Antibiotic Resistance Flashcards
Which 2 mechanisms are used in identification of antibiotic resistance?
- antibiotic sensitivity testing
- detection of antibiotic resistance genes
What is the background behind how antibiotic sensitivity testing works?
- try to grow the organism in the presence of an antibiotic
- if it grows in the presence of a high concentration (high MIC), it is resistant
- if it is killed at a low concentration (low MIC) it is sensitive
- the lower the MIC, the more sensitive the organism
What are you actually identifiying through antibiotic sensitivity testing?
you are looking at the phenotypic characteristics of the organism and whether it is sensitive to antibiotics
the phenotype is either resistant or sensitive
the phenotype is determined by the presence of resistance genes
What is show here?
microtitre plate
this is used in susceptibility testing to calculate MIC
How is the microtitre plate organised for susceptibility testing?
6 different antibiotics are added to the plate, along with 2 controls
- no antibiotic
- no bacteria
doubling dilutions are performed
the starting concentration is 32 micrograms/ml and this is halfed each time until you reach 0.015 mcg/ml
How much bacteria is added to each of the wells in susceptibility testing?
the same amount of microorganism from liquid culture is added to each of the wells, despite the concentration of antibiotic
the plate is then incubated for 24 hours
What is shown by these results?
where the organism has grown, the concentration of the antibiotic in that well is insufficiently high to inhibit the growth of organism in that well
e.g. for antibiotic 1, the MIC (minimum inhibitory concentration) is 0.125 mg/L
What does the MIC show about the resistance of the bacterium?
MIC is the minimum concentration at which growth of the bacteria is inhibited
How can you determine whether an organism is sensitive or resistant from a microtitre plate?
you need to compare the MIC to the break point value
MIC doesn’t show the availability of the antibiotic within the human body
the breakpoint is the MIC value which has been determined as sufficiently low, that if the MIC is this value or less, then the organism is sensitive
What does the result of the susceptibility test show?
it shows whether the bacteria causing an infection is likely to be sensitive or resistant to an antibiotic
this doesn’t mean that the patient will respond to an antibiotic to which the organism is sensitive
it just means that they are more likely to respond
What are the stages involved in disk sensitivity testing?
- add organism
- add antibiotics
- incubate
- compare zone sizes against published breakpoint zone sizes
- interpret and report results
How does a disk sensitivity test work?
the organism is spread evenly over the surface of the agar plate
antibiotics in the form of filter paper discs that are impregnated with known concentrations of 6 different antibiotics are placed on the surface
the antibiotic diffuses into the agar and the agar plate is incubated
What is meant by the zone of inhibition?
it is the area around the antibiotic disc where the bacteria has not grown
if the zone of inhibition is large then the antibiotic has diffused a long way out and is still killing the organism
this bacterium is senstive to the antibiotic
What is meant by breakpoints in disk sensitivity testing?
it refers to the sizes of the zone of inhibition
the size of the zone must be measured to determine whether the antibiotic is sensitive or resistant
Which test is used in detection of antibiotic resistance genes?
nucleic acid amplification tests
e.g. polymerase chain reaction
Which samples are typically used in detection of antibiotic resistance genes?
it can be applied to any sample type
- nose / skin is used in MRSA testing
- rectal / faecal is used to detect carbapenemase producing Enterobacteriales (CPE)
What are the advantages and disadvantages to detection of antibiotic resistance genes?
advantages:
- sensitive and fast
disadvantages:
- expensive
- presence of a gene doesn’t always correlate with resistance
- you can only test for resistance genes that are already known, but a new one could be present
What is meant by innate resistance mechanisms?
this is a fundamental property of the bacterium / antibiotic combination
it usually relates to the permeability / entry of the antibiotic into the cell
What are examples of innate resistance mechanisms?
gram-negatives:
- glycopeptides
- daptomycin
gram-positives:
- aztreonam
- colistin
anaerobes:
- aminoglycosides
streptococci:
- aminoglycosides
the antibiotic will never work against the organism no matter how it is used
Why can aminoglycosides not be used against anaerobes?
the process of taking an aminoglycoside into the cell is an aerobic process
anaerobic bacteria cannot take the aminoglycoside into the cell, so it cannot exert its effects
What is meant by an acquired resistance mechanism?
this is the acquisition of a gene that encodes an antibiotic resistance mechanism
it can be a new mutation or acquired through horizontal transfer
What are the most common acquired resistance mechanisms?
the new gene usually codes for an antibiotic-modifying enzyme or a target alteration
What are the 5 ways in which a bacterium can become resistant to an antibiotic?
- absent target
- decreased permeability
- target modification
- enzymatic degradation
- drug efflux
What is meant by “absent target” as a mechanism of resistance?
this is an innate mechanism
there is no target for the antibacterial to bind to, so it cannot exert an effect
e.g. treating a virus or fungus with an antibacterial
What is meant by “decreased permeability” as a mechanism of resistance?
the antibacterial agent must be able to enter the cell in order to exert an effect
organisms can have decreased permeability to prevent the antibiotic from entering the cell
this can be innate or acquired (e.g. developing thicker cell walls)
What are 2 examples of decreased permeability as a mechanism of resistance?
vancomycin & gram-negative bacteria:
- gram-negative bacteria have an outer membrane that is impermeable to vancomycin
gentamicin & anaerobic organisms:
- uptake of aminoglycosides requires an O2 dependent active transport mechanism
What is the main example of ‘target modification’ as a mechanism of resistance?
flucloxacillin & MRSA
the altered penicillin-binding protein does not bind to B-lactams
if the carboxypeptidases and transpeptidases (PBPs) are altered, then antibiotics can no longer bind to them but they can maintain their functions
What are other examples of target modification as a mechanism of resistance?
vancomycin and VRE:
- altered peptide sequence in gram-positive peptidoglycan
- this reduces the binding of vancomycin
trimethoprim & gram-negative bacilli:
- mutations in dihydrofolate reductase gene
What is meant by “enzymatic degradation” as a mechanism of antimicrobial resistance?
the antibiotic enters the cell, an enzyme binds to it and breaks it down
the antibiotic cannot have any effect on killing the cell
What are examples of enzymes that are produced that can break down antibiotics?
B-lactamases:
- these will break down penicillins and cephalosporins
- includes penicillinase, extended spectrum B-lactamases (ESBL) and carbapenemases
- other enzymes inactivate aminoglycosides and chloramphenicol
What is meant by an extended-spectrum B-lactamase (ESBL)?
these are capable of breaking down a large range of B-lactam antibiotics
What is meant by “drug efflux” as a mechanism of resistance?
the efflux pump will actively pump the antimicrobial out of the cell so it cannot have any effect
these are especially present in Gram-negative organisms
How are most resistance mechanisms encoded?
many resistance mechanisms are encoded by single genes
e.g. antibiotic-modifying enzymes
Resistance genes are encoded in plasmids
these are circular DNA sequences that are transmitted within and between species
What is meant by horizontal transfer?
What enables this process?
this is enabled by transposons and integrons
DNA sequences are designed to be transferred from plasmid to plasmid and/or from plasmid to chromosome
often contain cassettes with multiple resistance genes
What is meant by vertical transfer as a mechanism of spreading resistance?
chromosomal or plasmid-borne resistance genes transferred to daughter cells on bacterial cell division
What are the stages involved in the development of resistance?
- bacteria are constantly subject to spontaneous gene mutations
- mutations that confer a survival advantage favour the growth and propagation of the mutant strain
- if the organism is growing in the presence of a sub-inhibitory concentration of antibiotics the development of a resistance gene will confer a survival advantage
- the resistant mutation will have a survival advantage and out-compete other strains
How are resistant bacteria established within the human microbiome?
resistant bacteria gain access to the human microbiome
once established in the microbiome, resistance genes can be transferred between bacteria of different species
the result is a human microbiome that contains a mixture of sensitive and resistant bacteria
What happens if the human microbiome containing sensitive and resistant bacteria is exposed to antibiotics?
the sensitive bacterial strains will die out and strains carrying resistance genes will become dominant strains
subsequent endogenous infection is more likely to be caused by antibiotic resistant organisms
What are the 2 distinct ways in which antibiotic resistance can arise in a patient?
Which methods are in place to prevent this?
- exposure to antibiotics
- transmission of resistant organisms
the methods used to prevent antibiotic resistance are:
- minimising use of antibiotics (“antibiotic stewardship”)
- effective infection prevention and control
What is the positive aspect to antibiotic resistance?
resistance mutations usually affect vital bacterial cell functions
e.g. cell wall construction, DNA synthesis
antibiotic resistant organisms are often less fit than the strains that do not carry resistance genes
if antibiotic exposure is ceased, the resistant organisms are often out-competed and replaced with sensitive organisms
What are the practical consequences of antibiotic resistance?
bacterial infections become resistant to antibiotics that are traditionally used to treat them
What are the implications of resistance on empiric therapy?
there is a risk of undertreatment if a traditional antibiotic is used and treatment is not changed in accordance to developing resistance
there is a risk of excessively broad spectrum treatment if the risk of resistance is taken into account
