Despite efficient host immune system, the implant surface can be rapidly occupied by bacteria, resulting in infection persistence, implant failure, and even death of the patients. It is difficult to cope with these problems because bacteria exhibit complex adhesion mechanisms to the implants that vary according to bacterial strains.

Different biomaterial coatings have been produced to release antibiotics to kill bacteria. However, antibiotic resistance occurs very frequently. In each bacterial and fungal culture studied, adding nanomaterials increased protein production more than tenfold over the control condition.

Cell membrane rupture and cellular constituent depletion are two crucial factors influencing cellular malfunction and mortality [ 46 ]. Further, the formation of reactive oxygen species ROS as free radicals, from used nanomaterials can trigger the mitochondrial dysfunction, enzyme syntheisis blocking and rebuture of cell wall that discharge of the cellular fluid [ 47 , 48 ].

The vigrous antibacterial activity was attributed to the constant production of ROS, which caused the breakdown of proteins via disrupted cellular membranes, resulting in cell destruction [ 23 ]. The present research stated that ROS formed could successfully penetrate the cell membranes and wall in which degrade the cell wall and leakage protein outside damaged cells.

The amounts of discharged protein from destructed cells before and after subjecting to explored nanomaterials. The antimicrobial potential of multifunctional nanoparticles is an emerging field of study, with underlying mechanisms still not fully elucidated. However, recent research suggests that the antibacterial activity of these nanoparticles may be influenced by their spontaneous polarization and magnetization.

Spontaneous polarization refers to the permanent electric dipole moment present in a material even in the absence of an external electric field. In the case of multifunctional nanoparticles, they possess both ferroelectric and ferromagnetic properties, exhibiting both spontaneous polarization and magnetization concurrently.

The existence of spontaneous polarization in these multifunctional nanoparticles could impact bacterial growth through multiple mechanisms. One such mechanism involves the disruption of the bacterial cell membrane, potentially leading to cell death.

Additionally, it is proposed that the presence of spontaneous polarization might cause alterations in the electrochemical balance within the bacterial cells, further contributing to the antimicrobial effect [ 49 ]. Despite these promising findings, further research is necessary to gain a comprehensive understanding of the intricate interactions between multifunctional nanoparticles and bacteria.

Investigating these mechanisms could pave the way for novel antimicrobial strategies and applications in various fields, such as biomedicine and environmental protection. These ROS species are highly reactive and can induce oxidative stress in bacterial cells, leading to damage to cellular components and ultimately bacterial cell death.

The nanoceramics can interact with the bacterial cell membranes, causing disruption and destabilization [ 50 ]. This disruption can lead to increased permeability of the membranes, loss of membrane potential, and leakage of cellular contents, ultimately resulting in bacterial cell death.

BTF-xNFO nanoceramics can release metal ions, such as iron Fe and nickel Ni , which have antimicrobial properties.

These ions can interfere with bacterial cell processes, disrupt enzymatic activities, and induce cellular oxidative stress, leading to bacterial cell death. Biofilm populations threaten human well-being in healthcare, food processing plants and drinking water infrastructure [ 52 ].

Floating planktonic cells inhabited eventually develop into a multispecies biofilm, leading to an impairment of antibacterial activities overall.

In the detrimental microhabitats of bacterial populations as a biofilm, a full matrix of favorable circumstances for protracted biofilm life while protecting against antibacterial agents, as well as the mechanical stability of a strong EPS coating encircling persistent harmful bacteria, can frequently usually be found [ 53 ].

Several nanomaterials have indeed been postulated as possible candidates for ignoring long-term contagious diseases in the natural biofilm settings addressed previous section.

Many bacterial and fungal strains produce biofilm, making it more challenging to treat them with medical and healthcare equipment and instruments [ 56 ]. They are significant causes of a wide range of human illnesses, from minor skin infections to fatalities [ 48 ]. As shown in Fig.

Following a hour incubation period, the biofilm prevention of K. Under comparable conditions, P. Exopolysaccharides EPS must be produced and secreted by microorganisms for biofilm to form and become a microbial biofilm [ 57 ].

External signals cause bacteria to generate EPS. The EPS in the bacterium is produced in response to external signals. This can reduce biofilm formation when EPS synthesis is suppressed [ 58 ].

Gurunathan et al. aeruginosa and S. Dielectric studies have revealed that BTF-7NF nanoceramics possess specific dielectric constants and conductivity values.

In biological studies, it was observed that BTF-7NF nanoceramics exhibited strong antibacterial activity against pathogens like Pseudomonas aeruginosa and Staphylococcus aureus. Correlation: Higher dielectric constants may indicate increased charge accumulation and surface interactions with bacterial cells.

This could lead to enhanced adhesion and disruption of bacterial membranes, contributing to the observed antibacterial effect. Dielectric studies have shown that BTF-7NF nanoceramics exhibit specific impedance characteristics. This correlation suggests that dielectric characteristics influence ROS production, contributing to antibacterial effects.

Biological studies demonstrated that BTF-7NF nanoceramics effectively disrupted biofilms formed by bacterial species. Correlation: Dielectric relaxation frequencies may be associated with the ability of BTF-7NF to weaken the structural integrity of biofilms. The XRD patterns prove the formation of both the ferroelectric hexagonal iron barium titanate and the ferromagnetic nickel ferrite phases.

The FTIR revealed the successively formation of chemical bonds of the different phases in the multifunctional nanocomposites. The dielectric properties of BTF-xNF nanoceramics show superior properties.

The dielectric constant reaches more than 10 3 at low frequency and shows a relaxation behavior with frequency, it proves that the nanonanocomposite samples are frequency dependent.

The dielectric and magnetic properties of the samples demonstrate the possibilities of using the prepared materials in many industrial applications such as the electronics and mechanical industries. Low-cost BTF-7Ni nanoparticles have antimicrobial and antibiotic properties against a variety of species and may find application in biomedical applications such as dental units and surgical instruments.

The results obtained for BTF-7Ni superparamagnetic perovskite nanoparticles indicate their commercial potential as antibacterial agents and antibiotics.

The synthesis and characterization of multifunctional BaTi 2 Fe 4 O 11 - x NiFe 2 O 4 nanoceramics demonstrate their potential as highly effective materials for combating microbial infections and biofilm-related issues.

The developed BaTi 2 Fe 4 O 11 - x NiFe 2 O 4 nanoceramics showed promising antimicrobial and antibiofilm performance due to their unique microstructure and spectroscopic properties. This research opens new avenues for the design and application of multifunctional nanoceramics in various antimicrobial and antibiofilm treatments, with potential implications in biomedical and environmental fields.

Further studies are required to understand the underlying mechanisms and optimize the composite for specific applications. All data that support the findings of this study are included within the article and any supplementary files.

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Leaf extract and their charge density distribution, electrochemical and biological performance. Chem Phys Lett There are three types of AMEs; acetyltransferases AACs , nucleotidyltransferases ANTs and phosphorlyl transferases APHs. All of these decrease the binding affinity of the antibiotic to its target by transferring a functional group onto the aminoglycoside Azucena and Mobashery, All three types of AMEs have been observed in P.

aeruginosa and are common determinants of aminoglycoside resistance in non-CF isolates Poole, AMEs are not, however, the most common aminoglycoside resistance mechanism in people with CF. Instead, impermeability and efflux are the more commonly observed events Islam et al.

However, studies have observed a variable prevalence of these enzymes in between 5. These enzymes are carried on plasmids and are thought to have come from an aminoglycoside producing organism that utilizes these enzymes to protect its own ribosomes from aminoglycoside action Doi and Arakawa, Strains carrying 16s rRNA methylase genes show a high level of resistance to most clinically relevant aminoglycoside antibiotics.

The prevalence of these enzymes is not common in CF and other respiratory associated infection isolates Yokoyama et al. aeruginosa have been reported in Africa, Asia, Europe and South America and are, along with ESBLs, often associated with a pan-resistant genotype Fontes et al.

DNA gyrase and topoisomerase IV are both involved in DNA replication and management of chromosome integrity Drlica and Zhao, DNA gyrase functions to cleave double stranded DNA, introduce negative supercoiling and re-ligate the strand in an ATP-dependent reaction Levine et al.

Topoisomerase IV functions by decatenating and relaxing positively supercoiled DNA Corbett et al. Fluoroquinolones like ciprofloxacin act on DNA gyrase and topoisomerase IV to prevent the re-ligation step, leaving the DNA cleaved and thereby acting as a bacteriostatic antibiotic, blocking DNA replication Drlica, Mutations in gyrA and gyrB are commonly identified in CF associated infections and are likely to arise during prolonged antibiotic treatment with ciprofloxacin Fothergill et al.

The most frequently observed mutations in both CF and non-CF isolates, as well as in in vitro experiments, are the substitution of an amino acid at position 83 in GyrA from threonine to isoleucine and an amino acid substitution of serine to leucine at position 87 in ParC Higgins et al.

The second most common mutation is GyrA position 87, where tyrosine is substituted by asparagine, glycine or tyrosine Wydmuch et al.

These positions of GyrA are important for the binding of ciprofloxacin to DNA gyrase and the amino acid substitutions greatly lower the affinity of this binding Willmott and Maxwell, ; Aldred et al. Resistance to ciprofloxacin is higher when both mutations in gyrA and parC are present, in comparison to only gyrA mutation Pasca et al.

However, it is thought that gyrA mutation is necessary for high level resistance against ciprofloxacin, due to the strong affinity of the native form of the protein for this antimicrobial. When only gyrB, parC or parD mutations are present or when efflux is the main mechanism of ciprofloxacin resistance, only low level resistance is observed Rehman et al.

A combination of efflux, gyrAB and parCD mutation leads to a higher level of resistance and again highlights that resistance in P. aeruginosa is complex, often involving multiple mechanisms Dunham et al.

Mutations arising in the fusA1 gene lead to amino acid substitutions in a critical part of the translational machinery, elongation factor G EF-G. Mutations within different domains of EF-G have been identified in clinical strains and are linked to decreased aminoglycoside susceptibility.

Mutations in fusA1 have been reported in both CF and non-CF isolates globally Del Barrio-Tofiño et al. The mutation is often paired with upregulation of efflux, mainly the MexXY-OprM efflux pump, leading to strong aminoglycoside resistance.

However, on its own, fusA1 mutation can cause strains to be 4- to 8- fold more resistant to aminoglycosides. This was shown in vitro , where three fusA1 mutants in domains II, III and IV were created Bolard et al. The molecular mechanism linking mutations within EF-G to increased aminoglycoside resistance remains unclear.

Aminoglycosides do not bind to or target EF-G, thus resistance to aminoglycosides must be an indirect effect of EF-G alteration Gutierrez et al.

Bacterial motility is a vital part of P. aeruginosa pathogenicity and plays a role in host colonization and establishment of infection Yeung et al. aeruginosa employs several different forms of motility that are environmentally dependent and often require the use of type IV pili, flagella or rhamnolipid surfactants Overhage et al.

RNA-seq and transposon mutant studies have shown that both swarming and surfing phenotypes are associated with significant changes in gene expression, leading to an increase in antibiotic resistance. Swarming motility was linked to the altered regulation of 1, genes, including regulatory genes, including transcription factors, TCSS and sigma factors Coleman et al.

Surfing motility was linked to altered regulation of up to 2, genes and out of these, 31 resistome genes were identified which were known to cause an increase in antibiotic susceptibility when mutated Sun et al.

Both swarming and surfing play a role in adaptive resistance. They are environmentally dependent and associated with conferring a high level of antibiotic resistance in comparison to alternative motility phenotypes Wang et al. Swarming cells have been shown to be significantly more resistant to aminoglycosides, β-lactams, chloramphenicol, ciprofloxacin, tetracycline, ethoprim, erythromycin and azithromycin Coleman et al.

Surfing cells however, showed significantly higher resistance to polymyxins, aminoglycosides, fluoroquinolones, tetracycline, chloramphenicol, trimethoprim and several β-lactams, but not to macrolides Sun et al. On semi-solid surfaces with low nitrogen concentrations, P. aeruginosa is known to utilize swarming motility.

The conditions under which P. aeruginosa exhibits swarming motility appear to mimic those of the lung, where semi-solid mucous overlays epithelial cells. Likewise, the CF and bronchiectasis lungs contain high levels of mucin, the component necessary for surfing motility Sun et al.

Although P. aeruginosa motility is often lost in long term chronic illness such as in CF, a plethora of genetic heterogeneity exists in the bacterial populations residing in the CF lung. Furthermore, swarming and surfing motility may occur during early colonization and adaptation to the CF lung.

The biofilm mode of growth is a major impediment in the struggle to eradicate P. aeruginosa infection from the CF lung, due to the increased ability of bacteria in biofilms to withstand antibiotic treatment Høiby et al.

Biofilms are composed of bacteria surrounded by extracellular polymeric substances like exopolysaccharides, extracellular DNA and polypeptides Rasamiravaka et al. This can lead to an increase in tolerance to antimicrobial agents of times, compared to planktonic cells Ceri et al.

Contributing features include quorum sensing, decreased ability to penetrate biofilm, presence of oxygen gradients, altered metabolism and slow bacterial growth rate Olsen, Biofilms are common in chronic P. aeruginosa infection of the CF lung but have also been shown to be of significance in patients with COPD, bronchiectasis and chronic wounds Costerton, ; Parameswaran and Sethi, ; Chalmers and Hill, ; Hassett et al.

Airway mucins are found in abundance in people with CF, COPD and bronchiectasis. In vitro studies have shown that the presence of airway mucins are fundamental for the development of biofilm structures with enhanced tolerance to antimicrobials.

Further, mucin may serve as a suitable attachment surface for P. aeruginosa biofilm formation Landry et al. Iglesias et al. investigated antibiotic pharmacodynamics within biofilm structures in the context of CF. Bacterial counts, metabolic activity and biomass of PAO1 biofilms grown in artificial sputum media ASM or trypticase soy-based medium were compared.

They found that bacteria in ASM reached the same CFU and metabolic activity as biofilms formed in trypticase soy-based medium, although ASM biofilms grew slower and had a marginally higher biomass. When both biofilms were subjected to antibiotic treatment, ASM grown biofilms were substantially more resistant to tobramycin and colistin, but not ciprofloxacin and β-lactams Diaz Iglesias and Van Bambeke, These studies indicate that environmental factors play an important role in determining biofilm structure and antimicrobial resistance.

For comprehensive reviews on P. aeruginosa biofilms refer to: Olsen, and Maurice, Bedi and Sadikot, Olsen, ; Maurice et al. Combining resistance genes encoding mechanisms previously described here, along with intrinsic resistance, can lead the development of MDR and XDR P.

MDR and XDR P. aeru ginosa arise due to the compilation of resistance mechanisms, amassing building blocks into a barrier of resistance strategies that protect the bacteria from antimicrobial assault. Intrinsic resistance forms the base layer of this barrier, with additional acquired and adaptive resistance mechanisms forming further layers, collectively constructing a robust barrier against antimicrobials Figure 3.

Antibiotic resistance genes can quickly and easily be disseminated through P. Megaplasmids contain dynamic accessory genomes where frequent recombination and duplication events take place, leading to diverse and adaptive multidrug resistance traits Cazares et al.

Accordingly, resistance to antimicrobial agents in P. aeruginosa clinical isolates is highly complex, with frequent interplay between intrinsic, adaptive and acquired resistance mechanisms. AmpC, low outer membrane permeability and efflux systems often work together in resistance to carbapenems, chloramphenicol, fluoroquinolones, macrolides, penicillins, tetracyclines and β-lactams and resistance may be enhanced through the accumulation of mutations leading to up- or down- regulation of each of these systems.

The outer membrane porin OprH works in conjunction with the two-component signaling systems PhoPQ and PmrAB in modifying the bacterial LPS to regulate protection to polymyxin antibiotics. Aminoglycoside resistance is achieved through mechanisms such as aminoglycoside modifying enzymes, fusA1 mutation, 16S rRNA methylation, along with MexXY-OprM upregulation.

In addition, the presence of β-lactamases such as ESBLs and MBLs may further enhance carbapenem, cephalosporin and penicillin resistance. A perfect storm of highly resistant P. Counteracting these mechanisms could prolong the life of existing antimicrobials. Figure 3 Multi-layered, interacting resistance mechanisms in P.

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 β-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.

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. Increasing OM permeability to hydrophobic and amphiphilic compounds challenges the issue of intrinsic low-outer membrane permeability Tümmler, For example, polymyxin B nonapeptide causes a 2- to fold increase in susceptibility to ciprofloxacin, norfloxacin, and ofloxacin and to fold increase in susceptibility to nalidixic acid Kubesch et al.

Nalidixic acid is the precursor to ciprofloxacin, norfloxacin, and ofloxacin, and is not ordinarily more efficacious than the optimized antibiotics currently used in clinical practice.

However, when the membrane becomes more permeable through polymyxin B nonapeptide treatment, nalidixic acid becomes a more powerful anti-pseudomonal Tümmler, Polymyxin B nonapeptide is highly toxic and has therefore never been considered for clinical application Tsubery et al. However, more than 30 years after the first report of OM sensitizers, three have been approved for clinical studies; the anti-protozoal pentamidine Stokes et al.

None of these OM sensitizers, with the exception of SPR, have been proven to have promising anti-pseudomonal activity. SPR is active against P. aeruginosa with a similar potency to polymyxin B and is currently undergoing phase-one clinical trial Zhang et al.

OM sensitizers appear to be a promising approach to resistance that can by-pass intrinsic, acquired and spontaneous resistance Macnair and Brown, However, further investigation into additional non-toxic compounds that act on the P.

aeruginosa OM is essential, especially for the treatment of polymyxin resistant isolates. The most widely researched P. aeruginosa efflux-pump inhibiter EPI is Phe-Arg-β-naphthylamide PAβN , a broad-spectrum peptidomimetic compound capable of interfering with all four clinically relevant P. aeruginosa RND efflux pumps.

PAβN potentiates chloramphenicol, fluoroquinolones, macrolides, ketolides, oxazolidinones and rifampicin but not aminoglycosides or β-lactams Lomovskaya and Bostian, The proposed mechanism of action for PAβN is that it functions as a substrate of the Mex-series efflux pumps and outcompetes the antibiotic for extrusion, preventing the antibiotic from leaving the cell Mahmood et al.

However, PAβN and derivatives of this compounds are not yet approved, as adverse toxicology and pharmacokinetic profiles were identified during phase 1 clinical trials Renau et al. The pyridopyrimidine derivative D is active against the MexAB-OprM efflux pump Mahmood et al. D has shown promising in vitro and in vivo activity, as well as high solubility and low-toxicity profiles Yoshida et al.

D 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 D hydrophilic component and the substrate binding channel of MexB Nakashima et al.

EPIs will need to be broad-spectrum if they are to be used as an adjuvant to antibiotics that are substrates of several efflux-pumps. The specificity of D would limit its usage to co-administration with antibiotics extruded exclusively by MexAB-OprM Nakashima et al.

However, a study by Ranjitkar et al. found that there are several mechanisms of resistance to D potentiator activity in P. aeruginosa , when the agent is used together with carbenicillin, an antibiotic that is substrate specific to MexAB-OprM.

Loss of potentiating activity of D occurred rapidly due to a FL substitution in mexB , which is known to play an important role in inhibitor binding Ranjitkar et al. 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 viability and in the functioning of a wide array of organ systems, including, the nervous and immune systems Sánchez-Jiménez et al.

Fleeman et al. identified a polyamine scaffold as a strong efflux pump inhibitor with no direct antimicrobial activity. Five lead agents were found to potentiate aztreonam, chloramphenicol and tetracycline by causing a 5- to 8-fold decrease in the MIC90 Fleeman et al.

In addition, the polyamine derivatives did not disrupt the bacterial membrane, unlike other polyamines, which can lead to the identification of false positives for EPIs Fleeman et al. Moreover, polyamines did not display toxicity to mammalian cell lines and did not inhibit calcium channel activity in human kidney cells Fleeman et al.

Phage therapy, the use of bacteriophages to infect and lyse bacterial cells, has been widely discussed Chan et al. Traditional phage therapy involves the administration of one, or a mixture, of phages that will invade the bacterial cell and clear infection Waters et al.

A different approach to phage therapy has been proposed, whereby phages would be used to steer antibiotic resistance evolution, selecting for phage resistance and antibiotic susceptibility. For example, the lytic Myoviridae bacteriophage, OMKO1, utilizes OprM of the multidrug efflux systems MexAB and MexXY as a receptor-binding site.

Selection for resistance to OMKO1 bacteriophage attack creates an evolutionary trade-off in MDR P. aeruginosa , by changing the efflux pump mechanism, leading to an increased sensitivity to ciprofloxacin, tetracycline, ceftazidime and erythromycin, four drugs from different antibiotic classes Chan et al.

Phage steering can be achieved when the binding receptor for the bacteriophage is implicated in both antibiotic resistance and phage resistance. The advantage of this approach lies in the two distinct, and opposing, mechanisms leading to bacterial eradication Gurney et al.

The prototypical example of successful anti-resistance therapeutics are the β-lactamase inhibitors. β-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam are widely used to combat resistance mediated by β-lactamases Tooke et al.

However, the majority of clinically used β-lactamase inhibitors have a limited spectrum and mainly target Ambler class A β-lactamases, excluding KPC-type β-lactamase. Progress has been made in the development of novel β-lactamase inhibitors with a wider spectrum of activity.

Three novel β-lactamase inhibitors, avibactam, vaborbactam and relebactam, function against Ambler class A, C and D β-lactamases Wong and Duin, However, only avibactam and relebactam are efficacious against P. aeruginosa infection Aktaş et al. Lamut et al. designed 4,5,6,7-tetrahydrobenzo[d]thiazole-based DNA gyrase B inhibitors and incorporated these inhibitors with siderophore mimics.

The siderophore mimic served as an inducer for increased uptake of the gyrase B inhibitors into the bacterial cytoplasm. Out of the ten gyrase B inhibitors tested against P. Several more attempts have been made at 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. Murepavadin is a novel, non-lytic, species specific, outer-membrane protein targeting antibiotic for the treatment of P. aeruginosa infections, including those caused by MDR strains Dale et al. Murepavadin is derived from the β-hairpin host defense molecule protegrin 1 PG-1 and optimized to counteract unfavorable absorption, distribution, metabolism, excretion and toxicity ADMET properties normally associated with PG-1 Obrecht et al.

It is a macrocycle compound consisting of PG-1 loop sequences linked to a D-proline-L-proline sequence, the latter of which is important for its stability and subsequent strong antibacterial potential Srinivas et al. Murepavadin functions through binding to the LPS transport protein D LptD , an OMP necessary for LPS biogenesis in Gram-negative bacteria.

The interaction between murepavadin and LptD causes inhibition of LPS transport, which leads to alterations of the LPS on the bacterial OM and eventually, cell death Werneburg et al. Murepavadin derivatives have been screened for activity against Gram-negative ESKAPE pathogens, including P.

Therefore, compounds were generated consisting of β-hairpin macrocycles linked to the peptide macrocycle of polymyxin B. One of these compounds, compound 3, showed strong antimicrobial activity MIC 0.

aeruginosa isolates , low toxicity to mammalian cells, low plasma protein binding, good human plasma stability and no lytic activity towards human red blood cells. This compound was shown to perturb and permealise the bacterial membrane through interacting with the β-barrel domain of BamA in E.

coli ATCC Luther et al. BamA is part of the β-barrel assembly machinery BAM complex, which serves to fold and insert outer membrane proteins in the OM Gu et al. The binding interaction between BamA and compound 3 locks BamA in its closed state through changing the conformational composite in the β-barrel lateral gate between open and closed states.

It is not known what causes compound 3 to permeabilize the membrane. It may inhibit the folding activity of the BAM complex, leading to incorrectly folded proteins being misplaced in the inner membrane. Alternatively, BamA may only serve as an extra binding site for compound 3, thereby evading the LPS-modification resistance mechanism of Gram-negative pathogens Luther et al.

Quorum sensing regulates a wide range of genes involved in virulence and bacterial adaptation Kalia, For instance, QS is required for the surfing and swarming motility phenotypes associated with increased resistance to antimicrobials.

The surfing phenotype is regulated via three QS systems in P. aeruginosa ; Las, Rhl and Pqs Sun et al. In addition, QS has been found to influence tolerance to antibiotics in P. aeruginosa biofilms. QS provides structural rigidity through the regulation of Pel polysaccharides and eDNA release necessary for the extracellular polysaccharide matrix.

In addition, the production of rhamnolipids, surfactants important for the establishment and maintenance of biofilms, is controlled under QS de Kievit, Therefore, QS has been recognized as a significant potential target for developing anti-resistance therapies.

Strategies to combat antimicrobial resistance by targeting adaptive resistance mechanisms have significant potential for reversing antibiotic resistance in P.

Adaptive resistance is often mediated through complex global regulatory systems, such as the QS system, and regulate an extensive set of genes involved in resistance.

Targeting these regulatory systems may prevent the activation of expression of these resistance genes that would normally be expressed under the environmental conditions of infection.

Ajoene is a natural sulphur-containing compound extracted from garlic Yoshida et al. rsmZ and rsmY bind the global regulatory protein RsmA, and unbound RsmA represses the translation of genes by preventing ribosome binding to the Shine-Dalgarno site.

Several genes involved in QS are under RsmA regulation, and low expression of rsmZ and rsmY in the presence of ajoene promotes RsmA mediated repression of these target genes Jakobsen et al.

However, the therapeutic applicability of ajoene is limited due to availability, instability, hydrophobicity and relatively high MIC values. Efforts are being undertaken to overcome these issues through modification, the use novel delivery systems and a targeted route of administration and through the development of synthetic ajoene analogues Fong et al.

Another novel QS-inhibitor derived from a natural source is the plant flavonoid naringenin. Naringenin diminishes the production of QS-regulated virulence factors in P.

aeruginosa by binding directly to LasR, thereby competing with the activator of LasR, N- 3-oxo-dodecanoyl -l-homoserine lactone HSL. It is ineffective at outcompeting HSL when the activator is already bound to LasR.

Thus, the QS-inhibitor will only sufficiently interfere with the QS response when administered during early exponential growth, when naringenin can compete with unbound HSL for LasR binding. Naringenin is only suitable for combatting P. aeruginosa populations at low cellular densities, which often does not represent the clinical infection scenario.

The full potential of QS-inhibition will only be realized if an inhibitor is developed that is capable of targeting P. Bacterial biofilms pose a physical barrier for drug penetration, which is one of the reasons that bacteria in a biofilm mode of growth are more resistant to antimicrobials.

This phenomenon may be subverted with the use of nanocarriers that encapsulate antimicrobials and facilitate drug diffusion through the bacterial biofilm. In addition, nanocarriers can also protect drugs from degradation, ensure controlled drug release, and cause increased uptake by the drug target, leading to an overall higher efficiency 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 drug counterpart Alhariri et al.

Table 1 Recent efforts in the design of drug delivery methods for anti-pseudomonal therapies. Another promising antibiotic adjuvant targeting biofilms is the non-bactericidal, inhaled adjuvant, nitric oxide NO. Exposure of P. aeruginosa biofilms to low-dose NO has been shown to cause dispersal of biofilms, rendering the infection susceptible to subsequent antibiotic treatment Cai, NO functions by increasing bacterial phosphodiesterase activity which, in turn, leads to a reduction in the vital secondary signaling messenger, cyclic di-GMP.

Cyclic di-GMP is vital for intracellular regulation of biofilm formation. Howlin et al. carried out in vitro biofilm studies using CF sputum clinical samples. Biofilms treated with NO showed a relative decrease in biofilm biomass and surface bound thickness in comparison to the untreated control.

There is some evidence for the safety of NO administration in CF patients in vivo and NO is currently undergoing clinical trials to measure clinical efficacy Howlin et al. Poly-acetyl-arginyl-glucosamine PAAG , also called SNSP, is a novel inhaled adjuvant therapy currently undergoing phase one clinical trials.

PAAG is a polycationic glycoprotein that functions by permeabilizing the bacterial membrane and is active against methicillin resistant Staphylococcus aureus MRSA , Burkholderia spp. and E. PAAG has been shown to effectively disperse Burkholderia cepacia complex biofilm structures extracted from the CF lung Narayanaswamy et al.

aeruginosa , PAAG is has been shown to effectively eradicate persister cells, which is important for the prevention of recurrent P. aeruginosa infections and subsequent exacerbations in people with CF Narayanaswamy et al.

In addition to serving as an effective antibiotic adjuvant, PAAG also reduces inflammation and promotes viscoelasticity and mucociliary clearance, making it a suitable drug candidate to improve the quality of life for patients with a variety of mucus diseases Fernandez-Petty et al.

The global overuse and misuse of antibiotics during the last 80 years has led to a profound increase in antimicrobial resistance. AMR is a complex, One Health issue, involving human, animal and environmental factors.

The solution to AMR is therefore also likely to be a complex one, involving multiple strategies; maintaining AMR surveillance, containing AMR transmission, reducing selection pressure, developing novel antimicrobials or reverting antibiotic resistant microbes back to the susceptible phenotype with the use of antibiotic adjuvants Hernando-Amado et al.

Although progress in the development of naturally derived and peptide-based antimicrobials has been made Mok et al.

The conservation of existing antibiotics through careful stewardship is paramount to help mitigate the gap between the demand for new drugs and the diminishing supply pipeline. Antibiotic adjuvants will also play an important role in extending the shelf life of our existing antimicrobial therapeutic agents.

Adjuvant strategies targeting resistance mechanisms in P. aeruginosa could rejuvenate traditional antibiotic therapy by potentiating drug activity as well as slowing the development of antibiotic resistance.

As described in this review, antimicrobial resistance in P. aeruginosa is regulated through a complex interplay of mechanisms. Resistance encoded in the core genome of P. aeruginosa , such as low outer membrane permeability, Mex-type efflux pumps and AmpC β-lactamase amount to the basal level of resistance against antimicrobials.

This intrinsic resistance is present in the all P. aeruginosa strains and serves as a foundational level, which can be expanded upon.

This expansion can be induced by environmental influences, such as host factors and signalling molecules, that switch on adaptive resistance mechanisms. Acquired resistance mechanisms, such as antibiotic target modifications generated via mutation, and antibiotic modifying enzymes or resistance plasmids, acquired by gene transfer, may serve as additional building blocks to expand the arsenal of resistance mechanisms a particular strain might carry.

Several novel therapeutic strategies, targeting one or more of these mechanisms, have been described in this review. In light of recent findings, OM perturbants capable of sensitizing the Gram-negative bacterial membrane to previously non-active antibiotics seem an opportune strategy to combat resistance.

OM perturbants can by-pass intrinsic as well as acquired and spontaneous resistance mechanisms, making them highly promising drug candidates, for which the development of resistance would be unlikely.

However, efforts to finding perturbants suitable for targeting the P. aeruginosa membrane must be increased. A second promising strategy is phage steering, which uses the natural predators of bacteria and the forces of evolutionary pressure to our advantage.

Counterbalancing antibiotic resistance with phage susceptibility creates a double edged sword to circumvent key AMR mechanisms. In addition to these strategies, adjuvants targeting adaptive resistance mechanisms are worthy of consideration, due to the potential to disrupt multiple bacterial resistance and virulence processes with agents targeting a single regulator.

Targeting global regulatory systems that would normally control the expression of resistance genes under infection conditions will prevent the activation of those genes with potential knock-on advantages in inhibition of virulence mechanisms.

QS and two-component signaling systems are particularly attractive targets from this perspective, as are the regulators of biofilm formation.

There are several challenges in developing resistance-breaking therapy for P. aeruginosa infection. Firstly, due to its comparatively large genome and highly adaptive nature, a plethora of regulatory systems, as well as limited drug penetration and active efflux, many antibiotic adjuvants designed for Gram-negative pathogens do not show efficacy against P.

Secondly, toxicity has been proven to be the major hurdle for adjuvants designed against P. aeruginosa , leading to many being abandoned at early phases of development. Drug safety assessment is a long, expensive, but crucial process and toxicity is most likely where drug targets share structural similarity with human proteins.

In this respect, bacterial signaling systems are good candidates, as prokaryotic and eukaryotic signaling systems are highly divergent, with eukaryotes lacking TCSS or phosphorelay systems.

As with all newly developed drugs designed to be used as combination therapy, care must be taken in determining the correct dosing and investigating clear synergy profiles. Drug levels necessary for synergy in vitro may not be achievable in vivo.

Synergy in vivo may be affected by failure to obtain desired levels of drugs in the target tissue, drug metabolism or plasma protein binding. In addition, it is of paramount importance to evaluate drugs in relevant models that reflect the environmental conditions of infection. This will increase the predictive power of preclinical testing, reducing the costly progression of unpromising agents to clinical trials.

The lack of well-validated in vivo models for testing CF anti-infective therapeutics limits the speed of development of new drugs.

Murine models using cystic fibrosis transmembrane conductance regulator CFTR knockout animals, or transgenics in which the severe gut phenotype associated with loss of CFTR has been corrected are available, and have proved useful, but do not develop the characteristic features of acute and chronic P.

aeruginosa infection seen in those with CF Bragonzi, ; Semaniakou et al. Progress on the use of ferret and porcine infection models with mutated CFTR has been made, although these are limited by the availability of suitable immunobiology reagents Keiser and Engelhardt, Intranasal administration of P.

aeruginosa into the healthy murine lung often leads to either rapid clearance or sepsis. To create a model of persistent infection, it is usually necessary to immobilize P.

This can be achieved by encapsulating the bacteria into agar or alginate beads, where the bacteria are protected from clearance by immune effectors and where their mode of living more closely mimics bacterial biofilms present in chronic infection Cigana et al. However, this bead model is technically demanding and requires surgical transtracheal instillation of the bead suspension, leading to additional complications and mortality not representative of bacterial infections in CF Van Heeckeren and Schluchter, Alternatively, long term lung infection can be achieved using P.

aeruginosa isolates from CF, some of which naturally establish chronic infection in mice, without the need for implantation into beads Fothergill et al. This model has the advantages of using a natural infection route and having no requirement for surgical intervention, and offers opportunity to study lung infection over prolonged periods.

However, the density of infection achieved in the lung is low, making some analyses challenging. Questions remain regarding the commitment of governments 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 agents and vaccines, it is important that we do not lose ground in the fight against AMR.

Indeed, emerging evidence suggests that antibiotic use has increased dramatically in the COVID era Hsu, On top of this, increased usage of sanitizers and disinfectants globally may induce the development of cross-resistance to antibiotics.

The ESKAPE pathogens, for which new medicines are urgently needed, continue to cause serious community-acquired and nosocomial infections, and if investment into research and drug development for these bacterial pathogens is diminished, it will exacerbate the global health and economic costs associated with the ongoing pandemic.

FL wrote the manuscript with supervision and input from all others. All authors contributed to the article and approved the submitted version. FL is supported by a PhD studentship from the Rosetrees Trust M The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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