nature reviews microbiology https://doi.org/10.

1038/s41579-022-00820-y

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Molecular mechanisms of
antibiotic resistance revisited
Elizabeth M. Darby    1, Eleftheria Trampari    2, Pauline Siasat    1, Maria Solsona Gaya    2, Ilyas Alav    1,
Mark A. Webber    2,3  & Jessica M. A. Blair    1 
Abstract Sections

Antibiotic resistance is a global health emergency, with resistance Introduction

detected to all antibiotics currently in clinical use and only a few novel Reduced permeability
drugs in the pipeline. Understanding the molecular mechanisms that Active transport of antibiotics
bacteria use to resist the action of antimicrobials is critical to recognize
Target alteration, modification
global patterns of resistance and to improve the use of current drugs, and protection
as well as for the design of new drugs less susceptible to resistance
Inactivation and modification
development and novel strategies to combat resistance. In this Review, of the drug
we explore recent advances in understanding how resistance genes Target bypass
contribute to the biology of the host, new structural details of relevant
The promise of resistance
molecular events underpinning resistance, the identification of breakers
new resistance gene families and the interactions between different Conclusions and future
resistance mechanisms. Finally, we discuss how we can use this perspectives
information to develop the next generation of antimicrobial therapies.

1
College of Medical and Dental Sciences, Institute of Microbiology and Infection, University of Birmingham,
Birmingham, UK. 2Quadram Institute Bioscience, Norwich Research Park, Norwich, UK. 3Medical School,
University of East Anglia, Norwich Research Park, Norwich, UK.  e-mail: Mark.Webber@quadram.ac.uk;
J.M.A.Blair@bham.ac.uk

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Introduction in our understanding of how antibiotics work and the major mecha-
Antimicrobial resistance (AMR) is a major global health challenge, nisms by which bacteria can resist the inhibitory or killing effects of
causing substantial morbidity and death globally. Understanding the antibiotics (Fig. 1), including the importance of context for many resist-
molecular mechanisms that underlie resistance can aid in the design ance mechanisms in determining their effects (Box 1). For example, the
of novel strategies to treat infectious diseases. Bacteria use various expression of resistance genes and targets can substantially change
mechanisms of resistance1, some are ‘intrinsic’, whereby the cell can use between diverse growth conditions2. New technological advances
genes it already possesses to survive antibiotic exposure, and some are have revealed the structural details of many resistance mechanisms,
‘acquired’, whereby gain of new genetic material provides new capaci- including complex, multi-component structures of efflux systems
ties that mediate survival (Table 1). There has been substantial progress that might point towards possible routes for inhibitor development3,4

Table 1 | Major classes of antibiotics, primary targets and mechanisms of resistance

Antibiotic class (examples) Mechanism of action Mechanism of resistance

Aminoglycosides (gentamicin, Interact with the 30S ribosomal subunit of 16S rRNA Aminoglycoside-modifying enzymes, for example, acetyl­
streptomycin, kanamycin) causing misreading and/or truncated proteins and transferases, phosphotransferases and nucleotidyltransferases131;
cell death75; positively charged, attach to outer 16S ribosomal methylases170; mutations in the 16S rRNA gene171;
membrane causing pores to increase accumulation169 decreased influx and/or increased efflux172
β-Lactams (penicillins, cephalosporins, Target peptidoglycan crosslinking by inhibiting Production of β-lactamases173; modification of penicillin-binding
cephamycins, carbapenems, penicillin-binding proteins, which crosslink the proteins174; reduced permeability and increased efflux174
monobactams) peptide chain in the cell wall107, leading to lysis of
the cell75
Cationic peptides (colistin) Bind to lipid A in lipopolysaccharide175; permeabilizing Modification or removal of lipid A176,177
the outer membrane causing cell death175
Glycopeptides (vancomycin) Inhibit crosslinking and therefore synthesis of Intrinsic resistance in Gram-negative cells by impermeable
peptidoglycan by binding to D-alanyl-D-alanine in outer membrane178; in Gram-positive cells, enzymes can modify
the peptide chain178 and hydrolyse peptidoglycan precursors179; intermediate
susceptibility phenotype conferred by mutations leading to
thickened membrane and low permeability180
Lincosamides (clindamycin) Target the translation of proteins, specifically Methyltransferases that modify 23S rRNA182; expression of
23S rRNA of the 50S ribosomal subunit, causing proteins that inactivate lincosamides and efflux183
truncated peptide chains181
Lipopeptides (daptomycin) Insert in the cell membrane and cause Thickening of and increasing the positive charge in the cell
depolarization, reducing the ability to create ATP wall185; reducing the depolarization of membranes induced by
and cell death184 lipopeptides185
Macrolides (azithromycin, Inhibit the translation of proteins by targeting rRNA methyltransferases, which methylate 23S rRNA187;
erythromycin) 23S rRNA of the 50S ribosomal subunit, causing mutations in the ribosome188; efflux188; macrolide phospho­
truncated peptide chains186 transferases and esterases135; ribosomal protection by 
ATP-binding cassette F (ABC-F) proteins189
Oxazolidinones (linezolid) Limit translation by binding to 23S rRNA of the 50S Modifications of 23S rRNA, for example, by methyltransferases191;
subunit and preventing the formation of a functional protection of the ribosome via ABC-F proteins191
70S subunit190
Phenicols (chloramphenicol) Inhibit translation by binding to the A site of the 50S Mutations within 23S rRNA of the 50S ribosomal subunit192;
ribosomal subunit, inhibiting protein synthesis192 enzymatic inactivation via acetyltransferases and efflux192
Pyrimidines (trimethoprim) Affect C1 metabolism and folate synthesis by The modification or acquisition of novel dihydrofolate reductase
inhibiting dihydrofolate reductase, blocking genes and efflux of trimethoprim194
production of tetrahydrofolate193
Quinolones and fluoroquinolones Inhibit DNA replication by DNA gyrase and Mutations in DNA gyrase or topoisomerase IV195; the efflux
(ciprofloxacin) topoisomerase IV, which are involved in DNA of quinolones or proteins that protect DNA gyrase and
supercoiling, strand cutting and ligating195 topoisomerase IV195
Rifamycins (rifampicin) Inhibit transcription, specifically DNA-dependent Mutations in the drug target rpoB196; enzymatic ribosylation or
RNA synthesis, by binding to RNA polymerase196 inactivation of rifampicin144
Streptogramins (dalfopristin) Target protein translation by binding to 23S rRNA Mutations in 23S rRNA192; modification of streptogramins by
of the 50S ribosomal subunit at the peptidyl- acetyltransferases192; efflux out of the cell192
transferase domain causing truncated peptides197
Sulfonamides (sulfamethizole) Stop dihydrofolate acid synthesis by inhibiting Mutations in the dihydropteroate synthase gene and sul1/2
dihydropteroate synthase and arresting cell growth198 genes, which encode distinct dihydropteroate synthases that are
less susceptible to sulfonamides198
Tetracyclines (tigecycline, tetracycline) Inhibit translation by binding to 16S rRNA of the 30S Efflux199; protein-mediated ribosome protection199; ribosomal
ribosomal subunit, preventing tRNA binding to 30S mutations199; enzymatic inactivation of the drug199
at the A site199

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Active efflux
Periplasm Inner membrane
Outer membrane

Target protection protein Efflux pump

Drug
Antibiotic
target
A B
Target protection
Antibiotic- New protein with
modifying the same metabolic
enzyme capacity as antibiotic
Chemical
moiety target protein
Target site
modification
Target bypass
Inactivation Decreased influx
of antibiotic

Downregulation Porin

Fig. 1 | Overview of the molecular mechanisms of antibiotic resistance. not inhibited by the antibiotic, making the original target redundant and the
Inactivation of antibiotics is mediated by enzymes that either degrade or modify antibiotic ineffective203. Decreased influx is mediated by changes to membrane
the antibiotic molecule. Enzymatic degradation involves hydrolysis of the structure, for example, the downregulation of porins, which are transmembrane
functional group of the antibiotic, thereby rendering it ineffective200. Antibiotic- proteins that allow the passive transport of various compounds, such as antibiotics,
modifying enzymes transfer various chemical groups to the antibiotic, which into the bacterial cell13. Active efflux is facilitated by transmembrane efflux
prevent binding of the antibiotic to its target201. Target site alteration involves pumps, which export antibiotics out of bacterial cells to reduce their intracellular
alteration of the antibiotic target to reduce binding of the antibiotic. This can concentration204. Target protection generally involves the physical association
involve mutations in the gene encoding the protein target of the antibiotic of a target protection protein with the antibiotic target, thereby relieving it from
molecule or enzymatic alteration of the binding site102,202. During target bypass, antibiotic-mediated inhibition205.
the function of the antibiotic target is accomplished by a new protein that is

and a proposed mechanism of action for the newly identified ABC-F revolutionized the study of AMR (Box 3) and how together this information
ribosome rescue proteins. More is also known about the importance can aid in developing the next generation of antimicrobial therapies.
and hierarchy of multiple molecular mechanisms working together to
generate high-level resistance (for example, the underpinning role of Reduced permeability
efflux to support all other resistance mechanisms; Box 2). For many antimicrobials to exert their activity, they need to cross
In addition, the costs and benefits associated with acquiring the bacterial cell envelope to reach their target. This is particularly
resistance are now better understood. In particular, the interplay important in Gram-negative bacteria, which have a double-membrane
between plasmids, hosts and AMR genes is important in determining structure that makes the cellular envelope relatively impermeable,
the expansion of genes, vectors and strains5–7. Examples of successful providing intrinsic resistance to many antibiotics that work against
acquisition of specific resistance genes by globally dominant clones Gram-positive pathogens and presenting a major challenge to the devel-
of a species have been identified, for example, acquisition of the opment of novel antimicrobials that can penetrate the cell envelope.
extended-spectrum β-lactamase CTX-M-15 by Escherichia coli ST131, In addition, alterations to envelope structure, such as porin loss or
or the carbapenemase KPC by Klebsiella pneumoniae ST258, both of changes to phospholipid and fatty acid content of the cytoplasmic
which spread globally8,9. membrane, can affect the ability of a drug to penetrate the cell and
In this Review, we explore the mechanisms of resistance and out- can contribute to the emergence of AMR. Gram-positive bacteria lack
line the clinical relevance of different mechanisms. Specifically, we the outer membrane, which makes them naturally more permeable to
discuss the most recent progress in understanding antibiotic resistance many antibiotics; however, changes in composition of the cytoplasmic
via reduction in intracellular drug accumulation (reduced permeability membrane, which affect fluidity, have been shown to be important
and antibiotic efflux), modification or alteration of the bacterial antibi- in reducing permeability to antibiotics10. Mycobacteria produce an
otic target, modification or destruction of the drug itself, and bypass of extensive outer lipid layer and a polysaccharide capsule coat, thereby
whole metabolic pathways. Finally, we also discuss how genomics has preventing the entry of hydrophilic molecules into the cell11.

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Box 1

The role of the environment and lifestyle on survival

Bacteria live in varied conditions, where different genes are exposure to these drugs rapidly selects for cell wall-deficient bacteria
required for survival212. One major common lifestyle for bacteria (figure, part c)216.
is growth in biofilms, which are aggregated communities of cells A lifestyle change that can be induced by environmental
producing a protective extracellular matrix. Biofilms are inherently conditions is the formation of persisters, which are dormant cells
tolerant to the action of antibiotics and some exhibit resistance due that are often growth arrested but viability is maintained. Persister
to multiple factors213. During the planktonic lifestyle, bacteria are cells are highly resistant to the killing action of bactericidal antibiotics
inhibited by the drug (see the figure, part a,i). However, cells within but regain normal sensitivity once replication resumes217. Various
a biofilm exhibit a large degree of heterogeneity of metabolic mechanisms for persister cell formation have been suggested,
states and gene expression, which results in individual cells being including toxin–antitoxin systems and production of (p)ppGpp,
resistant within a biofilm due to reduced permeability (part a,ii), although there remains much debate about the importance of
low metabolic activity resulting in reduced target expression each218. Recent data have shown that cells with low levels of ATP
(part a,iii) and production of large numbers of persister cells213 are likely to become persisters219,220.
(part a,iv). Biofilms can also experience high rates of genetic Tolerance refers to populations of cells temporarily able to survive
exchange, allowing movement of antimicrobial resistance (AMR) antibiotic exposure at concentrations that exceed the minimum
genes (figure, part b)214. inhibitory concentration221. Tolerant cells exhibit slow growth (but not
A striking example of how a lifestyle change can result in complete arrest) due to mutation within genes that impact growth.
resistance is seen in ‘L-form’ bacteria, which are cells that lose their This slower growth allows cells to survive antibiotic exposure and
cell wall after stress exposure215. L-form bacteria are consequently favours the accumulation of resistance mutations when exposed
resistant to cell-wall targeting agents (for example, β-lactams) and to drugs222.

a b Movement of AMR genes

i Antibiotic
AMR gene

Antibiotic target

ii Decreased permeability
Antibiotic

c Cell-wall targeting antibiotic

Surface
iii Low expression of target proteins i
Cell wall

Cell lysis

L-form bacterium

iv Persister cells
ii

Cell survival

Persister cell

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The outer membrane of Gram-negative bacteria is a complex electric charge within the pore and, in turn, affect the permeability of
organelle that has evolved to provide protection and has a barrier antibiotics such as cefotaxime, gentamicin or imipenem17.
function, while still allowing the uptake of nutrients. Recent evidence Porin expression in Enterobacterales is actively regulated in
from Enterobacterales shows how permeability of the outer membrane response to environmental stimuli (Fig. 1). Perhaps the best-studied
dynamically changes during bacterial growth, which, in turn, affects examples are, again, OmpC and OmpF in E. coli, the expression of
how much drug can penetrate the membrane2. The outer membrane which is under the control of the EnvZ–OmpR two-component system.
contains porins, which are β-barrel protein channels categorized, based EnvZ is a periplasmic sensor protein that senses changes in the envi-
on their function and architecture, into families: general nonspecific ronment and controls the phosphorylation state of OmpR, its cog-
channels (for example, OmpF and OmpC); substrate-specific chan- nate response regulator18. High levels of phosphorylated OmpR in
nels (for example, PhoE and LamB) and small β-barrel channels (for the cell lead to reduced ompF and increased ompC transcription. This
example, OmpA and OmpX)12. Generally, porins allow the influx of differential regulation of the two porins allows appropriate porin
hydrophilic compounds <600 kDa into the cell, including many anti- expression: in high-osmolarity environments, where nutrients are
biotics13. OmpF and OmpC found in E. coli and related organisms are abundant, the small pore is predominant. Mutants that only produce
trimeric β-barrel structures through which different antibiotic classes the smaller pore survive antibiotic exposure better. It has been shown
are known to be able to pass. OmpF has a larger pore size than OmpC that carbapenems select for multiple first-step mutations within OmpR,
(6.5–7 Å versus 5.5–6 Å, respectively), which makes it generally easier conferring reduced porin expression19. Small regulatory RNAs (sRNAs)
for substrates to pass through OmpF compared with OmpC14. Despite can also control outer membrane structure at a post-transcriptional
earlier beliefs that porins exhibited selectivity towards certain sub- level. The sRNA micF is encoded by a sequence divergent to ompC and,
strates, recent studies suggest that diffusion occurs in a spontaneous, when transcribed, it inhibits expression of ompF by direct base pair-
passive way, without an active interaction observed15. ing to the ribosome-binding site and start codon of the ompF mRNA,
Recently, alterations to porin structure have also been identified thus preventing translation20. Expression of this sRNA is conditional
as important contributors to antibiotic resistance, demonstrating that and subject to various environmental stimuli such as high osmolar-
these are not evolutionarily static structures, and our knowledge of the ity conditions21. More recently, the micC sRNA has been identified
structure–function relationship of porins has improved. For example, and observed to repress OmpC translation by directly binding to the
carbapenem resistance in Klebsiella pneumoniae is partially mediated 5’ untranslated region of the ompC mRNA22. Transcription of these
by modification of the non-selective porins OmpK35 and OmpK36; sRNAs is co-regulated in response to antimicrobial stress, and β-lactams
a selected insertion (Gly115-Asp116) into loop 3 of OmpK36 causes actively induce the transcription of micC23.
significant constriction of the pore and, thus, increased tolerance to Nonspecific porins, such as OmpF and OmpC, are characteristic
carbapenems16. Multidrug-resistant E. coli isolates from patients were of Enterobacterales. However, other important pathogens, such as
recently found to carry multiple mutations within OmpC that alter the Pseudomonas aeruginosa and Acinetobacter baumannii, are instead

Box 2

Underpinning role of efflux and synergies with other resistance

mechanisms
High-level multidrug resistance (MDR) is often conferred by a resistance via the accumulation of point mutations63,226. Similarly, in
combination of multiple interacting mechanisms. In particular, there Staphylococcus aureus, amplification of the NorA efflux pump led
is a growing body of evidence indicating that many mechanisms of to more rapid evolution, whereas inhibition of the pump prevented
antibiotic resistance rely on or interact with the intrinsic resistance resistance evolution. Interestingly, positive epistasis was also
provided by efflux pumps. detected, whereby increased NorA expression interacted positively
First, efflux activity can cause altered expression of other with mutations conferring ciprofloxacin resistance in S. aureus to
genes involved in intrinsic antibiotic resistance. For example, in further increase resistance225.
Enterobacterales, the deletion or inhibition of acrAB can lead to Finally, there is also evidence that resistance–nodulation–division
decreased expression of the outer membrane porin OmpF, thereby efflux affects horizontal gene transfer. It was shown, in Escherichia
reducing membrane permeability and limiting intracellular drug coli, that, in the presence of the translation-inhibiting antibiotic
accumulation223. tetracycline, the AcrAB–TolC efflux pump was required for the
Second, the efflux status of cells affects the rate of evolution acquisition of plasmids carrying TetA, which confers high-level
of resistance in a population. Lack of efflux function has been tetracycline resistance. This is because the efflux pump reduces
shown to decrease the frequency with which antibiotic-resistant intracellular levels of the drug, thus allowing time to translate
mutants are selected41,224,225. Furthermore, there is single-cell proteins encoded on the newly acquired plasmid227. Furthermore,
level heterogeneity in acrAB expression, and cells with higher the acquisition of an MDR plasmid in Klebsiella pneumoniae has
levels of expression of acrAB had lower levels of expression of the been shown to cause increased transcription of efflux genes,
DNA mismatch repair gene mutS and subsequently an increased thus further strengthening the link between MDR plasmids and
mutation rate, which enables the rapid evolution of high-level efflux228.

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including antibiotics, across bacterial membranes in an energy-

Box 3 dependent manner. While all bacteria contain multiple efflux pumps,
they are particularly important mediators of AMR in Gram-negative
bacteria. They work synergistically with the impermeable double mem-
Genomics and the study of brane to make these pathogens intrinsically resistant to many antibi-
otics. The impact of different efflux systems on specific drugs varies,
antimicrobial resistance with some providing high levels and others low levels of resistance;
however, efflux acts as a crucial ‘platform’ mechanism that enables
The study of bacteria has moved from the first reported genomes of most other resistance mechanisms to have an impact28. Efflux transport-
pathogenic species, to comparisons of the first hundred, to today ers are categorized into six families, with members of the resistance–
where for some species, hundreds of thousands of individual strains nodulation–division (RND) family conferring the most clinically
have been sequenced229. Analysis of large numbers of genomes of a relevant levels of resistance in Gram-negative bacteria3. As inner mem-
species now allows epidemiology of antimicrobial resistance to be brane proteins, RND transporters associate with both periplasmic
studied and can show how the acquisition of resistance genes and adaptor proteins (PAPs) and an outer membrane factor (OMF) in a
elements interplay with host fitness in globally dominant clones. 3:6:3 protomer stoichiometry to form tripartite complexes that span
For example, the acquisition of carbapenem-resistant plasmids in the entire Gram-negative cell envelope29 (Fig. 2). RND pumps can export
dominant clones of Escherichia coli has been facilitated by mutation a broad range of structurally and chemically dissimilar antibiotics, and
in core metabolic genes230. the overexpression of these pumps contributes to multidrug resistance
Studying large isolate panels has been used to predict novel (MDR) in clinical isolates30,31.
mechanisms of resistance. An analysis of 95 Staphylococcus aureus Understanding of the structure and assembly of RND tripartite
isolates was used to identify genes involved in daptomycin resistance, systems has been revolutionized by technological improvements,
which was previously not well understood185, and a genome-wide particularly in cryo-electron microscopy (cryo-EM)3. Cryo-EM has
association study suggested that convergent evolution has selected allowed us to visualize efflux transporters in bound and unbound
for daptomycin-resistant mprF mutants on multiple occasions231. states, in addition to providing completely assembled tripartite efflux
Genomic sequencing following laboratory evolution experiments complexes in situ32,33.
is a simple and accessible tool to identify routes to resistance. RND transporters are homotrimeric proteins that recognize
For example, parallel selection of mutations within fusA1 and ptsP ligands and transduce electrochemical energy from the proton (H+)
in response to tobramycin in Acinetobacter baumannii grown in gradient across the inner membrane3,30. PAPs are elongated periplas-
different conditions demonstrated the primary importance of these mic proteins that mediate the assembly and stabilization of the tri-
mutations in resistance232. partite complex via six PAP monomers directly interacting with both
Functional genomics approaches have also become highly the inner membrane protein and OMF components. The assembled
advanced, CRISPR methods now allow defined mutants to be complex constitutes a continuous tunnel through which substrates
generated in species previously not tractable for manipulation, are exported3. As the exterior portion of the channel, OMFs are outer
and extremely high-density transposon mutant libraries have been membrane-bound, homotrimeric structures that protrude deep into
used to screen whole genomes for changes related to antibiotic the periplasmic space and serve as the final conduit of the tripartite
susceptibility at almost base pair resolution233. This allows the complex from which drugs exit the channel and are released into the
identification of many genes that have small contributions to
resistance but together form the ‘secondary resistome’. Using Glossary
this approach, a study of genes involved in colistin sensitivity in
Klebsiella pneumoniae showed that inactivation of a non-essential
gene (dedA) reversed antimicrobial resistance in isolates with high β-Lactamase Minimum inhibitory
colistin minimum inhibitory concentrations234. Enzymes produced by bacteria that concentration
degrade β-lactam antibiotics. (MIC). The lowest concentration of
antibiotic that prevents growth of
equipped with multiple, specific porins that allow the entry of mol- Epistasis bacteria.
ecules no larger than 200 Da (ref.24). This lack of larger, general porins Where a mutation can exert a
results in highly impermeable membranes, particularly towards hydro- phenotypic effect but only in concert Topoisomerases
philic molecules25. In the case of P. aeruginosa, loss of OprD porins with other genes, making the impact Essential enzymes involved in DNA
is very commonly reported as a mechanism of clinically important, conditional on the genetic background replication.
high-level carbapenem resistance26. This is often seen in conjunction of where it occurs.
with other mechanisms, and a recent analysis of the impacts of inacti- Two-component system
vating all porins of P. aeruginosa showed that the loss of no single porin Horizontal gene transfer A system that allows bacteria to
could completely abolish drug entry, demonstrating the importance The movement of genetic information respond to specific environmental
of synergy between porin loss and other mechanisms27. between bacterial cells. stimuli. Usually, it consists of a
membrane-bound histidine kinase
Active transport of antibiotics Insertion sequences that senses the stimuli and activates
As well as preventing drugs from entering the cell, bacteria can actively Small pieces of DNA that encode their a response regulator that alters the
export them in a process known as efflux. Efflux pumps are transmem- own recombination machinery and can expression of relevant genes.
brane proteins that can transport a wide variety of toxic compounds, move within or between genomes.

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a b
Outer
membrane

TolC
Distal BP
Exit
channel
Switch
loop
Distal BP
CH2 CH3 (closed)
AcrA
Proximal BP
Vestibule

CH4
AcrB CH1

T protomer
O protomer
Inner
membrane

AcrZ
Fig. 2 | Structure and entry channels of RND efflux systems. a, Shown is the (not indicated) and is open to the periplasmic space208. High-molecular-mass
crystal structure of the tripartite AcrAB(Z)–TolC efflux complex (Protein Data drugs (for example, erythromycin or rifampicin) preferentially enter through
Bank (PDB) ID: 5O66)34. The trimeric inner membrane resistance–nodulation– CH2 and bind to the proximal BP49. The switch loop separates the proximal BP and
division (RND) transporter AcrB has a crucial role in substrate recognition the distal binding pocket (distal BP) and is important for the translocation of high-
and energy transduction206. The AcrB trimer interacts with six copies of the molecular-mass drugs from the proximal BP to the distal BP209. Low-molecular-
periplasmic adaptor protein AcrA, which form a sealed tubular structure that mass drugs (for example, chloramphenicol and linezolid) preferentially enter
links AcrB to the outer membrane factor TolC37. The AcrA hexameric ring interacts through CH1 and bypass the proximal BP and the switch loop and bind directly
with the TolC trimer in a tip-to-tip fashion207. The TolC trimer is a cannon-shaped to the distal BP48. CH3 is open to the central cavity formed by the vestibules
channel, of which one end is inserted in the outer membrane with the other end between the protomer interfaces and is preferentially used by planar aromatic
extending into the periplasmic space and interacting with AcrA37. The small cations (for example, berberine or ethidium bromide) for transport directly to the
protein AcrZ interacts with AcrB and is proposed to play a role in allosterically distal BP47. The entrance of CH4 is situated at the groove formed by TM1 and TM2
modulating the activity of AcrB51. b, The AcrB trimer consists of multiple substrate and leads directly to the distal BP. CH4 is preferentially accessed by carboxylated
entry channels (L protomer not shown for clarity). The entrance of channel 1 drugs (for example, β-lactams or fusidic acid)50,210. During the transition of the
(CH1) is open to the outer leaflet of the inner membrane. Substrates entering AcrB protomers from tight to open (T-to-O), all substrate channels are closed, and
CH1 are guided to the proximal binding pocket (proximal BP). The entrance an exit channel is created in the O protomer. The exit channel is connected to the
of CH2 is situated at the cleft formed by the PC1 and PC2 subdomains of AcrB closed distal BP, allowing substrates to be exported211.

extracellular space, thereby completing substrate extrusion3,34. Cryo- initially assigned, it is now clear that the PAPs have a crucial and active
EM structures of AcrAB–TolC from E. coli, at near-atomic resolution, role in both pump assembly and substrate expulsion3,40. This impor-
have shown that a closed-state complex is formed upon tripartite tance has led to PAPs being considered as effective targets to combat
assembly when substrates are absent34,35. In the presence of substrates, efflux-mediated antibiotic resistance41,42.
such as puromycin, the periplasmic tip of the OMF TolC is dilated, RND efflux pumps contribute to the MDR phenotype of all clini-
while the three protomers of the RND transporter AcrB assume an cally relevant Gram-negative pathogens, including E. coli, P. aeruginosa,
asymmetric conformation — loose, tight and open — presumably in Neisseria gonorrhoeae and A. baumannii, partly due to their extraordi-
preparation for drug efflux34 (for a graphical representation see ref.30). narily large substrate range (see, for example, refs.43,44). The structural
The quaternary structural changes involved with the apo-to-active state basis for the poly-specificity of RND transporters is not fully under-
transition of AcrB are reflected onto TolC via the PAP AcrA34, which also stood, although they do contain both proximal binding pockets and
seals the pump channel to prevent leakage during substrate export34,36. distal binding pockets (distal BPs), which can accommodate different
Based on the most recent cryo-EM structures of tripartite pumps, substrates (Fig. 2). In AcrB, the surface of its distal BP allows for multi-
successful opening of the OMF conduit has been shown to depend site binding due to the presence of weakly hydrophobic and weakly
on the disruption of ‘primary gates’, formed by predominantly ionic polar residues45,46. In addition, multiple entry pathways to AcrB provide
interactions, at its periplasmic tip by the α-helical hairpin domain of different access routes to the distal BP for substrates with different
PAPs in a ‘tip-to-tip’ cogwheel manner3,34,37–39. PAPs have also been pro- chemical properties47,48. Entrances at the membrane–periplasm inter-
posed to enforce the directionality of cycling between RND transition face (channel 1) and at the periplasm (channel 2) permit entry to drugs
states based on functional and cryo-EM structural analysis of MexAB– with low and high molecular mass, respectively30,49, whereas an opening
OprM of P. aeruginosa3,39. Far from the simple bridging role they were between the periplasm and central cavity of the AcrB trimer (channel 3)

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is favoured by planar, aromatic cations such as ethidium bromide47,50. function and regulation of these transporters3,30,70,71. Indeed, this would
Most recently, a fourth channel located between transmembrane heli- also be applicable to members of other efflux families, such as NorA
ces 1 and 2 of AcrB has been proposed as an entry point for carboxylated of Staphylococcus aureus and P55 of Mycobacterium tuberculosis, from
substrates such as fusidic acid and hydrophobic β-lactams50. the major facilitator superfamily72–74.
The substrate preference of AcrB is also modulated by the effects
of a fourth transmembrane component, AcrZ51. Located within the Target alteration, modification and protection
inner membrane, AcrZ is a small α-helical protein that binds to AcrB Central to the selective toxicity of most antibiotics against bacteria
(Fig. 2) and promotes the preferential extrusion of antibiotics, such is their high specificity for important bacterial cellular targets. Many
as chloramphenicol, tetracycline and puromycin, by AcrAB–TolC34,51. antibiotics bind a primary target with high affinity, which generally
Recent cryo-EM data have suggested that lipids work with AcrZ in a syner- inhibits an essential cellular function and leads to inhibition of growth
gistic manner to allosterically modulate AcrB activity52. Understanding or death75. If the structure of the primary target is altered or protected
the mechanisms that support the activity of RND transporters would by decoration with other chemical moieties, then antibiotic binding
be beneficial for the rational design of efflux pump inhibitors to inhibit can become inefficient, conferring resistance to the antibiotic (Fig. 3a).
drug sequestration and restore antibiotic susceptibility46,53,54. For example, the quinolone antibiotics inhibit essential topoisomerase
Efflux pump production is carefully regulated, and most systems enzymes by binding near the active site. Amino acid substitutions in the
are controlled by a repressor encoded adjacent to the structural efflux target proteins, which result in lower binding efficiency while still allow-
system genes. Mutations in these transcriptional regulator genes are ing the enzyme to function, confer resistance76. Similarly, decreased
frequently observed to promote pump overexpression in clinical iso- susceptibility to β-lactam antibiotics is conferred by mutations in genes
lates (see, for example, refs.43,44,55). For example, MtrR is responsible for coding for penicillin-binding proteins (PBPs). For example, mutation
the repression of mtrCDE, the only RND pump of N. gonorrhoeae56. Point of PBP3 in E. coli has been identified to be almost ubiquitous in clinical
mutations within the MtrR-binding site or its DNA-binding domain isolates from India resistant to aztreonam and avibactam77. Altered tar-
are common and lead to increased mtrCDE expression and resistance get sites can be generated by random point mutations that accumulate
against structurally diverse antimicrobial agents, including penicil- during growth and can expand to dominance under drug pressure.
lin, tetracycline, azithromycin and third-generation cephalosporins Alternatively, mutant alleles of target genes can be generated at high
(see, for example, refs.56–58). The mtrCDE–mtrR locus was recently frequency by recombination between alleles if multiple copies exist
reported to be a hotspot for genetic recombination between multiple within the cell (for example, mutation and recombination between
Neisseria species, with epistatic interactions at the mtr region confer- homologues of the 23S ribosomal RNA (rRNA) gene can rapidly confer
ring resistance against azithromycin, polymyxin B and crystal violet59,60. linezolid resistance in Gram-positive species78) or by transformation,
Indeed, overexpression of MtrR has been shown to increase gonococcal whereby alternative alleles can be gained from related species and
susceptibility towards penicillin and ceftriaxone57. The relationship mosaic genes are generated by recombination, an approach common
between impaired regulatory factors and efflux-mediated resistance in competent species such as members of the Neisseria genus79.
has also been observed in other clinically relevant pathogens, including Mycobacteria are unusual in that carriage of plasmids seems to be
P. aeruginosa61, Campylobacter jejuni62 and Salmonella enterica subsp. rare80 and of minor importance as a mechanism of AMR. Treatment of
enterica serovar Typhimurium63. tuberculosis requires combination therapy, with isoniazid, rifampin,
The induction of expression of each RND component involves pyrazinamide and ethambutol being common first-line treatments.
both local and global transcription regulators responding to various For each drug, a target-site mutation can cause resistance81; for exam-
environmental signals. For instance, acrAB–tolC expression in S. Typh- ple, a recent study documented the structural basis for resistance to
imurium is regulated by RamA which, in turn, is under the control of pyrazinamide, whereby mutation of residues near the active site of
RamR. Recently, RamR was co-crystallized with bile components cholic PanD impair affinity and residence time of the active prodrug, resulting
acid and chenodeoxycholic acid, compounds typically found in the liver in resistance82.
and further metabolized in the intestinal tract; ligand binding to RamR Moreover, the addition of moieties to the drug target can pre-
reduced its DNA-binding affinity, resulting in increased acrAB–tolC vent antibiotic access and thus protect the target. This is a well-known
expression through the upregulation of ramA64. RamR was also shown mechanism of resistance to macrolides, whereby the 16S rRNA target
to interact with other substrates, although in a different manner, sug- can be methylated by ribosomal methyltransferases, thereby pre-
gesting that different recognition mechanisms permit transcriptional venting binding of macrolides (as well as of lincosamines and strep-
regulators to respond to several inducing signals. togramins)83. Methylation of the 16S rRNA is an emerging mechanism
Additionally, the activities of these regulators are not simply con- of high-level resistance to aminoglycoside antibiotics84. Resistance to
strained to efflux regulation. Indeed, MarA, SoxS and Rob — global the polymyxin colistin has also been recognized to result from target
regulators of acrAB–tolC expression — have important roles, including modification. As resistance to traditional first-line therapies for Gram-
in membrane integrity, DNA repair, biofilm formation, quorum sens- negative pathogens has increased in prevalence, colistin, an old drug, is
ing and virulence65–68. The multifactorial activities of these regula- often used, being one of the few efficacious agents remaining. Colistin
tors therefore indicate that efflux pump expression is part of a wider has a complex mode of action, but central to its efficacy is its ability to
network of genes that promote bacterial survival in diverse stressful target lipopolysaccharide (LPS), which results in membrane damage
conditions65,67. The identification of key regulatory features for efflux and cell death85,86. Resistance can occur by decorating LPS with moie-
pump expression would serve as useful therapeutic targets to combat ties that alter the charge of the overall molecule and inhibit interaction
MDR in clinically relevant pathogens69. between the drug and its target. Transfer of phosphoethanolamine
The evident contributions of RND efflux pumps towards MDR, par- (pEtN) by pEtN transferase enzymes from phosphatidylethanolamine to
ticularly in clinically relevant Gram-negative pathogens, present a clear LPS results in a modified LPS conferring resistance. The action of pEtN
opportunity to reinstate drug susceptibility by targeting the structure, transferases is controlled by the regulatory systems PmrAB or PhoPQ87,

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a Fig. 3 | Antibiotic resistance via target protection, drug

Target protection protein inactivation and target bypass. Schematic diagram of
i antibiotic resistance mechanisms mediated by target protection,
drug inactivation and target bypass. a, Mechanisms of target
protection. Target protection proteins can bind to the drug target
Drug and sterically remove the drug from the target (part a,i). Target
Antibiotic
target protection proteins can bind to the drug target and mediate
allosteric dissociation of the drug from its target (part a,ii).
Target protection protein
Target protection proteins can bind to the drug target and cause
ii conformational changes to allow the target protein to function
even in the presence of the drug (part a,iii). b, Mechanisms of
drug alteration. Enzymes can hydrolyse the functional group
of the drug, thereby destroying its antibacterial activity (part b,i).
Enzymes (such as acetyltransferases, methyltransferases or
phosphotransferases) can modify the drug by covalent transfer
iii of various chemical groups to prevent it from binding its target
(part b,ii). c, Mechanisms of target bypass. The drug target (such
as an enzyme) becomes redundant due to acquisition of a gene
that encodes an alternative enzyme that fulfils the function of
the drug target (part c,i). The drug target can be replaced by an
b alternative target that sequesters the drug, thereby allowing the
drug target to resume its function (part c,ii). The drug target can
i Hydrolysis be overproduced and thus there is insufficient amount of drug to
inhibit the increased available target (part c,iii). Part a adapted
from ref.205, Springer Nature Limited.
Enzyme
O
Acetyltransferase
CH3

ii Modification Methyltransferase
CH3
O
Phosphotransferase
P O
O
c
i Alternative enzyme

Target
enzyme

ii Alternative target

Target
iii overproduction

which are chromosomally encoded in many Gram-negative species analysis highlighted a family of mcr genes that have now been identi-
and resistant mutants are vertically inherited but not shared between fied in a wide range of species around the world88. MCR enzymes act in a
strains. However, in 2015, a novel mobilizable pEtN transferase family similar manner to chromosomal systems89. In isolation, the acquisition
was identified and named mcr (mobile colistin resistance). This was an of an mcr gene may confer a limited increase in the minimum inhibitory
extremely concerning finding given the importance of colistin and the concentration (MIC) of colistin and therefore the clinical importance of
potential for rapid spread of mobile colistin resistance. Subsequent mcr has been debated. There is a high prevalence of carriage of mcr in

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colistin-resistant isolates90 and strains carrying mcr have been shown to This study confirmed that the Sal proteins are responsible for resist-
be protected from low-level colistin exposure, although the same study ance to pleuromutilins, group A streptogramins and lincosamides in
found that carriage of mcr actually inhibited the selection of mutants a panel of staphylococci isolates. The structure of the SalB–ribosome
with very high MICs of colistin91. Expression of pEtN transferases results complex was analysed and, thus, an allosteric mechanism of resistance
in the production of diacylglycerol (DAG) as a by-product. DAG is a dead- was proposed, whereby SalB binding alters the structure of 23S rRNA
end metabolite that can become toxic; to arrest this, the cell relies on and results in the release of bound pleuromutilins100. While pleuromuti-
diacylglycerol kinase A (DgkA) to recycle DAG into useful precursor lin resistance currently seems to be rare, ABC-F proteins are often found
molecules. DgkA has recently been shown to be required for colistin on plasmids, making potential spread possible, and may likely result in
resistance to prevent toxic by-products inhibiting growth92. Together, clinically problematic rates of resistance in the future100.
these data support previous observations of a balance needed for pEtN
transferase expression that affects resistance and fitness93. Inactivation and modification of the drug
Protection of a target may confer a relatively mild increase in the A widespread mechanism of resistance in many pathogenic bacteria
MIC of the relevant antibiotic; however, in combination with mutation is the modification or inactivation of the antimicrobial drug itself101
of the target site, very high MICs can be achieved. This is commonly (Fig. 3b). This is typically achieved by the action of enzymes and, there-
seen with the Qnr proteins — a family encoded by genes carried on fore, often does not involve changes to any core components of the
plasmids commonly seen in Gram-negative species which protect bacterial cell, which can give an advantage in that there are less likely
topoisomerases from inhibition by quinolones. Acquisition of a qnr to be associated fitness costs than for other mechanisms such as muta-
gene (there are now seven families recognized) alone has a mild impact tion or alteration of the drug target. The modification of antibiotics can
but quinolone-resistant isolates will often carry a qnr gene in concert be broadly split into two mechanisms: inactivation of the antibiotic
with chromosomal mutations in gyrA, which can together result in by degradation or modification by the transfer of a chemical group.
high-level resistance94. The variety of different quinolone-resistance Both of these mechanisms are widespread among bacteria due to the
mechanisms, each with different impacts on MICs and fitness, offers mobility of the encoding genes.
many possible evolutionary routes to high-level resistance94.
Target protection can also occur without prevention of drug Inactivation of antibiotics
binding, in which case the drug reaches the target but the impact The inactivation of antibiotics is a major mechanism of drug resistance,
can then be alleviated, resulting in protection. For example, fusidic whereby the structure of the drug is damaged or degraded rendering
acid-resistant S. aureus often express FusB-type proteins. Fusidic acid it less effective; therefore, it can contribute to reduced treatment suc-
inhibits translation by binding to elongation factor G (EF-G), the ribo- cess in the clinic102. Examples of the inactivation of antibiotics include
some translocase that is involved in processing mRNA and tRNAs and the hydrolysis of β-lactam antibiotics by β-lactamases and the binding
in preventing dissociation of the complex once RNA translocation is of tetracycline hydroxylases to inactivate tetracyclines. β-Lactamases
complete. FusB proteins contain a zinc finger domain that rescues are enzymes that provide resistance to β-lactam drugs by hydrolys-
translation by promoting dissociation of the stalled complex, even ing the amide bond of the β-lactam ring, thus degrading the drug103.
though drug binding has occurred95. β-Lactamases have evolved in nature in response to the production
Another recent example of target rescue also involves the ribo- of β-lactam antibiotics by microorganisms and have been studied
some. As mentioned earlier, macrolides, lincosamides, streptogramins since the 1940s. The list of characterized β-lactamases continues to
and pleuromutilins target translation and act by binding to the peptidyl- increase; at the time of writing, the Beta Lactamase DataBase records
transferase centre crucial in the elongation phase of protein synthesis. over 7,000 distinct β-lactamases104. Two main schemes for β-lactamase
Proteins belonging to the ATP-binding cassette F (ABC-F) family can classification have been developed, based either on sequence in the
confer resistance to these drugs. In contrast to ABC transporter pro- Ambler class105 or by function103,106. Functionally, there are four classes
teins, ABC-F proteins are not membrane bound, and members of this of β-lactamases (A–D); classes A, C and D are serine β-lactamases and
group provide resistance to antibiotics binding to the peptidyl trans- members of class B are zinc-dependent metallo-β-lactamases103.
ferase centre of the ribosome by binding to the ribosome–antibiotic Carbapenem resistance is of particular concern as carbapenems
complex96. Recent evidence from structural and genetic studies sug- are among the most potent antibiotics in use, and carbapenem resist-
gests that the ABC-F proteins contain ‘antibiotic-resistance domains’ ance in combination with resistance to other β-lactams can exclude the
that interact with the drug-binding domains of the target and cause use of an entire class of drugs107. Extended-spectrum β-lactamases pro-
release of the drugs97,98. There are diverse ABC-F families of proteins, vide resistance to extended-spectrum cephalosporins and monobac-
and it is now known that they may have evolved on multiple occasions tams108. Carbapenem resistance can be mediated by carbapenemases
in many species. These have varying structures thought to provide or by production of an extended-spectrum β-lactamase in combination
differential affinities for different drug classes99. A model for how Vga with porin loss. In 2017, WHO listed three ‘critical’ priority pathogens,
and Lsa ABC-F proteins can protect a stalled ribosome complex has all of which were carbapenem resistant109. Carbapenemases, such as
recently been proposed based on structural and genetic data96. The KPC (class A), NDM (class B) and OXA (class D) types, have the capac-
model suggests that ABC-F proteins recognize a stalled ribosome ity to hydrolyse penicillins, cephalosporins and carbapenems, greatly
complex and bind to the E-site of the ribosome, which then induces a reducing the number of drug treatment options for a given infec-
shift in the P-site tRNA conformation within the ribosome. This allows tion103,110. Novel variants of carbapenemase enzymes are continually
access of the antibiotic-resistance domain of the ABC-F to the pepti- being discovered, for example, KPC-55, which is particularly efficient at
dyl transferase centre of the ribosome, which results in expulsion of catalysing aztreonam and meropenem111, NDM-19, which can hydrolyse
the antibiotic. Further work has investigated the structural basis for β-lactams even in low-zinc conditions112, and OXA-679 in Acinetobacter
how the Sal ABC-F proteins in staphylococci confer resistance to lefa- calcoaceticus, conferring high-level resistance to carbapenems113.
mulin, the first pleuromutilin used for systemic disease in humans100. Of the carbapenemases, NDM enzymes have had a huge impact on

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resistance in the past decade. First discovered in 2009 in India114, they phosphotransferases in complex with macrolides has been elucidated,
are now found globally across several different species due to their showing that they are members of the same protein superfamily as
presence on multiple different plasmids, and they can confer resist- aminoglycoside phosphotransferases135. Esterases are a diverse set of
ance to all β-lactams except aztreonam115–118. It was shown that different modifying enzymes and there is yet to be a resolved structure for the
species within the same hospital intensive care unit frequently shared esterase–macrolide complex135.
antibiotic resistance genes and, of these, β-lactam resistance genes Phenicol and streptogramin antibiotics are both commonly modi-
were most common, with >40% having predicted carbapenemase activ- fied by acetyltransferases, which are widespread. The chloramphenicol
ity119. This, and other work120,121, shows how frequently β-lactamases acetyltransferase (CAT) enzyme transfers an acetyl group to coen-
can spread between bacteria. Multidrug-resistant plasmids are often zyme A, preventing chloramphenicol from binding to its target on
characterized by the presence of β-lactamases, enabling these genes the ribosome136. Streptogramin antibiotics can be classified into two
to spread more easily between different bacteria122. In addition to groups based on their structure: group A, which binds to the peptidyl
plasmids, β-lactamases are often found near insertion sequences. This transferase centre, and group B, which binds to the peptide exit tunnel.
can both mobilize the resistance gene and affect its expression lev- Streptogramins are enzymatically modified by virginiamycin acetyl-
els123,124. For example, in A. baumannii, the insertion sequence ISAba1 is transferases (Vats), which acetylate the alcohol, changing the conforma-
found upstream of the blaampC gene (which encodes AmpC β-lactamase) tion of the protein and thereby reducing the activity of the antibiotic137.
increasing its expression and, in turn, providing resistance to A recent study has developed a pipeline specifically to search for novel
extended-spectrum cephalosporins124. streptogramin antibiotics that are less affected by Vats, which could be
Another important example of the inactivation of antibiotics is important in developing more efficacious drugs in this class137.
exemplified by tetracycline-inactivating enzymes that catalyse the Finally, rifamycins can be inactivated and modified by ADP-
oxidation of tetracyclines. The best known is the Tet(X) family, which ribosyltransferases, glycosyltransferases, phosphotransferases and
has been characterized in many different classes of bacteria and can monooxygenases. Rifamycins are often the first line of defence against
move horizontally on transposable elements, conferring high-level M. tuberculosis infections138. ADP-ribosyltransferases in Mycobacterium
tetracycline resistance101,125. The tet(X/X2) genes are widely spread and smegmatis were first characterized in the late 1990s but, recently, ADP-
commonly found in multidrug-resistant bacteria from various environ- ribosyltransferases have also been found, conferring high levels of rifa-
ments, including isolates from patients, and the presence of them in an mycin resistance in Mycobacterium abscessus139. At the time of writing,
environment is positively correlated with tetracycline use125,126. Tet(X3/ there has yet to be an ADP-ribosyltransferase characterized in M. tuber-
X4/X5) enzymes can confer resistance to tigecycline and have been culosis. ADP-ribosyltransferases catalyse the transfer of ADP ribose
found in both Enterobacterales and Acinetobacter isolates in China127–129. to hydroxyl-linked C23 on the antibiotic, blocking its interaction with
While Tet(X) enzymes have the capacity to confer resistance, their RNA polymerase140. Rifamycin resistance by glycosyltransferases141 is
ability to degrade tetracyclines varies. Structural analysis compared similar to the action of ADP-ribosyltransferases, whereby the enzymes
the ability of different enzymes in the family to degrade substrates and glycosylate the hydroxyl position at C23 (ref.140). Phosphotransferases
suggested that defined substitutions in the most effective enzymes convert rifamycins to inactive phospho-rifamycins142 at C21 (ref.140). The
reduce turnover time, allowing faster processing of substrates126. expression of such enzymes has the potential to reduce the suscepti-
bility profile of the organism if the phosphotransferases are found on
Modification of antibiotics by the transfer of a chemical group mobile genetic elements, such as plasmids, where expression can be
Antibiotics can also be rendered ineffective by the transfer of a chemi- high143. More recently, a novel mechanism of rifamycin inactivation
cal group. Drug-modifying enzymes have been identified for several conferred by rifamycin monooxygenases (Rox) enzymes has been
antibiotics, including aminoglycosides, macrolides, rifamycins, described. Rox enzymes have been shown to linearize the rifamycin
streptogramins, lincosamides and phenicols. structure after oxygenation of a naphthyl group, which abolishes the
Aminoglycosides can be modified by acetyltransferases, phos- binding of the drug to RpoB144.
photransferases or nucleotidyltransferases, modifying the hydroxyl
or amino groups of the drug, which in turn substantially reduces the Target bypass
affinity of the drug to the target130,131. Many aminoglycoside-modifying Target bypass is a strategy that consists of producing an alternative
enzymes are encoded on mobile genetic elements, in addition to chro- pathway that bypasses the antibiotic by making the original target
mosomes, and they are found in both Gram-positive and Gram-negative redundant. This can occur via the acquisition of an alternative gene
species. A recent example of a novel aminoglycoside-modifying that can confer the required properties to the cell but is not efficiently
enzyme is ApmA, which is an acetyltransferase capable of inactivating inhibited by the original antibiotic145 (Fig. 3c).
apramycin, an antibiotic that can currently evade other mechanisms The best-known example of target bypass is probably the develop-
of aminoglycoside resistance132. ment of methicillin-resistant S. aureus (MRSA). β-Lactam antibiotics,
Lincosamide antibiotics can be modified by nucleotidyltrans- such as methicillin, bind to PBPs and inhibit the transpeptidase domain,
ferases, which add phosphate-containing groups to the antibiotic. causing disruption of the cell wall synthesis. S. aureus can acquire an
Nucleotidyltransferases are encoded by lnu genes, for example, lnu(A) exogenous PBP (PBP2a) that is homologous to the original target but
in Staphylococcus species133. Novel lnu genes are still being character- with lower affinity for β-lactam antibiotics145,146. This protein is encoded
ized, and while their clinical impact remains uncertain, it is concern- by the mecA gene (methicillin-resistant gene), located in the staphylo-
ing that they can often be found on mobile genetic elements with the coccal cassette chromosome mec (SCCmec), a mobile genetic element
capacity to disseminate across a number of different bacteria134. that confers methicillin resistance146.
Phosphotransferases and esterases can modify and confer When methicillin binds to this alternative target site, inhibition
resistance to macrolides because the modified macrolides cannot of cell wall synthesis is prevented as the transpeptidase activity of
bind as efficiently to the 50S ribosome. The structure for macrolide PBP2a is maintained145. Native PBPs are also required as PBP2a does

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not possess a transglycosylase domain. With this mechanism, S. aureus sugar recycling and catabolism, is absent in many Gram-negative bac-
can bypass the action of methicillin to ensure cell survival146. Recent teria158. An ‘anabolic recycling pathway’, which bypasses the first steps
evidence has suggested that common lineages of MRSA often seen in in peptidoglycan biosynthesis, has been reported159,160 in most Gram-
human infection first appeared in European hedgehogs in response negative bacteria (absent in E. coli)160. This recycling pathway chan-
to β-lactams produced by endogenous dermatophytes. This was well nels MurNAc-6P directly into peptidoglycan biosynthesis in the form
before human antibiotic use and illustrates the broad diversity of of UDP-MurNAc160. As the UDP-MurNAc pool is affected, the target of
selective pressure operating in different contexts, which can have fosfomycin (MurA), which is present in the first steps of peptidoglycan
implications for human health147. biosynthesis, is also altered. Therefore, this pathway also provides
Another recently characterized example of target bypass is seen intrinsic resistance to fosfomycin159,161.
in E. coli, whereby the production of an alternative crosslinking mecha-
nism, which includes the L,D-transpeptidase YcbB, can bypass the The promise of resistance breakers
D,D-transpeptidase activity of PBPs and lead to β-lactam resistance148. An important reason to understand the molecular mechanisms of
In this example, besides the L,D-transpeptidase activity of YcbB, addi- resistance is to use this information to design novel strategies that
tional factors, such as high-level synthesis of the alarmone (p)ppGpp, interfere with or inhibit the resistance mechanism. So-called antibiotic
class C monofunctional PBP5 and the glycosyltransferase activity of resistance breakers are compounds that can disrupt or inhibit a specific
class A PBP1b, are also needed149. Although YcbB is not inhibited by mechanism of antibiotic resistance to restore the clinical efficacy of
β-lactam antibiotics, it remains susceptible to carbapenem antibiotics a specific antibiotic. This is an attractive strategy because it could be
such as meropenem and imipenem149. used to potentiate the use of existing antibiotics, and there is extensive
Vancomycin is a glycopeptide antibiotic widely used for the treat- proof-of-principle that this strategy works clinically as inhibitors of
ment of enterococcal and MRSA infections150. This drug also inhibits cell β-lactamase enzymes have been used successfully since the introduc-
wall synthesis but, instead of directly binding to PBPs as do β-lactams, tion of clavulanic acid in 1981. Generally, β-lactamase inhibitors work
vancomycin binds to the terminal D-alanine-D-alanine from the pen- by modifying β-lactamase enzymes, rendering them inactive. Many,
tapeptide precursors. This inhibits peptidoglycan crosslinking, which including clavulanic acid, sulbactam and tazobactam, are themselves
is essential for the synthesis of the bacterial cell wall. Vancomycin β-lactam compounds with minimal antibacterial effect but with the abil-
resistance in enterococci is mediated by the acquisition of the van ity to modify the β-lactamase enzyme, creating an inactive inhibitor–
cluster, with the vanA gene cluster being the most prevalent in clini- enzyme complex. Most recently, new β-lactamase inhibitors belonging
cal vancomycin-resistant strains. Expression of the genes in the vanA to the diazabicyclooctane (for example, avibactam, relebactam and
gene cluster, located on a transposon (Tn1546), leads to the abnormal durlobactam) or boronic acid (vaborbactam) classes have been devel-
synthesis of peptidoglycan precursors, which is the target site. Con- oped with regulatory approvals for meropenem–vaborbactam and
sequently, instead of binding to D-alanine-D-alanine, vancomycin imipenem–relebactam showing viable clinical potential162. Although
now binds, with reduced affinity, to terminal D-alanine-D-lactate or β-lactamase inhibitors have been hugely successful, there is now a
D-alanine-D-serine151,152. growing problem with resistance; just as there has been resistance
This bypass strategy confers resistance to vancomycin in reported to all clinically available antibiotics, there has now also been
Enterococcus species and other Gram-positive bacteria. Some MRSA resistance reported to all β-lactam–β-lactamase inhibitor combina-
strains can also carry the Tn1546 transposon acquired from vancomycin- tions available. Resistance is often mediated by high-level expression
resistant Enterococcus faecalis150. This led to the appearance of MRSA of the targeted enzymes or mutations in the enzymes. Although they
with vancomycin resistance conferred by the vanA gene cluster, with the have not entered clinical use, promising aminoglycoside-modifying
first case being reported in 2002 in the USA153. Although vancomycin- enzyme inhibitors have also been developed163.
resistant S. aureus infections are rare, a methicillin-resistant and Our increased understanding of how resistance mechanisms inter-
vancomycin-resistant S. aureus strain was isolated in Europe154. act with core physiology also offers opportunities, for example, the
The combination of the two antibiotics trimethoprim and sul- observation that colistin resistance conferred by pEtN transferases
famethoxazole (known as trimethoprim–sulfamethoxazole (TMP–SMX) results in toxic DAG production as a by-product, which is alleviated
or co-trimoxazole) is commonly used to treat urinary tract infections by DkgA. Loss of this control mechanism results in repression of pEtN
and prophylaxis for pneumonia caused by Pneumocystis carinii, which transferases and loss of resistance; therefore, targeting DkgA could
occurs in individuals with HIV155. The two active agents block the bio- potentiate colistin activity92.
synthesis of bacterial folic acid, which is essential for nucleic acid and An alternative resistance breaker strategy is to target the imperme-
protein synthesis. TMP acts as a dihydrofolate reductase (DHFR) inhibi- able Gram-negative outer membrane to increase drug uptake. However,
tor and SMX is a sulfonamide that inhibits dihydropteroate synthetase compounds that do this are often themselves bactericidal while also
(DHPS)156,157. In general, resistance emerges more slowly to combina- potentiating the activity of other antibiotics. For example, polymyxins
tion therapy, making it an attractive strategy. However, resistance to including colistin are potent antimicrobials and work by permeabilizing
TMP–SMX can occur when additional novel alleles of DHFR and/or the membrane but there is evidence that, along with other antimicro-
DHPS are acquired and/or overproduced. This causes an increase in bial peptides, they could be used in combination with other drugs to
target availability and a decrease in the binding activity of TMP–SMX, increase activity164.
maintaining folic acid production and ensuring cell survival145,157. Finally, the concept of inhibiting efflux pump activity is particu-
Bacteria possess different ways to recycle components of their larly attractive, not only because efflux underpins many other mecha-
own cell wall. In E. coli, the enzyme MurNAc-6P etherase (MurQ) is nisms of resistance (Box 2) but also because many efflux pumps are
used to recover uridine diphosphate (UDP) N-acetylmuramic acid also required for virulence and biofilm formation. The idea is that, by
(UDP-MurNAc), the first precursor of peptidoglycan de novo biosyn- blocking or otherwise inhibiting major efflux pumps, susceptibility
thesis. However, the MurQ enzyme, which is also required for amino to antimicrobials would increase because the extrusion of antibiotics

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