Drug Tolerance

Question 1

Persistence

Answer 1

• persister bacteria are non-replicating, multi-drug-tolerant bacteria
• killing susceptible cells leaves a population consisting only of persistent cells

Question 2

Resistance versus tolerance

Answer 2

treatment duration against [antibiotic] graph
- vertical asymptote = concentration below which culture will not be killed (MIC)
- horizontal asymptote = time needed to kill 99% of the culture

• as increase in resistance translates into a shift in MIC to higher concentrations
• an increase in tolerance manifests itself as a shift in the MDK99 (min duration to kill 99%) to longer treatment durations

Question 3

Lag time

Answer 3

• changes in single cell lag time distributions underlie antibiotic tolerance
• first adaptive change to antibiotic stress is development of tolerance through major adjustment in single cell lag time distribution without a change in resistance
• bacteria optimise the lag phase before regrowth to match duration of antibiotic exposure interval
• from a clinical POV, tolerance by lag presents a major challenge:
• the tolerance by lag phenotype confers a survival advantage against a broad spectrum of drugs and stresses
• the tbi genes are most likely associated with increased levels of deacetylated tRNA and then activation of toxin-antitoxin modules

Question 4

Toxin-antitoxin activity

Answer 4

• during pathogenesis, bacteria often form biofilms that increase their survival during harsh conditions
• deep within biofilms, bacteria have limited access to nutrients
• in stringent response that follows, bacteria increase levels of (p)ppGpp
• drug tolerance of E.coli in biofilms depends on (p)ppGpp
• high (p)ppGpp inhibits PPX activity
• polyphosphate stimulation Lon protease to degrade E.coli antitoxins
• activated toxins now inhibit mRNA synthesis or translation –> causes reversible inhibition of cell growth
• toxins work by inhibition of enzymes, RNA or DNA degradation, ADP-ribosylation of DNA and depolarisation

Question 5

Toxin-antitoxin activity - persister cells

Answer 5

persister cells exposed to bactericidal antibiotic, above MBC:
- protein synthesis inhibited by toxins (kinases and endonucleases)
- energetics of cells is inhibited by toxins that dissipate the electrochemical proton gradient and cause depletion of ATP pool
- metabolic pathways targeted by bactericidal antibiotics are less active
- drugs do not exert their toxic actions

Question 6

Tolerance vs persistence

Answer 6

• drug tolerance and persistence are the same phenomenon but persistence refers to a subpopulation of cells with tolerance properties
• cells can be drug tolerant or drug resistant

Question 7

Resistance vs hetero-resistance

Answer 7

• when choosing antibiotics for use in the clinic, we assume the susceptibility of bacteria is reflected by the lab estimates of MIC
• the caveat of this is hetero-resistance
• hetero-resistance refers to the presence of a resistant sub population within a main population of susceptible cells
• densities of these minority populations are not usually sufficiently high to be reflected in the MIC
• but in the presence of antibiotic, their frequencies increase and population MIC approaches that of the minority

Question 8

Measuring hetero-resistance

Answer 8

• epsilometer test = growth of colonies within the inhibition zone of gradient diffusion strips
• population analysis profile = bacterial cells are plated at different concentrations to determine the fraction of cells that grow at each concentration
• spontaneous duplications form and disappear at high rate through homologous recombination between directly repeated sequences
• gene duplications = 2 sister chromatids behind a replication fork
• antibiotic hetero-resistance is caused by amplifications, deletions and point mutations of genes encoding efflux pumps, antibiotic targets or antibiotic-modifying or degrading enzymes

Question 9

Example of hetero-resistance

Answer 9

unstable tandem gene amplification generates hetero-resistance to colistin in Salmonella

molecular mechanism of colistin resistance:
- PhoPQ and PmrAb are sensor kinase/response regulator systems that regulate lipid A modification machinery of amino-arbinose and phosphoethanolamine reduces the net negative charge of lipid A = colistin response
- additions of these to lipid A are regulated through the transcriptional regulators PmrA that increase transcription of amBCADTEF