The problem with every successful antibiotic target is that bacteria can, and do, evolve resistance. When a drug kills most of the population but leaves alive any bacteria that happen to carry a resistance mutation or gene, those survivors reproduce and pass resistance on. This is natural selection working as it always does, and it is why the global rise of antimicrobial resistance (AMR) is directly linked to antibiotic use. Understanding the mechanism of action of each antibiotic class makes it easier to understand why resistance emerges through specific routes, which antibiotics are likely to remain effective when resistance to related drugs is present, and which combinations of drugs might work synergistically.
This page covers every major class of antibiotic currently in clinical use, explaining the mechanism of action of each class in plain language, the key drugs in each class, the bacterial targets involved, whether each class is bacteriostatic or bactericidal, and how bacteria evade the mechanism.
Cell Wall Synthesis Inhibitors
Beta-lactam antibiotics are the largest and most widely used antibiotic class. They include penicillins (amoxicillin, flucloxacillin, piperacillin), cephalosporins (cefalexin, cefuroxime, ceftriaxone, cefepime), carbapenems (meropenem, imipenem, ertapenem), and monobactams (aztreonam). All share a core four-membered beta-lactam ring that mimics the D-Ala-D-Ala terminus of the peptidoglycan precursor. Penicillin-binding proteins (PBPs), the enzymes that cross-link peptidoglycan chains, mistake the beta-lactam for their normal substrate and irreversibly bind to it, becoming permanently inactivated. This stops new peptidoglycan cross-links from forming, and since growing bacteria continuously break down and rebuild their cell wall, inhibiting new synthesis causes the cell wall to weaken and eventually lyse. Beta-lactams are bactericidal against actively growing cells. They have no effect on stationary-phase or non-growing cells, and they cannot penetrate gram-negative outer membranes without the help of porin proteins.
The main resistance mechanism is the production of beta-lactamase enzymes that hydrolyse the beta-lactam ring, rendering the drug inactive before it reaches the PBPs. Extended-spectrum beta-lactamases (ESBLs), particularly CTX-M, SHV, and TEM variants, hydrolyse most penicillins and cephalosporins. Carbapenemases (KPC, NDM, OXA-48, VIM, IMP) hydrolyse even the carbapenems. A different resistance mechanism operates in MRSA: the mecA gene encodes an alternative PBP (PBP2a) with very low affinity for all beta-lactams. The bacteria continue to cross-link their cell wall using PBP2a regardless of beta-lactam concentration.
Glycopeptides (vancomycin, teicoplanin) work by a different mechanism. They are large molecules that bind directly to the D-Ala-D-Ala terminus of the peptidoglycan precursor lipid II, physically blocking the transglycosylation and transpeptidation reactions needed to incorporate the precursor into the growing peptidoglycan layer. Because they work by steric blockade rather than enzyme inhibition, bacteria cannot evade glycopeptides by producing a modified enzyme. Instead, vancomycin-resistant enterococci (VRE) modify the target itself: the vanA and vanB gene clusters reprogram the peptidoglycan synthesis pathway to substitute D-Ala-D-Lac for D-Ala-D-Ala at the precursor terminus, dramatically reducing glycopeptide binding affinity. Glycopeptides cannot penetrate gram-negative outer membranes and are therefore only active against gram-positive bacteria.
Protein Synthesis Inhibitors
The bacterial ribosome (70S, made of 30S and 50S subunits) differs from the human ribosome (80S) in ways that several antibiotic classes exploit. This is why protein synthesis inhibitors can work without harming human cells, though some toxicity does occur because mitochondrial ribosomes are structurally similar to bacterial 70S ribosomes.
Aminoglycosides (gentamicin, tobramycin, amikacin, streptomycin) bind irreversibly to the 16S rRNA of the 30S ribosomal subunit. Binding causes misreading of the mRNA codons, inserting incorrect amino acids into the growing protein chain. These misfolded proteins insert into the bacterial cell membrane and increase its permeability, allowing more aminoglycoside to enter in a self-amplifying cycle. Aminoglycosides are concentration-dependent bactericidal drugs: killing correlates with the peak drug concentration relative to the MIC (Cmax/MIC ratio). The main clinical use is in combination with beta-lactams for serious gram-negative infections. Resistance is primarily by aminoglycoside-modifying enzymes (acetyltransferases, phosphotransferases, adenylyltransferases) that chemically alter the drug, reducing its ribosomal binding.
Tetracyclines (tetracycline, doxycycline, minocycline, tigecycline) bind reversibly to the 30S ribosomal subunit at a different site, blocking the attachment of aminoacyl-tRNA to the ribosomal A site and preventing elongation of the growing protein chain. They are bacteriostatic. Their broad spectrum (gram-positive, gram-negative, atypicals, rickettsiae, chlamydiae) makes them useful across many infection types. Tigecycline has extended activity against organisms resistant to older tetracyclines because it overcomes the main resistance mechanisms (efflux pumps and ribosomal protection proteins). Resistance via efflux pumps (tet genes) is common.
Macrolides (erythromycin, azithromycin, clarithromycin) and lincosamides (clindamycin) bind to the 23S rRNA of the 50S ribosomal subunit in overlapping binding sites. They block the exit tunnel through which the growing peptide chain emerges from the ribosome, causing premature termination of translation. They are primarily bacteriostatic for most organisms but can be bactericidal against some at high concentrations. Macrolides cover atypical organisms (Mycoplasma, Legionella, Chlamydia) that are invisible to beta-lactams, making them essential for atypical pneumonia treatment. The main resistance mechanism is ribosomal methylation encoded by erm genes, which modifies the 23S rRNA binding site so macrolides and lincosamides can no longer bind. Importantly, erm-based resistance can be constitutive or inducible (D-zone test detects inducible resistance to clindamycin in organisms exposed to erythromycin).
Chloramphenicol binds to the 50S subunit and blocks the peptidyl transferase reaction, preventing formation of the peptide bond between amino acids. Bacteriostatic against most organisms. It has excellent CNS penetration and was historically important for bacterial meningitis. Now rarely used in high-income countries due to serious toxicity (aplastic anaemia) but still used in low-income settings. Resistance by chloramphenicol acetyltransferase (CAT) enzymes is the most common mechanism.
Linezolid and tedizolid (oxazolidinones) bind to 23S rRNA of the 50S subunit at a different site from macrolides, preventing formation of the initiation complex at the start of translation. They are active against MRSA, VRE, and multidrug-resistant gram-positive organisms that are resistant to older classes. Bacteriostatic. Resistance mutations in the 23S rRNA target gene are documented but remain uncommon.
DNA and RNA Synthesis Inhibitors
Fluoroquinolones (ciprofloxacin, levofloxacin, moxifloxacin) target two bacterial enzymes: DNA gyrase (topoisomerase II) and topoisomerase IV. Both are essential for managing the supercoiling state of bacterial DNA during replication, transcription, and cell division. Fluoroquinolones stabilise the complex formed when the enzyme cuts DNA, trapping the cut in place and preventing re-ligation. This converts the enzymes from helpers into weapons, generating double-strand DNA breaks that trigger the bacterial SOS response and lead to cell death. Fluoroquinolones are concentration-dependent bactericidal drugs. Resistance is primarily by chromosomal mutations in the genes encoding the enzyme target (gyrA, gyrB, parC, parE) and by efflux pumps. Plasmid-mediated quinolone resistance (PMQR, by qnr genes) reduces susceptibility but alone does not usually reach high-level resistance.
Rifampicin (rifampin) binds to the beta subunit of bacterial RNA polymerase (encoded by rpoB) and physically blocks the RNA exit channel, preventing elongation of the RNA transcript. Rifampicin is bactericidal and very rapidly achieving high intracellular concentrations, making it valuable for infections within cells (tuberculosis, legionellosis, brucellosis). It must never be used as monotherapy because single-step point mutations in rpoB occur at a frequency of 10^-8 per cell division, and in a large bacterial population (as in active TB), resistant mutants are reliably selected within days of starting treatment.
Metronidazole is uniquely active against anaerobic bacteria and protozoa (Clostridioides difficile, Bacteroides, Giardia, Trichomonas, and Entamoeba). Under the low redox potential conditions inside anaerobic organisms, metronidazole's nitro group is reduced to a nitro radical anion. This reactive intermediate causes single-strand DNA breaks, fragment DNA, and disrupt the helical structure of DNA. Metronidazole is therefore completely selective for organisms growing in anaerobic conditions: it has no activity against obligate aerobes or most facultative anaerobes in which the low-redox intermediate does not form.
Membrane-Active Antibiotics
Polymyxins (polymyxin B, colistin/polymyxin E) are cyclic lipopeptides that act as cationic detergents on the bacterial outer membrane. They displace divalent cations (Mg2+ and Ca2+) that stabilise the LPS layer of the gram-negative outer membrane, disrupt membrane integrity, and cause leakage of cell contents. Bactericidal and concentration-dependent. Polymyxins are used as last-resort drugs for carbapenem-resistant gram-negative organisms (CRE, carbapenem-resistant Acinetobacter, Pseudomonas). Plasmid-mediated resistance by the mcr-1 gene, first identified in Chinese livestock, modifies the LPS lipid A component so polymyxins no longer bind effectively.
Daptomycin is a cyclic lipopeptide active exclusively against gram-positive bacteria. It inserts into the bacterial cytoplasmic membrane in a calcium-dependent manner, oligomerises, forms ion channels, causes membrane depolarisation, and rapidly arrests DNA, RNA, and protein synthesis without cell lysis. Bactericidal and concentration-dependent. It is a key treatment option for MRSA, VRE, and other resistant gram-positive organisms. It is inactivated by pulmonary surfactant and therefore cannot be used for pneumonia. Resistance can emerge on treatment through chromosomal mutations affecting membrane phospholipid composition.
Metabolic Pathway Inhibitors
Sulfonamides and trimethoprim work sequentially on the same pathway: folate biosynthesis. Bacteria must synthesise their own folic acid (humans obtain folate from diet). Sulfonamides inhibit dihydropteroate synthase (DHPS), blocking the first step. Trimethoprim inhibits dihydrofolate reductase (DHFR), blocking the second step. Inhibiting either step depletes the pool of tetrahydrofolate needed for nucleotide synthesis, stopping bacterial replication. Each drug is bacteriostatic alone but the combination (co-trimoxazole: trimethoprim-sulfamethoxazole) is bactericidal against many organisms through sequential blockade of the same pathway. Resistance develops through mutations in dhps (sulfonamide resistance) and dfr genes (trimethoprim resistance).
Frequently Asked Questions
What is the difference between bacteriostatic and bactericidal antibiotics?
Bacteriostatic antibiotics inhibit bacterial growth without killing bacteria. Bactericidal antibiotics kill bacteria directly. For most infections in patients with functional immune systems, bacteriostatic activity is sufficient because the immune system kills the static bacteria. For serious infections in immunocompromised patients, for endocarditis, and for meningitis, bactericidal activity is generally preferred.
Why do beta-lactams not work on MRSA?
MRSA carries the mecA gene (or the closely related mecC gene), which encodes an alternative penicillin-binding protein called PBP2a. PBP2a has very low affinity for all beta-lactam antibiotics and continues to cross-link peptidoglycan even when the other PBPs are saturated by beta-lactam drugs. Bacteria with PBP2a therefore grow normally in the presence of beta-lactam concentrations that would inhibit susceptible strains.
What is the mechanism of fluoroquinolone resistance?
The most common mechanism is chromosomal point mutations in the genes encoding the target enzymes (gyrA and parC). These mutations change the structure of DNA gyrase or topoisomerase IV so fluoroquinolones can no longer bind effectively. Additional mechanisms include overexpression of efflux pumps (Mex pumps in Pseudomonas, AcrAB-TolC in Enterobacteriaceae) that actively expel fluoroquinolones from the cell, and plasmid-mediated quinolone resistance (PMQR) by Qnr proteins that protect the topoisomerase from drug binding.
What is vancomycin resistance and how does it develop?
Vancomycin-resistant enterococci (VRE) carry the vanA or vanB gene cluster. These gene clusters encode enzymes that reprogram the peptidoglycan synthesis pathway to substitute D-Ala-D-Lac (D-alanine-D-lactate) for the normal D-Ala-D-Ala terminus of the peptidoglycan precursor. Vancomycin cannot bind D-Ala-D-Lac with significant affinity (binding is reduced approximately 1000-fold), so the drug cannot block cell wall synthesis in VRE.
Why does metronidazole only work on anaerobes?
Metronidazole's antimicrobial activity depends on the formation of a reactive nitro radical anion intermediate, which only forms under the low oxidation-reduction potential conditions inside anaerobic cells. Aerobic bacteria and aerobic human cells do not reduce metronidazole to this active intermediate, so the drug is essentially non-toxic in aerobic conditions. This selectivity makes metronidazole uniquely useful for anaerobic infections while being safe for human cells.
Why is rifampicin never used as monotherapy for tuberculosis?
Rifampicin is highly active against Mycobacterium tuberculosis but resistance emerges through single-point mutations in the rpoB gene encoding the RNA polymerase beta subunit. These mutations occur at a frequency of approximately 10^-8 per cell division, which sounds rare but in an active TB infection with billions of bacteria, resistant mutants exist in the population before treatment starts. Rifampicin monotherapy rapidly selects for these pre-existing mutants. Combination therapy with isoniazid, pyrazinamide, and ethambutol ensures that resistant mutants selected by any one drug are killed by the other drugs in the regimen.
What is the significance of daptomycin and pulmonary surfactant?
Daptomycin is inactivated by pulmonary surfactant, the phospholipid-rich fluid that lines the alveoli. When daptomycin contacts surfactant, the surfactant phospholipids competitively bind to daptomycin's lipid tail, preventing it from inserting into the bacterial membrane and destroying its antimicrobial activity. This is why daptomycin cannot be used to treat pneumonia even though it is highly active against gram-positive organisms in other sites: adequate drug concentrations cannot be achieved in the lung.
What is colistin and why is mcr-1 resistance so significant?
Colistin (polymyxin E) is a last-resort antibiotic used for carbapenem-resistant gram-negative bacteria when no other options remain. MCR-1 is a plasmid-borne gene encoding a phosphoethanolamine transferase that modifies the lipid A component of LPS, reducing the negative charge that polymyxins depend on for binding. What makes mcr-1 critical is that it is carried on a mobile plasmid that can spread between bacterial species by horizontal gene transfer, including between livestock bacteria and human pathogens. First reported in China in 2015, mcr-1 has since been found globally.
What are ESBLs and why do they matter clinically?
Extended-spectrum beta-lactamases (ESBLs) are enzymes, primarily in E. coli and Klebsiella pneumoniae, that hydrolyse most penicillins and cephalosporins, including third-generation cephalosporins like ceftriaxone and cefotaxime. ESBL-producing organisms cause significant treatment challenges because many common antibiotics are ineffective. Treatment options are limited to carbapenems (meropenem, ertapenem) for serious infections, or to specific oral options (nitrofurantoin, fosfomycin, pivmecillinam) for uncomplicated ESBL urinary tract infections. The most common ESBL globally is CTX-M-15.
How do aminoglycosides cause nephrotoxicity?
Aminoglycosides accumulate in the proximal tubule cells of the kidney, where they disrupt cell membrane phospholipids, cause mitochondrial dysfunction, and trigger apoptosis. Nephrotoxicity is dose-related and cumulative. Risk factors include prolonged treatment duration, existing kidney impairment, concomitant nephrotoxic drugs (vancomycin, NSAIDs, contrast agents), and older age. Once-daily aminoglycoside dosing is designed partly to reduce nephrotoxicity by allowing a drug-free interval during which tubular cells can recover, compared to multiple smaller doses that maintain continuous high concentrations in tubular cells.