Microbiology Flashcards
The Human Microbiome
- The microbiome is the genetic material of all the microbes- bacteria, fungi, Protozoa, and viruses- that live on and inside the human body.
- The number of genes in all the microbes in one person’s microbiome is 200 times the number of genes in the human genome.
Key features of microbes
• Boundary -Barrier from the environment. -Cell wall (in some), membrane • Cytoplasm -Aqueous mixture of macromolecules -Proteins, lipids, nucleic acids polysaccharides, other organic and inorganic molecules. -Organelles (in some) • Transport requirements -Nutrients in, products out. -Membrane permeability and mechanisms of transport.
Fungi
- Eukaryotes
- Candida: a yeast- unicellular, reproduce by budding
- Aspergillus: mould- multicellular, reproduce by spores
Protozoa
Huge family of single-celled eukaryotic parasites. Major tropical and zoonotic diseases.
Helminths
- Huge family of single celled eukaryotic parasites.
- Major tropical and zoonotic diseases
Bacterial morphology
-Shape: Round (coccus, cocci), Long (bacillus/bacilli), A few are spiral/branched (filamentous), comma shaped
Gram positive and Gram negative
Based on the ability to take up the stain based on the thickness and accessbility of cell wall peptidoglycans.
Gram-pog: cell wall with peptidoglycan so turns out purple
Gram neg: thin peptidoglycan layer and an outer membrane. turns out pink
Bacterial growth requirements-physical:
• Temperature
• PH
• Salt content
All used for selection of bacteria in the lab.
Peptidoglycan structure and synthesis
A 3D polymer
• N-acetylated sugars
-glucosamine (NAG) and muramic acid (NAM)
And
• 3-5 amino acid peptides
-AA s peculiar to peptidoglycans- resistant to enzymatic destruction
• Cross-linked by transpeptidase enzymes
Synthetic pathways- unique to bacteria. • Polymerisation of sugars -To make the back bone • Elongation of aa side-chains -To add the peptides • Transpeptidase -To cross link
Mycobacteria
- Basically a Gram positive cell wall
- Dont stain Gram positive
- Very thick lipid membrane (mycolic acid mycomembrane) anchored to peptidoglycan layer- intracellular survival
Other important features of bacteria
• Capsule
-Polysaccharide coat
-‘hides’ immunogenic cell wall
-immunity requires antibodies to the capsule
-metabolic burden on the bacterium
-confers virulence e.g., haemophilia influenzae
• Ribosomes
-engines of protein synthesis
-70S (sedimentation rate): smaller than in eukaryotes 80S
-Subunits 50S and 30S
-Each contains RNA and proteins
-Bacterial RNA: target antibiotics and diagnostic tests
• Mobile genetic elements
• Spores
• Gene regulation
Mobile genetic elements: Plasmids vs Transposons
Plasmids: -circular ‘extra-chromosal’ DNA -independently replicating -present in many bacteria -can code for dozens of genes -like viruses: passes down to progeny, some transmitted between bacteria.
Transposons
-DNA sequences that are able to move location in the genome. -Encode transposase -Plus other genes -Mobile between: genomic and plasmid DNA, plsmids, plasmids and genomic DNA.
Code for toxins and antibiotics resistance genes.
Glycopeptides
Vancomycin, Teicoplanin, Telavancin- only active in G+
Used (intravenous) for serious gram pos organisms which produce beta-lactamases or are not responding to other treatments
Oral: not absorbed but used to treat Clostridium difficile anaerobic) associated with diarrhoea (vancomycin)
Important: Nephrotoxicity (renal toxicity)
Metronidazole
- Prodrug: only anaerobic organisms can metabolite to its active form
- Metabolites produced are toxic to DNA-bactericidal
- Considered potentially mutagenic, carcinogenic and teratogenic.
Rifampicin
-Bactericidal-Mycobateria (M.tuberculosis, M.leprae)
-Binds to RNA polymerase- inhibits mRNA synthesis
Metabolic interactions: strong induction of CP450, Orange colour: saliva, tears, and sweat
Resistance Mechanisms
- Inactivation or modification of the antibiotic
- Alteration of the microbial enzymes that transform pro-drugs to the effective moieties
- Alteration of the target
- Reduced uptake of the antibiotics
- Enhanced export of the antibiotic (efflux pumps)
- Development of alternative pathways
Ribosomes of bacteria
- engines of protein synthesis.
- 70S (sedimentation rate): smaller than in eukaryotes.
- subunits 50S and 30S
- each contains RNA and proteins.
- bacteria RNA: target for antibiotics and diagnostics.
Bacteria that produce spores?
Clotridia and Bacillus.
- Non replicating dormant form.
- Resistant to drying, temperature, disinfection, digestion.
- Important in clinical disease pattern, infection control.
Gene regulation-bacterial growth
- The lag phase: no increase in cell numbers, adjustment to new environment, gene regulation
- The exponential phase: cell doubling, slope of the curve= growth rate environment.
- The stationary phase: nutrients become depleted, metabolites build up, division stops, gene regulation
- The death phase: -exhaustion of resourced, toxicity of environment.
Virus latency
Virus lies dormant (latent) within a cell
Viral pathogenesis
a process by which viral infection results in disease
two components of viral disease
- effects of virus replication on the host
- effects of host reponse on the virus and host.
three requirements for a successful infection
- Enough virus
- Cells accessible, susceptible, permissive
- Local antiviral defence absent or overcome
horizontal transmission
between members of the same species (zoonotic different species)
Latrogenic transmission
activity of health care worker leads to infection of patient
nosocomial transmission
when an indiivdual is infected whilst in hospital or healthcare facility
vertical transmission
transfer of infection between parent and offspring.
Germ line transmission
agent is transmission as part of the genome, e.g., proviral DNA
Respiratory tract affected by virus
- most common route of viral entry
- barriers to infection: swallowing, ciliary acion from lower tract, macrophages in alveoli (no cilia or mucous)
- can enter by aeorosolised droplets from cough or sneeze or contact with saliva
- large droplets lodge in nose; smaller in airways or alveoli
Ailmentary tract affected by virus
- eating, drinking, social activities introduce viruses into the ailmentary tract
- designed to mix, digest, absorb food, so content are always in motion, good opportunities for virus-cell interactions.
- extremely hostile environment: stomach is acidic, intestine is alkaline, presence of digestive enzymes, bile detergents, mucous, antibodies, phagocytic cells.
- viruses have evolved to infect are resistant, e.g., enteroviruses, reoviruses
Urogenital tract affected by virus
- Protected by mucous, low pH
- Minute abrasions from sexual activity may allow viruses to enter.
- Some viruses produce local lesions (HPV)
- Some viruses spread from urogenital tract (HIV, HSV)
Eye
- Sclera and conjunctiva are route of entry
- Every few seconds, eyelid passes over sclera, washing away foreign particles; little oportunity for infection
- Infection usually occurs after injury: grit, opthalmologic procedures, improperly sanitised swimming pools
- Localised infection:conjunctivitis
- Disseminated infection: enterovirus 70 spreads to CNS
- HSV-1 can infect cornea, blindness may result, virus spread to sensory ganglia.
Skin
- Outer layer of dead, keratinised cells cannot support viral infection; entry usually occurs by breaks or punctures
- Skin abrasions; insect or animal bites; needle punctures
- Epidermis is devoid of blood or lymphatics; local replication
- Dermis and sub-dermal tissues are highly vascularised; infection may spread.
Viral spread
After replication at the site of entry, viruses may remain localised; virus spreads within the epithelium and is contained by tissue structure and immune system.
- Some viruses spread beyond the primary site: disseminated, if many organs are infected, systemic.
- Physical and immune barriers must be breached.
- Below the epithelium is the basement membrane; integrity can be compromised by epithelial inflammation and destruction.
- Below basement membrane are subepithelial tissues, where virus encounters tissue fluids, lymphatic system, and phagocytes, all play roles in clearing and spreading infection.
- Role of directional release of virus from polarised cells e.g., epithelial cells, neurons, etc.
Hematogenous spread of virus
- Viruses that produce disseminated infection often do so by entering the blood.
- Viruses may enter blood directly through capillaries, by replicating in endothelial cells, or through vector bite.
- Viruses in the extracellular fluids is taken by the lymphatic capillaries, which are more permeable than circulatory capillaries, then spread to the blood.
- Once in the blood, the virus has access to almost every tissue in the body.
Viremia
• Presence of infectious virus in the blood
Active vs Passive
- Active viremia: results from virus replication (measles)
* Passive viremia: results from virus introduced into the blood without replication (Dengue virus)
Neural spread of viruses
- Many viruses spread from primary site of infection by entering local nerve endings.
- For some viruses (rabies, alpha herpesviruses) neural spread is definitive characteristic of pathogenesis.
- For other viruses, invasion of the CNS is an infrequent diversion from normal replication and hematogenous spread.
Neurovirulent virus
Can cause disease of nervous tissue.
Tissue tropism
- the spectrum of tissues infected by virus, e.g., an enteric virus, replicated in the gut and not in the lung; a neurotropic virus replicates in cells of the nervous system and not in hematopoeitic cells.
- The tropism of some viruses is limites; other viruses are pan tropic, e.g, can replicate in many organs.
viral virulence and how can it be quantified.
- the capacity of a virus to cause disease in an infected host.
- a virulent virus causes significant disease, while an a virulent or attenuated virus causes reduced or not disease.
- can be quantified: LD50 (lethal dose 50%- amt of virus needed to kill 50% of infected animals), the mean time of death, the mean time to appearance of symptoms, measurement of fever or weight loss, measurement of pathological lesions (poliovirus), reduction in blood CD4 + lymphocytes (HIV-1)
What makes viruses virulent?
• A major goal of virology is to identify viral and host genes that determine virulence.
• Virulence genes are usually identifies by mutation: deletion or disruption of one of these genes results in a virus that causes reduced or no disease in a specified system
• Viral genes affecting virulence fall into four classes:
-those that affect the ability of the virus to replicate
-those that modify the host’s defence mechanisms
-those that enable the virus to spread in the host
-those which have intrinsic cell killing effects
virokines
secreted proteins that mimic cytokines, growth factors, or similar extracellular immune regulators
Viroreceptors
homologs of host receptors or cell surface immune molecules
Toxic viral proteins (NSP4)
- NSP4 non structural glycoprotein of rotaviruses: a viral enterotoxin
- When expressed in cells, causes increase in intracellular calcium
- When fed to young mice, causes diarrhoea by potentiating chloride secretion. Thus, NSP4 triggers a signal transduction pathway in intestinal mucosa.
Virulence factors:
Molecules produced by pathogens (bacteria, viruses, fungi…) to help them: • Colonise the host • Evade the host’s immune system • Suppression of host’s immune system • Extr/exit in and out of the cells • Obtain nutrition from host
Virus Host interactions:
• As obligate intracellular parasites restricted by limited genomic capacities, all viruses have evolved to hijack host factors to facilitate their replication
• Meanwhile, host cells have also developed intricate signalling networks to detect, control, and eradicate intruding viruses
-e.g., interferons are signalling proteins released by host cells in response to viruses (to increase anti-viral response)
• There is therefore an ongoing evolutionary arms race between viruses and hosts at the molecular and cellular level
Electron microscopy (virus)
- needs a very high viral copy number (106) in the sample
- needs expert personnel
- expensive and big machine
Cell culture (virus)
- Not all viruses can grow in cell culture
- Need multiple cell lines- different viruses grow in different cell lines
- Slow method of diagnosis- some viruses, e.g., CMV need> 1 week to grow.
- Currently of limited use.
Detection of viral antigen-immunofluoroscence
- Not applicable for all viruses- need manufacturing of specific monoclonal antibodies, e.g., currently antibodies only available against 6 of 15 respiratory viruses.
- Needs fluoroscent microscope set up
- Need expert personnel
Detection of antibody (or antigen) using ELISA
- Serology
- ELISA: enzyme-linked immunosorbent assay (ELISA)
- Chromogen or substrate which changes colour when cleavage by the enzyme attached to the second antibody.
- Anti human immunoglobulin (2nd antibody) coupled to an enzyme
- Patient serum contains antibodies.
- Purified antigens pre-coated onto an ELISA plate
Viral nucleic acid detection
- For all viruses, but particularly helpful for non-cultivable viruses, e.g., papillomaviruses.
- For viruses in low copy numbers, e.g., neurological infections such as herpes viruses
- Commonest test used is PCR
- Quick test answer can be within 24 hours.
- Relatively costly test but the cost has come down
- Can also quantify virus e..g, HIV viral load.
Measures of viral prevention
- General measures, e.g, gloves, masks, etc.
- Specific measures (long-term or short-term prevention).e.g., vaccines, chemoprophylaxis, immunoprophylaxis.
Vaccines in viruses
Vaccines inhibit the virus and reduce symptoms in individuals affected by viral infections
Anti-viral drugs
HIV- HAART drugs
HBV- lamivudine
HCV- ribavirin and PEG IFN
Infection
- An infection is the colonisation of a host by Microbial species
- Infecting microbes seek to use the host’s resources to reproduce, often resulting in disease.
- Viruses, prions, bacteria, and viroids, though larger organisms like macro parasites and fungi can also infect.
Basis of classification of infections
• They are classified by the causative agent as well as by the constellation of symptoms and medical signs that are produced.
- an infection that produces symptoms is an apparent infection - an infection that is active, but does not produce noticeable symptoms, may be called in apparent, silent or sub clinical. - an infection that is inactive or dormant is called a latent infection
The chain of infection
Causative agent -> Reservoir -> Portal of exit-? Mode of transmission -> Portal of entry -> Susceptible host
Causative agent
- any microorganism capable of producing disease (bacteria, viruses, fungi, and protozoa).
- some organisms are part of the normal flora but can cause infection in immunocompromised host. these are known as endogenous infections.
- Infections which are acquired from external sources are called exogenous infections.
Reservoir
- the place where the agent survives, frows, and/or multiplies: human, animal or environment. e.g., pseudomonas spp survive and multiply in nebulisers and hepatitis B virus survives but does not multiply on the surface of haemodialysis machines.
- reservoirs abound in healthcare settings, and may include everything from patients, visitors, and staff members to furniture, medical equipment, food, water and blood.
- human reservoir: case is a patient with an acute clinical infection, carrier is a person who is colonised with a specific pathogenic microorganism but shows no signs or symptoms of infection, e.g., HBV.
Portal of exit
- Ear (ear wax)
- Eyes (tears)
- Broken skin (blood)
- Skin (flakes)
- Anus (faeces)
- Seminal vesicles (semen)
- Urethra (urine)
- Vagina (secretions, blood)
- In females: mammary glands (milk, secretions)
- Mouth (saliva, sputum)
- Nose (secretions)
Mode of transmission (Direct contact)
-Direct contact: person to person spread of the microorganism through physical contact
Mode of transmission (Indirect contact)
-Indirect contact: occurs when a susceptible person comes in contact with a contaminated object
Mode of transmission (droplet transmission)
-Droplet transmission: results from contact with contaminated respiratory secretions. A person with a droplet-spread infection coughs, sneezes or talks, releasing infected secretions that spread through the air to the oral or nasal mucous membranes of a person nearby.
Mode of transmission (Airborne transmission)
Occurs when microbial particles or dust particles containing pathogens remain suspended in the air for a prolonged period, and then are spread widely by air currents and inhaled. The tiny particles remain suspended in the air for several hours and may cause infection when a susceptible person inhales them. e.g., pulmonary tuberculosis, measles.
different modes of transmission
- contact transmission
- droplet transmission
- airborne transmission
- ingestion (through gastrointestinal tract)
- inoculation (through accidental sharp injury or bites)
- Transplacental transmission (microbes may cross placenta from mother to foetus)
Portal of entry
the portal entry for tuberculosis and diptheria is through the respiratory tract. Salmonella enters through the GI tract. Other portals of entry include the skin, mucous membranes, bloodstream or body fluids. Invasive devices, e.g., IV line
Susceptible host
- a person who can become infected by infectious agent, e.g., patients, health care personnel, visitors from the community.
- risk factors that accelerate infection: age, nutrition and sociocultural condition, open wounds/invasive procedures, suppressed immune system/weak health condition, presence and/or number of infectious microorganism.
- Hospitalised patients are more prone to develop infection as a result of surgery, invasive procedures and devices, organ transplants, microorganisms flourish in healthcare setting and with breaks in infection control procedures and practices, patient’s weakened defense mechanism, help set the stage for nosocomial infections.
Breaking the chain of infection
• Rapid and accurate identification of organism:
-Routinely send blood cultures, urine culture, skin swabs, throat swabs, tracheal aspirate culture
-Send endotracheal tube tip, urinary catheter tip and central line tip for culture after removal
• Control or eliminate infectious agents:
-Proper cleaning with water/mechanical action with detergents
-Disinfection: high level (2%% activated glutaldehyde, 1% sodium hypochloride, 5% carbolic solution, 1% bleach powder), low level (70% methylated spirits, 10% betadine solution, 1% savlone)
-Sterlisation (autoclave, 121 degree Celsius for 20 mins)
- Protecting the susceptible host: protecting the normal defense system by regular oral hygiene, maintaining adequate intake, encouraging deep breathing exercise and encouraging proper immunisation of children and adult patients.
- Maintaining healing process: promote intake of balanced diet, measures to improve appetite of patient, help patients to reduce stress.
3 different types of infection
Acute: virus infects susceptible host, non equilibrium process host response and virus infection change continually until resolution
Chronic: continues infection beyond time when immune system should have cleared it. equilibrium between virus and host balance until equilibrium changes
Latent infection: persistent of viral DNA integrated within the host genomes and may result in reactivation (herpes) or transformation (retrovirus)
endogenous viruses (latency)
endogenous viruses can insert themselves into the human genome, e.g., shingles, recurrent oral and genital herpes, CMV infections. immunosurveillance controls these latent viruses. Aging or immunosupression can lead to virus reactivation
EBV infection and latency
- initial diagnosis: glandular fever due to EBV at 21yrs.
- Virus infects B cells that expand in the host.
- T cells expand to control the infection
- Latent infection of memory B cells established
- Level stable until immunosuppression
- Persistence of EBV infection can cause various cancers of B cells, e.g., B cell lymphoma
S.Aureus
-Commensal of anterior nares
-20%-60%
-highly adapted
-Golden colonies on blood agar
-Gram positive cocci in clusters
Virulence:
Adhesins: bind host proteins- tissue adherence, immune evasion
Protein A: binds the Fc portion of the immunogloblulins
Coagulase: stimulates clotting, role in immune evasion
Staphylococcal toxins
Cytotoxins: pore forming toxins, lyse host cells, paton-valentine leukocidin (PVL)- lyses polymorphs (immune cells)
Exofoilative toxins: proteases, target epidermal structural proteins
Enterotoxins (superantigens): stimulate massive T cell activation and immune evasion, ingestion-> vomiting.
Many of these are phage encoded- only present on a proportion of stains.
Staphylococcal capsule
Polysaccharide capsule: micro capsule thin compared to other encapsulated bacteria.
Specific Abs to capsular antigens required.
Lipopolysaccharide recognition
- Innate immune system is very sensitive to LPS: evolution (probably) gram negative predominnce in bites, contaminated wounds, etc., allows very early recognition of infection.
- LPS interacts with Toll-like receptors (esp. TLR4) on monocyte/macrophage lineage cells, endothelium.
- Results in activation of inflammatory pathways, coagulation and clotting pathways and changes in endothelial integrity.
