Efficacy of Liposomal Bismuth-Ethanedithiol-Loaded Tobramycin after Intratracheal Administration in Rats with Pulmonary Pseudomonas aeruginosa Infection

We sought to investigate alterations in quorum-sensing signal molecule N-acyl homoserine lactone secretion and in the release of Pseudomonas aeruginosa virulence factors, as well as the in vivo antimicrobial activity of bismuth-ethanedithiol incorporated into a liposome-loaded tobramycin formulation (LipoBiEDT-TOB) administered to rats chronically infected with P. aeruginosa. The quorum-sensing signal molecule N-acyl homoserine lactone was monitored by using a biosensor organism. P. aeruginosa virulence factors were assessed spectrophotometrically. An agar beads model of chronic Pseudomonas lung infection in rats was used to evaluate the efficacy of the liposomal formulation in the reduction of bacterial count. The levels of active tobramycin in the lungs and the kidneys were evaluated by microbiological assay. LipoBiEDT-TOB was effective in disrupting both quorum-sensing signal molecules N-3-oxo-dodeccanoylhomoserine lactone and N-butanoylhomoserine lactone, as well as significantly (P < 0.05) reducing lipase, chitinase, and protease production. At 24 h after 3 treatments, the CFU counts in lungs of animals treated with LipoBiEDT-TOB were of 3 log₁₀ CFU/lung, comparated to 7.4 and 4.7 log₁₀ CFU/lung, respectively, in untreated lungs and in lungs treated with free antibiotic. The antibiotic concentration after the last dose of LipoBiEDT-TOB was 25.1 μg/lung, while no tobramycin was detected in the kidneys. As for the free antibiotic, we found 6.5 μg/kidney but could not detect any tobramycin in the lungs. Taken together, LipoBiEDT-TOB reduced the production of quorum-sensing molecules and virulence factors and could highly improve the management of chronic pulmonary infection in cystic fibrosis patients.     

Overexpression of MexCD-OprJ Reduces Pseudomonas aeruginosa Virulence by Increasing Its Susceptibility to Complement-Mediated Killing

We evaluated the resistance to complement-mediated killing of a collection of isogenic Pseudomonas aeruginosa strains expressing different antimicrobial resistance phenotypes. Only the nfxB mutant demonstrated increased susceptibility to complement compared with that for the wild-type strain. This increment was due to the overexpression of MexCD-OprJ, which led to increased C3 opsonization and a reduced ability to infect the lungs of mice. Our results show that the acquisition of antibiotic resistance may alter the interplay of P. aeruginosa with the host immune system.     

Virulence of metallo-beta-lactamase-producing Pseudomonas aeruginosa in vitro and in vivo

We evaluated the virulence of Pseudomonas aeruginosa carrying bla(IMP), a metallo-beta-lactamase gene, and the efficacy of ceftazidime, imipenem-cilastatin, and ciprofloxacin in the endogenous bacteremia model. The presence of bla(IMP) did not practically change the virulence of the parent strain, and ciprofloxacin was effective against infection with P. aeruginosa carrying bla(IMP).     

Clarithromycin has an immunomodulatory effect on ERK-mediated inflammation induced by Pseudomonas aeruginosa flagellin

OBJECTIVES: Pseudomonas aeruginosa exoproducts are potent triggers of immune responses in eukaryotic cells. Clarithromycin initially decreases, then increases and finally produces a sustained suppression of interleukin (IL)-8 secretion from normal human bronchial epithelial (NHBE) cells through inhibition and activation of extracellular signal-regulated kinase (ERK). This polyphasic immune response is referred to as immunomodulation. METHODS: We studied the effects of P. aeruginosa flagellin and alginate on IL-8 secretion from NHBE cells and how this was affected by clarithromycin or dexamethasone. We also assessed the upstream kinase cell signalling intermediates that appear to be responsible for flagellin-induced IL-8 secretion. ELISA was used to measure IL-8 in culture supernatants, and western blots were used to measure kinase and phosphokinase levels. RESULTS: Flagellin dose-dependently increased IL-8 secretion in NHBE cells at 24 h, whereas alginate had no effect on IL-8. Clarithromycin significantly decreased flagellin-induced IL-8 over the first 9 h but not at 24 h. A 60 min exposure to clarithromycin decreased flagellin-induced ERK phosphorylation, but at 24 h, clarithromycin increased phospho-ERK1/2 beyond the effect of flagellin alone. Pre-treatment with PD98059 (MEK inhibitor) decreased flagellin-induced IL-8 secretion by 47.7% (P < 0.0001) compared with control flagellin exposure and decreased basal IL-8 in the absence of flagellin by 27.9% compared with untreated control cells (P < 0.0001). Dexamethasone and PD98059 together had an additive suppressive effect on flagellin-induced IL-8 secretion. CONCLUSIONS: P. aeruginosa flagellin, but not alginate, stimulates IL-8 secretion in NHBE cells in part through ERK1/2. This effect is modulated by clarithromycin, whereas suppression of IL-8 secretion by dexamethasone probably occurs through different pathways.     

Efficacy of oral immunization with Pseudomonas aeruginosa lipopolysaccharide

Oral administration of extracted lipopolysaccharide (LPS) from Pseudomonas aeruginosa was used to achieve serotype specific protection against P. aeruginosa in an ironloaded mouse model and a burned mouse model of infection. A significant delay to death could also be achieved using a neutropenic mouse model. In the iron-loaded mouse infection model protection could be achieved against three serotypes but not a fourth (serotype G). Protection against two serotypes could be achieved by simultaneous but not sequential feeding with both serotypes. Several oral immunization protocols were examined in the iron-loaded mouse model for efficacy and for toxicity. In protection studies, concentrations of LPS in the drinking water ranging from 2 to 200 μg/ml were used for periods from 1 to 40 days resulting in total intakes of 145 μg to 5·8 mg LPS. All protocols were successful in achieving protection provided that challenge occurred later than 2 days after the initial exposure to LPS. Toxicity studies were performed with similar amounts of LPS including an additional high dose (2 mg/ml LPS for 1 day, 14 mg total intake). No evidence of toxicity was observed for any dosage using a variety of criteria including pathological examination of tissue except for small temperature elevations in all but the lowest dose. Aerosol administration of LPS was also shown to provide protection against infection with the homologous strain. Passive transfer experiments demonstrated that protection was associated with specific antibody and not by cell mediated immunity.     

Effect of clarithromycin on Pseudomonas aeruginosa biofilms

Using an experimental in vitro culture system, we investigated the effect of clarithromycin on biofilm formation by a leucine-requiring Pseudomonas aeruginosa mutant strain (HU1). Biofilm formation on celldesks in a chemically defined medium was assessed by viable cell count as well as by measurement of glycocalyx production and scanning electron-microscopic observation. Cells proliferated exponentially until day 3 and remained stationary afterwards. The amount of glycocalyx, simultaneously semiquantified, showed a linear increase from day 1 to day 12. Scanning electron microscopy revealed firm biofilms on day 5. Three different concentrations of Clarithromycin (CAM) (minimum inhibitory concentration MIC 64 microg/ml) were added continuously at the early and late phases of biofilm formation, and the antibiofilm effect of CAM was evaluated by the changes in cell count and glycocalyx production. CAM was effective on biofilms at 100 microg/ml but neither at 1 nor at 10 microg/ml. It is suggested that glycocalyx production started following bacterial multiplication and continued even after the cells had entered the stationary phase to form mature biofilms. No antibiofilm effect of CAM was observed at sub-MIC.     

Characteristics of R931 and Other Pseudomonas aeruginosa R Factors

R factors were detected in 3.3% of 233 hospital isolates of Pseudomonas aeruginosa using P. aeruginosa recipients in conjugations. Transferred markers included streptomycin, tetracycline, and sulfonamide resistance. Gentamicin resistance was transferred from two strains previously shown to acetylate gentamicin. A group of R factors exemplified by R931 were characterized by failure to transfer to Escherichia coli recipients. Such R factors formed a single compatibility group when examined in a P. aeruginosa recipient. Other P. aeruginosa R factors, including RP4, showed stable coexistence with the R931 group. It is proposed that RP4 and similar R factors be members of the P-1 compatibility group and that R931, R3108, R209, and R130 be members of a group termed P-2. The buoyant densities of all R factors examined were similar, about 1.716 to 1.719 g/cm(3). The content of R-factor deoxyribonucleic acid (DNA) relative to the total DNA varied among the different R factors, ranging from about 18 +/- 2% in log-phase cells of 931 (R931) to undetectable for 679 (R679). However, R679, which transferred from strain 679 at extremely low and irregular frequencies to an E. coli host, was shown to represent about 4% R-factor DNA in that host. The relative DNA content of R931 appeared to decline in the stationary growth phase of 931 (R931) or 280 (R931). R931 covalently closed circular DNA was isolated by ethidium bromide-CsCl gradient centrifugation and examined by electron microscopy. Two major molecular distributions existed, having contour lengths of 0.5 and 12.4 mum. The molecular weights were estimated to be 10(6) and 25 x 10(6). Both molecules were under relaxed replication control. R factor R931 exists as a naturally occurring high-frequency transfer system in P. aeruginosa strains 931 and 1310. However, in strain 280 it acts as if subject to fertility repression. Other members of the P-2 compatibility group also are high-frequency transfer systems in the natural host and in strain 1310. RP4 is restricted from recipient strain 1310. Some additional recipient effects were noted in that strains 1310 or 280 sometimes differed in recipient effectiveness with a given donor. Agglutination reactions with absorbed antiserum were able to distinguish between two members of the same R-factor compatibility group, R931 and R3108.     

Quorum-Quenching Acylase Reduces the Virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans Infection Model

The Pseudomonas aeruginosa PAO1 gene pvdQ encodes an acyl-homoserine lactone (AHL) acylase capable of degrading N-(3-oxododecanoyl)-l-homoserine lactone by cleaving the AHL amide. PvdQ has been proven to function as a quorum quencher in vitro in a number of phenotypic assays. To address the question of whether PvdQ also shows quorum-quenching properties in vivo, an infection model based on the nematode Caenorhabditis elegans was explored. In a fast-acting paralysis assay, strain PAO1(pMEpvdQ), which overproduces PvdQ, was shown to be less virulent than the wild-type strain. More than 75% of the nematodes exposed to PAO1(pMEpvdQ) survived and continued to grow when using this strain as a food source. Interestingly, in a slow-killing assay monitoring the survival of the nematodes throughout a 4-day course, strain PAO1-ΔpvdQ was shown to be more virulent than the wild-type strain, confirming the role of PvdQ as a virulence-reducing agent. It was observed that larval stage 1 (L1) to L3-stage larvae benefit much more from protection by PvdQ than L4 worms. Finally, purified PvdQ protein was added to C. elegans worms infected with wild-type PAO1, and this resulted in reduced pathogenicity and increased the life span of the nematodes. From our observations we can conclude that PvdQ might be a strong candidate for antibacterial therapy against Pseudomonas infections.Pseudomonas aeruginosa is an opportunistic gram-negative pathogen of vertebrates and a primary pathogen of insects (17). It mainly infects individuals who are immunocompromised, such as human immunodeficiency virus-infected patients, as well as those who have cystic fibrosis. In addition, those having disruptions in normal barriers caused by severe burns or indwelling medical devices are at risk. Hospital-acquired P. aeruginosa pneumonias and septicemias are frequently lethal (2, 3). To facilitate the establishment of infection, P. aeruginosa produces an impressive array of both cell-associated and extracellular virulence factors, such as proteases and phospholipases, and also small molecules, including rhamnolipid, phenazines, and cyanide (17). Expression of many of the extracellular factors is cell density controlled, does not occur until the late logarithmic phase of growth, and is mediated through specific quorum-sensing signal molecules (23). Two of these molecules, N-butanoyl-l-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), have been studied in great detail. In our laboratory, we previously demonstrated that PA2385(pvdQ) from P. aeruginosa PAO1 encodes an acyl-homoserine lactone (AHL) acylase. Analysis of the gene product showed that the posttranslational processing of the acylase as well as the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PvdQ protein is a member of the N-terminal nucleophile hydrolase superfamily. The main AHL signaling molecule of P. aeruginosa PAO1, 3-oxo-C12-HSL, is degraded by PvdQ (16). Addition of the purified protein to PAO1 cultures completely inhibited accumulation of 3-oxo-C12-HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when pvdQ was overexpressed in P. aeruginosa (16). These results demonstrate that this protein has in situ quorum-quenching activity. This AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.To test our hypothesis that PvdQ can exert its beneficial functions also in vivo, we chose to study its effect on the infection of the nematode Caenorhabditis elegans. This model has been used before in multiple pathogenicity studies of Cryptococcus neoformans (13) and gram-positive (6) and gram-negative (9, 10, 11) bacteria. Infection with P. aeruginosa strain PA14 was found to result in fast (hours) or slow (days) killing, depending on the growth medium used (19, 20). When Darby and colleagues (2) used the system to study P. aeruginosa PAO1, a lethal paralysis of the worms was observed, indicating another mechanism by which P. aeruginosa can kill C. elegans. It was shown that quorum-sensing-dependent hydrogen cyanide production on rich medium by P. aeruginosa PAO1 is the causative agent for the fast paralysis (5). Under the same conditions, an attenuation of paralysis by an AHL acylase from Ralstonia sp. strain XJ12B upon expression in P. aeruginosa PAO1 was observed (12). Those authors performed the assays with a mixed population of worms only and did not test for slow killing. In this study we show that P. aeruginosa PAO1 can also elicit a slow-killing response when grown in low-nutrient medium. Moreover, we report that not only overexpression of the gene from its host but also external addition of the purified PvdQ renders P. aeruginosa PAO1 less pathogenic to C. elegans and increases the life span of the infected animals in both slow- and fast-killing assays. As the correlation between the bacterial virulence factors required for pathogenesis in mammals and in C. elegans has been shown to be very high (20), we propose that the external addition of purified AHL acylases may be developed into a novel quorum-quenching therapy.     

Safety and Efficacy of Multilamellar Liposomal Nystatin against Disseminated Candidiasis in Persistently Neutropenic Rabbits

The activity of liposomal nystatin (L-Nys) against subacute disseminated candidiasis was investigated in persistently neutropenic rabbits. Antifungal therapy was administered for 10 days starting 24 h after intravenous inoculation of 10(3) blastoconidia of Candida albicans. Responses to treatment were assessed by the quantitative clearance of the organism from blood and tissues. Treatments consisted of L-Nys at dosages of 2 and 4 mg/kg of body weight/day (L-Nys2 and L-Nys4, respectively) amphotericin B deoxycholate at 1 mg/kg/day (D-AmB), and fluconazole at 10 mg/kg/day (Flu). All treatments were given intravenously once daily. Compared to the results for untreated but infected control animals, treatment with L-Nys2, L-Nys4, D-AmB, and Flu resulted in a significant clearance of the residual burden of C. albicans from the kidney, liver, spleen, lung, and brain (P < 0.0001 by analysis of variance). When the proportion of animals infected at at least one of the five tissue sites studied was evaluated, a dose-dependent response to treatment with L-Nys was found (P < 0.05). Compared to D-AmB-treated rabbits, mean serum creatinine and blood urea nitrogen levels at the end of therapy were significantly lower in animals treated with L-Nys2 (P < 0.001) and L-Nys4 (P < 0.001 and P < 0.01, respectively). L-Nys was less nephrotoxic than conventional amphotericin B and had dose-dependent activity comparable to that of amphotericin B for the early treatment of subacute disseminated candidiasis in persistently neutropenic rabbits.     

Blue Light Rescues Mice from Potentially Fatal Pseudomonas aeruginosa Burn Infection: Efficacy,Safety, and Mechanism of Action

Blue light has attracted increasing attention due to its intrinsic antimicrobial effect without the addition of exogenous photosensitizers. However, the use of blue light for wound infections has not been established yet. In this study, we demonstrated the efficacy of blue light at 415 nm for the treatment of acute, potentially lethal Pseudomonas aeruginosa burn infections in mice. Our in vitro studies demonstrated that the inactivation rate of P. aeruginosa cells by blue light was approximately 35-fold higher than that of keratinocytes (P = 0.0014). Transmission electron microscopy revealed blue light-mediated intracellular damage to P. aeruginosa cells. Fluorescence spectroscopy suggested that coproporphyrin III and/or uroporphyrin III are possibly the intracellular photosensitive chromophores associated with the blue light inactivation of P. aeruginosa. In vivo studies using an in vivo bioluminescence imaging technique and an area-under-the-bioluminescence-time-curve (AUBC) analysis showed that a single exposure of blue light at 55.8 J/cm², applied 30 min after bacterial inoculation to the infected mouse burns, reduced the AUBC by approximately 100-fold in comparison with untreated and infected mouse burns (P < 0.0001). Histological analyses and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assays indicated no significant damage in the mouse skin exposed to blue light at the effective antimicrobial dose. Survival analyses revealed that blue light increased the survival rate of the infected mice from 18.2% to 100% (P < 0.0001). In conclusion, blue light therapy might offer an effective and safe alternative to conventional antimicrobial therapy for P. aeruginosa burn infections.     

Factors Involved in Beta-Lactam Antibiotic Resistance in Pseudomonas aeruginosa

A temperature-sensitive mutant, strain SS, which is assumed to be devoid of beta-lactamase activity and deficient in a permeability barrier to antibiotics, was isolated from a beta-lactamase-less mutant, strain L-2, derived from Pseudomonas aeruginosa IFO 3080. By comparing the beta-lactam antibiotic susceptibility between strains IFO 3080, L-2, and SS, the involvement of both beta-lactamase and a permeability barrier in determining the beta-lactam antibiotic resistance was estimated.     

Efficacy of rifampicin in experimental Bacteroides fragilis and Pseudomonas aeruginosa mixed infections

Experimental intraabdominal abscesses were produced in mice by intraperitoneal injection of Bacteroides fragilis and Pseudomonas aeruginosa. The therapeutic efficacy of rifampicin and cefsulodin alone, and in combination was investigated in this in-vivo experimental mixed intraabdominal abscess model. Treatment with rifampicin at 10, and 25 mg/kg or cefsulodin at 50, and 100 mg/kg singly or in combinations prevented mortality as compared to 68% mortality rate occurring in the untreated mice. Rifampicin, at 25 mg/kg dose, was very effective in preventing abscess formation and produced bacterial eradication. It prevented abscess formation in 80% of the mice and eradicated both Bacteroides and Pseudomonas in 100% and 75% of the abscesses of the mice. Cefsulodin failed to reduce the incidence of abscess formation, and to eradicate Bact. fragilis from the abscesses, although it significantly decreased Ps. aeruginosa in the abscesses. The combination of rifampicin at 10 mg/kg and cefsulodin at 100 mg/kg was more effective than either of the antibiotics alone and was as effective as rifampicin alone at 25 mg/kg levels. This combination was bactericidal against both organisms in the infected mice.     

Identification of Biofilm-Associated Cluster (bac) in Pseudomonas aeruginosa Involved in Biofilm Formation and Virulence

Biofilms are prevalent in diseases caused by Pseudomonas aeruginosa, an opportunistic and nosocomial pathogen. By a proteomic approach, we previously identified a hypothetical protein of P. aeruginosa (coded by the gene pA3731) that was accumulated by biofilm cells. We report here that a ΔpA3731 mutant is highly biofilm-defective as compared with the wild-type strain. Using a mouse model of lung infection, we show that the mutation also induces a defect in bacterial growth during the acute phase of infection and an attenuation of the virulence. The pA3731 gene is found to control positively the ability to swarm and to produce extracellular rhamnolipids, and belongs to a cluster of 4 genes (pA3729–pA3732) not previously described in P. aeruginosa. Though the protein PA3731 has a predicted secondary structure similar to that of the Phage Shock Protein, some obvious differences are observed compared to already described psp systems, e.g., this unknown cluster is monocistronic and no homology is found between the other proteins constituting this locus and psp proteins. As E. coli PspA, the amount of the protein PA3731 is enlarged by an osmotic shock, however, not affected by a heat shock. We consequently named this locus bac for biofilm-associated cluster.     

Factors influencing the accumulation of ciprofloxacin in Pseudomonas aeruginosa.

Ciprofloxacin accumulation in Pseudomonas aeruginosa was measured by a bioassay. Drug accumulation in strain PAO2 was compared with that of three spontaneous ciprofloxacin-resistant mutants selected with 0.5 micrograms of ciprofloxacin per ml. PAO4701 cfxA2 contains a mutation in the gyrA gene, PAO4742 cfxB5 may represent a permeability mutant based on pleiotropic drug resistance, and PAO4700 cfxA1 cfxB1 contains both types of mutations. In all strains, drug accumulation was similar, reaching steady state during the first minute of exposure. Drug accumulation was unsaturable over a range of 5 to 80 micrograms/ml, suggesting that ciprofloxacin accumulates by diffusion in P. aeruginosa. Although all four strains accumulated two- to sevenfold more ciprofloxacin in the presence of the inhibitor carbonyl cyanide m-chlorophenylhydrazone, the cfxB mutants accumulated two- to fourfold less drug than either PAO2 or the cfxA2 mutant. Polyacrylamide gel analysis revealed a protein common to cfxB mutants only, while all strains had similar lipopolysaccharide profiles. The results suggest that ciprofloxacin accumulation in P. aeruginosa is a complex phenomenon that may be affected by both an energy-dependent drug efflux process and outer envelope composition.     

Silver Sulfadiazine: Effect on the Ultrastructure of Pseudomonas aeruginosa

Pseudomonas aeruginosa exposed to silver sulfadiazine (AgSu) were examined in an electron microscope. The treated cells were distorted in shape, and structures (blebs) protruded from the cell surface. These "blebs" appeared to arise from the cell wall. A strain of P. aeruginosa resistant to AgSu did not display these changes. Upon exposure of P. aeruginosa to silver nitrate, none of these changes was seen; rather, such cells are characterized by large, central aggregations of nuclear material. The results are consistent with previous findings which suggested that AgSu acted at the cell surface.     

Antibacterial Efficacy of Phages against Pseudomonas aeruginosa Infections in Mice and Drosophila melanogaster

Phage therapy against Pseudomonas aeruginosa infections has received renewed attention owing to the increasing prevalence of antibiotic resistance in this bacterium. Here, we isolated and characterized two new potentially lytic bacteriophages (MPK1 and MPK6), which produced large and clear plaques on P. aeruginosa strain PAO1. Based on their morphology, MPK1 belongs to the Myoviridae, while MPK6 belongs to the Podoviridae. The group B polysaccharide of lipopolysaccharide was required for infection, suggesting that their host spectra are associated with the serotypes of P. aeruginosa strains. Intramuscular and intraperitoneal administration of MPK1 and, to a lesser extent, MPK6 significantly protected mice from mortality caused by PAO1-induced peritonitis-sepsis (P < 0.01). Mice treated with either phage also had lower bacterial burdens in their livers, lungs, and spleens. The antibacterial efficacy of MPK1 and MPK6 was also evaluated based on Drosophila melanogaster systemic infection caused by P. aeruginosa, for which phages were administered by feeding. Both phages significantly delayed the PAO1-induced killing of D. melanogaster (P < 0.001), although MPK1 persisted longer than MPK6 in uninfected D. melanogaster tissue samples. These results suggest that a mini-scale experiment using D. melanogaster infection is valid for evaluating the antibacterial efficacy of phage therapy against P. aeruginosa infections.Pseudomonas aeruginosa is an opportunistic human pathogen that is ubiquitously found in various biotic and abiotic environments. It is frequently isolated from human patients afflicted with cystic fibrosis, otitis media, keratitis, and burn wound infections as an etiological agent of septicemia in immunocompromised individuals. This bacterium, generally from the environmental reservoir, can colonize large numbers of child patients before the age of 3 years (27) and adversely affect their pulmonary function (23). Furthermore, P. aeruginosa is also commonly found in peritonitis-sepsis cases secondary to ruptured appendices in otherwise healthy children (4). Peritonitis by P. aeruginosa is a serious threat also to the patients undergoing continuous ambulatory peritoneal dialysis (CAPD) (15), accounting for 10% of fatality cases associated with CAPD. The bacterial intoxication usually leads to high morbidity, CAPD failure, and late complications in those cases (14, 15, 17). Rodent models of P. aeruginosa peritonitis have been developed for understanding the pathophysiology implicated in peritonitis (32). The pathological consequences are generally bacteremia and infected livers, with interleukin-6 levels elevated within 6 h postinfection, and ultimately cause mortality within 48 h depending on P. aeruginosa strains and infection doses (5).Although antibiotics are still widely used to control the bacterial infections, they are increasingly ineffective due to the emergence of antibiotic resistance. Selection and dissemination of intrinsic and acquired antibiotic resistance mechanisms increase the proclivity to resist the chemotherapy involving various antibiotics and promote the emergence of bacterial strains with multiple antibiotic resistances, which are associated with the mortality and morbidity in infected patients nowadays (22, 28). Hence, the development of new therapeutic and prophylactic strategies is compulsory for the control of bacterial infections.An alternative and/or supplementary anti-infective modality for combating infections caused by antibiotic-resistant microorganisms which is currently being revisited in various countries is bacteriophages that are able to specifically target their host bacterial infections, and this modality is called phage therapy (29). Phage therapy is a method of harnessing phages as bioagents and was first introduced before the discovery of the first antibiotic, penicillin (30). Phages continue to be used in place of antibiotics for the treatment of bacterial infections in the former Soviet Union and Eastern Europe (25). Much more attention has recently been paid to phage therapy, as more and more bacteria have evolved antibiotic resistance. Thus, phage therapy may be a valuable alternative modality to antibiotics and has already been proven to be medically superior to antibiotics in certain cases (3, 19).P. aeruginosa is a highly adaptable bacterium that could enhance its ecological fitness even in the presence of conventional antibiotic therapy. The rapid emergence of new P. aeruginosa strains as well as the persistence of the existing antibiotic-resistant clinical isolates has led to an urgent need to explore more sustainable alternative strategies such as phage therapy to manage P. aeruginosa-mediated infections. Recently, the therapeutic mixture of six lytic phages was shown to be effective in clinical ear infections caused by P. aeruginosa (26). The efficacy of phage therapy using a genetically modified filamentous phage (Pf3R) (10), lytic phage isolates, or phage cocktails has been also investigated against various experimental mouse infection models with P. aeruginosa that include burn wound infection (20) and gut-derived sepsis (31). Because the pathophysiology caused by P. aeruginosa infections is quite complicated, more and more relevant infection models need to be tested for the efficacy and relevancy of the antibacterial therapies.In this study, we evaluated the therapeutic efficacy of newly isolated Caudovirales phage strains, MPK1 and MPK6, against mouse peritonitis-sepsis caused by intraperitoneal (i.p.) infection with P. aeruginosa strain PAO1 that may resemble the clinical pathophysiology of P. aeruginosa-induced peritonitis in humans. Based on the fact that P. aeruginosa is able to infect hosts of multiple phylogenetic backgrounds, such as plants, worms, and insects, and the fact that a set of virulence factors identified from a Drosophila melanogaster systemic infection-based screen are also required for mouse peritonitis (16), we here established a D. melanogaster model to evaluate phage therapy and verified the antibacterial efficacy of both MPK1 and MPK6, using this nonmammalian infection model.