Medical and general microbiology Flashcards
Disease definition
conditions that impair normal tissue function
What is Cystic fibrosis?
is due to a specific genotype that results in impaired transport of chloride ions across cell membranes – genetic or metabolic disease
What’s Atherosclerosis?
disease of aging, because it typically becomes a problem later in life after plaques of cholesterol have built up and partially blocked arteries
What’s measles?
infectious disease because it occurs when an individual contracts an outside agent
What’s an infection?
when a pathogen invades and begins growing within a host.
Disease results only if and when, as a consequence of the invasion and growth of a pathogen, tissue function is impaired.
What is a true pathogen and a opportunistic pathogen?
A true pathogen is an infectious agent that causes disease in virtually any susceptible host.
Opportunistic pathogens are potentially infectious agents that rarely cause disease in individuals with healthy immune system, lots of people don’t believe in them
What’s Pathogenicity
the capacity of a microbe to cause damage in a (susceptible) host.
So its a discontinuous variable
Interactions between microbes and hosts?
commensalism - an association between two organisms in which one benefits and the other derives neither benefit nor harm
colonisation
latency - Of an infection, a period in which the infection is present in the host without producing overt symptoms.
disease.
What counts as damage from a pathogen?
Tissue Pathology
Loss of organ function
Growth in normally sterile sites
What’s virulence?
is one of a number of possible outcomes of host-microbe interaction.
Virulence is a continuous variable, that is, it is defined by the amount of damage or disease that is manifest
How is virulence is measured?
See what dosage produces an ID50 (when 50% of the population is infected) or LD (When 50% of population is killed)
What is a virulence factor (Kochs molecular postulates)?
Easy definition is a microbial trait which causes disease
What’s the Limitations of the virulence factor concept?
The view that pathogenicity is conferred by virulence factors is difficult to apply to many microbes whose pathogenicity is limited mostly to immunocompromised hosts,
How can bacteria hide themselves?
Form a biofilm which protects them from the immune system
Also allows them to replicate
What could influence virulence and pathogenicity?
Microbiome Inoculum Sex Temperature Environment Age Chance History Immunity Nutrition Genetics
Misteaching
Gnotobiotic means?
microbe free
What are microbiota?
The microorganisms that typically inhabit a specific environment
What is the microbiome?
The totality of microbes, their genomes and environmental interactions in a defined environment. e.g. the gut of a human, soil sample
What is dysbiosis?
Microbial imbalance on or within the body
How many more microbial genes are there compared to ours?
100 times more
2 phyla that dominate fecal matter?
Firmicutes and Bacteroidetes
What do microbes do?
Involved in immune system regulation
remove toxins and carcinogens,
crowd out pathogens (colonisation resistance),
improve intestinal functions,
gut-brain links in communication,
as important to us as a liver- because of metabolites produced in gut.
Considered as a vital organ
Feature of silver?
Its an antimicrobial
How does Naturally microbial succession occur?
from birth to later years (critical inoculation in infancy/birth).
Features of microbes and external organs?
, surface tissues (skin & mucous membranes and gut) are constantly exposed to environmental microorganisms and are readily colonized by diverse microbial (primarily bacterial) species
Whats the human microbiome project?
Highly parallel DNA sequencers combined with high-throughput mass spectrometers enable characterization of whole microbial communities
including Genomes, proteins and metabolic products
What were the aims of the human microbiome project?
Determine whether individuals share a core human microbiome
Can changes in the human microbiome be correlated with changes in human health?
What Significant advances were required in order to fully analyze the microbiome
?
Improved culture methods
Perform genome sequencing in the absence of culture
Features of the normal healthy human microbiota?
There is the core part which majority of all humans have this set of genes
Then there is a variable area which smaller subsets of humans have these genes
What’s The most heavily colonized organ in the human body?
The GI tract, contains more than 70% of microbes in the body
Overview of gut microbiota?
Strict anaerobes outnumber facultative aerobes & aerobes
Gut microbiota is dominated by just two phyla
Bacteroidetes (e.g. Bacteroides)
Firmicutes (e.g. Clostridium)
Bacterial abundance increases as we progress from the stomach to the colon
Colonization begins at birth, during passage through birth canal
What does endogenous mean?
All over us
eg. Staphylococcus aureus
What does exogenous?
Get from the exterior environment
eg.Clostridium spp.
Main cause of infection in hospitals?
When the skin is broken
Gram negative bacteria pathogen?
Pink on gram stain
Thin layer of peptidoglycan doesn’t trap crystal violet so it goes pink
Have 2 phospholipid bi layers the cytoplasmic membrane (phospholipid) and the outer membrane (carbohydrate) the sandwich the peptidoglycan layer
Features of Neisseria meningitides?
It’s a beta Proteobacteria
Gram negative
Carriage in the nasopharynx in 10-15% of the population
Epidemics occur every 10-12 years
Meningitidis belt in Africa where rates of infection can be 1 in 100
Classified by serogroup – reactivity to a bacterial polysaccharide capsule
Can turn their capsule on and off
There are 12 serogroups, 6 of which can cause epidemics
Carriage is facilitated by downregulation or loss of capsule expression, as this sterically inhibits adherence and biofilm formation.
However, survival in the bloodstream and in epithelial cells is enhanced by capsule expression
Why was meningitis B unable to have an effective vaccine produced for it?
The carbohydrate capsule was too similar to our carbohydrates on our cells
They fixed it by not basing vaccine on capsule rather a surface lipoprotein on it
Features of Gram positive bacteria?
Example is Corynebacterium diphteriae
Gram positive, immotile rod
Colonises upper respiratory tract
Route of infection: Airborne droplet
Produces exotoxin
Kills surrounding host cells
Toxin spreads systemically
Principally affects: Heart and Lungs
Main cause of mortality
Classic AB toxin - the main virulence factor (B is the binding factor)
What are emerging infections?
infections that are rapidly increasing in incidence and/or geographic range
The process of disease emergence can be divided into two steps?
Introduction
Establishment and dissemination
What are zoonotic diseases?
Capable infecting animal hosts other than humans
They tend to show no disease on their natural host but transferring to a different species will cause disease
Example is Streptococcus suis infects pigs then humans,
Course assessment includes up to 15th October lecture and also includes the practicals
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What causes IBD?
Intestinal microbiota and genetics
Is there a link between obesity and intestinal microbiota?
Yes
What is H. pylori?
first formally recognized bacterial carcinogen
ability to induce gastritis, peptic ulcers, gastric cancer and mucosa-associated lymphoid tissue lymphoma has been confirmed.
Susceptibility is multifactorial e.g. H. pylori virulence, environmental factors, genetic susceptibility of the host and status of host immune system.
Difficult to eradiciate
Gastric carcinoma development may be a result of host genetic, environmental and microbiological factors.
What’s Nod2?
intracellular sensor for bacterial peptidoglycan. 3 polymorphisms in nod2 associated with Chroms Disease, each polymorphism results in reduced activation of NF-kB (protein complex that controls transcription of DNA, cytokine production and cell survival) in response to bacterial peptidoglycan
Nod2 function is required for optimal defensin expression –very important antimicrobial peptides = a critical component of the innate immune system.
What’s Faecalibacterium?
Anti-inflammatory commensal bacterium identified by gut microbiota analysis of chronic disease patients
Obesity-related shift in human microbiota?
A shift in the relative abundance of Bacteroidetes and Firmicutes has been observed in obese humans
Furthermore, abundance of Bacteroidetes increased when dieting, with the change in abundance correlating with weight lost
What are the requirements for disease?
Portals of entry
Establishment
Avoiding host defences
Damaging the host
How do we protect portals of entry?
Washing them with secretions eg. tears
Filter hairs in nasal passages prevents entry of large particles
Cillia in respiratory tract push mucous and microbes upwards
Keratinised surface of skin - tough so acts as an effective barrier against entry
Features of mucous membranes?
Form a protective covering that resists penetration and traps many mircrobes
Are often bathed in antimicrobial secretions which contain a variety of antimicrobial substances
Contain mucosal associated lymphoid tissue
Reasons why fluids such as blood are not suitable for microbial growth?
Iron isn’t available in blood or breast milk
Long chains of fatty acids which are lethal to bacteria found on skin
Lactenin- proteins in breast milk are anti bacterial and protect against inflammation
What is lysozyme?
Found in tears and saliva
Hydrolyses bond connecting sugars in peptidoglycan
Affects gram positive bacteria
Features of the GI tract that wipe out gram negative bacteria?
Gastic acid in stomach
Paneth cells, Pancreatic enzymes, bile, intestinal enzymes, GALT, and peristalsis from the intestines
Intestines also produce lysozyme and cryptins
Features of the genitourinary tract?
Low pH of urine and vaginal epithelia
Urea and other toxic metabolic end products in urine
Hypertonic nature of kidney medulla
Flushing with urine and mucus
What are defensins?
Peptides that are open ended, rich in arginine and cysteine and disulphide linked.
Found in neutrophils, intestinal panteth cells and intestinal and respiratory epithelial cells
They defend from pathogens
Shape microbiota
Protect stem cells
What are bacteriocins?
Peptides produced by bacteria which are lethal to close related species
How do bacteria establish themselves?
Have adherence factors
Normally multi factorial
Examples of secreted enzyme defences?
Leukocidins - Kill white blood cells including neutrophils and macrophages
Produced by Stapylococcal and Streptococcal spp
Coagulase - Causes fibrin clots to form in the blood of a host. Advantageous to the bacteria in evasion
Example of damaging chemicals bacteria release? (Exotoxins)
Neourotoxins - cause paralysis
Enterotxins - cause sickness and diarrhoea
Cytotoxins - cause cell death
Bacterial anatomy of the bacterial cell wall?
Gram positive:
Thick layer of peptidoglycan on outside, has associated proteins as well as lipoteichoic and wall teichoic acid
Plasma membrane inside, has integral and peripheral proteins
Gram negative:
Outer membrane is made up of lipopolysaccharides, has porin gaps and a periplasmic gap between it and the middle peptidoglycan wall and has lipoproteins connecting them
Then inside is the plasma membrane
Stuctures outside the bacterial cell wall?
Glycocalyx - polysaccharides and proteins. If loosely attached referred to as slime layer. If they from a highly organised structure more commonly known as capsule. Common in preventing phagocytosis and allowing infection to continue
Fimbriae an pili are involved in adherence and mostly found in gram negative bacteria, aid motility in gliding or twitching motility. Potential vaccine candidates. Used in immune evasion through a process of antigenic variation
Flagella and Axial filaments-
Aids in movement to distal tissues
Use flagella to penetrate through gastric mucous
Bacterial anatomy inside the cell wall?
Plasma membrane - Target for therapeutic strategies
DNA - Spread of antibiotic resistance on plasmids Target of antibiotic therapy.
Ribosomes - Target for antibiotics
Historical workflow for determining an infection?
Stain based metrologies
Microbial culture the bug
Biochemical or antigenic techniques
Antimicrobial susceptibility testing (which one will be effective)
need to know steps of a gram stain
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Stains for microscopy?
Gram
Ziegl-neelson stain - specific, no need for culture, used mainly for tb, performed directly on sputum
PAS (periodic acid-Schiff) - stain for glycoproteins often used for fungi. disadvantage is stains for hosts cells as well
medias in a microbial culture?
Selective media- eg mannitol salt agar prevents gram negatives growing, it’s specific for staphylococci
Differential media - MacConkey used to select for gram negative
What are API strips?
20 biochemical tests done simultaneously
What are agglutination assays?
Don’t require cultures
Used frequently for detection of viral infections
create beads with antigen for a virus
take some blood if antibody is present from virus the beads will bind and form clumps which we can see
Describe agglutination assay using red blood cells?
Have red blood cells and add virus bug
Haemoglutination occurs so they bind to each other form a clump
Then add serum from someone who has infection you think it is, the antibodies will bind to the virus and haemoglutination won’t occur anymore with the red blood cells
Describe ELISA - (Enzyme Linked ImmunoSorbent Assay)
good because very sensitive
Direct - have a primary antibody conjugate attached to an enzyme which will bind to the substrate
Indirect - the same as direct but also have a secondary antibody conjugate, increases specificity, also wash so on indicator doesn’t give positive to anything else
Capture assay - the same as indirect but also have an initial capture assay
How to determine antibiotic resistance?
Use Etest strips, Antibiotic present at a gradient of concentrations on a Petri dish
Types of modern clinical microbiology techniques?
Next generation sequencing
PCR
Mass spectrometry
Types of total cell count?
Shows both living and dead cells
Direct microscopic count - difficult due to size of cells
Turbidity - measured using a calorimeter where the culture absorbance is proportional to the cell numbers and cell weight
Dry weight - involves analysis involves drying the cell mass after centrifuging a bacterial culture, the cell mass dried at 100 degrees is about 20-30% less than the wet weight
Chemical estimates - involves measuring the amount of ATP or protein to estimate the total amount of cells
The dry weight of prokaryotic cells ranges from?
10 ^-15 g to 10 ^-11 g
What are viable cell counts?
Measured through plate or colony count, based on the assumption that one bacteria cell produces one CFU
The culture is diluted several times and either spread onto agar plates or incorporated into molten agar and poured into plates
CFU’s that grow on agar can now be used to calculate bacteria per ml
The miles and Misra method uses smaller drops (10ul) of each dilution so more can be plated on one plate
Questions to ask in next lab?
Is measuring biomass and turbidity a good quantitive measurement enumerating bacteria in the probiotic sample?
For what other purposes would you make sequential measurements of optical density of a cell suspension at 600nm
How come there was a difference between bio mass and turbidity with the amount of CFU
CFU that are viable to count?
30-300
Why do we measure cell number at 600nm? and what wavelength is used to measure protein or DNA
Any more UV light would start killing bacteria
260nm DNA
280nm protein
Bacteria involved in the fermentation of milk?
Lactobacillus bulgaricus
Lactococcus lactis
Streptococcus thermophilus
Most commonly added culture after sterilisation in yoghurt is?
L.caseli, member of the genus Lactobacillus
How to work out CFU from a serial dilution?
Multiply the amount you counted so you have the correct amount fluid in your formula
Then multiply by the correct power of 10
In an isolated colony are all the bacteria genetically identical?
yes
Forms of bacteria going from circle shaped to very chain like?
Circular
Irregular
Filamentous
Rhizoid
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Different types of elevation of bacteria?
Raised - small dome Convex - higher dome Flat Umbonate - nipple Crateriform 0 crater
Rick Clapped Feebly Under Caren
Different types of margin?
Entire Undulate - few bobbles Filiform - thin hairs Curled - dart board Lobate -few thick hairs
Eat udders for cuban leprocorns
What causes filamentous appearance?
When bacteria have flagella and swarm across the plate
What appearance do bacteria that produce capsules or expolysaccharides show?
Mucoid or glossy appearance
Mostly are circular or raised
Bacteria that have a dry rough appearance are sometimes referred to as?
Ground glass appearance
Steps of gram stain?
Fix bacteria passing through flame
Crystal violet
Iodine
Alcohol
Counter stain with safranin
Observe under immersion oil 100 x
positive = purple
negative = pink
How to test for biochemical activities of a microbial isolate?
Testing the fermentation of different sugars
Members of the lactic acid bacteria when they ferment sugars they produce lactic acid so the pH goes down, hence lowering the pH
Can test this with the indicator bromocresol purple
Yellow media occurs when the pH is bellow 5.2
Purple when it’s more than 6.8
What type of antibiotic is Ampicillin?
B-Lactam
What type of antibiotic is kanamycin?
Aminoglycoside
neomycin phosphotransferase ii gene encodes to resistance for what antibiotic?
confers resistance to various aminoglycoside antibiotics, including kanamycin
What does B-lactamase gene (bla) do?
Encodes for an enzyme that confers resistance to B-lactam antibiotics
Does this by catalysing the hydrolysis of a broad range of β-lactam
What is MIC?
The minimum inhibitory concentration
The abundance of Faecalibacterium within the gastrointestinal tract is believed to influence Crohn’s disease because?
Faecalibacterium promotes the production of anti-inflammatory cytokines
Describe restriction endonucleases?
Cut DNA at specific target sequences
Their mode of action is DNA sequence dependent
Widely used in genotyping to identify genetic differences between bacterial strains without the need for sequencing
Techniques that use them include Pulsed-field gel electrophoresis (PFGE) and Single nucleotide polymorphism (SNP)
To look at plasmid DNA sequences use restriction fragment length polymorphism (RFLP)
Describe RFLP?
Used on 2 different strains
Method based on the principle that the plasmid circular DNA will be linearised if cut at a single location and will be cut into fragments if cut twice
The difference in length (length polymorphism) of the restriction fragments will be seen after gel electrophoresis and will indicate which plasmid has been isolated
Identification is done via a plasmid map which shows where the restriction enzymes recognition sites are located on the circular molecule and allows you to identify the plasmid
eg. which has the black (ampicillin) resistant plasmid and which has (Neo) kanamycin resistant
