Targeting bacterial outer-membrane remodelling to impact antimicrobial drug resistance

How porins are assembled into the bacterial outer membrane is now understood in molecular detail. We also have knowledge of the signals that dictate which porin-encoding genes are activated under specific environmental stimuli, including the presence of antimicrobial drugs. These signals change the protein-specific composition of the outer membrane, a process referred to as outer-membrane remodelling.The general mechanisms by which mutations and/or adaptations confer AMR phenotypes on bacteria are known. One of these mechanisms is outer-membrane remodelling. Its impact on AMR, particularly carbapenem resistance, is a central feature of several of the bacterial pathogens currently rated as being in urgent need of research and new treatments.New therapies are canvassed here and warnings around phage therapy, based on considerations of outer-membrane remodelling, are made clear. The cell envelope is essential for survival and adaptation of bacteria. Bacterial membrane proteins include the major porins that mediate the influx of nutrients and several classes of antimicrobial drugs. Consequently, membrane remodelling is closely linked to antimicrobial resistance (AMR). Knowledge of bacterial membrane protein biogenesis and turnover underpins our understanding of bacterial membrane remodelling and the consequences that this process have in the evolution of AMR phenotypes. At the population level, the evolution of phenotypes is a reversible process, and we can use these insights to deploy evolutionary principles to resensitize bacteria to existing antimicrobial drugs. In our opinion, fundamental knowledge is opening a new way of thinking towards sustainable solutions to the mounting crisis in AMR. Here we discuss what is known about outer-membrane remodelling in bacteria and how the process could be targeted as a means to restore sensitivity to antimicrobial drugs. Bacteriophages are highlighted as a powerful means to exert this control over membrane remodelling but they require careful selection so as to reverse, and not exacerbate, AMR phenotypes. The cell envelope is essential for survival and adaptation of bacteria. Bacterial membrane proteins include the major porins that mediate the influx of nutrients and several classes of antimicrobial drugs. Consequently, membrane remodelling is closely linked to antimicrobial resistance (AMR). Knowledge of bacterial membrane protein biogenesis and turnover underpins our understanding of bacterial membrane remodelling and the consequences that this process have in the evolution of AMR phenotypes. At the population level, the evolution of phenotypes is a reversible process, and we can use these insights to deploy evolutionary principles to resensitize bacteria to existing antimicrobial drugs. In our opinion, fundamental knowledge is opening a new way of thinking towards sustainable solutions to the mounting crisis in AMR. Here we discuss what is known about outer-membrane remodelling in bacteria and how the process could be targeted as a means to restore sensitivity to antimicrobial drugs. Bacteriophages are highlighted as a powerful means to exert this control over membrane remodelling but they require careful selection so as to reverse, and not exacerbate, AMR phenotypes. The global impact in loss of human life from increases in AMR has distracted many interested parties from the obvious, simple message that a key definition of AMR is that it is a phenotype. As with other phenotypes, it is a transient description of a given population of bacteria (or fungi, or parasites, but here we focus on bacteria) which is subject to evolution, based on selection under a given set of selective pressures. When we push the evolution of a bacterial population by increasing the exposure to antimicrobials we are selecting for an AMR phenotype. Any strategy that would slow or select against the AMR phenotype would be a better idea. For bacteria, four broad mechanisms are recognized by which AMR phenotypes are driven to evolve (Box 1). Firstly, mutations that alter the target of the antimicrobial drug to inhibit drug-binding to said target will generate an AMR phenotype. Secondly, the modification of existing genes or acquisition of new genes encoding efflux pumps (see Glossary) provides an AMR phenotype. Thirdly, the acquisition of new genes encoding enzymes that hydrolyze or modify the drug provides an AMR phenotype, with perhaps the most salient example seen in the multigenerational β-lactam developments towards carbapenems (Box 1). Fourthly, membrane remodelling to prevent drug influx at the cell surface, thereby protecting the internal compartments where most drug targets reside.Box 1Mechanisms for acquiring an AMR phenotypeMutations that alter the target of the antimicrobial drug to decrease (or inhibit) drug-binding to the target will generate an AMR phenotype. The drug developers’ solution to this scenario involves attempts to design drugs that would hit their target in such a way that any change in the target would result in loss-of-function. An example of this is the way in which linezolid binds to bacterial ribosomes: only one other escape conformation is possible in the ribosome and a second 'linezolid-like' drug has been proposed to block this site [55.Belousoff M.J. et al.cryoEM-guided development of antibiotics for drug-resistant bacteria.ChemMedChem. 2019; 14: 527-531Crossref PubMed Scopus (16) Google Scholar].The modification of existing genes, or acquisition of new genes encoding drug-efflux pumps, provides an AMR phenotype. The drug developers’ solution to this scenario involves drugs that would inhibit the efflux pump, to be used in combination with the primary antibiotic. Current inhibitors have issues with toxicity, but recent examples suggest progress both in the types of inhibitors that could be identified and in dosing strategies that could enter future clinical trials [56.Ferrer-Espada R. et al.A permeability-increasing drug synergizes with bacterial efflux pump inhibitors and restores susceptibility to antibiotics in multi-drug resistant Pseudomonas aeruginosa strains.Sci. Rep. 2019; 9: 3452Crossref PubMed Scopus (51) Google Scholar, 57.Marshall R.L. et al.New multidrug efflux inhibitors for Gram-negative bacteria.mBio. 2020; 11e01340-20Crossref Scopus (18) Google Scholar, 58.Zwama M. Nishino K. Ever-adapting RND efflux pumps in Gram-negative multidrug-resistant pathogens: a race against time.Antibiotics (Basel). 2021; : 10PubMed Google Scholar].The acquisition of new genes encoding enzymes that hydrolyse the drug provides an AMR phenotype. The drug developers’ solution to this scenario involves attempts to design new-generation drugs that are resistant to existing enzymes. An example of this is provided by the four generations of β-lactam drugs currently in clinical use. The acquisition of genes encoding penicillinases led to the development of cephalosporins. The acquisition of genes encoding cephalosporinases led to the development of later generations of cephems, such as cefotaxime. The acquisition of genes encoding extended-spectrum β-lactamases led to the development of carbapenems. The acquisition of genes encoding carbapenemases is currently without solution.Remodelling of the cell surface to prevent drug influx protects the internal compartments where most drug targets reside. The drug developers’ solution to this scenario is to design new-generation drugs that target surface-exposed features. Examples of this are the recent successes in targeting features exposed on the outer surface of Gram-negative bacteria, such as the β-barrel assembly machinery [59.Steenhuis M. et al.A ban on BAM: an update on inhibitors of the beta-barrel assembly machinery.FEMS Microbiol. Lett. 2021; 368fnab059Crossref PubMed Scopus (10) Google Scholar]. Mutations that alter the target of the antimicrobial drug to decrease (or inhibit) drug-binding to the target will generate an AMR phenotype. The drug developers’ solution to this scenario involves attempts to design drugs that would hit their target in such a way that any change in the target would result in loss-of-function. An example of this is the way in which linezolid binds to bacterial ribosomes: only one other escape conformation is possible in the ribosome and a second 'linezolid-like' drug has been proposed to block this site [55.Belousoff M.J. et al.cryoEM-guided development of antibiotics for drug-resistant bacteria.ChemMedChem. 2019; 14: 527-531Crossref PubMed Scopus (16) Google Scholar]. The modification of existing genes, or acquisition of new genes encoding drug-efflux pumps, provides an AMR phenotype. The drug developers’ solution to this scenario involves drugs that would inhibit the efflux pump, to be used in combination with the primary antibiotic. Current inhibitors have issues with toxicity, but recent examples suggest progress both in the types of inhibitors that could be identified and in dosing strategies that could enter future clinical trials [56.Ferrer-Espada R. et al.A permeability-increasing drug synergizes with bacterial efflux pump inhibitors and restores susceptibility to antibiotics in multi-drug resistant Pseudomonas aeruginosa strains.Sci. Rep. 2019; 9: 3452Crossref PubMed Scopus (51) Google Scholar, 57.Marshall R.L. et al.New multidrug efflux inhibitors for Gram-negative bacteria.mBio. 2020; 11e01340-20Crossref Scopus (18) Google Scholar, 58.Zwama M. Nishino K. Ever-adapting RND efflux pumps in Gram-negative multidrug-resistant pathogens: a race against time.Antibiotics (Basel). 2021; : 10PubMed Google Scholar]. The acquisition of new genes encoding enzymes that hydrolyse the drug provides an AMR phenotype. The drug developers’ solution to this scenario involves attempts to design new-generation drugs that are resistant to existing enzymes. An example of this is provided by the four generations of β-lactam drugs currently in clinical use. The acquisition of genes encoding penicillinases led to the development of cephalosporins. The acquisition of genes encoding cephalosporinases led to the development of later generations of cephems, such as cefotaxime. The acquisition of genes encoding extended-spectrum β-lactamases led to the development of carbapenems. The acquisition of genes encoding carbapenemases is currently without solution. Remodelling of the cell surface to prevent drug influx protects the internal compartments where most drug targets reside. The drug developers’ solution to this scenario is to design new-generation drugs that target surface-exposed features. Examples of this are the recent successes in targeting features exposed on the outer surface of Gram-negative bacteria, such as the β-barrel assembly machinery [59.Steenhuis M. et al.A ban on BAM: an update on inhibitors of the beta-barrel assembly machinery.FEMS Microbiol. Lett. 2021; 368fnab059Crossref PubMed Scopus (10) Google Scholar]. In this opinion piece we explore the relationships between bacterial membrane remodelling and AMR. As our awareness grows that many of the worst-case scenarios of bacterial superbug lineages spreading globally are composite phenotypes, addressing the issue of membrane remodelling could be an effective means to resensitize bacteria to existing antibiotics. It is worthy of discussion: the 20th century has taught us that the silver bullet approach of a new antibiotic drug can never be a sustainable solution to AMR, with each new drug failing due to AMR after just a few years in clinical use. This remains true in this first part of the 21st century. Yet we almost know enough about bacterial cell biology to be inventive in how we might push back on evolution to restore susceptibility to some existing drugs within our antibacterial arsenal. It is our opinion that understanding just a few more features of the fundamental biology of bacterial outer membranes will both assist in the development of novel therapies and restore our ability to use existing drugs to treat what would otherwise be AMR infections. All of the antibiotic drugs in widespread clinical use in the 20th century targeted bacterial cell processes via binding to targets in internal compartments of the bacterial cell (Figure 1). In order to do so in Gram-negative bacteria, these drugs have to permeate the bacterial outer membrane [1.Pages J.M. et al.The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria.Nat. Rev. Microbiol. 2008; 6: 893-903Crossref PubMed Scopus (636) Google Scholar,2.Prajapati J.D. et al.How to enter a bacterium: bacterial porins and the permeation of antibiotics.Chem. Rev. 2021; 121: 5158-5192Crossref PubMed Scopus (62) Google Scholar]. The means by which this otherwise highly impermeable lipopolysaccharide (LPS)–phospholipid barrier can be breached by drugs is via the major porins. Almost all of the Gram-negative bacteria studied to date express a major porin: 'major' in the sense that it is the most abundant protein integrated in the outer membrane, and 'porin' in the sense that it has a central luminal space that serves as an aqueous pore through which nutrients and water-soluble drugs can pass. In the case of Escherichia coli, the major porin represents more than 50% of the total protein integrated in the outer membrane [2.Prajapati J.D. et al.How to enter a bacterium: bacterial porins and the permeation of antibiotics.Chem. 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Major porins have also been studied in Klebsiella pneumoniae [7.Jasim R. et al.A comparative study of outer membrane proteome between paired colistin-susceptible and extremely colistin-resistant Klebsiella pneumoniae strains.ACS Infect. Dis. 2018; 4: 1692-1704Crossref PubMed Scopus (13) Google Scholar,8.Rocker A. et al.Global trends in proteome remodeling of the outer membrane modulate antimicrobial permeability in Klebsiella pneumoniae.mBio. 2020; 11e00603-20Crossref PubMed Scopus (24) Google Scholar], in Neisseria gonorrhoeae [9.Deo P. et al.Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis.PLoS Pathog. 2018; 14e1006945Crossref PubMed Scopus (79) Google Scholar,10.Marzoa J. et al.Analysis of outer membrane porin complexes of Neisseria meningitidis in wild-type and specific knock-out mutant strains.Proteomics. 2009; 9: 648-656Crossref PubMed Scopus (17) Google Scholar], and so on. There are exceptions of course, such as Caulobacter crescentus that facilitates small-molecule uptake with a series of TonB-dependent β-barrel proteins instead of any major porin [11.Anwari K. et al.A modular BAM complex in the outer membrane of the alpha-proteobacterium Caulobacter crescentus.PLoS One. 2010; 5e8619Crossref PubMed Scopus (52) Google Scholar]. However, for all of the Gram-negative species that feature as urgent pathogens, as judged by the WHO and the US-CDC, major porins are used for nutrient and drug entry into the bacterium. Studies on E. coli showed that while there are two genes (i.e., ompC and ompF) that encode the major porins, only one of these is expressed under any given set of environmental conditions [12.Martinez J.L. Rojo F. Metabolic regulation of antibiotic resistance.FEMS Microbiol. Rev. 2011; 35: 768-789Crossref PubMed Scopus (186) Google Scholar]. The outer-membrane proteome is subject to remodelling wherein either OmpC or OmpF is the major porin. Grown in hyperosmotic conditions or nitrogen-limited media, OmpC is the major porin in the E. coli outer membrane, while under hypo-osmotic conditions, or in glucose-limited media, OmpF is the major porin expressed in the membrane [13.Pratt L.A. et al.From acids to osmZ: multiple factors influence synthesis of the OmpF and OmpC porins in Escherichia coli.Mol. Microbiol. 1996; 20: 911-917Crossref PubMed Scopus (265) Google Scholar]. Structurally, OmpC and OmpF are almost identical, with the very limited sequence variation confined to a few of the interstrand loops that sit at the cell surface [14.Cowan S.W. et al.Crystal structures explain functional properties of two E. coli porins.Nature. 1992; 358: 727-733Crossref PubMed Scopus (1339) Google Scholar], and we therefore tend to refer to these porins with the compound term OmpC/F. Studies in E. coli also showed that OmpF expression is turned on (and OmpC turned off) in response to carbapenems and tetracycline [15.Chetri S. et al.Transcriptional response of OmpC and OmpF in Escherichia coli against differential gradient of carbapenem stress.BMC Res. Notes. 2019; 12: 138Crossref PubMed Scopus (20) Google Scholar,16.Lin X. et al.Differential regulation of OmpC and OmpF by AtpB in Escherichia coli exposed to nalidixic acid and chlortetracycline.J. Proteom. 2012; 75: 5898-5910Crossref PubMed Scopus (31) Google Scholar]. Conversely, OmpC expression is turned on (and OmpF turned off) in response to nalidixic acid or 3% ethanol [16.Lin X. et al.Differential regulation of OmpC and OmpF by AtpB in Escherichia coli exposed to nalidixic acid and chlortetracycline.J. Proteom. 2012; 75: 5898-5910Crossref PubMed Scopus (31) Google Scholar,17.Zhang D.F. et al.Identification of ethanol tolerant outer membrane proteome reveals OmpC-dependent mechanism in a manner of EnvZ/OmpR regulation in Escherichia coli.J. Proteom. 2018; 179: 92-99Crossref PubMed Scopus (10) Google Scholar]. The OmpC/F major porin system in E. coli is a useful model for this outer-membrane remodelling phenomenon, and it is not unique. For example, K. pneumoniae has four genes (i.e., ompK35, ompK36, ompK37, and ompK38) encoding major porins that are structurally near-identical [8.Rocker A. et al.Global trends in proteome remodeling of the outer membrane modulate antimicrobial permeability in Klebsiella pneumoniae.mBio. 2020; 11e00603-20Crossref PubMed Scopus (24) Google Scholar]. It is not yet clear which environmental conditions induce expression of each of these genes, although ompK35 is sensitive to osmotic conditions [18.Hernandez-Alles S. et al.Porin expression in clinical isolates of Klebsiella pneumoniae.Microbiology (Reading). 1999; 145: 673-679Crossref PubMed Scopus (167) Google Scholar]. Through enforced remodelling of the outer membrane, these porins (i.e., OmpK35, OmpK36, OmpK37, and OmpK38) were shown to differ in which drugs they permit to enter into K. pneumoniae, with consequences and causality for AMR phenotypes [1.Pages J.M. et al.The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria.Nat. Rev. Microbiol. 2008; 6: 893-903Crossref PubMed Scopus (636) Google Scholar,8.Rocker A. et al.Global trends in proteome remodeling of the outer membrane modulate antimicrobial permeability in Klebsiella pneumoniae.mBio. 2020; 11e00603-20Crossref PubMed Scopus (24) Google Scholar]. Of greatest concern, it is becoming clear that many clinical strains of Klebsiella have remodelled their outer membrane to express no major porin, and this makes these strains highly resistant to carbapenems [1.Pages J.M. et al.The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria.Nat. Rev. Microbiol. 2008; 6: 893-903Crossref PubMed Scopus (636) Google Scholar,8.Rocker A. et al.Global trends in proteome remodeling of the outer membrane modulate antimicrobial permeability in Klebsiella pneumoniae.mBio. 2020; 11e00603-20Crossref PubMed Scopus (24) Google Scholar,19.Tian X. et al.First description of antimicrobial resistance in carbapenem-susceptible Klebsiella pneumoniae after imipenem treatment, driven by outer membrane remodeling.BMC Microbiol. 2020; 20: 218Crossref PubMed Scopus (11) Google Scholar,20.Pitout J.D. et al.Carbapenemase-producing Klebsiella pneumoniae, a key pathogen set for global nosocomial dominance.Antimicrob. Agents Chemother. 2015; 59: 5873-5884Crossref PubMed Scopus (561) Google Scholar]. Remodelling the proteome of the outer membrane depends on two biomolecular processes: the removal and degradation of existing membrane proteins, and the integration of molecules of new proteins into the outer membrane (Figure 2, Key figure). Relatively little is known about the mechanism for membrane protein removal from the outer membrane, although a set of proteases – BepA, DegP, YcaL – do degrade damaged outer-membrane proteins [21.Chang Z. The function of the DegP (HtrA) protein: Protease versus chaperone.IUBMB Life. 2016; 68: 904-907Crossref PubMed Scopus (27) Google Scholar, 22.Daimon Y. et al.The TPR domain of BepA is required for productive interaction with substrate proteins and the beta-barrel assembly machinery complex.Mol. 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The fellowship of the rings: macrocyclic antibiotic peptides reveal an anti-Gram-negative target.Biochemistry. 2020; 59: 343-345Crossref PubMed Scopus (14) Google Scholar, 31.Srinivas N. et al.Peptidomimetic antibiotics target outer-membrane biogenesis in Pseudomonas aeruginosa.Science. 2010; 327: 1010-1013Crossref PubMed Scopus (441) Google Scholar] that may be developed into a new class of antimicrobial drugs. As far as is known, both the integration process and the degradation process are constitutive without selectivity, and so the selective changes needed to have new protein components in the outer membrane are likely to occur at a transcriptional level: the induction of transcripts encoding the proteins to be introduced into the proteome, or mechanisms that silence transcription for proteins that would, thereby, no longer be integrated into the outer membrane. At a transcriptional and post-transcriptional level, the genes encoding porins are regulated by a complex system of regulators, including two-component signalling systems (e.g., OmpR, CpxR), small noncoding RNAs (e.g., micF, micC, micA), and other factors. These transcriptional and post-transcriptional signals regulate porin production in response to several environmental stimuli [13.Pratt L.A. et al.From acids to osmZ: multiple factors influence synthesis of the OmpF and OmpC porins in Escherichia coli.Mol. Microbiol. 1996; 20: 911-917Crossref PubMed Scopus (265) Google Scholar,32.Chen S. et al.MicC, a second small-RNA regulator of Omp protein expression in Escherichia coli.J. Bacteriol. 2004; 186: 6689-6697Crossref PubMed Scopus (200) Google Scholar,33.De la Cruz M.A. Calva E. The complexities of porin genetic regulation.J. Mol. Microbiol. Biotechnol. 2010; 18: 24-36Crossref PubMed Scopus (46) Google Scholar], creating a genuine systems-level of control. By way of example, the two-component EnvZ–OmpR regulatory switch responds to environmental sensing of osmolarity, or ethanol, or various antimicrobial drugs, to remodel the porin composition of the outer membrane [21.Chang Z. The function of the DegP (HtrA) protein: Protease versus chaperone.IUBMB Life. 2016; 68: 904-907Crossref PubMed Scopus (27) Google Scholar,34.Fernandez L. Hancock R.E. Adaptive and mutational resistance: role of porins and efflux pumps in drug resistance.Clin. Microbiol. Rev. 2012; 25: 661-681Crossref PubMed Scopus (527) Google Scholar,35.Kenney L.J. Anand G.S. EnvZ/OmpR two-component signaling: an archetype system that can function noncanonically.EcoSal Plus. 2020; 9 (Published online March 20, 2020. http://doi.org/10.1128/ecosalplus.ESP-0001-2019)Crossref PubMed Scopus (38) Google Scholar]. CpxR and other components of the Cpx envelope stress response also signal into this system, sensing osmolarity or antimicrobial drugs [36.Delhaye A. et al.Fine-tuning of the Cpx envelope stress response is required for cell wall homeostasis in Escherichia coli.mBio. 2016; 7e00047-16Crossref PubMed Scopus (75) Google Scholar,37.Raivio T.L. Everything old is new again: an update on current research on the Cpx envelope stress response.Biochim. Biophys. Acta. 2014; 1843: 1529-1541Crossref PubMed Scopus (136) Google Scholar], and the expression of micC increases in the presence of drug treatment, acting synergistically with the Cpx envelope stress response to remodel the outer membrane in response to β-lactam antibiotics [38.Dam S. et al.Dual regulation of the small RNA MicC and the quiescent porin OmpN in response to antibiotic stress in Escherichia coli.Antibiotics (Basel). 2017; 6: 33Crossref Scopus (18) Google Scholar]. These small RNAs can be manipulated to remodel the outer membrane in several species of bacteria, and biotech applications are already exploiting this membrane remodelling system [39.Hao M. et al.Porin deficiency in carbapenem-resistant Enterobacter aerogenes strains.Microb. Drug Resist. 2018; 24: 1277-1283Crossref PubMed Scopus (24) Google Scholar,40.Negrete A. Shiloach J. Improving E. coli growth performance by manipulating small RNA expression.Microb. Cell Factories. 2017; 16: 198Crossref PubMed Scopus (12) Google Scholar]. There is also the prospect that the remodelling system signals out to the protein integration machinery: intriguing proteomics analysis of E. coli in response to either β-lactam drugs or tetracycline treatment suggested that two components of the BAM complex, BamC (aka NlpB) and BamD (aka YfiO), are upregulated coordinately when the ompC gene is induced to remodel the outer membrane [41.Xu C. et al.Analysis of outer membrane proteome of Escherichia coli related to resistance to ampicillin and tetracycline.Proteomics. 2006; 6: 462-473Crossref PubMed Scopus (112) Google Scholar]. Outer-membrane remodelling matters because it is a tractable target to override AMR phenotypes and/or effectively reverse the evolutionary trend towards AMR. The question then becomes 'How' can the remodelling of bacterial outer membranes be controlled to resensitize bacteria in an infection site or a biofilm to be susceptible to existing drugs. Encouragement that this is a worthy question and a tractable issue comes from the use of this strategy in biotech applications [39.Hao M. et al.Porin deficiency in carbapenem-resistant Enterobacter aerogenes strains.Microb. Drug Resist. 2018; 24: 1277-1283Crossref PubMed Scopus (24) Google Scholar,40.Negrete A. Shiloach J. Improving E. coli growth performance by manipulating small RNA expression.Microb. Cell Factories. 2017; 16: 198Crossref PubMed Scopus (12) Google Scholar] coupled with a growing awareness that AMR phenotypes are costly to maintain, suggesting that so-called 'superbugs' are not as super as they might appear. Experimental studies have demonstrated that changes in the outer-membrane proteome of K. pneumoniae and Acinetobacter baumannii confer antibiotic resistance, but with an associated fitness cost and physiological burden for these clinically important bacteria [42.Alonso A. et al.Overexpression of the multidrug efflux pump SmeDEF impairs Stenotrophomonas maltophilia physiology.J. Antimicrob. Chemother. 2004; 53: 432-434Crossref PubMed Scopus (85) Google Scholar, 43.Garcia-Sureda L. et al.OmpK26, a novel porin associated with carbapenem resistance in Klebsiella pneumoniae.Antimicrob. Agents Chemother. 2011; 55: 4742-4747Crossref PubMed Scopus (44) Google Scholar, 44.Sherrard L.J. et al.Emergence and impact of oprD mutations in Pseudomonas aeruginosa strains in cystic fibrosis.J. Cyst. Fibros. 2021; (Published online March 25, 2021)https://doi.org/10.1016/j.jcf.2021.03.007Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar, 45.Smani Y. et al.Association of the outer membrane protein Omp33 with fitness and virulence of Acinetobacter baumannii.J. Infect. Dis. 2013; 208: 1561-1570Crossref PubMed Scopus (81) Google Scholar]. Under selection, in an environmental condition where drugs are present, strains that remodel the outer membrane to become drug-resistant would have a competitive advantage against drug-sensitive bacteria. What is less clear is whether, in an environment where drugs are absent, the remodelled outer membrane would impose sufficient cost on the strain to make it less able to compete against other, drug-sensitive bacteria. In theoretical modelling, mutations conferring AMR are very costly in the absence of the drug [46.Moura de Sousa J. et al.Multidrug-resistant bacteria compensate for the epistasis between resistances.PLoS Biol. 2017; 15e2001741Crossref PubMed Scopus (42) Google Scholar]. The predicted reversal of an AMR phenotype could occur by either of two means: (i) a genetic change, by which the outer membrane is restored through mutations that reverse (or modify) the outer-membrane proteome to allow evolution of a drug-sensitive phenotype, or (ii) a population- level change, where the carbapenem-resistant strain fails to compete against a new, carbapenem-sensitive strain that can thrive in a drug-free environment. The gene-regulatory mechanisms that control membrane remodelling are worthy of further investigation since, for example, factors that can switch on porin expression would be a valuable means of restoring carbapenem sensitivity in an infection site. The unmet need to drive these further investigations is here: many carbapenem-resistant strains of Klebsiella, Enterobacter, Acinetobacter, and Pseudomonas are evolving in hospitals to have an inactivated gene (e.g., ompK36) but with a functional albeit silent gene (e.g., ompK35) that – if switched on – would provide for drug-resensitization [1.Pages J.M. et al.The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria.Nat. Rev. Microbiol. 2008; 6: 893-903Crossref PubMed Scopus (636) Google Scholar,8.Rocker A. et al.Global trends in proteome remodeling of the outer membrane modulate antimicrobial permeability in Klebsiella pneumoniae.mBio. 2020; 11e00603-20Crossref PubMed Scopus (24) Google Scholar,39.Hao M. et al.Porin deficiency in carbapenem-resistant Enterobacter aerogenes strains.Microb. 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The current move towards phage therapy promises a powerful means to treat infections with AMR phenotypes since the drug-resistance mechanisms (Box 1) do not impact bacterial susceptibility to phage infection and ensuing bacterial death [50.Gordillo Altamirano F.L. Barr J.J. Phage therapy in the postantibiotic era.Clin. Microbiol. Rev. 2019; 32: e00066-18Crossref PubMed Scopus (408) Google Scholar]. However, we note one issue that must be taken into account when selecting which phages to use in these treatments. In our opinion, phages that use major porins as their receptor must be avoided. If used in therapy, these phages would place selective pressure on the bacterial strain to become porin-defective in order to become phage-resistant, since the prime cause of phage-resistance is the downregulation of the surface receptor [51.Gordillo Altamirano F. et al.Bacteriophage-resistant Acinetobacter baumannii are resensitized to antimicrobials.Nat. Microbiol. 2021; 6: 157-161Crossref PubMed Scopus (107) Google Scholar,52.Gordillo Altamirano F.L. Barr J.J. Unlocking the next generation of phage therapy: the key is in the receptors.Curr. Opin. Biotechnol. 2021; 68: 115-123Crossref PubMed Scopus (51) Google Scholar]. The unintended corollary therefore is that phages that use a major porin as their receptor would be mediators to select for AMR phenotypes in situ, a terrible prospect that can be avoided by consideration of outer-membrane remodelling (Figure 3). Advances are being made in this direction, resensitization to antibiotics shown in the case of phage treatment of multidrug-resistant A. baumannii strains [51.Gordillo Altamirano F. et al.Bacteriophage-resistant Acinetobacter baumannii are resensitized to antimicrobials.Nat. Microbiol. 2021; 6: 157-161Crossref PubMed Scopus (107) Google Scholar,53.Wang X. et al.Phage resistance mechanisms increase colistin sensitivity in Acinetobacter baumannii.bioRxiv. 2021; (2021.07.23.453473)Google Scholar]. The increase in prevalence of AMR is a major concern worldwide. According to the WHO it is 'one of the 10 top global public health threats' and carbapenem-resistant Gram-negative bacteria of various species are listed as urgent threats by the CDCi,ii. The economic burden related to AMR is estimated to be US$4.6 billion in the USA per year, and €1.4 billion in the EU/EEA per yeari,iii. The costs for other continents are not available but will be at least as high. Globally, the economic impact in healthcare costs is predicted to increase in a range equivalent to US$300 billion to US$1 trillion each year by 2050 and with a corresponding 10 million deaths annually by the same year [54.Ahmad M. Khan A.U. Global economic impact of antibiotic resistance: A review.J. Glob. Antimicrob. Resist. 2019; 19: 313-316Crossref PubMed Scopus (94) Google Scholar]iv. Discovery and development of effective drug treatments is declining dramatically. In addition, the 20th century has taught us that a new antibiotic drug will never be a sustainable solution to AMR. We must expand novel therapeutic approaches, such as phage therapy, to combat the ever-evolving mechanisms that multidrug-resistant bacteria use to evade current treatments. In our opinion, deep knowledge of bacterial membrane remodelling and the use of evolution and selection for resensitization to existing drugs are important considerations that need to be in place alongside the current moves for development of new antimicrobial drugs and other new therapies (see Outstanding questions). We believe that only with multidisciplinary action will the world combat the urgent threat of AMR.Outstanding questionsCan antimicrobial drugs that target essential features at the outer surface of the outer membrane be used to circumvent the mechanisms for the evolution of AMR?Is there a mechanism for regulated removal of specific outer-membrane proteins? If such a pathway exists, small molecules applied extracellularly could have a therapeutic effect by interfering with the removal of drug-entry porins.Can phages be selected so that the collateral effect of phage resistance in an infection site results in an increase in drug sensitivity? This would provide a one-two punch to kill most of the bacteria in the infection site population by phage-induced lysis, followed by killing any remaining phage-resistant individuals with existing antimicrobial drugs. Can antimicrobial drugs that target essential features at the outer surface of the outer membrane be used to circumvent the mechanisms for the evolution of AMR? Is there a mechanism for regulated removal of specific outer-membrane proteins? If such a pathway exists, small molecules applied extracellularly could have a therapeutic effect by interfering with the removal of drug-entry porins. Can phages be selected so that the collateral effect of phage resistance in an infection site results in an increase in drug sensitivity? This would provide a one-two punch to kill most of the bacteria in the infection site population by phage-induced lysis, followed by killing any remaining phage-resistant individuals with existing antimicrobial drugs. We thank Rhys Dunstan, Manasa Bharathwaj, and Rebecca Bamert for critical comments on the manuscript. N.R. was supported by the Monash Biomedicine Discovery Scholarship. Figures were created using BioRender.com. No interests were declared. iwww.cdc.gov/drugresistance/major-threats.html iiwww.who.int/news-room/fact-sheets/detail/antimicrobial-resistance iiiwww.oecd.org/health/health-systems/AMR-Tackling-the-Burden-in-the-EU-OECD-ECDC-Briefing-Note-2019.pdf ivhttps://apo.org.au/sites/default/files/resource-files/2016-05/apo-nid63983.pdf this complex structure is formed from a population of bacteria enmeshed in a matrix of secreted macromolecules: extracellular DNA and/or polysaccharides and/or structural proteins. The extracellular matrix adheres to an abiotic or biological surface and protects bacteria from environmental stresses, including antimicrobial drugs. antimicrobial drugs belonging to the β-lactam family. They inhibit cell wall synthesis by binding to the penicillin-binding proteins that catalyze the formation of the peptidoglycan cell wall. Carbapenems have broad-spectrum activity against both Gram-negative and Gram-positive bacteria. spanning across the inner and outer membranes, efflux pumps are transporters that export toxic solutes such as organic solvents, detergents, and antimicrobial drugs. Exporting a wide range of antibiotics, the activity, and upregulation, of efflux pumps is an important mechanism by which an AMR phenotype is acquired. bacteria with an outer membrane are not permeant to Gram stain, and so are deemed Gram-negative. Most of the pathogens identified for urgent attention by the WHO and the US CDC are Gram-negative, including the extended-spectrum β-lactam-resistant and carbapenem-resistant forms of E. coli, K. pneumoniae, A. baumannii, P. aeruginosa, and others. modification or removal of membrane components, that is, specific proteins or lipids, to adapt to a new environmental condition. This opinion article focuses on the remodelling of membranes by changes in the membrane protein composition. the proteome is the totality of proteins expressed by an organism, and the outer-membrane proteome is therefore that fraction of the proteome located in the outer membrane. the therapeutic use of viruses that infect bacteria, that is, bacteriophages, also called phages, to treat bacterial infections in human patients and agriculturally important animals. outer-membrane proteins that are structured as a barrel, antiparallel β-strands forming the barrel wall and leaving a central pore in the β-barrel structure. Porins assist the envelope integrity of Gram-negative bacteria and serve as water-filled pores to facilitate diffusion of small molecules, including antimicrobial drugs. The 'major porin' is the protein that dominates the outer-membrane surface area, sometimes reaching 50% of the total protein integrated in the outer membrane.