Antimicrobials and resistance Flashcards
what are antimicrobials?
Natural or synthetic agents which kill or inhibit the growth of microorganisms
* Killing = bactericidal
* Inhibiting growth = bacteriostatic
what are the 3 types of antimicrobials?
- Antiseptics = skin
- Disinfectants – inanimate surfaces
- Antibiotics
what are antiseptics and disinfectants?
- Most kill bacteria, viruses and protozoa – indiscriminatory/non-selective
- Too toxic for internal use
- Antiseptics = skin, Disinfectants = inanimate surfaces
- Disinfectants are stronger than antiseptics so are used on surfaces, but cannot kill spores
what are antibiotics?
Low molecular mass compounds that kill (bactericidal) or inhibit (bacteriostatic) growth of bacteria
- Can be ingested or injected into the human body with minimal side effects
- Around 190 million doses of antibiotics are administered every day in hospitals worldwide
what are the 3 main requirements for antibiotics?
- Killing/inhibition
- For intact immune system, bacteriostatic compounds can be used
- For defective immune system, bactericidal compounds are needed - Selective toxicity - Needs minimal side effects to the host
- Exploit differences between host and pathogen – selectively interfere with the bacterium
- Glycopeptides cause deafness so only used for serious infections - Pharmacokinetics
- Distribution of compound in the body so it reaches the infection
- Some compounds do not reach all areas of the body if ingested/injected
what makes a good antibiotic?
- few side effects
- broad spectrum - symptoms often common between causes
what are the issues with antibiotics?
May destroy natural flora, allowing other organisms in (e.g. yeast vaginitis)
what is the ideal target of an antibiotic?
- something that is present in the bacterium but not in human
- something that is essential for the life of the bacterium
what are the main targets of antibiotics?
- Cell wall synthesis
- Protein synthesis - ribosomes
- Cell membrane
- Nucleic acid function – transcription/translation
- Intermediary metabolism
what is peptidoglycan?
Peptidoglycan is unique to bacteria and is essential for bacteria cell viability
- Polymers inside the cell form a sugar which is converted into glucosamine, amino acid attaches to glucosamine
- The oligomeric unit is flipped across the cytoplasmic membrane to the outside of the cell, where transglycosylase connects the carbohydrate backbone and transpeptidase to join the peptides to form peptidoglycan
why do some antibiotics target peptidoglycan?
they target peptidoglycan to inhibit bacterial cell wall biosynthesis
- peptidoglycan is essential for bacterial cell viability, so inhibiting its synthesis will lead to bacterial death
- If peptidoglycan cannot form, the osmotic pressure in the cell increases too much, leading to bacterial cell rupture
what are the main inhibitors of cell wall biosynthesis?
- cycloserine
- phosphomycin
- bacitracin
- beta-lactams: penicillin, methicillin, cephalosporins
- glycopeptides: vancomycin, teichoplanin
how does cycloserine inhibit cell wall biosynthesis?
inhibits incorporation of alanine into cell wall precursor
- Cycloserine is a structural analogue of D-ala, so it prevents alanine being built into the pentapeptide, halting the process of peptidoglycan synthesis
how does phosphomycin inhibit cell wall biosynthesis?
prevents UDP-NAG»UDP-NAM
- In the process, it interacts with enzyme MurA and prevents it from converting UDP-NAG to UDP-NAM, and stops the process
how does bacitracin inhibit cell wall biosynthesis?
stops recycling of bactoprenol (quite toxic so only topical use)
- Bactoprenol is very hydrophobic so sits within the cytoplasmic membrane and can be phosphorylated to form an adaptor molecule to flip substrates to the outside
- prevents bactoprenol recycling so that substrates cannot be flipped to the outside of the cell, and therefore peptidoglycan synthesis cannot continue
what are beta-lactams?
- Some of the most useful antibiotics (over half of all antibiotics used)
- All have 4-membered beta-lactam ring
- Structural alterations effect pharmacokinetics, spectrum of activity and bacterial resistance mechanism
- All bind and inhibit Penicillin Binding Proteins (PBPs) in the bacteria
how do beta-lactams inhibit cell wall biosynthesis?
All bind and inhibit Penicillin Binding Proteins (PBPs) in the bacteria
- PBPs are cytoplasmic membrane proteins involved in transpeptidation and transglycosylase reactions for peptidoglycan
- Beta-lactams inhibit PBPs by preventing them from taking up the peptide substrates
are beta-lactams more effective for gram-positive or gram-negative bacteria?
Work for gram-positive as they don’t have to cross outer membrane
Less effective in gram-negative as they have to cross outer membrane
how do glycopeptides inhibit cell wall biosynthesis?
vancomycin and teichoplanin bind to D-ala-D-ala and prevent transpeptidation and transglycosylase reactions
Vancomycin is the drug of last resort for some may cause multiplication of antibiotic-resistant strains of Staphylococcus aureus
what are the main inhibitors of nucleic acid synthesis?
- sulfonamides and trimethoprim - inhibit synthesis of precursors
- quinolones - inhibit DNA replication
- rifampin - inhibit RNA synthesis
how do sulfonamides and trimethoprim inhibit nucleic acid synthesis?
These inhibit tetrahydrofolic acid production by acting as substrate analogues
- THF is necessary for nucleic acid synthesis
- Mammals cannot make THF (required in diet) and so the host is resistant to this antibiotic
- Sulfonamides competitively inhibits an enzyme as it is a structural analogue of THF, so the enzyme catalyses the sulfonamide instead, meaning nucleic acid cannot be formed
- Trimethoprim is metabolised and interferes with the second step of nucleic acid synthesis
how do quinolones prevent nucleic acid synthesis?
- Quinolones bind to and inhibit DNA gyrase, the negative supercoiling Topoisomerase II enzyme
- They stabilise the complex in which the DNA is permanently bound to the gyrase, so the topoisomerase is blocked
how does rifampin inhibit nucleic acid synthesis?
- Rifampin inhibits RNAP enzyme
- Used primarily on drug resistant Mycobacterium tuberculosis
why are ribosomes a good antibiotic target?
Good target as ribosomes of bacteria are different from humans - different subunit composition
what are the main inhibitors of protein synthesis by ribosomes?
- macrolides (erythromycin), lincosamides (clindamycin) and chloramphenicol - bind 50S subunit
- aminoglycosides: kanamycin, gentamycin - bind 30S subunit and prevent 50S binding
- Tetracyclines - bind 30S subunit
how do macrolides (erythromycin), lincosamides (clindamycin) and chloramphenicol prevent protein synthesis by ribosomes?
- These bind 50S subunit of ribosome to block transfer of peptides
- Widely used
what is the complication with clindamycin in inhibiting ribosomes?
Complication with clindamycin due to killing of resident anaerobic microflora (allows Clostridium difficile to grow and produce toxins) – can be toxic
how do aminoglycosides inhibit protein synthesis by ribosomes?
- Bind 30S subunit and prevent 50S binding.
- Effective bactericidal but side effects (kidney function, hearing loss)
how do tetracyclines inhibit protein synthesis by ribosomes?
- Bind 30S subunit and prevent alignment of aminoacylate-tRNA with mRNA
- Extremely widely used -> resistance is very common
why is targeting of ribosomes more susceptible to antimicrobial resistance than targeting cell wall biosynthesis?
- Structure of the ribosome can be altered via mutations as it is a simple amino acid protein structure, so bacteria can find ways to overcome antibiotics
- Peptidoglycan synthesis is very specific and cannot be replaced by bacteria, so is a constant, unchanged target
what are the main inhibitors of cytoplasmic membrane function?
- Gramicidin
- Polymyxins
- Daptomycin
how does gramicidin inhibit cytoplasmic membrane function?
Gramicidin forms cation channel, H+ leaks so cell cannot maintain PMF
- Peptides exist on skin of mammals fight off bacteria by forming channels in cytoplasmic membranes, leading to proton leaks
- Gramicidin is the use of specific peptides to form cation channels for specific bacteria
how do polymyxins inhibit cytoplasmic membrane function?
act on gram-negative bacteria (same function as gramicidin)
how does daptomycin inhibit cytoplasmic membrane funciton?
Daptomycin is structural analogue of cyclic lipopeptides found in bacteria (ribosomally-synthesised post-translational peptides (RIPPs))
- Daptomycin relies on Ca2+ dependent insertion into cell membrane
- It forms oligomeric structures to form a transmembrane channel, leading to potassium efflux from the bacterial cell
- Leads to electrochemical gradient issues with the cell
- The cell dies with minimal lysis, so contents are not released
- Useful against gram-positive bacteria
what is the function of synercid antibiotic?
- Mixture of dalfopristin and quinupristin.
- Both streptogramins (bind to 50S subunit of ribosome and prevent elongation of peptide chain).
However used for many years in animal feeds so resistant bacteria already developed.
what is the function of linezolid?
Oxazolidinone - bind to 50S subunit of ribosome preventing initiation of protein synthesis
However resistance already found.
what is the definition of a resistant organism?
A resistant organism is one that will not be inhibited or killed by an antibacterial agent at concentrations of the drug achievable in the body after normal dosage
why can’t resistant organisms be treated with high concs of antimicrobials?
High concentration of antimicrobial can be used to kill a resistant microbe, but this high conc would be dangerous to apply to a patient (side effects)
what are persistors?
Persistors – antibiotics cannot kill all bacteria in a culture, meaning some of the microbes survive, these are called persistors of the antibiotic
- Some genes influence the percentage of persistors
what factors can contribute to antimicrobial resistance?
- healthcare
- agriculture
- culture
how does healthcare contribute to antimicrobial resistance?
- overprescription - no diagnosis or misdiagnosis
- some patients are prescribed antibiotics, but it may be a viral infection, so all the flora of the body are unnecessarily exposed to antibiotics, leading to resistance - availability - some countries do not require prescriptions to get antibiotics and instead can be bought over the counter
- easy accessibility, less regulation - improper use by patients - some patients may prematurely stop taking antibiotics and not complete the full course
what evidence is there of healthcare contributing to antimicrobial resistance?
As prescription of penicillin increases, resistance increases
why are antibiotics used in agriculture?
- for growth promotion - when antibiotics are applied, animals gain weight 16% faster so can be slaughtered sooner for food
- By subduing the no. bacteria in the animal’s gut, the animals ability to take up nutrients is maximised - prophylaxis - removal of harmful bacteria from the intestine so animals don’t fall ill
how does agriculture contribute to antimicrobial resistance?
in US, 70% of medically imported antibiotics are consumed by farm animals
- animals are highly exposed to antibiotics, and bacteria inside them may develop resistance
- this resistance may be spread to humans
how does antimicrobial resistance in agricultural animals spread to humans?
-Direct Contact – cuts can be infected with resistant bacteria when working with livestock
- Consumption – can become a reservoir of resistant bacteria if somebody is immunocompromised
- Environment
what are the 2 types of antimicrobial resistance?
- innate/natural resistance
- acquired resistance
what is innate/natural resistance? give examples:
- when there is a lack of target in the pathogen
- e.g. Mycoplasma have no peptidoglycan and are resistant to penicillin
- Persistors may convert into forms which do not have peptidoglycan, changing their entire cell shape based on osmotic conditions
- They can then return to their rod shape form and remake their peptidoglycan - when the pathogen is impermeable to antibiotic
- e.g. most gram-negative bacteria are impermeable to pen G
what are the forms of acquired antimicrobial resistance?
- reduced uptake of the antibiotic
- efflux of the drug
- inactivation of the drug
- alteration of the target
how may pathogens acquire resistance through reduced reuptake of the antibiotic?
- Outer membrane porins are non-specific channels and control passive entry of small molecules such as nutrients and small drugs
- Larger molecules such as drugs cannot pass through
- Single mutation can increase resistance 5-10 fold for many drugs
how may pathogens acquire resistance through efflux of the antibiotic?
example: multidrug resistance (MDR) exporter - AcrB system in E. coli
- MDR protein is responsible for excluding substances from cell including some antibiotics
- it is made up of the AcrB pump which uses PMF to translocate molecules across cytoplasmic membrane into periplasm
- a large funnel RdrA domain in periplasm pipelines substances out the cell via outer membrane channel called TolC
- therefore MDRs can transport antibiotics out of the cell
other examples are tetA and tetG in gram-negative bacteria against tetracyclines, and staphylococcus against macrolides
The pumps are not very specific and interact with many hydrophobic substances, so can expel substances like dyes as well as antibiotics
how may pathogens acquire resistance through inactivation of the antibiotic?
- beta-lactamases - hydrolyse lactam ring of penicillin, causing its inactivation
- common in gram-negatives such as S. aureus - enzymes which chemically alter the drugs via cleavage/modification
-Chloramphenicol acetyl transferase – transfer an acetyl group on drug to inhibit it
- Streptogramin acetyl transferase
- Tetracycline oxidation
- Aminoglycoside modification
how may pathogens acquire resistance through alteration of the target?
- PBPs: Alteration in penicillin binding proteins (PBP) via point mutations so that they have low affinity for beta-lactam antibiotics
- e.g. Mosaic PBP encoding genes in Streptococcus pneumoniae – genes swap domains between the proteins - Methicillin-Resistant Staphylococcus aureus (MRSA) - MecA (PBP2a) can replace normal PBPs and is not inhibited by useful concentrations of beta-lactams
how can resistance occur against glycopeptide antibiotics such as vancomycin?
Vancomycin binds to terminal D-ala-D-ala on muramyl peptide and prevents cross-linking in the peptidoglycan formation
- Resistance involves incorporation of D-ala-D-lac, due to van genes in Enterococcus
- Vancomycin used as drug of last resort for MRSA BUT VRSA (Vancomycin Resistant Staphylococcus aureus) first case 2002
how can resistance occur against tetracyclines?
Resistance due to ribosome protection by another protein preventing Tetracycline binding (Neisseria, Haemophilus etc.)
- Can be due to point mutation in ribosome amino acids, meaning drug cannot interact
- Can be due to other proteins which shield the ribosome to block the drug from binding
how can resistance occur against macrolides, streptogramins and lincosamides?
Due to RNA methylase which methylates the adenine residues within the binding sites of the drugs, causing them to be inactive
how can resistance occur against quinolones and rifampin?
Due to point mutations in DNA gyrase and RNA polymerase, respectively, rendering the drug useless
how do bacteria gain resistance properties?
- point mutations e.g. DNA gyrase
- during cell stress, bacteria undergo many mutations to form genetic variability within the population to increase chances of survival - uptake of new genetic material
- Transduction (phage) - Narrow host range due to phage receptor
- Transformation (linear DNA) - require homologous recombination - conjugation - direct transfer of DNA between cells via a protein complex
- Can occur between unrelated organisms
- Conjugative plasmids: self-transmissible (>25kb), mobilizable
- Can carry multiple resistance genes at one time
- Plasmids and transposons carry drug resistance
what are the current problems of antimicrobial resistance?
MRSA and VRSA
- Mycobacterium tuberculosis multidrug resistance cost 1 billion dollars to bring under control in New York alone
colistin resistance Colistin peptide drugs are the last resort for multidrug resistant Enterobacteriaceae:
- They are very hydrophobic and destroy membranes in gram-positive bacteria
- mcr-1 gene alters the outer membrane, plasmid-mediated
- mcr1 encodes phosphatidyl-ethanolamine-transferase
- mcr-1 gene mutation enables resistance as it transfers phosphoethanolamine to the headgroup of lipids, meaning the bilayer no longer is affected by colistin
how may future drugs against antimicrobial resistance be developed?
- Empirical screening of environmental samples
- Alter and modify existing drugs to enhance activity: Combinatorial chemistry
- Alter biosynthetic genes
- Rational drug design: use crystal structure of target molecule and design inhibitor (saquinavir (HIV))
what are the problems with drug development against antimicrobial resistance?
- Cost > $1 billion per drug
- Takes 10-12 years
- Identification -> pharmacokinetics -> trials
what method may help tackle antimicrobial resistance?
vaccination could help combat drug resistance
- Veterinary vaccines – vaccinate livestock?
- Improved vaccination rate will reduce need for antibiotics as there will be a reduction in the no. resistant infections
- will also be effective against other pathogens such as viruses and fungi
how can antibiotic use be improved so it is more effective?
We can sequence bacterial strains, which can help select which antibiotic should be used and at what dose
- Next-generation sequencing can indicate which species, which strain, what resistance will be encountered and what is that strain susceptible to
- Helps to avoid the wrong drug being administered
what is required for bacterial strain sequences to diagnose infections?
- Rapid
- Point of Care
- Cheap
