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New perspectives in the management of Pseudomonas aeruginosa infections

Dianella Savoia*

ABSTRACT: Infections with Pseudomonas aeruginosa are a major health problem, especially for immune-compromised and cystic fibrosis patients, owing to the particular drug resistance of the microorganism. The aim of this review is to provide recent insights into strategies under investigation for prevention and therapy of these infections. In this survey, the approach directed against bacterial biofilm formation and quorum-sensing systems was focused, along with the evaluation of the treatment with bacteriophages. New interesting, developmental studies and clinical trials to prevent or treat infections due to this opportunistic pathogen are based on active and passive immunotherapy. Some monoclonal antibodies and different vaccines against this microorganism have been developed in the last few decades, even though to date none of them have obtained market authorization. Pseudomonas aeruginosa (PA) is a Gram-negative opportunistic microorganism that can be found in the environment. It is an important nosocomial pathogen, which causes different severe acute and chronic infections. Moreover, infections with PA are a major health problem for immunecompromised patients and individuals with cystic fibrosis (CF) [1,2] . In fact, given the ubiquitous presence of this microorganism, it is understandable that the healthy immune system is quite capable of controlling infections. However, susceptible individuals, particularly those affected by HIV infection, recipients of transplanted organs, cytotoxic drug addicts or burn patients with vascular damage hindering localized phagocytosis, frequently suffer from infections caused by this pathogen [3,4] . Impaired respiratory clearance mechanisms are present in patients with bronchiectasis, a condition that predisposes to colonization and infection by PA. Chronic infections of the respiratory tract are a major cause of the increased morbidity and mortality of individuals with CF [5–7] . The prevalence of PA infection varies widely among CF centers [8] . According to our previous results obtained by assessing microorganisms isolated from the expectorations of CF adult patients attending a CF referral center over a period of 5 years [9] , the mean prevalence of PA infection during the period of the study was 44%, similar to percentages found in Naples (Italy) by Lambiase et al. [10] and in Germany by Valenza et al. [11] . PA, which can survive in different physical conditions, can be responsible for infections determined by medical devices and related to hospital environments. This microorganism is particularly resistant to the current antibiotic arsenal; in fact, it displays intrinsic multidrug resistance (MDR) and has a tremendous capacity to acquire further resistance mechanisms [12,13] . Recently, widespread extensively drug-resistant PA clones have been reported [14] . Moreover, during chronic infections, theorganism can sometimes adopt a mucoid phenotype and is also thought to adopt a biofilm-like mode of growth, resulting in protection from host immune and antibiotic attack, with an oxygen limitation and low bacterial metabolic activity [15] .

KEYWORDS 

• bacteriophages • biofilm • cystic fibrosis • immunotherapy • nanoparticles • Pseudomonas aeruginosa • quorum sensing

*Department of Clinical & Biological Sciences, University of Torino, S Luigi Gonzaga Hospital, Regione Gonzole 10, 10043 Orbassano, Torino, Italy; Tel.: +39 011 670 5427; Fax: +39 011 236 5427; [email protected]

10.2217/FMB.14.42 © 2014 Future Medicine Ltd

Future Microbiol. (2014) 9(7), 917–928

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Review Savoia Virulence factors PA harbors an impressive range of virulence factors, many of which are controlled by the quorum-sensing (QS) system [16] . Among these virulence-related factors, there are type II, III and VI secretion systems and their associated effector proteins, such as extracellular proteases and phospholipases and the type III secreted toxins (type III secretion system [TTSS]) ExoU, S, T and Y. Our results, obtained by analyzing PA strains sequentially isolated from the expectorations of CF adult patients [9] , noted the longtime persistence of the same strain, as previously reported [17–19] . We observed that in some PA strains collected from patients at different times, ExoU (both gene and secretion) was present only at the first diagnosis (initial strains) and absent in subsequently collected bacteria (unpublished). Other authors [20,21] have shown the importance of ExoU in acute invasive infections and Manos et al. [22] recently demonstrated lower levels of most virulence factors in PA strains subsequently collected (persister strains) from infants with CF. This decrease in virulence correlated with some differentially expressed genes such as oprG, lasB, rsaL and lecB [23] . PA, which produces flagella and type IV pili involved in motility and host cell adhesion, regulates the gene expression of these appendices and of genes involved in different metabolic functions [24] . Moreover, Workentine et al. [19] collected multiple isolates from individual sputum samples of a patient chronically colonized with PA and assayed them for different phenotypes, such as colony morphology, motility, QS, protease activity and others. They revealed that each phenotype displayed significant variations, indicating the presence of a large degree of ­individuality across phenotypes. Recently, Neidig et al. [25] defined the role of TypA, also named BipA, belonging to a superfamily of ribosome-binding GTPases (enzymes that can bind and hydrolyze guanosine triphosphate) and key proteins in many critical biological processes, in the pathogenesis and virulence of PA. These enzymes are involved in many chronic infections contributing to the formation of microcolonies on tissue surfaces. The maturation of biofilm determinates in bacteria resistance to antimicrobial agents and protection from the immune response of the host causing a difficult eradication [26] . Therefore, in CF patients, lung diseases are responsible for frequent hospitalization and intravenous

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antibiotic therapy determining a selective pressure of bacteria that adapt by changing different characteristics [27–29] . The QS is a cell-to-cell, density-dependent communication process that microorganisms use to regulate collective behaviors; this mechanism only regulates biofilm production under some laboratory conditions [16,30–31] . However, it can favor the tolerance to the action of antimicrobial agents, the production of virulence factors and the capacity to evade host defences [32] . The CF milieu directly and indirectly affects innate host defence, with effects such as volume and electrolyte imbalance, an altered mucus b­arrier and impairment of phagocytosis [6] . Therapy Recently, guidance for treatments and new formulations of old antibiotics, such as aztreonam, quinolones and amikacin in liposomal formulation, has been reported [32–34] . An efficacy was also observed by using other inhaled antibiotic therapies, such as colistin, levofloxacin, liposomal amikacin, fosfomycin/tobramycin and aztreonam lysine [33–38] . Other innovative aerosol formulations, such as liposomes and microspheres, were found to be effective for therapy and patients’ compliance [39] . A new mechanism of action on PA has been studied by Srinivas et al. [40] ; they synthesized a family of peptidomimetic antibiotics based on the antimicrobial peptide protegrin I, which revealed a nonmembrane-lytic mechanism of action. Recently, the inhibitory effect of 2-bromohexanoic acid on enzymes responsible for rhamnolipid and polyhydroxyalkanoic acid synthesis in PA was evaluated [41] , indicating the possible utilization of this compound in screening for antimicrobial agents based on new antimicrobial targets. The development of biofilm also affects accessibility to clearance by immune mechanisms, which also includes vaccine-induced immunity. Several novel therapeutic alternatives are under investigation to combat PA [39,42–44] , such as those directed against biofilm formation and QS systems along with bacteriophages and immunotherapies. The purpose of this article is to provide the reader with a panoramic view of the updated literature on the strategies of contemporary antiPseudomonas research, paying due attention to the already published reviews, and focusing

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New perspectives in the management of Pseudomonas aeruginosa infections  on the new perspectives that are in the most advanced stage of development. Biofilm & QS inhibitors As previously reported, the presence of bacterial biofilm has been considered an important factor responsible for chronic human infections. Recently, the effect of silver nanoparticles in the formation of biofilm in multidrugresistant strains of PA was assessed [45] , revealing its potential as an alternative therapy to reduce severity of disease due to these infections. The human innate immune system has developed to recognize and block biofilm development through the action of lactoferrin, which chelates iron prompting the bacteria to increase motility, rather than form a biofilm. Generally, bacterial cells link, aggregate and form the biofilm by using lectins, outer membrane proteins that recognize sugar residues, forming a barrier that prevents the process of bacterial clearance. Competitive inhibitors of the two specific lectins LecA and LecB (fucose and galactose moieties, respectively) block binding, dissolving or preventing biofilm formation [46] . The activity of these sugars was observed after inhalation treatment in a small randomized trial effected in CF patients with chronic PA infection. Therapeutic activity was demonstrated using multivalent dendrimers with fucose/ galactose attached [47] , linked to the binding of C-fucosyl group of the dendrimers in the same mode as fucose itself, establishing contacts with the lectin. The aqueous solubility of the fucosyl peptide dendrimers is generally very good and an enhanced binding of the dendrimers compared with monovalent ligand is caused by ­multivalency in architecture. These peptide dendrimers, in particular the LecB-specific glycopeptide dendrimers FD2 and D-FD2 and the LecA-specific glycopeptide dendrimers GalAG2 and GalBG2, also efficiently block PA biofilm formation and induce biofilm dispersal in vitro. They might eventually result in novel treatments as inhibitors of PA adhesion and biofilm production and these lines of research are currently in progress [47,48] . Some galactotripeptides were studied to optimize the peptide-lectin interactions; two of these bind strongly to the PA lectin LecA, and inhibit PA biofilms, as well as dispersing already established ones. Dendrimers based on these tripeptide ligands showed improved PA biofilm inhibition. The possibility of retaining and even improving

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Review

the biofilm inhibition in several analogs suggests that it should be possible to fine-tune this dendrimer toward therapeutic use by adjusting the pharmacokinetic parameters in addition to the biofilm inhibition [47–49] . Compared with non-CF sputa, the sputa of patients affected by CF are a rich source of iron, which correlates with chronic PA infection [50] . Iron acquisition is important for infection because this element is a micronutrient that PA can acquire by secreting pyoverdine and pyochelin, iron-chelating compounds (siderophores). Siderophore-mediated iron uptake is generally considered a key factor in the ability of PA to cause infection and in the CF airway the affinity iron-siderophores is higher in respect to serum proteins [51] . Therefore, a perturbation of iron acquisition systems has been proposed as a novel therapeutic approach to treat PA biofilm infection. To modify iron metabolism, iron chelation activity by gallium has been assayed. A safety study in patients with CF (NCT01093521) is underway using gallium nitrate (ganite) by intravenous infusion [52] . Coencapsulation of gallium with gentamycin in liposomes enhances in vitro activity of gentamycin against CF c­linical i­solates of PA [53] . In PA, QS regulates the bacterial expression of specific genes whose products modify the local host environment favoring the invasion and persistence of the pathogen. It is, therefore, considered an attractive therapeutic target and currently many compounds are being evaluated as QS antagonists representing a new family of anti-infective agents [42,54–55] . Recently, Migiyama et al. [56] demonstrated in a mouse model of acute pneumonia that some N-acyl homoserine lactone (AHL) molecules, regulating the production of various virulence factors in PA, were inactivated by AHL-lactonase enzymes, thus reducing the levels of proinflammatory cytokines and myeloperoxidase activity. Therefore, this hydrolase could be a potential therapeutic agent. Also ambroxol [2-amino3,5-dibromo-N-(trans-4-hydroxycyclohexyl) benzylamine], a mucolytic agent, interferes with PA QS reducing the content of extracellular polymeric substances produced by PA and consequently also biofilm thickness [57] . Tan et al. [58] , using a structure-based virtual screening for QS inhibitors in 3040 natural compounds and their derivatives, found five compounds, and in particular GI (5-imino-4,6-dihydro-3H-1,2,3-triazolo[5,4-d]pyrimidin-7-one),

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Review Savoia able to inhibit QS-regulated gene expression of virulence factors in PA, in particular protease IV, chitinase, elastase and pyoverdine synthetases. Also O’Loughlin et al. [59] recently found that some synthetic molecules derived from chlorolactone, and in particular meta-bromo-thiolactone, inhibited the two PA QS receptors, LasR and RhlR, and contemporarily the production of the virulence factor pyocyanin and biofilm formation. Anti-QS activity can be detected among drugs, such as niclosamide and flucytosine, already used in humans and that could easily be used in clinical applications. The anthelmintic drug niclosamide [60] strongly inhibits the QS response and production of acyl-homoserine lactone QS signal molecules, suppresses surface motility and production of the secreted virulence factors elastase, pyocyanin and rhamnolipids, and reduces biofilm formation. The antimycotic agent flucytosine [61] inhibits the expression of the iron-starvation σ-factor PvdS, thereby repressing the production of major PA virulence factors, namely pyoverdine, PrpL protease and exotoxin A. Besides the strategies reported in the past, recently, Christiaen et al. [62] assessed the biotherapeutic potential of quorum quenching bacteria previously isolated from environmental samples [63] , producing and secreting small compounds that inhibit QS, and others that quench QS by enzymatic degradation of AHLs. Some quorum-quenching bacteria, or the active compounds they produce, could be useful to attenuate virulence of PA PAO1 and possibly also other Gram-negative pathogens that use AHLs to regulate the production of virulence factors. Bacteriophages & phage-derived molecules Since the pioneer work of d’Herelle, several studies, mostly in eastern European countries [64–66] , have demonstrated that bacteriophages can be successfully used in the therapy of animal and human bacterial infections. Phage therapy is considered a potential treatment for some selected infections, such as MDR PA lung infection in CF patients; it would be advantageous as a kind of personalized medicine that could complement antibiotic treatment also because it reaches bacteria trapped inside biofilms. In Georgia purulent skin, wound and lung infections are often treated with therapeutic cocktails such as the ‘pyophage’ formulation that contains

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different phages against different target pathogens [67] . In wound infections caused by PA, there are significant problems and phage therapy has been proposed as a possible application due to the direct contact of bacteria and their viruses [68] . However, Essoh et al. [69] emphasize the difficulty in preparing a phage cocktail efficient on all bacterial species, and in particular on PA, owing to different lysis susceptibility of the strains. In fact, Henry et al. [70] demonstrated that the bacteriophages isolated directly on the targeted host were the most efficient in vivo, supporting the efficacy of a personalized approach. Another trend of research involves the use as anti-infective agents of purified phage products, such as lysins, bacterial cell-wall hydrolytic enzymes that kill Gram-positive and Gram-negative bacteria [71,72] . Bacteriophage endolysins are mureine-degrading enzymes, originally studied and developed to control mucous membrane infections. These are also denominated ‘enzybiotics’ [73] , phage-encoded PGH proteins, endolysins and virion-associated peptidoglycan hydrolases, enzymes that locally degrade the peptidoglycan of the bacterial cell wall during infection. The recent construction of a database and catalog of therapeutic enzybiotics [74,75] has increased the amount of available information on the potential of these bacteriolytic antimicrobials in research and preclinical trials. Immunotherapy To prevent or treat infections due to drugresistant bacteria, in particular in presence of impaired host defense, potential adjuvant therapies can be assessed. In nature’s repertoire of bactericidal compounds, hypothiocyanite and lactoferrin are deficient in CF subjects, thus altering local innate immunity. A product (MEVEOL), consisting of these two antimicrobial molecules, produced by ALAXIA (orphan drug designation) and scheduled for administration by inhalation [76] , has revealed a potential benefit for the treatment of lung infections [77,78] . In the group of proteins and peptides important in the innate immunity, lysozyme is an effective antimicrobial component present in airway secretions. Teneback et al. [79] recently constructed a charge engineered variant of human lysozyme that in a murine model revealed antibacterial and anti-inflammatory activity. Starting from the natural antimicrobial peptide protegrin I, a novel class of antibiotics

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New perspectives in the management of Pseudomonas aeruginosa infections  with a unique spectrum of antimicrobial activity was discovered by applying Protein Epitope Mimetics technology. These peptidomimetic antibiotics, synthesized by Srinivas et al. [40] , were effective against PA by targeting a protein, called LptD, involved in the formation of the outer membrane of Gram-negative pathogens, responsible for a perturbation of the critical lipopolysaccharide (LPS) transport function of LptD. Some compounds, characterized by a broad spectrum of antimicrobial activity similar to that of protegrin I, but reduced capacity to lyse human blood cells, were optimized by iterative cycles of peptidomimetic library synthesis and screened for antimicrobial activity. The compounds POL7001 and POL7080 proved to be peptidomimetics with high activity against PA, including a large panel of MDR clinical isolates, but only minimal activity against other Gram-negative or Gram-positive bacteria. These molecules, and in particular POL7080, which showed potent antimicrobial activity in a mouse septicemia infection model, in a Phase I clinical trial demonstrated antimicrobial activity, safety and tolerability [80] . Since a blockade in the autophagy pathway in CF patients was recognized which contributes to the killing of internalized PA in mast cells, thus increasing susceptibility to infection, the development of specific autophagy modulators, as well as therapeutic strategies for restoring normal autophagy in CF patients, might be an essential step. Therefore, pharmacological interventions targeting this pathway, in particular using rapamycin, could have therapeutic potential in the bacterial clearance [81] . ●●Anti-Pseudomonas IgY

An interesting approach to prevent PA colonization of the oropharynx and successive establishment of lung infection in CF patients is the oral prophylactic treatment with egg yolk antibodies against PA, anti-Pseudomonas IgY, which revealed a preventive potential in CF patients [82] and is presently in clinical studies (IMPACTT-FP7 projects-rare ­diseases-medical research, and European Cystic Fibrosis Society – clinical trials network). ●●Monoclonal antibodies

In many MDR microorganisms, a common surface polysaccharide, poly-b-1,6-N-acetylglucosamine, which mediates biofilm formation, has been recognized [83] . Antibodies to this

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Review

component determinate a protective immunity. A human monoclonal antibody (MAb F598) to poly-b-1,6-N-acetylglucosamine is currently in a Phase II trial in healthy adults for future use in therapy in patients infected by poly-b-1,6-Nacetylglucosamine-producing MDR bacteria or prophylaxis in patients at risk of developing MDR infections [83,84] . Different research groups recently studied the development of an innovative therapeutic strategy, an antibacterial drug in the form of an mAb targeting an exposed virulence factor. Adawi et al. [85] reported that a fully human IgG1 mAb, indicated as LST-007, targeting PA flagellin type b, recombinantly expressed and characterized in vitro and in an infection model, demonstrated a highly specific binding toward an MDR PA strain. Another mAb was designed using singlechain variable fragment phage libraries derived from healthy individuals and patients convalescing from PA infections [86] , approaching three distinct epitopes of the exopolysaccharide Ps1, important for PA attachment to mammalian cells, and for the formation and maintenance of biofilms. In multiple protection animal models, Ps1 was found to be an accessible serotype-independent surface feature and a promising novel protective antigen for preventing PA infections. Other interesting results have been obtained using a different particular approach on PA TTSS to prevent the killing of immune cells and to reduce inflammatory cytokine release, without developing resistance, unlike the results with antibiotics. The Mab166, a murine mAb against PcrV, a protein located at the tip of the injectisome of PA TTSS, which enables bacteria to kill immune cells by direct puncture (oncosis) or injection of protein toxins, has been found to be effective against PA infection [87] . This mAb, in association with an antibiotic, improves the survival of PA-infected mice. Another anti-PcrV study was realized by KaloBios with the Mab KB001, which demonstrated safety and tolerability [88,89] . At present, there are randomized, double-blind, placebo-controlled clinical trials in Phase II using the Mab KB001-A, a humaneered, high-affinity, PEGylated Fab’ antibody [90] . This product was developed by KaloBios in association with the partner Sanofi Pasteur and is under evaluation in CF patients chronically infected with PA and for the prevention of mechanical ventilator-associated pneumonia caused by this microorganism. A fully human monoclonal anti-LPS IgM, Panobacumab, contributes to the

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Review Savoia clearance of PA also in combination with antibiotics; this antibody proved to be effective on acute pneumonia in mice [91] and safe and useful in critical patients in a Phase IIa clinical trial [92] . ●●Vaccines

A vaccine against PA has long been sought after, but is so far unavailable. Several vaccine candidates have been developed and assessed in the past in experiments with animals and humans, but only a few have reached clinical phases and none of these vaccines has obtained market authorization. Some recent reviews [93–95] report the current state of vaccine development for this ubiquitous pathogen to provide in particular mucosal immunity against infections of the respiratory tract in susceptible individuals with CF. Some years ago, the most promising vaccine antigens included the LPS O antigen, polysaccharide-protein conjugates, flagella, outer membrane proteins, pili, exotoxins and proteases, as reported in a review by Doring and Pier [96] . Purified alginate was injected into a large number of healthy volunteers in the 1990s, but only low opsonic antibody titers were produced and the nonconjugated alginate vaccine is no longer being investigated. The eight-valent conjugate vaccine Aerugen developed by Berna Biotech was used for the prevention of infection and the preservation of lung function in CF patients in 46 centers in four European countries, but the study was stopped in 2006 by the manufacturer Crucell (Berne, Switzerland), because the result of this Phase III study failed to confirm its efficacy. Another vaccine LPS-based (Pseudogen) was found to be ineffective both in CF patients already infected and in other patients without the pathogen; owing to the adverse reactions, its development has been stopped [96,97] . Therefore, purified PA polysaccharides and alginate vaccine candidates proved to be poorly immunogenic, whereas the LPS antigens induce a high level of opsonophagocytic and protective immunity differing for each serotype; the outer membrane proteins present mostly conserved antigens, even if no large clinical trials were effected [96] . Flagellin monomers were found to be more immunogenic even if a good immunity to PA required additional component proteins [97] . Bumann et al. [98] in a clinical trial assessed systemic, nasal and oral live vaccines against PA

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based on attenuated live Salmonella followed by a systemic booster vaccination. Nasal and oral outer membrane protein F (OprF)-OprI (recombinant fusion protein of the highly conserved PA outer membrane proteins) vaccines induced a specific antibody response in the lung. However, a recent Cochrane Database [99] , comparing different trials on PA vaccines (oral, parenteral or intranasal) with control vaccines or no intervention in CF, concluded that vaccines against PA cannot be recommended. Different approaches were studied by other authors. Staczek et al. [100] , using one OprFbased vaccine, called F/I, containing carboxy oprF sequences fused to oprI in an expression vector, delivered three-times biolistically by gene gun in mice, demonstrated that this vaccine induced protection and that the protective role was played by cell-mediated immunity. Wu et al. [101] , using a library of in vitro transcribed and translated PA Th17-stimulating protein antigens, identified that PopB, one of the hydrophobic translocator subunits of the TTSS for the injection of toxins directly into host cells, was a promising vaccine candidate per PA. Recently, Ad vectors were studied [102] as delivery vehicles for genetic vaccines. A good epitope-specific immunity was shown using epitopes incorporated into the Ad fiber protein owing to their ability to act as immune system adjuvants evoking good immune responses against the transgene product and viral capsid proteins. In particular, studies effected in mice using Ad vectors expressing the 14-mer PA immune-dominant OprF epitope 8 in five particular sites of the Ad5 fiber proved to be a promising vaccine strategy. Other Ad vectors, using less prevalent human or nonhuman Ad serotypes, including nonhuman primates, such as AdC7, have been developed [103,104] . These vectors induced a superior protective immunity in the lung, in particular after a mucosal immunization. However, only a few mucosal vaccines have been developed for human use [105] owing to the need of adjuvants. The immunogenicity of a AdC7 mucosal vaccine with the addition of integrin-binding arginine-glycine-aspartic acid motif to the fiber was recently evaluated [106] , revealing the induction of a protective mucosal immunity against PA. Nanovaccines are a novel approach to combat diseases; this area of research has been particularly developed in recent years. Some characters of the nanosized particles, such as size and

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New perspectives in the management of Pseudomonas aeruginosa infections  surface properties including surface charge and hydrophobicity [107,108] , condition the interaction of nanovaccines and cells of the immune system. Taranejoo et al. [109] prepared chitosan microparticles with tripolyphosphate by spraydrying loaded with exotoxin A subunit antigen for intranasal vaccination against PA. This carrier system seems to be clinically acceptable for mucosal vaccine delivery to mucosal associated lymphoid tissue. Recently, a bovine serum albumin nanoparticle vaccine with several entrapped antigenic proteins derived from various parts of the bacterial cell extracted from PA, was tested on mice; this formulation revealed the capability to protect mice from nasal infection by PA, inducing a functional response and a reduction of inflammatory signs [110] . Conclusion Infections with PA represent a major health problem, in particular for immune-compromised patients and individuals affected by CF. This microorganism, which produces a large quantity of virulence factors, many of which are controlled by the QS system, displays a very large drug resistance. Moreover, during chronic infection, it can form a protective biofilm that further reduces the efficacy of existing antibiotics. Therefore, several novel therapeutic alternatives are under investigation, such as therapies directed against biofilm formation and QS systems. Among these are the peptide dendrimers, which act as inhibitors of PA adhesion and ­biofilm production. Since siderophore-mediated iron uptake is generally considered a key factor in the ability of PA to cause infection, and in the CF airway, the affinity iron-siderophores is higher in respect to serum proteins, a perturbation of iron acquisition systems, the use of gallium nitrate (ganite) by intravenous infusion has been proposed as a novel therapeutic approach to treat PA biofilm infection, and a safety study in patients with CF is underway. Moreover, phage therapy may be considered a potential treatment, especially bacteriophage endolysins, which are mureine-degrading enzymes, developed to control mucous membrane infections, and denominated ‘enzybiotics.’ Other new types of approaches to prevent or treat PA infections are based on active and passive immunotherapy. The bactericidal compounds of the local innate immunity, hypothiocyanite and lactoferrin, deficient in CF subjects, may be

future science group

Review

used as a drug product (MEVEOL, orphan drug designation) by inhalation for the treatment of lung infections. The benefits of this drug consist in the synergic antibacterial local mechanism of action with antibiofilm and anti-inflammatory activity associated to a low risk of toxicity. Starting from the natural antimicrobial peptide protegrin I peptidomimetic antibiotics were produced. These are effective against PA by targeting a protein involved in the formation of the outer membrane of Gram-negative pathogens. Recently, the peptidomimetic POL7080 successfully completed Phase I and will start Phase II clinical trial by Polyphor and Roche, a joint effort to target the ‘superbug’ PA [111] . To prevent PA colonization of the oropharynx and successive establishment of lung infection in CF patients, oral treatment with egg yolk antibodies against PA, anti-Pseudomonas IgY, which revealed a preventive potential in CF patients, is presently in clinical studies. A protective immunity has also been shown (and is currently in a Phase II trial in healthy adults) by using a human mAb (MAb F598) to a common surface polysaccharide that mediates biofilm formation. The product Mab KB001-A Panobacumab, a fully human monoclonal anti-LPS IgM [90] , developed by KaloBios in association with the partner Sanofi Pasteur, is under evaluation in CF patients chronically infected with PA and for the prevention of mechanical ventilator-associated pneumonia caused by this microorganism. A vaccine against PA is so far unavailable; several vaccine candidates have recently been developed and assessed in experiments with animals and humans, but only a few have reached clinical phases and none of these vaccines has obtained market authorization. A recent Cochrane Database review comparing different trials regarding PA vaccines concluded that to date vaccines against PA cannot be recommended. A further evolution to induce a higher mucosal and protective immunity in the respiratory tract utilizes Ad vectors expressing an immune-dominant OprF epitope suited as respiratory mucosal vaccines that seem to elicit robust and persistent antitransgene responses. Also the application of nanotechnology in vaccine development promoting mucosal antibody response and reducing inflammatory signs might give new strategies for the prevention or the treatment of diseases caused by PA, above all in immunosuppressed or CF patients.

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Review Savoia EXECUTIVE SUMMARY Background ●●

The increasing incidence of drug-resistant Pseudomonas aeruginosa (PA) observed in recent years suggests the need of alternative strategies to prevent and cure infections by this opportunistic microorganism.

●●

This article reports a panoramic view of the updated literature on the strategies of contemporary anti-Pseudomonas research.

Biofilm & quorum-sensing inhibitors ●●

Silver nanoparticles, lactoferrin and peptide dendrimers recently demonstrated a biofilm inhibitory activity.

●●

Gallium nitrate (ganite) is underway in a safety study in patients with cystic fibrosis (CF) to modify iron metabolism of microorganisms.

●●

A N-acyl homoserine lactone hydrolase, such as some natural compounds, reducing biofilm thickness, could be therapeutic agents.

●●

The antimycotic agent flucytosine and the anthelmintic drug niclosamide demonstrated antiquorum-sensing activity.

Phage therapy ●●

Phage therapy could be a complement of antibiotic therapy in the form of cocktails, such as the ‘pyophage’ formulation or ‘enzybiotics,’ bacteriophage endolysins that are mureine-degrading enzymes.

Immunotherapy ●●

In the presence of impaired host defense or in CF patients, there is a resurgence of interest for vaccination and immunotherapy.

●●

A product (MEVEOL), consisting of hypothiocyanite and lactoferrin, nature’s repertoire of bactericidal compounds, deficient in CF subjects, has revealed a potential benefit for the treatment of lung infections.

●●

A novel class of antibiotics, indicated as peptidomimetics, was discovered. Among these the compounds POL7001 and POL7080 proved to have high activity against PA.

●●

Anti-Pseudomonas IgY, egg yolk antibodies, represent an interesting approach to prevent PA colonization of the oropharynx and are presently in clinical studies.

●●

Among the monoclonal antibodies, the Mab KB001, an antibody anti-PcrV, demonstrated safety and tolerability.

At present, there are clinical trials using the Mab KB001-A, a humaneered, high-affinity, PEGylated Fab’ antibody. This product is under evaluation in CF patients chronically infected with PA and for the prevention of mechanical ventilator-associated pneumonia caused by this microorganism. Panobacumab, a fully human monoclonal antilipopolysaccharide IgM, is in a clinical trial in critical patients. Vaccines ●●

Several vaccine candidates have been developed and assessed in the past, but only a few have reached clinical

phases and none of these vaccines has obtained market authorization. Nasal and oral outer membrane protein F-OprI (recombinant fusion protein of the highly conserved PA outer membrane proteins) vaccines induced a specific antibody response in the lung. However, a recent Cochrane Database concluded that vaccines against PA cannot be recommended. ●●

A promising vaccine candidate per PA has been obtained using a library of in vitro transcribed and translated PA Th17stimulating protein antigens.

Future perspective ●●

Recently, Ad vectors were studied as delivery vehicles for the induction of a protective mucosal immunity against PA.

●●

Nanovaccines are a novel approach for the prevention and therapy of PA infections by inducing a functional response of the host and a reduction of inflammatory signs.

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future science group

New perspectives in the management of Pseudomonas aeruginosa infections  Future perspective The management of PA infections represents a major health problem owing to the particular drug resistance of the microorganism. Therefore, there is an urgent need to research novel strategies for discovering bioactive products that in the near future could become useful preventive and therapeutic tools. In nature’s repertoire of bactericidal compounds, a product (MEVEOL, orphan drug designation), consisting of hypothiocyanite and lactoferrin, two antimicrobial molecules that are deficient in CF subjects altering local innate immunity, is scheduled for administration by inhalation having revealed potential for the treatment of lung infections. Moreover, the use of some drugs, such as niclosamide and flucytosine, already used in humans for different purposes, or of peptide dendrimers, appears to be very interesting. References

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Williams BJ, Dehnbostel J, Blackwell TS. Pseudomonas aeruginosa: host defence in lung diseases. Respirology 15, 1037–1056 (2010).

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Cohen TS, Prince A. Cystic fibrosis: a mucosal immunodeficiency syndrome. Nat. Med. 18, 509–519 (2012).

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Gross J, Welch M. Why is Pseudomonas aeruginosa a common cause of infection in individuals with cystic fibrosis? Future Microbiol. 8, 1–3 (2013). 

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Summarizes the adaptive modifications of the Pseudomonas aeruginosa (PA) population in cystic fibrosis patients. Ciofu O, Hansen CR, HØiby N. Respiratory bacterial infections in cystic fibrosis. Curr. Opin. Pulm. Med. 19, 251–258 (2013).

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In particular, a peptidomimetic antibiotic, indicated as POL7080, and the human monoclonal anti-LPS antibody Panobacumab, are both presently being tested in clinical trials. Other studies are being developed to evaluate a personalized approach to therapeutic bacteriophages, and to apply nanotechnology in vaccine production promoting mucosal antibody response. Financial & competing interests disclosure The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. Leone I, Chirillo MG, Raso T, Zucca M, Savoia D. Phenotypic and genotypic characterization of Pseudomonas aeruginosa from cystic fibrosis patients. Eur. J. Clin. Microbiol. Infect. Dis. 27, 1093–1099 (2008).

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Microbiology of airway disease in a cohort of patients with cystic fibrosis. BMC Infect. Dis. 6, 4 (2006).

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C, Hancock RE. Pseudomonas aeruginosa: all roads lead to resistance. Trends Microbiol. 19, 419–426 (2011).  Authors report the adaptations and genetic switches of PA associated with drug resistance and maintenance of a chronic infection state.

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MA et al. Genetic markers of widespread extensively drug-resistant Pseudomonas aeruginosa high-risk clones. Antimicrob. Agents Chemother. 56, 6349–6357 (2012). 15 HØiby N, Ciofu O, Bjamsholt T.

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Authors evaluated the importance of biofilm formation in the persistence of chronic PA infection.

16 Girard G, Bloemberg GV. Central role of

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Review

18 Hogardt M, Heesemann J. Microevolution of

Pseudomonas aeruginosa to a chronic pathogen of the cystic fibrosis lung. Curr. Top. Microbiol. Immunol. 358, 91–118 (2013). •

Authors report the evolution of PA to maintain a chronic infection state.

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et al. Treatment of lung infection in patients with cystic fibrosis: current and future strategies. J. Cyst. Fibros. 11, 461–479 (2012).  •

Discusses the present status of antibiotic therapy for the major pathogens in cystic fibrosis airways and outline measures to optimize treatments using novel drug formulations.

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Pseudomonas aeruginosa infections in cystic fibrosis. BMC Med. 9, 32 (2011). 34 Okusanya OO, Bhavnani SM, Hammel J

et al. Pharmacokinetic and pharmakodynamic evaluation of liposomal amikacin for

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inhalation in cystic fibrosis patients with chronic pseudomonas infection. Antimicrob. Agents Chemother. 53, 3847–3854 (2009).

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IG et al. Inhibition of Pseudomonas aeruginosa biofilm formation by LecA-binding polysaccharides. Int. J. Antimicrob. Agents 42, 471–472 (2013).

35 Trapnell BC, McColley SA, Kissner

DG et al. Fosfomycin/tobramycin for inhalation in patients with cystic fibrosis with pseudomonas airway infection. Am. J. Respir. Crit. Care Med. 185, 171–178 (2012).

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et al. Glycopeptide dendrimers with high affinity for the fucose-binding lectin LecB from Pseudomonas aeruginosa. ChemMedChem 4, 562–569 (2009).

36 Hutchinson D, Barclay M, Prescott WA,

Brown J. Inhaled aztreonam lysine: an evidence-based review. Expert Opin. Pharmacother. 14, 2115–2124 (2013).

48 Reymond JL, Bergmann M, Darbre T.

Glycopeptide dendrimers as Pseudomonas aeruginosa biofilm inhibitors. Chem. Soc. Rev. 42, 4814–4822 (2013).

37 Gaspar MC, Couet W, Olivier JC. Pais AA,

Sousa JJ. Pseudomonas aeruginosa infection in cystic fibrosis lung disease and new perspectives of treatment: a review. Eur. J. Clin. Microbiol. Infect. Dis. 32, 1231–1252 (2013). •• Some innovative aerosol formulations, such as liposomes and microspheres, useful to prevent bacterial resistance, are reviewed.

49 Kadam RU, Bergmann M, Garg D et al.

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38 Stockmann C, Sherwin CM, Ampofo K,

Spigarelli MG. Development of levofloxacin inhalation solution to treat Pseudomonas aeruginosa in patients with cystic fibrosis. Ther. Adv. Respir. Dis. 8, 13–21 (2014).

51 Reid DW, Carroll V, O’May C, Champion A,

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39 Hurley M, Càmara M, Smyth AR. Novel

approaches to the treatment of Pseudomonas aeruginosa infections in cystic fibrosis. Eur. Respir. J. 40, 1014–1023 (2012).

52 A Pharmacokinetic and Safety Study of IV

Gallium Nitrate (Ganite) in Cystic Fibrosis Patients. http://clinicaltrials.gov/show/NCT01093521

40 Srinivas N, Jetter P, Ueberbacher BJ et al.

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53 Halwani M, Yebio B, Suntres ZE, Alipour M,

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41 Gutierrez M, Choi MH, Tian B et al.

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54 Bhardwaj AK, Vinothkumar K, Rajpara N.

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Antibiotic adjuvant therapy for pulmonary infection in cystic fibrosis. Cochrane Database Syst. Rev. 6, CD008037 (2013). 45 Palanisamy NK, Ferina N, Amirulhusni

AN et al. Antibiofilm properties of chemically synthesized silver nanoparticles found against Pseudomonas aeruginosa. J. Nanobiotechnol. doi:10.1186/1477-3155-12-2 (2014) (Epub ahead of print).

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Describes recent patents on various quorum-sensing inhibitors useful as alternatives for controlling different pathogens.

55 Jakobsen TH, Bjarnsholt T, Jensen PO

et al. Targeting quorum sensing in Pseudomonas aeruginosa biofilms: current and emerging inhibitors. Future Microbiol. 8, 901–921 (2013).  •• Reports state-of-the-art research of quorum-sensing inhibitors against PA. 56 Migiyama Y, Kaneko Y, Yanagihara K et al.

Efficacy of AiiM, an N-acylhomoserine

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  lactonase, against Pseudomonas aeruginosa in a mouse model of acute pneumonia. Antimicrob. Agents Chemother. 57, 3653–3658 (2013).

therapeutic phages to fight Pseudomonas aeruginosa in wound infections. Viruses 5, 15–53 (2013). susceptibility of Pseudomonas aeruginosa strains from cystic fibrosis patients to bacteriophages. PLoS ONE 8, e60575 (2013).

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Repurposing the antimycotic drug flucytosine for suppression of Pseudomonas aeruginosa pathogenicity. Proc. Natl Acad. Sci. USA 110, 7458–7463 (2013). 62 Christiaen SE, Matthijs N, Zhang

XH et al. Bacteria that inhibit quorum sensing decrease biofilm formation and virulence in Pseudomonas aeruginosa PAO1. Pathog. Dis. 70, 271–279 (2014). 63 Christiaen SE, Brackman G, Nelis HJ,

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M et al. European regulatory conundrum of phage therapy. Future Microbiol. 2, 485–491 (2007). 65 Harper DR, Morales S. Bacteriophage

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Bacteriophage endolysins as novel antimicrobials. Future Microbiol. 7, 1147–1171 (2012). 67 Chan BK, Abedon ST, Loc-Carrillo C. Phage

cocktails and the future of phage therapy. Future Microbiol. 8, 769–783 (2013). •• Discusses the commercial and medical context of phage cocktails as therapeutic agents. 68 Krylov V, Shaburova O, Krylov S et al. A

genetic approach to the development of new

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www.collegehill-lifesciences.com  81 Junkins RD, Shen A, Rosen K, McComick C,

Lin TJ. Autophagy enhances bacterial clearance during P. aeruginosa lung infection. PLoS ONE 8, e72263 (2013). 82 Nilsson E, Larsson A, Olesen HV, Wejaker

70 Henry M, Lavigne R, Debarbieux L.

of five structurally quorum-sensing inhibitors of Pseudomonas aeruginosa from a naturalderivative database. Antimicrob. Agents Chemother. 57, 5629–5641 (2013). 59 O’Loughlin CT, Miller LC, Siryaporn A,

80 Instinctif Partners.

69 Essoh C, Blouin Y, Loukou G et al. The

57 Lu Q, Yu J, Yang X et al. Ambroxol interferes

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Results obtained showed that the bacteriophages isolated directly on the targeted host were the most efficient in vivo, supporting a personalized approach of treatment.

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71 Pastagia M, Schuch R, Fischetti VA

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85 Adawi A, Bisignano C, Genovese T et al.

In vitro and in vivo properties of a fully human IgG1 monoclonal antibody that combats multidrug resistant Pseudomonas aeruginosa. Int. J. Mol. Med. 30, 455–464 (2012).

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86 DiGiandomenico A, Warrener P, Hamilton

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87 Song Y, Srinivasan R, Lima J et al. PcrV

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Suggests beneficial effects of cotreatment with panobacumab and meropenem also in immunocompromised patients.

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Review Savoia 92 Lu QL, Rouby JJ, Laterre

99 Johansen HK, GØtzsche PC. Vaccines for

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preventing infection with Pseudomonas aeruginosa in cystic fibrosis. Cochrane Database Syst. Rev. 6, CD001399 (2013). 100 Staczek J, Gilleland LB, van der Heyde HC

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adenovirus vector expressing Pseudomonas aeruginosa OprF. Clin. Exp. Immunol. 173, 230–241 (2013). 107 Zaman M, Good MF, Toth I. Nanovaccines

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Applications of nanotechnology for immunology. Nat. Rev. Immunol. 13, 592–605 (2013). •• The application of nanotechnology in the field of immunology will enable new generations of vaccines, adjuvants and immunomodulatory drugs to improve clinical outcome in response to infectious and noninfectious diseases. 109 Taranejoo S, Janmaleki M, Rafienia

M, Kamali M, Mansouri M. Chitosan microparticles loaded with exotoxin A subunit antigen for intranasal vaccination against Pseudomonas aeruginosa: an in vitro study. Carbohydrate Polymers 83, 1854–1861 (2011). 110 Rodrigues NF, van Tilburg Bernardes E,

Rocha RP et al. Bovine serum albumin nanoparticle vaccine reduces lung pathology induced by live Pseudomonas aeruginosa infection in mice. Vaccine 31, 5062–5066 (2013). 111 Polyphor. 

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