Pseudomonas aeruginosa: Infections, Animal Modeling, and Therapeutics

Pseudomonas aeruginosa is an important Gram-negative opportunistic pathogen which causes many severe acute and chronic infections with high morbidity, and mortality rates as high as 40%. What makes P. aeruginosa a particularly challenging pathogen is its high intrinsic and acquired resistance to many of the available antibiotics. In this review, we review the important acute and chronic infections caused by this pathogen. We next discuss various animal models which have been developed to evaluate P. aeruginosa pathogenesis and assess therapeutics against this pathogen. Next, we review current treatments (antibiotics and vaccines) and provide an overview of their efficacies and their limitations. Finally, we highlight exciting literature on novel antibiotic-free strategies to control P. aeruginosa infections.


Introduction
Charles-Emmanuel Sédillot, a French military physician, was the first to reference an infection involving Pseudomonas aeruginosa in 1850. Sédillot described how surgical dressings of patients both in the field and hospital beds often became colored with a blue-green substance having a grape-like sweet odor, which we now know as the diagnostic hallmarks of P. aeruginosa infections, due to pyocyanin and 2-aminoacetophenone productions, respectively [1]. Thirty years later in 1882, a French pharmacist by the name of Carle Gessard reported the successful isolation of P. aeruginosa which he initially termed Bacillis pyocyaneus, in a publication entitled "On the blue and green coloration of bandages" [2,3]. It is not surprising that the site of the infection these authors described were wound dressings of surgical incisions given that wounds are preferred niches for P. aeruginosa infection [4][5][6][7][8][9][10][11][12][13][14]. After many taxonomic revisions over the past 100 years, at present, the Pseudomonas aeruginosa species is identified on the basis of 16S rRNA and genomic sequence comparisons, analysis of the cellular fatty acids, virulence factors, and differentiating physiological and biochemical tests [15][16][17][18][19][20][21][22][23][24][25][26].
P. aeruginosa is a rod-shaped Gram-negative bacterium of the class γ-proteobacteria and family Pseudomonadaceae [27]. It is a facultative aerobe that prefers to use oxygen as the final electron acceptor during aerobic respiration, although it is also capable of anaerobic respiration using other alternative electron acceptors such as nitrate [28]. P. aeruginosa can also catabolize a wide-range of organic molecules for nutrients, making it one of the most biochemically versatile and ubiquitous bacterium found in many environments such as soil, water, vegetation, and even human skin and oral mucosa [29][30][31]. The ability P. aeruginosa to thrive in diverse environments increases P. aeruginosa reservoirs and the possibility for exposure, leading to higher incidence of infections. P. aeruginosa has been isolated from locations such as hot tubs, humidifiers, and soil [1,32]. While in hospitals, P. aeruginosa has been isolated from respirators, physical therapy pools, sinks, and mops [33,34]. Patients infected with P. aeruginosa can also act as sources for new infections in hospitals [35]. These large reservoirs in both community and hospital settings allow for significant rates of infections. Because of its metabolic versatility and an arsenal of virulence factors it possesses [25], P. aeruginosa is responsible for many serious and life-threatening acute and chronic infections, particularly in the setting of immunocompromised hosts with mortality rates reaching as high as 40% [36][37][38][39].
P. aeruginosa is a killer of immunocompromised patients, a leading cause of bacteremia and sepsis in neutropenic cancer patients undergoing chemotherapy, and the number one cause of hospital-acquired pneumonia and respiratory failure [36][37][38][39]. P. aeruginosa infections are also common in diabetic ulcers, burn wounds, corneal ulcers, and surgical wounds [4][5][6][10][11][12][13][14]37]. Chronic infection by P. aeruginosa is a characteristic of individuals afflicted with cystic fibrosis (CF) and accounts for the pulmonary failure that leads to death in these individuals [40]. Life-threatening infections with P. aeruginosa are also becoming increasingly frequent in patients with AIDS [4][5][6][10][11][12][13][14]37,[41][42][43]. Exacerbating the challenge with P. aeruginosa infections is this organism's high intrinsic and acquired resistance to many current antibiotics [44,45]. Because of this challenge and the severity of infections caused by P. aeruginosa, it has been placed amongst the priority pathogens, known as the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), for which new antimicrobial development is urgently needed according to the World Health Organization (WHO) and the Center for Disease Control (CDC) [45][46][47]. In this review, we review the important acute and chronic infections caused by this pathogen. We provide an overview of current antibiotic treatments and their failure due to development of antibiotic resistance in P. aeruginosa. Finally, we discuss various animal models, developed to assess P. aeruginosa acute and chronic infections, and highlight exciting literature on the development of novel antibiotic-free strategies to combat P. aeruginosa infections.

Pseudomonas aeruginosa Infections
In this section, we discuss the various acute and chronic infections that are caused by P. aeruginosa (summarized in Table 1).

Pseudomonas aeruginosa Acute Infections
P. aeruginosa can cause serious acute infections including, respiratory tract infections [48,49], hospital acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP) [50,51], keratitis and corneal ulcers in contact lens wearing individuals [52,53], urinary tract infection (UTI), blood stream infections (BSIs) [54][55][56], osteomyelitis [57,58], and endocarditis [59,60]. Between 1997-2008, P. aeruginosa was reported to be responsible for 21.8% of the hospital acquired pneumonia (HAP) or the ventilator associated pneumonia (VAP), second only to Staphylococcus aureus which accounted for 28% [61]. A more recent report in 2016 also attributed similar HAP and VAP infection rates to P. aeruginosa [62]. In a meta-analysis of 11 studies of VAP cases after post-cardiac surgery, P. aeruginosa was the causative agent in 23.2%, followed by S. aureus (20.2%) [63]. A prospective study of adult patients with nosocomial pneumonia involving 75 hospitals in 11 countries between 2008-2009, found P. aeruginosa to be the leading cause of HAP (15.6%) and the secondleading cause of VAP (25.9%) behind only Acinetobacter spp. (35.6%) [64]. In a meta-analysis of 50 studies in China between 2010 to 2014, P. aeruginosa was responsible for 19.4% (95% confidence interval (CI) 17.6-21.2%) of all isolates in VAP and 17.8% (95% CI 14.6-21.6%) in HAP [65]. P. aeruginosa infections in VAP and HAP can be deadly with mortality rates ranging between 13% to nearly 50%. In one retrospective study involving 110 patients with P. aeruginosa VAP from 2008 to 2013 in Intensive Care Unit (ICU) in Italy, the mortality rate was reported to be 44.5%, the highest amongst all other pathogens [66]. The higher mortality rates have been associated with the extent of antibiotic resistance of infecting P. aeruginosa strains (such as multidrug resistance (MDR) clinical isolates) or specific viru-tion with P. aeruginosa occurs early in the life of CF patients, either through community or hospital exposure, and remains chronic throughout their lifespan [91].
Wounds are also highly vulnerable to P. aeruginosa infections. P. aeruginosa is the most abundant and frequently reported Gram-negative pathogenic bacterium in all chronic wounds-(wounds that do not heal within 3 months)-including diabetic foot ulcers, venous leg ulcers, and pressure ulcers [13,[92][93][94][95][96][97][98]. Moreover, presence of P. aeruginosa in these wounds correlates with poor prognosis for healing [11][12][13][14]99]. P. aeruginosainfected wounds have been reported to be significantly larger than wounds containing other bacteria-including Staphylococcus aureus, which is the most abundant Gram-positive bacterial pathogen reported in these wounds [11,13,100-102]-suggesting that P. aeruginosa infection may be more detrimental to the process of tissue repair and wound healing than other pathogens. Corroborating these reports, P. aeruginosa has been demonstrated to prevent wound healing and exacerbate tissue damage in cell culture-based in vitro and in various in vivo animal wound infection models [99,[103][104][105][106]. In fact, a number of in vitro and in vivo studies indicate that wound is a preferred niche for P. aeruginosa [4][5][6]10,106].
Similarly, P. aeruginosa causes a variety of important infections (e.g., pneumonia, blood stream infections, UTI, and wound) with high morbidity and mortality rates in patients suffering from drug-induced neutropenia (reduced neutrophil count), drug-induced qualitative neutropenia (defects in neutrophil function), or drug-induced immunosuppressed patients [115,122,123]. P. aeruginosa infections are reported to be more common in neutropenic patients with malignancy, particularly those with leukemias [124,125]. Over 21% of bacteremia in patients with acute leukemia were reported to be due to P. aeruginosa infection [126,127]. P. aeruginosa was also the most common cause of pneumonia in patients with solid tumors [125].
Another immunocompromised patient group that is highly susceptible to P. aeruginosa infection are the burn patients. Hyperinflammatory cytokine response and hypo innate and adaptive immunity are the hallmark of immunosuppression following major burn traumas in human [138][139][140]. P. aeruginosa has been reported as the most frequently recovered infectious pathogen in burn units and the clinical isolates display high resistance to antibiotics. In a recent large study involving 17119 patients at a burn ward in China between 2006 and 2019, S. aureus and P. aeruginosa were the predominant clinical pathogens responsible for bacterial infections in these patients [141]. It is worth noting that while the rates of S. aureus infections appeared to remain stable during this study (17.06% in 2006 to 18.54% in 2019); the rates of P. aeruginosa infections rose from 10.59% in 2006 to 17.68% in 2019, suggesting an upward trajectory in P. aeruginosa infections among burn patients at least in this burn unit. In another retrospective study assessing bacteria from wounds, catheters, blood, feces, urine and sputum of 10,276 hospitalized patients in burn wards between 2007 and 2014 in China; 3005 pathogenic strains were isolated and identified [142]. While S. aureus was the predominant strain in the beginning, its annual detection rates declined significantly over these years. In contrast, the annual detection rates of P. aeruginosa increased significantly during this period. Alarmingly, the detection rate increases in P. aeruginosa were associated with increased incidence of MDR bacteria in the culture, prompting the authors to caution against the use of ciprofloxacin, ceftazidime and cefoperazone/sulbactam to counter the related increase in resistance levels in P. aeruginosa. In another study involving 184 positive cultures from burn patients in burn unit in Iran, 205 different bacterial strains were isolated and identified, of which P. aeruginosa was the most prevalent, accounting for 57% of all clinical isolates; followed by Acinetobacter (17%), E. coli (12%), S. aureus (8%) and other organisms (6%) [143]. Importantly, over 90% of P. aeruginosa isolates displayed resistance to gentamicin, ceftizoxime, carbenicillin, cephalothin and ceftazidime. P. aeruginosa has also been the primary infective agent reported in other studies involving burn patients [143][144][145][146][147].

P. aeruginosa Infection Animal Models
In this section, we review the animal models which have been developed to study various infections caused by P. aeruginosa and assess the effectiveness of conventional and emerging therapies against this pathogen (summarized in Table 1).

Acute and Chronic Pneumonia Infection Models
Animal models have been extremely useful in advancing our understanding of P. aeruginosa pathogenesis and for the development and the therapeutic assessment of new antibiotics or novel biologicals to control this pathogen. Although most studies involving P. aeruginosa infections rely on mouse or rat models due to the cost and availability of reagents, larger animal modeling is also performed usually to fulfill the requirement by organizations-such as Food and Drug Administration (FDA)-to evaluate the efficacy and the safety profiles of new investigative biologics in two animal models that approximate human responses with respect to the condition under investigation [154]. As was discussed above, P. aeruginosa is an important bacterial pathogen in acute and chronic pneumonia, including the ventilated-associated pneumonia (VAP) and hospital acquired pneumonia (HAP). The first animal model of chronic pulmonary infection was a rat model in which P. aeruginosa infection was initiated by intratracheal inoculation of P. aeruginosa bacteria enmeshed in agar beads [155]. In this chronic model of infection, P. aeruginosa was detected during the 35 days of observation. Importantly, infected lungs in these rats exhibited lesions resembling those seen in lung tissues of humans with acute or chronic P. aeruginosa pneumonia, including the presence of goblet-cell hyperplasia, focal areas of necrosis, and acute and chronic inflammatory infiltrate [155]. Since then, various similar animal models (in mouse, rat, rabbit, porcine, dog, cat, etc.) of P. aeruginosa infections for acute and chronic pneumonia (including VAP and HAP) have also been described, albeit with modifications in the P. aeruginosa strain, initial inoculum levels, and in the rout of P. aeruginosa delivery into animal [156][157][158][159][160][161].

Urinary Tract and Kidney Infection Models
P. aeruginosa also causes urinary tract and kidney infections as discussed above. The initial animal models to assess UTI caused by P. aeruginosa involved intravenous injection of P. aeruginosa into mice [162,163]. In these systemic infection models, large doses (close to lethal doses) of P. aeruginosa-were needed to establish infection in the urinary tract and kidney. However, the high rate of mortality, due to systemic infection, made these murine models impractical [162,163]. To overcome this difficulty, artificial manipulations, (e.g., administration of bromoethylamine hydrobromide or ferric sorbitol citrate), were used to make kidneys in animals more susceptible to P. aeruginosa colonization and growth [164,165]. However, these artificial means made the interpretation of the data unreliable [166]. These limitations then led to the development of methods (e.g., surgical implantation of glass beads laden with P. aeruginosa, or transvesical ureteral catheterization) to directly deliver P. aeruginosa into the rat kidney in order to cause infection in this organ [167][168][169]. At present, urinary tract and kidney infection models frequently instill bacteria into the bladder using a catheter, based on the UTI protocol that was developed for Uropathogenic Escherichia coli (UPEC) by Hung et al. [170,171].

Blood Stream and Systemic Infection Models
Different approaches have been used to cause blood and systemic infection in animals with P. aeruginosa. For example, intravenous (i.v.), intraperitoneal (i.p.), or tail vein injections have been used as technical means to cause systemic infection with P. aeruginosa [172][173][174]. P. aeruginosa has also been delivered retro-orbitally to cause systemic infection and sepsis [175]. P. aeruginosa systemic infection has been shown to increase pro-inflammatory cytokines both in the blood and tissues, leading to other morbidities such as septic arthritis and gallbladder damage [172,176,177].

Keratitis and Corneal Ulcers Infection Models
As this was discussed above, keratitis and corneal ulcer infections are relatively rare but they are very serious medical conditions requiring urgent medical care because of the possibility that they can lead to vision loss in the affected eye(s). In the murine models for corneal ulcer, 2-3 parallel scratches (~1 mm) are usually made by sterile 25-gauge needle on the cornea of anesthetized animal prior to bacterial inoculation [178][179][180][181]. Animal models have been informative in showing the potency of antimicrobial activities in human tear [178]; in establishing the crucial roles for IL-16 pro-inflammatory cytokine and cathelicidin antimicrobial peptide in corneal defenses against P. aeruginosa [179,180]; and in demonstrating the therapeutic potential of the broad host range bacteriophage KPP12 in P. aeruginosa clearance and corneal healing [181].

Endocarditis Infection Models
Animal models of S. aureus infective endocarditis (IE) [182,183], are commonly used to investigate the underlying pathogenesis, disease progression, potential diagnostic approaches, and therapeutic treatment for endocarditis caused by P. aeruginosa [184]; Rabbits [185,186]. These models are based on surgical valve trauma followed by intravenous injection of bacteria within 10-24 h following the surgical valve trauma. In a rabbit model of endocarditis, with sterile right ventricular cardiac vegetations, Archer et al. demonstrated 78% mortality within 3 weeks, following P. aeruginosa infection [185]. In a follow-up study, the same group demonstrated that 14-day treatment with high dose gentamycin (7.5 mg/kg) and carbenicillin (400 mg/kg) was significantly more effective than either therapy alone, resulting in 64% sterilization of cardiac vegetations in this rabbit model of P. aeruginosa endocarditis [186]. In a rat model of P. aeruginosa endocarditis, Oechslin et al. demonstrated that a combination of systemic vancomycin and phage therapy was highly effective against P. aeruginosa endocarditis [184].

Wound and Surgical Site Infection Models
Skin is a formidable barrier against invading pathogens, including P. aeruginosa [187]. As an opportunistic pathogen, P. aeruginosa cannot colonize or cause infection in the skin of normal animal unless this barrier is breached by injury [4][5][6]10]. Therefore, animal wound and surgical site models of infections for P. aeruginosa (and other pathogens) usually involve full or partial thickness excisional wounding, or addition of bacteria directly to implants or stents before or after their insertion into animals [7,9,[188][189][190][191][192][193][194][195]. In the settings of injury, P. aeruginosa can efficiently colonize and cause infection [11][12][13][14]196]. In a recent study, P. aeruginosa was shown to thrive in wound environment, (in a mouse model of wound infection), by dampening the host innate immune responses in wound tissue via inhibition of the NLRC4 inflammasome mediated by its most conserved virulence factor, ExoT [7].
Chronic wounds are particularly vulnerable to P. aeruginosa infection [100,197,198]. In a recent study involving db/db type 2 diabetic mouse, it was shown that impairment in the formyl peptide chemokine receptors (FPR) in diabetic neutrophils results in a delay in neutrophil response, rendering diabetic wounds vulnerable to colonization and infection by P. aeruginosa [8]. Macrophage response has also been shown to be delayed in db/db diabetic wounds, due to dysregulation in IL-10 expression and signaling [199,200], further dampening innate immune responses and diabetic wound's ability to prevent P. aeruginosa infection [9]. In other studies, P. aeruginosa has been demonstrated to several other virulence factors (e.g., biofilm, type 3 secretion system (T3SS), pyocyanin, extracellular proteases, and Exotoxin A) to prevent wound healing and exacerbate tissue damage [99,103-106, 201,202]. In a burn wound model of infection, P. aeruginosa infection was shown to lead to bacteremia in a manner that was dependent on superoxide response regulator (soxR) expression and function in P. aeruginosa [203]. In another report, quorum sensing (QS) was shown to be involved in biofilm maturation and P. aeruginosa colonization and pathogenesis a pressure ulcer infection model in rat [204].

Immunocompromised Infection Models
As was discussed above, immunocompromised people are highly vulnerable to infection with P. aeruginosa. Not surprisingly, animal models have been developed to assess the impact of P. aeruginosa infection in immunocompromised hosts. For example, Takase et al. demonstrated that P. aeruginosa infection in the calf muscle of immunocompromised mice, (generated by cyclophosphamide), caused high mortality in these mice, in a manner that was mediated by pyoverdine and pyochelin siderophore production in P. aeruginosa [205]. In another study, P. aeruginosa was shown to induce death within 46 to 59 h in a leukopenic immunosuppressed mouse model [206]. Similarly, Mahmoud et al. demonstrated that wounds in the neutropenic immunocompromised C57BL/6 mice are vulnerable to P. aeruginosa enhanced infection [188]. In another study, P. aeruginosa infection was proved to be highly lethal in an immunosuppressed guinea pig model of pneumonia [207].

Cystic Fibrosis Infection Animal Models
Cystic Fibrosis (CF) is a genetic disorder caused by null mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes for the chloride channel [208][209][210]. Not surprisingly, different transgenic animal species, (i.e., mice, rats, rabbits, ferrets, pigs, and sheep), harboring similar mutations in the CFTR gene have been constructed to model various CF pathologies [211][212][213][214][215][216]. In one report, endobronchial infection with a mucoid P. aeruginosa strain was shown to elicit production of TNF-α, MIP-2, and KC/N51 inflammatory cytokines in bronchoalveolar lavage fluid and cause 80% mortality in CF mice (harboring the S489X mutation of the CFTR gene), thus phenocopy-ing some of the CF hallmark pathologies observed in human [217]. Corroborating these studies, van Heeckeren et. al., demonstrated that infection with P. aeruginosa resulted in significantly higher mortality rates, weight loss, higher lung pathology scores, and higher inflammatory mediator and neutrophil levels in the lungs of CF mice as compared to wildtype littermates [218]. However, murine models do not completely develop human CF disease severity in the pancreas, lung, intestine, liver, and other organs [219,220], thus necessitating the need for the development of larger animal models for CF, such as newborn pigs and ferrets [214][215][216]219,221]. For example, CF pigs were demonstrated to develop airway inflammation, mucus accumulation, and impaired bacterial clearance [222]. CF pig lungs contained multiple bacterial species, suggesting impaired immune defenses against bacteria [222].

Current Treatments for P. aeruginosa Infections
Here, we provide an overview of conventional antibiotic treatments for P. aeruginosa infections. Full description on these therapies can be found in the guidelines from the Infectious Diseases Society of America (IDSA) [223,224]. Regardless of the antibiotic agent(s) administered, patients infected with P. aeruginosa should be closely monitored as P. aeruginosa can rapidly acquire additional resistance mechanisms while exposed to antibiotic therapy as discussed in Section 4 below and discussed previously [44]. A summary of the antibiotic treatments, their limitations, and the mechanisms of resistance to these antibiotics can be found in Table 2.

β-Lactam Antibiotics (Alone and Combination Therapies)
β-lactam antibiotics generally are bactericidal antibiotics that destroy bacterial pathogens by disrupting their peptidoglycan cell wall via covalent binding to essential penicillin-binding proteins (PBPs) in bacteria [225]. Non-carbapenem β-lactam antibiotics (e.g., ceftazidime, cefepime, piperacillin-tazobactam, aztreonam, etc.) are the preferred first line of therapy where P. aeruginosa clinical isolate tests susceptible to these antibiotics [223,224]. The rational for their use is their clinical effectiveness using empiric regimes with fixed doses, their high potency and efficacy against a wide therapeutic range, and the low cytotoxicity and side-effects associated with their use [226,227]. Ceftazidime may be considered a preferred first-line βlactam therapy because its use has been associated with the lowest risk of resistance while on therapy [228]. Carbapenems are a class broad spectrum β-lactam antibiotics broad spectrum of activity that are recommended to treat infections caused by bacteria, including P. aeruginosa, resistant to traditional β-lactams or fluoroquinolones (discussed below) [224,229].
Cefiderocol is a novel injectable cephalosporin which was approved by FDA in 2019 for the treatment of complicated UTIs [230]. It is also a preferred option for the treatment of uncomplicated and difficult-to-treat (DTR) cystitis caused by P. aeruginosa infection [223,224,231,232]. Cefiderocol has been shown to be more potent than both ceftazidime-avibactam and meropenem against all resistance phenotypes of Pseudomonas aeruginosa because of its unique siderophore-like property (which enhances its entry into the bacterial periplasmic space [233]) and because of its high stability to a variety of βlactamases, including AmpC and extended-spectrum β-lactamases (ESBLs) [234,235].
More novel β-lactam combination antibiotic regiments (e.g., ceftazidime/avibactam, ceftolozane/tazobactam, imipenem/cilastatin/relebactam, etc.) are also used but only as alternative options for MDR or extensively-drug resistant (XDR) P. aeruginosa strains [223,224]. By definition, the MDR strains display acquired resistance to at least one agent in three antimicrobial categories, whereas the XDR strains show susceptibility to antibiotics in 2 or fewer categories and display resistance to at least one agent in the rest of available antibiotic categories [236,237]. Ceftazidime, (a third-generation broad-spectrum cephalosporin antibiotic [238]) is also used in combination with avibactam (a synthetic non-β-lactam, β-lactamase inhibitor which inactivates β-lactamase targets via covalent acylation [239]), and has shown to be highly effective against MDR and XDR P. aeruginosa strains (>90% effectiveness) [240,241]. However, P. aeruginosa strains resistant to ceftazidime/avibactam have been reported and are on the rise, adding to the challenges associated with P. aeruginosa therapeutics [242,243]. Ceftolozane, (a semi-synthetic broad-spectrum fifth-generation cephalosporin β-Lactam antibiotic [244]), in combination with tazobactam, (an irreversible β-lactamase inhibitor) [245], has been shown to be effective against most MDR and XDR resistant P. aeruginosa strains [245,246]. Not surprisingly, resistance to this combination therapy in P. aeruginosa has also been reported [247,248]. Combination therapy, imipenem (a β-lactam antibiotic belonging to the carbapenem subgroup [249]), cilastatin (an inhibitor of imipenem-degrading human renal dehydropeptidase and bacterial metalloβ−lactamase [250,251]), and relebactam (an inhibitor of β-lactamases [252]); has been recently approved in the USA and Europe, and shown to be highly effective for the treatment of complicated urinary tract infections including pyelonephritis, complicated intraabdominal infections, and hospital-acquired bacterial pneumonia caused by MDR and XDR Gram-negative bacterial pathogens including P. aeruginosa [253][254][255]. Antibiotic resistance in P. aeruginosa will be discussed in the next section.

Fluoroquinolones
Fluoroquinolones (e.g., ciprofloxacin and levofloxacin) are broad-spectrum antibiotics that destroy bacteria by inhibiting DNA gyrase and topoisomerase IV (essential enzymes for DNA synthesis in bacteria) [256]. Fluoroquinolones are also recommended for the first line of treatment in acute otitis externa or skin and soft tissue infections and complicated UTI caused by P. aeruginosa strains that are found to be susceptible to these agents [223,224,[257][258][259][260]. Limitations with the use of fluoroquinolones include rapid development of resistance in bacteria [261] and the sensitivity of fluoroquinolones to acidic conditions, which adversely affect their cell uptake [262,263], although recently developed novel fluoroquinolones (i.e., delafloxacin) show improved cellular uptake and maintain their antibacterial activities in acidic conditions [263].

Eravacycline (Tetracyclin)
Generally, tetracycline antibiotics are not very effective against P. aeruginosa infections and may even enhance its virulence by stimulating its T3SS virulence function [264]. However, eravacycline (a novel fully synthetic fluorocycline) is different in that it exhibits potent activity against a broad spectrum of clinically relevant Gram-positive and Gramnegative aerobic and anaerobic bacteria, including P. aeruginosa, and has been recently approved in several countries for the treatment of complicated intra-abdominal infections in adult patients [265][266][267]. It owes its potency to 2 modifications in the tetracyclic D ring at position C7 (fluorine atom addition) and C9 (pyrrolidinoacetamo group addition) [268], which allow it to be effective even in pathogens that express tetracycline-specific efflux and ribosomal protection mechanisms in clinical isolates [269]. Like all tetracyclines, it exerts its antimicrobial activities by inhibiting protein synthesis machinery in bacteria through its interaction with the 30S (and to a lesser extent 50S) ribosomal subunits [266].

Aminoglycosides
Aminoglycosides (e.g., gentamycin, amikacin, tobramycin) are broad-spectrum bactericidal antibiotics that destroy bacterial pathogens by targeting the 30S subunit of bacterial ribosomes, thus inhibiting protein synthesis [270]. They are also routinely prescribed against P. aeruginosa infections. A meta-analysis of publications until 2018 found that a single intravenous dose of aminoglycosides was highly effective (94.5 ± 4.3%) for uncomplicated cystitis with the recurrence-free and cure rate of >73%, with minimal toxicity [271]. In a large phase III clinical trial, plazomicin (a next-generation semisynthetic aminoglycoside) was shown to be noninferior to meropenem in the treatment of complicated urinary tract infections, even against Gram-negative pathogens, (including P. aeruginosa) with aminoglycoside-modifying enzymes that impart resistance to most aminoglycosides [272]. Other reports have confirmed the effectiveness of plazomicin against hard-to-treat resistant Gram-negative pathogens [273]. In another randomized trial involving seven U.S. centers for the treatment of cystic fibrosis, the short-term aerosol administration of a high dose of tobramycin in patients with clinically stable cystic fibrosis was shown to significantly increase lung function (as assessed by forced expiratory volume, forced vital capacity, and forced expiratory flow at the midportion of the vital capacity), and decrease the density of P. aeruginosa in sputum by a factor of 100, without any ototoxicity nor nephrotoxicity [274]. Although, nebulized amikacin/fosfomycin was shown to be ineffective in improving clinical outcomes in pneumonia in 3 clinical trials, despite reducing bacterial burden in these patient cohorts [275][276][277].

Polymyxins (Colistin, Polymyxin B)
Therapeutic use of polymyxins as one of the last lines of therapy against MDR/XDR P. aeruginosa infections have skyrocketed during the past decade [278,279]. Polymyxins kill Gram-negative bacterial pathogens by disrupting their membrane via targeting the Lipid A moiety of the lipopolysaccharide (LPS) in the outer membrane of these bacteria [280]. Therapeutic use of polymyxins has been associated with relatively higher toxicity side-effects, (e.g., nephrotoxicity and neurotoxicity), as compared to other antimicrobials [281,282], thus dosing and the treatment protocol should be carefully designed to reduce these sideeffects [283]. IDSA recommends polymyxin B (over colistin) in combination with the β-lactam and β-lactamase inhibitor for the treatment of non-urinary tract infections, if no aminoglycoside is effective against P. aeruginosa isolate in vitro [223,224]. The rational for this recommendation is that polymyxin B is not administered as a prodrug and therefore can achieve more reliable plasma concentrations than colistin which is administered as a prodrug and it converts to its active form in the urinary tract [284]. For precisely the same reason, colistin (not polymyxin B) monotherapy has been recommended as an alternate consideration for treating urinary tract infections caused by MDR/XDR P. aeruginosa [279], although IDSA also warns that clinicians should remain cognizant of the associated risk of nephrotoxicity in these patients [223,224].

Antibiotic Resistance in P. aeruginosa
The rate of antimicrobial resistance among P. aeruginosa clinical isolates has climbed sharply over the past 5 decades worldwide [285][286][287][288]. P. aeruginosa clinical isolates frequently exhibit resistance to various classes of antibiotics including β-lactams, aminoglycosides, fluoroquinolones, and even polymyxins with strains isolated from the Intensive Care Units (ICUs) demonstrating the highest incidence of resistance to these antibiotics [289][290][291]. A retrospective 10-year study reported a 10% increase in antibiotic resistance rates in P. aeruginosa clinical isolates in ICU within a decade [292]. Another study investigated the profile of antimicrobial resistance of Gram-negative bacteria in blood cultures in a university-affiliated hospital in China and found resistance rate to carbapenem among blood culture isolates of P. aeruginosa to increase significantly from 2004-2011 [293]. Of note, between 2000-2010, consumption of antibiotics (particularly carbapenems) increased by nearly 35% in China [294], thus establishing a direct correlation between antibiotic use and the emergence of antibiotic resistance in P. aeruginosa clinical isolates. Additionally, the incidence and the rate of infections with the multidrug resistant (MDR) P. aeruginosa strains-(resistant to at least one antibiotic in 3 or more antibiotic classes)-have been steadily rising over the past 20 years [286,[295][296][297][298], as a direct consequence of increased antibiotic consumption [294]. A meta-analysis report assessing the clinical and economic impacts of hospital-acquired resistance and MDR P. aeruginosa infections between the years 2000 to 2013, indicated a greater than 2-fold increased risk of mortality in patients infected with MDR P. aeruginosa strains and a 24% increased risk of mortality in patients infected with resistant P. aeruginosa strains as compared to patients infected with antibiotic susceptible P. aeruginosa strains [297]. Not surprisingly, the same study found longer hospital stay and increased cost associated with resistant and MDR P. aeruginosa infections as compared to susceptible P. aeruginosa and control patients. Corroborating these data, an international multicenter retrospective study in 2015 reported significant increased association between mortality in nosocomial pneumonia and MDR P. aeruginosa infections [299].
Another frightening concern is the emergence of extensively drug resistant (XDR) P. aeruginosa infections among HAIs. XDR P. aeruginosa strains are defined as the strains that remain susceptible to only one or two classes of anti-Pseudomonas drugs and show resistance to at least 1 antibiotic in the other antibiotic groups [300,301]. A retrospective study of a cohort of adult, hospitalized patients with P. aeruginosa in Thailand reported that 22% of P. aeruginosa infections were due to XDR strains, resulting in significantly higher mortality [302]. Another prospective study involving 1915 ICU patients between 2014-2015 in India reported 47.7% of P. aeruginosa infections to be due to MDR and XDR P. aeruginosa strains [303]. Recently, a nosocomial outbreak of P. aeruginosa infection with XDR strains was reported among ICU patients subjected to aromatherapy in Austria, which was successfully addressed through the implementation of restriction of oil sharing among patients [304]. A systemic review and meta-analysis of 54 articles between 2000 and 2016 reported prior use of antibiotics and prior hospital or ICU stay as the most significant risk factors for nosocomial infections with MDR and XDR P. aeruginosa strains [305].
The emergence of multi-drug resistant P. aeruginosa poses even more serious threat in immunocompromised patients and complicates treatment options for these patients [306,307]. A retrospective study involving 7386 clinical specimens collected from HIV patients, reported increasing antibiotic resistance rates to aztreonam, cefepime, levofloxacin, meropenem, piperacillin, piperacillin-tazobactam, ticarcillin, and tobramycin in P. aeruginosa clinical isolates in this patient cohort [308]. Similar to HIV patients, in a 10-year study involving 149 solid organ transplant recipients, 43% of P. aeruginosa clinical isolates from transplant recipients were reported to be MDR [309].

The Mechanisms of Antibiotic Resistance
The mechanisms of antibiotic resistance in P. aeruginosa have been reviewed extensively elsewhere [310][311][312][313]. In brief, resistance to antibiotics in P. aeruginosa is multifactorial involving various intrinsic (inherent) and extrinsic (acquired) mechanisms. The intrinsic mechanisms of antibiotic resistance in P. aeruginosa include: (i) expression of porin molecules (e.g., OprF) that are considerably more restrictive to antibiotics entry into P. aeruginosa as compared to other Gram-negative bacteria such as E. coli [314,315]; (ii) reduction in the expression of outer-membrane porins, which further renders bacterial cell wall less permeable to antibiotics [313,316]; (iii) expression of various efflux pumps (e.g., MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM), which impart resistance to β-lactams, fluoroquinolones, and aminoglycosides by pumping out these antibiotics thus reducing their effective concentrations in P. aeruginosa cytosol [313,[317][318][319]; (iv) biofilm production which imparts resistance to antibiotics through various mechanisms-(discussed in [320][321][322][323][324])-including (a) biofilm-specific protection against oxidative stress induced by bactericidal antibiotics, (b) biofilm-specific expression of efflux pumps; (c) the protection provided by matrix polysaccharides through chemical interactions between the chemical functional groups in biofilm and antibiotics, thus limiting their accessibility to biofilm bacteria, and (d) the reduced metabolic state of biofilm bacteria; (v) emergence of persister and antibiotic tolerant phenotypes-(due to metabolic slowdown, oxygen limitations, and stress conditions)-that substantially increases resistance to antibiotics in otherwise sensitive bacteria, particularly in chronic infections [325][326][327][328][329]; (vi) mutations and polymorphism in the gene targets of antibiotics which reduce drug/target interactions, such as, quinolone resistance due to mutation in DNA Gyrase gene [330][331][332].
The extrinsic mechanisms of antibiotic resistance include acquisition of resistance genes through horizontal gene transfer (HGT) mechanisms, such as transformation, transduction, conjugation, transposons, outer membrane vesicles (OMVs), and insertion elements [333,334]). These resistance genes can inactivate antibiotics by hydrolysis, such as metallo or extended spectrum β-lactamases; or by changing antibiotics through struc-tural modifications, such as aminoglycoside modifying enzymes (e.g., acetyltransferases, nucleotidyltransferase, and phosphotransferases) [289,335].

Emerging Therapies to Combat P. aeruginosa Infections
As discussed above, antibiotics are routinely prescribed for the treatment of P. aeruginosa infections. While antibiotics have saved numerous lives over the past 9 decades, their use is not without its problems. First, the widespread use of antibiotics has led to an explosion of antibiotic resistance [336][337][338]. Second, currently, there is no antibiotic in the market that is effective against all bacterial pathogens, therefore, choosing the right antibiotic for prophylaxis use is crucial [339]. The choice of prophylaxis antibiotic is empirical in that it is based on the most probable cause of infection [340,341]. Prophylaxis antibiotics could fail if the patient encounters a different pathogen or a pathogen that is resistant to the administered antibiotic [342]. Third, antibiotics have many undesirable and dangerous side-effects; including nephrotoxicity, ototoxicity, hepatotoxicity, acute renal failure, and dysbiosis in the gut microbiota which itself has been associated with obesity, diabetes, and immunological and neurological diseases such as Parkinson disease [343][344][345]. Forth, prophylactic antibiotic use is associated with increased risk of infection with Clostridium difficile which is one of the deadliest causes of nosocomial infections, costing approximately $1.5 billion annually in USA alone [346][347][348][349]. Fifth, antibiotic use has been shown to interfere with healing processes at surgical sites [350]. In this section, we discuss the antibiotic-free strategies that are gaining tractions in dealing with P. aeruginosa infections and possible limitations associated with them (summarized in Table 3).

Immune System-Based Approaches against P. aeruginosa Infections
Innate immune system possesses powerful cellular, (e.g., neutrophils and macrophages), and humoral (e.g., antimicrobial peptides and complement), components that destroy pathogens via many mechanisms; such as phagocytosis, bursts of reactive oxygen and nitrogen species (ROS and RNS), antimicrobial peptides (AMPs) and complement-mediated direct microbial killing via membrane attack complex (MAC), and indirect microbial killing through opsonization and phagocytosis, and neutrophil extracellular trap (NET) [351][352][353][354][355]. Below, we discuss the two main immune system-based approaches to control infection.

Antimicrobial Peptides (AMPs)-Based Approaches against Bacterial Infections
AMPs are natural peptide-based antibiotics which are expressed by almost all life forms, including humans [356]. Generally, AMPs destroy pathogens by attacking and permeabilizing their membranes but there are some AMPs that kill their targets via different mechanisms such as modulation of membrane fluidity, and inhibition of intracellular pathways such as DNA replication and protein synthesis [356,357]. AMPs have been studied as promising new therapies to combat infections and some of them are even in clinical trials [358,359]. In one cell-culture based study, LL-37 (also known as cathelicidin) or cecropin(1-7)-melittin A(2-9) amide (CAMA) AMPs were shown to reduce the minimum biofilm eradication concentrations (MBEC) against biofilm P. aeruginosa by 8-fold [360]. In another in vitro study, immobilized Melimine and Mel4 chimeric cationic AMPs were shown to reduce biofilm P. aeruginosa viability by 82% and 63%, respectively [361], highlighting their therapeutic potential against device associated P. aeruginosa infections, such as catheter-associated pneumonia. In a rat model of systemic infection with P. aeruginosa, the AMPs (Magainin II and Cecropin A) exerted strong antimicrobial activity and achieved a significant reduction in bacterial levels, plasma endotoxin, and TNF-α concentrations when compared with control and rifampicin-treated groups [362]. Rifampicin and Magainin II or Cecropin A combined therapies showed synergistic effect in reducing infection and mortality rates when compared with singly treated and control groups. In another set of in vitro and in vivo lung infections, peptide ZY4 (a cathelicidin mimetic) was shown to be highly effective against standard and clinical MDR P. aeruginosa and Acinetobacter baumannii, strains in a mouse septicemia infection model [363]. Importantly, ZY4 showed low propen-sity to induce resistance, eradicated biofilm bacteria, (even killing persister bacteria), and decreased susceptibility to lung infection by P. aeruginosa and suppressed dissemination of P. aeruginosa and A. baumannii to other organs in mice. However, notable limitations have also hampered AMPs' therapeutic use (including AMPs' inherent cellular and tissue toxicities, potential limitations with their spectrum of activities against pathogens, and emergence of resistance to AMPs [359,364,365]. 7.1.2. Immunomodulator-Based Approaches against P. aeruginosa Infections In this approach, immunomodulators-primarily proinflammatory cytokines, such as CCL3, or bacterial products, such as N-Formylmethionine-leucyl-phenylalanine (fMLF)-are used to mobilize and activate innate immune responses at the possible site(s) of infection [8,188,189]. Immunomodulator-based approaches to control infection theoretically have many advantageous over antibiotics, although they could also be used in combination with antibiotics to further enhance their effectiveness. First, it is highly unlikely for a pathogen to develop resistance to all antimicrobial weapons that our immune system has at its disposal, including phagocytosis, bursts of reactive oxygen species (ROS), hypochlorous acid (HOCl), neutrophil and macrophage extracellular traps and antimicrobial peptides as discussed above. Second, immunomodulator-based therapies would not be empirically based because, once mobilized to the site of infection, immune system could in theory be effective against majority of infections regardless of their origin (bacterial, fungal, or viral). Third, many pathogens, (including P. aeruginosa), possess stealth mechanisms that allow them to establish infection even in immunocompetent healthy individuals by dampening host's immune responses [7,[366][367][368][369][370]. Immunomodulators could potentially overcome at least some of these stealth strategies and fortify tissue's defenses against invading pathogens by mobilizing immune responses to the site of these stealth pathogens. Forth, immunomodulators likely have fewer undesirable side-effects and may be safer than antibiotics. For example, it is unlikely that topical application of immunomodulators would result in development of bacterial resistance, gut dysbiosis, or organ damage (all side-effects associated with the use antibiotics as discussed above).
In a wound model of infection in diabetic mice, Roy et al. demonstrated that one-time topical treatment with low level CCL3 reduced P. aeruginosa infection by >99% by enhancing the neutrophil response in diabetic wounds [8]. Importantly, CCL3 treatment did not lead to persistent non-resolving inflammation in diabetic wounds, which is the hallmark of diabetic foot ulcers [371,372]. Rather, inflammation subsided over time and CCL3-treated diabetic wounds healed substantially better even in the presence of P. aeruginosa infection, which has been shown to exacerbate healing and contribute to heightened inflammatory environment in diabetic wounds as they age and become chronic [9,103,373]. In another wound infection model in immunocompetent C57BL/6 mice, Mahmud et al. recently demonstrated that one-time topical applications of CCL3, fMLF, or LPS immunomodulators were as effective, (if not more), as prophylactic tobramycin, in reducing P. aeruginosa infection (by nearly 90%) in wound [188]. Interestingly, these immunomodulators did not adversely impact healing processes. Rather, they even modestly stimulated wound healing in this immunocompetent animal even in the absence of infection. In another study involving periprosthetic implant joint infection with Staphylococcus aureus, Hamilton et al. demonstrated treatment with fMLP significantly reduced S. aureus infection levels by >90%. In addition, fMLF therapy reduced infection-induced peri-implant periosteal reaction, focal cortical loss, and areas of inflammatory infiltrate in mice distal femora. Importantly, fMLP treatment reduced pain behavior and increased weight-bearing at the implant leg in infected mice. These reports highlight the therapeutic potential of immunomodulators in reducing infections and potentially as stimulants in wound healing even in uninfected animals. The possible drawback against this approach is their possible ineffectiveness in severely immunocompromised patients who may lack the ability to mount effective immune responses to immunomodulators.

Phage-Based Therapeutics against P. aeruginosa Infections
Lytic bacteriophages (also known as phages) are viruses that selectively target and kill bacteria by lysis [374,375]. Phage effective killing of P. aeruginosa was first reported in 1957 by Kellenberger et al. [376]. Since then, various animal models and clinical studies have demonstrated the effectiveness of phages as alternative or adjunctive therapies with antibiotics to treat P. aeruginosa infections particularly when infections with MDR or XDR P. aeruginosa strains are suspected [374,[377][378][379][380]. In a mouse burn wound model of fatal infection with P. aeruginosa, McVay et al. demonstrated that phage cocktail consisting of three different P. aeruginosa phages was effective in increasing survival rates by 87%, (when administered via intraperitoneal (i.p.) route), as compared to only 6% in the sham control group [379]. In a CF zebra fish model, phage therapy was shown to substantially lower bacterial burden, reduce proinflammatory response, and significantly decrease lethality, caused by P. aeruginosa infection [380]. In a case report involving a patient with a pan drug-resistant P. aeruginosa spinal abscess, local and intravenous injections of a purified personalized phage cocktail adjunct therapy with antibiotics was able to heal this patient, despite the strain's resistance to all antibiotics including ceftazidime/avibactam, ceftolozane/tazobactam, and colistin [377]. In another case report involving prosthetic vascular graft P. aeruginosa infection, single application of phage OMKO1 and ceftazidime was sufficient to resolve infection with no signs of recurrence [381]. Despite these successful cases, clinical trials of phage therapies show low to moderate efficacy with large variations in infection clearance between subjects within the studies [382]. In addition, phage therapies also have their own set of limitations, including development of resistance to phages in bacteria, the requirement for high dose of viral particles, and the potential serious but not life-threatening side-effects [382][383][384].

Therapeutics Targeting P. aeruginosa Virulence Factors
P. aeruginosa pathogenesis is due to an arsenal of cell-associated and secreted virulence factors [385]. Therefore, disarming P. aeruginosa by reducing its virulence in vivo is a logical and promising therapeutic strategy that is gaining momentum [386][387][388][389][390][391]. For example, the T3SS virulence structure is an essential virulence structure for T3SS-expressing P. aeruginosa strains during infection and without it, this organism is rendered avirulent and cannot cause disease [9,392,393]. In a recent study, INP0341, a T3SS inhibitor, was shown to attenuate corneal infection by P. aeruginosa in an experimental model of murine keratitis [394]. In another study, monoclonal antibodies against T3SS structural component PcrV provided strong prophylactic protection in several murine infection models and a post-infection therapeutic model [389]. Quorum sensing (QS) is another major virulence mechanism that regulates the expression of many virulence factors and biofilm in P. aeruginosa [395]. In one study, sitagliptin (a QS inhibitor) was shown to inhibit pyocyanin, hemolysin, protease, and elastase in addition to blocking swimming, swarming and twitching motilities, and biofilm formation in P. aeruginosa [390]. In another recent report, the FDA-approved drug allopurinol showed anti-QS activity against P. aeruginosa and reduced the infiltration of P. aeruginosa and leucocytes and diminished the congestion in the liver and kidney tissues of infected mice [396]. Other QS inhibitors have also shown effectiveness in the treatment of drug resistant P. aeruginosa [397]. In another report, inhibition of Pseudomonas aeruginosa secreted virulence factors reduced lung inflammation in CF mice [398]. Other virulence factors (e.g., pyoverdine, AlgR, CdpR, RpoN, CysB, and AnvM) have also been investigated as potential drug targets to inhibit P. aeruginosa virulence during infection [399].

Vaccine Development against P. aeruginosa
With the rapid rise in the MDR and XDR P. aeruginosa clinical strains, it is absolutely vital to develop effective vaccines against P. aeruginosa. To date, there have also been several vaccine candidates developed against P. aeruginosa membrane and LPS O antigens, outermembrane porin proteins, the T3SS structural components (PcrV) and secreted effector peptides (ExoU), and combinations of these factors [400][401][402][403][404][405]. One study assessed the effectiveness of oral and intraperitoneal (i.p.) vaccinations with attenuated Salmonella enterica serovar Typhimurium SL3261 expressing P. aeruginosa serogroup O11 O antigen against P. aeruginosa infection in an acute fatal pneumonia model in BALB/c mice [406]. They reported that while both modes of immunization elicited O11-specific serum immunoglobulin G (IgG) antibodies, IgA was observed only after oral immunization. They further showed that oral vaccination significantly increased survival in an acute fatal P. aeruginosa pneumonia model. In another study, a multivalent live-attenuated mucosal vaccination (combining up to 4 attenuated strains having different LPS serogroups) elicited opsonophagocytic antibodies, which were directed not only to the LPS O antigens but also to the LPS core and surface proteins, correlated with protective immunity to P. aeruginosa lung infections [407]. In another study, vaccination with Pseudomonas aeruginosa outer membrane vesicles (PA_OMVs), formulated with aluminum phosphate adjuvant, was shown to reduce bacterial colonization, cytokine secretion and tissue damage in the lung tissue, thus protecting mice from lethal challenge of P. aeruginosa [408].
As for vaccine candidates against the T3SS, active and passive immunization with the PcrV antigen was shown to protect mice against P. aeruginosa-induced lung inflammation and injury and ensured the survival of challenged mice [403]. Interestingly, antibodies to PcrV also inhibited the translocation of type III toxins, thus reducing P. aeruginosa pathogenesis. In a follow up study, intramuscular injection of PcrV NH (a chimeric derivative of PcrV which contains the N-terminal domain (Met1-Lys127) and H12 domain (Leu251-Ile294) of PcrV)-elicited a multifactorial immune response and conferred broad protection in an acute P. aeruginosa pneumonia model and was equally effective to full-length PcrV [405]. Moreover, passive immunization with anti-PcrV NH antibodies also showed significant protection, at least based on inhibition of the T3SS and mediation of opsonophagocytic killing activities. An important limitation with T3SS-directed vaccines is the emergence of PA7-like clade of P. aeruginosa strains which lack T3SS [409][410][411].
Vaccine candidates against recombinant outer membranes OprF-OprI conjugates have been shown to be well-tolerated in healthy volunteers patients following mucosal administration [401]; or in patients with severe burns following intramuscular injection [402]. These vaccines have also been shown to elicit specific serum IgG and s-IgA antibodies in these patients, respectively [401,402]. Supporting these findings, in a clinical trial study involving 48 volunteers in six vaccination groups with either a systemic, a nasal, or four newly constructed oral live vaccines based on attenuated live Salmonella, expressing P. aeruginosa OprF-OprI recombinant as antigen, it was reported that while systemic and mucosal vaccines induced a comparable rise of serum antibody titers, a significant rise of IgA and IgG antibodies in the lower airways was only noted after nasal and oral vaccinations [412]. Disappointingly, in a randomized placebo-controlled phase II study in ventilated ICU patients, OprF-OprI vaccination (via intramuscular injection) produced a significant immunogenic effect (increases specific IgG), but it did not reduce infection rates [404]. Despite these considerable efforts, no vaccine has yet been found to be clinically efficacious against this pathogen. The main obstacles to achieve this goal include poor immunogenicity of the protective epitopes, a large variant subtype antigens, leading to high degree of serologic variability, a large genome facilitating adaptation to new environments, phenotypic plasticity between acute and chronic infection, and variations in animal responses to P. aeruginosa that make determination of the optimal vaccine formulations difficult from such studies [400,413]. 7.5. Other Antibiotic-Free Therapies for P. aeruginosa Infection 7.5.1. Silver Silver has been recognized for its antimicrobial properties for centuries and because it is considerably less toxic to human cells than pathogens, it has been used in a variety of applications to prevent infection [414]. Silver has a broad spectrum of antibacterial, antifungal and antiviral properties. Many mechanisms have been postulated to underlie silver antimicrobial action, including cell wall and cytoplasmic membrane disruption, inhi-bition of protein synthesis machinery by denaturation of ribosomes, interference with ATP production and chemiosmosis, production of ROS, and interference with DNA replication machinery (reviewed in [415]). Silver has been shown to be very effective against both planktonic and biofilm P. aeruginosa, although higher silver concentrations are needed for it to be effective against biofilm P. aeruginosa [416]. In a murine model of wound infection, silver nanoparticles (AgNPs) were shown to reduce P. aeruginosa infection by nearly 5 log-orders as compared to control wounds on day 4 post-infection and stimulate complete wound closure by day 12, although AgNPs in combination with tetracycline was more effective [417]. In another study, silver nanocomposite was used to deliver two pharmaceutical compounds (alginate lyase and ceftazidime) to degrade the alginate and eradicate biofilm P. aeruginosa from the lungs. Silver nanocomposites displayed a high dispersity, good biocompatibility, and high ceftazidime-loading capacity, and a low pH-dependent drug release and degradation profiles [418]. Importantly, they were highly effective in eradicating P. aeruginosa from the mouse lungs and decreasing the lung injuries. In another wound infection studies in diabetic mice, combination of AgNPs-immobilized on chitin-nanofiber sheet (CNFS)-and weakly acidic hypochlorous acid (HClO) was shown to significantly reduce P. aeruginosa infection burden and stimulate healing in an otherwise impaired healing animal model [419]. Because of its potent antimicrobial property, silver is routinely used as an adjunct in wound dressings [420]. However, because it can also be toxic to keratinocytes, it also has the potential to impair healing [420]. The application of silver as an adjunct therapy on medical devices may not be suitable. In a large randomized clinical trial involving 1309 hospitalized patients, coating urinary catheter with silver was not only ineffective in reducing the incidence of bacteriuria, it actually increased infection rates in male patients, particularly with S. aureus [421].

Honey
Honey has long been known for its antimicrobial and healing properties, dating back to ancient times, well before we gained the knowledge that microorganisms could cause infection and disease [422]. Honey exhibits a broad-spectrum of antibacterial activity against both Gram-positive bacteria and Gram-negative bacteria [423]. The antibacterial activity of honey has been attributed to several factors, including its high viscosity, its simple and complex sugar contents, its mild acidic nature, its hydrogen peroxide content, and its high levels of phenolic compounds with antimicrobial properties [424,425]. Lu et al. recently demonstrated that honey, at substantially lower concentrations compared to those found in honey-based wound dressings, inhibited P. aeruginosa biofilm formation and significantly reduced established biofilms [426]. In another recent investigation, Bouzo et al. demonstrated that no single component of honey can account for its total antimicrobial action against P. aeruginosa [427]. They further showed that honey affects the expression of many genes, particularly the genes involved in the electron transport chain maintenance, causing proton leakage across membranes, and inducing membrane depolarization and permeabilization in P. aeruginosa. A randomized clinical trial (105 patients) to compare a medical grade antibacterial honey (Medihoney™) with conventional treatments in wound care, reported clinical benefits from using honey in wound care with 23% fewer episodes of infection [428].

Hyperbaric Oxygen Therapy (HBOT)
HBOT is used as an adjunctive therapy in the management of infections such as diabetic foot, osteomyelitis, gas gangrene, necrotizing fasciitis, and fungal infections [429]. The underlying mechanisms of HBOT's bactericidal functions involve activation of innate immune system (via restoration of neutrophils' bactericidal functions under hypoxic environment or diabetic condition), and enhancement of ROS production in targeted bacteria [191,429,430]. In another study to evaluate the therapeutic potential of HBOT as an adjuvant to tobramycin treatment, HBOT was able to significantly enhance the effect of tobramycin against aggregates of all the P. aeruginosa isolates from CF patients in vitro.
The effect was attributed to increased O 2 levels [431]. In experimental subcutaneous and pulmonary rat infection models, oxygen therapy was shown to significantly reduce P. aeruginosa titers in the blood and bronchial aspirates, and protected animals against infection-induced mortality and morbidity as compared to control group [432]. In combination with amikacin antibiotic, these effects were even more impressive, highlighting HBOT's potential as an adjunctive therapy. Although the impact of HOBT on P. aeruginosa infection has not been directly examined, two systemic reviews of reports on randomized controlled trials comparing the effects of therapeutic regimens which include HBOT with those that exclude HBOT (with or without sham therapy)-have found HBOT to be beneficial for the treatment of necrotizing infections and to improve wound healing in diabetic ulcers and ischemic leg ulcers [433,434].

Negative Pressure Wound Therapy (NPWT)
NPWT has been shown to reduce surgical site infections (SSIs) by reducing fluid accumulation within the avascular dead space in a closed wound [435,436]. NPWT was shown to reduce infection and decrease mortality in a murine model of burn wound sepsis with P. aeruginosa [437]. In a rabbit wound model of infection, NPWT was shown to be significantly more effective than the control treatment (a sterile gauze dressing) in reducing the expression of virulence factors and P. aeruginosa bacteria counts [438]. In another report, NPWT was shown to reduce the mobility of P. aeruginosa and enhance wound healing in a rabbit ear biofilm infection [439]. NPWT was also shown to inhibit the invasion and proliferation of P. aeruginosa in burn-wounded tissue and decrease mortality in a murine model of burn-wound sepsis [437].

Concluding Remarks
Pseudomonas aeruginosa is a serious pathogen that can cause deadly acute and chronic infections particularly in immunocompromised hosts and CF patients. What makes this pathogen so successful in colonizing diverse environments within its host are its large genome, which gives this pathogen the metabolic flexibility to quickly adapt to changes within its environment; and its large arsenal of cell-associated and secreted virulence factors, which protect this pathogen from recognition and attack by host's immune responses. What is alarming about P. aeruginosa is its high intrinsic and acquired resistance to many available antibiotics which further highlights the need and the impetus for the development of novel strategies to deal with this organisms. Intratracheal inoculation in various animal models infection [155,156,[158][159][160][161][162]440] Keratitis and corneal ulcers [52,53] 6.8% to 55% [72][73][74][75][76] Eye infection in murine models [178][179][180][181].

Therapy Target Therapy imitations & Resistance Mechanisms
Antimicrobial peptides (AMPs) Membrane integrity, DNA replication, protein synthesis [356,357] Cellular toxicity, limited spectrum of activity, Multiple resistance mechanisms, including alteration in cell wall & degradation by proteases [359,364,365] Immunomodulators Activation of host cellular immunity [8,188] No cellular toxicity; Resistance not reported but highly unlikely as they activate multiple immune responses [8,188,189] Phage-based therapeutics Membrane lysis [374,375] -Low clinical efficacy, Development of resistance, & side-effects in patients [382][383][384] Therapies against Virulence factors -T3SS inhibition by small molecule or antibody [389,394] -Quorum sensing activity [390,394] -Secreted virulence factors [398,399] Not reported but bacteria can potentially become resistant to these therapies in similar mechanisms to antibiotics -Cytotoxic to keratinocytes [420] -Potentially ineffective as medical device coating [421] Honey Various [424,425] Not Reported

Hyperbaric oxygen therapy
Activation of innate immunity and enhanced ROS production in bacteria [191,429] Not Reported