lecture 2 Flashcards
explain the evolution of antimicrobial resistance.
This schematic describes the process of a sensitive population becoming a resistant one.
Within any bacterial population you have mutations-
the process of copying the genome introduces mistakes-
These mutations don’t occur often: 1 in every 106- 108 cells-
However a single colony of bacteria can contain millions of cells.
In a population of millions of cells some of these mutations will confer resistance to antibiotics.
Under normal conditions these mutations may be detrimental to the bacteria- it may slow down their growth,
and so under normal conditions these mutated bacteria are at a disadvantage.
The problem comes when you treat this population with an antibiotic- you select for the cells which have resistance-
The cells which have resistance survive, and then divide, and you end up with a resistant population-
you have turned a sensitive population into a resistant one.
Over time bacteria will carry on accumulating more mutations, and gain greater resistance to an antibacterial or resistance to another antibacterial drugs.
(For context – E.coli has a doubling time of 20 mins, and a typical stationary phase culture culture will have a cell number of 1x109- or more )
UK government 5 year plan and global concern on antimicrobial resistance
The UK Government is concerned about the development of resistance
They have developed a 5 year action plan- the current strategy takes us to 2024
The latest strategy focus’ on reducing the use of antibiotics. – by lowering the burden of human infection.
Eg improving sanitation and water quality- resistance is a global problem- and the strategy is acknowledging that global action is needed to resolve it.
Another major concern is the use of antimicrobials in farming. Eg using antibiotics as a food supplement to improve yields- this is still common practices in some countries .
Optimising the use of antibiotics eg
better diagnostic tests and surveillance, so we are only prescribing antibiotics where necessary- and these are the correct antibiotics. and using good antibiotic stewardship
Investing in innovation –developing new antibiotics isn’t particularly profitable for drugs companies-
If we develop new antimicrobial drugs we are likely to want to reserve them, until we need them to treat the most significant multi drug resistant strains-
so changes in the profitability, or patenting might help in the development of new therapeutics
Innovation Also includes the development of new vaccines and testing –
reduces the need for antibiotics, and targeting them for use only when necessary
what factors promote resistance
overuse, overprescribing, aging population, use in farming, lack of development of new classes of drug
Organisms currently of particular concern are
Multi-drug resistant Gram negative pathogens:
Klebsiella pneumoniae
Pseudomonas aeruginosa
Escherichia coli
Multi drug resistant Gram +ve – MRSA
Multi-drug resistant M. tuberculosis
explain different classes of antimicrobial resistance, with example, in bacteria, with examples.
Methicillin Resistant Staphylococcus aureus =MRSA
2 types of antimicrobial resistance-
Intrinsic resistance and acquired.
Intrinsic resistance is a bacteria’s natural resistance to an antimicrobial- and all strains of one particular species will be resistant to a certain class of antimicrobial.-
Mycoplasma sp are an intracellular bacteria -they often live inside other cells- and have NO cell – and so has intrinsic or innate resistance to classes of antibacterials which target the cell wall including the beta lactams.
So all strains of E.coli are resistant to Vancomycin- it’s a large molecule- it can’t get through the small porin channels on E.coli’s outer membrane-
E.coli has intrinsic resistance to Vancomycin
Acquired resistance is when bacteria develop resistance – only some bacteria in a species will be resistant.
There are 2 type of acquired resistance-
genetic (irreversible) and
phenotypic which is reversible-
phenotypic –resistancethat can be achieved without any genetic alteration- so it’s an effect of how the cells are growing;
Phenotypic resistance is associated with specific processes such as growth in biofilms.
-if cells are growing in a biofilm – they are encased in an extracellular matrix they have resistance to antibiotics, which they will lose if they are no longer growing in a biofilm.
Genetic resistance – is irreversible
The resistance is transferred to daughter cells
Genetic resistance can develop through
the acquiring of mutations in the chromosomal DNA or
Acquiring new DNA such as a plasmid-
Mutations occur naturally in a population- 1 in every 106 cells will have a mutation- if that mutation provides resistance- then selective pressure will make that population expand.
Sometimes a single base pair change in a gene can confer resistance to some antimicrobials, and this will usually result in a low level of resistance.
An exception to that is that a single base pair change can result in a high level of resistance to the antibiotic Streptomycin,
and similarly only a single base pair change results in a high level of Trimethoprim resistance.
These single mutations can accumulate overtime – and the bacteria can become resistant to even high concentrations of an antibiotic-
THIS IS WHY CONSTANT EXPOSURE TO LOW LEVELS OF AN ANTIBIOTIC CAN SIGNIFICANTLY DRIVE THE INCREASE IN ANTIMICROBIAL RESISTANCE.
Bacteria can also acquire new genetic material in the form of Plasmids which contain resistance genes- they usually acquire these plasmids through bacterial conjugation- which can occur between species.
a plasmid encoded Ampicillin resistance gene, = 40% of E.coli contain this plasmid and so are resistant to Ampicillin (ampicillin is a type of penicillin)
insertion of new genetic material into the chromosome-
The MecA gene is present on a piece of DNA called a mobile genetic element which can insert itself into the bacterial chromosome- this is the gene which produces MRSA
multi-drug resistance plasmids
A single Plasmids can contain multiple antibiotic resistance genes- they can have more than one gene on them
if a bacteria acquired this plasmid then they would instantly become resistant to all the antimicrobial drugs encoded by it.
This example of a multidrug resistant plasmid- encodes for resistance to 6 different antibiotics, and also mercury- = 7 different antimicrobial agents.
Bacteria transfer plasmids by CONJUGATION, this is both within species, and also depending on the plasmid, between different species. This means resistance can spread very easily
explain bacterial conjugation
bacterial conjugation is the transfer of plasmids between different species which increases the spread of antimicrobial resistance
The donor bacteria can transfer a copy of the plasmid to the recipient cell via a structure called the F-pilli, this can occur between between different species of bacteria. antibiotic resistance easily spreads
what are the mechanisms of antibiotic resistance
1-inactivate or modify the drug itself.
2-alter the drugs target site (for example by altering the site where the drugs binds to the enzyme but without effecting how the enzyme functions,- If the drug can’t bind it can’t work.)
3-altered transport- either prevent the drug entering the cell or remove the drug from the cell before it can reach an effective concentration within the bacteria
how are bacteria are able to be resistant to Beta lactams
Bacteria are able to deactivate the beta lactam antibiotics by producing enzymes called beta lactamases, which hydrolyze beta lactams by breaking open the beta lactam ring and rendering them inactive. there are 4 classes of beta lactamases, A to D. The enzymes can be encoded on the bacterial chromosome for example in Stapphalococus auerus and Pseudomonas aeruginosa. They can also be encoded on a plasmid, and this is the most common-
explain the classification of plasmid-mediated Beta- lactamases
slide 15 of the lecture TI-2
explain the difference between gram+ive and gram-ive based on how they release Beta-lactamases
Gram negative bacteria accumulate beta lactamases intracellularly,
gram positive bacteria excrete the beta lactamases from the cell.-
This means the Gram negative bacteria are more effective at disabling our beta lactam antibiotics..
how to inhibit beta lactamases
include beta lactamase inhibitor in our beta lactam drug. No antimicrobial activity but inactivate beta lactamase enzymes to allow beta lactam drug to reach its target
We give them in combination with a beta lactam drug,
The inhibitors extend the life of the beta lactam drugs
A common example is : clavulanic acid (a beta lactamase inhibitor) which is given in combination with amoxicillin- the beta lactam drug. Together this is Co-amoxiclav. Tax-o-cin is a combination of piperacillin (a penicillin) and tazobactam the inhibitor.
Taxocin is widely used for treating gram -negative bacteria
We also have beta lactam antibiotics which have side chains which make them resistant to some beta lactamases
Flucloxacillin is an example - the presence of an Isoxazoyl group on a side chain inhibits some beta lactamases activity.
explain resistnace ot Beta lactams via altered target sire
You can also get resistance to beta lactam drugs by altering the drugs target site. –
The target site for the beta lactams - is the Penicillin Binding Protein PBP on the outside of the bacteria’s cytoplasmic membrane.
The beta lactams bind to the PBP and prevent it working –if you have a change in the PBP- if you change its structure - the beta lactam can no longer bind, and so can’t inhibit cell wall biosynthesis.
The change does not effect the PBPs enzyme function .
It just prevents the beta lactam drug from binding
Just to note bacteria don’t contain just one version of PBPs they have 5 or 6 different types. Different types of bacteria have different amounts and versions of the PBPs
They get named PBP1 or PBP2 etc)
Bacteria which have altered PBPs
Methicillin resistance staphylococcus aureaus, known as MRSA. Methicillin is a type of penicillin, but MRSA bacteria are resistant to all beta lactam drugs.
They have a change in PBP2 which occurs because the bacteria acquire a MecA gene-
the MecA gene codes for different version of PBP2 known as PBP2a and this has an altered drug a target site.
explain Resistance to b-Lactams: altered uptake
The third type of mechanism to resistance to our beta lactams is altered uptake.
This example we are looking at here only occurs in Gram negative bacteria as it involves altering the porin channels
,
if the bacteria reduce the number of channels or alter the size of the channels it prevents the antibacterial’s from entering the cell and reaching their target.
explain resistnace in vancomycin
Vancomycin is a glycopeptide and it inhibits both the crosslinking of peptides, and formation of the glycosidic bonds by binding to the terminal amino acids of the peptide side chain.
Before you get crosslinking of the monomer you have 5 amino acids in that peptide side chain and the final 2 are D-alanine, D-Alanine.
The terminal D-alanine is removed during the crosslinking step. Vancomycin binds to this side chain, and prevents this happening.
You can get resistance through chromosomal and plasmid encoded resistance-
one of the most clinically important is plasmid encoded Van A resistance.
This alters the terminal amino acid on the peptide side chain on the peptidoglycan monomer. from alanine to a lactate-
Vancomycin can no longer form a stable complex – and so we have an altered target site.
Vancomycin can still bind- but it can now only form 4 hydrogen bonds instead of 5-
This reduces the binding affinity by about 100 fold. And so its unstable, and the drug becomes ineffective.
The VanA plasmid –casues resistance in our enterocccal bacteria- which cause UTI , blood stream infections and endocarditis (infections in the heart), - eg enterococcus faecalis
VRE - Particularly problematic as Vancomycin is the first choice if someone has a penicillin allergy-
and VRE strains are resistant to both Vancomycin and a commonly used alternative -Teicoplanin.
explain Resistance to aminoglycosides
Bacteria produce a number of enzymes known as ‘aminoglycoside modifying enzymes’,
These enzymes are produced by the bacteria and they modify the aminoglycosides and make them inactive
A lot of these enzymes have been identified-
Classified into 3 types based on how they modify the aminoglycoside-
These modifying enzymes are usually expressed from plasmids
this is another example of bacteria gaining resistance by inactivating the drug.-
so we had our beta lactamases which hydrolysed the beta lactam ring,
and now we have aminoglycoside modifying enzymes which inactivate the aminoglycosides by adding extra groups which prevents them binding to their target.
