scholarly journals TonB-Dependent Receptor Repertoire of Pseudomonas aeruginosa for Uptake of Siderophore-Drug Conjugates

2018 ◽  
Vol 62 (6) ◽  
Author(s):  
Alexandre Luscher ◽  
Lucile Moynié ◽  
Pamela Saint Auguste ◽  
Dirk Bumann ◽  
Lena Mazza ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Genetic Locus ◽  
Drug Uptake ◽  
Beta Lactam ◽  
Strain Pao1 ◽  
Content Type ◽  
Drug Molecules ◽  
Drug Conjugates ◽  
Receptor Repertoire ◽  
Outer Membrane Permeability

ABSTRACT The conjugation of siderophores to antimicrobial molecules is an attractive strategy to overcome the low outer membrane permeability of Gram-negative bacteria. In this Trojan horse approach, the transport of drug conjugates is redirected via TonB-dependent receptors (TBDR), which are involved in the uptake of essential nutrients, including iron. Previous reports have demonstrated the involvement of the TBDRs PiuA and PirA from Pseudomonas aeruginosa and their orthologues in Acinetobacter baumannii in the uptake of siderophore-beta-lactam drug conjugates. By in silico screening, we further identified a PiuA orthologue, termed PiuD, present in clinical isolates, including strain LESB58. The piuD gene in LESB58 is located at the same genetic locus as piuA in strain PAO1. PiuD has a similar crystal structure as PiuA and is involved in the transport of the siderophore-drug conjugates BAL30072, MC-1, and cefiderocol in strain LESB58. To screen for additional siderophore-drug uptake systems, we overexpressed 28 of the 34 TBDRs of strain PAO1 and identified PfuA, OptE, OptJ, and the pyochelin receptor FptA as novel TBDRs conferring increased susceptibility to siderophore-drug conjugates. The existence of a TBDR repertoire in P. aeruginosa able to transport siderophore-drug molecules potentially decreases the likelihood of resistance emergence during therapy.

scholarly journals Multicenter Evaluation of the Etest Gradient Diffusion Method for Ceftolozane-Tazobactam Susceptibility Testing of Enterobacteriaceae and Pseudomonas aeruginosa

2018 ◽  
Vol 56 (9) ◽  
Author(s):  
Adam L. Bailey ◽  
Tom Armstrong ◽  
Hari-Prakash Dwivedi ◽  
Gerald A. Denys ◽  
Janet Hindler ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Susceptibility Testing ◽  
Clinical Isolates ◽  
Diffusion Method ◽  
Beta Lactam ◽  
Content Type ◽  
Gradient Diffusion ◽  
Tract Infections ◽  
Abdominal Infections ◽  
Inhibitor Combination

ABSTRACT Ceftolozane-tazobactam (C/T) is a novel beta-lactam–beta-lactamase inhibitor combination antibiotic approved by the U.S. Food and Drug Administration in 2014 for the treatment of complicated intra-abdominal infections (in combination with metronidazole) and complicated urinary tract infections. In this study, we evaluated the performance of the C/T Etest, a gradient diffusion method. C/T Etest was compared to broth microdilution (BMD) for 51 Enterobacteriaceae challenge isolates and 39 Pseudomonas aeruginosa challenge isolates at three clinical sites. Essential agreement (EA) between the methods ranged from 47 to 49/51 (92.2 to 96.1%) for the Enterobacteriaceae, and categorical agreement (CA) ranged from 49 to 51/51 (96.1 to 100.0%). EA and CA for P. aeruginosa were 100% at all sites. The C/T Etest was also compared to BMD for susceptibility testing on 966 clinical isolates (793 Enterobacteriaceae, including 167 Klebsiella pneumoniae and 159 Escherichia coli isolates, in addition to 173 P. aeruginosa isolates) collected at four clinical sites. EA between Etest and BMD was 96.9% for Enterobacteriaceae isolates and 98.8% for P. aeruginosa isolates. Within the Enterobacteriaceae, isolates from each species examined had >96% CA. For the clinical isolates, no very major errors were identified but two major errors were found (one for K. pneumoniae and one for Providencia rettgeri). By BMD, 47.0% of Enterobacteriaceae and 46.2% of P. aeruginosa challenge strains were nonsusceptible to C/T by CLSI breakpoint criteria; 8.2% of clinical Enterobacteriaceae isolates and 12.1% of clinical P. aeruginosa isolates were nonsusceptible to C/T by CLSI breakpoint criteria. In conclusion, Etest is accurate and reproducible for C/T susceptibility testing of Enterobacteriaceae and P. aeruginosa.

scholarly journals Structure and Function of the PiuA and PirA Siderophore-Drug Receptors from Pseudomonas aeruginosa and Acinetobacter baumannii

2017 ◽  
Vol 61 (4) ◽  
Author(s):  
Lucile Moynié ◽  
Alexandre Luscher ◽  
Dora Rolo ◽  
Daniel Pletzer ◽  
Antoni Tortajada ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Acinetobacter Baumannii ◽  
Rational Design ◽  
Transport Systems ◽  
Gram Negative ◽  
Content Type ◽  
Drug Conjugates ◽  
Membrane Complex ◽  
And Function ◽  
Drug Receptors

ABSTRACT The outer membrane of Gram-negative bacteria presents an efficient barrier to the permeation of antimicrobial molecules. One strategy pursued to circumvent this obstacle is to hijack transport systems for essential nutrients, such as iron. BAL30072 and MC-1 are two monobactams conjugated to a dihydroxypyridone siderophore that are active against Pseudomonas aeruginosa and Acinetobacter baumannii. Here, we investigated the mechanism of action of these molecules in A. baumannii. We identified two novel TonB-dependent receptors, termed Ab-PiuA and Ab-PirA, that are required for the antimicrobial activity of both agents. Deletion of either piuA or pirA in A. baumannii resulted in 4- to 8-fold-decreased susceptibility, while their overexpression in the heterologous host P. aeruginosa increased susceptibility to the two siderophore-drug conjugates by 4- to 32-fold. The crystal structures of PiuA and PirA from A. baumannii and their orthologues from P. aeruginosa were determined. The structures revealed similar architectures; however, structural differences between PirA and PiuA point to potential differences between their cognate siderophore ligands. Spontaneous mutants, selected upon exposure to BAL30072, harbored frameshift mutations in either the ExbD3 or the TonB3 protein of A. baumannii, forming the cytoplasmic-membrane complex providing the energy for the siderophore translocation process. The results of this study provide insight for the rational design of novel siderophore-drug conjugates against problematic Gram-negative pathogens.

scholarly journals Mutations in β-Lactamase AmpC Increase Resistance of Pseudomonas aeruginosa Isolates to Antipseudomonal Cephalosporins

2015 ◽  
Vol 59 (10) ◽  
pp. 6248-6255 ◽  
Author(s):  
M. Berrazeg ◽  
K. Jeannot ◽  
Véronique Yvette Ntsogo Enguéné ◽  
I. Broutin ◽  
S. Loeffert ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Reference Strain ◽  
Binding Pocket ◽  
Amino Acid Substitutions ◽  
Substrate Binding Pocket ◽  
Strain Pao1 ◽  
Gram Negative ◽  
Content Type ◽  
Omega Loop ◽  
New Mutations

ABSTRACTMutation-dependent overproduction of intrinsic β-lactamase AmpC is considered the main cause of resistance of clinical strains ofPseudomonas aeruginosato antipseudomonal penicillins and cephalosporins. Analysis of 31 AmpC-overproducing clinical isolates exhibiting a greater resistance to ceftazidime than to piperacillin-tazobactam revealed the presence of 17 mutations in the β-lactamase, combined with various polymorphic amino acid substitutions. When overexpressed in AmpC-deficientP. aeruginosa4098, the genes coding for 20/23 of these AmpC variants were found to confer a higher (2-fold to >64-fold) resistance to ceftazidime and ceftolozane-tazobactam than did the gene from reference strain PAO1. The mutations had variable effects on the MICs of ticarcillin, piperacillin-tazobactam, aztreonam, and cefepime. Depending on their location in the AmpC structure and their impact on β-lactam MICs, they could be assigned to 4 distinct groups. Most of the mutations affecting the omega loop, the R2 domain, and the C-terminal end of the protein were shared with extended-spectrum AmpCs (ESACs) from other Gram-negative species. Interestingly, two new mutations (F121L and P154L) were predicted to enlarge the substrate binding pocket by disrupting the stacking between residues F121 and P154. We also found that the reported ESACs emerged locally in a variety of clones, some of which are epidemic and did not require hypermutability. Taken together, our results show thatP. aeruginosais able to adapt to efficacious β-lactams, including the newer cephalosporin ceftolozane, through a variety of mutations affecting its intrinsic β-lactamase, AmpC. Data suggest that the rates of ESAC-producing mutants are ≥1.5% in the clinical setting.

scholarly journals Performance of Ceftolozane-Tazobactam Etest, MIC Test Strips, and Disk Diffusion Compared to Reference Broth Microdilution for β-Lactam-Resistant Pseudomonas aeruginosa Isolates

2017 ◽  
Vol 56 (3) ◽  
Author(s):  
Romney M. Humphries ◽  
Janet A. Hindler ◽  
Paul Magnano ◽  
Annie Wong-Beringer ◽  
Robert Tibbetts ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Los Angeles ◽  
Performance Characteristics ◽  
Disk Diffusion ◽  
Broth Microdilution ◽  
Beta Lactam ◽  
Content Type ◽  
Test Strips ◽  
Santa Ana

ABSTRACT The performance characteristics of the ceftolozane-tazobactam (C-T) Etest (bioMérieux, Marcy l'Etoile, France), MIC test strips (MTS; Liofilchem, Italy), and disk diffusion (Hardy, Santa Ana, CA) were evaluated for a collection of 308 beta-lactam-resistant isolates of Pseudomonas aeruginosa recovered from three institutions in Los Angeles, CA. Reference testing was performed by the reference broth microdilution (rBMD) method. MIC and disk results were interpreted using Clinical and Laboratory Standards Institute breakpoints. Overall, 72.5% of the isolates were susceptible to C-T by rBMD. Etest and disk diffusion demonstrated acceptable performance, whereas MTS yielded a greater than acceptable percentage of minor errors. Categorical agreement was 96.8% for Etest, 87.0% for MTS, and 92.9% for disk diffusion. No very major errors were observed by any test, and no major errors (ME) were observed by Etest or disk diffusion. Two ME (0.9% of susceptible isolates) were observed by MTS. The incidence of minor errors was 3.2%, 12.3%, and 7.1% for Etest, MTS, and disk diffusion, respectively. Essential agreement (EA) for Etest was excellent, at 97.7%, whereas the MICs obtained by MTS tended to be 1 to 2 dilutions higher than those obtained by rBMD, with an EA of 87.0%.

scholarly journals Activity of Ceftolozane-Tazobactam and Ceftazidime-Avibactam against Beta-Lactam-Resistant Pseudomonas aeruginosa Isolates

2017 ◽  
Vol 61 (12) ◽  
Author(s):  
Romney M. Humphries ◽  
Janet A. Hindler ◽  
Annie Wong-Beringer ◽  
Shelley A. Miller
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Los Angeles ◽  
Beta Lactam ◽  
Content Type

ABSTRACT Ceftolozane-tazobactam (C/T) and ceftazidime-avibactam (CZA) MICs were evaluated for a collection of 309 beta-lactam-resistant isolates of Pseudomonas aeruginosa recovered from three institutions in the area of Los Angeles, CA. Overall, 12.0% of isolates were susceptible to imipenem, 15.9% were susceptible to meropenem, 20.7% were susceptible to piperacillin-tazobactam, 24.6% were susceptible to ceftazidime, 25.9% were susceptible to cefepime, 72.5% were susceptible to C/T, and 61.8% were susceptible to CZA. Among C/T-resistant isolates, 9.1% were CZA susceptible, whereas 36.4% of CZA-resistant isolates were susceptible to C/T.

scholarly journals Analyses of Short-Term Antagonistic Evolution of Pseudomonas aeruginosa Strain PAO1 and Phage KPP22 (Myoviridae Family, PB1-Like Virus Genus)

2016 ◽  
Vol 82 (15) ◽  
pp. 4482-4491 ◽  
Author(s):  
Jumpei Uchiyama ◽  
Masato Suzuki ◽  
Koji Nishifuji ◽  
Shin-ichiro Kato ◽  
Reina Miyata ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Bacterial Resistance ◽  
Phage Therapy ◽  
Open Reading Frames ◽  
Missense Mutations ◽  
Short Term ◽  
Strain Pao1 ◽  
Content Type ◽  
Phage Adsorption

ABSTRACTPseudomonas aeruginosacauses serious intractable infections in humans and animals. Bacteriophage (phage) therapy has been applied to treatP. aeruginosainfections, and phages belonging to the PB1-like virus genus in theMyoviridaefamily have been used as therapeutic phages. To achieve safer and more effective phage therapy, the use of preadapted phages is proposed. To understand in detail such phage preadaptation, the short-term antagonistic evolution of bacteria and phages should be studied. In this study, the short-term antagonistic evolution of bacteria and PB1-like phage was examined by studying phage-resistant clones ofP. aeruginosastrain PAO1 and mutant PB1-like phages that had recovered their infectivity. First, phage KPP22 was isolated and characterized; it was classified as belonging to the PB1-like virus genus in theMyoviridaefamily. Subsequently, three KPP22-resistant PAO1 clones and three KPP22 mutant phages capable of infecting these clones were isolated in three sets ofin vitroexperiments. It was shown that the bacterial resistance to phage KPP22 was caused by significant decreases in phage adsorption and that the improved infectivity of KPP22 mutant phages was caused by significant increases in phage adsorption. The KPP22-resistant PAO1 clones and the KPP22 mutant phages were then analyzed genetically. All three KPP22-resistant PAO1 clones, which were deficient for the O5 antigen, had a common nonsense mutation in thewzygene. All the KPP22 mutant phage genomes showed the same four missense mutations in the open reading framesorf060,orf065, andorf086. The information obtained in this study should be useful for further development of safe and efficient phage therapy.IMPORTANCEPseudomonas aeruginosacauses serious intractable infections in humans and animals; bacteriophage (phage) therapy has been utilized to treatP. aeruginosainfections, and phages that belong to the PB1-like virus genus in the familyMyoviridaehave been used as therapeutic phages. The preadapted phage is trained in advance through the antagonistic evolution of bacteria and phage and is proposed to be used to achieve safer and more effective phage therapy. In this study, to understand the phage preadaptation, thein vitroshort-term antagonistic evolution was studied usingP. aeruginosastrain PAO1 and the newly isolated PB1-like phage KPP22. Phage KPP22 was characterized, and the molecular framework regarding the phage preadaptation of KPP22 was elucidated. The importance of study of antagonistic evolution of bacteria and phage in phage therapy is discussed.

scholarly journals Dynamics of Mutations during Development of Resistance by Pseudomonas aeruginosa against Five Antibiotics

2016 ◽  
Vol 60 (7) ◽  
pp. 4229-4236 ◽  
Author(s):  
Yanfang Feng ◽  
Martijs J. Jonker ◽  
Ioannis Moustakas ◽  
Stanley Brul ◽  
Benno H. ter Kuile
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quantitative Relationship ◽  
Opportunistic Pathogen ◽  
Cellular Systems ◽  
Beta Lactam ◽  
Cellular Functions ◽  
Lower Growth ◽  
Content Type ◽  
Development Of Resistance ◽  
Considerable Morbidity

ABSTRACTPseudomonas aeruginosais an opportunistic pathogen that causes considerable morbidity and mortality, specifically during intensive care. Antibiotic-resistant variants of this organism are more difficult to treat and cause substantial extra costs compared to susceptible strains. In the laboratory,P. aeruginosarapidly developed resistance to five medically relevant antibiotics upon exposure to stepwise increasing concentrations. At several time points during the acquisition of resistance, samples were taken for whole-genome sequencing. The increase in the MIC of ciprofloxacin was linked to specific mutations ingyrA,parC, andgyrB, appearing sequentially. In the case of tobramycin, mutations infusA,HP02880,rplB, andcapDwere induced. The MICs of the beta-lactam compounds meropenem and ceftazidime and the combination of piperacillin and tazobactam correlated linearly with beta-lactamase activity but not always with individual mutations. The genes that were mutated during the development of beta-lactam resistance differed for each antibiotic. A quantitative relationship between the frequency of mutations and the increase in resistance could not be established for any of the antibiotics. When the adapted strains are grown in the absence of the antibiotic, some mutations remained and others were reversed, but this reversal did not necessarily lower the MIC. The increased MIC came at the cost of moderately reduced cellular functions or a somewhat lower growth rate. In all cases except ciprofloxacin, the increase in resistance seems to be the result of complex interactions among several cellular systems rather than individual mutations.

scholarly journals In Vitro Evaluation of Biofilm Dispersal as a Therapeutic Strategy To Restore Antimicrobial Efficacy

2017 ◽  
Vol 61 (10) ◽  
Author(s):  
Dan Roizman ◽  
Celine Vidaillac ◽  
Michael Givskov ◽  
Liang Yang
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Therapeutic Strategy ◽  
Antimicrobial Efficacy ◽  
Direct Impact ◽  
Proof Of Concept ◽  
Strain Pao1 ◽  
Content Type ◽  
Biofilm Dispersal ◽  
Engineered Strain

ABSTRACT As a proof-of-concept study, the direct impact of biofilm dispersal on the in vitro efficacy of imipenem and tobramycin was evaluated against 3-day-old biofilms of Pseudomonas aeruginosa. Arabinose induction of biofilm dispersal via activation of the phosphodiesterase YhjH in the P. aeruginosa engineered strain PAO1/p BAD -yhjH resulted in increased antimicrobial efficacy and synergy of the imipenem-tobramycin combination. These results support the use of biofilm dispersal to enhance antimicrobial efficacy in the treatment of biofilm-associated infections, representing a promising therapeutic strategy.

scholarly journals Antibacterial Activity of Human Simulated Epithelial Lining Fluid Concentrations of Ceftazidime-Avibactam Alone or in Combination with Amikacin Inhale (BAY41-6551) against Carbapenem-Resistant Pseudomonas aeruginosa and Klebsiella pneumoniae

2018 ◽  
Vol 62 (7) ◽  
Author(s):  
Safa S. Almarzoky Abuhussain ◽  
Joseph L. Kuti ◽  
David P. Nicolau
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Combination Therapy ◽  
Klebsiella Pneumoniae ◽  
Combination Regimen ◽  
Epithelial Lining ◽  
Beta Lactam ◽  
Chemostat Model ◽  
Epithelial Lining Fluid ◽  
Content Type ◽  
Carbapenem Resistant

ABSTRACT The role of inhalational combination therapy when treating carbapenem-resistant Pseudomonas aeruginosa and Klebsiella pneumoniae with newer beta-lactam/beta-lactamase inhibitors has not been established. Using a 72-h in vitro pharmacodynamic chemostat model, we simulated the human exposures achieved in epithelial lining fluid (ELF) following intravenous treatment with ceftazidime-avibactam (CZA) 2.5 g every 8 h (q8h) alone and in combination with inhaled amikacin (AMK-I) 400 mg q12h, a reformulated aminoglycoside designed for inhalational administration, against three P. aeruginosa isolates (CZA [ceftazidime/avibactam] MICs, 4/4 to 8/4 μg/ml; AMK-I MICs, 8 to 64 μg/ml) and three K. pneumoniae isolates (CZA MICs, 1/4 to 8/4 μg/ml; AMK-I MICs, 32 to 64 μg/ml). Combination therapy resulted in a significant reduction in 72-h CFU compared with that of CZA monotherapy against two of three P. aeruginosa isolates (−4.14 log 10 CFU/ml, P = 0.027; −1.42 log 10 CFU/ml, P = 0.020; and −0.4 log 10 CFU/ml, P = 0.298) and two of three K. pneumoniae isolates (0.04 log 10 CFU/ml, P = 0.963; −4.34 log 10 CFU/ml, P < 0.001; and −2.34 log 10 CFU/ml, P = 0.021). When measured by the area under the bacterial growth curve (AUBC) over 72 h, significant reductions were observed in favor of the combination regimen against all six isolates tested. AMK-I combination therapy successfully suppressed CZA resistance development in one K. pneumoniae isolate harboring bla KPC-3 that was observed during CZA monotherapy. These studies suggest a beneficial role for combination therapy with intravenous CZA and inhaled AMK when treating pneumonia caused by carbapenem-resistant Gram-negative bacteria.

scholarly journals Detection of the KPC Gene in Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii during a PCR-Based Nosocomial Surveillance Study in Puerto Rico

2011 ◽  
Vol 55 (6) ◽  
pp. 2968-2970 ◽  
Author(s):  
Iraida E. Robledo ◽  
Edna E. Aquino ◽  
Guillermo J. Vázquez
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Puerto Rico ◽  
Klebsiella Pneumoniae ◽  
Acinetobacter Baumannii ◽  
Surveillance Study ◽  
Beta Lactam ◽  
Content Type ◽  
Geographical Regions ◽  
Clinically Significant

ABSTRACTA 6-month, PCR-based, island-wide hospital surveillance study of beta-lactam resistance inEscherichia coli,Klebsiella pneumoniae,Pseudomonas aeruginosa, andAcinetobacter baumanniiwas conducted in Puerto Rico. Of 10,507 isolates, 1,239 (12%) unique, multi-beta-lactam-resistant isolates from all geographical regions were identified. The KPC gene was detected in 61E. coli, 333K. pneumoniae, 99P. aeruginosa, and 41A. baumanniiisolates, indicating the widespread dissemination of the KPC gene in clinically significant nosocomial isolates.

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