Efficacy of AiiM,an N-Acylhomoserine Lactonase,against Pseudomonas aeruginosa in a Mouse Model of Acute Pneumonia

Quorum sensing (QS) in Pseudomonas aeruginosa regulates the production of many virulence factors and plays an important role in the pathogenesis of P. aeruginosa infection. N-acyl homoserine lactones (AHL) are major QS signal molecules. Recently, a novel AHL-lactonase enzyme, AiiM, has been identified. The aim of this study was to evaluate the effect of AiiM on the virulence of P. aeruginosa in a mouse model of acute pneumonia. We developed a P. aeruginosa PAO1 strain harboring an AiiM-expressing plasmid. The production of several virulence factors by the AiiM-expressing strain was examined. Mice were intratracheally infected with an AiiM-expressing PAO1 strain. Lung histopathology, bacterial burden, and bronchoalveolar lavage (BAL) fluid were assessed at 24 h postinfection. AiiM expression in PAO1 reduced production of AHL-mediated virulence factors and attenuated cytotoxicity against human lung epithelial cells. In a mouse model of acute pneumonia, AiiM expression reduced lung injury and greatly improved the survival rates. The levels of proinflammatory cytokines and myeloperoxidase activity in BAL fluid were significantly lower in mice infected with AiiM-expressing PAO1. Thus, AiiM can strongly attenuate P. aeruginosa virulence in a mammalian model and is a potential candidate for use as a therapeutic agent against P. aeruginosa infection.     

Efficacies of Ceftazidime-Avibactam and Ceftazidime against Pseudomonas aeruginosa in a Murine Lung Infection Model

This study aimed to determine the efficacy of human-simulated plasma exposures of 2 g ceftazidime plus 0.5 g avibactam every 8 h administered as a 2-h infusion or a ceftazidime regimen that produced a specific epithelial lining fluid (ELF) percentage of the dosing interval in which serum free drug concentrations remain above the MIC (fT>MIC) against 28 Pseudomonas aeruginosa isolates within a neutropenic murine pneumonia model and to assess the impact of host infection on pulmonary pharmacokinetics. The fT>MIC was calculated as the mean and upper end of the 95% confidence limit. Against the 28 P. aeruginosa strains used, the ceftazidime-avibactam MICs were 4 to 64 μg/ml, and those of ceftazidime were 8 to >128 μg/ml. The change in log₁₀ CFU after 24 h of treatment was analyzed relative to that of 0-h controls. Pharmacokinetic studies in serum and ELF were conducted using ceftazidime-avibactam in infected and uninfected mice. Humanized ceftazidime-avibactam doses resulted in significant exposures in the lung, producing reductions of >1 log₁₀ CFU against P. aeruginosa with ceftazidime-avibactam MICs of ≤32 μg/ml (ELF upper 95% confidence limit for fT>MIC [ELF fT>MIC] of ≥19%), except for one isolate with a ceftazidime-avibactam MIC of 16 μg/ml. No efficacy was observed against the isolate with a ceftazidime-avibactam MIC of 64 μg/ml (ELF fT>MIC of 0%). Bacterial reductions were observed with ceftazidime against isolates with ceftazidime MICs of 32 μg/ml (ELF fT>MIC of ≥12%), variable efficacy at ceftazidime MICs of 64 μg/ml (ELF fT>MIC of ≥0%), and no activity at a ceftazidime MIC of 128 μg/ml, where the ELF fT>MIC was 0%. ELF fT>MICs were similar between infected and uninfected mice. Ceftazidime-avibactam was effective against P. aeruginosa, with MICs of up to 32 μg/ml with an ELF fT>MIC of ≥19%. The data suggest the potential utility of ceftazidime-avibactam for treatment of lung infections caused by P. aeruginosa.     

Pharmacodynamics of Levofloxacin in a Murine Pneumonia Model of Pseudomonas aeruginosa Infection: Determination of Epithelial Lining Fluid Targets

The dose choice for Pseudomonas aeruginosa remains a matter of debate. The actual exposure targets required for multilog killing of organisms at the primary infection site have not been delineated. We studied Pseudomonas aeruginosa PAO1 using a murine model of pneumonia. We employed a large mathematical model to fit all the concentration-time data in plasma and epithelial lining fluid (ELF) as well as colony counts in lung simultaneously for all drug doses. Penetration into ELF was calculated to be approximately 77.7%, as indexed to the ratio of the area under the concentration-time curve for ELF (AUC_ELF) to the AUC_plasma. We determined the ELF concentration-time profile required to drive a stasis response as well as 1-, 2-, or 3-log₁₀(CFU/g) kill. AUC/MIC ratios of 12.4, 31.2, 62.8, and 127.6 were required to drive these bacterial responses. Emergence of resistance was seen only at the two lowest doses (three of five animals at 50 mg/kg [body weight] and one of five animals at 100 mg/kg). The low exposure targets were likely driven by a low mutational frequency to resistance. Bridging to humans was performed using Monte Carlo simulation. With a 750-mg levofloxacin dose, target attainment rates fell below 90% at 4 mg/liter, 1 mg/liter, and 0.5 mg/liter for 1-, 2-, and 3-log kills, respectively. Given the low exposure targets seen with this strain, we conclude that levofloxacin at a 750-mg dose is not adequate for serious Pseudomonas aeruginosa pneumonia as a single agent. More isolates need to be studied to make these observations more robust.Nosocomial pneumonia, particularly when caused by pathogens such as Pseudomonas aeruginosa, remains a major cause of morbidity and mortality in intensive care units (ICUs). Part of the difficulty surrounding this disease entity is the uncertainty of the adequacy of therapy. There are many variables that have an impact on this uncertainty. In the ICU environment, bacterial burdens in patients are often high, making the selection of antibiotic dose choice important (2, 5, 13, 15).Part of the uncertainty revolves around an understanding of the drug concentration-time profile for the primary infection site. Over the last two decades, important inroads have been made in the understanding of the relationship between drug concentration-time profile and the outcome of infections (8). This understanding has come from preclinical in vitro and animal studies and from clinical trials (1, 14, 21). Much of this insight has been guided by preclinical animal models, particularly the mouse thigh infection model (3, 6). The mouse thigh model is an excellent surrogate for exploring dose and schedule choice for infections of the skin and skin structure. Generally, drug penetration is good at this site, and the drug concentration-time profile for plasma is a good guide to the concentration-time profile for the infection site.Much less investigation has been done for pneumonia, and little understanding is available regarding the drug concentration-time profile for the lung and how this relates to the killing of the pathogen at this site. In this investigation, we employed a well-validated murine pneumonia model (11) using strain PAO1 of Pseudomonas aeruginosa. We chose levofloxacin as the antibiotic probe into this system. As part of the approach, we developed a novel mathematical model where murine plasma concentrations, epithelial lining fluid (ELF) concentrations, and colony counts were simultaneously modeled for all dosing regimens examined. Drug exposure targets in ELF were then calculated and bridged to humans by employing Monte Carlo simulation and ELF penetration data for humans, which were previously reported by this laboratory (9).(This work was recently presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 25 to 28 October 2008 [10].)     

Elucidation of the Pharmacokinetic/Pharmacodynamic Determinant of Colistin Activity against Pseudomonas aeruginosa in Murine Thigh and Lung Infection Models

Colistin is increasingly used as last-line therapy against Gram-negative pathogens. The pharmacokinetic (PK)/pharmacodynamic (PD) index that best correlates with the efficacy of colistin remains undefined. The activity of colistin against three strains of Pseudomonas aeruginosa was studied in neutropenic mouse thigh and lung infection models. The PKs of unbound colistin were determined from single-dose PK studies together with extensive plasma protein binding analyses. Dose-fractionation studies were conducted over 24 h with a dose range of 5 to 160 mg/kg of body weight/day. The bacterial burden in the thigh or lung was measured at 24 h after the initiation of treatment. Relationships between antibacterial effect and measures of exposure to unbound (f) colistin (area under the concentration-time curve [fAUC/MIC], maximum concentration of drug in plasma [fC_max]/MIC, and the time that the concentration in plasma is greater than the MIC [fT > MIC]) were examined by using an inhibitory sigmoid maximum-effect model. Nonlinearity in the PKs of colistin, including its plasma protein binding, was observed. The PK/PD index that correlated best with its efficacy was fAUC/MIC in both the thigh infection model (R² = 87%) and the lung infection model (R² = 89%). The fAUC/MIC targets required to achieve 1-log and 2-log kill against the three strains were 15.6 to 22.8 and 27.6 to 36.1, respectively, in the thigh infection model, while the corresponding values were 12.2 to 16.7 and 36.9 to 45.9 in the lung infection model. The findings of this in vivo study indicate the importance of achieving adequate time-averaged exposure to colistin. The results will facilitate efforts to define the more rational design of dosage regimens for humans.Infections caused by multidrug-resistant Gram-negative bacteria, in particular, Pseudomonas aeruginosa, are presenting a critical challenge, and they are associated with a high mortality rate if they are not treated promptly and effectively (3). The Antimicrobial Availability Task Force of the Infectious Diseases Society of America, the FDA, and other organizations have highlighted the urgent need to develop new antibiotics with activity against Gram-negative organisms, including P. aeruginosa (4, 27). Unfortunately, novel agents with activity against P. aeruginosa may not be available in the next 9 to 11 years (24). In the meantime, colistin (polymyxin E) is often the only available active antibiotic and is increasingly used as the last line of therapy against Gram-negative “superbugs” (8, 13, 18).Colistin is a cationic lipopeptide antibiotic with concentration-dependent bactericidal activity against P. aeruginosa and other Gram-negative bacteria (18). It is administered parenterally in the form of its inactive prodrug, colistin methanesulfonate (CMS) (2). CMS/colistin entered clinical use in the late 1950s, but it fell out of favor in the 1970s due to concerns about the potential for nephrotoxicity and neurotoxicity (8, 18). Since CMS/colistin was never subjected to contemporary drug development procedures, there has been a substantial lack of preclinical and clinical pharmacokinetic (PK) and pharmacodynamic (PD) data that may be used to guide the selection of appropriate dosage regimens (18). As a result, the dosage regimens in use today were selected empirically and are not based upon solid PK/PD data. The urgency to establish optimized dosage regimens is highlighted by recent reports of colistin resistance (11, 27).To assist with the optimization of dosage regimens, the aims of the present study were to elucidate the PK/PD index of colistin, the antibacterially active form of CMS (2), that correlates best with efficacy against P. aeruginosa by using mouse thigh and lung infection models and to determine the target values of the index for various magnitudes of antibacterial effects.(This study was presented, in part, at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, September 2007, abstr. A-5.)     

Bioengineered Lysozyme Reduces Bacterial Burden and Inflammation in a Murine Model of Mucoid Pseudomonas aeruginosa Lung Infection

The spread of drug-resistant bacterial pathogens is a growing global concern and has prompted an effort to explore potential adjuvant and alternative therapies derived from nature''s repertoire of bactericidal proteins and peptides. In humans, the airway surface liquid layer is a rich source of antibiotics, and lysozyme represents one of the most abundant and effective antimicrobial components of airway secretions. Human lysozyme is active against both Gram-positive and Gram-negative bacteria, acting through several mechanisms, including catalytic degradation of cell wall peptidoglycan and subsequent bacterial lysis. In the infected lung, however, lysozyme''s dense cationic character can result in sequestration and inhibition by polyanions associated with airway inflammation. As a result, the efficacy of the native enzyme may be compromised in the infected and inflamed lung. To address this limitation, we previously constructed a charge-engineered variant of human lysozyme that was less prone to electrostatic-mediated inhibition in vitro. Here, we employ a murine model to show that this engineered enzyme is superior to wild-type human lysozyme as a treatment for mucoid Pseudomonas aeruginosa lung infections. The engineered enzyme effectively decreases the bacterial burden and reduces markers of inflammation and lung injury. Importantly, we found no evidence of acute toxicity or allergic hypersensitivity upon repeated administration of the engineered biotherapeutic. Thus, the charge-engineered lysozyme represents an interesting therapeutic candidate for P. aeruginosa lung infections.     

OligoG CF-5/20 Disruption of Mucoid Pseudomonas aeruginosa Biofilm in a Murine Lung Infection Model

Biofilm growth is a universal survival strategy for bacteria, providing an effective and resilient approach for survival in an otherwise hostile environment. In the context of an infection, a biofilm provides resistance and tolerance to host immune defenses and antibiotics, allowing the biofilm population to survive and thrive under conditions that would destroy their planktonic counterparts. Therefore, the disruption of the biofilm is a key step in eradicating persistent bacterial infections, as seen in many types of chronic disease. In these studies, we used both in vitro minimum biofilm eradication concentration (MBEC) assays and an in vivo model of chronic biofilm infection to demonstrate the biofilm-disrupting effects of an alginate oligomer, OligoG CF-5/20. Biofilm infections were established in mice by tracheal instillation of a mucoid clinical isolate of Pseudomonas aeruginosa embedded in alginate polymer beads. The disruption of the biofilm by OligoG CF-5/20 was observed in a dose-dependent manner over 24 h, with up to a 2.5-log reduction in CFU in the infected mouse lungs. Furthermore, in vitro assays showed that 5% OligoG CF-5/20 significantly reduced the MBEC for colistin from 512 μg/ml to 4 μg/ml after 8 h. These findings support the potential for OligoG CF-5/20 as a biofilm disruption agent which may have clinical value in reducing the microbial burden in chronic biofilm infections.     

Microbial Growth Inhibition by Alternating Electric Fields in Mice with Pseudomonas aeruginosa Lung Infection

High-frequency, low-intensity electric fields generated by insulated electrodes have previously been shown to inhibit bacterial growth in vitro. In the present study, we tested the effect of these antimicrobial fields (AMFields) on the development of lung infection caused by Pseudomonas aeruginosa in mice. We demonstrate that AMFields (10 MHz) significantly inhibit bacterial growth in vivo, both as a stand-alone treatment and in combination with ceftazidime. In addition, we show that peripheral (skin) heating of about 2°C can contribute to bacterial growth inhibition in the lungs of mice. We suggest that the combination of alternating electric fields, together with the heat produced during their application, may serve as a novel antibacterial treatment modality.The 20th century was the golden era of the antibacterial agents, with millions of people owing their lives to the discovery of and treatment with the numerous antibiotic families used today. Surely, antibacterial agents will continue to play a major role in the battle against pathogenic bacteria in the 21st century; however, the extensive use of antibiotics holds a threat of a far less optimistic future due to the rapid rise of multidrug-resistant bacteria. Recently, the potential use of physical means as an aid to antibiotics in the battle against bacterial pathogens has been studied: photodynamic therapy (12, 21, 35), ultrasound wave therapy (7, 23, 25), thermotherapy (26), and weak electric currents (4, 6, 24, 32, 33) are all being tested as treatment modalities against pathogenic microorganisms. The major drawback of the methods mentioned above is their low therapeutic index due to the high levels of heating produced by ultrasonic waves, thermotherapy, and photodynamic therapy (36) and the activated oxygen generated by photodynamic therapy, both of which can damage the tissues in and around the target area (22). In addition, the use of conductive electrodes for the generation of electric currents is associated with the release of metal ions and free radicals at the electrode surface, all of which are toxic to living cells (18). Indeed, as of today, none of the above-mentioned means has matured into an approved treatment modality against bacterial pathogens.Recently, we demonstrated that low-intensity alternating electric fields of high frequencies (antimicrobial fields [AMFields]) have an in vitro inhibitory effect on the growth of pathogenic bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa (8). AMFields were generated using electrically insulated electrodes and therefore were not associated with the production of free radicals, toxic metal ions, or electrolysis at the electrode surface. Fields of the relatively high frequency at which the AMFields effects were observed (with an optimum at 10 MHz) have no known effect on human cells (13, 14). Indeed, we found that AMFields have no effect on the growth of cell cultures in vitro (unpublished results). Furthermore, the high frequencies of the AMFields allow for the application of relatively high intensities without nerve and muscle stimulation, and their nonattenuated spread in tissues makes deep treatment realistic.In view of the above-described factors, the objective of the present study was to test the feasibility of using AMFields generated by insulated electrodes for the inhibition of P. aeruginosa proliferation in the infected lungs of mice.     

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.     

Relationship Between Gentamicin Susceptibility Criteria and Therapeutic Serum Levels for Pseudomonas aeruginosa in Mouse Infection Model

In this study estimations of in vivo and in vitro gentamicin susceptibility for a series of strains of Pseudomonas aeruginosa were compared. The series included an extremely susceptible strain, typically susceptible strains by current susceptibility criteria, and strains with enzymatic and permeability-mediated resistance. In vivo testing was done by using a mice protection test involving six 1-h doses of gentamicin and an inoculum of 50 50% lethal doses of P. aeruginosa. Both normal mice and cyclophosphamide-treated mice were used. It was found that peak serum levels and serum levels of gentamicin obtained just prior to the sixth dose (fifth dose trough levels) required for protection were much higher than minimal inhibitory concentrations (MICs) or minimal bactericidal concentrations (MBCs) obtained in high-cation medium. However, first dose trough levels were similar to MICs or MBCs. Only an extremely susceptible strain, 280, could be treated at antibiotic dosages and serum levels which are considered likely to be safe in humans. A distinct inoculum effect was found in the mice tests, with a 10-fold increase in inoculum producing a 4-fold increase in the amount of gentamicin required, but no inoculum effect was found for MICs. These results suggest that current susceptibility criteria in use for gentamicin and P. aeruginosa overestimate gentamicin susceptibility, particularly when low-cation growth medium is used for susceptibility testing and when treating disseminated infection.     

A Novel Anti-PcrV Antibody Providing Enhanced Protection against Pseudomonas aeruginosa in Multiple Animal Infection Models

Pseudomonas aeruginosa is a major cause of hospital-acquired infections, particularly in mechanically ventilated patients, and it is the leading cause of death in cystic fibrosis patients. A key virulence factor associated with disease severity is the P. aeruginosa type III secretion system (T3SS), which injects bacterial toxins directly into the cytoplasm of host cells. The PcrV protein, located at the tip of the T3SS injectisome complex, is required for T3SS function and is a well-validated target in animal models of immunoprophylactic strategies targeting P. aeruginosa. In an effort to identify a highly potent and protective monoclonal antibody (MAb) that inhibits the T3SS, we generated and characterized a panel of novel anti-PcrV MAbs. Interestingly, some MAbs exhibiting potent inhibition of T3SS in vitro failed to provide protection in a mouse model of P. aeruginosa infection, suggesting that effective in vivo inhibition of T3SS with anti-PcrV MAbs is epitope dependent. V2L2MD, while not the most potent MAb as assessed by in vitro cytotoxicity inhibition assays, provided strong prophylactic protection in several murine infection models and a postinfection therapeutic model. V2L2MD mediated significantly (P < 0.0001) better in vivo protection than that provided by a comparator antibody, MAb166, a well-characterized anti-PcrV MAb and the progenitor of a clinical candidate, KB001-A. The results described here support further development of a V2L2MD-containing immunotherapeutic and may suggest even greater potential than was previously recognized for the prevention and treatment of P. aeruginosa infections in high-risk populations.     

Triclosan Resistance of Pseudomonas aeruginosa PAO1 Is Due to FabV,a Triclosan-Resistant Enoyl-Acyl Carrier Protein Reductase

Triclosan, a very widely used biocide, specifically inhibits fatty acid synthesis by inhibition of enoyl-acyl carrier protein (ACP) reductase. Escherichia coli FabI is the prototypical triclosan-sensitive enoyl-ACP reductase, and E. coli is extremely sensitive to the biocide. However, other bacteria are resistant to triclosan, because they encode triclosan-resistant enoyl-ACP reductase isozymes. In contrast, the triclosan resistance of Pseudomonas aeruginosa PAO1 has been attributed to active efflux of the compound (R. Chuanchuen, R. R. Karkhoff-Schweizer, and H. P. Schweizer, Am. J. Infect. Control 31:124-127, 2003). We report that P. aeruginosa contains two enoyl-ACP reductase isozymes, the previously characterized FabI homologue plus a homologue of FabV, a triclosan-resistant enoyl-ACP reductase recently demonstrated in Vibrio cholerae. By deletion of the genes encoding P. aeruginosa FabI and FabV, we demonstrated that FabV confers triclosan resistance on P. aeruginosa. Upon deletion of the fabV gene, the mutant strain became extremely sensitive to triclosan (>2,000-fold more sensitive than the wild-type strain), whereas the mutant strain lacking FabI remained completely resistant to the biocide.Fatty acids are major components of cell membrane as well as metabolic intermediates in bacteria (9, 20, 25). The bacterial fatty acid synthesis system (FAS II), which is carried out by a series of discrete enzymes, differs significantly from the mammalian and fungal system (FAS I), which uses a large complex multifunctional enzymes (25, 43). The differences between the FAS I and FAS II systems make the FAS II enzymes good targets for antibacterial inhibitors (19, 20, 32, 45). The enoyl-acyl carrier protein (ACP) reductase (ENR) components of the type II system catalyze the last step of the elongation cycle in the synthesis of fatty acids (28). ENRs catalyze the reduction of trans-2-acyl-ACPs (an enoyl-ACP) to the fully saturated acyl-ACP species (Fig. ​(Fig.1A).1A). In bacteria four ENR isozymes have been reported. These are FabI (3, 39), FabL (18), FabV (29), and FabK (17, 27). FabI, FabL, and FabV are members of the short-chain dehydrogenase/reductase (SDR) superfamily, whereas FabK, a TIM barrel flavoprotein, is unrelated to this family (28). Most bacteria contain an easily identifiable fabI gene in their chromosomes; the expressed proteins are about 40% identical to Escherichia coli FabI and contain a conserved Tyr-156-(Xaa)₆-Lys-163 (E. coli numbering) catalytic dyad (19, 29). In E. coli, FabI has been shown to be the site of action of triclosan (30), a biocide used in hand soaps and a large variety of other everyday products. Since FabI is the only E. coli ENR, it acts in each step of the fatty acid elongation cycle and thus is essential for cell growth and survival (2, 3, 15). Soon after the identification of FabI as the triclosan target, the existence of a number of bacterial species naturally resistant to triclosan was recognized. FabL was identified in Bacillus subtilis by the presence of the Tyr-(Xaa)₆-Lys dyad, and although it shares a degree of sequence similarity with FabI proteins, it is not a member of the FabI family (18). FabL is moderately resistant to triclosan and uses NADPH as the coenzyme (18). More recently, FabV, the third SDR family ENR, was discovered in Vibrio cholerae. FabV is unlike the other SDR ENRs in that it is completely refractory to triclosan inhibition, is 60% larger than the typical SDR family member (which are generally about 250 residues long), and has an eight-residue space between the active-site tyrosine and lysine residues (Tyr-X₈-Lys) (29). This spacing has two more residues than those in the FabI and FabL active sites and one more than the maximum reported for other SDR proteins (33). However, like FabI, FabV has the classical Rossman fold motif (29). The only non-SDR-family ENR reported to date is Streptococcus pneumoniae FabK which is as refractory to triclosan as FabV (17, 27). The crystal structure of FabK has been reported recently (36). Unlike the SDR enzymes, FabK has a TIM barrel (α8-β8) structure and is an FMN-dependent oxidoreductase of the NAD(P)H-dependent flavin oxidoreductase family (28, 36).Open in a separate windowFIG. 1.The ENR reaction and alignment of P. aeruginosa FabV with V. cholerae FabV. (A) The ENR reaction. Depending on the enzyme, the reductant can be NADPH or FMNH₂ (coupled to NADH oxidation) rather than NADH. (B) The V. cholerae FabV (top line) and P. aeruginosa FabV sequences have 58% identity and 79% similarity. The active-site tyrosine and lysine residues are asterisked.The important opportunistic pathogen Pseudomonas aeruginosa contains a fatty acid synthase system very similar to that of E. coli (21, 22, 24). A fabI gene was identified in the P. aeruginosa genome and disrupted (21). The fabI-null mutant strain grew normally, and thus, it was concluded that unlike E. coli, P. aeruginosa must contain another ENR (21). Indeed, assay of extracts of the fabI-null mutant strain showed that it retained 62% of the activity seen in wild-type extracts and that this activity was resistant to triclosan (21). Searches of the P. aeruginosa genome database revealed at least 18 possible FabI paralogs (averaging 25% identity and 40% similarity), but all lacked the signature motifs found in FabI proteins (21). Hence, Heath and Rock (17) suggested that the second triclosan-resistant ENR of P. aeruginosa was a FabK homologue, three of which are encoded in the P. aeruginosa genome. However, we have expressed all three putative FabK homologues and found that none possessed ENR activity (data not shown). The discovery of Vibrio cholerae FabV suggested another possibility for the highly triclosan resistant ENR of P. aeruginosa. In this report we describe the characterization of the P. aeruginosa fabV gene and its product, the second ENR of this bacterium.     

Italian Survey on Comparative Levofloxacin Susceptibility in 334 Clinical Isolates of Pseudomonas aeruginosa

A national survey on susceptibility patterns of 334 Pseudomonas aeruginosa isolates from intensive care units and hematology and oncology wards from 13 Italian hospitals compared the in vitro activity of levofloxacin, an injectable oral fluoroquinolone, to those of ciprofloxacin, ofloxacin, ceftazidime, imipenem, amikacin, and gentamicin. Amikacin and imipenem had the best susceptibility profiles. The activity of levofloxacin was superior to those of the other quinolones and was comparable to that of ceftazidime. The effect of levofloxacin in vitro on P. aeruginosa clinical isolates suggests that further clinical investigations are warranted.     

Experimental Keratitis Due to Pseudomonas aeruginosa: Model for Evaluation of Antimicrobial Drugs

An improved method for experimental keratitis due to Pseudomonas aeruginosa is described. Essential features of the method are use of inbred guinea pigs, intracorneal injection of bacteria, subconjunctival injection of antibiotics, “blind” evaluation of results, and statistical analysis of data. Untreated ocular infections were most severe 5 to 7 days after infection. Sterilized bacterial suspensions caused no abnormalities on day 5. Tobramycin and polymyxin B were more active than gentamicin against two strains of Pseudomonas. This model is suitable for many types of quantitative studies on experimental keratitis.     

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.     

Efficacy of tachyplesin III, colistin, and imipenem against a multiresistant Pseudomonas aeruginosa strain

An experimental study has been performed to compare the in vitro activity and the in vivo efficacy of tachyplesin III, colistin, and imipenem against a multiresistant Pseudomonas aeruginosa strain. In vitro experiments included MIC determination, time-kill, and synergy studies. For in vivo studies, a mouse model of sepsis has been used. The main outcome measures were bacterial lethality, quantitative blood cultures, and plasma levels of lipopolysaccharide, tumor necrosis factor alpha, and interleukin-6. The combination of tachyplesin III or colistin with imipenem showed in vitro synergistic interaction. A significant increase in efficacy was also observed in vivo: combination-treated groups had significantly lower levels of bacteremia than did groups treated with a single agent. Tachyplesin III combined with imipenem exhibited the highest efficacy on all main outcome measurements. These results highlight the potential usefulness of these combinations and provide therapeutic alternatives for serious infections caused by gram-negative bacteria in the coming years.     

In Vivo Activity of Ceftobiprole in Murine Skin Infections Due to Staphylococcus aureus and Pseudomonas aeruginosa

Ceftobiprole, a broad-spectrum cephalosporin with activity against methicillin-resistant Staphylococcus aureus (MRSA) (P. Hebeisen et al., Antimicrob. Agents Chemother. 45:825-836, 2001), was evaluated in a subcutaneous skin infection model with Staphylococcus aureus Smith OC 4172 (methicillin-susceptible S. aureus [MSSA]), S. aureus OC 8525 (MRSA), Pseudomonas aeruginosa OC 4351 (having an inducible AmpC β-lactamase), and P. aeruginosa OC 4354 (overproducing AmpC β-lactamase). In the MSSA and MRSA infection models, ceftobiprole, administered as the prodrug ceftobiprole medocaril, was more effective in reducing CFU/g skin (P < 0.001) than were cefazolin, vancomycin, or linezolid based on the dose-response profiles. Skin lesion volumes in MSSA-infected animals treated with ceftobiprole were 19 to 29% lower than those for cefazolin-, vancomycin-, or linezolid-treated animals (P < 0.001). In MRSA infections, lesion size in ceftobiprole-treated mice was 34% less than that with cefazolin or linezolid treatment (P < 0.001). Against P. aeruginosa, ceftobiprole at similar doses was as effective as meropenem-cilastatin in reductions of CFU/g skin, despite 8- and 32-fold-lower MICs for meropenem; both treatments were more effective than was cefepime (P < 0.001) against the inducible and overproducing AmpC β-lactamase strains of P. aeruginosa. Ceftobiprole was similar to meropenem-cilastatin and 47 to 54% more effective than cefepime (P < 0.01) in reducing the size of the lesion caused by either strain of P. aeruginosa in this study. These studies indicate that ceftobiprole is effective in reducing both bacterial load and lesion volume associated with infections due to MSSA, MRSA, and P. aeruginosa in this murine model of skin and soft tissue infection.Antimicrobial resistance is becoming more problematic in the health care setting, with infections mediated by drug-resistant pathogens being associated with increased morbidity and mortality and corresponding increased health care costs and longer hospital stays (12). β-Lactams, including cefazolin, have been used for many years to treat methicillin-susceptible Staphylococcus aureus (MSSA) skin and soft tissue infections (9, 52). The rising proportion of methicillin resistance in staphylococcal infections in hospitals and in the community has compromised the effectiveness of this antibiotic class for staphylococci and resulted in the increased use of vancomycin to treat patients with these infections (21, 41). As a consequence, vancomycin-intermediate S. aureus (VISA) strains continue to increase in prevalence, and vancomycin-resistant S. aureus (VRSA) strains have begun to appear (24, 33, 34, 49, 51). Linezolid, an oxazolidinone antibiotic with a gram-positive antibacterial spectrum of activity including methicillin-resistant S. aureus (MRSA) (27), was introduced in 2000 to address the growing problem of MRSA, but safety concerns have limited its utility (4, 29).Prior to the advent of methicillin resistance (and therein resistance to all approved β-lactams), cephalosporins were considered the drugs of choice to treat staphylococcal infections (45). Ceftobiprole is a new cephalosporin that, unlike currently marketed β-lactams, has high binding affinity for penicillin-binding protein 2a (PBP2a) (19), resulting in potent in vitro and in vivo activity against MRSA. The prodrug form of this agent, ceftobiprole medocaril, has completed two phase III complicated skin and skin structure infection (cSSSI) clinical trials in which efficacy was demonstrated against a broad spectrum of bacteria, including MRSA (1, 44).Antimicrobial resistance in gram-negative organisms is also a serious problem that has limited the number of effective antimicrobial agents available to physicians to treat infection in the hospital setting. While skin infections with Pseudomonas aeruginosa are found with less frequency (6.2%) than are staphylococcal skin infections (19.1%), they remain a common cause of morbidity in surgical site and burn infections (2) and with immunocompromised patients. (26, 52). Antipseudomonal cephalosporins are frequently used for the treatment of P. aeruginosa; however, the overproduction of AmpC β-lactamases reduces the antibiotic choices available for treatment (40).Ceftobiprole binds tightly to PBP3 in P. aeruginosa, as do other cephalosporins (20), and consequently has also been shown to have in vitro and in vivo activity against this bacterium. MICs for ceftobiprole against recent clinical isolates are similar to those of cefepime and meropenem (10, 25). The in vivo antipseudomonal activity of ceftobiprole has previously been characterized in the mouse septicemia and the neutropenic thigh infection models (23, 30, 32), but the effect of AmpC β-lactamases on in vivo activity has not been reported.In this study, ceftobiprole medocaril was evaluated in a murine skin and soft tissue infection model utilizing methicillin-susceptible and -resistant strains of S. aureus and strains of P. aeruginosa that express various levels of AmpC. In these models, the broad-spectrum (gram-positive and gram-negative) activity of ceftobiprole was demonstrated against these important skin pathogens.     

Epinecidin-1 Has Immunomodulatory Effects,Facilitating Its Therapeutic Use in a Mouse Model of Pseudomonas aeruginosa Sepsis

Antimicrobial peptides (AMPs) are garnering attention as possible alternatives to antibiotics. Here, we describe the antimicrobial properties of epinecidin-1 against a multidrug-resistant clinical isolate of P. aeruginosa (P. aeruginosa R) and a P. aeruginosa strain from ATCC (P. aeruginosa ATCC 19660) in vivo. The MICs of epinecidin-1 against P. aeruginosa R and P. aeruginosa ATCC 19660 were determined and compared with those of imipenem. Epinecidin-1 was found to be highly effective at combating peritonitis infection caused by P. aeruginosa R or P. aeruginosa ATCC 19660 in mouse models, without inducing adverse behavioral effects or liver or kidney toxicity. Taken together, our results indicate that epinecidin-1 enhances the rate of survival of mice infected with the bacterial pathogen P. aeruginosa through both antimicrobial and immunomodulatory effects.     

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.     

Bacterial and Clinical Characteristics of Health Care- and Community-Acquired Bloodstream Infections Due to Pseudomonas aeruginosa

Health care-associated infections, including Pseudomonas aeruginosa bloodstream infection, have been linked to delays in appropriate antibiotic therapy and an increased mortality rate. The objective of this study was to evaluate intrinsic virulence, bacterial resistance, and clinical outcomes of health care-associated bloodstream infections (HCABSIs) in comparison with those of community-acquired bloodstream infections (CABSIs) caused by P. aeruginosa. We conducted a retrospective multicenter study of consecutive P. aeruginosa bacteremia patients at two university-affiliated hospitals. Demographic, clinical, and treatment data were collected. Microbiologic analyses included in vitro susceptibility profiles and type III secretory (TTS) phenotypes. Sixty CABSI and 90 HCABSI episodes were analyzed. Patients with HCABSIs had more organ dysfunction at the time of bacteremia (P = 0.05) and were more likely to have been exposed to antimicrobial therapy (P < 0.001) than those with CABSIs. Ninety-two percent of the carbapenem-resistant P. aeruginosa infections were characterized as HCABSIs. The 30-day mortality rate for CABSIs was 26% versus 36% for HCABSIs (P = 0.38). The sequential organ failure assessment score at the time of bacteremia (hazard ratio [HR], 1.2; 95% confidence interval [CI], 1.1 to 1.3) and the TTS phenotype (HR 2.1; 95% CI, 1.1 to 3.9) were found to be independent predictors of the 30-day mortality rate. No mortality rate difference was observed between CABSIs and HCABSIs caused by P. aeruginosa. Severity of illness and expression of TTS proteins were the strongest predictors of the 30-day mortality rate due to P. aeruginosa bacteremia. Future P. aeruginosa bacteremia trials designed to neutralize TTS proteins are warranted.