Abstract
Pseudomonas aeruginosa is one of the most clinically important Gram-negative bacteria and is related to many severe and life-threatening infections worldwide. It presents intrinsic resistance against many antibiotics and has the ability to acquire or develop additional mechanisms to overcome the action of all anti-pseudomonal drugs. Formerly abandoned antibiotics and new compounds such as cefiderocol and combinations of β-lactams with new β-lactamase inhibitors are considered for the treatment of infections due to multi- or extensively-drug-resistant strains. In the present review, the antimicrobial resistance mechanisms of P. aeruginosa and the potential treatment options for the difficult to treat P. aeruginosa infections are discussed in an attempt to correlate microbiological and laboratory data to the choice of optimal treatment in everyday clinical practice.
Keywords
Introduction
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that may cause considerable morbidity and mortality. 1 Infections caused by P. aeruginosa range from acute to chronic; and from community to mainly hospital-acquired, commonly affecting immunocompromised patients. The most frequent P. aeruginosa infections in clinical practice include respiratory tract infections especially in cystic fibrosis patients, ventilator-associated pneumonia, sepsis, urinary tract infections, otitis externa, and infections of the skin and soft tissues, including burn and wound infections. 2 Moreover, P. aeruginosa is commonly present in intensive care units where it is known for its limited treatment options due to a plethora of intrinsic, adaptive and acquired resistance determinants. 3
The intrinsic resistance of P. aeruginosa is established by a variety of genetically encoded mechanisms, while acquired resistance occurs through the acquisition of additional genes or mutations under selective pressure. Adaptive resistance is expressed through the formation of biofilms in infection sites such as cystic fibrosis patients’ lungs. 4 With one of the largest bacterial genomes, P. aeruginosa has many molecular pathways for environmental adaptability, while it can also receive horizontally transferable genetic elements, further enhancing its potential to survive under adverse circumstances. 5
Antimicrobial resistance (AMR) has become a global issue of great importance, as the extraordinary adaptability of some clinically significant bacteria combined with the overuse and/or misuse of antimicrobials in some counties contributed to the rapid emergence and worldwide spread of high-risk clones.6,7 Resistant strains are classified as multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) as follows: MDR is determined as non-susceptibility to ⩾1 agent in ⩾3 antimicrobial categories; the term XDR is used when susceptibility is limited to ⩽2 antimicrobial categories; and PDR in case of non-susceptibility to all agents in association with all antimicrobial categories.8,9
According to WHO’s 2024 update, carbapenem-resistant P. aeruginosa (CRPA) is characterized as a “high” priority. 10 In addition, numerous studies have shown that difficult to treat infections, particularly caused by MDR bacteria, lead to prolonged hospital stays, higher hospital costs, and increased mortality rates, underscoring the urgent need for new treatment plans and enhanced infection control measures.11,12
This review aims to summarize the intrinsic, adaptive, and acquired resistance mechanisms of P. aeruginosa and to discuss the range of therapeutic options available for managing these infections. Particular emphasis is placed on acquired resistance pathways and their implications for antibiotic selection, as well as an overview of established and emerging treatment strategies designed to address multidrug-resistant P. aeruginosa.
Genomic pool and plasticity
P. aeruginosa is a highly adaptable opportunistic pathogen whose clinical relevance is largely attributed to its expansive genomic pool, encompassing a wide array of virulence and antibiotic resistance genes. 13 Comparative genomic analyses have revealed that P. aeruginosa possesses a large and plastic genome, typically ranging between 5.5 and 7 Mb, which includes a conserved core genome and a variable accessory genome enriched with pathogenicity islands, resistance cassettes, and mobile genetic elements such as integrons and plasmids. 14 This genomic flexibility facilitates the acquisition of resistance genes contributing to antibiotic-resistant phenotypes commonly found in nosocomial settings.15,16
Among the virulence factors, the lipopolysaccharide causes tissue damage, mediates immune recognition, promotes host cell adhesion, and has been linked to the development of biofilms.17,18 Exotoxin A inhibits eukaryotic protein synthesis and promotes tissue necrosis. 19 Additionally, outer membrane proteins play roles in nutrient uptake, adherence, and antimicrobial resistance. 20 Biofilm-associated drug resistance is supported by structural appendages such as flagella, pili, and other adhesins, which promote surface attachment and colonization, while exopolysaccharides further enhance biofilm structure and reduce bacterial clearance by the host. 21 P. aeruginosa employs six distinct secretion systems (T1SS, T2SS, T3SS, T4SS, T5SS, T6SS) that are crucial for host colonization, motility, and evasion of immune responses. Other virulence factors include pyocyanin, which exacerbates tissue damage and organ dysfunction, and a variety of lytic enzymes—such as LasA and LasB elastases, AprA alkaline protease, LipC lipase, phospholipase C, and esterase A—that regulate host-pathogen interactions and modulate other virulence mechanisms.22,23 Rhamnolipids disrupt lung surfactant and epithelial tight junctions, directly damaging respiratory tissues. 24 Siderophores like pyoverdine and pyochelin aid in iron acquisition under iron-limited conditions, thereby enhancing pathogenicity. 25 Moreover, P. aeruginosa utilizes antioxidant enzymes, including catalases, alkyl hydroperoxide reductases and superoxide dismutases to neutralize reactive oxygen species and survive within phagocytic cells. 26
P. aeruginosa exhibits remarkable genomic plasticity that supports the acquisition and dissemination of both virulence factors and antimicrobial resistance genes, primarily through horizontal gene transfer mechanisms such as conjugation, transformation, and transduction. 27 This capacity enables the incorporation of mobile genetic elements, including plasmids, transposons, integrons, and pathogenicity islands, which collectively enhance its adaptability in diverse environments. 28
Among these elements, class 1 and 2 integrons are key platforms that capture and express resistance genes on plasmids, facilitating their horizontal spread.29,30 Simultaneously, pathogenicity islands such as PAPI-1 and PAPI-2 (P. aeruginosa pathogenicity islands) are commonly associated with epidemic, high-risk clones and harbor important virulence determinants. 31 These mobile elements often carry genes encoding exotoxins, elastases, proteases, and biofilm-related polysaccharides, which contribute to host damage and immune evasion. In parallel, resistance genes such as β-lactamases, aminoglycoside-modifying enzymes, and multidrug efflux pumps are frequently co-located on the same mobile elements. 32
The emergence of resistance to frontline antipseudomonal drugs reflects the pathogen’s exceptional ability to combine intrinsic, acquired, and adaptive mechanisms. Resistance may arise under therapeutic pressure, often through the accumulation of multiple mechanisms within the same isolate, leading to reduced efficacy of agents that were once reliable. Although novel antimicrobials and inhibitor combinations have expanded treatment options, their effectiveness could be undermined by the development of resistance. Moreover, biofilm-associated tolerance and the dissemination of high-risk clones further complicate therapy and infection control. These challenges highlight the importance of mechanism-guided treatment, robust antimicrobial stewardship, and integration of rapid diagnostics.
Intrinsic resistance to antimicrobials
The intrinsic resistance of P. aeruginosa refers to the innate feature of the species to remain unaffected by the action of certain antimicrobials. This is either the result of antimicrobial-target incompatibility or is due to the expression of chromosomally encoded resistance genes. 33 These genes may encode enzymatic or non-enzymatic mechanisms (Table 1). Noticeably, P. aeruginosa presents intrinsic resistance to more antimicrobials than most other clinically relevant species, limiting a priori the available treatment options even for wild-type or non-MDR isolates. 34
Summarized P. aeruginosa resistance mechanisms.
Examples of P. aeruginosa lower permeability include tetracyclines, erythromycin, and ertapenem. Efflux pumps expel a wide array of substrates, including fluoroquinolones, β-lactams, macrolides, tetracyclines, and aminoglycosides.
Antibiotic-inactivating enzymes
P. aeruginosa commonly produces antibiotic-inactivating enzymes. Among them, some class C and D β-lactamases as well as aminoglycoside-modifying enzymes are of clinical importance. These enzymes hydrolyze susceptible bonds like amides and esters in certain β-lactams and aminoglycosides.
P. aeruginosa is classified as part of the SPICE group of bacteria (Serratia spp., P. aeruginosa, Indole-positive Proteus, Citrobacter spp., Enterobacter spp.). 35 These microorganisms harbor an inducible ampC gene that encodes class C β-lactamases and can be induced by the presence of specific inducers such as imipenem, cefoxitin, and clavulanic acid. AmpC β-lactamases are serine cephalosporinases that hydrolyze the β-lactam ring of many penicillins and cephalosporins. 36 They are not affected by traditional β-lactamase inhibitors such as clavulanic acid and sulbactam but newer β-lactamase inhibitors such as avibactam and relebactam are stable to AmpC hydrolysis.37–39 Another endogenous β-lactamase that this microorganism expresses is the class D oxacillinase PoxB that commonly hydrolyzes oxacillin, methicillin, and cloxacillin. Even though it has been shown that overexpression of PoxB may lead to increased meropenem MICs without a concomitant increase for imipenem, 40 this enzyme is not considered as an important contributor to the widespread clinical resistance observed in P. aeruginosa infections because it is rarely overexpressed in clinical isolates, and its hydrolytic activity is substantially lower than that of the clinically relevant carbapenemases. 41 Moreover, P. aeruginosa may produce enzymes such as phosphotransferases (APHs), acetyl transferases (AACs), and nucleotidyl transferases (ANTs), which modify and inactivate aminoglycosides by targeting hydroxyl or amino groups.42,43 Different enzymes, however, may have variable inactivating activity on different compounds of the aminoglycoside family.
Low outer membrane permeability
Many antimicrobials enter the periplasmic space of Gram-negative bacteria through porins located in their outer membrane. General porins allow many different substances to pass, whereas specific porins are specialized and allow entrance only to specific molecules. 44 Unlike most other Gram-negative bacteria, P. aeruginosa has predominantly specific rather than general porins, which makes its outer membrane less permeable to antibiotics. A characteristic example is the intrinsic resistance of P. aeruginosa to ertapenem, a carbapenem with a larger molecular weight than imipenem and meropenem. Ertapenem has no effect on P. aeruginosa simply because its molecule cannot pass through the porins and enter the periplasmic space where its target is located. 45
In contrast, antibiotics such as aminoglycosides and colistin are not affected by this mechanism, as they diffuse through the outer membrane of Gram-negative bacteria by interacting with their lipopolysaccharides and changing the membrane’s permeability. 39
Efflux systems
P. aeruginosa expresses various efflux pumps. These are protein complexes that pump harmful substances like antibiotics or toxins out of the cell. Each complex consists of three proteins: an outer membrane porin channel (e.g., OprD, OprM, OprN, OprJ), a periplasmic linker protein (e.g., MeXA, MeXX, MeXC, MeXE), and an inner membrane transporter that uses proton-motive force as its source of energy (e.g., MeXB, MeXY, MeXD, MeXD, MeXF). 39 Particularly in P. aeruginosa, this excretion mechanism offers resistance to most antibiotics, except polymyxins, through four main efflux pumps: MexAB-OprM, MexXY-OprM, MexCD-OprJ, MexEF-OprN. 46 This multidrug efflux ability justifies the name Mex of the proteins on the periplasmic and inner membrane, while the Opr stands for the outer membrane porin. Thus, the efflux pumps’ proteins are named so along with a letter (e.g., MexAB and OprM). 47
Adaptive resistance (biofilms)
Biofilms are communities of microorganisms that adhere to surfaces and form a matrix of extracellular polymeric substances. 48 Examples of these substances are exopolysaccharides and other macromolecular components such as proteins, lipids, and biosurfactants. This matrix formation is responsible for additional resistance to antimicrobials in many species such as P. aeruginosa 49 whereas, antibiotic resistance genes and biofilm formation determinants may co-exist. 50 The biofilm development is a multicellular process driven by environmental signals like nutrition, messengers, and low antibiotic concentrations. At the molecular level, it emanates by gene regulatory networks that offer ecological versatility in hostile environments. 51 As cells shift to biofilm growth, gene regulation changes, causing various physiological and phenotypic alterations to the bacteria. 52 Specifically, the biofilm-mediated resistance protects bacteria from antibiotics through various mechanisms such as hindering antibiotics’ entrance, establishing a microenvironment that slows cell growth, activating an adaptive stress response, and promoting the differentiation of persister cells. 53 Persister cells that make up about 1% of biofilm cells, are slow-growing, metabolically inactive, and highly tolerant to antibiotics despite the fact that they do not have genetically encoded AMR mechanisms. 54 Biofilm formation in P. aeruginosa is important due to its main role in many persistent infections, particularly in the chronic lung infection of cystic fibrosis patients, and in chronic leg ulcers. Furthermore, P. aeruginosa possesses flagellum proteins on its surface that, at the early phase of infection, are responsible for its swarming and twitching motility that is essential for biofilm development. 39
Treating biofilm-forming P. aeruginosa infections is challenging55,56 therefore, in such cases, antibiotics may be used in combination (e.g., gentamicin/ciprofloxacin 57 and tobramycin/clarithromycin 58 ) or together with other substances to destroy biofilms. 59 Interestingly, significant differences have been observed in vitro regarding the effects of tobramycin when combined with different macrolides, azithromycin, and clarithromycin, against P. aeruginosa biofilm infections. Azithromycin is often selected in clinical practice due to its favorable pharmacokinetic profile, particularly its superior tissue penetration. 60 Moreover, azithromycin has demonstrated beneficial genomic effects, including inhibition of quorum sensing and suppression of biofilm formation by P. aeruginosa. However, emerging evidence suggests that these same properties may paradoxically antagonize the antimicrobial activity of agents such as tobramycin. 61 In contrast, clarithromycin appears to be a more compatible macrolide when combined with tobramycin. Experimental studies have shown that clarithromycin not only avoids antagonizing tobramycin's activity but may also prevent or even reverse biofilm-associated resistance to tobramycin, while this drug combination exhibits synergistic effects against P. aeruginosa biofilms, significantly reducing biofilm volume and enhancing bactericidal activity. 62 Therefore, the combination of tobramycin and clarithromycin is considered a more effective therapeutic approach for treating P. aeruginosa infections than the tobramycin–azithromycin combination, particularly in infections affecting the respiratory tract. However, when tobramycin is used at very high concentrations (100–500 µg/mL), its effect becomes so dominant that any additional benefit from clarithromycin cannot be clearly distinguished, highlighting the need for further in vivo investigations. 63
Antimicrobial peptides are used in order to interact with the bacterial cell membrane and penetrate to cause bacterial death. 64 Quorum-sensing inhibitors inhibit the quorum-sensing system and interfere with signaling molecules and receptor proteins. 65 Specific enzymes target extracellular polymeric substances such as exopolysaccharides and matrix proteins to disrupt biofilms 66 whereas bacteriophages may be implemented to encode enzymes to destroy the extracellular matrix. 67
Acquired resistance mechanisms
Acquired mechanisms confer resistance to P. aeruginosa even against the available anti-pseudomonal drugs. This type of resistance is achieved by: mutations that alter the drug target sites; through the acquisition of mobile genetic elements (MGEs) such as plasmids, transposons, and integrons encoding resistance determinants and; by over-expression or down-regulation of pre-existing genes. Subedi et al. 33 showed that P. aeruginosa acquires resistance genes (e.g., blaCTX-M) from other microorganisms through genetic transfer via self-transmissible megaplasmids or insertion of DNA fragments into highly variable genomic regions like PA0069. PA0069 is a spot of high genomic plasticity in the genome of P. aeruginosa that allows the insertion of large DNA fragments including those harboring multiple β-lactamase genes. 68 Overproduction of AmpC β-lactamases, along with acquired antibiotic-inactivating enzymes, over-expression of pumps, and lower expression of specific porins, contribute to a complicated interplay of antimicrobial resistance. Particularly, in P. aeruginosa several mutations may result in the overexpression of endogenous β-lactamases. For instance, clavulanic acid induces ampC expression, leading to the reduction of ticarcillin's effectiveness. 69 Furthermore, the deficiency or downregulation of specific porin proteins diminishes further the outer membrane permeability, as these porins are essential for the influx of antibiotics such as quinolones and β-lactams.70,71 Other mutations lead to overexpression of efflux pumps that expel various antibiotics such as quinolones, antipseudomonal penicillins, cephalosporins, and aminoglycosides. 72
Mutations in PBPs (penicillin-binding proteins), the target of β-lactams, reduce their affinity for these antibiotics. PBPs are essential for cell wall synthesis and cell division, and especially in P. aeruginosa, PBP3 variants are common in clinical isolates with reduced β-lactam susceptibility. These mutations, particularly in and around the active site of PBP3, may prevent the effective binding of β-lactams, with different variants affecting the resistance to specific antibiotics like aztreonam, ceftazidime or meropenem. In a recent study, some PBP3 variations reduced susceptibility to a variety of β-lactam antibiotics, including meropenem, ceftazidime, cefepime, and ticarcillin with different variations affecting different antibiotics but none of the tested variations reduced susceptibility to imipenem or piperacillin. However, these variations were correlated with fitness cost for bacterial cells by inhibiting cell division. 73 PBP5 is one of the most abundant PBPs in P. aeruginosa and although its main function is that of a cell wall dd-carboxypeptidase, it seems to possess some β-lactamase activity to contribute to the ability of P. aeruginosa to resist the antibiotic activity of some β-lactams. This is due to its unique structure that includes features closely resembling those of the class A β-lactamases. 74 More research, however, is needed to fully understand the impact of PBPs on β-lactam resistance.75,76
Resistance to fluoroquinolones
Resistance against fluoroquinolones in P. aeruginosa is commonly related to efflux pump overexpression (MexAB-OprM, MexCD-OprJ, MexEF-OprN) and to mutations in the genes of DNA gyrase and topoisomerase IV (ParC and ParE). DNA gyrase genes (GyrA and GyrB) are the primary targets, while mutations in gyrA and parC are common in highly resistant strains.77,78 Khan et al. 79 observed resistance to fluoroquinolones in strains that carried both crpP and qnrVC1 resistance genes, as well as mutations in the quinolone resistance-determining regions (QRDRs).
Resistance to aminoglycosides
P. aeruginosa presents acquired resistance against aminoglycosides, driven by mutations in the mexZ, fusA1, parRS, and armZ genes, by transferable aminoglycoside-modifying enzymes (AMEs), rRNA methylases, and by overexpression of efflux systems (mainly MexXY-OprM).43,80 AMEs inactivate aminoglycosides through acetylation (AACs), adenylation (ANT), and phosphorylation (APHs) with AAC(3) and AAC(6′) subfamilies being common in P. aeruginosa. 81 Nucleotidyl transferases like ANT(2′)-I contribute to resistance to gentamicin and tobramycin. Phosphoryl transferases, although rare in clinical isolates, inactivate less commonly used aminoglycosides (e.g., kanamycin and neomycin). 81 Additionally, 16S rRNA methylases, such as RmtA, RmtB, and ArmA, hinder aminoglycoside binding, leading to high-level resistance to all aminoglycosides. 82 The enzymes of this family that inactivate anti-pseudomonal aminoglycosides are APH(3′)-VI, APH(3′)-IIb-like, and APH(2”). 82 Noticeably, aminoglycoside resistance is commonly acquired with resistance determinants to other antibiotic classes such as β-lactams, contributing to multi-drug resistance in clinical isolates. For example, the gentamicin resistance gene aac(3) is associated with a chromosomal transposon (Tn801), which also carries the blaTEM-21 β-lactamase gene. 83 Similarly, genes like aph(3'), which provide resistance to kanamycin, neomycin, and streptomycin, and ant(3"), which offers resistance to streptomycin and gentamicin, are carried on MGEs and are often associated with β-lactam resistance determinants. 81
Acquired resistance to β-lactams
β-lactams are the most frequently used antibiotics globally, and they include the penicillins, cephalosporins, carbapenems, and monobactams. β-lactams exhibit bactericidal activity by prohibiting the formation of the bacterial cell wall by binding to and inactivating the PBPs (Figure 1). P. aeruginosa may harbor β-lactamase genes located either on the chromosome (endogenous β-lactamases) or on MGEs (acquired β-lactamases). P. aeruginosa has at least three regulatory ampD genes and may present increased AmpC β-lactamase production. 84 Juan et al. 85 showed that ampD inactivation is the most common cause of AmpC hyperproduction in P. aeruginosa but also indicated that another regulator (ampE) may indirectly contribute to β-lactam resistance, with the contribution of additional unknown genes, located possibly near the ampDE operon. Acquired ampC genes on transposable elements confer resistance to monobactams. 86
Cell targets of anti-pseudomonal drugs.
An important acquired β-lactamase type in P. aeruginosa is cefotaximase-München (CTX-M), with variants like CTX-M-1, CTX-M-2, and CTX-M-43 commonly detected in MDR isolates. 87 CTX-M enzymes exhibit a clear substrate preference for cefotaxime and ceftriaxone over ceftazidime. Their strong cefotaximase activity is related to the unique geometry of the β-lactam-binding site, which allows efficient recognition of penicillins, narrow-spectrum cephalosporins, and cefotaxime, but not of the bulkier ceftazidime molecule even though some CTX-M variants with enhanced ceftazidimase activity have also been detected. 88 They are typically inhibited by clavulanic acid, sulbactam, and tazobactam though some variants may present reduced susceptibility to these inhibitors. 89 Additional extended-spectrum β-lactamases (ESBL) include Pseudomonas Extended Resistance (PER). These are Ambler class A enzymes that hydrolyze penicillins and cephalosporins including oxyimino-β-lactams (third- and fourth-generation of cephalosporins) and aztreonam and are inhibited by β-lactamase inhibitors, such as clavulanic acid, sulbactam, or tazobactam. 90
Resistance to carbapenems
Carbapenems are the most effective β-lactams 91 exhibiting significant advantages compared to other antibiotics of the same or different categories. More precisely, carbapenems have a broad spectrum of activity, 92 are not susceptible to many β-lactamases including AmpC and ESBLs 93 and present less adverse effects than other last line therapeutic choices such as the polymyxins. 94 These important advantages make carbapenems and particularly imipenem and meropenem, a safe treatment option for infections due to P. aeruginosa. On the other hand, similar to other beta-lactam antibacterials, nephrotoxicity, neurotoxicity, gastrointestinal effects, and immunomodulation have been reported with the use of carbapenems, and thus predisposing factors should be considered when administering any carbapenem. 91
Carbapenem resistance in P. aeruginosa is mediated by four mechanisms or by various combinations of them expressed in a single strain. 95 These mechanisms are: (i) the diminished expression or loss of the OprD porin, (ii) the overexpression of efflux pumps, (iii) the overexpresion of AmpC β-lactamases, and (iv) the production of carbapenem-hydrolyzing enzymes named carbapenemases.
The diminished permeability of the outer membrane does not allow carbapenems to reach the PBPs that are located in the periplasmic space. The OprD porin is an essential protein for the entrance of imipenem in P. aeruginosa cells. Thus, downregulation of the oprD gene leads to imipenem resistance that may occur during treatment with this compound. Meropenem is less affected even though it is not completely impervious to porin loss. In this regard, a recent study showed a high correlation between predicted OprD loss and meropenem nonsusceptibility irrespective of the presence of carbapenemases. 96 In addition to facilitating carbapenem resistance, the loss of oprD enhances the capacity of P. aeruginosa to colonize mucosal surfaces and increases its resistance to acidic environments in mouse infection models.97–99
Overexpression of efflux pumps results in increased expulsion of carbapenems, mostly meropenem (mainly through MexAB-OprM) out of the cell.100,101 Imipenem is less affected by the efflux system alone unless this overexpression is combined with OprD loss. 102
Class C β-lactamases (AmpC) are not considered carbapenemases because they have only a low potential for carbapenem hydrolysis. 36 Their overexpression, however, in P. aeruginosa may contribute to carbapenem resistance especially when it is combined with one or more other carbapenem resistance mechanisms.
The production of carbapenemases is the most clinically important type of carbapenem resistance because these enzymes inactivate all or almost all β-lactams together with the carbapenems, they confer high levels of carbapenem MICs (minimum inhibitory concentrations), are encoded by genes that are horizontally transferable by mobile genetic elements thus spreading rapidly and effectively and; are commonly located with other resistance genes encoding for resistance determinants to different antibiotic categories.
Carbapenemases in P. aeruginosa
Carbapenem-resistant P. aeruginosa clinical isolates from around the world have been reported to harbor many different carbapenemase-encoding genes over the last years and interestingly, the first detection for many of the most successful metallo-β-lactamases (MBLs) occurred in this species. 103
Commonly, carbapenem-hydrolyzing enzymes are classified into Ambler A, B (also known as MBLs), and D classes. 104 Class A includes the plasmid-encoded KPC (Klebsiella pneumoniae Carbapenemase) and GES (Guiana Extended Spectrum) that hydrolyze penicillins, cephalosporins, monobactams, and carbapenems and are only partially inhibited by clavulanic acid, boronic acid, and tazobactam.105,106
Class B MBLs bear zinc in their active center; 107 they inactivate almost all β-lactams (except aztreonam) and are resistant to all administrable β-lactamase inhibitors. MBLs are inhibited only through metal ion chelation by EDTA (Ethylene diamine tetraacetic acid). It has been proven over the last decades that P. aeruginosa is probably the species that is associated with the most MBL-type enzymes than any other. Indeed, IMP (Imipenemase), VIM (Verona Integron-encoded MBL), NDM (New Delhi MBL), SPM (Sao Paulo MBL), GIM (German Imipenemase), SIM (Seoul Imipenemase), DIM (Dutch Imipenemase), AIM (Adelaide Imipenemase), HMB (Hambourg MBL), and CAM (Canada MBL) have been detected in P. aeruginosa isolates around the world. 108
Class D carbapenemases such as OXA-48 and OXA-198 hydrolize oxacillin and cloxacillin are resistant to various older β-lactamase inhibitors but are susceptible to novel inhibitors such as avibactam. 109 Whereas class A and B enzymes confer the highest level of carbapenem resistance compared to other mechanisms, OXA-type carbapenemases are commonly less potent in terms of carbapenem MIC increase.
Among the aforementioned carbapenemases, KPC, IMP, VIM, NDM, and OXA-48 types have spread through many countries and different bacterial species. Therefore, these are the targets of many diagnostic molecular panels and lateral flow immunochromatographic assays. Especially though in P. aeruginosa, there is a clear geographic distribution of clones harboring specific carbapenemase-encoding genes. IMP, VIM, GES, and OXA types present global distribution, KPC-producing isolates are mainly located in Latin America whereas NDM-producers are spread in the Indian subcontinent, Australia and are recently expanding rapidly in Greece.108,110 All other carbapenemases detected in P. aeruginosa remain contained in their specific geographic area of first emergence, as designated by their nomenclature.
Resistance to polymyxins
Polymyxins are bactericidal cationic peptides with a lipophilic fatty acyl side chain, primarily interacting with the negatively charged lipopolysaccharides (LPS) on the outer membrane of Gram-negative bacteria. This electrostatic interaction destabilizes the outer membrane, increasing permeability and leading to cytoplasmic leakage and cell death. 111 Polymyxins were formerly abandoned due to their severe adverse effects, including nephrotoxicity and neurotoxicity. 112 Nowadays, however, they are considered last-resort antibiotics for treating life-threatening infections caused by MDR and XDR strains. 113 Indeed, polymyxins remain one of the few available treatment options against MDR P. aeruginosa and commonly the only option against XDR isolates. Polymyxin B and polymyxin E (colistin) in the forms of colistin sulfate and colistin methanesulfonate are the only available antibiotics of this category. 114 Although P. aeruginosa does not exhibit intrinsic resistance against polymyxins, it has the potential to develop acquired resistance against them. Acquired resistance of P. aeruginosa against polymyxins consists of two distinct mechanisms: chromosomal mutations and plasmid-mediated resistance. Chromosomal mutations on regulatory genes lead to the dysregulation of two-component regulatory systems (TCSs), such as pmrA/pmrB, phoP/phoQ, ColRS, and CprRS.115,116 These systems result in modification of the LPS through the addition of 4-amino-4-deoxy-L-arabinose (Lara4N) and phosphoethanolamine (PEtN) or the incorporation of galactosamine into the lipid A of the LPS core. Consequently, the reduced negative charge of the phosphate groups diminishes polymyxin’s binding affinity to the LPS. 117
Plasmid-mediated resistance is mediated by horizontally transferable mobile colistin resistance (mcr) genes. The mcr-1 gene and its variants encode a PEtN transferase that attaches PEtN to lipid A reducing colistin efficacy.118,119 The mcr-1 gene, which was first identified in 2015, has been found in polymyxin-resistant strains of P. aeruginosa and displays a rapid worldwide spread. 120
In addition to these mechanisms, the overexpression of the outer membrane protein OprH enhances the acquired resistance to polymyxins in P. aeruginosa by occupying potential binding sites for polymyxin B on the LPS. 121
Resistance to fosfomycin
Fosfomycin is another old antibiotic that was revived recently because of the worldwide spread of multi-drug resistance. The drug is commercially available as disodium salt for intravenous administration and as calcium or trometamol salt for oral administration. Oral fosfomycin is primarily used to treat lower urinary tract infections whereas intravenous fosfomycin is prescribed for a variety of infections, including respiratory tract infections, bacteremia/sepsis, urinary tract infections, bone and joint infections, intra-abdominal infections, and skin/soft tissue infections. It reaches high concentrations in urine, but there are challenges in determining reliable breakpoints for P. aeruginosa and doubts about its effectiveness in intravenous administration for systemic infections. 122
Fosfomycin enters the P. aeruginosa cell through the glucose-3-phosphate transporter (GlpT) by mimicking its natural substrate, glycerol-3-phosphate (G3P). 123 Inside the cell, fosfomycin mimics phosphoenolpyruvate and blocks the enzyme UDP-N-acetyl glucosamine enolpyruval transferase (MurA) that normally catalyzes peptidoglycan biosynthesis for the formation of bacterial cell wall. More precisely, fosfomycin prevents MurA from synthesizing the peptidoglycan precursor UDP-N-acetylmuramic acid (UDP-MurNAc). 124
P. aeruginosa may overcome the action of fosfomycin by three distinct mechanisms. First, by producing Fos enzymes that modify the structure of the drug and inactivate fosfomycin. Second, by peptidoglycan recycling enzymes that salvage UDP-MurNAc and bypass the de novo synthesis of peptidoglycan and third; through mutations of the GlpT-encoding gene that result in reduced fosfomycin permeability. 124
According to the European Committee on Antimicrobial Susceptibility Testing (EUCAST), the majority of P. aeruginosa have FosA enzymes. 125 Moreover, fosfomycin monotherapy against P. aeruginosa infections is not recommended, and breakpoints are not available for both EUCAST 126 and Clinical and Laboratory Standards Institute (CLSI) 127 documents. Of note also, fosfomycin breakpoints referred to Enterobacterales should not be extrapolated to P. aeruginosa because different fosfomycin resistance mechanisms are involved.
P. aeruginosa high-risk clones
Genetic studies have shown that P. aeruginosa contains a conserved core and an accessory genome consisting of extrachromosomal elements, including plasmids and DNA blocks inserted into the chromosome at several loci. This accessory genome has been most probably acquired through horizontal gene transfer (mostly through phages) from different sources. 128
Bacterial population studies showed that P. aeruginosa has a non-clonal epidemic population structure meaning that it is composed of a limited number of widespread clones, which are selected from a background of a large number of unrelated genotypes that are recombining at high frequency. 129 In this context, worldwide reports revealed the existence of MDR and XDR global clones that are disseminated in health-care facilities in all continents. These are denominated high-risk clones and among them ST235, ST111, and ST175 seem to be likely those more widespread. As expected, there is a clear association of such clones, particularly ST235, with transferable resistance. 130 ST235 strains frequently carry MBL genes like blaNDM and blaVIM and are commonly resistant to carbapenems, fluoroquinolones, and aminoglycosides.131,132 In some cases, ST235 can be XDR being resistant to almost all available antibiotics. 133 ST111 is also known for fluoroquinolone, aminoglycoside and carbapenem resistance mainly attributed to the presence of VIM and IMP-encoding genes. 134 Similarly, ST175 presents high-level resistance to fluoroquinolones, aminoglycosides, and certain β-lactams. Carbapenem resistance is mainly via oprD inactivation and mexXY overexpression 135 but these strains can also carry horizontally acquired MBLs like VIM. 136 Moreover, some ST175 strains exhibit mutations in the AmpC active site leading to resistance to newer β-lactam/β-lactamase inhibitor combinations like ceftolozane/tazobactam and ceftazidime/avibactam. 137
Common anti-Pseudomonas treatment
Despite the fact that P. aeruginosa exhibits intrinsic resistance to many antimicrobial agents, some compounds are adequate for the treatment of infections due to isolates without the respective acquired resistance mechanisms. 138 In such cases, the drug is commonly selected from the following antibiotic classes: (i) β-lactams, (ii) quinolones, and (iii) aminoglycosides. The β-lactams that are active against P. aeruginosa are mainly piperacillin and ticarcillin as anti-pseudomonal penicillins even though ticarcillin is currently not widely available; ceftazidime and cefepime (a third and a fourth generation cephalosporin, respectively); aztreonam (monobactam) and; imipenem and meropenem among the carbapenems. When allowed by the local ecology, ciprofloxacin and levofloxacin are the fluoroquinolones that are used to treat P. aeruginosa infections whereas aminoglycosides with proven anti-pseudomonal action are amikacin and tobramycin. Gentamicin is no longer considered to be an antipseudomonal aminoglycoside per both CLSI 127 and EUCAST. 126 Aminoglycosides, however, present narrower therapeutic range and are often associated with adverse effects such as ototoxicity and nephrotoxicity. 139 Therefore, they are used in combination with other antimicrobials except for the treatment of urinary tract infections where they reach high concentrations.140,141
It has to be mentioned that lately EUCAST is using arbitrary breakpoints for key anti-pseudomonal drugs such as piperacillin, piperacillin-tazobactam, ticarcillin-clavulanic acid, ceftazidime, cefepime, imipenem, ciprofloxacin, levofloxacin, and aztreonam. 126 Following these breakpoints, it is impossible to obtain a “susceptible” result during the antimicrobial susceptibility testing, and this was done to assist clinicians with antimicrobial dosing since current guidelines recommend treating this pathogen with increased doses of the aforementioned antibiotics. 142 Thus, the otherwise susceptible isolates now fall into the “I” category that after 2019 is interpreted as “susceptible, increased exposure” by EUCAST. This means that there is a high likelihood of therapeutic success when exposure to the agent is increased by adjusting the dosing regimen or by its concentration at the site of infection. The adjusted dosing regimens for each compound are available in the EUCAST breakpoint document. 126
New antibiotics
Recently approved β-lactam/β-lactamase inhibitor combination antibiotics have been designed to treat infections from MDR or even XDR Gram-negative bacteria by protecting the β-lactam antibiotic from bacterial β-lactamase enzymes. Those already available include ceftolozane/tazobactam and combinations with new inhibitors such as ceftazidime/avibactam, imipenem/relebactam, meropenem/vaborbactam, and aztreonam/avibactam.
Ceftolozane/tazobactam presents high affinity for specific penicillin-binding proteins of P. aeruginosa (PBP1b, PBP1c, PBP3) and higher stability against AmpC β-lactamases. Additionally, it is not affected by efflux pump overexpression and porin mutations and inhibits β-lactamase activity via tazobactam. Tazobactam though, does not inhibit the action of carbapenemases. 38
The use of antibiotic combinations with novel β-lactamase inhibitors depends strongly on the underlying resistance mechanisms of P. aeruginosa and practically, the type of carbapenem resistance determined by molecular techniques or rapid immunochromatographic assays combined with clinical data and antimicrobial susceptibility results is used for the subsequent treatment choice (Table 2). Indeed, the new inhibitors commonly inhibit ESBLs, AmpC, some class D carbapenemases, and class A carbapenemases including KPC but are ineffective against all MBLs. Consequently, these combinations are not active on metallo-enzyme-producing isolates. More precisely, ceftazidime/avibactam inhibits the action of class A carbapenemases and other non-metalloenzyme β-lactamases via avibactam. 143 Imipenem/relebactam is not affected by pump overexpression whereas relebactam enhances the action of imipenem even upon AmpC overexpression or OprD loss and inhibits the action of class A carbapenemases. 144 Meropenem/vaborbactam does not offer any particular advantages over meropenem alone for the treatment of P. aeruginosa infections and in the presence of other alternatives for class A carbapenemase producers, it is suggested to be reserved for use in KPC-producing Enterobacterales. 145 Aztreonam/avibactam is a recently introduced combination that potentially can treat various resistant Gram-negative pathogens with diverse resistance mechanisms, including carbapenemase-producers. 146 The use of aztreonam/avibactam can be justified by the fact that aztreonam commonly escapes from the hydrolysis of MBLs (Table 3). The usefulness of the new combination however is limited by the main resistance mechanism for aztreonam alone, the expression of the MexAB-OprM efflux pump. Thus, practically, the activity of aztreonam/avibactam in P. aeruginosa does not offer many advantages over aztreonam. 147 Unfortunately, the effectiveness of the novel inhibitors and especially of avibactam is threatened by the emergence and rapid spread especially in K. pneumoniae, of new KPC variants that are resistant to the action of avibactam. 148 P. aeruginosa harboring KPC variants have already been detected in China. 149
Treatment options for carbapenem and quinolone-resistant P. aeruginosa non-urinary tract infections.
Characteristics of the newer commercially available β-lactam/β-lactamase inhibitor combinations for the treatment of P. aeruginosa infections.
The only antimicrobial that reliably retains its activity against P. aeruginosa even in the presence of MBLs to date is the new siderophore cephalosporin cefiderocol. 150 Cefiderocol incorporates the carboxypropanoxymino group from ceftazidime on the C-7 chain and the pyrrolidinium group from cefepime on the C-3 side chain and presents increased stability against all β-lactamases. 151 However, its MIC determination by broth microdilution is problematic and requires iron-depleted cation-adjusted Mueller–Hinton broth, while the disk diffusion method currently presents an area of technical uncertainty according to EUCAST. 126 Moreover, and despite some very encouraging laboratory data, some authors consider that there may not yet be enough clinical data to support its use against XDR P. aeruginosa.152,153 The 2022 European Society of Clinical Microbiology and Infectious Diseases (ESCMID) guidelines against carbapenem-resistant P. aeruginosa are on the same page 154 whereas, the 2024 Infectious Diseases Society of America (IDSA) guidelines against difficult to treat P. aeruginosa recommend cefiderocol for the treatment of infections due to MBL-producing P. aeruginosa. 155 Interestingly, there is a small percentage of resistant NDM-producing P. aeruginosa to cefiderocol 137 as well as sporadic reports for the emergence of mutations in iron transport systems and the Ω-loop of AmpC or horizontally acquired ESBLs leading to cefiderocol resistance. 156 Because of the presence of resistance and the challenges related to reliable antimicrobial susceptibility testing, the use of cefiderocol in combination with other antimicrobial agents has been proposed. 157
Recently, a new quinolone with intravenous and oral administration named delafloxacin was evaluated for its in vitro activity against MDR and quinolone-resistant strains of P. aeruginosa. Noticeably, the new compound was active in vitro against 30 out of 101 MDR quinolone-resistant P. aeruginosa. 158 Future research will show if delafloxacin can play an important role at the clinical level to overcome quinolone resistance at least for some selected cases.
Older revived antibiotics
Older, revived antibiotics that are effective against P. aeruginosa include polymyxins and fosfomycin. These drugs were abandoned or used less frequently in the past due to side effects or lack of perceived need, but they have regained importance as treatment options in the absence of better alternatives.
Polymyxins have been used widely for the treatment of XDR P. aeruginosa infections in areas where carbapenemases have become endemic. Special attention however has to be shown on the method used for susceptibility testing of these drugs. Polymyxins are large molecules that do not diffuse well in agar therefore; all methods involving agar diffusion (including gradient strips) are considered inadequate. Moreover, the semi-automatic systems for minimum inhibitory concentration (MIC) determination do not perform impeccably. Thus, the only reliable method for polymyxin susceptibility testing is broth microdilution. Moreover, polymyxins are unequivocally nephrotoxic, and increasing concentrations increase the risk of kidney damage. 159
Fosfomycin reaches high concentrations in urine, but there are challenges in determining reliable breakpoints and doubts about its effectiveness in intravenous administration for systemic infections. For these reasons, some authors have proposed its co-administration with carbapenems, aminoglycosides, colistin, and even ceftazidime/avibactam or ceftolozane/tazobactam. 160
Additional strategies to enhance treatment efficacy
Two of the most impactful strategies to enhance treatment efficacy against P. aeruginosa are prolonged infusion and combination therapy. Administering β-lactam antibiotics via prolonged infusion (over 3–4 h) or continuous infusion is considered particularly advantageous for critically ill patients and those infected with high-MIC P. aeruginosa strains.161,162 β-Lactams exhibit time-dependent killing, so prolonged infusion, by maintaining free drug concentrations above the MIC for an extended portion of the dosing interval (fT > MIC), is crucial to maximize their bactericidal effectiveness. 163 This approach minimizes the need for repeated venipuncture in patients with limited venous access, improves pharmacokinetic/pharmacodynamic (PK/PD) performance, suppresses the emergence of resistant mutants, and enhances therapeutic effectiveness—all without increasing the total antibiotic dose.164–166 Despite its advantages, prolonged infusion of β-lactam antibiotics presents several challenges. It necessitates dedicated intravenous access, often requiring a separate lumen on a central venous catheter, which can complicate administration. 167 This method also elevates the risk of catheter-related infections, particularly in critically ill patients.168,169 Additionally, it limits patient mobility due to the continuous infusion setup and complicates the administration of drugs unstable at room temperature, such as meropenem and ceftazidime.170,171
While there is no compelling evidence that two agents improve survival over monotherapy, combination therapy is considered necessary for P. aeruginosa by some clinicians due to potential synergistic effects, which some have hypothesized may improve outcomes. Combinations of β-lactam and aminoglycoside antibiotics are among the most frequently used treatments for P. aeruginosa infections, with many in vitro studies demonstrating synergistic interactions.172–174 Extensive in vitro studies have also shown varying degrees of synergy between quinolones and β‑lactam antibiotics, whereas combinations of aminoglycosides with fluoroquinolones rarely exhibit synergistic effects.175–177 Nevertheless, studies in cystic fibrosis exacerbations and MDR P. aeruginosa diabetic foot infections found no difference in outcomes between colistin monotherapy and colistin combination therapy, although nephrotoxicity increased with combination treatment.178,179
Combination antimicrobial therapy for Gram‑negative infections is often justified initially, since it increases the likelihood that at least one drug will be effective, may deliver synergistic effects, and can potentially slow resistance development. In critically ill patients, empirical use of multiple agents is believed to ensure broad coverage and to reduce the risk of delaying effective treatment. 180 However, once the pathogen and its antibiotic susceptibilities are identified, there is no clear evidence that adding a second agent improves clinical outcomes beyond what can be achieved with a single, appropriate antibiotic, while the drug-related adverse effects are increased.181,182 Similarly, though in vitro synergy may appear promising, especially when treating highly resistant organisms, optimizing antibiotic selection, dosage, frequency, and duration is likely more effective at preventing resistance than relying on combination therapy alone. 175
Alternative approaches
Given the limited efficacy of antibiotics against certain P. aeruginosa infections, especially when biofilms are involved, alternative therapies such as phage therapy and nanoparticles have been investigated. 183 Bacteriophages act by degrading the extracellular matrix, enhancing antibiotic penetration into biofilm layers, and inhibiting biofilm formation through quorum-sensing disruption. 184 Synergistic effects have been demonstrated when bacteriophages are combined with other agents, including nanoparticles, 185 enzymes, and natural compounds, resulting in enhanced biofilm eradication compared to monotherapies. Phage therapy offers several advantages over conventional antimicrobials, including minimal toxicity to humans and non-target microorganisms, and its efficacy against antibiotic-resistant strains. 186 Additionally, phages can replicate autonomously in the presence of suitable host bacteria and are naturally cleared in their absence. 53 However, a major limitation is their high specificity, which necessitates detailed knowledge of phage-host dynamics and precise identification of the infective phage for effective treatment. 187 Although relatively rare, bacteria may develop resistance to phages at high frequencies, often through single-point mutations in surface receptors. Beyond that, bacteria can deploy multiple defense mechanisms such as CRISPR-Cas systems, restriction-modification enzymes, or modifications in surface structures like the O-antigen to evade phage predation. 53 To address these limitations, phage cocktails (combinations of multiple bacteriophages) are used, as they demonstrate superior bacterial reduction and increased phage efficacy in vitro.188–190
Furthermore, the combined use of specific bacteriophages and antibiotics has been shown to enhance antimicrobial efficacy against P. aeruginosa, while simultaneously suppressing the emergence and persistence of resistant bacterial populations within biofilms. 191 Notably, the most effective strategy involves applying phages prior to antibiotics, as the initial high bacterial density allows for greater phage replication, thereby enhancing their therapeutic impact. 192
Interestingly, the application of nanoparticles in veterinary medicine is expanding, particularly due to the potential antibacterial properties of silver nanoparticles (Ag-NPs) against both susceptible and MDR pathogens including P. aeruginosa.193,194 Nanomaterials have been suggested to have a promising role that allows them to interact with microbial membranes because of their distinctive physical and chemical functions, such as their large surface area to volume ratio. Consequently, there has been interest in the medical applications of some metal nanoparticles, particularly in their usage as alternatives to antibiotics against MDR isolates. Recent studies have shown promising results, 195 even though their use by humans in the future requires more research.
Concluding remarks
P. aeruginosa is a remarkable example of survival and adaptability and as such, it is intrinsically resistant to many antibiotics while it is capable of developing resistance mechanisms to every other available antimicrobial agent. Understanding the complexity and interplay of mechanisms against anti-pseudomonal drugs should have a major role in optimizing treatment choices, developing new antimicrobials, and organizing appropriate antibiotic stewardship strategies in health care settings.
The introduction of novel β-lactam/β-lactamase inhibitor combinations and other innovative agents is promising; however, their efficacy is threatened by the rapid emergence of resistant strains. This underscores the urgent need for a multifaceted approach that integrates rational antibiotic use, robust infection control measures, and the continuous development of new therapeutic strategies.
The strength of the present review lies in its comprehensive synthesis of the molecular resistance mechanisms of P. aeruginosa, correlated with a discussion of currently available treatment options. By combining mechanistic insights with therapeutic perspectives, the review provides a valuable resource for both researchers and clinicians seeking to understand and address this critical public health threat.
Nonetheless, some limitations should be acknowledged. While the review integrates recent developments, the field is rapidly evolving, and the continuous emergence of resistance to newly introduced agents may not be fully captured. In addition, most of the therapeutic strategies discussed are based on literature data; thus, real-world effectiveness and long-term outcomes may vary. Furthermore, the focus on antimicrobial resistance and pharmacological interventions limits discussion of complementary approaches such as diagnostics and stewardship programs, which also play key roles in combating P. aeruginosa infections.
In conclusion, the battle against P. aeruginosa requires sustained research efforts, global surveillance, and multidisciplinary strategies. This review aims to contribute to that effort by outlining both the biological underpinnings of resistance and the available therapeutic choices.
