The Building Blocks of Antimicrobial Resistance in Pseudomonas aeruginosa: Implications for Current Resistance-Breaking Therapies PDF

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REVIEW published: 16 April 2021 doi: 10.3389/fcimb.2021.665759 The Building Blocks of Antimicrobial Resistance in Pseudomonas aeruginosa: Implications for Current Resistance-Breaking Therapies R. Frèdi Langendonk *, Daniel R. Neill and Joanne L. Fothergill Institute of Infection, Veterinary and Ecological Science, University of Liverpool, Liverpool, United Kingdom P. aeruginosa is classified as a priority one pathogen by the World Health Organisation, Edited by: and new drugs are urgently needed, due to the emergence of multidrug-resistant (MDR) Melanie Ghoul, strains. Antimicrobial-resistant nosocomial pathogens such as P. aeruginosa pose University of Oxford, United Kingdom unwavering and increasing threats. Antimicrobial stewardship has been a challenge Reviewed by: Oana Ciofu, during the COVID-19 pandemic, with a majority of those hospitalized with SARS-CoV2 University of Copenhagen, Denmark infection given antibiotics as a safeguard against secondary bacterial infection. This Cramer Nina, increased usage, along with increased handling of sanitizers and disinfectants globally, Hannover Medical School, Germany Cristina Cigana, may further accelerate the development and spread of cross-resistance to antibiotics. In IRCCS San Raffaele Scientific addition, P. aeruginosa is the primary causative agent of morbidity and mortality in people Institute, Italy with the life-shortening genetic disease cystic fibrosis (CF). Prolonged periods of selective *Correspondence: R. Frèdi Langendonk pressure, associated with extended antibiotic treatment and the actions of host immune [email protected] effectors, results in widespread adaptive and acquired resistance in P. aeruginosa found colonizing the lungs of people with CF. This review discusses the arsenal of resistance Specialty section: This article was submitted to mechanisms utilized by P. aeruginosa, how these operate under high-stress environments Molecular Bacterial Pathogenesis, such as the CF lung and how their interconnectedness can result in resistance to multiple a section of the journal antibiotic classes. Intrinsic, adaptive and acquired resistance mechanisms will be Frontiers in Cellular and Infection Microbiology described, with a focus on how each layer of resistance can serve as a building block, Received: 08 February 2021 contributing to multi-tiered resistance to antimicrobial activity. Recent progress in the Accepted: 29 March 2021 development of anti-resistance adjuvant therapies, targeting one or more of these building Published: 16 April 2021 blocks, should lead to novel strategies for combatting multidrug resistant P. aeruginosa. Citation: Langendonk RF, Neill DR Anti-resistance adjuvant therapy holds great promise, not least because resistance and Fothergill JL (2021) against such therapeutics is predicted to be rare. The non-bactericidal nature of anti- The Building Blocks resistance adjuvants reduce the selective pressures that drive resistance. Anti-resistance of Antimicrobial Resistance in Pseudomonas aeruginosa: adjuvant therapy may also be advantageous in facilitating efficacious use of traditional Implications for Current antimicrobials, through enhanced penetration of the antibiotic into the bacterial cell. Resistance-Breaking Therapies. Front. Cell. Infect. Microbiol. 11:665759. Promising anti-resistance adjuvant therapeutics and targets will be described, and key doi: 10.3389/fcimb.2021.665759 remaining challenges highlighted. As antimicrobial stewardship becomes more Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 1 April 2021 | Volume 11 | Article 665759 Langendonk et al. Resistance-Breaking Therapies for P. aeruginosa challenging in an era of emerging and re-emerging infectious diseases and global conflict, innovation in antibiotic adjuvant therapy can play an important role in extending the shelf- life of our existing antimicrobial therapeutic agents. Keywords: multidrug-resistance (MDR), resistance mechanism interplay, antimicrobial resistance (AMR), resistance-breaking therapy, adjuvant therapies, Pseudomonas aeruginosa P. AERUGINOSA THE PATHOGEN induced through external stimuli, such as stress factors and the presence of certain antibiotics. This is different from acquired P. aeruginosa is a ubiquitous environmental bacterium capable of mutational resistance as adaptive resistance is transient and establishing opportunistic infections in both plants and animals, unstable. Adaptive resistance mechanisms are not permanent, including humans (Mathee et al., 2008). It is the primary cause of unlike mutational events, and become inactive upon removal of Gram-negative nosocomial infections and of lung infections in the stress factor (Moradali et al., 2017). Adaptive resistance often people with cystic fibrosis (CF) (Pendleton et al., 2013) (WHO involves regulatory pathways and leads to altered gene publishes list of bacteria for which new antibiotics are urgently expression, changes in protein production or target alteration. needed), chronic obstructive pulmonary disease (COPD) and For example, two-component signaling systems (TCSS), MexXY non-CF bronchiectasis (Streeter and Katouli, 2016). P. induction and biofilm formation (Hocquet et al., 2003; aeruginosa has a large genome of 5.5-7 million base pairs, with Ferná ndez et al., 2010; Sun et al., 2018a; Coleman et al., 2020). remarkable plasticity (Abril et al., 2019; Stover et al., 2000). Its In this review, we will discuss the different resistance ability to adapt to a range of environmental niches and its high mechanisms P. aeruginosa employs to evade antibiotic action. nutritional versatility stems from this genome plasticity. In Intrinsic resistance mechanisms, such as low-outer membrane addition, a large variety of intrinsic and acquired resistance permeability, efflux, lipopolysaccharide (LPS) modification and mechanisms exist within the P. aeruginosa population (El the bacterial enzyme AmpC, will be described, with a focus on Zowalaty et al., 2015). Understanding these mechanisms of how these contribute to high-level resistance via interplay with resistance and their interplay can help develop targeted acquired mutations and adaptive mechanisms. In addition, we therapies for combatting MDR P. aeruginosa infections. will outline the roles of bacterial enzymes and commonly acquired resistance mechanisms, as well as phenotypic contributions to resistance, such as motility and biofilm formation. Novel therapeutic efforts that directly target these RESISTANCE IN P. AERUGINOSA resistance mechanisms will be reviewed and future priorities discussed. Antibiotic resistance mechanisms can be broadly divided into three categories; intrinsic, acquired and adaptive. Intrinsic resistance mechanisms are those genetically encoded in the core genome of the organism, whereas adaptive resistance SURFACE PROTEINS AND mechanisms are those induced by environmental stimuli, and SYSTEMS: PORINS acquired resistance arises from gain of resistance genes from other organisms or as a consequence of selection of advantageous Antibiotics must cross the bacterial membrane in order to act on mutations (Tenover, 2006). intracellular targets (Figure 1) (Tipper, 1985; Tenson et al., 2003; Intrinsic resistance mechanisms of P. aeruginosa include its Jacoby, 2005; Shakil et al., 2008). Accumulation of antibiotics in low outer membrane permeability (12- to 100- fold lower than the cell is intrinsically diminished in P. aeruginosa due to its low that of Escherichia coli), the presence of antibiotic efflux pumps outer membrane permeability (Lambert, 2002). The relative and b-lactamases, such as OXA-50 and AmpC (Bellido et al., dearth of porins within the outer membrane (OM) of P. 1992; Slama, 2008; Lister et al., 2009; Szczepanowski et al., 2009). aeruginosa decreases the rate at which antibiotics can penetrate Acquired resistance mechanisms from horizontal gene transfer the cell (Trias et al., 1989). Porins not only function in include acquisition of transferable aminoglycoside modifying transporting nutrients and other molecules across the enzymes and b-lactamases, while acquired resistance as a result membrane, but they also play a role in signaling, adhesion and of de novo mutational events often takes the form of stability of the membrane (Achouak et al., 2006). P. aeruginosa overexpression of efflux pumps and b-lactamases, along with has 26 different types of porin, of which OprF is the most decreased expression or modification of target sites and porins abundant in P. aeruginosa and has been implicated in a variety (Meletis and Bagkeri, 2013). Acquired resistance through of functions (Chevalier et al., 2017). adaptive mutations is common in CF isolates due to the prolonged periods of selective pressure during extended OprF – A Major Porin With a Key Role in antibiotic treatment of chronic infection and the actions of Lung Infections host immune effectors (Oliver et al., 2000; Dettman et al., OprF is present in two forms, the highly abundant two-domain 2016). Adaptive resistance mechanisms are those that are closed channel and the single domain open channel. The open Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 2 April 2021 | Volume 11 | Article 665759 Langendonk et al. Resistance-Breaking Therapies for P. aeruginosa FIGURE 1 | Antibiotic uptake in P. aeruginosa. Aminoglycosides diffuse through the outer membrane due to electrostatic interactions between the positively charged aminoglycoside and the negatively charged LPS. They undergo rapid energy-dependent accumulation into the cell with the use of electron transport and ATP hydrolysis. Once inside the cell, aminoglycosides bind to the 30S subunit of ribosomes to inhibit protein synthesis. b-lactam antibiotics target trans-peptidases on the cytoplasmic membrane that play a vital role in the assembly and cross-linking of cell wall peptidoglycan. Macrolides diffuse across the lipid bilayer due to their hydrophobic nature. They bind to the 50S ribosomal subunit and cause the dissociation of peptidyl-tRNA from the ribosome inside the cell. Quinolones inhibit DNA gyrase and DNA topoisomerase IV in Gram-negative and Gram-positive bacteria, respectively, leading to double stranded DNA breaks. Protein and drug structures were generated with Protein Imager (Tomasello et al., 2020). channel conformation occurs in 420kb). Biofilms Megaplasmids contain dynamic accessory genomes where The biofilm mode of growth is a major impediment in the frequent recombination and duplication events take place, struggle to eradicate P. aeruginosa infection from the CF lung, leading to diverse and adaptive multidrug resistance traits due to the increased ability of bacteria in biofilms to withstand (Cazares et al., 2020). Accordingly, resistance to antimicrobial antibiotic treatment (Høiby et al., 2010). Biofilms are composed agents in P. aeruginosa clinical isolates is highly complex, with of bacteria surrounded by extracellular polymeric substances like frequent interplay between intrinsic, adaptive and acquired exopolysaccharides, extracellular DNA and polypeptides resistance mechanisms. AmpC, low outer membrane (Rasamiravaka et al., 2015). This can lead to an increase in permeability and efflux systems often work together in tolerance to antimicrobial agents of 100-1000 times, compared to resistance to carbapenems, chloramphenicol, planktonic cells (Ceri et al., 1999). Contributing features include fluoroquinolones, macrolides, penicillins, tetracyclines and b- quorum sensing, decreased ability to penetrate biofilm, presence lactams and resistance may be enhanced through the of oxygen gradients, altered metabolism and slow bacterial accumulation of mutations leading to up- or down- regulation growth rate (Olsen, 2015). Biofilms are common in chronic P. of each of these systems. The outer membrane porin OprH aeruginosa infection of the CF lung but have also been shown to works in conjunction with the two-component signaling be of significance in patients with COPD, bronchiectasis and systems PhoPQ and PmrAB in modifying the bacterial LPS to chronic wounds (Costerton, 2001; Parameswaran and Sethi, regulate protection to polymyxin antibiotics. Protection against 2012; Chalmers and Hill, 2013; Hassett et al., 2014; Rybtke polymyxin antibiotics is additionally mediated via TCSS ParRS et al., 2015). Airway mucins are found in abundance in people and/or CprRS in the presence of cationic antimicrobial peptides. with CF, COPD and bronchiectasis. In vitro studies have shown Aminoglycoside resistance is achieved through mechanisms that the presence of airway mucins are fundamental for the such as aminoglycoside modifying enzymes, fusA1 mutation, development of biofilm structures with enhanced tolerance to 16S rRNA methylation, along with MexXY-OprM upregulation. antimicrobials. Further, mucin may serve as a suitable Likewise, gyrA and parC mutation leading to altered DNA attachment surface for P. aeruginosa biofilm formation gyrase and topoisomerase IV leads to fluoroquinolone (Landry et al., 2006; Müller et al., 2018). resistance which can be heightened through the cooperation Iglesias et al. investigated antibiotic pharmacodynamics of MexAB-OprM and/or MexCD-OprJ, as well as MexEF- within biofilm structures in the context of CF. Bacterial counts, OprN. In addition, the presence of b-lactamases such as metabolic activity and biomass of PAO1 biofilms grown in ESBLs and MBLs may further enhance carbapenem, artificial sputum media (ASM) or trypticase soy-based medium cephalosporin and penicillin resistance. A perfect storm of were compared. They found that bacteria in ASM reached the highly resistant P. aeruginosa and a “dry pipeline” of same CFU and metabolic activity as biofilms formed in trypticase traditional antimicrobials are driving innovation in novel soy-based medium, although ASM biofilms grew slower and had therapeutic approaches that directly target resistance a marginally higher biomass. When both biofilms were subjected mechanisms. Counteracting these mechanisms could prolong to antibiotic treatment, ASM grown biofilms were substantially the life of existing antimicrobials. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 9 April 2021 | Volume 11 | Article 665759 Langendonk et al. Resistance-Breaking Therapies for P. aeruginosa FIGURE 3 | Multi-layered, interacting resistance mechanisms in P. aeruginosa. Innate (intrinsic) resistance mechanisms are encoded in the core genome of the organism, such as low outer membrane permeability, Mex-type efflux pumps and AmpC b-lactamase. Collectively, these comprise basal level resistance to antimicrobials, a foundation on which a variety of adaptive and acquired mechanisms of resistance may serve as building blocks to further enhance AMR in P. aeruginosa. Adaptive resistance mechanisms, including two-component regulatory systems, are environmentally dependent and will be expressed under certain conditions only. Mechanisms of resistance that are acquired, such as antibiotic modifying enzymes or mutations leading to antibiotic target modifications are strain dependent. The building blocks of innate, adaptive and acquired mechanisms of resistance contribute to a strong and multi-faceted protection against antimicrobial activity. Hexagon building blocks of resistance mechanisms are colored according to mechanism type; direct action on antibiotic, permeability, global regulation, modification of antibiotic target. Upper labels on hexagon building blocks describe resistance mechanism whilst lower labels define examples of each such systems. Protein and drug structures were generated with Protein Imager (Tomasello et al., 2020). ANTI-RESISTANCE THERAPIES FOR becomes a more powerful anti-pseudomonal (Tümmler, 2019). P. AERUGINOSA Polymyxin B nonapeptide is highly toxic and has therefore never been considered for clinical application (Tsubery et al., 2000). Outer Membrane Sensitizers However, more than 30 years after the first report of OM Increasing OM permeability to hydrophobic and amphiphilic sensitizers, three have been approved for clinical studies; the compounds challenges the issue of intrinsic low-outer membrane anti-protozoal pentamidine (Stokes et al., 2017) and the permeability (Tümmler, 2019). For example, polymyxin B polymyxin B analogues SPR206 and SPR741 (Corbett et al., nonapeptide causes a 2- to 40-fold increase in susceptibility to 2017; Vaara, 2019). None of these OM sensitizers, with the ciprofloxacin, norfloxacin, and ofloxacin and 80- to 200-fold exception of SPR206, have been proven to have promising anti- increase in susceptibility to nalidixic acid (Kubesch et al., 1987). pseudomonal activity. SPR206 is active against P. aeruginosa Nalidixic acid is the precursor to ciprofloxacin, norfloxacin, and with a similar potency to polymyxin B and is currently ofloxacin, and is not ordinarily more efficacious than the undergoing phase-one clinical trial (Zhang et al., 2020). OM optimized antibiotics currently used in clinical practice. sensitizers appear to be a promising approach to resistance that However, when the membrane becomes more permeable can by-pass intrinsic, acquired and spontaneous resistance through polymyxin B nonapeptide treatment, nalidixic acid (Macnair and Brown, 2020). However, further investigation Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 10 April 2021 | Volume 11 | Article 665759 Langendonk et al. Resistance-Breaking Therapies for P. aeruginosa into additional non-toxic compounds that act on the P. tetracycline by causing a 5- to 8-fold decrease in the MIC90 aeruginosa OM is essential, especially for the treatment of (Fleeman et al., 2018). In addition, the polyamine derivatives did polymyxin resistant isolates. not disrupt the bacterial membrane, unlike other polyamines, which can lead to the identification of false positives for EPIs (Fleeman Efflux Pump Inhibitors et al., 2018; Reza et al., 2019). Moreover, polyamines did not display Phe-Arg-b-naphthylamide (PAbN) toxicity to mammalian cell lines and did not inhibit calcium channel The most widely researched P. aeruginosa efflux-pump inhibiter activity in human kidney cells (Fleeman et al., 2018). (EPI) is Phe-Arg-b-naphthylamide (PAbN), a broad-spectrum peptidomimetic compound capable of interfering with all four Bacteriophage OMKO1 clinically relevant P. aeruginosa RND efflux pumps. PAbN Phage therapy, the use of bacteriophages to infect and lyse bacterial potentiates chloramphenicol, fluoroquinolones, macrolides, cells, has been widely discussed (Chan et al., 2013; Gordillo ketolides, oxazolidinones and rifampicin but not Altamirano and Barr, 2019). Traditional phage therapy involves aminoglycosides or b-lactams (Lomovskaya and Bostian, the administration of one, or a mixture, of phages that will invade 2006). The proposed mechanism of action for PAbN is that it the bacterial cell and clear infection (Waters et al., 2017). A different functions as a substrate of the Mex-series efflux pumps and approach to phage therapy has been proposed, whereby phages outcompetes the antibiotic for extrusion, preventing the would be used to steer antibiotic resistance evolution, selecting for antibiotic from leaving the cell (Mahmood et al., 2016). phage resistance and antibiotic susceptibility. For example, the lytic However, PAbN and derivatives of this compounds are not yet Myoviridae bacteriophage, OMKO1, utilizes OprM of the multidrug approved, as adverse toxicology and pharmacokinetic profiles efflux systems MexAB and MexXY as a receptor-binding site. were identified during phase 1 clinical trials (Renau et al., 2003). Selection for resistance to OMKO1 bacteriophage attack creates Instead, PAbN is solely utilized for research on EPI’s and AMR an evolutionary trade-off in MDR P. aeruginosa, by changing the in vitro, rather than as a therapeutic, and may be used to validate efflux pump mechanism, leading to an increased sensitivity to the discovery of future EPI’s. ciprofloxacin, tetracycline, ceftazidime and erythromycin, four drugs from different antibiotic classes (Chan et al., 2016). Phage D13-9001 steering can be achieved when the binding receptor for the The pyridopyrimidine derivative D13-9001 is active against the bacteriophage is implicated in both antibiotic resistance and MexAB-OprM efflux pump (Mahmood et al., 2016). D13-9001 phage resistance. The advantage of this approach lies in the two has shown promising in vitro and in vivo activity, as well as high distinct, and opposing, mechanisms leading to bacterial eradication solubility and low-toxicity profiles (Yoshida et al., 2007). D13- (Gurney et al., 2020). 9001 obstructs normal functioning of MexAB-OprM in two ways. Firstly, it prevents conformational changes by binding tightly to the hydrophobic trap. Secondly, it prevents substrate binding to MexB, through the interaction of the D13-9001 BACTERIAL ENZYME INHIBITORS hydrophilic component and the substrate binding channel of b-lactamase Inhibitors MexB (Nakashima et al., 2013). EPIs will need to be broad- The prototypical example of successful anti-resistance therapeutics spectrum if they are to be used as an adjuvant to antibiotics that are the b-lactamase inhibitors. b-lactamase inhibitors such as are substrates of several efflux-pumps. The specificity of D13- clavulanic acid, sulbactam and tazobactam are widely used to 9001 would limit its usage to co-administration with antibiotics combat resistance mediated by b-lactamases (Tooke et al., 2019). extruded exclusively by MexAB-OprM (Nakashima et al., 2013). However, the majority of clinically used b-lactamase inhibitors have However, a study by Ranjitkar et al. found that there are several a limited spectrum and mainly target Ambler class A b-lactamases, mechanisms of resistance to D13-9001 potentiator activity in P. excluding KPC-type b-lactamase. Progress has been made in the aeruginosa, when the agent is used together with carbenicillin, an development of novel b-lactamase inhibitors with a wider spectrum antibiotic that is substrate specific to MexAB-OprM. Loss of of activity. Three novel b-lactamase inhibitors, avibactam, potentiating activity of D13-9001 occurred rapidly due to a vaborbactam and relebactam, function against Ambler class A, C F628L substitution in mexB, which is known to play an and D b-lactamases (Wong and Duin, 2017). However, only important role in inhibitor binding (Ranjitkar et al., 2019). avibactam and relebactam are efficacious against P. aeruginosa infection (Aktaş et al., 2012; Barnes et al., 2018). Polyamine Derivatives Polyamines are aliphatic carbon chains containing several amino groups and are essential organic polycations present in every form of life. Polyamines are implicated in cell maintenance and DNA GYRASE AND TOPOISOMERASE IV viability and in the functioning of a wide array of organ systems, TARGETING THERAPIES including, the nervous and immune systems (Sá nchez-Jimé nez et al., 2019). Siderophore Mimic Bound DNA Fleeman et al. identified a polyamine scaffold as a strong efflux Gyrase B Inhibitors pump inhibitor with no direct antimicrobial activity. Five lead Lamut et al. designed 4,5,6,7-tetrahydrobenzo[d]thiazole- agents were found to potentiate aztreonam, chloramphenicol and based DNA gyrase B inhibitors and incorporated these Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 11 April 2021 | Volume 11 | Article 665759 Langendonk et al. Resistance-Breaking Therapies for P. aeruginosa inhibitors with siderophore mimics. The siderophore mimic compound 3 to permeabilize the membrane. It may inhibit the served as an inducer for increased uptake of the gyrase B folding activity of the BAM complex, leading to incorrectly inhibitors into the bacterial cytoplasm. Out of the ten gyrase B folded proteins being misplaced in the inner membrane. inhibitors tested against P. aeruginosa, four were able to inhibit Alternatively, BamA may only serve as an extra binding site ≥50% but only under iron-supplemented conditions, which is for compound 3, thereby evading the LPS-modification not reflective of the host environment during infection (Lamut resistance mechanism of Gram-negative pathogens (Luther et al., 2020). Several more attempts have been made at et al., 2019). designing broad-spectrum anti-bacterial and anti-biofilm therapies targeting DNA gyrase or topoisomerase but none have shown good activity for P. aeruginosa (Dubey et al., 2012; Masih et al., 2020). QUORUM SENSING, BIOFILM AND MOTILITY ATTENUATION Quorum sensing regulates a wide range of genes involved in LPS MODIFICATION virulence and bacterial adaptation (Kalia, 2013). For instance, QS is required for the surfing and swarming motility phenotypes Murepavadin associated with increased resistance to antimicrobials. The Murepavadin is a novel, non-lytic, species specific, outer- surfing phenotype is regulated via three QS systems in P. membrane protein targeting antibiotic for the treatment of P. aeruginosa; Las, Rhl and Pqs (Sun et al., 2018b). In addition, aeruginosa infections, including those caused by MDR strains QS has been found to influence tolerance to antibiotics in P. (Dale et al., 2018). Murepavadin is derived from the b-hairpin aeruginosa biofilms. QS provides structural rigidity through the host defense molecule protegrin 1 (PG-1) and optimized to regulation of Pel polysaccharides and eDNA release necessary for counteract unfavorable absorption, distribution, metabolism, the extracellular polysaccharide matrix. In addition, the excretion and toxicity (ADMET) properties normally production of rhamnolipids, surfactants important for the associated with PG-1 (Obrecht et al., 2011). It is a establishment and maintenance of biofilms, is controlled under macrocycle compound consisting of PG-1 loop sequences QS (de Kievit, 2009). Therefore, QS has been recognized as a linked to a D-proline-L-proline sequence, the latter of which significant potential target for developing anti-resistance is important for its stability and subsequent strong therapies. Strategies to combat antimicrobial resistance by antibacterial potential (Srinivas et al., 2010). Murepavadin targeting adaptive resistance mechanisms have significant functions through binding to the LPS transport protein D potential for reversing antibiotic resistance in P. aeruginosa. (LptD), an OMP necessary for LPS biogenesis in Gram- Adaptive resistance is often mediated through complex global negative bacteria. The interaction between murepavadin and regulatory systems, such as the QS system, and regulate an LptD causes inhibition of LPS transport, which leads to extensive set of genes involved in resistance. Targeting these alterations of the LPS on the bacterial OM and eventually, regulatory systems may prevent the activation of expression of cell death (Werneburg et al., 2012). these resistance genes that would normally be expressed under Murepavadin derivatives have been screened for activity the environmental conditions of infection. against Gram-negative ESKAPE pathogens, including P. aeruginosa. These derivatives were initially shown to be Ajoene effective, although the majority of leads were found to have Ajoene is a natural sulphur-containing compound extracted high MIC values in the presence of 50% human serum and from garlic (Yoshida et al., 1998) that has been shown to showed signs of lytic activity in human red blood cells. modulate biofilm formation by inhibiting QS-induced Therefore, compounds were generated consisting of b- production of virulence factors (Holm Jakobsen et al., 2012). It hairpin macrocycles linked to the peptide macrocycle of targets the Gac/Rsm component of QS, leading to a decreased polymyxin B. One of these compounds, compound 3, showed expression of rsmZ and rsmY small regulatory RNAs. rsmZ and strong antimicrobial activity (MIC 0.5-2 mg/mL for P. rsmY bind the global regulatory protein RsmA, and unbound aeruginosa isolates), low toxicity to mammalian cells, low RsmA represses the translation of genes by preventing ribosome plasma protein binding, good human plasma stability and no binding to the Shine-Dalgarno site. Several genes involved in QS lytic activity towards human red blood cells. This compound are under RsmA regulation, and low expression of rsmZ and was shown to perturb and permealise the bacterial membrane rsmY in the presence of ajoene promotes RsmA mediated through interacting with the b-barrel domain of BamA in E. repression of these target genes (Jakobsen et al., 2017). coli ATCC 25922 (Luther et al., 2019). BamA is part of the b- However, the therapeutic applicability of ajoene is limited due barrel assembly machinery BAM complex, which serves to fold to availability, instability, hydrophobicity and relatively high and insert outer membrane proteins in the OM (Gu et al., MIC values. Efforts are being undertaken to overcome these 2016). The binding interaction between BamA and compound issues through modification, the use novel delivery systems and a 3 locks BamA in its closed state through changing the targeted route of administration and through the development of conformational composite in the b-barrel lateral gate synthetic ajoene analogues (Fong et al., 2017; Vadekeetil between open and closed states. It is not known what causes et al., 2019). Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 12 April 2021 | Volume 11 | Article 665759 Langendonk et al. Resistance-Breaking Therapies for P. aeruginosa Naringenin tobramycin and ceftazidime led to a 243% and 155% increase in Another novel QS-inhibitor derived from a natural source is the biofilm biomass and a 199% and 174% increase in biofilm plant flavonoid naringenin. Naringenin diminishes the thickness, respectively, when compared to the untreated production of QS-regulated virulence factors in P. aeruginosa biofilm control. There is some evidence for the safety of NO by binding directly to LasR, thereby competing with the activator administration in CF patients in vivo and NO is currently of LasR, N-(3-oxo-dodecanoyl)-l-homoserine lactone (HSL). undergoing clinical trials to measure clinical efficacy (Howlin It is ineffective at outcompeting HSL when the activator is et al., 2017). already bound to LasR. Thus, the QS-inhibitor will only sufficiently interfere with the QS response when administered PAAG (SNSP113) during early exponential growth, when naringenin can compete Poly-acetyl-arginyl-glucosamine (PAAG), also called SNSP113, with unbound HSL for LasR binding. Naringenin is only suitable is a novel inhaled adjuvant therapy currently undergoing phase for combatting P. aeruginosa populations at low cellular one clinical trials. PAAG is a polycationic glycoprotein that densities, which often does not represent the clinical infection functions by permeabilizing the bacterial membrane and is scenario. The full potential of QS-inhibition will only be realized active against methicillin resistant Staphylococcus aureus if an inhibitor is developed that is capable of targeting P. (MRSA), Burkholderia spp., Mycobacterium spp. and E. coli. aeruginosa QS signaling regardless of bacterial density and/or PAAG has been shown to effectively disperse Burkholderia QS status (Hernando-Amado et al., 2020). cepacia complex biofilm structures extracted from the CF lung (Narayanaswamy et al., 2019). In P. aeruginosa, PAAG is has Anti-Pseudomonal Drug Delivery Methods been shown to effectively eradicate persister cells, which is Bacterial biofilms pose a physical barrier for drug penetration, important for the prevention of recurrent P. aeruginosa which is one of the reasons that bacteria in a biofilm mode of infections and subsequent exacerbations in people with CF growth are more resistant to antimicrobials. This phenomenon (Narayanaswamy et al., 2018). In addition to serving as an may be subverted with the use of nanocarriers that encapsulate effective antibiotic adjuvant, PAAG also reduces inflammation antimicrobials and facilitate drug diffusion through the bacterial and promotes viscoelasticity and mucociliary clearance, making biofilm. In addition, nanocarriers can also protect drugs from it a suitable drug candidate to improve the quality of life for degradation, ensure controlled drug release, and cause increased patients with a variety of mucus diseases (Fernandez-Petty uptake by the drug target, leading to an overall higher efficiency et al., 2019). of encapsulated drugs. Drug delivery methods can be diverse in chemical structure and nature (Table 1). Most published studies concur that encapsulated antibiotics are more effective at preventing or eradicating biofilm formation than their free PERSPECTIVES AND drug counterpart (Alhariri et al., 2017; Shaaban et al., 2017; FUTURE DIRECTIONS Zahra et al., 2017; Dai et al., 2018; Li et al., 2019), although several concluded that the activity of encapsulated and free The global overuse and misuse of antibiotics during the last 80 antibiotic was equal (Severino et al., 2017; Wang et al., 2018; years has led to a profound increase in antimicrobial resistance. Hill et al., 2019). Between 2000 and 2010, global antibiotic consumption increased by nearly 70% and antibiotic resistant infections have Nitric Oxide accordingly become more pervasive, according to global Another promising antibiotic adjuvant targeting biofilms is the epidemiological antimicrobial resistance surveillance networks non-bactericidal, inhaled adjuvant, nitric oxide (NO). Exposure (European Centre for Disease Prevention and Control, 2018) of P. aeruginosa biofilms to low-dose NO has been shown to (Christaki et al., 2020). AMR is a complex, One Health issue, cause dispersal of biofilms, rendering the infection susceptible to involving human, animal and environmental factors. The subsequent antibiotic treatment (Cai, 2020). NO functions by solution to AMR is therefore also likely to be a complex one, increasing bacterial phosphodiesterase activity which, in turn, involving multiple strategies; maintaining AMR surveillance, leads to a reduction in the vital secondary signaling messenger, containing AMR transmission, reducing selection pressure, cyclic di-GMP. Cyclic di-GMP is vital for intracellular regulation developing novel antimicrobials or reverting antibiotic resistant of biofilm formation. Howlin et al. carried out in vitro biofilm microbes back to the susceptible phenotype with the use of studies using CF sputum clinical samples. Biofilms were treated antibiotic adjuvants (Hernando-Amado et al., 2019). Although with NO only, tobramycin only, tobramycin and ceftazidime, progress in the development of naturally derived and peptide- NO + tobramycin and NO + tobramycin and ceftazidime. based antimicrobials has been made (Mok et al., 2020; Upert Biofilms treated with NO showed a relative decrease in biofilm et al., 2021). The conservation of existing antibiotics through biomass and surface bound thickness in comparison to the careful stewardship is paramount to help mitigate the gap untreated control. Further, treatment of biofilms with NO + between the demand for new drugs and the diminishing tobramycin and NO + tobramycin and ceftazidime led to supply pipeline. Antibiotic adjuvants will also play an complete eradication of biofilm biomass and surface bound important role in extending the shelf life of our existing thickness. In comparison, biofilms treated with tobramycin or antimicrobial therapeutic agents. Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 13 April 2021 | Volume 11 | Article 665759 Langendonk et al. Resistance-Breaking Therapies for P. aeruginosa TABLE 1 | Recent efforts in the design of drug delivery methods for anti-pseudomonal therapies. Chemical nature Avr. Particle size Cell type Effect (nm) Anionic liposome 100 Planktonic No difference between capsulated and free Wang et al., 2018 abx 100 Biofilm Enhanced biofilm eradication Zahra et al., 2017 625 - 806.6 Planktonic and Prevents biofilm formation Alhariri et al., 2017 biofilm Graphene-oxide conjugates N/A Biofilm Enhanced biofilm eradication Dai et al., 2018 Poly(lactic-co-glycolic) acid nanoparticles 229.4 - 469.2 Planktonic No difference between capsulated and free Hill et al., 2019 abx 132 - 348 Planktonic and Prevents biofilm formation Shaaban et al., biofilm 2017 Solid lipid nanoparticles 200 - 500 Planktonic No difference between capsulated and free Severino et al., abx 2017 Water-soluble chitosan oligosaccharide N/A Biofilm Enhanced biofilm eradication Li et al., 2019 conjugates Adjuvant strategies targeting resistance mechanisms in P. activation of those genes with potential knock-on advantages in aeruginosa could rejuvenate traditional antibiotic therapy by inhibition of virulence mechanisms. QS and two-component potentiating drug activity as well as slowing the development signaling systems are particularly attractive targets from this of antibiotic resistance. As described in this review, antimicrobial perspective, as are the regulators of biofilm formation. resistance in P. aeruginosa is regulated through a complex There are several challenges in developing resistance- interplay of mechanisms. Resistance encoded in the core breaking therapy for P. aeruginosa infection. Firstly, due to its genome of P. aeruginosa, such as low outer membrane comparatively large genome and highly adaptive nature, a permeability, Mex-type efflux pumps and AmpC b-lactamase plethora of regulatory systems, as well as limited drug amount to the basal level of resistance against antimicrobials. penetration and active efflux, many antibiotic adjuvants This intrinsic resistance is present in the all P. aeruginosa strains designed for Gram-negative pathogens do not show efficacy and serves as a foundational level, which can be expanded upon. against P. aeruginosa. Secondly, toxicity has been proven to be This expansion can be induced by environmental influences, the major hurdle for adjuvants designed against P. aeruginosa, such as host factors and signalling molecules, that switch on leading to many being abandoned at early phases of adaptive resistance mechanisms. Acquired resistance development. Drug safety assessment is a long, expensive, but mechanisms, such as antibiotic target modifications generated crucial process and toxicity is most likely where drug targets via mutation, and antibiotic modifying enzymes or resistance share structural similarity with human proteins. In this respect, plasmids, acquired by gene transfer, may serve as additional bacterial signaling systems are good candidates, as prokaryotic building blocks to expand the arsenal of resistance mechanisms a and eukaryotic signaling systems are highly divergent, with particular strain might carry. Several novel therapeutic strategies, eukaryotes lacking TCSS or phosphorelay systems. targeting one or more of these mechanisms, have been described As with all newly developed drugs designed to be used as in this review. In light of recent findings, OM perturbants combination therapy, care must be taken in determining the capable of sensitizing the Gram-negative bacterial membrane correct dosing and investigating clear synergy profiles. Drug to previously non-active antibiotics seem an opportune strategy levels necessary for synergy in vitro may not be achievable in to combat resistance. OM perturbants can by-pass intrinsic as vivo. Synergy in vivo may be affected by failure to obtain desired well as acquired and spontaneous resistance mechanisms, levels of drugs in the target tissue, drug metabolism or plasma making them highly promising drug candidates, for which the protein binding. In addition, it is of paramount importance to development of resistance would be unlikely. However, efforts to evaluate drugs in relevant models that reflect the environmental finding perturbants suitable for targeting the P. aeruginosa conditions of infection. This will increase the predictive power of membrane must be increased. preclinical testing, reducing the costly progression of A second promising strategy is phage steering, which uses the unpromising agents to clinical trials. The lack of well-validated natural predators of bacteria and the forces of evolutionary in vivo models for testing CF anti-infective therapeutics limits pressure to our advantage. Counterbalancing antibiotic the speed of development of new drugs. Murine models using resistance with phage susceptibility creates a double edged cystic fibrosis transmembrane conductance regulator (CFTR) sword to circumvent key AMR mechanisms. In addition to knockout animals, or transgenics in which the severe gut these strategies, adjuvants targeting adaptive resistance phenotype associated with loss of CFTR has been corrected are mechanisms are worthy of consideration, due to the potential available, and have proved useful, but do not develop the to disrupt multiple bacterial resistance and virulence processes characteristic features of acute and chronic P. aeruginosa with agents targeting a single regulator. Targeting global infection seen in those with CF (Bragonzi, 2010; Semaniakou regulatory systems that would normally control the expression et al., 2019). Progress on the use of ferret and porcine infection of resistance genes under infection conditions will prevent the models with mutated CFTR has been made, although these are Frontiers in Cellular and Infection Microbiology | www.frontiersin.org 14 April 2021 | Volume 11 | Article 665759 Langendonk et al. Resistance-Breaking Therapies for P. aeruginosa limited by the availability of suitable immunobiology reagents disinfectants globally may induce the development of cross- (Keiser and Engelhardt, 2011). Intranasal administration of P. resistance to antibiotics. The ESKAPE pathogens, for which new aeruginosa into the healthy murine lung often leads to either medicines are urgently needed, continue to cause serious rapid clearance or sepsis. To create a model of persistent community-acquired and nosocomial infections, and if infection, it is usually necessary to immobilize P. aeruginosa. investment into research and drug development for these This can be achieved by encapsulating the bacteria into agar or bacterial pathogens is diminished, it will exacerbate the global alginate beads, where the bacteria are protected from clearance health and economic costs associated with the ongoing pandemic. by immune effectors and where their mode of living more closely mimics bacterial biofilms present in chronic infection (Cigana et al., 2016). However, this bead model is technically demanding and requires surgical transtracheal instillation of the bead AUTHOR CONTRIBUTIONS suspension, leading to additional complications and mortality not representative of bacterial infections in CF (Van Heeckeren FL wrote the manuscript with supervision and input from all and Schluchter, 2002). Alternatively, long term lung infection others. All authors contributed to the article and approved the can be achieved using P. aeruginosa isolates from CF, some of submitted version. which naturally establish chronic infection in mice, without the need for implantation into beads (Fothergill et al., 2014; Bricio- Moreno et al., 2018). This model has the advantages of using a natural infection route and having no requirement for surgical FUNDING intervention, and offers opportunity to study lung infection over FL is supported by a PhD studentship from the Rosetrees Trust prolonged periods. However, the density of infection achieved in (M750). DN is supported by a Sir Henry Dale Fellowship funded the lung is low, making some analyses challenging. by the Wellcome Trust and the Royal Society (Grant number Questions remain regarding the commitment of governments 204457/Z/16/Z). and pharmaceutical manufacturers to ongoing investment in antibacterial drug development, particularly as financial and research priorities are reshuffled by the ongoing SARS-CoV2 crisis. Despite the understandable current emphasis on anti-viral ACKNOWLEDGMENTS agents and vaccines, it is important that we do not lose ground in the fight against AMR. Indeed, emerging evidence suggests that Figures were designed and illustrated by Dr. Eliza Wolfson, antibiotic use has increased dramatically in the COVID-19 era https://lizawolfson.co.uk using molecular renderings (Hsu, 2020). On top of this, increased usage of sanitizers and by Protein Imager. REFERENCES OprJ in the Pseudomonas aeruginosa quorum sensing response. Front. 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