Microbiology L1-11 Flashcards
Why are bacteria important?
-Largest producers of oxygen (~50-80%)
-Remove carbon
-Couldn’t digest food
-Helps trees grow
-Helps communication between certain organisms
-MOs make the best recyclers
-Produce food and chemicals
What extreme environments can bacteria be found in?
-Psychrophiles in the antarctic
-Hyperthermophiles in a hot spring (Taq pol for PCR)
What are the different types of bacteria that exist in different environments?
-Psychrophile
-Mesophile
-Thermophile
-Hyperthermophile
Which types of bacteria exist in high temperature extreme environments?
Hyperthermophiles
What are the similarities and differences between bacteria and archaea?
Similar:
Lack cell nuclei
Information-handling system resembles eukaryotes
Different:
No peptidoglycan in cell wall of archaea
Metabolic processes unique to archea
What are the different catabolic mechanisms bacteria can have?
-Chemoorganotrophy (fermentation)
-Chemolithotrophy
-Phototrophy (photoautotrophy and photoheterotrophy)
What is chemosynthesis?
Not using sunlight for energy (use CO2 and SO2)
Why is chemosynthesis advantageous?
It can be used to soak up harmful chemicals for the atmosphere and produce useful products
What can biotechnology produce?
-Recombinant proteins
-Biologic drugs
-Natural product drugs
-Fine chemicals
-Industrial enzymes
-Synthetic biology
What did Robert Koch discover?
-Microbiological methods
-Discovery of causative agents
What are Koch’s postulates?
1- suspected pathogen must be present in all cases of the disease (absent in healthy animals)
2- Suspected pathogen grown in pure culture
3- Cells from pure culture cause disease in healthy animal
4- Pathogen re-isolated and shown to be same as original
What was the highest cause of death in the 1900s?
Influenza and pneumonia
What is the highest cause of death today?
Heart disease
What is the microbiota?
Microbial organisms living everywhere that aren’t apart of our own bodies
What is the microbiome?
Genes harboured by the microbiota
What roles does the microbiome have in the body?
-Protection against pathogens
-Synthesis of vitamins
-Immune system development
-Promotion of intestinal angiogenesis
-Promotion of fat storage
-SCFA production by fermentation of dietary fibre
-Modulation of the CNS
What first establishes the gut microbiome in a baby?
Milk oligosaccharides from breast milk
How do Bifidobacterium infantis benefit the baby?
They lower the gut pH enhancing the epithelial barrier allowing immune modulation
Who are two of the first people to revolutionise the microscope?
Robert Hooke and Antonie van Leeuwenhoek
What is the resolution limit of the light microscope?
200nm
What are the different types of commonly used microscopes?
-Light microscope
-Transmission electron microscope
-Scanning electron microscope
What are the different types of light microscopy techniques?
-Brightfield
-Darkfield
-Phase contrast
-Differential interference contrast (DIC)
-Fluorescence
-Confocal
-Two-photon
What is the process of gram staining?
1- Application of 1st stain crystal violet
2- Application of iodine
3- Wash with alcohol
4- Application of safranin
What shape and colour do gram-negative bacteria appear?
Rod shaped and pink once stained
What shape and colour do gram-positive bacteria appear?
Circular shaped and purple once stained
What are the differences between gram-positive and negative bacteria?
Gram-negative bacteria:
Contains small layer of peptidoglycan
Contains a cytoplasmic membrane and an outer membrane
Gram-positive bacteria:
Contains large layer of peptidoglycan
Contains cytoplasmic membrane only
How is the bacterial cell wall adapted?
-Provides important protection against osmotic & environmental stresses and helps with uniform cells
-Target for immune defences (lysozymes)
-Target for many antibiotics
How is green fluorescent protein (GFP) used?
-Used in genetic manipulation, enables imaging
-Many colour variants allows visualisation of different proteins
What are downfalls of green fluorescent protein?
Fusion to proteins malfunction and localisation can be misleading
How was he complexity of the bacterial cell wall revealed?
By fluorescence microscopy
Which protein in bacteria is equivalent to eukaryotic tubulin?
FtsZ protein
Which protein in bacteria is equivalent to eukaryotic actin?
MreB
What is an S-layer?
It is the outermost layer of a bacterial cell
What is the composition of the S-layer?
It is a crystalline lattice of a single rotein
What does the S-layer do?
It is protective and acts like a selective sieve
What are capsules made up of?
Polysaccharide
What are the features of capsules?
-Important in biofilms
-Immunogenic
-Avoidance of immune responses
What are the differences between pili and fimbriae?
Pili - longer
Fimbriae - shorter
What are pili and fimbriae?
Surface appendages
What are pili and fimbriae made of?
Protein polymers
Why are pili and fimbriae important?
In pathogenesis
Support gene transfer by conjugation
What are components of pili and fimbriae?
-Immunogenic
-Varied classes of appendage
What are flagella made up of?
Flagellin in filaments
How are flagella used?
In chemotaxis to give directionality
What rings are flagella made up of?
-L ring
-P ring
-MS ring
-C ring
How are endospores triggered?
By starvation
What are endospores resistant to?
Heat, solvents and lysozymes
What is the composition of endospores?
-Spore coats
-Outer membrane
-Cortex
-Inner membrane
-Spore core
What are the sporulation stages?
-Spore is engulfed into mother cell as a prespore
-Then the cortex of the spore is formed
-The spore takes nutrients from the mother cell
-Spore matures and mother cell lyses
What are biofilms?
Communities of bacteria (often multiple species together)
What holds the biofilm together and what is it made up of?
Held together by a matrix made up of:
Mainly polysaccharide
Protein
Nucleic acids
How do biofilms differentiate to survive?
Swim - senssile
Growth - sporulation
Multicellular structures (complex)
What is generation time?
The time needed for one call to divide and form 2 cells
What is bacterial growth limited by?
Nutrients become limiting or toxins accumulate
What takes place in the lag phase of bacterial ‘batch’ growth?
Cells adjust to the new environment, have to regulate enzymes and motabolites
What takes place during the exponential phase?
Optimal growth with regular doubling
What takes place during the stationary phase?
Growth limited due to nutrient depletion/ accumulation of toxin, rate of new cell production= cell death
What takes place during the death phase?
Complex gradual loss of viability but with some cell turnover
How can bacterial growth be measured?
-Plating methods
-Turbidity
-Direct microscopic counting
-Flow cytometry
How is bacteria counted in plating methods?
Sample is diluted using a serial dilution then the diluted broths are plated and the colonies found on the plates can be multiplied by the dilution factor to find the approximate amount of cells in the culture
What are disadvantages of using plating methods to count bacteria?
-There can be underestimates (cells in chains/clusters)
-Number of colonies is dependent on growth conditions
What are advantages of using plating methods to count bacteria?
-Only measures viable cells
-Highly sensitive (even living cell can be detected)
-Growth conditions customised
How is bacteria counted in turbidity methods?
Uses spectrophotometer to scatter cells and measure the optical density
What are the disadvantages of using turbidity methods to count bacteria?
-Measures living AND dead cells
-Low sensitivity
-Turbidity has to be in a certain range
What are the advantages of using turbidity methods to count bacteria?
-Simple and convenient
-Non-destructive done continuously
How is bacteria counted in direct counting methods?
A coverslip containing a grid is placed on a slide to be observed under a microscope the cells are counted then multiplied by the magnification and the area of the grid
What are the disadvantages of using direct counting methods to count bacteria?
Doesn’t discriminate (living and dead cells in sample)
Laborious
What are the advantages of using direct counting methods to count bacteria?
Accommodates clumping and chaining
How is bacteria counted using flow cytometry and FACS (fluorescent activated cell sorting)?
Measures the particles in a microfluidic flow like spectrophotometry
What are the disadvantages using flow cytometry and FACS to count bacteria?
Requires the right equipment, reagents and expertise (can be expensive)
What are the advantages using flow cytometry and FACS to count bacteria?
-Highly automated
-measures at multiple wavelengths
-Cells sorting possible (using FACS)
What are the steps of cell division in bacteria?
1) Cell grows, structures duplicated and chromosome replicated
2) Daughter chromosomes segregate to different ends of cell
3) Septum forms at middle as Z-ring constricts, new cell poles form as this happens
4) Cell division occurs in the middle resulting in 2 identical daughter cells
What are the factors of bacterial DNA replication?
-Replisome forms which opens the strand to make new DNA
-Replication fork to form 2 new strands
-Lagging strand slower as okazaki fragments form
What are the components of chromosome replication in bacteria?
Chromosomes circular
Bidirectional (starts at oriC and finishes at terC)
What structure is formed when replication is taking place on the bacterial chromosome?
Theta structure
How does bacteria allow quicker chromosome replication?
Replication is initiated in the previous cycle
What protein is key in the Z-ring?
FtsZ protein
What is the function of the Z-ring?
Contracts which causes septum formation
What are heterocytes?
Non-dividing cells that allow N2 fixation
What are Akinetes?
They are cells containing spores that do not divide as they contain many nutrients for bacterias life so in unfavourable conditions the bacteria has sufficient nutrients
How does Bdellovibrio work?
It grows inside another bacteria causing lysis of the infected cell
What is the function of Myxococcus bacteria?
It eats other bacteria then makes complex fruiting bodies which then allows more cell growth
What is a catabolic reaction?
Energy releasing reactions
What are anabolic reactions?
Building enery into food
What are the 2 different types of nutrients bacteria require?
Macronutrients and micronutrients
What are some examples of the nutrients bacteria need in the lab?
Sugars, Amino acids, water, oxygen, nucleic acids
What are the 2 different types of media bacteria can be grown in?
Chemically defined and undefined media
What are some of the different defined medias bacteria can grow in?
Glucose, NH4Cl, NaCl etc
What are the undefined media bacteria can grow in?
Tryptone, yeast extract and NaCl
What can be measured in bacterial growth?
Population density and number
What can be calculated in bacterial growth?
Growth rate and generation time
How is the final cell number calculated?
N=N02^n
N - final cell number
N0 - initial cell number
n - number of generations
How is generation time calculated?
g=t/n
g - generation time
t - duration of exponential growth
n - number of generation
Why is generation time useful?
-Allows comparison of different bacterial species growing in the same conditions
-Allows comparison of one species grown in different conditions
-Can b used to show if data is significantly different or not
What is optimum pH based on for bacterial growth?
Extracellular environment only
What is the main influence on bacterial growth?
Salinity (salt concentration)
What are chemostats?
Known as bioreactor, it is a continuous culture
Fresh media added as cells are removed so growth is at a steady state
What culture parameters remain constant in a chemostat?
Culture volume
Dissolved oxygen concentration
Nutrients
Waste concentrations
pH
Cell density
etc
How is dilution rate calculated?
D=F/V
D - dilution rate
F - flow rate
V - volume
What is microfluidics?
It is the study of bacteria that involves holing the cells in an order
What are the different shapes bacteria can have?
Coccus
Rod
Spirillum
Spirochete
Budding & appendaged
Filamentous
What is genetically programmed in bacteria?
Shape, growth, cell division and survival in environments
How many base pairs does E.coli have?
4.6 million
How long would the E.coli genome be if unwound?
~1.56mm
How is the genome of E.coli wound?
Into a nucleoid of ~1micrometer
Not double membrane bound
What are components of E.coli’s genome?
Circular
oriC and terC
Genes organised in operons
Clusters of genes with related functions are transcribed together in a single mRNA
What is the average distance between E.coli genes?
~118 bp
How do bacteriophages act?
In a lysogenic cycle - stable insertion in host chromosome
Can transfer genes in transduction
What are transposons and how do they work?
They are jumping genes and hop in and out of chromosomes and plasmids, often contains resistance genes
What are integrons?
They can pick up and accumulate ‘useful’ genes
How do plasmids confer diverse phenotypes?
Extrachromosomal but can be integrates
Varied size range <5kbp to >1 Mbp
Often mobile (conjugation)
Can confer accessory functions
What is shigella?
It is a bacteria related to E.coli and causes dysentery
Plasmid encodes key virulence genes including a Type 3 Secretion System
How do bacterial genomes vary between strains of the same species?
Core genomes - housekeeping genes possessed by all strains
Accessory genome - mobile elements of different origin
Vary between strains of species
What is the pangenome?
The totality of genes found across different isolates of a species
How many genes are in E.coli pangenome?
~15000 genes
What are the different habitats bacteria can inhabit?
The human microbiome
Soybean roots
Extreme environments
What is the bacterial genome known as?
The complete set of genetic material in a bacterial cell
Approximately how long is E.coli’s genome?
4.6 million base pairs
What are components of the E.coli chromosome?
-Circular
-Contains an origin of replication
-Genes organised in operons
-Clusters of genes with related functions form single mRNA
What is the average distance between genes in E.coli?
~118bp
What are common mobile genetic elements?
Plasmids -small extrachromosomal circular DNA
-Bateriophage (bacterial virus)
-Transposons and insertion sequences- transfer genes - jumping genes
-Integrons - pick up useful genes
What are transposons and insertion sequences?
Jumping genes
Hop in and out of chromosomes and plasmids
Often carry resistance genes
What are examples of phenotypes conferred by plasmids in prokaryotes?
Antibiotic production
Conjugation
Metabolic functions
Resistance
Virulence
What is contained in the core genome of bacteria?
Housekeeping genes possessed by all strains of a species
Approximately how many genes are in the E.coli pangenome?
~15000 genes
What are pathogenicity islands?
Clusters of genes of “foreign” appearance present only in certain strains and corrected with virulence
What was the first sequencing developed?
Sanger sequencing 1977
How is Sanger sequencing conducted?
DNA denatured then amplified with PCR
Dideoxynucleotides used to terminate PCR and produce different length chains
Sequencing
How is illuminate sequencing conducted?
Library preparation - fragment, denature and legate adaptors
Cluster amplification - addition to flow cell and cluster amplification
Sequencing - fluorescent ply labelled nucleotides
What is sequence assembly with bioinformatics?
Overlapping sequences aligned
Repetitive sequences interfere with assembly
Complete genome assembly - compare with known sequence, long read sequencing methods
What is the oxford nanopore?
Has very long reads
10-30kbp genomic libraries common
generally less accurate
What are the profound implications of genome sequencing?
Bacterial genetics
Epidemiology of infectious disease
Comparative genomics
How has sequencing benefitted infectious disease and epidemiology?
-Pre screen patients for problem organisms
-Identify all of the virulence factors and antibiotic resistances in a patient sample
-Sequence isolates from multiple patients
How is metagenomics beneficial?
Gives new ways to study the diversity of bacteria including unknown bacteria
What is metagenomics?
The study of genetic material recovered directly from environmental samples
What is bioremediation?
A process that uses mainly microorganisms, plants or microbial or plant enzymes to detoxify contaminants in the soil and other environments
What is an example of bioremediation?
Hydrocarbon degrading bacteria cleaning up oil spills
What type of pollutants are often resistant to natural degradation?
Xenobiotic pollutants
What is an example of bioremediation of xenobiotic pollutants?
Pesticide dechlorination by Burkholderia using oxygenates to generate TCA intermediates
How does Idoeonella sakaiensis bacteria help in bioremediation?
It expresses PETase enzyme that hydrolyses and degrades PET-based plastic
What are bacterial “cell factories”?
They use living organisms that produce medically or comercially useful biomolecules
What is the main advantage of cell factories?
-Bacterial cultures grow quickly and easily and materials are cheap and environmentally friendly
What is synthetic biology?
The design and construction of new biological parts, devices and systems, and the re-design of existing, natural biological systems for useful purposes
What are the key steps to synthetic biology and bio-engineering?
Understanding
Standard parts
Bacterial systems
Microorgansims is produced
What are the advantages of biological components?
-Modular and well characterised
-Put together in any order
-Complex designs
How are DNA encoding genes controlled?
Using the promotor, RBS, terminator and DNA binding transcription factors
What is a key example of gene regulation?
The Lac repressor
What are optogenetics?
Using light to control gene expression by modifying biological components
What is protein engineering?
Adding new functions to proteins or improving current functions
What is metabolic engineering?
Gene circuits and engineered proteins combined to alter or create new metabolic processes in bacteria
What is transcriptomics?
Measures gene expression of all genes under any specified condition
What does CRISPR stand for?
Clustered
Regularly
Interspaced
Short
Palindromic
Repeats
What is the approximate ratio of bacteria to human erythrocytes?
1:1
How does DNA/RNA sequencing help detect and find microbes in the GIT?
Helps determine microbial community fingerprint, at a rapid and high output
What is the most common way of sequencing bacteria in the microbiome?
16S rRNA gene profiling
How is 16S rRNA profiling conducted?
Genes are clustered them mapped to find OUTs
What are culturomics?
They help identify unknown microbes
Why are culturomics important?
TO improve reference databases for further NGS approaches
Phenotypic and mechanistic studies
Culture collections
Therapy development
What are the main functions of the bacteria in the microbiome?
They are mainly used for the digestion of carbohydrates
Where are human milk oligosaccharides abundant?
In breast milk
What are the components of the microbiota in a baby?
Pioneer microbes
Low diversity
High instability
What are the components of the microbiota in a toddler?
New species out compete early ones
Rapid increase in diversity
High instability
What are the components of the microbiota in an adult?
Highly distinct and diverse microbiota
Microbial community may continue to change but at a slower rate
What are the components of the microbiota in the elderly?
Substantially different microbiota than younger adults
Lower diversity
How does pregnancy influence the microbiome of the foetus?
Intra-uterine environment - amniotic fluid
Maternal exposures - e.g. stress
Length of gestation - term vs. preterm
How does birth influence the baby microbiome?
Mode of delivery
Contact with mother or healthcare professional
Environment straight after birth
How does the baby’s microbiome become influenced after birth?
Feeding modality
Antibiotics
Weaning or food supplementation
Home/family setting
Home structure
What is colonisation resistance?
Resistance to colonisation by ingested bacteria or inhibition of overgrowth of resident bacteria normally present at low levels within the intestinal tract
How does the microbiota help immune development?
By allowing mucosal priming and systemic priming
What can cause microbiota disturbances?
Diet
Antibiotics
Birth mode
Infections
Genetics
Etc
How do pathogenic microbes cause infection?
By gaining foothold in a particular niche
What is colonisation?
Growths of a microbe after gaining access to host tissue
What is pathogenicity?
The ability of a pathogen to inflict host damage
What is virulence?
The degree of pathogenicity of an infecting pathogen
What is the main mechanism of microbial pathogenesis?
Exposure
Adherence
Invasion
Colonisation + growth
Toxicity - to tissue damage/ disease
Invasiveness - to tissue damage/ disease
What are the key components of infections of microbial pathogenesis?
Can be invasive but not exclusive
Symptomatic or asymptomatic
Highly acute or chronic (long term complications)
What are the components of respiratory infection?
Bacterial/ viral
Upper respiratory tract has abundant microbiome
Lower respiratory tract typically devoid of microbes
What are the components of mycobacterium tuberculosis?
> 1 million deaths/year
Mortality rate 2 in 3 without treatment
Rising drug resistance (cell wall structure and efflux)
Transmission risk due to respiratory expulsion
Survives within immune cells (inactive for a while)
What are the components legionella pneumonphila?
10 cases per million
Grows in stagnant water
Cause pneumonia leads to high mortality
Survives in immune cells (protective vacuole)
Amino acid auxotroph (promotes proteasomal degredation)
What are the components of gastroenteritis?
Diverse causes and infectious characteristics
-bacterial - ingestion, feacal-oral, contamination
- viral - winter vomiting bugs
- parasitic - water-borne
Acute - rapid content ejection, self limiting
Chronic - long lasting
What are examples of gastroenteritis?
Salmonella
Campylobacter
Vibrio
Shigella
Enterotoxigenic E.coli
C.difficle
What are the components of Escherichia coli?
Gram -ive bacteria in mammaliam gut
Diverse array of pathotypes
Intestinal pathogenic E.coli
Extraintestinal pathogenic E.coli
What is the most common serotype of intestinal pathogenic E.coli?
O157:H7
What are the components of the E.coli serotype O157:H7?
Encodes shiva toxin - causes HUS, massive health and economical burden
What are the components of vibrio cholera?
Gram -ive, motile, commonly associated with aquatic reservoirs
4 million cases
Phage-encoded CTX results in massive loss of fluid
What are the components of helicobacter pylori?
Gram -ive, motile, spiral
Very common
Symptoms - severe stomach pain
Aetiological agent of gastric and peptic ulcers
What are the main components of UTIs and bloodstream infections?
Many pathogens cause UTI and systemic infection
Gut colonisation is asymptomatic but adapted
Specific virulence and fitness factors promote UTI and bloodstream dissemination
Recurrent and drug resistant
Bloodstream infection can lead to sepsis
What are most skin infections caused by?
Gram +ive cocci i.e. staphylococcus & streptococcus sp.
What are common symptoms with skin infections?
Skin lesions, inflammation can disseminate to infection of throat, middle ear, blood and lungs
What are the different types of immunity?
Innate and adaptive
What is innate immunity?
Immediate, rapid response
What is adaptive immunity?
Exposure, targeted defence weapons
What is inflammation?
A nonspecific reaction driven by neutrophil accumulation
What are the main signs of acute infection?
Rubor
Calor
Tumor
Dalor
Change in function
What are the methods of adaptive immunity?
Specificity and memory (host mediated response and repeat responses)
Humoral immunity (B cells)
Cellular immunity (T cells)
ADCC ( antibody dependent cell mediated cytotoxicity)
What is bacteraemia?
The presence of bacteria in the bloodstream
What is sepsis?
Extreme, system-wide inflammatory response to blood poisoning
What is systemic shock?
Systemic drop in blood pressure leading to mass organ failure
What are the different types of pathogen reservoir?
Environmental - V.cholerae
Zoonotic - EHEC
Human - UPEC
Why is global awareness important?
Health and wellbeing of global population
Economical burden of infections diseases
What are the different diagnostic media and what are the components?
General purpose - non-selective, establish growth
Enrichment - selective certain species
Selective - certain media
Differential - between 2 bacterial species
What type of agar does general purpose media use?
LB (lurid-bertani)
What types of bacterial does general purpose media grow?
Fastidious and non-fastidious
What bacteria is enrichment media for?
Fastidious bacteria often supplement media with sheep blood
How is selective media used?
Mixed bacterial sample taken
Incubated in microaerobic conditions
Uses media and growth conditions as selection
What does differential media often test for?
Presence/absence of enzymes
What does MacConkey agar detect?
Bacilli and enteric bacteria
What is contained in MacConkey agar?
Bile salts and crystal violet (inhibit gram+ive bacteria)
What does MacConkey agar traditionally contain?
Lactose, identifies lactose fermenting and non-fermenting
What are pathogenicity or virulence factors?
Structures, molecules or regulatory systems that enable the disease process
What is pathogenicity driven by?
Adhesion to epithelial surface or invasion of underlying tissue
How many bacteria make up the microbiome?
~10^13 cells
How can pathogens adhere to mucosal surfaces?
By either subvert host cell function (extracellular) or invade underlying tissue (invasive) during pathogenesis
How is adherence aided in animal cells?
Using polysaccharide capsules which double up as physical barriers to desiccation and immune masks
What are main components of the bacterial capsule?
Key virulence determinant
Polymer of repeating sugar units
Encases bacterial cell
Provides resistance against immune recognition, phagocytosis and complement killing
Exclude hydrophobic detergents
Important precursor to biofilm formation
What are the advantages of biofilm formation?
Physical defence
Nutrition
Intracellular communication
Exchange of genetic material
What are the main stages of biofilm formation?
Absorption
Irreversible attachment
Growth and division
Mature macrocolony
What are fimbriae?
Filamentous cell surface protein structures
Capped by sugar binding, lectin-like proteins
What are components of type 1 fimbriae?
CUP type adhesion
Found widely in gram-ive
FimH has a specific affinity for mannose (bladder colonisation)
How are pili different to fimbriae?
Pili are longer, less abundant and involved in genetic exchange (conjugation)
What is the fibril catch-bond theory?
Mechanistic benefit in natural environments subject to fluid shear stress
Strength of binding dictated by an allosteric switch in the Fish-sugar interaction
What are the 2 general pathways of transport across the inner membrane for bacterial protein secretion?
SEC- unfolded, common
TAT- folded, elusive substrates
What is the system for bacterial protein secretion in gram -ives?
2 step systems following SEC translocation to the periplasm followed by a specific system for outer membrane transport
What are examples of different diverse secretion systems in bacteria?
T3SS - required for epithelial attachment and in some cases invasion
T6SS - used for inter-bacterial warfare as well as host-cell subversion
T4SS - primarily involved in genetic exchange (conjugation)
What are key points of bacterial toxins?
Cause damage to host tissue/cells
Damage is typically distal to site of infection
Toxins offer range of advantages, access to tissue and nutrients, facilitating transmission and can enhance other virulence processes
What are the properties of exotoxins?
Chemical - secreted proteins
Action - Cytotoxin, enterotoxin, neurotoxin, superantigens
Toxicity -high
Immunogenicity - high
Toxoid potential - destroyed but remain immunogenic
Fever - no
What are the properties of endotoxins?
Chemical - lipopolysaccharide
Action - fever, diarrhoea, vomiting
toxicity - weak
Immunogenicity - poor
Toxoid potential - none
Fever - yes
What are the mechanisms of action of exotoxins?
Cytolytic
Disruptive
Stimulatory
What are examples of toxins that are highly site-specific?
Enterotoxins affect small intestine resulting in fluid loss (Ctx)
Neurotoxins of nerve cells (tetanus and botulinum)
Renal toxins (Stx)
What is Stx?
It is a phage triggered in response to certain stresses
EHEC T3SS is essential for colonisation
Has a repressive effect on T3SS while also stimulating cell receptor expression in host
What are endotoxins?
Lipopolysaccharides of gram -ive bacteria
Only release when cells lyse
What are the different ways to identify the specimen of a pathogen a patient has presented?
Antibody assays
Rapid testing (antigen assays)
Culturing
Serology
What is a CFU?
Colony Forming-unit
How can CFUs be used to quantify specimens in order for diagnosis and treatment?
Urine can be used to grow and dilute in order to find colonies, they also use selective media to find the right treatment for the diagnosis
Machines quantify quickly
In mixed culture probable microbe can be extracted
What is latex agglutination?
Surface antigen binds to latex beads causing agglutination which allows for rapid diagnosis
What are the advantages of latex agglutination?
Confirms the identity of isolates
Identifies known pathogen-associated antigens in specimen when isolation fails
How is serology used in treatment and diagnostics?
It relies on diagnostic identification of antibodies in the serum
How is serotyping used in treatment and diagnostics?
It determines the subtype of organism
How is finding the serotype advantageous?
It provides information of how serious the infection is, meaning it can help with treatment of the disease
How can biochemical assays help diagnosis?
It finds information of different properties the bacteria may have helping with identification subsequently helping with treatment
What are the advantages of molecular diagnosis of infections?
Bacterial genomes are unique
Genetic material can be extracted from infected specimens
DNA is easy to detect and even quantify
Extremely sensitive 1-10 CFU/ reaction
What are the disadvantages of molecular diagnosis of infections?
Technology is still being developed
Some tests require the bacteria to be isolated first
Some tests are too sensitive (false positive)
Standardisation from lab to lab can be problematic
How can detecting DNA sequences help provide diagnosis?
By adding fluorescent DNA intercalating agent to the finished reaction
What type of PCR can a dye be added to without inhibiting it?
Real-time PCR
What are the advantages of real-time PCR?
It is quicker (1 hour) than adding dyes to the end of PCR (3-4 hours)
Which diseases has RT-PCR helped diagnose?
Meningitis and Whooping cough
Why was RT-PCR more advantageous for Whooping cough diagnosis?
As culture growth is very slow whereas RT-PCR diagnoses within hours
What has happened to antibiotics as the years progress?
There are shortages of new antibiotics as natural antibiotics are repeating and there is little investment into new ones
What approach has been used to regulate use of antibiotics in organisms?
The one health approach
How are antibiotics used on farms?
They are put into feed to prevent infection in the animals however this causes resistance
What does a bacteriostatic drug do?
It prevents replication, when used the immune system can clear the bacteria
What does a bactericidal drug do?
It kills the bacteria, typically used on invasive/aggressive infections
What do bacteriolytic drugs do?
They burst the bacteria open interfering with structural integrity causing everything to pop
What are the main antibiotic targets?
Cell wall
DNA/RNA synthesis
Folate synthesis
Cell membrane
Protein synthesis
What are the main areas of antibiotic resistance?
Efflux - pump bacteria out
Immunity and bypass - proteins destroy Abs and hide from them
Target modification
Inactivating enzymes
What are the main targets for antibiotic protein synthesis drugs?
Bind to the subunits, preventing peptide from forming, block the tRNAs from binding and impair the proof-reading of proteins all leading to either faulty or no protein formation
Which type of antibiotic blocks the bacterial 30s ribosomal subunit?
Tetracyclines
What is MIC?
Minimum inhibitory Concentration
What does MIC do?
Allows an approximation of how much antibiotic is required to kill the bacteria using specific antibiotics
What are the different approaches for treating infections (preventing antibiotic resistance)?
Limiting antibiotic use
Last resort antibiotics
Combination therapy
Phage therapy
Faecal transplant
What is needed when an outbreak is suspected?
Reports of whom and where
Type of suspected outbreak
Who is affected
Population at risk
Why is global monitoring needed?
Health and education can limit spread
Prevents an epidemic
Field diagnostics are now available
Transient population and air travel mean no infection ‘barriers’
What are notifiable diseases?
They are diseases doctors are required to notify public health England about
What do epidemiologists look doe in patters of cases?
What the normal/ seasonal diseases are
Any changes in distribution
What is the difference between endemic and epidemic?
Endemic: Among a particular group/ area of interest
Epidemic: Infects a slightly later number of people , may spread through several communities
How can DNA help with tracing infection?
It provides understanding of the serotype of the organism allowing us to tailor treatment
Genome sequencing is rapid and RT-PCR is very fast
Sharing data shows patterns of spread
Why is the study of eukaryotes important?
To find ancestors, suggesting important model systems of cell biology, genomics and evolution
Important players in the food web
Important in food industry and biotechnology
Microbial eukaryotes as symbionts
What are the theoretical aspects of phylogeny?
Evolutionary history of a group of organisms
Inferred indirectly from nucleotide/ amino acid sequence data
Gene/proteins globally distributed allowing investigations of global phylogenies (tree of life)
Organelles posses a genome/ genes (mitochondria, plastids)
What is the most widely used phylogenetic marker?
Small subunit ribosomal RNA (SSUrRNA)
What are the components of SSUrRNA being used in evolution?
16S rRNA in prokaryotes and 18S rRNA in eukaryotes
12S rRNA in mitochondria and plastics
Functionally constant
Sufficiently conserved allows cloning with degenerate provers and generate informative alignments
What do eukaryotic organelles with a genome allow?
They can encode rRNAs and some proteins
Contain necessary machinery for protein synthesis (ribosomes, tRNAs and other components for TLN)
What is the archezoa hypothesis?
It suggests the nucleus came first prior to the mitochondrion acquisition through endosymbiosis
How has the tree of life evolved?
More phylogenetic methods developed and richer sampling changing understanding
New genomes, like from environmental metagenomic surveys
-resolve important nodes
-refined out understanding of host cell
refine phylogenetic relationships between major lineages of eukaryotes
What are hydrogenosomes?
Produce hydrogen
Produce ATP through substrate level phosphorylation
Genome with mitochondrial signature
Nuclear genomes encode reduced fractions of mitochondria proteins set
What are mitosomes?
Reduced in size
No evidence for any capacity to produce ATP
No genome
Nuclear genomes encode reduced mitosomal/mitochondrial proteins set
What are components of Cryptosporidium species of protozoan?
Disease: Acute watery diarrhoea
Transmission: Water-borne outbreaks
Importance: Prevalence ranging from 1-10% life threatening in AID patients
What are components of Plasmodium species of protozoan?
Disease: Malaria, acute febrile disease
Transmission: Mosquitoes
Importance: 40% worlds population at risk; 300-500 million clinical cases with 1-1.5 million deaths per year
What are the components of the Taxoplasma gondii of protozoan?
Disease: Birth defects or encephalitis
Transmission : Ingestion of undercooked meat
Importance: Seroprevelence ranging from 6 to 75%, UK: ~20%
What are the components of the Trichomonas vaginalis of protozoan?
Disease: Inflammation of urogenital tract
Transmission: Sexually transmitted disease (STD)
Importance: Most common non viral STD with 174 million new cases per year
What is the difference between extra and intracellular parasites?
Extracellular: thrive on and in tissues without entering their host cells
Intracellular: Need to penetrate their host cells to complete their life cycle (developed specific mean to invade host cell without killing them and exploit nutrients)
What are examples of parasites with different invasion mechanisms?
Apicomplexan
Kinetoplastids
Microsporidia
What is the invasion mechanism of Apicomplexan?
Apical organ orchestrating moving junction mediated entry
What is the invasion mechanism of Kinetiplastids?
Lysosome mediated entry
Phagocytosis
What is the invasion mechanism of Microsporidia?
Polar tube mediated entry
In combination with endocytosis and/or phagocytosis
How do parasitic microbial eukaryotes help understanding of evolution?
Broaden understanding of eukaryote genomic diversity
-Distributed across diversity of eukaryotes
-Genome sequences of parasites have led to different findings
Important data source to discover and study traits underlying host-parasite interactions
-Virulence factors
-Vaccine and diagnostic developments, identification of potential drug targets
How are fungi and Microsporidia important?
Important decomposers of dead animals and plants
Form important mutualistic interactions with plants
Include members of the human microbiota
Many are pathogenic species to animals and plants
What are the factors of Microsporidia infecting humans?
Worldwide distribution
Opportunistic pathogens (AID & HIV)
2 most common : Enterocytozoon bienusi
Encephalitozoon intestunalis
Oral-faecal route - zoonotic origins
Have mitosomes
How do microsporidia replicate?
They depend on one or more host to proceed through their life cycle
Mostly animal hosts
Zoonotic origins for many
What are Stramenopiles?
Many free-living species
Some are symbionts/parasites
-oomycetes: Phytophtora, plant pathogen, Blastocytis
What are Alveolates?
Dinoflagellates
Cillates
Apicomplexa (parasites of animals and humans) - malaria
What is the general organisation of the Apicomplexa?
Mitochondrion
Apicoplast
Nucleus
Dense granules
Polar ring
Micronemes
Rhoptries
Microtubules
Inner membrane complex
How was the apicoplast discovered?
Using molecular data - unexpected small genome 35000bp
Genome organisation - phylogeny linked to plastics
Ultrastructural - identification of a new organelle
What are 3 examples of Apicomplexa infecting humans?
Plasmodium falciparum: human-anopheles mosquito life cycle
Toxoplasma gondii - cat-prey life cycle, common infections with humans (zoonotic)
Cryptosporidium paves, C.hominis - only one host required, human or animal hosts (zoonotic)
What are components of the Toxoplasma gondii?
Worldwide distribution
Seropositive prevalence rates vary greatly
Thought to cause benign disease in immune-competent adults
Tissue cyst forming coccidia
-preditor-prey life cycle
-feline host
With mitochondria and apicoplast with oeganellar genomes
What are examples of Excavata?
Parabasalia - obligate symbionts +parasitic forms, in invertebrates, anaerobes
Diplomonada - host depended = parasitic, anaerobes with hydrogenosomes or mitosomes
Euglenoza - Free-living + parasitic, kinetoplastids, possess secondary plastids, complex mitochondrial genomes
What are components of the Parabasalia, Trichomonas vaginalis?
Most common sexually transmitted cellular pathogen
Very common in resource-limited conditions
Strongly linked with HIV, HPV and Mycoplasma
Linked with prenatal and post-data complications
Positively correlated with cervical and possibly aggressive prostate cancers
