipment (PPE): masks, gloves,
Bed nets in zones of malaria;
Increasing host defenses:
Vaccination,
Prophylactic use of medication - antimalarials,
Herd immunity.
What are the Standard Infection Control Precautions?
Hand hygiene, PPE, Linen management, Equipment, Environment, Patient placement, Occupational Exposure, Respiratory & cough hygiene, Waste management, Blood & body fluid spillage
What are transmission based precautions in infection control?
Clinician:
Investigations,
Antimicrobial treatment
Infection control:
Isolation in health care environment,
Standard Infection Control precautions (SICPS),
Outbreak detection
Public health:
Contact tracing,
Containment in the community,
Post-exposure prophylaxis
Vaccination
What is disease surveillance?
Continuous and systematic collection and analysis of data and subsequent reporting of significant findings to effect change.
Aim: Recognition and initial management of outbreaks.
Examples: Influenza virus, Salmonella, E.coli 0157 outbreaks in food.
What are the primary, secondary, and tertiary aims of disease prevention?
Primary: prevent occurrence of disease by preventing exposure to hazards that cause poor health (Prevention of smoking, Vaccination…)
Secondary: Minimize impact of a poor health outcome that has already occurred (Screening tests at early stages fo disease - Hepatitis C…)
Tertiary: Lessen the impact of a poor health outcome with lasting effects (Early antiretroviral therapy for HIV…)
What are public health actions in disease control?
Aim to contain the infected patient or deal with the source of infection in the community:
Depend on the particular organism,
Isolation of infected individual – exclusion from work during incubation or symptomatic periods,
Environmental assessment, decontamination of sources,
Post-exposure prophylaxis,
Vaccination.
Quaratine: Separating and restriciting movement of individuals exposed to a communicable disease.
Isolation: Separating and restricting movement of individuals suspected or confirmed to be infected with a communicable disease.
How is immunity from infection developed?
Immunity is the ability of the body to protect itself from infectious disease. Can be innate or acquired.
Acquired: specific to a single organism or a group to closely related organisms. Can be active or passive.
Active - involves cellular and antibody responses, produced by patient’s own immune system, long lasting, can be acquired by natural disease or vaccination;
Passive - protection by transfer of antibodies from immune individuals across the placenta or from transfusion of blood or blood products such as immunoglobulin.
How do vaccines work?
Induce active immunity and provide immunological memory;
Antibodies can be detected in blood or serum;
Even in the absence of detectable antibodies, immunological memory may still be present.
Types of vaccines:
Inactivated organisms,
Secreted products,
Recombinant components – cell walls, mRNA
What does clinical management of an infected patient involve?
Establish a diagnosis: Clinical signs and symptoms, Relevant exposures – risk factors, Request appropriate investigations;
Antimicrobial treatment,
Source control,
Infection control precautions to limit spread in the hospital environment,
Public health interventions,
Occupational health interventions
What are the recommendations for routine pneumococcal vaccination?
As part of infant immunisation programme;
Routine older adult programme (>65 years);
Clinical risk groups:
Asplenia,
Chronic respiratory disease,
Chronic heart disease,
Chronic kidney disease,
Chronic liver disease,
Diabetes,
Immunosuppression,
Individuals with cochlear implants,
Individuals with cerebrospinal fluid leaks,
Ocupational risk: exposure to metal fumes - welders
How are UTIs diagnosed?
Urinary tract infection (UTI) commonly results from the presence and the multiplication of bacteria in one or more structures of the urinary tract with consequent tissue invasion.
Can cause variety of clinical syndromes (acute uncomplicated cystitis, acute urethral syndrome, acute pyelonephritis, chronic pyelonephritis, perinephric abscess, renal abscesses, urethritis and prostatitis).
Bacteriuria refers to the presence of bacteria in the urine sample.
Significant bacteriuria is defined as >10^5 of colony-forming units (CFU) of a single species of bacteria per millilitre in a freshly voided midstream sample of urine.
The presence of bacteriuria is NOT synonymous with UTI; the patient MUST have clinical symptoms of infection (dysuria and frequency, urgency of micturition, suprapubic discomfort, haematuria, pyrexia, loin pain and rigours). Asymptomatic bacteriuria is not a UTI and does not usually require antimicrobialtreatment (exception: pregnancy, renal transplant).
Colony count and significance of the culture:
>100,000 colonies/ml of one colony type - significant finding (from result using 1µl loop - plate shows >100 colonies of 1 type),
10,000-100,000 colonies/ml - indeterminate (from result using 1µl loop - plate shows 10-100 colonies of 2+ types),
<10,000 colonies/ml - likely not a significant finding (from result using 1µl loop - plate shows <10 colonies).
How are urine culture results for suspected UTI interpreted?
If using 1µl loop:
Plate shows <10 colonies - No significant growth,
Plate shows 10-100 colonies of two or more types of colonies - Report as mixed organisms with colony count (10,000-100,000 cfu/ml),
Plate shows >100 colonies of one colony type - Report the organism with colony count (>100,000 cfu/ml) and perform sensitivities,
Plate shows >100 colonies of more than two types of
colonies - Report as mixed organisms with the colony count (>100,000 cfu/ml), Add comment (for example “Doubtful significance. Please consult microbiologist if advice is required. Isolate(s) will be held for 7 days.”).
Colony count and significance of the culture:
>100,000 colonies/ml of one colony type - significant finding,
10,000-100,000 colonies/ml - indeterminate,
<10,000 colonies/ml - likely not a significant finding.
Culture media:
MacConkey agar (pink) is a selective medium useful for the culture of coliforms, contains bile salts, lactose, and a pH indicator;
The bile salts inhibit growth of non-intestinal bacteria,
The pH indicator, neutral red, helps to distinguish the lactose-fermenting coliforms from the non-lactose fermenting organisms - some coliforms (for example E. coli) ferment lactose (catabolism), producing acid which acts on the neutral red pH indicator, causing a colour change,
Lactose-fermenting colonies grown on MacConkey agar are therefore pink,
Examples of non-lactose fermenters include Salmonella and dysentery groups.
What causes Bacteraemia (bacteria in the bloodstream) and sepsis in hospitalised patients?
Bacteraemia (bacteria in the bloodstream) and sepsis caused by Gram-negative bacteria are common problems in hospitalised patients.
The cell wall of Gram-negative bacteria contains a component called endotoxin - this is responsible for much of the classical pathophysiology of septic shock; essentially, endotoxin causes immune cells to overproduce huge amounts of cytokines that result in increased vascular permeability and hypotension.
The most encountered Gram-negative bacteria are members of the Enterobacteriaceae (also known as “coliforms”), including Escherichia coli.
Other non-coliform Gram-negative species, such as Pseudomonas aeruginosa, are also important in hospitalised patients.
Pseudomonas aeruginosa is resistant to many of the normally used antimicrobials (e.g. ampicillin) but is usually sensitive to other antimicrobials (including piperacillin-tazobactam, ciprofloxacin and gentamicin). In the laboratory, Pseudomonas aeruginosa is easily recognised - it often forms characteristic flat colonies with a metallic sheen on blood agar, does not ferment lactose on MacConkey agar, and is oxidase test positive.
In hospitalised patients, common sources of Gram-negative bacteraemia include urinary tract infections (most common, particularly in those with a urinary catheter), abdominal infections and hospital-acquired pneumonia.
Initial antimicrobial treatment should be chosen to cover the possible sources, considering previous microbiology results from the patient, your hospital’s antimicrobial prescribing policy, as well as any allergies your patient may have. It is also important to consider the source of the infection and remove it if possible (e.g. if the source is an abscess full of pus this will need surgical drainage, if the source is a urinary catheter - the catheter should be removed if possible or changed if still required.)
Urinary catheters should NOT be used unless there is no alternative for the patient, as they are a definite risk factor for hospital-acquired infection.
What is Antimicrobial resistance?
Since the first widespread clinical use of penicillin in the mid-20th Century, bacteria have developed mechanisms to become resistant to antimicrobials.
Although resistance in Gram positive bacteria, such as Staphylococci (e.g., Methicillin Resistant Staph. aureus - MRSA) is a worry, several new antimicrobials are available that have overcome this resistance - for now.
More worrying at present is the rapid rise in resistance seen in Gram-negative bacteria (e.g. E. coli, Pseudomonas aeruginosa).
In the UK, infections are still usually treatable with antimicrobials, but in some parts of the world infection with bacteria that are resistant to almost all antimicrobials have started to occur. The worry is that this resistance will spread to other parts of the world, including the UK.
With few or no new antimicrobials that work against these resistant Gram-negative bacteria, we may be heading full circle back to an era when some bacterial infections are no longer treatable with antimicrobials.
What is the Mandatory Data Set Requirements for Laboratory Requests?
Mandatory patient demographics on request form:
1. Patient Identifier Number (CHI/PAS)
2. Surname
3. Forename
4. Date of birth
5. Gender
6. Location (for hospital name of ward, clinic… For GP’s this must be the GP practice)
NB. If the patient identifier is unavailable then the MDS includes: First line of patient address, Postcode.
Mandatory information required on the sample:
1. Surname
2. Forename (preferably) or initial
3. Date of birth
4. Sample date/time
Other information required: Consultant or GP, Specimen Type, Clinical details.
What is septic arthritis?
Septic arthritis is the inflammation of a joint, which is caused by microorganisms (mostly bacteria but also other organisms for example fungi).
The signs and symptoms include pain, swelling, redness of the joint and fever.
The organisms most frequently isolated are Staphylococcus aureus and Streptococci.
Investigations must include aspiration of joint fluid and blood cultures prior to the initiation of antimicrobials.
Methicillin-resistant Staphylococcus aureus (MRSA) can also cause septic arthritis.
MRSA is resistant to beta-lactam antimicrobials e.g., flucloxacillin and standard antimicrobial treatment would fail if infection is caused by MRSA.
It is very important to be aware of risk factors that should alert you towards this aetiology like previous history of MRSA colonisation/infection, recent surgery, admission to hospital or nursing home, previous exposure to antimicrobials, renal dialysis, permanent indwelling catheter, skin ulcers and some underlying diseases like diabetes mellitus.
If MRSA is suspected, a glycopeptide antimicrobial (e.g. vancomycin) should be prescribed until identification and susceptibility testing results from the investigations are available.
What is the Staphaurex procedure?
Slide agglutination test for Identification of Staphylococcus aureus:
(You can use the positive and negative control if result is difficult to interpret.)
- Shake the latex to obtain an even suspension and dispense a drop into a circle on the reaction card for each culture to be tested.
- Take a loop and pick up some of the culture by touching it with the flat end of the loop. As a guide, an amount of growth roughly equivalent to 1-2 average-sized colonies should be picked.
- Emulsify the sample of culture in a drop of latex by rubbing with the flat end of the loop. Rub thoroughly, but not too vigorously or the surface of the card may be damaged. Some strains, particularly of species other than S. aureus remain difficult to emulsify and this should be noted, since lumps of unemulsified culture can make the latex appear ‘rough’ or ‘stringy’ on reading. Spread the latex over approximately half the area of the circle. Discard the mixing stick for safe disposal.
- Rotate the card gently and examine for agglutination for approximately 20 seconds, holding the card at normal reading distance (25-35 cm) from the eyes. Do not use a magnifying lens. The patterns obtained are clear-cut and can be recognised under any normal lighting conditions.
A Positive test is indicated by agglutination within 20 seconds.
A Negative test shows no agglutination.
NB An uninterpretable result shows agglutination in the negative control.
What is a genome?
Complete set of genetic informationin an organism,
Provides all the information an organism requires to function,
Genome is stored in long molecules of DNA packaged in chromatin andformed into chromosomes,
In eukaryotic cells, the genome is contained within the nucleus.
What is the basic building block of DNA?
The nucleotide:
5-Carbon sugar - 2-deoxyribose,
Phosphate,
Base - nitrogen containing ring - either pyrimidines (cytosine/thymine {- all got Ys}) or purines (adenine/guanine)
Base pairs: A pairs with T, G pairs with C
(Just the base + sugar is called a nucleoside)
What are chains of DNA and how are they formed?
DNA is a polynucleotide chain.
Sugars are joined by a phosphodiester linkage - linkage is via the 5’ and 3’ groups on the sugar.
Chain has distinct directionality or polarity.
The free 5’ phosphate is the 5’ end of the chain (the start),
The free 3’ OH is the 3’ end of the chain (the end)
How is DNA packed?
DNA is packed into chromosomes by first packing into nucleosomes (beads on a string arrangement).
DNA is associated with proteins: histones & other nonhistone chromosomal proteins, known as chromatin,
First and most fundamental ‘level’ of chromatin packing is the nucleosome,
Histones are DNA-binding proteins (small positively charged proteins). Two each of: H2A, H2B, H3 & H4 (octamer),
Histone tails are significant (+ charge, provide driving force for folding by mediating favorable internucleosomal interactions and screening DNA repulsion),
Histones are highly conserved in evolution - only 2 differences in amino acid sequence between pea and cow (histone H4).
Highly-ordered:
Short region of DNA double helix (2nm),
“Beads-on-a-string” form of chromatin (11nm),
Chromatin fiber of packed nucleosomes (30nm),
Chromatin fiber folded into loops (700nm),
Entire mitotic chromosome (1400nm),
Net result: each DNA molecule has been packaged into a mitotic chromosome that is 10,000-fold shorter than its fully extended length.
What are histones?
Histones are DNA-binding proteins (small positively charged proteins). Two each of: H2A, H2B, H3 & H4 (octamer),
Histone tails are significant (+ charge, provide driving force for folding by mediating favorable internucleosomal interactions and screening DNA repulsion),
Histones are highly conserved in evolution - only 2 differences in amino acid sequence between pea and cow (histone H4)
What is the human karyotype?
23 chromosomes;
46 chromosomes in diploid cells;
3 billion nucleotide (base) pairs;
22 autosomes;
1 pair sex chromosomes;
Average chromosome = 4.8 cm length, 140 million nucleotides per strand = a lot of DNA in a very small space.
How does DNA replication work?
Each strand of a DNA molecule can act as a template for the synthesis of its complementary strand. Base-pairing enables DNA replication.
DNA replication is semiconservative (original strand remains intact through many rounds of replication).
Strands must be separated before replication can begin;
Initiator proteins recognise replication origins & open up the helix locally;
Provides single-stranded templates ready for DNA synthesis.
~10,000 replication origins in human genome
DNA synthesis occurs at replication forks, two forks form at each origin;
Replication proceeds bidirectionally, unzipping the DNA strands as it goes;
Most important enzyme in DNA replication is DNA polymerase.
DNA is synthesised in the 5’ to 3’ direction;
DNA polymerase adds deoxyribonucleotides to the 3’ end of the growing chain;
Added as deoxyribonucleotide triphosphates (dNTPs);
Base-pairing dictates which nucleotide is added;
Energy required for synthesis reaction comes from hydrolysis of the dNTP’s high-energy phosphate bond.
At a replication fork, the two newly synthesised
DNA strands are of opposite polarities - this creates a problem for DNA polymerase, which can only synthesise DNA in a 5’-3’ direction;
Solution: Semidiscontinuous replication;
Leading strand synthesised continuosly,
Lagging strand synthesised discontinuosly - short sections (Okazaki fragments) are subsequently joined together by enzyme DNA ligase.
DNA polymerase can only continue an existing strand, not initiate new ones;
An RNA polymerase known as primase makes RNA primer first (~10 bases long);
DNA polymerase can then extend the RNA chain;
Only one RNA primer needed for leading strand, but lagging strand has continuous requirement;
RNA later removed by nuclease activity.
DNA synthesis is carried out by a group of proteins that act together as a DNA synthesis machine:
Single stranded binding proteins keep the helix unwound,
DNA helicase helps unwind the double helix.
DNA polymerase proofreads its own work:
Replication must be very, very accurate (although mutations must arise at some frequency, or evolution couldn’t occur);
Consequences of errors can be fatal;
Allowing for proofreading, DNA polymerase makes around 1 error in 10^7 bases.
What Mechanisms exist that can correct mistakes made by DNA polymerase?
DNA mismatch repair:
Mismatch repair corrects ~99% of errors made by DNA polymerase;
Mutations in mismatch repair genes predispose to cancer;
Must repair new strand.
Why do we need DNA repair?
Replication errors, UV light, Chemicals, Radiation, Cellular metabolism all lead to DNA damage, so need DNA repair mechanisms.
DNA continuously suffers damage in cells - spontaneous intracellular chemical reactions (eg depurination, deamination), exposure to ultraviolet light can form thymine dimers.
What are the DNA repair pathways?
Many different repair pathways exist, each recognising and correcting a specific type of damage.
Most often, information in the undamaged strand is used to correct the damaged strand;
Repair relies on the redundancy of information, if one strand is damaged, the encoded information is not lost.
What is Xeroderma pigmentosum?
Genetic defect:
Affected individuals cannot repair thymine dimers,
Leads to severe skin lesions, including skin cancer.
What is the difference between mitochondrial and nuclear DNA?
Mitochondrial:
Circular,
16,569 base pairs,
37 genes - 2 rRNAs, 22 tRNAs and 13 core subunits of the cellular respiratory chain that drives oxidative phosphorylation (ATP),
Genome is not enveloped and not packaged into chromatin,
Inheritance is strictly maternal,
Replication via DNA polymerase γ
Nuclear:
Linear,
3.3 billion base pairs,
~21,000 genes,
Genome is enveloped and packaged into chromatin,
Inheritance equal from both parents.
Why do we need proteins?
Genes contain information to make proteins;
Proteins:
Serve as building blocks for cell structures,
Form enzymes to catalyze the cells chemical reactions,
Regulate activity of genes,
Enable the cells to move and communicate with each other.
What’s the difference between DNA and RNA?
DNA:
Very long - many genes (250 million nucleotides),
Double stranded,
‘Simple’ 3-D structure (double helix),
Chemically stable,
Contains deoxyribose,
Contains thymine
RNA:
Short - copy of a single gene (500-2000 nucleotides),
Single stranded,
Complex 3-D structure (more like protein),
Less stable,
Contains ribose,
Contains uracil in place of thymine
What is transcription?
Transcription produces an RNA copy of the coding strand of DNA (except that it contains U, not T)
What part of DNA is mRNA synthesised from?
DNA has a coding (sense) strand and noncoding (template, antisense) strand. The non-coding strand of DNA provides the template for mRNA synthesis.
How is transcription carried out?
DNA has a coding (sense) strand and noncoding (template, antisense) strand. The non-coding strand of DNA provides the template for mRNA synthesis.
Transcription is carried out by RNA polymerase. Three types; mRNA is made by RNA polymerase II:
Makes an RNA copy of one DNA strand (= mRNA),
Requires a DNA template and activated precursors (nucleoside triphosphates - ATP, GTP, UTP and CTP),
Synthesises in 5’ - 3’ direction,
Does not require a primer,
Error rate ~1 in 10^4,
Many RNA polymerases can transcribe a gene at the same time
What is control of gene expression?
Although all cells in a human contain the same set of genes, each different cell type expresses a different complement of genes.
Genes may be either on (expressed) or off in a given cell - not all genes are expressed in all tissues at all times - gene expression is highly controlled.
Genes may be expressed in a: tissue-specific pattern; developmentally regulated pattern; combination of these; “on” all the time, in all tissues (constitutive - e.g. housekeeping genes).
How is gene expression controlled?
There are 6 possible control arenas to modulate/control gene expression:
Control of the activation of gene structure (open chromatin/euchromatin) - promoters,
Control of the initiation of transcription and transcriptional elongation - enhancers,
Processing the RNA transcript,
Transport of mRNA to the cytoplasm from the nucleus,
Translation of mRNA,
Degradation and turnover of RNA.
Promoters and enhancers:
A promoter is made up of the sequence elements found immediately 5’ to the gene that interact with RNA polymerase & other components of the transcription machinery;
Enhancers increase transcription from a nearby gene but can operate over considerable distances;
Promoters and enhancers contain sequences to which transcription factors specifically bind.
What are transcription factors?
Transcription factors are proteins which bind to specific DNA sequences (upstream/downstream to RNA polymerase complex) within the promoter or enhancers so as to increase or decrease gene expression
How is expression of a single gene controlled to give different products?
One gene may encode more than one product
Expression of a single gene can be controlled at various levels to give different products:
• alternative promoters
• alternative splicing
• alternative polyadenylation
What is splicing?
Most eukaryotic genes are discontinuous, being split into exons and introns - exons contain coding sequences; introns (‘intervening sequences’) are found between exons.
Introns are removed from the primary transcript by splicing to give a functional RNA molecule.
Alternative splicing means can express >1 product from one single gene (e.g. the alpha-tropomyosin gene can be spliced in many different ways)
What happens between transcription and translation?
Transcription occurs in the nucleus;
Translation occurs in cytosol.
Before RNA can be exported from the nucleus it must go through RNA processing steps:
• Slicing
• Capping
• Polyadenylation
What is RNA capping?
Modifies the 5’ end of a RNA transcript.
The RNA cap includes an atypical nucleotide: a guanine nucleotide that has a methyl group attached to the 5’ end of the RNA in an unusual way.
What is Polyadenylation?
A polyA tail is a string of adenylate residues added to the 3’ end of an mRNA.
Not found on rRNAs or tRNAs;
Transcription proceeds past polyA site, transcript is cleaved, the polyadenylated (i.e. termination of transcription is distinct from polyadenylation).
What are the 5’ and 3’ untranslated regions that are included in the mRNA?
5’ untranslated (5’UTR) is the region of an mRNA that is found upstream of the translated region;
Function of 5’UTRs is mostly unclear, may affect translational control.
3’ untranslated (3’UTR) is the region of an mRNA that is found downstream of the translated region;
3’UTRs can determine the stability of the mRNA.
What are the three major classes of RNA?
mRNA: messenger RNA, encodes proteins
tRNA: transfer RNA, adaptor molecules
rRNA: ribosomal RNA, component of ribosome
Three major classes of RNA, all 3 encoded by DNA, not all RNAs encode proteins.
What are non-coding RNAs?
Majority of genes specify amino acid sequences;
Final products of other genes is the RNA itself: non-coding RNAs.
mRNA (messenger RNA) encodes proteins,
rRNA (ribosomal RNA) form core of ribosome structure and catalyse protein synthesis,
miRNAs (micro RNAs) regulate gene expression,
tRNA (transfer RNA) serve as adaptors between mRNA and amino acids during protein synthesis,
Other noncoding RNAs are used in RNA splicing, gene regulation, telomere regulation…
What are circular RNA species?
Class of RNA molecules with closed loops,
High stability,
Abundantly expressed in eukaryotic organisms.
What is the polymerase chain reaction?
Based on DNA replication, in vitro.
Applications include genotyping– e.g. to detect mutations in the DNA of patients affected by a genetic disorder.
Get region of double stranded DNA to be amplified, then heat to separate strands, cool to anneal primers (+ pair of primers), allow for DNA synthesis (+ DNA polymerase + dATP + dGTP + dCTP + dTTP). And you will get the products of the first cycle.
Products of first cycle is 2 double-stranded DNA molecules,
Products of second cycle is 4 double-stranded DNA molecules,
Products of third cycle is 8 double-stranded DNA molecules…
What is DNA?
Deoxyribonucleic acid is a nucleic acid polymer, formed by two polynucleotide chain strands usually arranged in adouble-helix,
The nucleotides are joined to one another by a covalent (phosphodiester) bond;
The individual bases of the two separate polynucleotide strands are associated with each other according to the rules ofcomplementary base pairing (a pyrimidine always binds to a purine): adenine with thymine via two hydrogen bonds and cytosine with guanine via three hydrogen bonds to form double stranded DNA;
Both strands of double-stranded DNA store information that is complementary.
It has a direction: the phosphodiester bonds form between the third and the fifth carbon of adjacent deoxyribose sugar molecules. Therefore, any DNA strand normally has one end (the 5’ end) where there is a terminal phosphate group attached to the 5′ carbon of a ribose (the 5′ phosphoryl); while at the other end of the chain (the 3’ end), there is a free hydroxyl group attached to the 3′ carbon of a ribose (the 3′ hydroxyl).
The 3’-hydroxyl is one of the most important groups in all biology: in a DNA double helix, the two complementary strands of DNA run in opposite directions to each other (anti-parallel); each complementary strand runs from 5’ to 3’ but in opposite directions in the helix.
What is involved in the cell cycle?
Interphase (most time spent here):
The cell grows and copies its DNA;
G1 - Cell growth,
S: DNA synthesis,
G2: More growth, preparation for mitosis
Mitosis:
The cell divides its DNA and cytoplasm, forming two new cells;
Prophase,
Metaphase,
Anaphase,
Telophase
G0: Resting state where the cell performs its functions and is not preparing to divide (kinda like a pause G1 stage)
Checkpoints:
G1 checkpoint (restriction) - at end of first growth phase of interphase,
G2 checkpoint - at end of second cell growth phase in interphase,
M checkpoint - just after metaphase in mitosis
Control:
Cyclin D CDK4 - start of G1,
Cyclin E CDK2 - middle/end of G1,
Cyclin A CDK2 - during S,
Cyclin B CDK1 - between G2 and Mitosis
How is DNA ‘primed’ for replication?
DNA replication occurs in S phase of cell cycle;
Before DNA replication can begin the super-coiled DNA in the chromosome must be relaxed, this occurs in segments and begins at multiple origin of replication sequences (and requires the action of topoisomerase) to transiently separate the two strands of the parental DNA to create a ‘replication bubble’.
The enzymes that can synthesise the new DNA strand from a template are called DNA polymerases (and in all eukaryotes and prokaryotes, different DNA polymerases share the same fundamental type of synthetic activity (anti-parallel synthesis in the 5’ to 3’ direction).
DNA polymerases can only elongate a DNA strand, not initiate synthesis, so a primer (providing the critical 3’OH group) is needed to initiate DNA synthesis;
This function is provided by a special RNA polymerase (called a primase) which synthesis a short complementary RNA chain that provides the 3’OH priming end from the DNA template (both the lagging strand and the leading strands require primers).
What is semi-conservative replication?
In semi-conservative replication, each parental strand of DNA serves as the template for the synthesis of a new strand so that, eventually, the old parental duplex is replaced by two parental duplexes (each formed by one parental strand and one newly-synthesised strand).
The enzymes that can synthesise the new DNA strand from a template are called DNA polymerases (and in all eukaryotes and prokaryotes, different DNA polymerases share the same fundamental type of synthetic activity (anti-parallel synthesis in the 5’ to 3’ direction).
What enzymes are most important in DNA replication?
Most important enzymes in DNA replication are the DNA polymerases (nuclear DNA replication in eukaryotes requires multiple proteins including three DNA polymerases: α {alpha}, δ {delta}, ε {epsilon});
Two forks form at each origin; replication proceeds bi-directionally, unzipping the DNA strands as it goes
What direction is DNA synthesised?
DNA is synthesised in the 5’ to 3’ direction.
DNA polymerase (as part of a large multi-protein ‘holo-enzyme’ complex adds deoxyribonucleotides to the 3’ end of the growing chain);
Added as deoxyribonucleotide triphosphates (dNTPs).
Base-pairing dictates which nucleotide is added; the energy required for the synthesis reaction comes from hydrolysis of the dNTP’s high-energy phosphate bond.
But the fork exposing more 5’ creates a problem for DNA polymerase, which can only synthesise DNA in the 5’ to 3 direction - solution is semi-discontinuous replication.
How does semi-discontinuous replication work?
DNA polymerase a/primase binds to the initiation complex at the origin of replication and synthesizes a short strand (of approximately 10 bases of RNA followed by 20 to 30 bases of DNA) - forming a different primer on both the leading strand and the lagging strand.
DNA polymerase a/primase is then replaced by a different DNA polymerase which will extend the DNA chain from the 3’-OH - DNA polymerase e on the leading strand and DNA polymerase d on the lagging strand (polymerase-switch).
The leading strand at each replication fork is synthesised continuously (requiresonly one primer) while the lagging strand of each replication fork requires a series of initiation events/primers and is synthesised discontinuously (requiring multiple primers)
(But the replication fork moves)
What are Okazaki fragments?
Fragments formed by semi-discontinuous replication on the lagging strand of the DNA fork.
1000-2000 bases in length;
Synthesis of the next upstream Okazaki fragment displaces the original RNA primer,
An enzyme (flap endonuclease 1, FEN1) cleaves the RNA primer,
All of the fragments are joined together by the enzyme DNA ligase.
What is Lynch Syndrome?
‘Hereditary non-polyposis colorectal cancer’ syndrome.
Most colo-rectal cancers are sporadic but inherited cancer syndromes account for 5-10%, most commonof these is Lynch syndrome:
The individual has an 80% life-time risk of colo-rectal cancer and a 60% risk, if female, of endometrial cancer;
There is also an increased risk of malignancy at other sites (e.g. stomach, ovary, small intestine).
Lynch syndrome is due to a germline mutation in one of 4 mismatch repair genes: MLH1, MSH6, PMS2 and MSH2,
These proteins are necessary for repairing incorrectly-paired nucleotide bases during DNA replication;
Individuals with Lynch syndrome have one function alallele and one non-functional allele of the pertinent DNA repair gene;
Their risk of cancer increases when the previously functional allele acquires a mutation that inactivates its function;
Lynch syndrome can be inherited as autosomal dominant;
But acquired mutations can also occur sporadically, in both alleles of the pertinent genes, in individuals without an inherited pre-disposition.
What is Apopotosis?
Tightly-regulated process of cell death (‘programmed cell death’) in multi-cellular organisms,
Almost all of the genes involved in its regulation are alternatively spliced: the different forms can exhibit opposite functions: pro-apoptosis vs anti-apoptosis.
It can be initiated via two pathways:
Intrinsic pathway - due to intrinsic cell stress, including DNA damage, and
Extrinsic pathway - because of signals from other cells;
Both pathways, eventually, induce cell death by activating proteases called caspases (initiator and execution caspases) that lead to cell death by indiscriminate protein degradation.
What happens if apoptosis is defective?
Defective apoptosis is associated with pathological processes as the death of a cell with too high a DNA-damage burden may not occur.
Mutations in certain apoptosis-associated genes can be seen in neoplastic cells…
e.g. BCL2 (an anti-apoptotic gene): if over-expressed (follicular lymphoma) it leads to a shift in the sensitivity of the cell to apoptotic stimuli (apoptosis evasion),
e.g. p53 (a transcription factor that influences the regulation of >900 genes in many processes, including apoptosis): has a complex role in both the intrinsic and extrinsic apoptotic pathways. If the gene encoding p53 (TP53) is damaged, tumour suppression is severely compromised; individuals who inherit only one functional copy of the TP53 gene have a high risk of neoplasia in early adulthood (Li-Fraumeni syndrome).
What is a gene?
For DNA-based organisms, a gene could be considered to be the part of a DNA nucleotide sequence which is copied (transcribed) into a corresponding RNA nucleotide sequence that either encodes a functional protein (if the transcript is mRNA) or a functional structural RNA (eg. tRNA or rRNA).
The sequence of a particular gene can differ between individuals of the same species. These variants are known as alleles and can be associated with different phenotypic traits (because they can encode slightly different proteins).
What is an allele?
A variant in the sequence of nucleotides, at a particular location (locus), in a DNA molecule.
Alleles can differ at a single position through a single nucleotide polymorphism (SNP) or through larger insertions or deletions.
Different alleles often result in little or no change in function of the gene product encoded but sometimes they can result in a different phenotype.
In diploid organisms, every cell in an individual has two full sets of somatic chromosomes, meaning that there are two alleles for any gene (one is inherited from each parent);
If these two alleles are the same = homozygous,
If these two alleles are different = heterozygous.
What is the genotype and phenotype?
Genotype is the complete set of genetic material;
Phenotype is the observable traits and characteristics of an organism/individual.
The genotype contributes to, and can significantly determine, the phenotype but the environment is also influential in the phenotype.
Some alleles are ‘dominant’ over others and, therefore, in certain scenarios, it is possible to have a different genotype but the same phenotype.
How do DNA sequences vary between individuals?
DNA sequences vary considerably between individuals: these variations are sometimes described as mutations and sometimes as polymorphisms.
A mutation is any change in a DNA sequence different to ‘normal’ - this implies that there is a normal allele (wild-type) that is prevalent in the population and that the mutation changes this to a rare and abnormal variant.
A polymorphism is a DNA-sequence variation that is, in contrast, ‘common’ in the population (and, in this case, may be no single allele is regarded as the standard ‘normal’ sequence).
The arbitrary cut-off-point between a mutation and apolymorphism is 1% (i.e. to be classed as a polymorphism, the least common allele must have a frequency of 1% per or more in the population); if that frequency is lower than this, then that allele is regarded to represent a mutation.
However, a rare disease allele in one population can become a polymorphism in another if it confers a selective advantage and, as a consequence, increases in frequency in that population.
E.g. sickle-cell disease occurs when codon 6 of the beta globin gene is changed from GAG to GTG, causing a Glutmate to Valine substitution (E6V); the haemoglobin produced is referred to as Haemoglobin S. In Caucasian populations this is a rare sequence variant of the beta-globin gene but in certain parts of Africa (etc.), the same allele is polymorphic because it is much more common, as it confers some resistance to blood-borne malaria parasites.
What is chromatin?
DNA is packaged in chromatin; chromatin has a compact organisation in which most DNA sequences are structurally-inaccessible and functionally-inactive.
The fundamental subunit of chromatin has the same type of design in all eukaryotes:
The nucleosome is formed by a core histone octamer - there are two copies of each of the small basic histone proteins H2A, H2B, H3 & H4 organised as an H3 2 - H4 2 octamer and two H2A-H2B dimers;
These are associated with about 145-147 bp of DNA wrapped around the outside of the octamer,
This is further packaged.
How is transcription related to the structure of chromatin?
Gene expression is mainly controlled at the level of the initiation of transcription; this is associated with the opening of chromatin (open chromatin = euchromatin), histone H3 & H4 acetylation and CpG-island demethylation.
Active or potentially-active (‘poised’) genes are normally found in open chromatin.
In contrast, heterochromatin is usually associated with inactive genes, where there is methylation of lysine residues on histone H3 and methylation of CpG islands in the upstream DNA-regulatory sequences of the gene.
How do promoters work?
RNA polymerases have about 12 subunits.
No RNA polymerase recognises a promoter directly - first, the chromatin must be ‘opened’.
An RNA polymerase II promoter consists of a variety of short sequence elements in the region of the transcriptional start site;
Each of these elements is bound by one or more transcription factors including multiple ‘basal’ transcription factors (which include members of the TFII family);
These forma pre-initiation complex, assemble at the promoter and provide a target for RNA polymerase II.
A unifying principle is that transcription factors have primary responsibility for recognising and binding many of the characteristic regulatory sequences in a promoter and they, in turn, serve to bind RNA polymerase II and position it correctly at the transcriptional start point.
How does RNA splicing and processing work?
RNA is the central player in gene expression; all RNAs require processing of the primary transcript to become functional and mature.
The 5’ end of eukaryotic mRNA is capped, during transcription, by the addition of G residue, by guanylyl transferase; this is subsequently methylated.
The cap has an influences: mRNA stability, mRNA splicing, mRNA transport and mRNA translation.
A typical gene has many introns and these are removed from the RNA transcript by splicing (RNA splicing).
Most introns are associated with a GU…AG consensus.
The mRNA is spliced by the spliceosome (which has over 100 proteins).
The transcription and splicing machinery are physically and functionally-integrated, therefore, transcription and RNA processing are highly coordinated in multi-cellular eukaryotes.
3’ end processing: the addition of a poly A tail (poly-adenylation) influences mRNA stability by protecting against 3’ to 5’ exonuclease degradation, signals the termination of RNA polymerase II transcription, and also influences translation efficiency.
What is alternative splicing?
Alternative splicing results in the production of mRNAs with different sequences (even although they have been generated from the same RNA transcript); when these are translated different proteins (or variations on the same protein) can be produced.
Multiple gene products can therefore be produced from the same locus (structural diversity) and possibly more than 90% of genes in mammals are differentially spliced.
What are codons?
An mRNA transcript carries a sequence that can be divided into groups of 3 (each group of 3 is a codon and specifies a specific amino acid in a polypeptide).
The codons interact with the anti-codons on the tRNA component of an amino-acyl tRNA.
There are 64 possible codons (4^3);
61/64 codons specify for one of the 20 amino acids;
3/64 are STOP codons (terminate translation);
The 61 amino acid-specifying codons are each recognised by a specific aminoacyl-tRNA with an anti-codon complementary to the codon and carry the amino acid specified by that codon.
The order of the codons in the mRNA determines the order of amino acids incorporated into a polypeptide chain.
Almost all amino acids are coded for by more than one codon except methionine (MET, M) and tryptophan (TRP, W);
As a consequence, there is intrinsic redundancy to the genetic code;
In contrast to the 61 codons specifying an amino acid, the 3 termination codons are each recognised by protein release factors.
What are the special codons?
AUG = start (Met, M),
UAA = stop (ochre),
UGA = stop (opal),
UAG = stop (amber)
How are codons read?
Code is read from 5’-3’,
An mRNA can be translated in three different reading frames,
Critical that translation starts at the correct point and in the correct reading-frame.
How is translation initiated?
Most mRNAs need a 5’cap to be translated efficiently (there are some exceptions e.g. polio virus RNAs are uncapped).
Translation occurs in three stages (and different sets of multiple accessory proteins are required to assist the ribosome at each of these stages).
Initiation:
Prior to the start of translation, the pre-initiation complex (PIC) scans the mRNA for the translational start site;
The PIC consists of the 40S ribosome bound to elongation initiation factor 2 (eiF2), GTP and the initiator MET-tRNA;
Multiple other proteins bind the PIC and aid in its binding to the m7G 5’ cap;
This protein complex scans the mRNA until it reaches the AUG initiation codon (this is usually the first AUG but it is also part of a longer specialised sequence);
To eukaryotes, the Kozak (Consensus) sequence usually functions as the site of mRNA translation initiation - the AUG codon is recognised, and bound by, the the anti-codon of the aminoacyl-initiator-tRNA (MET-tRNA);
This binding leads to a structural re-arrangement that results in the PIC binding the large 60S ribosome (to form the ribosomal complex, 80S);
Once the 80S ribosomal complex is formed the elongation phase of translation begins.
What is the elongation stage of translation?
Elongation: all the reactions involved from the formation of the first peptide bond to the addition of the last amino acid; amino acids are added one-at-a-time and in the order specified by the mRNA sequence.
tRNAs (transfer RNAs) are the key adaptors required for protein synthesis.
