Recent advances in therapeutic targets identification and development of treatment strategies towards Pseudomonas aeruginosa infections

Abstract

The opportunistic human pathogen Pseudomonas aeruginosa is the causal agent of a wide variety of infections. This non-fermentative Gram-negative bacillus can colonize zones where the skin barrier is weakened, such as wounds or burns. It also causes infections of the urinary tract, respiratory system or bloodstream. P. aeruginosa infections are common in hospitalized patients for which multidrug-resistant, respectively extensively drug-resistant isolates can be a strong contributor to a high rate of in-hospital mortality. Moreover, chronic respiratory system infections of cystic fibrosis patients are especially concerning, since very tedious to treat. P. aeruginosa exploits diverse cell-associated and secreted virulence factors, which play essential roles in its pathogenesis. Those factors encompass carbohydrate-binding proteins, quorum sensing that monitor the production of extracellular products, genes conferring extensive drug resistance, and a secretion system to deliver effectors to kill competitors or subvert host essential functions. In this article, we highlight recent advances in the understanding of P. aeruginosa pathogenicity and virulence as well as efforts for the identification of new drug targets and the development of new therapeutic strategies against P. aeruginosa infections. These recent advances provide innovative and promising strategies to circumvent infection caused by this important human pathogen.

Background

Pseudomonas aeruginosa is a heterotrophic, motile, Gram-negative bacterium, which clinical isolates can be highly diverse regarding their genetic backgrounds and antimicrobial resistance profiles. P. aeruginosa is an opportunistic pathogen causing nosocomial and ventilator-associated infections with a high mortality rate [1]. Infections by this pathogen are of especially high importance for immunocompromised and cystic fibrosis (CF) patients. CF is a genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator that provoke an abnormal thickening of mucus, impaired ciliary function, and weakening of pulmonary immune response. Those alterations create an ideal microenvironment for lung infection by P. aeruginosa [2].

P. aeruginosa is intrinsically resistant to many antimicrobials, having a limited outer membrane permeability, expressing a wide variety of efflux pumps and producing AmpC, an inducible cephalosporinase. It can quickly develop antibiotic resistance through chromosomal mutations or horizontal gene acquisition. For example, the hypermutability of pmrB (10³–10⁴ times the background mutation rate) in PmrAB regulatory system facilitates a rapid adaptation to colistin [3]. Antibiotics having to cross the cell wall to reach their targets, such as aminoglycosides or polymyxins, have limited diffusion through P. aeruginosa cell envelope due to restricted permeability of the outer membrane and, in some cases, to the overexpression of outer membrane proteins, which restrain interaction of lipopolysaccharides (LPS) with the antimicrobial agents [4]. Furthermore, small hydrophilic antibiotics such as ß-lactams and quinolones that are able to cross the outer membrane through porins are expelled by efflux pumps [5]. P. aeruginosa can get further resistance by overexpression of AmpC, acquisition of mutations causing AmpC hyperactivity or through modification of the antimicrobial target structures [6]. In addition, horizontal gene transfer plays an important role, allowing the acquisition of resistance genes towards carbapenems [7] or quinolones (qnrB, qnrA, and qnrS), among others [8].

The emergence of multidrug-resistant (MDR) P. aeruginosa isolates has become a public health threat worldwide as infection by these isolates restricts treatment options and augments morbidity and mortality [1]. The emergence of carbapenem resistance in P. aeruginosa is particularly concerning, predominantly among critically ill patients, since carbapenems are an important treatment option against drug-resistant Gram-negative bacteria. P. aeruginosa is thus listed by World Health Organization as a critical priority pathogen urgently requiring novel treatment options [9].

P. aeruginosa does not only develop resistance, but can also acquire antibiotic tolerance through formation of biofilms, which are complex clusters of bacteria attached to a surface and embedded in a self-produced matrix. Biofilm formation by P. aeruginosa can lead to the development of nosocomial urinary tract infections (UTIs), catheter-associated UTIs, surgical site infections, infections in burn-wound patients and bloodstream infections, all associated with high rates of morbidity and mortality [10]. Bacteria entrapped in biofilms can be up to 1000-fold more tolerant to antibiotics than free-living bacteria, making treatment of such infections highly tedious [11]. In addition, some strains of P. aeruginosa are hyperbiofilm-forming, exhibiting some rugose small colony variants (RSCVs), developing biofilm aggregates surrounded by an extracellular matrix containing fragmented extracellular DNA and responsible for persistent infections, resistance to disruption by DNaseI and enhancement of biofilm formation [12].

Despite being an important human pathogen, P. aeruginosa is also widely present in the environment. Interestingly, some strains can have beneficial effects. P. aeruginosa is frequently found in sediments from ditches and tributaries and it displays an important role in nitrogen cycling in agriculture through its utilization of nitrate and urea [13]. Moreover, P. aeruginosa shows potential applications in industrial processes. For example, the strain P. aeruginosa ISJ14 degrades low-density polyethylene with no side effects on health or the environment, illustrating the potential of P. aeruginosa in waste processing [14]. Other applications were developed: Rilda et al. took advantage of P. aeruginosa antibacterial features in the construction of anti-bacterial textile fibres based on ZnO–TiO₂ nanorods template [15].

In this review, we discuss recent discoveries regarding pathogenesis mechanisms of P. aeruginosa and how these discoveries may lead to the identification of novel drug targets. We then highlight recent breakthroughs in the development of antimicrobial agents targeting P. aeruginosa, and showcase the potential of vaccination as an alternative to strengthen host immune responses and counteract antibiotic tolerance or resistance from this bacterial pathogen.

Main text

Pathogenesis of Pseudomonas aeruginosa

P. aeruginosa mainly causes hospital-acquired respiratory infections, but can also infect wounds, surgical sites, urinary tract and even provoke bacteremia [1]. An essential step for P. aeruginosa pathogenesis is its adhesion to its host. This is mediated by flagella and pili that induce attachment to epithelial cells via respiratory mucins and glycolipid asialoGM1 [16]. Several host factors are important for efficient P. aeruginosa binding to host, including carbohydrate-binding proteins (lectins). It was recently shown that a fucose-binding lectin, LecB, plays an important role in the high-affinity host-cell binding of P. aeruginosa [17]. LecB contains a carbohydrate-binding site composed of two closely located calcium ions, which create a bridge between the ligand and the protein. The high-affinity binding of LecB relies on a low-barrier hydrogen bond, cooperative rings of hydrogen bonds, coordination contacts leading to a unique charge delocalization, and the mobility of trapped water molecules [17]. Importantly, adhesion of P. aeruginosa to respiratory epithelial cells is further increased in CF patients. Indeed, P. aeruginosa adheres to the CF epithelium at early stages of infection in a process that is facilitated by the apical overexpression of the Vav3 protein in CF airway epithelial cells. This protein stimulates β1 integrin and fibronectin production at the luminal side of epithelial cells, leading to enhanced P. aeruginosa adhesion [18].

Adhesion mechanisms are not the only contributors to the pathogenesis of P. aeruginosa. Interestingly, some clinical P. aeruginosa isolates found in diverse infected human body sites, encode a defective lasR, constitutively expressing a biofilm-adapted transcriptional profile without a need for environmental stimulus. The lasR gene encodes the major quorum-sensing (QS) regulator LasR and defective lasR genotype is suggested to contribute to the success of these clinical P. aeruginosa isolates [19].

Additional connections between the QS of P. aeruginosa and the regulation of its virulence have been provided. P. aeruginosa QS mechanism is initiated by the production of cell-to-cell signals, so-called quorum sensing autoinducer (QSAI) molecules [20]. The massive delivery of QSAI molecules by approximately two thousands cells is essential for P. aeruginosa aggregates to activate QS [21]. Several QS circuits are essential in this process: LasI and RhlI produce quorum sensing autoinducers (QSAI), which are sensed by LasR and RhlR. Binding of QSAI to LasR and RhlR regulate expression of large sets of genes, including virulence factors such as pyocyanin or rhamnolipids, among others [22]. Moreover, RhlS, a QS small noncoding RNA, triggers the translation of the global virulence regulator Vfr, the interacting partner of the RNA-binding protein Hfq [23].

During biofilm formation, P. aeruginosa secretes several exopolysaccharides (EPS), including alginate, Psl, and Pel. Detailed structure and expression regulation of these EPSs are reviewed elsewhere [24]. Psl encourages and sustains airway microbial community development, being important for attachment of bacteria to abiotic and biotic surfaces. Pel is a positively charged EPS, which plays an important role in cell to cell interactions in biofilm. Alginate is a high molecular weight polymer and is involved in the structural stability of biofilms and protects it from dehydration. These EPSs are also implicated in the development of microbial communities in the respiratory tract. For example, in the presence of P. aeruginosa environmental and clinical isolates, Streptococcus salivarius employs the maltose-binding surface protein MalE to interact with the P. aeruginosa Psl. This interaction initiates and sustains S. salivarius biofilm formation within the CF lung [25]. Bacterial biofilms still remain a challenge to the healthcare system due to their resilience against antimicrobial agents and immune system. Moreover, mechanisms involved in biofilm formation and/or biofilm antimicrobial tolerance in P. aeruginosa are only partially understood, making research in this field highly relevant. Recently, the small hypothetical protein encoded by the gene PA2146 and conserved in ɣ-proteobacteria has been shown to regulate biofilm architecture and antimicrobial tolerance of P. aeruginosa PAO1. The deletion of this gene did not impact growth rate of planktonic cells or minimal inhibitory concentrations of antibiotics against them, but seriously impaired P. aeruginosa PAO1 biofilm architecture and antimicrobial tolerance in presence of tobramycin [26].

It has been recently established that P. aeruginosa strains presenting a mucoid phenotype and playing a major role in the pathogenicity of P. aeruginosa towards CF patients [27] secrete two types of QSAI molecules: C4-HSL and PQS [28]. The C4-HSL molecule interacts with EPS solely through Van der Walls interactions and is thermodynamically stable within the vicinity of the EPS. The PQS molecule forms thermodynamically stable ionic complexes with EPS-bound Ca²⁺ and establishes a hydrogen bond with a single EPS chain [28]. Iron/siderophore acquisition systems also significantly contribute to virulence-related phenotype, such as biofilm formation and enhance the pathogenesis of hypervirulent P. aeruginosa in wound infection isolates [29]. In addition, P. aeruginosa produces virulence factors, such as phenazine, pyocyanin, pyoverdin, and rhamnolipid, regulated by multiple QS-pathways, to trigger pathogenicity [30].

Beside adhesion and biofilm formation, P. aeruginosa also needs to escape the immune response to efficiently infect its host. There has been tremendous interest in understanding the molecular mechanisms involved in the injection of effector proteins into eukaryotic host cells via the P. aeruginosa’s repertoire of secretion systems (type II, type III, type IV, and type VI) and their role in the disease onset. Type III Secretion System (T3SS) effectors contribute, for example, to P. aeruginosa pathogenesis in wounds. The P. aeruginosa T3SS effector protein Exotoxin T acts as an anti-inflammatory agent by interrupting phosphorylation cascade through tyrosine kinase Abl/PKCδ kinase/inflammasome subtype NLRC4. This impairs NLRC4 inflammasome activation by targeting CrkII, which is required for both Abl transactivation and NLRC4 inflammasome activation [31].

Moreover, human immune cells contain C-type lectins receptors, Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), mannose receptor (MR), and Dectin-2, that recognize and bind P. aeruginosa biofilm carbohydrates (e.g. Psl and/or Pel). DC-SIGN strongly recognizes P. aeruginosa biofilms and planktonic cells while MR and Dectin-2 weakly recognize biofilms. Yet, interference with the endocytic activity of cell-associated DC-SIGN and MR and hindrance of Dectin-2-mediated cellular activation by biofilm carbohydrates, especially those containing a high percent of mannose, can lead to immune evasion [30].

Riquelme et al. unveiled that P. aeruginosa escapes immune clearance in infected lungs and persists in the inflamed human airway by redirecting its metabolism to promote biofilm formation and significantly augment synthesis of EPS to the detriment of LPS in the presence of the host macrophages-derived immunometabolite itaconate [32]. EPS shelters P. aeruginosa from itaconate-triggered membrane stress and stimulates human myeloid cell metabolic reprogramming, both locally and in circulating monocytes, to trigger even greater itaconate delivery, making the host immune response permissive to chronic infection [32].

Beyond virulence-related factors, such as QS, flagella [33] and formation of biofilm, whose matrix proteome is enriched in proteins involved in oxidation–reduction processes, proteolysis, and transmembrane transport [34], the virulence of P. aeruginosa can also be controlled by multiple biological factors. These factors include pyoverdine production (siderophore), lasR gene presence, capsule, alginate D, elastase B, exotoxin A and Transcription factors (TFs), with noticeable regulatory roles during pathogenesis [35,36,37]. For example, RsaL, QscR, RhlR, CdpR, MvfR, PchR, PhoB and LasR were functionally unveiled as master regulators of QS, ExsA was notified as the master regulator of T3SS, and GacA was described as a key regulator of T6SS [37]. Moreover, a recent article detailed the key role played by AlgKX protein complex in alginate production and biofilm attachment in P. aeruginosa PAO1 [38].

Additional factors contributing to Pseudomonas aeruginosa survival and infections

P. aeruginosa genome encodes two functional DksA (DnaK suppressor protein) paralogs, which confer resistance to oxidative stress. DksA1, containing a zinc-finger motif, is essential for H₂O₂ tolerance in both planktonic and biofilm growing cells, and allows the escape of P. aeruginosa from macrophages-mediated killing activities via regulation of the genes katA and katE. DksA2, on its side, is expressed only under zinc starvation and can replace the protective function of DksA1 against oxidative stress [39].

It has also been proven that the acquisition of molybdate through the Type VI secretion system (T6SS), endowed with the ability to secrete an anion-binding protein, confers to P. aeruginosa a competitive advantage over the surrounding bacterial species under anaerobic conditions [40]. P. aeruginosa also possesses a T6SS toxin (Tse8) that interacts with VgrG1a, component of the VgrG1a-tip complex, for its delivery into target cells where it restricts their ability to synthesize proteins [41].

In the context of co-infecting pathogen communities, the augmented mutation rates (mutators) of P. aeruginosa bacteria infecting CF patients are found to be encouraged only in the absence of other species [42]. This illustrates the tremendous advantages that could be endorsed by polymicrobial infections in strategies aiming at P. aeruginosa eradication.

Additionally, frequency of resistance emergence in P. aeruginosa populations can shift within days based on the nature and duration of antibiotic therapy, since rare mutations not found using culture-based strategies can expand over 5–12 days in riposte to antibiotic changes, while mutations conferring resistance to antibiotics that were not administered decrease and undergo extinction [43]. Another study reported that the higher tolerance of P. aeruginosa biofilm cells towards multiple antibiotics, such as gentamicin and colistin, is due to the presence of strains with inactivated flgE gene that display cell aggregation, reduced ability to adhere to surfaces, and a faster biofilm growth [44].

Identification of new potential therapeutic targets against Pseudomonas aeruginosa infections

Rapid development of antimicrobial resistance in P. aeruginosa as well as the limited efficiency of current antimicrobial treatments on biofilms require the development of alternative antimicrobial strategies to combat P. aeruginosa infections. In this perspective, it is essential to identify pathways or mechanisms that are essential for proliferation and/or pathogenicity of this bacterium and that can potentially become new therapeutic targets. We provide here some examples of recent discoveries, which might pave the way to the development of novel antimicrobial strategies towards infections caused by P. aeruginosa.

It has been recently confirmed that P. aeruginosa possesses a complete denitrification pathway, generating nitric oxide (NO) from NO₂ supplementation, the supply of endogenous oxygen used in aerobic conditions being directed by NO [45]. Targeting nitrogen sources can be therefore integrated into the strategies designed to eradicate this pathogen.

Even if P. aeruginosa exhibits slow growth in CF lung infections [46], reversal to high growth rate in the airways of cystic fibrosis patients enhances antibiotic susceptibility, partially relying on reverse mutations or changes in the transcriptional profile of genes known to be associated with antibiotic resistance [47]. A strategy focusing on reverting the slow growth phenotype of P. aeruginosa clinical strains to a high growth rate could therefore be relevant for infections eradication.

Furthermore, to adapt to and thrive within the host system, P. aeruginosa modulates the transcription termination stage of its transcription cycle. Indeed, upon induction by small-molecule guanosine tetraphosphate (ppGpp) and in response to DNA damage, the processive antiterminator AlpA positively monitors the expression of the alpBCDE genes-encoded programmed cell death pathway by recognizing specific sites on the DNA, and interacts with the β-flap and/or region 1.1 of σ70 RNA polymerase, allowing it to bypass intrinsic termination sites positioned downstream of target promoters. The AlpA also positively monitors the expression of genes in a second putative operon, comprising genes PA0807–PA0829, to facilitate the survival of P. aeruginosa cells in the host [48].

On the other hand, P. aeruginosa contains some rhamnolipids (glycolipids molecules) forming micelles that transport both self-produced toxic compounds (pyochelin) and heterologous compounds (e.g. lincosamide antibiotics) for targeting and killing of competing bacterial species (e.g. Staphylococcus aureus) during inter-species competition and establishment of the pathogen in its niche [49]. A co-isolated pair of S. aureus and P. aeruginosa from patients with tracheobronchitis or bronchial colonization revealed that P. aeruginosa exoproducts impacted biofilm formation and decreased in vitro growth of its S. aureus counterpart, while S. aureus did not impair biofilm formation and triggered swarming motility in P. aeruginosa [50].

To eradicate infection, the host immune system must sense the presence of the pathogen. In line with this, it has been revealed in the nematode Caenorhabditis elegans model of infection that the gut efflux pump multidrug resistance-associated proteins MRP-1, belonging to the C-type family of ATP-binding cassette transporters and showing a high degree of sequence homology to human MRP-1, transports oxidized glutathione, acting as a signalling agent capable of warning C. elegans of changes in intestinal homeostasis initiated by the presence of P. aeruginosa infection [51].

All these recently described mechanisms involved in P. aeruginosa survival and pathogenicity may be exploited in the future as targets for the development of novel antimicrobial agents. Interestingly, targeting mechanisms that are specific to P. aeruginosa might allow the development of narrow spectrum antimicrobials, which would specifically inhibit P. aeruginosa infection and have no effect on commensal bacteria from the human microbiome.

Development of diagnostic tools for rapid identification of Pseudomonas aeruginosa

In order to efficiently and specifically combat P. aeruginosa infections, proper methods for pathogen identification and antimicrobial susceptibility testing (AST) are required. Diagnosis of P. aeruginosa infections is usually based on cultures from blood, urine or respiratory samples. AST can then be performed on the isolated strains. Alternatively, rapid tests using real-time quantitative polymerase chain reaction (qPCR) can be used, but cannot differentiate colonization from infection. Recently, based on four novel specific target gene sequences of P. aeruginosa identified through pangenome analysis, Wang et al. designed high-specificity and high-sensitivity PCR and qPCR assays for rapid detection of P. aeruginosa [52]. qPCR can also provide information about the presence of resistance genes, using a multiplex PCR targeting known resistance genes [1]. However, this does not replace a phenotypic AST, which directly measures the activity of antibiotics on the isolated bacteria. Nevertheless, an important limitation of AST is that it can take up to 48–72 h to identify the suitable antimicrobial treatment.

To respond to the lack of rapid diagnostic protocols for AST, that would allow a timely and rational antibiotic prescription, He et al. designed a specific and rapid reverse assaying protocol for detection and antimicrobial susceptibility testing of P. aeruginosa [53]. This method exploits tail fibre protein (TFP)-functionalized magnetic particles for a specific capture of P. aeruginosa and a fluorescein isothiocyanate (FITC) labeled magainin II applied as a fluorescent tracer. AST results can be reached within 4 h with this method, avoiding a time-consuming process of bacterial isolation and identification [53].

As failing to detect P. aeruginosa early enough is associated with high mortality in immunocompromised patients, the potential biomarkers specific for P. aeruginosa infection have been investigated. Xanthine was identified as a potential biomarker and its rapid detection may strongly reduce the time between the onset of symptoms and administration of suitable antimicrobials, which should help avoiding high mortality rates [54]. Moreover, based on the high level and preferential binding of the receptor binding protein GP12, from T4 bacteriophages to the LPS structures on the surface of P. aeruginosa cells, this protein has been proposed for P. aeruginosa detection in future diagnostic and therapeutic applications [55]. On its side, the Enc34 endolysin from bacteriophage Enc34, containing an N-terminal enzymatically active endolysin domain and a C-terminal transmembrane domain, displays a peptidoglycan-degrading activity towards outer membrane-permeabilized P. aeruginosa PAO1 [56]. These two aforementioned proteins can be valuable tools for clinical surveillance and medical-based research. On the other hand, a portable analyser using silica bead-based nucleic acid extraction, and 8-plex real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) could detect P. aeruginosa with high sensitivity in less than 2 h [57].

Antimicrobial methods to circumvent Pseudomonas aeruginosa infection

In the last few years, diverse treatment strategies have been developed in order to circumvent infections caused by P. aeruginosa (Table 1). Some antibiotics efficiency towards P. aeruginosa infections could be improved by context-specific actions. Indeed, in ventilator-associated pneumonia, combination of cephalosporin and beta-lactamase inhibitor ceftolozane/tazobactam (C/T) exhibited both efficacy and safety in treating extensively drug-resistant P. aeruginosa [58]. However, antibiotic resistance against such a combination has been reported when administered at suboptimal steady-state concentrations of 20 mg/L in the susceptible P. aeruginosa ST175 isolate [59]. It has also been established that the acquisition of OXA β-lactamases such as OXA-10, and OXA-50, ESBLs GES-1, GES-15, and VEB-1, as well as metallo-β-lactamases (IMP-15, NDM-1, and VIM-2) rendered P. aeruginosa isolates resistant to C/T [60].

Table 1 Promising methods for eradicating Pseudomonas aeruginosa infections stages

Methods increasing drug permeation of bacterial cell envelope

As cell envelopes of Gram-negative bacteria act as barriers against exogenous antimicrobial agents, some efforts have been directed toward understanding how small molecules may break through these barriers. A study focused on identifying the permeation potential of compounds towards P. aeruginosa and unveiled antibiotics fluoroquinolones and derivatives (prulifloxacin and norfloxacin), linezolid, sulfamethazine, the alkaloid ergotamine and the peptidase inhibitor sitagliptin, as permeators efficiently crossing the epithelial barrier and permeabilizing bacterial membranes [78].

Strategies for inhibiting biofilm formation and quorum sensing

Quorum sensing (QS) is an important regulator of virulence factors expression and biofilm formation. Many efforts have been done recently to develop so called “anti-virulence” therapies, by targeting QS signalling.

Indeed, the Dyer Ex Eichler extract (DSE) from the plant Dioon spinulosum, was reported to lower biofilm formation, cell surface hydrophobicity, and EPS accumulation of P. aeruginosa isolates. It also reduces the relative expression of four QS genes (lasI, lasR, rhlI, rhlR) and the biofilm-related gene ndvB [79]. On its side, the combination of cinnamaldehyde with gentamicin represses acyl-homoserine lactones production and downregulates the expression of critical QS genes, to substantially diminish pyocyanin, alginate, rhamnolipid, hemolysin, protease, and elastase production, to successfully suppress preformed biofilms and to impair biofilm formation by disrupting EPS production [80].

Other molecules with anti-biofilm and/or anti-QS features have also been described. For example, the newly synthesized molecules, pyridine derivative 3, amino benzenesulfonamide derivative 2, furothiazole derivative 4, and thienothiazole derivative 5, exhibit potent biofilm inhibition activity against P. aeruginosa ATCC 27853. However, the pyridine derivative 3 has a similar efficiency against P. aeruginosa ATCC 27853 to that of reference drugs, ampicillin, and ciprofloxacin [81]. Compounds (e.g. Aurine tricarboxylic acid) that may impair EPS protein-eDNA interaction can also be specifically effective in eradicating biofilms generated by RSCVs [12]. Furthermore, N-Aryl Malonamides (NAMs) were recently described as potent compounds inhibiting the QS transcriptional regulator MvfR and thus controlling virulence [82].

P. aeruginosa can escape antimicrobials threat by trapping the antibiotics in the biofilm extracellular matrix. To overcome this, dextran-based single-chain polymer nanoparticles (SCPNs) coupled with DNase I and associated with tobramycin have been applied to disperse the biofilm matrix. This enhances the activity of tobramycin and DNase I on P. aeruginosa biofilms by neutralizing the ionic interactions that retain this antibiotic in the biofilm periphery [83].

Natural molecules reducing Pseudomonas aeruginosa growth

Although synthetic compounds can drastically reduce P. aeruginosa growth (e.g. phosphate-based coacervates containing metallic antibacterial ions Ag⁺ [84]), natural molecules can be highly efficient, safe, cost-effective and environment friendly. The natural compounds 6-gingerol and curcumin have been unveiled to inhibit QS activation and production of biofilm, EPS, pyocyanin, and rhamnolipid along with enhancing the susceptibility of P. aeruginosa AM26 to antibiotics such as ciprofloxacin and ceftazidime hydrate [85]. On the other hand, pure compounds from plants have been reported to exert antimicrobial activities against clinical isolates of P. aeruginosa. Indeed, molecules extracted from the leaf of the plant Andrographis paniculata (Burm f.) Nees, andrographolide, 14-deoxyandrographolide, 14-deoxy-12-hydroxyandrographolide, and neoandrographolide, exert QS quenching activity, inhibiting biofilm formation, protease production, and swarming motility of the clinical isolates of metallo-β-lactamase producing P. aeruginosa PA22 and PA24 [86]. Paeonol, also known as 2′-hydroxy-4′-methoxyacetophenone, is a phenolic acid molecule isolated from the root bark of traditional Chinese medicinal herbs, such as Arisaema erubescens [87]. It has been demonstrated to exert a wide range of activities, spanning from immune regulatory activity [88], anti-inflammatory [89], to antibacterial effect [90, 91]. Paeonol efficiently reduces infection of macrophages by P. aeruginosa, as described recently [92]. Furthermore, it has the potential to drastically impede the expression of QS-regulated gene rhlI/R and pqsA/R [93]. Detailed effects of paeonol on infected macrophages are described in Fig. 1.

Fig. 1

Paeonol effects on Pseudomonas aeruginosa infected macrophages. Based on the work of Tang H et al. (2022) [92]. Pathogen bacteria and their genes are in yellow, macrophage is in blue and the components of immune system in other colours

Silver nanoparticles produced using extracts from the berries of the plant Ligustrum vulgare have also been proven to exhibit antibacterial ability against P. aeruginosa [94]. Moreover, naturally derived hydroquinone has been found to rapidly disrupt the cell membrane, increase permeability and provoke leakage of intracellular potassium ions in P. aeruginosa MTCC 741 [95]. Furthermore, the marine organism brittle star Ophiocoma dentata crude extract and derived sesquiterpenoids molecules 8,11-epoxy-9(15)-himachaladiene-4-ol(O8-ophiocomane) and 11-epoxy-9(15)-himachaladiene-4-ol (O7-ophiocomane), exert noticeable antimicrobial effects against P. aeruginosa [96]. It is worth noting that human body naturally produces antimicrobial peptides that can combat infections driven by bacterial pathogens. For example, the antimicrobial peptides S100A12 (calgranulin C) is significantly expressed in immune cells like neutrophils and macrophages in addition to corneal tissues of patients with Pseudomonas keratitis [97, 98]. Its roles on P. aeruginosa growth, biofilm formation, pyoverdine secretion and type VI secretion system have been depicted in Fig. 2.

Fig. 2

Genes or pathways affected in Pseudomonas aeruginosa PAO1 in response to S100A12 treatment. The exact mechanism of action of S100A12 is not known, but it causes a broad transcriptional response. The genes of pyoverdine biosynthesis are downregulated in P. aeruginosa PAO1 in response to S100A12 treatment. pvdA, encoding the enzyme L -ornithine N5-oxygenase, induces the initial step in the pyoverdine synthesis pathway, pvdP turns ferribactin into fluorescent pyoverdine, pvdF and pvdN are important genes in pyoverdine biosynthesis. Quorum sensing lasR system related genes are impeded. Metabolic and redox pathways along with virulence factors as well as transporter and membrane proteins are also impeded. Reduced expression of rhamnolipid biosynthesis related genes rhlG, encoding an NADPH dependent ketoacyl reductase and rhlA, rhlG, as well as QS rhlR system rhlR, rhll- are reduced. Extracellular polysaccharides genes pelA, pelF, and pelG as well as biofilm genes pslA, pslG, algD, and alg44 are downregulated. Oxidative stress defense pathway genes, oxyR, ospR, fpvL and katB are downregulated. Expression of genes implicated in phenazine synthesis pathway, phzA1, phzB1, phzA2, and phzB2 are drastically reduced. Expression of genes related to the structural component of type 6 secretion system (T6SS); pldA, icmF1, hcp1, and vrgG1, are also diminished. This figure is based on Mishra P et al. (2022) [98] publication

Additional methods for restricting Pseudomonas aeruginosa infections

Other methods for controlling P. aeruginosa infections implicate the use of bacteriophages whose host specificity and mode of infectivity depend on the interactions between the viral proteins and the surface of the host bacteria. Nodstrom et al. proposed bacteriophage therapy as a promising alternative to eradicate biofilms formed by genetically diverse P. aeruginosa clinical strains isolated from cystic fibrosis patients [99]. The lytic Pseudomonas phage LUZ19 targets QS of P. aeruginosa via a QS targeting protein, Qst. Qst interacts with PqsD, a key enzyme of the quinolone biosynthesis pathway. This causes a decrease in levels of Pseudomonas quinolone, allowing efficient LUZ19 infection [100]. Another mechanism, which might inspire the development of innovative antimicrobial strategies, is involved in Pseudomonas bacteriophage LUZ24 activity. This bacteriophage produces a peptide, Igy, that interacts with the gyrase GyrB, impairing its activity and blocking DNA replication even in fluoroquinolone-resistant P. aeruginosa isolates [101].

Another study describes the role of PP-007, a polyethylene-glycol-modified (PEGylated) bovine hemoglobin-based CO carrier, in priming monocytes/cystic fibrosis-affected macrophages to express high levels of heme oxygenase-1 to stimulate the resolution of neutrophilic pulmonary inflammation without compromising the clearance of P. aeruginosa [102].

Considering that antibiotic resistance of pathogens can lead to high mortality of the host, two main strategies have been envisioned to combat multiresistant bacteria: the restoration of efficiency of antibiotics that have been rendered ineffective due to the increasing rate of antibiotic resistance and the modification of existing antimicrobial agents to design new compounds with superior efficacy. For example, Hochvaldová et al. improved and restored the antibacterial activity of antibiotics (gentamicin, ceftazidime, ciprofloxacin, and colistin) by applying them together with a synthesized cyanographene/Ag nanohybrid [103]. They reported the antibacterial efficiency of this combined treatment against P. aeruginosa strains and determined that both antibiotic's modes of action and mechanisms of bacterial resistance strongly impacted the combined treatment’s efficacy. When the FDA-approved antispasmodic drug, Otilonium bromide (IUPAC: N,Ndiethyl-N-methyl-2-[(4-benzoyl)oxy]ethanaminium) was simultaneously applied with the last-line antibiotic colistin on P. aeruginosa strains, it restored the antimicrobial effects of colistin. The two compounds act synergistically to permeabilize bacterial cell membranes, dissipate proton motive force, inactivate efflux pumps, and induce membrane damages, cytosol leakage and cell death [104]. On their side, in order to improve efficiency of polymyxin B (PMB) and colistin and decrease their toxicity, Roberts et al. modified multiple non-conserved positions within the polymyxin scaffold to design the synthetic lipopeptide F365 (QPX9003) displaying a wider therapeutic window, reduced nephrotoxicity and toxicity, improved pharmacokinetics properties, and efficacy against lung infections caused by top-priority multi-drug resistant pathogens including P. aeruginosa [105]. Peptides are becoming increasingly important for many therapeutic areas [106], due to their capacity to permeate tissues and membranes and the low rate of resistance emergence towards them. The de novo-engineered cationic peptide antibiotic E35, for example, irreversibly damages cell membranes and kills the extensively-resistant isolate PA239 [107].

Polytherapy with an antibiotic and a lyotropic liquid-crystalline lipid-based nanoparticle carrier has been successfully applied to kill P. aeruginosa and the mechanism of action of the two members of this therapy has been described. Indeed, combined treatment of PMB and cubosomes intensifies P. aeruginosa bacterial killing as PMB initiates the disorganization of the outer membrane of the target bacteria, and thereafter, an influx of cubosomes further accelerates membrane disruption through a lipid exchange process [108].

Many potential antimicrobial agents that were developed in vitro did not reach clinical application, since their in vivo activity was strongly diminished compared to their in vitro efficacy. To understand the reduced efficacy of antibiotics against P. aeruginosa in vivo, a study focused on the impaired diffusion of the antibiotic colistin across an artificial sputum matrix/medium and quantified its antimicrobial activity against P. aeruginosa NH57388A biofilms. Stokniene et al. revealed that the binding of colistin to mucin-rich AS medium substantially diminished its diffusion rate and reduced its effectiveness. On the other hand, the addition of the low molecular weight alginate oligosaccharide OligoG CF-5/20, derived from the stem of brown algae Laminaria hyperborean, enhanced colistin diffusion in mucin-rich AS medium, enhanced mucus penetration by colistin, and significantly intensified colistin antimicrobial activity against mucoid P. aeruginosa biofilms [109]. Of note, colistin is known to bind to mucin in CF sputum or on the airway epithelium [110], and its bactericidal activity is mediated by the electrostatic interplay between its cationic amino groups and LPS anionic phosphate groups on the outer membrane of the targeted Gram-negative bacteria. O’Brien et al. further highlighted that the growth in a polymicrobial environment shields the target microorganism from the effect(s) of antimicrobial agents [111]. The same authors identified a single nucleotide polymorphism as well as indels in genes encoding LPS biosynthesis and/or pilus biogenesis in colistin-resistant P. aeruginosa isolates and also reported that loss-of-function mutations (e.g. frameshifts and nonsense mutations) in the genes implicated in LPS biosynthesis (eg. wzy gene) contributed to the resistance mechanism towards colistin.

While the search for efficient antibiotics continues, some works focused on compounds that do not induce antimicrobial resistance selection and are deprived of significant cytotoxicity on mammalian cells. For example, vitamin C administration is found to display remarkable antibacterial and anti-biofilm features against P. aeruginosa [112]. Furthermore, the combination of epsilon-poly-L-lysine and antibiotics ampicillin, gentamicin, tetracycline, or methicillin, inhibit formation of P. aeruginosa biofilm and improve preformed biofilm disruption in vitro with no significant cytotoxicity in fibroblasts [113]. On the other hand, microwave plasma-activated water was shown to exert bactericidal activities against P. aeruginosa with no harmful effects on normal skin cells [114]. This could therefore be used as an efficient and safe skin disinfectant. Antimicrobial hybrid peptide Lf-KR exhibits increased permeabilization and depolarization of microbial membranes, the ability to substantially impair expression and production of pro-inflammatory cytokines (nitric oxide and tumor necrosis factor‐α) in LPS-stimulated mouse macrophages and a powerful suppressing effect on preformed multidrug-resistant P. aeruginosa biofilms [115].

A method increasing the sensitivity to various antibiotics and that can be used as an alternative to conventional antibiotics has also been investigated. Indeed, a sensitizing approach employing cell-penetrating peptides conjugated with peptide nucleic acid and targeting bamB (encoding an outer membrane lipoprotein) and oprM, a tolC homolog encoding an outer membrane efflux protein, has been reported to enhance the uptake of vancomycin, erythromycin and carbenicillin by P. aeruginosa [116]. This finding confirms that manipulating outer membrane transport in P. aeruginosa can enhance its susceptibility to antibiotics.

Vaccine: a tool to prevent infections by antibiotic-resistant Pseudomonas aeruginosa

Development of vaccines can be an alternative to prevent infections and thus avoid excessive use of antibiotics leading to antibiotic resistance. Vaccines targeting T3SS translocons (V-antigen PcrV), exoenzymes (ExoS, ExoU), fimbrial components, flagella, core LPS [117], outer membrane vesicles components [118], recombinant lipoprotein I (OprI, [119]), and alginate deriving from a synthase-dependent exopolysaccharide secretion system [120], have received great attention as they can provide protection against infection and disease onset [121,122,123]. For example, a trivalent vaccine based on outer membrane proteins (OprF and OprI) and T3SS translocon protein (PopB) of P. aeruginosa, and formulated with or without Granulocyte–macrophage colony-stimulating factor (GM-CSF) as an adjuvant, is found to stimulate Th1 and Th2 responses, to increase the secretion of immunoglobulin A (IgA), and to induce proper level of IgG (G1, G2a, G2b) against P. aeruginosa in the burned rat models [124].

Strategies focusing on notorious virulence factors that aid in the evasion of the host immune response represented the predominant steps in vaccine development towards P. aeruginosa. In this regard, a report highlighted that LPS and Oligopolysaccharides (OPS) antigens conjugated with Poly Lactic-co-Glycolic Acid (PLGA) nanoparticles have the potential to be employed as nano-vaccines stimulating cellular and humoral immune systems against P. aeruginosa infections. Indeed, LPS-PLGA and OPS-PLGA conjugates triggered immunoglobulin M (IgM), IgA, immunoglobulin G (IgG), IgG1, IgG2b, IgG2a and IgG3 antibodies production and facilitated an effective immunity against P. aeruginosa in a conjugate dependent manner. In fact, mice vaccinated with LPS-PLGA conjugates produced higher levels of anti-LPS-PLGA antibodies and were more efficiently protected towards P. aeruginosa infections than mice vaccinated with OPS-PLGA conjugates [125]. By loading cytosolic antigens derived from bacterial lysates of P. aeruginosa PAO1 strain onto mesoporous silica nanospheres decorated with membrane antigens derived from double-layered membrane vesicles of the same bacterial strain (PAO1), an efficient nano-vaccine has been created. This nano-vaccine was reported to trigger humoral as well as cellular immune responses that significantly prevented infections in mice by the drug-resistant P. aeruginosa PAO1 and PA-XN-1 strains [126]. As alginate of P. aeruginosa mediates pathogenesis in host cells, one main target of efforts is to design therapeutic vaccines. Immunity against P. aeruginosa in mice has also been induced by using mannuronic acid tetrasaccharide, as antigen epitope for vaccine development [127].

Furthermore, hybrid proteins composed of the full-length V-antigen (PcrV) and C-terminal domain exoenzyme S (ExoS) from P. aeruginosa, coupled with adjuvants alum and monophosphoryl Lipid A, have been used as vaccine candidates to protect mice against urinary tract infections caused by P. aeruginosa strain PAO1. This treatment enhanced the levels of humoral (IgG1, serum anti-protein IgA, mucosal IgG), and IL-17 production in the vaccinated mice [128]. Noteworthy, whole-cell vaccine inactivated by X-ray irradiation, containing nucleic acids and 8-hydroxyguanosine, was reported to trigger a humoral response in dentritic cell (DCs) that prevented infection by P. aeruginosa PAO1 and multidrug-resistant clinical isolate W9 in mice model of pneumonia. In addition, this treatment induced cGAS-STING pathway triggering (innate immune response), modulation of Toll-like receptors, apoptosis, pyroptosis, CD8⁺ T-cell proliferation, Th1 and Th2 cytokine responses, and reduced levels of inflammatory factors (IL-6, TNF-α and IL-8) in DC [129]. Another report confirmed that vaccination of mice with P. aeruginosa’s outer membrane vesicles (PA-OMVs) conjugated with the diphtheria toxoid (DT) formulated with alum adjuvant, namely PA-OMVs-DT + adj conjugated, resulted in a lower bacterial load, drastic decrease of inflammatory cell infiltration with less tissue damage and conferred an efficient protection against P. aeruginosa in the mice burn model [130]. Of notes, OMVs are particularly attractive as a vaccine platform due to their non-replicating nature, ability to accumulate in lymph nodes, their natural and innate composition of pathogen-associated molecular patterns, and ability to be produced in large quantities by bacterial fermentation [131, 132].

Additionally, Rahbar et al. designed a triple-target antigen that could stimulate simultaneous protective and neutralizing antibodies against COVID-19 responsible virus, respiratory syndrome coronavirus 2 (SARS-CoV-2), and associated bacterial pathogens causing nosocomial infections, such as A. baumannii and P. aeruginosa [133]. The designed antigen was made by combining epitopes originating from A. baumannii outer membrane protein A (AbOmpA), OprF (P. aeruginosa outer membrane protein F), and foreign multi-epitopes (SARS-CoV-2 Spike glycoprotein).

Vaccines designed to antagonize the virulence of drug-resistant P. aeruginosa aimed at preventing chronic infection and pathogenesis exacerbation, and conferring post-exposure immunization. Although the biosecurity of these vaccines has not been fully elucidated for human, the available vaccines represent a promising approach for the prevention of P. aeruginosa infections. For example, purified recombinant fragment of the OprL (reOprL) of P. aeruginosa has been employed as vaccine to elicit a strong pulmonary response of specific effector T cells deriving from naive T cells, namely Th17 cells, leading to serotype-independent protection against acute lung infection of mice by P. aeruginosa [134]. In the meanwhile, the intranasal injection of the primate-based AdC7 vector AdC7OprF.RGD, expressing outer membrane protein F (OprF) of P. aeruginosa (AdC7OprF) and exhibiting an integrin-binding arginine–glycine–aspartic acid (RGD) sequence, immunized a mouse model of cystic fibrosis against established P. aeruginosa respiratory infection and eliminated P. aeruginosa from the lungs [135]. A trivalent combination DNA vaccine based on oprL, oprF and flgE genes of P. aeruginosa has also been unveiled to trigger a robust humoral immune response (higher levels of IFN-γ, IL-2, and IL-4), lymphocyte proliferation and protective efficacy in immunised chickens [136]. For reaching a protective efficacy in vaccinated chickens, divalent combination DNA vaccine (pOPRL and pOPRF) had to be employed at an optimal immunization dose (100 and 200 µg doses) to control infection driven by P. aeruginosa [137]. This finding illustrates vaccines limitations in achieving protective immunogenicity.

Vaccines can induce a protective response against a given pathogen via either active or passive immunization. It has been shown that vaccination prevents infections by other pathogens via the release of cross-reactive antibodies that bind closely-related antigens in other organisms. For example, mice vaccinated with recombinantly produced Bordetella pertussis OmpA protein are protected against P. aeruginosa PAO1 pneumonia and sepsis [138]. The same report demonstrated that B. pertussis whole cell vaccine (Bp-WCV) mitigated P. aeruginosa PAO1 bacterial burden in the airways of mice, triggered anti-P. aeruginosa IgG production, and antibodies generated against B. pertussis also recognized clinical P. aeruginosa strains [138]. This finding also suggests that whole cell vaccine targeting a given pathogen can train the immune system to develop immunological response upon exposure to a different pathogen.

The development of preventive therapies, including the design of novel vaccines, has been reported to require specific carbohydrates [139]. A recently developed P. aeruginosa glycoconjugate vaccine, containing bacterial core lipopolysaccharide tetrasaccharide Hep2Kdo2 attached via a chain linker to a diphtheria toxin mutant carrier protein, binds the cell-surface sugars of Pseudomonas aeruginosa and facilitates bacterial killing [117]. Glycoconjugate vaccines are highly promising for the clearance of many important human pathogens due to their ability to trigger both T-cell-dependent and T-cell-independent immune responses [140]. It is worth noting that carrier proteins utilization can be hindered by some limitations: (i) a restricted number of existing carrier proteins is applicable in licensed conjugate vaccines (e.g., DT, CRM197, Outer Membrane Protein Complex), and (ii) repeated exposure to the same carrier may provoke diverse immune interferences including carrier-specific enhancement of T cell help, carrier-induced epitope suppression and bystander interference [141, 142]. Fortunately, alternative carrier proteins candidates including recombinant non-toxic form of P. aeruginosa exotoxin A (rEPA) and recombinant proteins containing strings of universal CD4⁺ T-cell epitopes have been developed and can overcome those limitations [143].

Protein engineering of the natural polyhydroxyalkanoate (PHA) production system of P. aeruginosa PAO1 has been proven extremely relevant to display selected antigens that can be employed as vaccines candidates against P. aeruginosa infections. Indeed, the deletion of key genes coding for the synthesis of PHA inclusions, alginate, and pel polysaccharide, allowed to improve production of PHA beads coated with surface epitopes of vaccine candidates outer membrane proteins AlgE, OprF, and OprI. These PHA beads coated with OprI/F-AlgE fusion antigen elicited both Th1 type immune response illustrated by the production of IFN-γ and IgG2c isotype antibodies and opsonophagocytic killing mediated by sera antibodies in the vaccinated mice [144].

Monoclonal antibodies neutralizing the activity of P. aeruginosa strains

Aside from vaccines and antibiotics, monoclonal antibodies (mAbs) have been deployed to control the spread of P. aeruginosa and lower infection severity. mAbs are viewed as a treatment option for high-risk individuals for whom vaccination is not an option and passive administration of mAbs could have a major effect on P. aeruginosa pathogenesis by conferring immediate protection, thus complementing the effect of prophylactic vaccines. For example, to overcome canonical antimicrobial resistance of biofilm-resident bacteria, monoclonal antibodies that can release P. aeruginosa and its common co-pathogens from the protective biofilm for subsequent killing by antibiotics have been developed [145]. Indeed, monoclonal antibodies directed against DNABII protein epitopes or targeting type IV pilus from the respiratory tract pathogen Haemophilus influenzae significantly impaired biofilms of P. aeruginosa and related respiratory tract pathogens (Burkholderia cenocepacia, Staphylococcus aureus, Streptococcus pneumoniae or Moraxella catarrhalis [145]). Among the mAbs candidates that have been examined, chicken egg yolk immunoglobulins IgY antibodies, have drawn a special interest in passive immunization due to a wide range of features encompassing the absence of immunological cross-reactivity with mammalian IgG and the complement system, high levels of antigen-specific production yield without disease resistance, and ability to facilitate immunization methods without stress in human [146, 147]. More specifically, IgY raised against the T3SS translocating protein, recombinant PcrV from P. aeruginosa PAO1 strain, allowed to generate Anti-PcrV IgY for immunization of hen. This Anti-PcrV IgY augmented opsonophagocytic killing and repressed bacterial invasion in P. aeruginosa murine acute pneumonia and burn wound models [148]. A synergistic action between anti-P. aeruginosa IgY and beta-lactams (ceftazidime, imipenem, and meropenem) has been recently unveiled and raises the possibility to combine antibodies and antibiotics for treatment of infections by multi-drug resistant P. aeruginosa [149].

The mAbs can be coupled with antibiotics to achieve superior therapeutic efficacy for severe P. aeruginosa pneumonia. In this regard, the DNA-delivered monoclonal antibodies (DMAbs) produced in vivo by skeletal muscles and containing potent human IgG clones as well as non-natural bispecific IgG1 candidates targeting P. aeruginosa strain 6077 have been proven to protect mice against lethal pneumonia caused by aggressive P. aeruginosa strains [150]. DMAbs reduced bacterial colonization of organs (spleen, kidneys), prevented pulmonary oedema, acted synergistically with a commonly used carbapenem family antibiotic (meropenem), was temperature stable and is proposed to be suitable for treating high-risk patients with chronic diseases, and pathogens that are refractory to many broad-spectrum antibiotic regimens [150].

Although therapeutic monoclonal antibodies are reported as promising methods to restrict P. aeruginosa pathogenesis, they can display some limitations. For example, the bivalent, bispecific human immunoglobulin G1 kappa monoclonal antibody MEDI3902 (gremubamab) failed to mitigate P. aeruginosa nosocomial pneumonia incidence in P. aeruginosa-colonised mechanically ventilated subjects [151]. Moreover, passive immunization by monoclonal antibodies such as IgY raised against chimeric protein pilQ-pilA-DSL region in P. aeruginosa also failed to protect rabbits against sepsis [152]. Immunogenicity and protective efficacy of IgY antibodies can also be dose-dependent and non-type specific. In fact, IgY antibodies raised against recombinant type A flagellins of P. aeruginosa did not protect mice in burn wound model, but conferred full protection against P. aeruginosa PAK and PAO1 in acute pneumonia challenge [153]. Therefore, passive immunization by polyclonal antibodies or direct administration of higher dose of mAbs could be envisioned as a safer alternative against bacterial infections.

Conclusions

P. aeruginosa deploys a range of virulence-associated and adaptive mechanisms to subvert the host system during infection. New methods for developing treatments that can offer substantial benefits for patients with serious, unmet medical needs have been covered in this review. Efficient strategies to improve existing antimicrobial agents as well as the discovery of novel molecules with less toxicity have been reported. More viable approaches that may selectively kill bacteria upon contact yet remain nontoxic to mammalian cells or treatment focusing on how the tissue responds to biofilm proliferation, and not merely how effective the treatment is in eradicating the virulence-associated factors merits critical investigation. Natural compounds are of particular interest, since they are a promising source of antimicrobial agents that may allow to improve the clinical management of P. aeruginosa infections. Efforts have now to be made in order to bring the recently developed antimicrobial strategies to clinical application, in order to circumvent infections caused by MDR P. aeruginosa.