Antibiotic Resistance Lecture 17 (PDF)

Antibiotic resistance Lecture 16 Dr Sacha Pidot [email protected] www.pidotlab.com Lectures 16-18 Antibiotics Antibiotic resistance Antibiotic discovery and development Sources of information Papers! – Wright, 2010, BMC Biol., “Q&A: Antibiotic resistance...

Antibiotic resistance Lecture 16 Dr Sacha Pidot [email protected] www.pidotlab.com Lectures 16-18 Antibiotics Antibiotic resistance Antibiotic discovery and development Sources of information Papers! – Wright, 2010, BMC Biol., “Q&A: Antibiotic resistance: where does it come from and what can we do about it?” – The science of antibiotic discovery (https://www-sciencedirect- com.ezp.lib.unimelb.edu.au/science/article/pii/S0092867420302336?via%3Dihub ) By the end of this lecture you should be able to: To appreciate the importance of antibiotic resistance and key resistance mechanisms To appreciate the principles and practice of how antimicrobial susceptibility is determined  The problem of antibiotic resistance Antibiotic resistance is increasing WHO priority pathogens for R&D of new antibiotics Priority 1: CRITICAL Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, carbapenem-resistant Enterobacteriaceae, carbapenem-resistant, ESBL-producing Priority 2: HIGH Enterococcus faecium, vancomycin-resistant Staphylococcus aureus, methicillin-resistant, vancomycin-intermediate and resistant Helicobacter pylori, clarithromycin-resistant Campylobacter spp., fluorquinolone-resistant Salmonellae, fluorquinolone-resistant O’Neill, 2016, Tackling drug-resistant infections globally, UK Gov report Neisseria gonorrhoeae, cephalosporin-resistant, fluoroquinolone-resistant  What do we mean by “resistance” Organism that can grow in the presence of an antibiotic designed to kill/inhibit it Causes? 8 Important factors in resistance 9  Consequences of antibiotic resistance Why is this important? – Leads to untreatable infections – Longer hospital stays – Greater costs – more expensive drugs needed – Increased mortality Several infections are completely untreatable!! 10 Genetic basis of resistance Where does resistance come from? – intrinsic (de novo) mutation within organism – acquired resistance genes  Genetic basis of resistance Intrinsic resistance – G+ve/G-ve cell type Larger molecules cannot penetrate G-ve outer membrane – lack of target/mutated target – chromosomal resistance gene/mutations Genetic basis of resistance acquired – Horizontal Gene Transfer (HGT) Extrachromosomal DNA Move between cells by Plasmids Transformation, conjugation Transposons Transformation, conjugation Phage Transduction Where does acquired resistance come from? Origins of resistance = environmental bacteria Antibiotic producers must protect themselves from their products – Resistance genes co-evolved with production Estimated 200 – 800 million years old – Many producers resistant to multiple antibiotics (avg of 7-8 per strain) → more than they produce – Suggests genes are ancient!  Where does resistance come from? Emergence as evidenced by genomics: – Seq of 1915 Shigella flexneri – PenR, ErmR Predates introduction of antibiotics! – Permafrost sequencing Permanently frozen snow Plasmids/transposons with resistance markers found in permafrost bacteria (>30,000 yrs old) – CTX-M bla origins (cefoxitimeR) Emerged in Enterobacteriacae in 1980’s Found in environmental Kluyvera sp with 100% ID to ctx-m genes in Enterobacteriaceae plasmids - including flanking genes! Selection for resistant mutants Resistance mechanisms  Resistance mechanisms: efflux Many targets inside cytoplasm/inner membrane Efflux pumps actively pump antibiotics out of the cell 5 major families – wide or specific substrate tolerance Munita and Arias, 2016, Microb Spectr  Resistance mechanisms: efflux Eg - tetracycline resistance: – MFS family – >20 different tet genes described – Tet(K), Tet(L) – Some specific for tetracycline and doxycycline, but not tigecycline RND family: – P. aeruginosa » MexAB-OprM - also β-lactams, aminoglycosides, quinolones, macrolides, amphenicols, etc Li et al, 2015, Clin Microbiol Rev  Resistance mechanisms: inactivation Destruction of antibiotic β-lactamases – cleave β-lactam ring – Known as bla genes, >1000 different β-lactamases described – As new bla genes identified, new penicillins were developed and introduced → followed by resistance Serine beta-lactamase Metallo beta-lactamase - Have active site serine - Require Zn2+ for activity - Thought to have evolved - Thought to have evolved from DD-transpeptidase from RNase H  Resistance mechanisms: inactivation β-lactamases – Very complex classification/nomenclature Extended spectrum β-lactamases (ESBLs) – Active against penicillin, 3rd gen cephalosporins, monobactams – not carbapenems Carbapenemases – carbapenems – Some can be inhibited by β-lactamase inhibitors clavulanic acid avibactam  Resistance mechanisms: inactivation Transferases – Alter structure of compound – Example: Aminoglycoside modifying enzymes (AMEs) Covalently modify –OH or –NH2 groups Predominant mechanisms of aminoglycoside resistance Normally on mobile genetic elements Classified by: – Site of action – Type of modification – eg AAC(6’)-I  Resistance mechanisms: Target modification Methicillin (and β-lactam) resistance – mecA – S. aureus – Encodes PBP2a – low affinity for all β-lactams – Most β-lactams useless against MRSA  Resistance mechanisms: Target modification Vancomycin resistance – van genes E. faecium  Identification of resistance How do we test for resistant bacteria? Antibiotic susceptibility testing – “Decreased susceptibility” MIC/MBC – Methods Disk diffusion, e-test, VITEK, broth microdilution Genomics!  MIC and MBC Minimum inhibitory concentration (MIC) – The lowest concentration of an antibiotic that prevents visible growth of bacteria Minimum bactericidal concentration (MBC) – The minimum concentration of an antibacterial agent that results in bacterial death. The closer the MIC is to the MBC, the more bactericidal the compound. Identification of resistance Broth dilution https://www.intechopen.com/books/antibacterial-agents/current-approaches-for-exploration-of-nanoparticles-as-antibacterial-agents  Identification of resistance Disk diffusion – Antibiotic impregnated disks – Antibiotic diffuses outwards – concentration gradient – Size of zones of inhibition measured – Cannot compare between antibiotics Prescott’s Microbiology, 11th Ed, Ch 9  Identification of resistance Etest strips – Antibiotic concentration gradient – MIC determined at point of intersect Prescott’s Microbiology, 11th Ed, Ch 9 VITEK – Automated, rapid antimicrobial susceptibility – Continuously measures growth Identification of resistance Genomics – Sequence genomes to detect resistance markers – Compare reads/assemblies to databases Boolchandani et all, 2019, Nat Rev Genet Why do we have a resistance problem? Introduction of an antibiotic always followed by resistance The antibiotic discovery timeline Golden age of antibiotics No new antibiotic classes since 1990’s! What can we do about resistance? Antibiotic stewardship – Use fewer antibiotics – Use only when necessary – Stop unnecessary use in agriculture What can we do about resistance? Alternative treatments – Phage therapy Pros – highly specific – kill rapidly Cons – need lytic phages – highly specific - need to maintain cocktails of phages – difficult to commercialise What can we do about resistance? Find new antibiotics – But, there’s a problem… How do we find antibiotics? Whole cell assays Target based screening INSERT PICS Goal: Kill bacteria Goal: Inactivate enzymes - Know it can enter cells - Know molecular target - Molecular target? - Cell entry?  Why can’t we just make more? A case study in library screening – GSK – 7 years – 530,000 compounds – synthetic library – 70 high throughput screens – Cost >$70 million – 5 leads, 0 actual products “We are enthusiastic advocates of natural product screening to search for novel antibacterial leads.” Payne et al, 2007, Nat Rev Drug Discov  Antibiotic discovery from Nature Natural products/secondary metabolites – Primarily from Streptomyces, soil bacteria – Represent >70% antibiotics in clinical use Excellent source, but… – Identifying new structures is challenging – Complex structures – not easily modified/synthesised tetracycline New methods for antibiotic discovery 2 key problems with current antibiotic discovery – Same sources = same compounds (rediscovery) – Lots of chemical diversity, but hard to access (silent gene clusters) New methods to overcome this – iChips – access new bacteria = new sources – Induction – turn on silent genes – Clone and express genes Antibiotic resistance is a complex problem Summary Mechanisms of resistance – Efflux – Inactivation – Target modification Antibiotic resistance is a complex, multifactorial problem! Finally… Take 2 minutes and write down: – What was the most important thing you learned today? – What questions remain in your mind?