scholarly journals Ceftazidime-Avibactam Susceptibility Breakpoints againstEnterobacteriaceaeandPseudomonas aeruginosa

2018 ◽  
Vol 62 (11) ◽  
Author(s):  
Wright W. Nichols ◽  
Gregory G. Stone ◽  
Paul Newell ◽  
Helen Broadhurst ◽  
Angela Wardman ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Clinical Trials ◽  
Renal Function ◽  
Intravenous Infusion ◽  
Dosage Regimen ◽  
Resistance Mechanisms ◽  
Content Type

ABSTRACTClinical susceptibility breakpoints againstEnterobacteriaceaeandPseudomonas aeruginosafor the ceftazidime-avibactam dosage regimen of 2,000/500 mg every 8 h (q8h) by 2-h intravenous infusion (adjusted for renal function) have been established by the FDA, CLSI, and EUCAST as susceptible (MIC, ≤8 mg/liter) and resistant (MIC, >8 mg/liter). The key supportive data from pharmacokinetic/pharmacodynamic analyses,in vitrosurveillance, including molecular understanding of relevant resistance mechanisms, and efficacy in regulatory clinical trials are collated and analyzed here.

scholarly journals Multicenter Evaluation of Ceftazidime-Avibactam and Ceftolozane-Tazobactam Inhibitory Activity against Meropenem-Nonsusceptible Pseudomonas aeruginosa from Blood, Respiratory Tract, and Wounds

2017 ◽  
Vol 61 (10) ◽  
Author(s):  
Mordechai Grupper ◽  
Christina Sutherland ◽  
David P. Nicolau
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Inhibitory Activity ◽  
Clinical Utility ◽  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Broth Microdilution ◽  
Content Type ◽  
Carbapenem Resistant

ABSTRACT The recent escalation of occurrences of carbapenem-resistant Pseudomonas aeruginosa has been recognized globally and threatens to erode the widespread clinical utility of the carbapenem class of compounds for this prevalent health care-associated pathogen. Here, we compared the in vitro inhibitory activity of ceftazidime-avibactam and ceftolozane-tazobactam against 290 meropenem-nonsusceptible Pseudomonas aeruginosa nonduplicate clinical isolates from 34 U.S. hospitals using reference broth microdilution methods. Ceftazidime-avibactam and ceftolozane-tazobactam were active, with ceftolozane-tazobactam having significantly higher inhibitory activity than ceftazidime-avibactam. The heightened inhibitory activity of ceftolozane-tazobactam was sustained when the site of origin (respiratory, blood, or wound) and nonsusceptibility to other β-lactam antimicrobials was considered. An extensive genotypic search for enzymatically driven β-lactam resistance mechanisms revealed the exclusive presence of the VIM metallo-β-lactamase among only 4% of the subset of isolates nonsusceptible to ceftazidime-avibactam, ceftolozane-tazobactam, or both. These findings suggest an important role for both ceftazidime-avibactam and ceftolozane-tazobactam against carbapenem-nonsusceptible Pseudomonas aeruginosa. Further in vitro and in vivo studies are needed to better define the clinical utility of these novel therapies against the increasingly prevalent threat of multidrug-resistant Pseudomonas aeruginosa.

scholarly journals Ceftolozane-Tazobactam in the Treatment of Experimental Pseudomonas aeruginosa Pneumonia in Persistently Neutropenic Rabbits: Impact on Strains with Genetically Defined Mechanisms of Resistance

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Vidmantas Petraitis ◽  
Ruta Petraitiene ◽  
Ethan Naing ◽  
Thein Aung ◽  
Wai Phyo Thi ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Efflux Pump ◽  
Antimicrobial Effect ◽  
Resistance Mechanisms ◽  
Time Curve ◽  
Lung Weight ◽  
Content Type ◽  
Treatment Groups ◽  
Inoculation Treatment

ABSTRACT Ceftolozane-tazobactam (C/T) is a novel cephalosporin with in vitro activity against Pseudomonas aeruginosa that is resistant to extended-spectrum penicillins and antipseudomonal cephalosporins. In order to assess the antimicrobial effect of C/T in treatment of Pseudomonas pneumonia, we investigated the pharmacokinetics and efficacy of C/T in persistently neutropenic rabbits. Pseudomonas pneumonia was established by direct endotracheal inoculation. Treatment groups consisted of C/T, ceftazidime (CAZ), piperacillin-tazobactam (TZP), and untreated controls (UC). Rabbits received a dosage of C/T of 80 mg/kg every 4 h (q4h) intravenously (i.v.) (53 mg/kg ceftolozane/26 mg/kg tazobactam) to match the free drug time above the MIC as well as a comparable plasma area under the concentration-time curve (AUC) (humanized doses of ceftolozane-tazobactam of 3 g [2 g/1 g]) q8h, due to the more rapid elimination of ceftolozane in rabbits (0.75 h) than in humans (2.5 h). Four molecularly characterized clinical P. aeruginosa isolates from patients with pneumonia were studied, including one isolate from each classification group: pan-susceptible (PS), outer membrane porin D (OPRD) porin loss (OPRDPL), efflux pump expression (EPE), and AmpC hyperexpression (ACHE). Treatment was continued for 12 days. Treatment with ceftolozane-tazobactam resulted in a ≥105 reduction in residual pulmonary and bronchoalveolar lavage (BAL) fluid bacterial burdens caused by all 4 strains (P ≤ 0.01). This antibacterial activity coincided with reduction of lung weight (an organism-mediated pulmonary injury marker) (P < 0.05). CAZ was less active in ACHE-infected rabbits, and TZP had less activity against EPE, ACHE, and OPRDPL strains. Survival was prolonged in the C/T and CAZ treatment groups in comparison to the TZP and UC groups (P < 0.001). Ceftolozane-tazobactam is highly active in treatment of experimental P. aeruginosa pneumonia in persistently neutropenic rabbits, including infections caused by strains with the most common resistance mechanisms.

scholarly journals In VivoComparison of CXA-101 (FR264205) with and without Tazobactam versus Piperacillin-Tazobactam Using Human Simulated Exposures against Phenotypically Diverse Gram-Negative Organisms

2011 ◽  
Vol 56 (1) ◽  
pp. 544-549 ◽  
Author(s):  
Catharine C. Bulik ◽  
Pamela R. Tessier ◽  
Rebecca A. Keel ◽  
Christina A. Sutherland ◽  
David P. Nicolau
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Resistance Mechanisms ◽  
Infection Model ◽  
Gram Negative ◽  
Content Type ◽  
Comparison Groups ◽  
Gram Negative Organisms

ABSTRACTCXA-101 is a novel antipseudomonal cephalosporin with enhanced activity against Gram-negative organisms displaying various resistance mechanisms. This study evaluates the efficacy of exposures approximating human percent free time above the MIC (%fT > MIC) of CXA-101 with or without tazobactam and piperacillin-tazobactam (TZP) against target Gram-negative organisms, including those expressing extended-spectrum β-lactamases (ESBLs). Sixteen clinical Gram-negative isolates (6Pseudomonas aeruginosaisolates [piperacillin-tazobactam MIC range, 8 to 64 μg/ml], 4Escherichia coliisolates (2 ESBL and 2 non-ESBL expressing), and 4Klebsiella pneumoniaeisolates (3 ESBL and 1 non-ESBL expressing) were used in an immunocompetent murine thigh infection model. After infection, groups of mice were administered doses of CXA-101 with or without tazobactam (2:1) designed to approximate the %fT > MIC observed in humans given 1 g of CXA-101 with or without tazobactam every 8 h as a 1-h infusion. As a comparison, groups of mice were administered piperacillin-tazobactam doses designed to approximate the %fT > MIC observed in humans given 4.5 g piperacillin-tazobactam every 6 h as a 30-min infusion. Predicted piperacillin-tazobactam %fT > MIC exposures of greater than 40% resulted in static to >1 log decreases in CFU in non-ESBL-expressing organisms with MICs of ≤32 μg/ml after 24 h of therapy. Predicted CXA-101 with or without tazobactam %fT > MIC exposures of ≥37.5% resulted in 1- to 3-log-unit decreases in CFU in non-ESBL-expressing organisms, with MICs of ≤16 μg/ml after 24 h of therapy. With regard to the ESBL-expressing organisms, the inhibitor combinations showed enhanced CFU decreases versus CXA-101 alone. Due to enhancedin vitropotency and resultant increasedin vivoexposure, CXA-101 produced statistically significant reductions in CFU in 9 isolates compared with piperacillin-tazobactam. The addition of tazobactam to CXA-101 produced significant reductions in CFU for 7 isolates compared with piperacillin-tazobactam. Overall, human simulated exposures of CXA-101 with or without tazobactam demonstrated improved efficacy versus piperacillin-tazobactam.

scholarly journals Exogenous Alginate Protects Staphylococcus aureus from Killing by Pseudomonas aeruginosa

2019 ◽  
Vol 202 (8) ◽  
Author(s):  
Courtney E. Price ◽  
Dustin G. Brown ◽  
Dominique H. Limoli ◽  
Vanessa V. Phelan ◽  
George A. O’Toole
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Physical Space ◽  
Late Time ◽  
Protective Effects ◽  
Content Type ◽  
Alginate Production ◽  
The Impact

ABSTRACT Cystic fibrosis (CF) patients chronically infected with both Pseudomonas aeruginosa and Staphylococcus aureus have worse health outcomes than patients who are monoinfected with either P. aeruginosa or S. aureus. We showed previously that mucoid strains of P. aeruginosa can coexist with S. aureus in vitro due to the transcriptional downregulation of several toxic exoproducts typically produced by P. aeruginosa, including siderophores, rhamnolipids, and HQNO (2-heptyl-4-hydroxyquinoline N-oxide). Here, we demonstrate that exogenous alginate protects S. aureus from P. aeruginosa in both planktonic and biofilm coculture models under a variety of nutritional conditions. S. aureus protection in the presence of exogenous alginate is due to the transcriptional downregulation of pvdA, a gene required for the production of the iron-scavenging siderophore pyoverdine as well as the downregulation of the PQS (Pseudomonas quinolone signal) (2-heptyl-3,4-dihydroxyquinoline) quorum sensing system. The impact of exogenous alginate is independent of endogenous alginate production. We further demonstrate that coculture of mucoid P. aeruginosa with nonmucoid P. aeruginosa strains can mitigate the killing of S. aureus by the nonmucoid strain of P. aeruginosa, indicating that the mechanism that we describe here may function in vivo in the context of mixed infections. Finally, we investigated a panel of mucoid clinical isolates that retain the ability to kill S. aureus at late time points and show that each strain has a unique expression profile, indicating that mucoid isolates can overcome the S. aureus-protective effects of mucoidy in a strain-specific manner. IMPORTANCE CF patients are chronically infected by polymicrobial communities. The two dominant bacterial pathogens that infect the lungs of CF patients are P. aeruginosa and S. aureus, with ∼30% of patients coinfected by both species. Such coinfected individuals have worse outcomes than monoinfected patients, and both species persist within the same physical space. A variety of host and environmental factors have been demonstrated to promote P. aeruginosa-S. aureus coexistence, despite evidence that P. aeruginosa kills S. aureus when these organisms are cocultured in vitro. Thus, a better understanding of P. aeruginosa-S. aureus interactions, particularly mechanisms by which these microorganisms are able to coexist in proximal physical space, will lead to better-informed treatments for chronic polymicrobial infections.

scholarly journals Activity of Ceftazidime-Avibactam against Fluoroquinolone-Resistant Enterobacteriaceae and Pseudomonas aeruginosa

2015 ◽  
Vol 59 (6) ◽  
pp. 3059-3065 ◽  
Author(s):  
C. Pitart ◽  
F. Marco ◽  
T. A. Keating ◽  
W. W. Nichols ◽  
J. Vila
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Resistant Mutant ◽  
Fluoroquinolone Resistance ◽  
Cross Resistance ◽  
Content Type ◽  
E Coli ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
The Impact

ABSTRACTCeftazidime-avibactam and comparator antibiotics were tested by the broth microdilution method against 200Enterobacteriaceaeand 25Pseudomonas aeruginosastrains resistant to fluoroquinolones (including strains with the extended-spectrum β-lactamase [ESBL] phenotype and ceftazidime-resistant strains) collected from our institution. The MICs and mechanisms of resistance to fluoroquinolone were also studied. Ninety-nine percent of fluoroquinolone-resistantEnterobacteriaceaestrains were inhibited at a ceftazidime-avibactam MIC of ≤4 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference). Ceftazidime-avibactam was very active against ESBLEscherichia coli(MIC90of 0.25 mg/liter), ESBLKlebsiella pneumoniae(MIC90of 0.5 mg/liter), ceftazidime-resistant AmpC-producing species (MIC90of 1 mg/liter), non-ESBLE. coli(MIC90of ≤0.125 mg/liter), non-ESBLK. pneumoniae(MIC90of 0.25 mg/liter), and ceftazidime-nonresistant AmpC-producing species (MIC90of ≤0.5 mg/liter). Ninety-six percent of fluoroquinolone-resistantP. aeruginosastrains were inhibited at a ceftazidime-avibactam MIC of ≤8 mg/liter (using the susceptible CLSI breakpoint for ceftazidime alone as a reference), with a MIC90of 8 mg/liter. Additionally, fluoroquinolone-resistant mutants from each species tested were obtainedin vitrofrom two strains, one susceptible to ceftazidime and the other a β-lactamase producer with a high MIC against ceftazidime but susceptible to ceftazidime-avibactam. Thereby, the impact of fluoroquinolone resistance on the activity of ceftazidime-avibactam could be assessed. The MIC90values of ceftazidime-avibactam for the fluoroquinolone-resistant mutant strains ofEnterobacteriaceaeandP. aeruginosawere ≤4 mg/liter and ≤8 mg/liter, respectively. We conclude that the presence of fluoroquinolone resistance does not affectEnterobacteriaceaeandP. aeruginosasusceptibility to ceftazidime-avibactam; that is, there is no cross-resistance.

scholarly journals Within-Host Evolution of Pseudomonas aeruginosa Reveals Adaptation toward Iron Acquisition from Hemoglobin

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Rasmus Lykke Marvig ◽  
Søren Damkiær ◽  
S. M. Hossein Khademi ◽  
Trine M. Markussen ◽  
Søren Molin ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Iron Acquisition ◽  
Chronic Infections ◽  
Content Type ◽  
Mortality And Morbidity ◽  
Airway Infections ◽  
Host Evolution ◽  
Promoter Mutations

ABSTRACTPseudomonas aeruginosaairway infections are a major cause of mortality and morbidity of cystic fibrosis (CF) patients. In order to persist,P. aeruginosadepends on acquiring iron from its host, and multiple different iron acquisition systems may be active during infection. This includes the pyoverdine siderophore and thePseudomonasheme utilization (phu) system. While the regulation and mechanisms of several iron-scavenging systems are well described, it is not clear whether such systems are targets for selection during adaptation ofP. aeruginosato the host environment. Here we investigated the within-host evolution of the transmissibleP. aeruginosaDK2 lineage. We found positive selection for promoter mutations leading to increased expression of thephusystem. By mimicking conditions of the CF airwaysin vitro, we experimentally demonstrate that increased expression ofphuRconfers a growth advantage in the presence of hemoglobin, thus suggesting thatP. aeruginosaevolves toward iron acquisition from hemoglobin. To rule out that this adaptive trait is specific to the DK2 lineage, we inspected the genomes of additionalP. aeruginosalineages isolated from CF airways and found similar adaptive evolution in two distinct lineages (DK1 and PA clone C). Furthermore, in all three lineages,phuRpromoter mutations coincided with the loss of pyoverdine production, suggesting that within-host adaptation toward heme utilization is triggered by the loss of pyoverdine production. Targeting heme utilization might therefore be a promising strategy for the treatment ofP. aeruginosainfections in CF patients.IMPORTANCEMost bacterial pathogens depend on scavenging iron within their hosts, which makes the battle for iron between pathogens and hosts a hallmark of infection. Accordingly, the ability of the opportunistic pathogenPseudomonas aeruginosato cause chronic infections in cystic fibrosis (CF) patients also depends on iron-scavenging systems. While the regulation and mechanisms of several such iron-scavenging systems have been well described, not much is known about how the within-host selection pressures act on the pathogens’ ability to acquire iron. Here, we investigated the within-host evolution ofP. aeruginosa, and we found evidence thatP. aeruginosaduring long-term infections evolves toward iron acquisition from hemoglobin. This adaptive strategy might be due to a selective loss of other iron-scavenging mechanisms and/or an increase in the availability of hemoglobin at the site of infection. This information is relevant to the design of novel CF therapeutics and the development of models of chronic CF infections.

scholarly journals Development of Resistance in Wild-Type and Hypermutable Pseudomonas aeruginosa Strains Exposed to Clinical Pharmacokinetic Profiles of Meropenem and Ceftazidime Simulated In Vitro

2007 ◽  
Vol 51 (10) ◽  
pp. 3642-3649 ◽  
Author(s):  
Beate Henrichfreise ◽  
Irith Wiegand ◽  
Ingeborg Luhmer-Becker ◽  
Bernd Wiedemann
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Parent Strain ◽  
Resistance Mechanisms ◽  
In Vitro Models ◽  
Wild Type ◽  
Resistance Development ◽  
Development Of Resistance ◽  
Pharmacokinetic Profiles ◽  
Mutant Prevention Concentration

ABSTRACT In this study we investigated the interplay of antibiotic pharmacokinetic profiles and the development of mutation-mediated resistance in wild-type and hypermutable Pseudomonas aeruginosa strains. We used in vitro models simulating profiles of the commonly used therapeutic drugs meropenem and ceftazidime, two agents with high levels of antipseudomonal activity said to have different potentials for stimulating resistance development. During ceftazidime treatment of the wild-type strain (PAO1), fully resistant mutants overproducing AmpC were selected rapidly and they completely replaced wild-type cells in the population. During treatment with meropenem, mutants of PAO1 were not selected as rapidly and showed only intermediate resistance due to the loss of OprD. These mutants also replaced the parent strain in the population. During the treatment of the mutator P. aeruginosa strain with meropenem, the slowly selected mutants did not accumulate several resistance mechanisms but only lost OprD and did not completely replace the parent strain in the population. Our results indicate that the commonly used dosing regimens for meropenem and ceftazidime cannot avoid the selection of mutants of wild-type and hypermutable P. aeruginosa strains. For the treatment outcome, including the prevention of resistance development, it would be beneficial for the antibiotic concentration to remain above the mutant prevention concentration for a longer period of time than it does in present regimens.

Activities of Tobramycin and Six Other Antibiotics against Pseudomonas aeruginosa Isolates from Patients with Cystic Fibrosis

1999 ◽  
Vol 43 (12) ◽  
pp. 2877-2880 ◽  
Author(s):  
Ribhi M. Shawar ◽  
David L. MacLeod ◽  
Richard L. Garber ◽  
Jane L. Burns ◽  
Jenny R. Stapp ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Antimicrobial Agents ◽  
Active Drug ◽  
Resistance Mechanisms ◽  
Phase Iii ◽  
In Vitro Activity ◽  
Phase Iii Clinical Trials ◽  
Multiple Antibiotics

ABSTRACT The in vitro activity of tobramycin was compared with those of six other antimicrobial agents against 1,240 Pseudomonas aeruginosa isolates collected from 508 patients with cystic fibrosis during pretreatment visits as part of the phase III clinical trials of tobramycin solution for inhalation. The tobramycin MIC at which 50% of isolates are inhibited (MIC50) and MIC90 were 1 and 8 μg/ml, respectively. Tobramycin was the most active drug tested and also showed good activity against isolates resistant to multiple antibiotics. The isolates were less frequently resistant to tobramycin (5.4%) than to ceftazidime (11.1%), aztreonam (11.9%), amikacin (13.1%), ticarcillin (16.7%), gentamicin (19.3%), or ciprofloxacin (20.7%). For all antibiotics tested, nonmucoid isolates were more resistant than mucoid isolates. Of 56 isolates for which the tobramycin MIC was ≥16 μg/ml and that were investigated for resistance mechanisms, only 7 (12.5%) were shown to possess known aminoglycoside-modifying enzymes; the remaining were presumably resistant by an incompletely understood mechanism often referred to as “impermeability.”

scholarly journals Antimicrobial Activity of Ceftazidime-Avibactam Tested against Multidrug-Resistant Enterobacteriaceae and Pseudomonas aeruginosa Isolates from U.S. Medical Centers, 2013 to 2016

2017 ◽  
Vol 61 (11) ◽  
Author(s):  
Helio S. Sader ◽  
Mariana Castanheira ◽  
Dee Shortridge ◽  
Rodrigo E. Mendes ◽  
Robert K. Flamm
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Multidrug Resistant ◽  
Drug Resistant ◽  
Large Collection ◽  
Content Type ◽  
Negative Results ◽  
Medical Centers ◽  
Extensively Drug Resistant ◽  
Genes Encoding

ABSTRACT The in vitro activity of ceftazidime-avibactam and many comparator agents was determined against various resistant subsets of organisms selected among 36,380 Enterobacteriaceae and 7,868 Pseudomonas aeruginosa isolates. The isolates were consecutively collected from 94 U.S. hospitals, and all isolates were tested for susceptibility by reference broth microdilution methods in a central monitoring laboratory (JMI Laboratories). Enterobacteriaceae isolates resistant to carbapenems (CRE) and/or ceftazidime-avibactam (MIC ≥ 16 μg/ml) were evaluated for the presence of genes encoding extended-spectrum β-lactamases and carbapenemases. Ceftazidime-avibactam inhibited >99.9% of all Enterobacteriaceae at the susceptible breakpoint of ≤8 μg/ml and was active against multidrug-resistant (MDR; n = 2,953; MIC50/90, 0.25/1 μg/ml; 99.2% susceptible), extensively drug-resistant (XDR; n = 448; MIC50/90, 0.5/2 μg/ml; 97.8% susceptible), and CRE (n = 513; MIC50/90, 0.5/2 μg/ml; 97.5% susceptible) isolates. Only 82.2% of MDR Enterobacteriaceae (n = 2,953) and 64.2% of ceftriaxone-nonsusceptible Klebsiella pneumoniae (n = 1,063) isolates were meropenem susceptible. Among Enterobacter cloacae (22.2% ceftazidime nonsusceptible), 99.8% of the isolates, including 99.3% of the ceftazidime-nonsusceptible isolates, were ceftazidime-avibactam susceptible. Only 23 of 36,380 Enterobacteriaceae (0.06%) isolates were ceftazidime-avibactam nonsusceptible, including 9 metallo-β-lactamase producers and 2 KPC-producing strains with porin alteration; the remaining 12 strains showed negative results for all β-lactamases tested. Ceftazidime-avibactam showed potent activity against P. aeruginosa (MIC50/90, 2/4 μg/ml; 97.1% susceptible), including MDR (MIC50/90, 4/16 μg/ml; 86.5% susceptible) isolates, and inhibited 71.8% of isolates nonsusceptible to meropenem, piperacillin-tazobactam, and ceftazidime (n = 628). In summary, ceftazidime-avibactam demonstrated potent activity against a large collection (n = 44,248) of contemporary Gram-negative bacilli isolated from U.S. patients, including organisms resistant to most currently available agents, such as CRE and meropenem-nonsusceptible P. aeruginosa.

scholarly journals NirA Is an Alternative Nitrite Reductase from Pseudomonas aeruginosa with Potential as an Antivirulence Target

mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Samuel Fenn ◽  
Jean-Frédéric Dubern ◽  
Cristina Cigana ◽  
Maura De Simone ◽  
James Lazenby ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Nitrite Reductase ◽  
Disease Models ◽  
Attractive Alternative ◽  
Dependent Manner ◽  
Content Type ◽  
Isogenic Mutant ◽  
Virulence Traits ◽  
Infection Models

ABSTRACT The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro. Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA. IMPORTANCE The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs. The host-specific nature of P. aeruginosa virulence adds complexity to the discovery of these types of targets. Using a sequence of in vitro assays and phylogenetically diverse in vivo disease models, we have identified a PA4130 mutant with reduced production in a number of virulence traits and severe attenuation across all infection models tested. Characterization of PA4130 revealed that it is a ferredoxin-nitrite reductase and hence was named NirA. These results, together with attenuation of nirA mutants in different clinical isolates, high level conservation of its gene product in P. aeruginosa genomes, and the lack of orthologues in human genomes, make NirA an attractive antivirulence target.

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