L13 I&I Antimicrobial resistance

Overview

This lecture covers the molecular mechanisms of antibiotic resistance (restricted access, inactivation, target modification), how resistance spreads (selection plus horizontal gene transfer via mobile genetic elements), the epidemiological drivers of rising resistance (agricultural and human antibiotic use, inappropriate prescribing, increased spread and susceptibility), the current global and New Zealand burden (carbapenemase producers, colistin resistance), and the case for antimicrobial stewardship.

Mechanisms of Resistance: Overview

Three broad mechanism categories, all covered in turn below:

• Restricted access to the target: decreased permeability, increased efflux
• Inactivation of the antibiotic (e.g. by enzymes)
• Modification of the target

Restricting Access to the Target

• β-lactams must cross the outer membrane of gram-negative bacteria to reach their target; many other antibiotic classes must reach the cytoplasm.
• Gram-negative bacteria are intrinsically resistant to vancomycin because it cannot cross this extra outer membrane (gram-positives have only a single membrane, with lipoteichoic acid, lipoprotein and peptidoglycan; gram-negatives additionally have an LPS-containing outer membrane with porins, then peptidoglycan, then the inner membrane).
• Porins sit in the gram-negative outer membrane and allow selective diffusion of small molecules (including antibiotics) into the periplasm. Gram negatives encode multiple porins. Mutations can decrease porin expression or restrict the channel, reducing antibiotic entry; this often affects multiple antibiotic classes at once. Outcomes seen: porin loss, a narrowed channel, or decreased porin expression — all reducing diffusion into the periplasm.
• Efflux pumps actively pump small molecules (including metabolites, toxins and antibiotics) out of the cytoplasm. Bacteria encode multiple efflux pumps: some are highly specific (e.g. TetA, a tetracycline-specific efflux pump), others export many different compounds. Efflux-mediated resistance has now been described for almost every antibiotic class. Structurally, an efflux pump can be tripartite, spanning the outer membrane, periplasm and inner membrane to export molecules directly from the cytosol to the extracellular space.

Target Modification and Inactivation Affecting Cell Wall Synthesis

• Normal peptidoglycan synthesis: penicillin-binding protein (transpeptidase) cross-links the peptide side chains of peptidoglycan strands (NAM/NAG backbone), forming pentaglycine cross-links between adjacent strands.
• β-lactams inhibit the last step of peptidoglycan synthesis: they bind irreversibly to the penicillin-binding protein, forming a covalent bond with a serine residue in its active site, which blocks cross-linking of the peptidoglycan backbone and weakens the cell wall.
• Inactivation — β-lactamases:
  • Class A and C β-lactamases: a serine residue forms a covalent bond with the β-lactam, then the complex is attacked by water, which hydrolyses (inactivates) the β-lactam and frees the β-lactamase enzyme to repeat the cycle on another antibiotic molecule (catalytic, repeatable inactivation). Spectrum ranges narrow to broad (e.g. AmpC, ESBLs [extended-spectrum β-lactamases], KPC). Class C = AmpC.
  • Class B β-lactamases are zinc-dependent, broad spectrum (e.g. NDM carbapenemase), and especially common in gram-negative bacteria.
  • β-lactamase inhibitors: clavulanic acid inhibits class A β-lactamases by remaining bound to the enzyme. It does not inhibit AmpC (class C) β-lactamases produced by the ESCPM group of organisms (Enterobacter cloacae/aerogenes [now Klebsiella aerogenes], Serratia spp, Citrobacter freundii, Proteus vulgaris/Providencia spp, Morganella morganii). AmpC production in these organisms can be inducible or constitutive, and resistance can emerge during treatment through mutation — highest risk with Enterobacter cloacae, Klebsiella aerogenes and Citrobacter freundii.
• Target modification — altered penicillin-binding proteins: the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) encodes PBP2’, an alternative transpeptidase that is not inhibited by methicillin. PBP2’ replaces the normal transpeptidase and continues cross-linking peptidoglycan even in the presence of the antibiotic.
• Vancomycin target modification: vancomycin normally binds the D-alanine–D-alanine terminus of the peptide side chain, sterically blocking the cross-linking enzyme. Resistance arises when the terminal residue is changed to D-alanine–D-lactate, which vancomycin cannot bind effectively, so cross-linking proceeds despite vancomycin’s presence. This requires acquisition of an entire operon (vanHAX system), not a point mutation:
  • vanR/vanS act as sensor/regulator, activating the operon in response to vancomycin.
  • vanH converts pyruvate to D-lactate.
  • vanA synthesises D-Ala-D-Lac (vancomycin-resistant) instead of normal D-Ala-D-Ala.
  • vanX cleaves normal D-Ala-D-Ala dipeptides.
  • vanY removes the terminal D-Ala from the pentapeptide.
  • vanZ has an additional undefined role.

  Take-home message (explicitly flagged by the lecturer as the key point, mechanistic detail not required): vancomycin resistance requires acquisition of a whole operon (horizontal gene transfer), not a simple point mutation.

• Target modification through mutation — fluoroquinolones: resistance can develop by chromosomal mutation during therapy, in the quinolone resistance determining region (QRDR) of gyrA, gyrB and parE/parC. Resistance increases stepwise as mutations accumulate: isolates with no QRDR mutation cluster at low MIC (susceptible to ciprofloxacin), while combined mutations (especially gyrA+parC and gyrA+gyrB+parC) push MIC sharply upward to >8 µg/ml (highly resistant).

Intrinsic vs Acquired Resistance

• Intrinsic resistance: the bacterium is normally resistant to the antimicrobial; it is chromosomally encoded. Examples: gram-negative bacteria and vancomycin; chromosomal AmpC β-lactamase.
• Acquired resistance: the bacterium is normally susceptible; resistance is acquired through gene acquisition or mutation. Examples: vancomycin-resistant enterococci; MRSA; plasmid-encoded AmpC β-lactamase; ESBL.
• Historically, resistance to each new antibiotic class has tended to emerge relatively quickly after deployment, sometimes almost immediately (e.g. penicillin deployed ~1943, resistance observed by the ~1940s; methicillin deployed ~1960, resistance by ~1962).

Emergence and Spread of Resistance

How resistance happens (four-step model):

1. A population of germs contains a few that are already drug-resistant.
2. Antibiotics kill the bacteria causing illness as well as protective “good” bacteria (antibiotic-mediated selection).
3. The drug-resistant bacteria are no longer outcompeted and are free to grow and take over the population.
4. Some resistant bacteria pass their resistance genes to other bacteria via horizontal gene transfer, spreading the problem further.

Horizontal transfer is easier than acquiring resistance by mutation, and gene exchange is especially frequent within animal guts. Four mechanisms of horizontal transfer:

• Conjugation: direct cell-to-cell contact via a pilus, transferring a plasmid.
• Transformation: uptake of free DNA from the environment.
• Transduction: bacteriophage-mediated DNA transfer.
• (Mutation is a spontaneous chromosomal change within a single lineage — not horizontal transfer, included as a contrast.)

Mobile genetic elements form a nested hierarchy that mobilises and spreads resistance genes: a bacterial clone contains plasmids (spread), which contain transposons (spread), which contain integrons (maintenance), which contain insertion sequences and specific resistance genes such as CTX-M (mobilisation, expression).

Multiple resistance and genetic linkage:

• Multidrug resistance can arise from genetic linkage on plasmids, transposons and integrons — a single plasmid can carry genes conferring resistance to many different, unrelated antibiotics.
• Selection pressure from one antibiotic class can maintain resistance genes to unrelated antibiotic classes (cross-selection), because the genes are physically linked.
• Disinfectant resistance genes can also be linked to antibiotic resistance genes, meaning disinfectant use may inadvertently select for antibiotic-resistant bacteria.

Drivers of Rising Resistance

Three converging causal pathways drive increasing antibiotic resistance:

• Inappropriate use of antibiotics (from viral infections being treated, wrong doses, wrong duration, over-prescription of new antibiotics, and animal growth supplements), combined with selective pressure and the genetic plasticity of bacteria.
• Increased spread (from crowding, homelessness, poor sanitation, day care centres, hospitals, international travel).
• Increased susceptibility to infection and antibiotic use (from poor nutrition, immunosuppression, invasive medical procedures).

Evidence for the role of antibiotic use:

• Outpatient antibiotic use correlates strongly with resistance: across ~19 European countries, higher outpatient penicillin use tracked with a higher rate of penicillin-non-susceptible S. pneumoniae (r = 0.84, p < 0.0001) — e.g. France had both the highest penicillin use and the highest resistance rate (~46%), while the Netherlands had both the lowest use and lowest resistance (~1%).
• Human antibiotic consumption and agricultural antibiotic use both vary greatly by country; New Zealand has relatively low agricultural antibiotic use compared internationally (lowest alongside Norway and Iceland), but only mid-pack human consumption compared to other countries.
• In the US, far more antibiotics by weight are sold for use in livestock than to treat sick humans, illustrating a large agricultural contribution to overall antibiotic use (a One Health issue: resistant organisms/genes flow multidirectionally between livestock, meat, humans, manure, sewage, crops, soil, water and wildlife).

Global and New Zealand Burden of Resistance

• Carbapenemase-producing organisms (e.g. NDM-producing Enterobacteriaceae) can be almost pan-drug resistant: susceptibility approaches 0% for nearly all standard agents tested (imipenem, meropenem, piperacillin-tazobactam, cephalosporins, aztreonam, fluoroquinolones, aminoglycosides, minocycline), leaving only tigecycline (56–67% susceptible) and colistin (89–100% susceptible) as partially active last-resort options.
• Plasmid-mediated colistin resistance (the mcr-1 gene) has since been found combined with carbapenem resistance (blaNDM), including in carbapenem- and colistin-resistant E. coli causing a complicated urinary tract infection — i.e. resistance to the last-resort agents identified above.
• Newer antibiotics have variable activity against carbapenemase-producing gram-negative organisms: cefiderocol is active against all major resistant categories tested (ESBL, KPC, NDM, OXA-48-like carbapenemases, carbapenem-resistant Pseudomonas aeruginosa and Acinetobacter baumannii); ceftazidime-avibactam, imipenem-cilastatin-relebactam and meropenem-vaborbactam each have gaps (notably no activity against NDM); eravacycline lacks activity against carbapenem-resistant P. aeruginosa.
• Klebsiella pneumoniae carbapenem resistance is geographically variable and clinically significant: 67% resistant in Greece and Egypt, 57% resistant in India (2020 data), with a mortality risk ratio of 2.14 for resistant infection.
• New Zealand has seen a marked recent rise in carbapenemase-producing organisms (CPOs): numbers stayed near zero until about 2013, then rose steadily, with a sharp increase from ~2021 (~45–80 isolates) to ~2023–2024 (~220–315 isolates), and 193 isolates already recorded by 30 June 2025. The proportion acquired within New Zealand (rather than overseas) has also risen (31% acquired in NZ in 2022, versus only 11% in 2017), while of overseas-acquired cases, hospital acquisition abroad remained common (64% in 2017, 71% in 2022).
• WHO (2014) warning: “The problem is so serious that it threatens the achievements of modern medicine. A post-antibiotic era — in which common infections and minor injuries can kill — is a very real possibility for the 21st century.”

Antimicrobial Stewardship

• CDC data: more than half of antibiotic prescribing for selected events in US hospitals was not consistent with recommended prescribing practices. Antibiotic prescribing was not supported in 79% of community-acquired pneumonia cases and 77% of urinary tract infection cases; 47% of fluoroquinolone prescriptions and 27% of intravenous vancomycin prescriptions were also flagged as inappropriate.
• Hospital prescribers and pharmacists can improve prescribing by: optimising antibiotic selection, re-assessing treatment once diagnostic test results are available, and using the shortest effective duration of therapy.
• Antimicrobial stewardship is defined as making sure patients get the right antimicrobials, at the right time, only when they need them.

Self-test

1. Name and briefly describe the three broad categories of antibiotic resistance mechanism.
2. Describe how porin mutations reduce antibiotic entry into gram-negative bacteria, and list the three specific porin-related outcomes described.
3. Describe the structure and role of bacterial efflux pumps in antibiotic resistance, including one example of a highly specific pump.
4. Describe the normal mechanism by which β-lactam antibiotics inhibit peptidoglycan synthesis.
5. Describe the catalytic cycle by which class A and C β-lactamases inactivate β-lactam antibiotics.
6. Distinguish class A/C β-lactamases from class B β-lactamases in terms of mechanism and spectrum.
7. Explain why clavulanic acid fails to protect against resistance in ESCPM organisms, and name the three organisms at highest risk of AmpC-mediated resistance emerging during treatment.
8. Explain how mecA/PBP2’ confers methicillin resistance in MRSA.
9. Describe the molecular basis of vancomycin resistance, and explain why the lecturer said the mechanistic detail (the vanHAX operon) was not the key point to learn.
10. Explain how fluoroquinolone resistance develops via QRDR mutations, including how MIC changes with accumulating mutations.
11. Distinguish intrinsic from acquired antibiotic resistance, giving one example of each.
12. Describe the four-step model of how antibiotic resistance emerges and spreads in a bacterial population.
13. List the three main mechanisms of horizontal gene transfer of resistance genes, distinguishing them from mutation.
14. Describe the hierarchical organisation of mobile genetic elements that allows resistance genes to spread.
15. Explain what is meant by cross-selection and how genetic linkage on a single plasmid can produce multidrug resistance.
16. A patient in a New Zealand hospital develops a urinary tract infection with an organism resistant to carbapenems, colistin and tigecycline. Using the concepts in this lecture, explain what class of resistance mechanism(s) could account for carbapenem resistance and why treatment options are now so limited.
17. Summarise the three converging causal pathways that this lecture identifies as driving rising antibiotic resistance.
18. What evidence from outpatient antibiotic use data supports a causal link between antibiotic use and resistance?
19. Describe the recent trend in carbapenemase-producing organisms in New Zealand, including how the proportion acquired locally versus overseas has changed.
20. List the stewardship actions hospital prescribers and pharmacists can take to reduce inappropriate antibiotic prescribing, and state the definition of antimicrobial stewardship given in the lecture.

Answers

Reveal answers

1. (1) Restricted access to the target (decreased permeability, increased efflux); (2) inactivation of the antibiotic (e.g. by enzymes); (3) modification of the target.
2. Porins in the outer membrane normally allow antibiotics to diffuse into the periplasm; mutations can cause porin loss (no diffusion), a narrowed channel (restricted diffusion), or decreased porin expression (fewer porins, reduced diffusion) — all reducing antibiotic entry, often affecting multiple antibiotic classes.
3. Efflux pumps actively export small molecules (metabolites, toxins, antibiotics) from the cytoplasm; a tripartite pump can span the outer membrane, periplasm and inner membrane to move molecules directly from cytosol to outside the cell. TetA is a highly specific tetracycline efflux pump; other pumps export many compounds and efflux resistance has been described for almost every antibiotic class.
4. Penicillin-binding protein (transpeptidase) normally cross-links peptide side chains of peptidoglycan strands; β-lactams bind irreversibly to this protein via a covalent bond with a serine residue in its active site, blocking cross-linking and weakening the cell wall.
5. A serine residue on the β-lactamase forms a covalent bond with the β-lactam; water then attacks the complex, hydrolysing and inactivating the β-lactam and releasing the free enzyme, which can repeat the cycle on further antibiotic molecules.
6. Class A and C β-lactamases use a serine-based mechanism and range from narrow to broad spectrum (class C = AmpC); class B β-lactamases are zinc-dependent, broad spectrum (e.g. NDM carbapenemase) and especially common in gram-negative bacteria.
7. Clavulanic acid inhibits class A β-lactamases but not AmpC (class C) β-lactamases, which ESCPM organisms produce (inducibly or constitutively) and which can increase during treatment via mutation; highest risk organisms are Enterobacter cloacae, Klebsiella aerogenes (formerly Enterobacter aerogenes) and Citrobacter freundii.
8. mecA encodes PBP2’, an alternative transpeptidase not inhibited by methicillin; it replaces the normal transpeptidase and continues cross-linking peptidoglycan even when β-lactam antibiotics are present.
9. Vancomycin normally binds D-alanine–D-alanine at the peptide terminus, blocking cross-linking; resistance changes the terminus to D-alanine–D-lactate, which vancomycin cannot bind, so cross-linking proceeds. The lecturer emphasised that this requires acquisition of an entire operon (horizontal gene transfer) rather than a simple point mutation — that take-home message, not the individual gene functions, was the key learning point.
10. Chromosomal mutations accumulate in the quinolone resistance determining region (QRDR) of genes such as gyrA, gyrB and parC/parE during therapy; isolates with no mutation have low MIC (susceptible), while combined mutations (especially gyrA+parC and gyrA+gyrB+parC) sharply raise the ciprofloxacin MIC to >8 µg/ml.
11. Intrinsic resistance is a chromosomally encoded, natural resistance of a species (e.g. gram negatives to vancomycin, or chromosomal AmpC); acquired resistance arises in a normally susceptible organism through gene acquisition or mutation (e.g. MRSA, vancomycin-resistant enterococci).
12. (1) A population contains a few naturally drug-resistant germs; (2) antibiotics kill both the illness-causing and protective bacteria; (3) the drug-resistant bacteria, no longer outcompeted, grow and dominate; (4) resistant bacteria transfer their resistance genes to other bacteria via horizontal gene transfer.
13. Conjugation (plasmid transfer via direct cell contact/pilus), transformation (uptake of free environmental DNA), and transduction (bacteriophage-mediated transfer) — these move genes between bacteria, unlike mutation, which is a spontaneous change within a single lineage.
14. A bacterial clone contains plasmids (which spread), which contain transposons (which spread), which contain integrons (maintenance), which contain insertion sequences and specific resistance genes such as CTX-M (mobilisation and expression) — a nested architecture enabling resistance genes to move between plasmids, transposons and clones.
15. Genes for resistance to different, unrelated antibiotics can sit together on the same plasmid, transposon or integron; selecting for resistance to one antibiotic class therefore also selects for (maintains) the linked resistance genes to other classes, even without direct exposure to those other antibiotics — this is cross-selection, and it can also link disinfectant resistance to antibiotic resistance.
16. Carbapenem resistance in gram-negative organisms is commonly due to carbapenemase enzymes (inactivation), such as NDM (class B, zinc-dependent, broad spectrum); colistin resistance can arise via plasmid-mediated mcr-1; combined resistance to carbapenems, colistin and tigecycline leaves almost no active standard agents, since these are considered among the last-resort options once carbapenemase-producers emerge, illustrating near pan-drug resistance.
17. (1) Inappropriate antibiotic use (viral infections treated, wrong dose/duration, over-prescription, agricultural growth supplements) combined with selective pressure and bacterial genetic plasticity; (2) increased spread of resistant organisms (crowding, homelessness, poor sanitation, day care, hospitals, travel); (3) increased host susceptibility to infection and antibiotic use (poor nutrition, immunosuppression, invasive procedures).
18. Across ~19 European countries, higher outpatient penicillin use correlated strongly with higher rates of penicillin-non-susceptible S. pneumoniae (r = 0.84, p < 0.0001); France had both the highest use and highest resistance (~46%), the Netherlands the lowest use and lowest resistance (~1%).
19. CPO numbers in New Zealand stayed near zero until about 2013, then rose steadily, with a sharp increase from ~2021 (~45–80 isolates) to ~2023–2024 (~220–315 isolates) and 193 isolates recorded by mid-2025; the proportion of cases acquired within New Zealand (rather than overseas) has risen from 11% in 2017 to 31% in 2022.
20. Optimise antibiotic selection, re-assess treatment once diagnostic results are available, and use the shortest effective duration of therapy. Antimicrobial stewardship is defined as making sure patients get the right antimicrobials, at the right time, only when they need them.