Microbiology Flashcards
What are Protozoa
Unicellular eukaryotes
Many are Free-living
Often with parasites
What are eukaryotes
Cellular organisms with membrane bound organelles
DNA in the form of chromosomes in a nucleus
Complex metabolic function
example of protozoa
trypanosomiasis (chagas disease)
malaria
cryptosporidium
Toxoplasma gondii
how big are protozoa generally
2-100 micrometers
what does Bordetella pertussis cause
whooping cough
how to streptococcus cause infection
release Streptolysin O toxin
haemolysis
what shape is streptococci and staphylococci
strep = line of circles
staph = bunch of circles
what is a protkaryote
single-celled organism that lacks a nucleus, and other membrane-bound organelles.
compare gram positive and negative bacteria
Gram negative have 2 membranes separated by a very thin layer of peptidoglycan which cannot sustain the gram stain leaving it its natural histological pink colour.
Gram positive microbes have a very thick peptidoglycan wall which can hold the stain, leaving it purple.
explain the structure of peptidoglycan (3)
Peptidoglycan is a polymer of N-acetyl muramic acid (NAM) and N-acetyl glucosamine (NAG)
crosslinked via amino acid pentapeptides and anchored to the cell wall.
what part of bacteria does penicillin attack
the cross linking of the peptidoglycan in cell walls
compare flagella and Pili
Flagella rotate in different directions to propel the bacteria in a direction
Pilli have sticky ends that adhere to host surfaces and contract to bring closer
both for movement of bacteria
what is a pathogen
disease causing organism
what is a commensal
an organism that lives freely in a host that does not cause disease in normal circumstances
what is an opportunistic pathogen
a commensal that is usually harmless but under certain circumstances e.g. immunocompromised - leads to disease
name some commensals, where they are found and their opportunistic diseases
Staph aureus - found in nasal passages = MRSA, endocarditis
Staph Epidermidis - found on skin = catheter related sepsis
how many bacteria are in 1g of GI tract
1000,000,000
give the structure and gram stain of E. Coli
gram negative rod
commensal
how do we test for mycobacterium and why
It is impervious to gram staining due to waxy cell wall
unusual cell wall containing mycolic acid - higher melting point that have a waxy nature - can survive in lungs
Ziehl-Neelsen acid-fast staining procedure: mycobacteria in red.
An example of this would be M.Tuberculosis.
compare the structure of a gram negative wall and a acid fast wall
gram negative have 2 membranes separated by a thin layer of peptidoglycan
acid fast walls contain mycolic acid making it waxy
how would we test for M. Tuberculosis or M. Lepore
Ziehl-Neelsen acid-fast test under microscope
red rods
what was the first pathogen related to a disease
m. Leprae
what is the carrier state and why is it important
asymptomatic carriage of pathogenic bacteria
it increases the spread of the disease quickly before people realise they have the disease. Half of gonorrhoea and chlamydia is asymptomatic and even less so in women. But with larger effects on women.
why does washing hands prevent spread of typhoid fever and what causes this (3)
Salmonella Typhi causes Typhoid fever
Small amounts of this are kept in the gall bladder which is then excreted
Washing hands after going to the toilet stops spread
what is an Obligate intracellular parasites
can survive outside a cell but can only thrive and reproduce within cells.
describe the structure of a virus
Nucleic acid (DNA/RNA/Single/Double) encapsulated by proteinaceous capsid
Varying shape and symmetry
Some further coated with lipid coat - enveloped e.g. HIV derived from sometimes host and always virus proteins.
Spike proteins on the surface interact with human cell receptors e.g. Covid = ACE protein
what is a viruses Lytic Cycle
the cell cycle tat occurs whilst in the host cell
what is a lentivirus and how do they work
another name for retrovirus
inject mRNA and reverse transcriptase turns this into DNA
implemented into Host DNA
when host replicates cell, infectious DNA also replicated and transcribed
pathogen proteins created
how might some infections re-activate over time
some pathogens can hide in dorsal root ganglions e.g. chicken pox
when immunocomprimised, re-occurance can occur
why might a patient get a very small portion of shingles on the body
chicken pox zoster virus hides in dorsal root ganglions
when these particular nerves are re-activated, shingles occurs along this nerve
can be very smal
what is the most abundant organism on earth
bacteriophage 10^31
what are bacteriophages and how might we identify one
viruses that kill bacterial cells
place virus on bacterial colonies on agar
if clear, bacteriophage
what are prions and how do they work
Prions are proteins made from living organisms/viruses
They act against other proteins and stop them working properally
why are prions a problem for dental materials (endo especially)
they are small so very hard to filter
heat resistant so very hard to kill by heat
very sticky and stick to instruments
in animal studies have been found in pulp so endodontic equipment is disposed of
why do we dispose of endodontic equipment
animal studies have found prions in pulp
prions are very small and heat resistant
very hard to sterilise the equipment
what is BSE and how does it lead to CJD
BSE is a prion infection in the brain of cows
cow brain fed to other cows spreading the BSE
cow brain fed in human food
causing CJD infection in human brain
how does CJD work
BSE prion from cow brain
Inhibit nerve and brain function
normal PrPC converted into ‘Rogue’ PrPSC
Aggregate into long fibres and amyloid plaques (the picture on the right is 3 subunits that would stack on top to form a fibre)
Loose ability to move, speak and process information
what antigens can be found on the outside of gram negative bacterial walls
H - flagella
K - capsule
O - LPS on outer surface
Peptidoglycan -
what is the H antigen
flagella
what is the O antigen
LPS outer surface
what is the K antigen
capsule
describe the cell wall of a gram negative bacteria
Double membrane asymmetric phospholipid bilayer
Thin layer of peptidoglycan in between membranes held to the outer surface with lipoproteins
Lipid A attached to a polysaccharide chain = lipopolysaccharide LPS antigen on outer surface = antigen O which deters complement factors preventing MAC
Polymer capsule antigen K which Deter antibodies and evade antibody binding
Therefor stops opsonization
what is the function of a bacterial capsule Antigen K
Deter antibodies and evade antibody binding
Therefor stops opsonization
what is the function of LPS antigen O
Deters complement factors preventing MAC
what is the structure and gram stain and main virulence factor of Neisseria and give 2 main examples
Gram Negative Diplococci
variable capsule being virulence factor
N. Meningisitis
N. Ghonnorreha
what can N.meningitidis cause and what are the symptoms of this
meningitis
symptoms include:
Rash doesn’t blanch
Stiff neck
Cold feet
Fever
Bright light intolerance
Vomiting
what is meningitis
Infection of CSF and meninges
Commensal carriage in pharynx/nasopharynx can enter the bloodstream or go directly into the sub-arachnoid plexus
Grow in the CSF
Inflammation of capsules around the brain e.g. meninges put pressure on the brain
what vaccines are used against meningitis and what are the different types
N.meningitidis
type A,B,C,Y,W135 based on capsule variance
B vaccine and tetravalent A,C,Y,W135
must be above 2 years old
what is the most common capsule type of N.Meningitidis
B capsule serogroup
what % of people will carry meningitis in nasopharynx
10-25%
50% in winter seasons
what are the common age/place outbreaks of meningitis
1st year of life and 16-23 at university
2/3 of cases are under the age of 5
Sub-saharan - African meningitis belt through mid africa with high prevalence e.g. MECCA
what vaccine must people have before going on a MECCA pilgrimage
meningitis ACY135
how do we diagnose meningitis
very quick in first 24 hours
CSF has many Polymorphonucelated Leukocytes PMNLs e.g. neutrophils and presence of diplococci
Blood/CFS/nasopharyngeal swab cultures
White cultures on chocolate and blood agar
DNA PCR for meningitis
what is the chance of passing meningitis on to close contact
1 in 300
how do we treat meningitis
B-lactam antibiotics
sometimes accompanied by corticosteroids
VACCINATION
what are fastidious microorganissm
microorganisms that are difficult to grow in the laboratory because they have complex or restricted nutritional and/or environmental requirements
how do we grow Neisseria
heated blood chocolate agar with CO2
fastidious
what is the morphology and gram stain of haemophilus influenza
gram negative coccus-bacilli (oval shaped)
what can H. Influenza cause and how does it enter the blood
meningitis and pneumonia (4 months-2 years)
Non-invasive disease : otitis media, sinusitis (2 - 4 yrs old)
septacaemia
Invasion by penetration of submucosa of nasopharynx
how many survivors of H.Influenza have disability after
15-35%
what capsule types can we get with H. Influenza and how do they act
A-f and some without capsules = commensals
capsule major virulence factor avoidance of c3b binding
which capsule type of H.Influenza causes 99% of invasive disease
B
How can we tell the difference between H.Influenza meningitis and N.Meninigitis
We cannot tell clinically
would have to use microscopy or PCR
N. Miningiditis = gram negative diplococci and white colonies on heated chocolate agar
H. Influenza = gram negative bacilli oval shaped
which vaccine works against H.Influenza infections and when is it given
Hib - typhoid toxin polysacchardie- protein conjugate vaccine
Haemophilus Influenza Type B vaccine
given at 2, 3, 4 months
what 2 factors does H. Influenzas require to live
X (haemin) and V (NADH)
how do we treat meningitis or non-invasive H. INfluenza
meningitis: Ceftriaxone/ Cefotaxime
non-invasive: Amoxicillin
what is a pathogens R number
its rate of spread from patient to patient
compare the bubonic plague and the pneumonic plague
bubonic = 75% death with no person to person spread
Pneumonic= 90% death with high person to person spread
what pathogen causes the plague
Yersinia pestis
what are the virulence factors of Yersinia Pestis (4)
Gram negative- has LPS (antigen O)
Contains three large virulence plasmids
Encode type III secretion needle for injection of toxins into host cells: suppress immune response, promote bacterial invasion and survival inside host cells
Can degrade C3b and C3a
what pneumonic describes antibiotic resistant bacteria
ESKAPE
Enterococcus faecium (G+)
Staph aureus (MRSA) (G+)
Klebsiella pneumoniae (G-)> hospital pneumonia/ sepsis in UK> common in environment
Acinetobacter (G-)> more of a problem in developing countries (but multiply resistant)- common in environment
Pseudomonas (G-)
Enterobacter (G-)
G- are more of a worry as becoming resistant to last resort antibiotic: Carbepenem
what atibiotic are some G- bacteria becoming resistant to, and which bacteria are these?
last resort antibiotic Carbepenem
Klebsiella pneumoniae (G-)> hospital pneumonia/ sepsis in UK> common in environment
Acinetobacter (G-)> more of a problem in developing countries (but multiply resistant)- common in environment
Pseudomonas (G-)
Enterobacter (G-)
describe the shape/G stain and relevance of Pseudomonas aeruginosa
Gram Negative motile Rod
becoming antibiotic resistant to carbepenem and major killer of cystic fibrosis
where do we find Pseudomonas aeruginosa in the environment and humans
soil and ground water
commensal bacteria found in gut
how does Pseudomonas aeruginosa appear on Agar
green spreading colonies
grape smell
what is the major killer of cystic fibrosis patients
Pseudomonas aeruginosa
leads to septicaemia and gangrene
well adapted to warm, moist conditions e.g lung/burn
how many species of staphylococcus are there
40
what is the coagulase test
some bacteria produce an enzyme called coagulase that clots blood plasma
plasma is mixed with bacteria to determine if they are coagulase + or -
where do we find most staph
nose and skin
what type of staph are important in opportunistic disease
coagulase negative
Staph aureus
Staph epidermisis
what are carrier and shedders
they carry the same load of microorganism
shedders spread disease quicker as they ‘shed’ the pathogen more
how would we identify Staph. Areus
Mannitol salts agar
selective for Gram positive, S.aureus ferments mannitol → yellow droplets on agar
how would we describe staph aureus (3)
gram positive
coagulase positive
Beta Haemolytic
what are some virulence factors of Staph aureus
Pore-forming toxins (some strains) = penumonia
Proteases = Exfoliation = exfoliation of skin = scalded skin syndrome < 2 year olds
Toxic Shock Syndrome toxin (TSST) stimulates cytokine release- tampons
Protein A (surface protein which binds Ig’s in wrong orientation)
what produces toxic shock syndrome toxin
Staph. Arues
what causes scaled skin syndrome
staph. Aureus
proteases that reduce connections between cells
what are some pyogenic associations with all staphylococci
Wound infections
Abscesses - boils
Septicemia
Endocarditis
Pneumonia
osteomyelitis
what are some toxin mediated associations with Staph Aureus
Food poisoning
Toxic shock syndrome
Scalded skin syndrome
what are some associations with coagulase negative staphylococcus
Infected implants
Septicemia
Endocarditis
what is the most common coagulase negative and coagulase positive staph
+ve = staph aureus
-ve = staph. epidermidis
how would we identify staph epidermidis (4)
gram positive (gram stain)
Staphylococcus
Coagulase negative
white colonies on blood again (previously called staph albicans)
how does Staph Epidermidis act
opportunistic on skin
forms strong biofilm (main virulence factor) around prosthesis or catheters
someone has an infection around their catheter, resistant to antibiotics. What is the cause
staph epidermidis
strong biofilm made preventing antibiotic passage
what is staph epidermidis’ main virulence factor
ability to form strong thick biofilms
what are the two main coagulase negative staphyloccous
staph epidermidids
staph saprophyticus
what is haemolysis and give the types that bacteria can be and how can we test this
haemolysis is the breakdown of blood
Alpha Hemolysis is the process of incomplete destruction of red blood cells in the blood
Beta Hemolysis is the process of complete destruction of red blood cells in the blood. Gamma haemolysis is no break down
Grow on blood agar
what does alpha, beta and gamma haemolytic bacteria present as on blood agar
alpha = partial breakdown = green/yellow patches
beta = full breakdown = clear patches
gamma = no breakdown = red
what is the main beta haemolytic, gram positive staphylococcus
staph aureus
what is the main alpha haemolytic staphylococcus
Staph Intermedius (also coagulase positive )
what causes beta or alpha haemolysis
beta = release of haemolysins that break down blood
alpha= release of H202 and reacts with Hb to partially destroy RBC
how do we classify streptococci
Haemolysis
Lancefield Grouping
what is Lancefield grouping
a method of grouping coagulase negative bacteria based on bacterial carbohydrate cell surface antigens
Lancefield A-H and K-V (A and B most pathogenic)
Antiserum is added to suspension of bacterial colony and clumping indicates recognition = +ve
what Lancefield groups are there and which are most pathogenic
Lancefield A-H and K-V
A and B most pathogenic
A = Strep. Pyogenes
B = Strep .agalactiae
how would we identify Strep. Pyogenes
Gram positive
Streptococcus
Coagulase negative
Lancefield type A
what can Strep Pyogenes cause
Cellulitis
Pharyngitis
tonsillitis
Impetigo
Scarlet Fever
what is Impetigo
skin infection leading to sores on the face and skin that burst after about a week and cause yellow, crusty scabs
Strep. Pyogenes
what is scarlet fever
red rash that affects skin and tongue often in babies (red on white skin, darker skin no colour change but texture change)
Strep. Pyogenes
what can follow tonsillitis and what can cause this
Rheumatic Fever
Strep. Pyogenes
what is rheumatic fever and what can cause this
Inflammatory disease of heart, joints, skin, brain. Often follows Strep. throat infection - follows tonsillitis - Strep. Pyogenes
how can we test the likelihood of Strep. Pyogenes leading to more complications like rheumatic fever and Glomerulonephritis
anti SLO titre
what are some virulence factors of Strep. Pyogenes
Streoptolysins O and S for haemolysis
Streptokinase breaks down clots
C5a peptidase - reduces chemotaxis + vasodilation
Erythrogenic toxin - SPeA – exaggerated response = similar to TSST
explain the Anti SLO titre and what it is used for
used to test likelihood of Strep. Pyogenes patients developing rheumatic fever and Glomerulonephritis. Tests amount of anti SLO in patients plasma
ASLO – Antibody against SLO found in plasma
ASLO reacts with SLO (streptolysin O) to neutralise haemolytic activity
Patients serum is serially diluted with constant concentration of SLO & RBCs added
Dilution of serum at which there is is still prevention of hemolysis of RBC = Anti SLO titre
give 2 ways of testing for anti - SLO in blood plasma and what does this tell us
ASLO titre with serial dilution of plasa with constant SLO and RBC
ASO rapid test where ASO binds to latex particles and presence of an ASLO titer of >200 IU/mL in the serum = agglutination of the latex particles.
Presence of anti-SLO means recent infection of Group A strep
explain the ASLO rapid test
Streptococcal exoenzymes are bound to biologically inert latex particles
If streptococcal antibodies present in the test sample, agglutination occurs.
presence of an ASLO titer of >200 IU/mL in the serum = agglutination of the latex particles.
Pt has antibodies and has had recent infection
where is Strep. Pneumoniae found
oro-pharynx of 30% of population
what does Strep. Pneumoniae cause
pneumonia, otitis media, sinusitis, meningiti
what are some predisposing factors of Strep. Pneumoniae
impaired mucus trapping (e.g. viral infection)
Hypogammaglobulinemia - immunocompromised
Asplenia - immunocompromised
what is Strep. Pneumonia virulence factors
Pneumolysin cytotoxin - causes lysis of cells
teichoic acid (choline) - binds to choline helping to bind to host cells
Peptidoglycan - inflammatory
polysaccharide (84 types), antiphagocytic
what are the 2 main vaccines for Strep. Pneumoniae
PCV - Pneumococcal conjugate vaccine (protein-polysaccharide conjugate) for children e.g. PCV13 - 13 most common carbohydrates in capsule
PPSV – Pneumococcal polysaccharide vaccine eg ‘PPV23’
polyvalent vaccine – against 23 most common types of capsule polysaccharide
available to >2 year olds at risk of infection and adults
what are viridians strep
collective streptococci of the oral cavity
what organisms might be accountable for deep organ abscesses
Viridians Strep
what is the tell tale sign of Corynebacterium diphtheriae
Pesduomembranes grey/green on tonsils and thick ‘bull neck
how would we grow diptheria
potassium tellurite
how do we detect C. Diptheria
Elek plate - strip of filter paper with antitoxin and streak suspicious bacteria along the filter paper and form precipitates if the toxin is being released)
Or PCR
other than C. Diptheria, what else can cause Diptheria
C. Ulcerans
what is the main cause of osetomyelitis
Staph Aureus
what are draughtsman colonies and what causes them
white, drafts pieces, raised edges, dimple in middle, colonies on blood agar
S. Pneumoniae
what is blood agar selective for
aerobic bacteria
what are the three types of anaerobe
obligate anaerobes = harmed by oxygen
Facultative anaerobes = can do both aerobic and anaerobic respiration
Microaerophiles- grow in atmosphere of low oxygen <5%
what is the maximum ATP/mol of glucose possible with aerobic respiration
38
instead of entering the oxygen dependant TCA cycle, what happens to pyruvate in anaerobic respiration
organic electron acceptors to produce
2ATP
Acids (VolitileFAs) - lactate is produced under extreme exercise
Alcohols
A lot of anaerobes cannot survive in the presence of oxygen due to the radicals produced. How does P.gingivalis survive these radicals?
P.Gingivalis reduces superoxide→peroxide→water = survive in oxygen
what is a effective antibiotic for anaerobes? how does it work
metronidazole
pre-drug is metabolised by anaerobes and produced metronidazole to kill bacteria
what is the morphology and products of Clostridia
Large, straight, Gram-positive bacilli
Produce endospores - inside cell
Produce exo-toxins
how does Cl. Perfringens show on a red blood agar
double beta haemolytic
fully clear in middle
encircled by a ring of partial haemolysis
what does Cl. Perfringes cause
Gas gangrene
food sickness
what is the leading 3 causes of food poisoning
norovirus
salmonella
Cl. Perfringes
what is the incubation period for Cl. Perfringens
10-12 hours
what is the incubation period for Cl. Tetani
10-14 days
what is the morphology, agar and gram stain of Cl. Tetani
drumstick rod with terminal ball end
thin white film on blood agar
gram positive
what is tetanus and how is it caused
Acute spastic paralysis caused by a potent bacterial neuro-exotoxin
locked jaw and uncontrolled contraction of muscles in CNS
Cl. Tetani spores enter wound and travel along nerves
what is the gram stain, morphology of Cl. Botulinum
Bacillus with sub-terminal spores
Gram positive
what does Cl. Botulinum cause
botulism severe food poisining
what is Cl. Botulinum incubation period
1-2 days
what does Cl. Difficile cause
pseudomembranous colitis, hospital/antibiotic related diorrhea
what causes Pseudomembranous colitis
Cl. Difficile
why is Cl. Difficile present in hosptial
lots of antibiotics, outcompetes other bacteria after use of antibiotics
very heat resistant and resistant to sterilisation
Travels in Faeces
how many toxigenic types are there
5 A-E
how do we detect toxins
Nagler Reaction Plate
describe Tetanus Toxin
Classic A-B neurotoxin called Tetanospasmin
A domain contains active site - Zinc endopeptidase - inhibits release of inhibitory trasmitter = continuous excitement
B domain> Carbohydrate receptor binding- Sialic acid containing poly-sialic-gangliosides
how do we treat tetanus
anti toxin
penecillin
metronidazole
how do we prevent tetanus
toxoid vaccination
how many cases of tetanus every year
15-30,000
how do we get botulism (3)
ingestion of the pre-toxin
soil or reheated food that has been left to cool for too long or home tinned foods
what does the botulism toxin cause
Flaccid paralysis - uncontrolled relaxation
Drooping eyelids, progressive
motor loss, flaccid paralysis;
Neurological symptoms- dizziness
respiratory & cardiac failure
describe the botulism toxin
potent neurotoxin
AB toxin with 7 types A-g
A B and E most common
A active site Zinc endopeptidase
Affects peripheral cholinergic synapses
blocks release of acetylcholine
IRREVERSIBLE BINDING!
how many types of botulism toxin are there and which are most cmmon
7 types
A-G
A B and E most common
how does the botulism toxin cause uncontrolled relaxation
A site is the active site
Zinc Endopeptidase
Irreversibly blocks release of acetylcholine so no excitation
how do we treat botulism
polyvalent anti-toxin
what can we use the botulism toxin for therapeutically
t can weaken muscles temporarily when injected in small amounts, used for treatment of spasms and dystonias, e.g
Strabismus (Cross-eyes)
Bruxism (tooth grinding)> linked to bone loss
Torticolis (neck spasms)
Muscle spasms in cerebral palsy
BOTOX cosmetics
how do we diagnose Cl. Difficle
Stool sample
how do we treat C. Difficle
remove offending broad spectrum, selective antibiotic e.g. clindamycin/penicillin
vancomycin or metronidazole
what are the virulence factors of Cl. Difficle and how do they act
TcdA and TcdB
inactivates GTPase Rho family
causes apoptosis and cell damage of intestinal epithelial
describe the morphology and gram stain of porphymonamos e.g. P. Gingivalis
Gram-negative, non-motile, rod-shaped
why do black pigmented anaerobes appear black on blood agar
they store oxidised iron which is black
what is the morphology and staining of fusobacterium
LONG Rod shaped spindle-shaped bacilli, Gram negative
where would we find Eubacterium and Bifidobacterium
isolated from abdominal infections POST oral/ genital trauma.
what relevance does Fusobacterium Nucleate have to cancer
Oral infection of F.Nucleatum in the gut is linked to colon cancer
FadA adhesin stimulates oncogenic response in colon- BIG news in microbiology- READ about it….
describe the general structure and staining of actinomyces
filamentous sometimes branched
gram positive with uneven staining
which bacteria is most found in actinomycosis
A. Israeli 70%
what would A.Meyeri cause
brain abscess
what type of bacteria are likely to cause brain abscess
A. Meyeri
name 2 type of actinomycetes found in caries
A. odontolyticus
A. gerencseriae
how do we grow actinomyces and what does it appear as
‘molar colonies’ will grow in an anaerobic brain heart infusion broth due to them being fastidious and facultatively anaerobic
how many species of actinomyces are there
21
where are actinomyces found
mouth, gut and vagina
commensals
what is Actinomycosis
Rather rare infectious agent
Painful slow growing abscesses – months to develop
Affected people have often just had dental treatment, poor oral hygiene, periodontal disease
Large abscesses – can penetrate bone and muscle to the skin where they can break open leaking pus
where do most actinomycosis occur
55-65% cervicofacial
what type of pus do we get from actinomycosis
‘sulphur pus’ yellow
what does actinomycosis look like histologically (3)
Locules of pus and filametous gram positive bacteria
surrounded by fibrous eosinic amorphous club shaped material
surrounded by dense sheets of neutrophils
how does an Actinomycosis abscess defend itself (2)
Amorphous polysacchardie protein matrix
Splendore-Hoeplli Club shaped cationic protein complex including fibrin and Ag-Ab complexes
Both wall off from host immune system
The characteristic formation of the peri-bacterial or peri-fungal Splendore-Hoeppli reaction prevents phagocytosis and intracellular killing of the insulting agent leading to chronicity of infection.
what disease is associate with Splendore-Hoeppli Phenomenon
Actinomycosis
what are the virulence factors of actinomyces
induction of chronic inflammation
walling off from defences
slow growth as large aggregates in a matrix
what is treatment for actinomycosis
Surgical removal/drainage
6-8 week course of penicillin, amoxicillin o tetracycline
what is the structure of candida
Trimorphic
blastopore (yeast) - circles
Pseudohyphae - short chains
Hyphae (chlamydospores) - long chains
what agar can we use for growth of Candida
Sabouraud’s dextrose medium SAB - straw colour
doesnt row bacteria, Candida. Albicans grows white colones
what is chromatic agar used for
growth of multiple fungi, differentiating by enzyme
compare the 3 forms of Candida Albicans
Yeast with daughter spores and scars from past spores
Pseudohyphae with mother and rod daughter which are walled off due to environment
Hyphae - no wall between mother and daughter
what is the main fungus and bacteria to cause infective endoarditis
S. Aureus
C. Albicans
What is Candidosis
Systemic infection of Candida Albicans
50% fatal
What is Candidosis
Infection/Colonisation of Candida Albicans on skin/mucosa
Systemic infection of Candida Albicans is uncommon but
50% fatal
what causes chronic Atrophic Candidosis
Dentures
what causes chronic hyperplastic candidosis
smoking
what causes acute pseudomembranous Candidosis
Trush/HIV
what causes Erythromatus Candidosis
Red patches = HIV
give 4 pre-disposing factors of Candidosis
Prostheses - no exfoliation, no desquamation, candida proliferation, hyphae
Low saliva - no flow; low soluble defences
Antibiotics - reduced bacterial competition
Immunosuppression - no T-cell defence
diabetes, immunodeficiency
Smoking
which type of form is more dangerous in fungi
yeast forms = commensals
Hyphae = Pathogenic
what pH favour hyphae/Yeast
under 6 = yeast
over 6 = hyphae
how do candida albicans attach to epithelium
Agglutinin-Like Sequence (ALS)
8 family members, ALS3 appears to be most important for adhesion to cells – only expressed by hyphae
how do hyphae invaginate into epithelium
Secrete inactive candid lysin - A pore forming toxin that kills human cells and also initiates an immune response
Hyphae go into well they create and carry on
what is candidolysin
A pore forming toxin that kills human cells and also initiates an immune response
what is SAP
secreted Aspartyl Proteases
secreted by the tip of Hyphal Penetration
what are 2 mechanicams important for hyphal penetration
SAP - Secreted Aspartyl Proteases
Candidolysin
what is the most common cause of hospital-acquired-related death in the US (even greater than MRSA !)
systematic Candidiasis
50% mortality
why can we use zebra fish ethically and biologically (2)
they are not classed as animals and their endothelium is flourescent
how does candida cause death
Enters blood stream and epithelial attachment occurs ALS (ALS3)
Travels along endothelium until a bend but carries on through endothelium
with SAP and actinolycin
causing infection around body in organs causing organ failure
how do we treat candidiosis
Fluconazole - antifungals
removal of predisposing factors
describe the structure and gram stain of spirochetes
gram negative very thin spiral shaped rods
how do spirocheates move
internal flagella
how are different spirochetes identified
Identification by Sequence analysis of 16s rRNA gene which is well conserved due to need for function
what and where are axial filaments found
found in the periplasmic space of spirochaetes
internal flagella
how many people have candida albicans
40-60% have candida albicans in their mouth
what is wild type fungus
mixture of yeast forms and hyphae forms
what would happen if the internal flagella were removed from spirochaetes
this would lead to the spiral shape going and the bacteria becoming straight non-motile
do spirocheates have LPS?
no
what are the three major spirocheates that cause clinical problems
Leptospira = Weil’s disease Leptospirosis
Borella = Lyme Disease, Relapsing fever
Treponema = Syphilis, ANUG, Periodontitis
what are zoonotic infections
They have an animal reservoir of infection that can under certain circumstances infect humans via a zoonotic vector
what bacteria causes Lyme’s Disease
Borrelia burgdorferi - zoonotic, tics
how can we view Borrelia burgdorferi
EM or darkfield microscopy
how does lymes disease spread
spirocheate lives in the midgut of rodents
Uninfected larval ticks feed on rodents and get bacteria in saliva
live in midgut
further move to saliva
ticks bite humans and when they d, they release anaesthetic so we don’t feel it. In doing so, releasing Lymes infected saliva
what symptoms is specific to lymes disease
Bulls eye rash
what happens if a patient goes undetected with lymes disease
flu like symptoms
bulls eye rash
30% patients get rash somewhere else on body
1 week to 2 year post infection = arthiritis, neurological problems due to bacteria hiding in joints/nerves
how do we treat lymes disease
doxycycline 100 mg twice per day or 200 mg once per day 3 weeks
Borrelia burgdorferi
how can we prevent Lymes disease
wear long clothes
DEET repellents
vaccination (pulled due to antivaxx movement)
what is the main virulence factor of Borrelia burgdorferi
motility
how does Borrelia burgdorferi survive in low iron levels
Copes with low-iron levels by using Manganese instead of iron as an enzyme co-factor
what is Borrellia Recurrentis and what does it cause and how
Spirochaete
recurrent fever of 3-10 episodes
due to genetic variation of surface protein
what causes Wiels Disease
“Leptospira interrogans
describe the shape of Leptospira interrogans
very thin, coiled spirochaete
Two terminal Periplasmic flagella
where would we find “Leptospira interrogans
stagnant water
near dog/rat colonies as in their urine
what is Leptospirosis
infection of
what are the symptoms of Leptosperosis and what can it lead to
Febrile, flu-like illness with muscle pain, reddening of eyes, and other symptoms such as diarrhoea and in some cases meningitis and Haemorrhage in Aqueous humour of eye and CSF
10-15% of cases develop Weil’s disease:
kidney failure
Jaundice and liver failure, yellow eyes
Most symptoms caused by damage to blood vessel
what are most symptoms of Leptosperosis due to
damaged blood vessels
How do we reduce cases of Leptospirosis and Wiels Disease
Rodent control
Doxycycline
Avoid swimming in infected rivers
why do patients with Leproperosis get red eyes
Haemorrhage in Aqueous humour of eye due to damaged blood vessels
how do we treat necrotising ulcerative gingivitis
metronidazole or Hydrogen peroxide washes
deep removal of plaque and bacteria
what causes ANUG
acute necrotising ulcerative gingivitis
Treponema Vincentii , Rods and fusobacterium (fuso-spirocheatal complex)
what are the red complex bacteria
Porphyromonas gingivalis
Tannerella forsythia
Treponema denticola
describe the structure of T. Denticola
spirochete, gram negative, anaerobic
what are T. Denticola’s virulence factors
Co-adherence with other oral bacteria in mixed biofilm and to the basement membrane
good at invading tissues and oral mucosa
Proteases
Motility due to internal flagella
compare narrow and broad spectrum antibiotics
narrow only aerobic/anaerobic or only gram positive/negative
broad can be negative AND positive or aerobic AND anaerobic
what is selective toxicity
where a drug has a high affinity and toxicity to bacteria
but low toxicity to human cells
the larger to theraputic index…
the safer the drug is for use
ideal characteristics of an antimicrobrial
High Theraputic index
target is is not present in man
if microorganism has higher affinity for the drug than man
It should be non allergenic to the host.
It should not eliminate the normal flora of the host.
It should be able to reach the part of the human body where the infection is occurring.
It should be inexpensive and easy to produce.
It should be chemically-stable (have a long shelf-life).
Microbial resistance is uncommon and unlikely to develop
what three ways can we classify anti-biotics
by chemical structure e.g. β-lactam ring
by target site
according to whether they are bactericidal (kill) or bacteriostatic (inhibit growth)
how can we test the efficacy of an anti-biotic (2)
disc plates on agar
lquid MIC/MBC testing
explain MIC MBC testing
MIC = minimal inhibitory concentration
MBC = minimum bactericidal concentration
Standardised [bacteria] + different [drug]
MBC usually higher than MIC
what are the main targets for antibiotics
Cell wall - peptidoglycan
Protein synthesis - ribosomes or enzymes unique to bacteria
Metabolic pathways
DNA
Membranes
Enzymes
what is antibiotic resistance
an organism that is not inhibited or killed by an antibacterial agent at concentrations of the drug achievable in the body after normal dosage.
give three ways of spread of antibiotic resistance
Some resistance is by chromosomal mutation, some is coded for by plasmid DNA
Some plasmids are transmissible - between species
Transposons can carry resistance genes and jump between chromosome and plasmids
what mechanisms might cause an antibiotic to no longer work (4)
The target is structurally altered (by mutation) -Now has a lower affinity for the antibacterial (PBPs) Penicillin-binding proteins
The target is overproduced Dihydropteroate synthetase (bacterial target) and sulphonamide
The drug is not activatedAerobes and metronidazole - intrinsic, not acquired resistance
The drug is removed e.g. Β-lactamase - breaks beta lactam ring produced by bacteria
how can we gain a blood sample for testing infections
needle aspiration
what organisms are likely to be found in pharyngitis
Streptococcus pyogenes: beta haemolytic, Coagulase -ve, Lancefield Group A
EBV (glandular fever is virus with no medication)
what type of bacteria are likely in a dental abscess
Viridans group streptococci
Anaerobes
Gram negative rods
what is the centor criteria
set of criteria to determine if a sore throat is of viral or bacterial origin
what are the set of centor criteria (CENT)
Bacterial if:
Cough is absent.
Exudates are present on the tonsils.
Nodes are tender in the anterior cervical region.
Temperature of 100.4 F by history (i.e. fever by history).
what determins if the antibiotic can penetrate the site (2)
the pH of the site - abscess/infection is low pH
weather the drug is lipid soluble
why is infective endocarditis hard to treat (3)
build up and infection of bacteria on the heart valves
most antibiotics work via the blood stream
heart valves do not have any blood supply
give an example of intrinsic resistance
Naturally resistant
pseudomonas is naturally resistant to penicillin
give some ways of finding if an antibiotic is effective
sensitivity testing with discs
breakpoint testing
tube dilution
explain how we can find the MIC of an antibiotic
MIC minimum inhibitory concentration
agar plat with cultured bacteria
place strip of increasing [antibiotic]
will form a oval shape, lowest [antibiotic] with clear zone is MIC
what are the major 2 determinants of effectiveness of an antibiotic
the concentration at the binding sites
the time that the antibiotic remains on these binding sites to allow sufficient inhibition of metabolic processes
compare concentration and time dependant killing
time dependant is done at a lower concentration, closer to the MIC for a longer time
concentration is done is done or less time at the peak concentration (higher [])
give a few ways that horizontal gene transfer can occur
Conjugation is where plasmids are shared (not in nucleus) by exocytosis or endocytosis e.g. MRSA
Virus vector damages bacteria and then released DNA spreading bacterial DNA
Transformation when bacteria dies its DNA is released and can be picked up
Resistance start to occur in the gut and where antibiotics have their major effect
what is the structure of herpes virus (3)
Icosahedral capsid surrounding dsDNA Virus
tegument glycoproteins on outside very important for specificity e.g. binding to oral mucosa
what are the three alpha-herpese viruses
Type 1 Herpes Simplex Virus
Type 2 Herpes Simplex Virus
Varicella-Zoster Virus (VZV)
what are the 3 beta-herpes viruses
Cytomegalovirus (HCMV)
Human Herpesvirus 6
Human Herpesvirus 7
what are the 2 gamma-herpes viruses
B-lymphocytes Epstein-Barr virus (EBV)
Human Herpesvirus 8 (important in HIV)
what mucosa does HHV-1 and HHV-2 affect and why does this occur
1 = oral mucosa
2 = genital and oral due to cross contamination
due to different tegument glycoproteins on outside
what are the two stages of herpes viruses
infection
reactivation
what are the two stages of HHV-1 (3)
Herpes Simplex (infection)
Herpetic Gingivostomatitis
lesions around the tongue causing epithelial damage, becoming latent by transferring from epithelium to the nervous system to the trigemnical ganglion. This stays for ever and is never removed
50% of cases = retrograde infection –>
Herpes Simplex (reactivation)
Herpes Labialis (cold sores)
what can cause secondary herpes simplex re-activation
in 50% of patients, virus will travel down from the trigeminal ganglion and re-infect the neurones it spread from
this can be caused by many factors:
UV light
Stress
Illness
Immuno- suppression
of those infected with primary herpes infection, how many are symptomatic and what would they present with
1-10% are symptomatic
herpetic gingivostomatitis
what is the incubation, duration and symptoms of herpatic gingivostomatitis
Incubation period 3-10 days
Duration 5-14 days
Multiple vesicles - rupture to form extensive sloughing ulcers
Gingivitis with erythema and sloughing
Malaise, pyrexia, lymphadenopathy
how could we diagnose gingivostomatitis genetically and histologically
genetically = PCR
histologically = ballooning of epithelial cells due to intraepithelial vesicles
how do we treat herpatic gingivostomatitis (3)
Acyclovir (500mg 5 x daily for 5 days) if found early or in immunocompromised
Fluids and soft diet
Analgesics / antipyretics (paracetamol) Local antiseptics e.g. chlorhexidine Topical analgesics e.g.
what is added to cordysol toothpaste apart from flouride
CHX
how does Acyclovir work (4)
mimics structure of guanosine
when HSV replicates, TK enzyme phosphorylates Acyclovir
implemented into DNA translation and acts as a gene terminator
prevents formation of new proteins
What is herpes Labialis and what are its clinical features
Prodromal irritation - tingling (coming down neurones)
Vesicles at or near mucocutaneous junction of lips (or skin, chin)
Crusting lesions lasting 7-10 days Usually re-occurs at the same
what is management of Herpes Labialis
5% Acyclovir cream
prophylactic acyclovir very rare - only in immunocompromised patients or if patients are having very frequent problems
what are the effects of using prophylactic Acyclovir for Herpes 1
reduces duration of cold sores
reduces pain
Reduces re-occurance by 50%
what is herpatic whitlow and how can we prevent it
very painful herpetic infections of the fingers of those handling patients’ oral tissues with herpes simplex 1 Virus
prevent by wearing gloves and good cross contamination control
what is HSV encephalitis and what does it effect
swelling of frontal lobes of brains in 1 in 500,000 patients
Usually only people >50 years (HSV-1) and neonates (HSV-2) affected
what is the mortality and recovery rate of HSV encephalitis
70% - 80% mortality if untreated
Of survivors, only 3% return to normal
how might we tell if an adult has HSV encephalitis
headaches
change in behaviour over a few days
history of HSV infection
what are the two stages of VZV
varicella = chicken pox = primary infection
Zoster = shingles = secondary infection
at what age do 70% of shingles cases occur
> 50
what 4 factors lead to increased chances of developing shingles in the future
Age (70% >50yrs)
Stress
Illness
Immunosuppression
explain the progression of a shingles belt
blisters of epithelial balooning cells intracellular vesicles
turns to rash
release and heal
contagious throughout
what are the three phases of oral herpes zoster
Pre-herpetic neuralgia
Rash
Post-herpetic neuralgia
what is oral herpes zoster
infection of VZV along the trigeminal nerves from the trigeminal ganglion
explain pre-herpetic oral zoster
Pain in the distribution of the affected division of the trigeminal nerve
Prior to the development of the rash
May mimic dental pain = think shingles if tooth ache with sound teeth
if a patient has sound teeth but is complaining of tooth ache in a lower or upper reigon, what might this be
oral zoster pre-herpatic symptoms
explain the rash of oral zoster virus
Unilateral vesicles in the distribution of a branch of the trigeminal nerve: Ophthalmic, Maxillary, Mandibular
Vesicles break down to form
Ulcers (mucosa)
Crusting lesions (skin)
Last 2-3 weeks
which division of the trigeminal ganglion is oral zoster most likely to have severe implications
opthalmic divison:
glaucoma, cataract, double vision and scaring of the cornea
= shingles of the eye send to hospital immediately
how do we manage oral zoster
cyclovir 800mg 5 x daily for 7 days if seen soon after lesions develop
Analgesics
Ophthalmic referral if eye involved
Avoid contact with children
what is post-herpetic neuralgia of oral zoster
continuous pain after reactivation in the elderly
not well understood
10% of patients go on to get extremely unpleasant intractable burning pain in the distribution of the affected nerve
More common in the elderly
Effective early treatment of zoster may ↓ risk of neuralgia
Treat pain with tricyclic anti-depressants and neuropathic pain drugs
how do we treat post-herpetic oral zoster
Treat pain with tricyclic anti-depressants and neuropathic pain drugs
how many patients with shingles will get post-herpetic neuralgia
10%
what is EBV
Epstein Barr virus affecting B cells
beta-herpes virus
depending on the local environment, can cause 4 different diseases
depending on the local environment, can cause 4 different diseases. what are they
Infectious Mononucleosis (Galndular Fever) – acute 1º infection with EBV - kissing disease
Burkitt’s Lymphoma – a B-cell malignancy
Nasopharyngeal Carcinoma – an epithelial cell malignancy
Oral Hairy Leukoplakia – seen in AIDS patients and some transplant recipients
where does EBV initiate and then where does it remain latent
Primary infection EBV replicates in oro-pharyngeal epithelial cells but then establishes latency in B- lymphocytes.
how many people have EBV
95%
what are symptoms of symptomatic EBV
Symptoms include sore throat, swollen cervical lymph nodes and mild fever, Petichae on palate (red spots)
a young patient has a sore throat, cervicale lymphadenopathy and petechiae. what are they likely to have
Infectious mononucleosis (Oral) from EBV
where is Burkitts syndrome prelevent and what is it
b cell carcinoma caused by EBV
found in tropical Africa at elevations below 1500 metres where malaria is present
how do we treat burkitts syndrome and why is this not usually done
cyclophosphamide (chemo)
relatively cheap but not available where this disease is prelevent:
tropical Africa at elevations below 1500 metres where malaria is present
a child, recently migrated from tropical Africa, has a very swollen jaw/face and is generally ill. what are they likely to have
Burkitts syndrome
B cell carcinoma
caused by EBV
how does Burkitt’s syndrome often present
usually forms a bone tumour on the mandible
where is nasopharyngeal cancer related to EBV and why
japand
due to high soya eating
how might cytomegalovirus present
Glandular fever-like illness
Salivary gland swelling
rarely causes illness in healthy individuals
what might CMV cause in immunocompromised patients
Large ragged oral mucosal ulcers
Salivary gland swelling
Retinitis
what is the cause of kapsosis sarcoma in AIDs patients
HSV-8
what does HSV-8 cause
usually has no effect
in very immunocompromised patients (AIDS) the patient will get Kapsosi’s Sarcoma = very large, red swollen gingiva
what is HIV
human immunodeficiency virus
causes depletion of host immune system resulting in AIDs
what is HIV
human immunodeficiency virus
causes depletion of host immune system resulting in AIDs
type of lentivirus - slow growing retrovirus
what is AIDs
Acquired Immune Deficiency Syndrome
give 5 ways HIV is transferred
sexual contact
blood contamination
placental carriage to uterus
breast milk
infected blood products e.g. meat
what are, and compare breifly, the two types of HIV
HIV 1 = more common, more symptomatic and more easily tranferred
HIV 2 = less pathogenic and less easily tranferred, localised to western africa
why does HIV mutate readily
has no proofreading genes
if mistakes are made during reverse transcription, there is nothing to correct these mistakes
forms new genes and new proteins
what do M, O and N stand for e.g. HIV-1m
m = main/pandemic
n = new
o = outlier e.g. confined to cameroon
describe the structure of HIV (5)
2 single stranded RNA
reverse transcriptase
proteases
protein core Capsid
Capsule lipid membrane with glycoproteins:
-gp120
-gp41
(begin as one strand, then get cut in two by the proteas)
what are the major two glycoproteins on HIV
gp120 and gp41
(begin as one protein but then cut in two by a proteas)
explain how HIV binds to cells, and which cells
CD4 cells macrophages and T-cell
have receptors for gp120 and gp41 expressed by HIV
intial attachment CD4 - gp120
co-receptor attachment CCR5 receptor - gp41
how might someone be naturally immune to HIV
HIV attaches gp120 to CD4
then co-receptors need to attach gp41 - CCR5 (macrophages)
if pt has mutation in CCR5 protein, co-receptor cannot occur so HIV cannot attach to CD4 cells
how many people are immune to HIV and how
2-14% europeans (caucasian) , and 15% of Icelandic
mutatin in CCR5 gene that is used by HIV to attach via gp41
after binding, how does HIV proliferate
uncoated and release capsid releasing RNA strands
reverse transcriptase
into DNA double stranded
incorporated into genes of host immune cell
transcribed when the cell replicates
more replication occurs due to infection, making more HIV which destrosy CD4 cells
after binding, how does HIV proliferate
uncoated and release capsid releasing RNA strands
reverse transcriptase
into DNA double stranded
incorporated into genes of host immune cell
transcribed when the cell replicates
more replication occurs due to infection, making more HIV which destrosy CD4 cells
what is the most important post-translational modification with HIV
the snipping of glycoproteins into two seperate glyocproteins gp120 and gp41 for attachment to CD4 and CCR5 (on macropahges, or CXCR4 on T cells) respectivly
explain the virus variation during HIV infection
Isolates from early in infection- CCR5 (M) macrophage tropic and low cytopathic effect- more transmissable
Isolates from late infection- CXCR4 (T) high cytopathic ability – less transmissable, causes more problems
explain the course of HIV infection
initial infection peaks CD4 cells
viral load increases to peak at 6 weeks
= flu symptoms
viral load steady level
Cd4 slowly reduce over 3-10 years
when Cd4 < viral, viral laod increases massively
oppertunistic diseases show now
what are some AIDs defining illnesses
Candidia Albican oral infection
Kapsosis Carsoma HHV-8
HSV- Herpes simplex virus- chronic oral infection
EBV- Hairy leukoplakia, and B- cell lymphomas
Cryptosporidum (chronic diaarhoea)
Toxoplasma gondii (disseminated, including CNS- from Cats) - most common diagnostic disease, can get from cats (Avoiddealing with ctas if pregnant and lower immune system)
what is cryptosporidum
chronic diarrhea
aids defining
what is toxoplasma gondii
Common parasite, common in uncooked meats or cat litter
causes toxoplasmosis = gives un-noticed symptoms of flu syptoms for 7 days
in reduced immune system (pregnancy, HIV) can be life threatening
most common diagnostic tool for HIV
why should pregnnat women not handle cats
prengnancy = immunocompromised
more suseptable to toxoplasmosis caused by the common parasite toxoplasma gondii found in cat litter and blood
what would the mouth of a pt with HBV look like
hairy, striated leukoplakia on tongue
give 4 oral representations of AIDS
hairy, striated leukoplakia - HBV
oral candidiasis - thrush
erythematous candidosis - red markings
gingival erythema
why does HIV treatment rely on compliance (2)
drugs have a short half life so must take regularly
have to take in combination so multiple drugs
name some targets for HIV treatment
protease inhibitors (no gp120 and gp41)
Reverse transcriptase inhibitors (no DNA)
CCD5 entry inhibitors (no co-receptor)
what are NNRTI and NRTI
(non) nucleoside reverse transcriptase inhibitors
main tx for HIV
how do NRTIs work
nucleoside reverse transcriptase inhibitors
similar shape to a nucleotide
implemented into growing nucleotide chain
terminates the chain = no DNA formation
what is HAART
highly-active anti-retroviral treatment
what are zidovudine (AZT), lamivudine (3TC), emtricatabaine (FTC), stavudine (d4T)
NRTIs for HIV tx
what are Ritonavir, Indinavir (IDV), Fos-amprenavir (FPV) and what are there side effects
protease inhibitors
lipodystrophy- fat loss from legs, fat gain-pot belly
a pt is on treatment for HIV and also has a pot belly and thin legs. what are they taking
protease inhibitors e.g. ritonavir
which NRTI do we use less now
AZT - zidovudine
headaches and nausea, anaemia, neutropenia
what can be a side effect of NNRTIs
Stevens Johnson Syndrome: a severe disorder of mucous membranes
teratogenecity
what is Stevens Johnson Syndrome and what causes it
severe disorder of mucous membranes following flu like symptoms
caused by drugs e.g. Carbamazapine, NNRTIS for HIV and Phenytoin
how can we prevent dental contamination with HIV
Normal infectious control procedures
Gloves
Sterilise instruments
Dispose of sharps
Suction
Care if blood spillage
If at risk- contact Occupational health physician for prophylactic HAART and HIV testing
If needlestick of HIV + patient» PEP administration ASAP and within 72 hours at longest. (using this method NO NHS employee was infected (last 10y- no sero conversions – out of around 600 incidents).
what is PEP
post exposure prophylaxis
if we get a needlestick with a pt possibly have HIV what do we do
PEP < 72 hours
test at 3 months where virus would be at its highest
how do we test for HIV
ELISA or PCR
