antimicrobial drug resistance mechanisms

What is drug resistance?

A reduction in effectiveness of a drug in treating a disease - consequence of evolution. Microbes learn to adapt to their environments order to ensure their survival e.g. Microbial drug resistance

Intrinsic resistance

Particular characteristic of a microorganism that limits the action of a microbial agent:

Lipid bilayer outer membrane/ envelope with low permeability, acts as a strong antibiotic barrier, common to gram negative pathogens e.g. E. coli

The constitutive efflux pumps observed in many bacteria

What are antibiotics?

Agents that can either kill or inhibit the growth of a microorganism

Most modern anti-bacterials are semisynthetic modifications of various natural compounds e.g. penicillin derivatives - contain beta-lactam ring which interfere with bacterial cell wall synthesis by targeting penicillin-binding proteins

Moa of antibiotics in gram +ve and -ve bacteria

Inhibit the formation of peptidoglycan cross-links in the cell wall - covalently binds to the active site of penicillin-binding proteins, inactivating the enzymes - inhibits cross linking → weak peptidoglycan → osmotic instability → cell lysis and death

+ve: thick cell walls largely composed of a peptidoglycan layer (95%) - more susceptible

-ve: thin cell walls and their peptidoglycan layer as little as 5-10% - harder to access peptidoglycan layer

Microbial acquired drug resistance

bacteria that were once susceptible to an antibiotic gain the ability to resist it

Natural selection by spontaneous mutation (vertical evolution)

Gene transfer (horizontal evolution)

Natural selection by spontaneous mutation (vertical evolution)

random mutation occur naturally in bacterial DNA during replication which could have resistance to a specific antibiotic

when antibiotics are introduced, they kill susceptible bacteria but mutants with resistance survive → selection pressure

resistant bacteria multiply and pass the resistant mutation too their offspring - over time the population becomes predominantly resistant

Gene transfer (vertical evolution)

movement of genetic material between bacteria - resistant bacteria transfer their mutant DNA to non-resistant DNA

efficient, major way bacteria rapidly acquire antibiotic resistance

Three processes:

• Transformation

• Transduction

• Conjugation

Transformation gene transfer

Uncommon

Bacterial cells die and release exogenous DNA fragments which may contain resistant genes that can be taken up by recipient cells

Uptake requires the bacterium to enter competence (a transient physiological state - genetically encoded DNA internalisation process) where the bacterium expresses a series of competent genes needed for DNA uptake

Transduction

Uncommon

When a virus, bacteriophage infects a bacterium, picks up plasmid DNA and infects another bacterial cell with it

Conjugation

Most common gene transfer

Two bacteria in close proximity exchange DNA through a pilus in gram negative bacteria - adhesion molecules are likely involved among gram positive

often involves resistance (R) plasmids that carry multiple resistance genes

how are resistant genes transferred?

plasmids: small circular dsDNA - most drug resistance encountered is plasmid derived

transposons: jumping genes - segments of DNA that cannot self-replicate - carried by a plasmid during conjugation and inserts itself into the recipient bacterium’s DNA

integrons and gene cassettes are genetic systems used to capture, store, and express resistance genes - spread of multidrug resistance

cassettes: mobile genetic material containing one/two antibiotic resistant genes

integrons: incorporate cassettes using site-specific recombination mediated by an intergon-integrase at the attC of a cassette

molecular mechanisms of drug resistance

enzymatic drug inactivation

efflux/influx-mediated drug resistance in bacteria

modification of drug binding target

enzymatic drug inactivation

in penicillin-like agents, resistant bacteria produce beta-lactamase which hydrolyse the beta-lactam ring - disabling the drug

efflux/influx-mediated drug resistance

efflux: multidrug resistant (MDR) transporters pump the antibiotic out of the bacterium e.g. primary active ATP-binding cassette (ABC)-type MDR transporters - upon binding of the antibiotic to the intracellular domain, causes conformational change → ATP binds to the nucleotide-binding domains (NBDs) of the transporter on the cytoplasmic side → conformational change which opens the transporter to the outside of the cell which pushes the antibiotic out the cell. mutations can lead to increased expression or increased activity. intrinsic and acquired resistance

influx: reducing of drug entry into bacterial cell via porins - protein channels in the outer membrane of gram -ve bacteria. intrinsic resistance, mutation of the gene that encodes the porin → porin loss, modification of the size/conductance of the porin channel, lower expression of porin channel → slower diffusion of drug into the bacterium

modification of drug binding binding target

involving enzyme required for a vital metabolic pathway: dihydrofolate reductase (DHFR) is involved in folic acid metabolism to form thymidine. bacteria need DHFR to replicate DNA. Trimethoprim is an antibiotic which blocks the active site of DHFR so DHF cannot bind. resistance to this drug can arise from mutations causing gene amplification or changing DHFR binding site → reduced drug binding affinity

involving cell wall synthesis: penicillin-binding protein (PBP) binds to the D-ala-D-ala end of the peptidoglycan chain, one of the D-alanine residues is released and the enzyme attaches to the end of the peptide → cross linkage with lysine. methicillin binds to PBP, stopping it binding to D-ala → no cross linkage formed. resistance occurs via overproduction of a structurally altered PBP, PBP2a, which has a lower affinity for beta-lactam antibiotics → allowing normal PBP activity to persist

methicillin resistant Staph aureus (MRSA)

MRSA is a type of bacterium that has acquires resistance to beta-lactam antibiotics

infections in the skin, soft tissue, bloodstream, pneumonia

two groups: hospital-acquired (HA-MRSA) and community-acquired (CA-MRSA)

both types are linked to increased morbidity and mortality → can develop into sepsis if patient is left waiting for too long

staphylococcal cassette chromosome mec (SCCmec)

SCCmec is a mobile genetic element of Staph which carries the mecA gene that encodes for PBP2a

there is a growing number of SCCmec types - characterised by the mec complex (mecA and mecI/mecR1) and the ccr (cassette chromosome recombinase) complex.

the expression of mecA is regulated by regulator proteins encoded by the mecR1 (sensor-transducer) and mecI (repressor) genes - mutant mecR1 protein → no PBP2a expression, mutant mecI protein → overexpression of PBP2a

MRSA treatments

vancomycin: however becoming less effective due to resistance and it has a similar MOA to methicillin → VRSA

daptomycin: lipopeptide antibiotic, last-line treatment - inserts itself into the bacterial membrane in a calcium-dependent manner, forms pores → rapid depolarisation → loss of membrane potential and rapid cell death (bactericidal), resistance is emerging but still uncommon

what is colistin?

last-line defence against serious gram negative infections

effective but nephrotoxic

important to preserve antibiotics such as colistin in the fight against resistance

used often in agriculture

targets LPS in the outer membrane of gram -ve bacteria - binds to lipid A → displaces 2+ charged cations that stabilise the outer membrane → disrupts membrane integrity → cell lysis and death

colistin resistance

E.coli containing the plasmid mediated ‘mobilised colistin resistance gene’ (mcr-1) which codes for PPEA enzyme that catalyses the modification of lipid A of LPS, forming PPEA-4’-lipid A which has a low affinity for colistin

this resistance is known as target modification - similar to PBP2a

new future developments

teixobactin:

currently in late-stage preclinical development

small molecule antibiotic against gram +ve bacteria

prevents cell wall synthesis by binding to lipid II and III which are final intermediates in the formation of peptidoglycan