scholarly journals ThiL is a valid antibacterial target that is essential for both thiamine biosynthesis and salvage pathway in Pseudomonas aeruginosa

2020 ◽  
Author(s):  
Hyung Jun Kim ◽  
Hyunjung Lee ◽  
Yunmi Lee ◽  
Inhee Choi ◽  
Yoonae Ko ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Drug Target ◽  
Bacterial Load ◽  
Thiamine Pyrophosphate ◽  
Infection Model ◽  
Antibiotic Development ◽  
New Drug ◽  
Thiamine Biosynthesis

ABSTRACTThiamine pyrophosphate (TPP) is an essential cofactor for various pivotal cellular processes in all living organisms, including bacteria. As thiamine biosynthesis occurs in bacteria but not humans, bacterial thiamine biosynthesis is an attractive target for antibiotic development. Among enzymes in the thiamine biosynthetic pathway, thiamine monophosphate kinase (ThiL) catalyzes the final step of the pathway, phosphorylating thiamine monophosphate (TMP) to produce TPP. In this work, we extensively investigated ThiL in Pseudomonas aeruginosa, a major pathogen of hospital-acquired infections. We demonstrated that thiL deletion abolishes not only thiamine biosynthesis but also thiamine salvage capability, showing growth defects of the ΔthiL mutant even in the presence of thiamine derivatives except TPP. Most importantly, the pathogenesis of the ΔthiL mutant was markedly attenuated compared to wild-type bacteria, with lower inflammatory cytokine induction and 103~104 times decreased bacterial load in an in vivo infection model where the intracellular TPP level is in the submicromolar range. In order to validate P. aeruginosa ThiL (PaThiL) as a new drug target, we further characterized its biochemical properties determining a Vmax of 4.0±0.2 nomol·min−1 and KM values of 111±8 and 8.0±3.5μM for ATP and TMP, respectively. A subsequent in vitro small molecule screening identified PaThiL inhibitors including WAY213613 that is a noncompetitive inhibitor with a Ki value of 13.4±2.3 μM and a potential antibacterial activity against P. aeruginosa. This study proved that PaThiL is a new drug target against P. aeruginosa providing comprehensive biological and biochemical data that could facilitate to develop a new repertoire of antibiotics.

scholarly journals The ThiL enzyme is a valid antibacterial target essential for both thiamine biosynthesis and salvage pathways in Pseudomonas aeruginosa

2020 ◽  
Vol 295 (29) ◽  
pp. 10081-10091
Author(s):  
Hyung Jun Kim ◽  
Hyunjung Lee ◽  
Yunmi Lee ◽  
Inhee Choi ◽  
Yoonae Ko ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Drug Target ◽  
Biochemical Properties ◽  
Thiamine Pyrophosphate ◽  
Infection Model ◽  
Screening Assay ◽  
Antibiotic Development ◽  
Thiamine Biosynthesis

Thiamine pyrophosphate (TPP) is an essential cofactor for various pivotal cellular processes in all living organisms, including bacteria. Thiamine biosynthesis occurs in bacteria but not in humans; therefore, the enzymes in this pathway are attractive targets for antibiotic development. Among these enzymes, thiamine monophosphate kinase (ThiL) catalyzes the final step of this pathway, phosphorylating thiamine monophosphate to produce TPP. Here, we extensively investigated ThiL in Pseudomonas aeruginosa, a major pathogen responsible for hospital-acquired infections. We demonstrate that thiL deletion abolishes not only thiamine biosynthesis but also thiamine salvage capability and results in growth defects of the ΔthiL strain even in the presence of thiamine derivatives, except for TPP. Most importantly, the pathogenesis of the ΔthiL strain was markedly attenuated, compared with that of WT cells, with lower inflammatory cytokine induction and 103–104-fold decreased bacterial loads in an in vivo infection model in which the intracellular TPP level was in the submicromolar range. To validate P. aeruginosa ThiL (PaThiL) as a drug target, we further characterized its biochemical properties, determining a Vmax of 4.0 ± 0.2 nmol·min−1 and Km values of 111 ± 8 and 8.0 ± 3.5 μm for ATP and thiamine monophosphate, respectively. An in vitro small-molecule screening assay identified PaThiL inhibitors including WAY213613, a noncompetitive inhibitor with a Ki value of 13.4 ± 2.3 μm and potential antibacterial activity against P. aeruginosa. These comprehensive biological and biochemical results indicate that PaThiL represents a potential drug target for the development of an augmented repertoire of antibiotics against P. aeruginosa.

scholarly journals Contribution of swarming motility to dissemination in a Pseudomonas aeruginosa murine skin abscess infection model

2020 ◽  
Author(s):  
Shannon R Coleman ◽  
Daniel Pletzer ◽  
Robert E W Hancock
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Bacterial Load ◽  
Infection Model ◽  
Swarming Motility ◽  
Semisolid Medium ◽  
Low Concentrations ◽  
Complex Adaptive

Abstract Swarming motility in Pseudomonas aeruginosa is a multicellular adaptation induced by semisolid medium with amino acids as a nitrogen source. By phenotypic screening, we differentiated swarming from other complex adaptive phenotypes, such as biofilm formation, swimming and twitching, by identifying a swarming-specific mutant in ptsP, a metabolic regulator. This swarming-deficient mutant was tested in an acute murine skin abscess infection model. Bacteria were recovered at significantly lower numbers from organs of mice infected with the ∆ptsP mutant. We also tested the synthetic peptide 1018 for activity against different motilities and efficacy in vivo. Treatment with 1018 mimicked the phenotype of the ∆ptsP mutant in vitro, as swarming was inhibited at low concentrations (<2 μg/mL) but not swimming or twitching, and in vivo, as mice had a reduced bacterial load recovered from organs. Therefore, PtsP functions as a regulator of swarming, which in turn contributes to dissemination and colonization in vivo.

scholarly journals Drug Repurposing: In vitro and in vivo Antimicrobial and Antibiofilm Effects of Bithionol Against Enterococcus faecalis and Enterococcus faecium

2021 ◽  
Vol 12 ◽  
Author(s):  
Pengfei She ◽  
Yangxia Wang ◽  
Yingjia Li ◽  
Linying Zhou ◽  
Shijia Li ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Bacterial Load ◽  
Drug Repurposing ◽  
Infection Model ◽  
Dependent Manner ◽  
Antibiotic Development ◽  
Vancomycin Resistant ◽  
Conventional Antibiotics

Widespread antibiotic resistance has been reported in enterococcal pathogens that cause life-threatening infections. Enterococci species rapidly acquire resistance and the pace of new antibiotic development is slow. Drug repurposing is a promising approach in solving this problem. Bithionol (BT) is a clinically approved anthelminthic drug. In this study, we found that BT showed significant antimicrobial and antibiofilm effects against Enterococcus faecalis and vancomycin-resistant Entercococcus faecium in vitro, in a dose-dependent manner, by disrupting the integrity of the bacterial cell membranes. Moreover, BT effectively reduced the bacterial load in mouse organs when combined with conventional antibiotics in a peritonitis infection model. Thus, BT has shown potential as a therapeutic agent against E. faecalis- and vancomycin-resistant E. faecium-related infections.

scholarly journals Preclinical In Vitro and In Vivo Characterization of the Fully Human Monoclonal IgM Antibody KBPA101 Specific for Pseudomonas aeruginosa Serotype IATS-O11

2010 ◽  
Vol 54 (6) ◽  
pp. 2338-2344 ◽  
Author(s):  
Michael P. Horn ◽  
Adrian W. Zuercher ◽  
Martin A. Imboden ◽  
Michael P. Rudolf ◽  
Hedvika Lazar ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Treatment Options ◽  
Human Monoclonal Antibody ◽  
Human Monocyte ◽  
Peripheral Blood Lymphocytes ◽  
Infection Model ◽  
High Avidity ◽  
Burn Wound

ABSTRACT Pseudomonas aeruginosa infection in ventilator-associated pneumonia is a serious and often life-threatening complication in intensive care unit patients, and new treatment options are needed. We used B-cell-enriched peripheral blood lymphocytes from a volunteer immunized with a P. aeruginosa O-polysaccharide-toxin A conjugate vaccine to generate human hybridoma cell lines producing monoclonal antibodies specific for individual P. aeruginosa lipopolysaccharide serotypes. The fully human monoclonal antibody secreted by one of these lines, KBPA101, is an IgM/κ antibody that binds P. aeruginosa of International Antigenic Typing System (IATS) serotype O11 with high avidity (5.81 × 107 M−1 ± 2.8 × 107 M−1) without cross-reacting with other serotypes. KBPA101 specifically opsonized the P. aeruginosa of IATS O11 serotype and mediated complement-dependent phagocytosis in vitro by the human monocyte-like cell line HL-60 at a very low concentration (half-maximal phagocytosis at 0.16 ng/ml). In vivo evaluation of KBPA101 demonstrated a dose-response relationship for protection against systemic infections in a murine burn wound sepsis model, where 70 to 100% of animals were protected against lethal challenges with P. aeruginosa at doses as low as 5 μg/animal. Furthermore, a high efficacy of KBPA101 in protection from local respiratory infections in an acute lung infection model in mice was demonstrated. Preclinical toxicology evaluation on human tissue, in rabbits, and in mice did not indicate any toxicity of KBPA101. Based on these preclinical findings, the first human clinical trials have been initiated.

scholarly journals Systemic Antibacterial Activity of Novel Synthetic Cyclic Peptides

2005 ◽  
Vol 49 (8) ◽  
pp. 3302-3310 ◽  
Author(s):  
Véronique Dartois ◽  
Jorge Sanchez-Quesada ◽  
Edelmira Cabezas ◽  
Ellen Chi ◽  
Chad Dubbelde ◽  
...  
Keyword(s):  
Cyclic Peptides ◽  
Prolonged Exposure ◽  
Cyclic Peptide ◽  
Present Report ◽  
Bacterial Load ◽  
Pharmacokinetic Profile ◽  
Therapeutic Window ◽  
Infection Model

ABSTRACT Cyclic peptides with an even number of alternating d,l-α-amino acid residues are known to self-assemble into organic nanotubes. Such peptides previously have been shown to be stable upon protease treatment, membrane active, and bactericidal and to exert antimicrobial activity against Staphylococcus aureus and other gram-positive bacteria. The present report describes the in vitro and in vivo pharmacology of selected members of this cyclic peptide family. The intravenous (i.v.) efficacy of six compounds with MICs of less than 12 μg/ml was tested in peritonitis and neutropenic-mouse thigh infection models. Four of the six peptides were efficacious in vivo, with 50% effective doses in the peritonitis model ranging between 4.0 and 6.7 mg/kg against methicillin-sensitive S. aureus (MSSA). In the thigh infection model, the four peptides reduced the bacterial load 2.1 to 3.0 log units following administration of an 8-mg/kg i.v. dose. Activity against methicillin-resistant S. aureus was similar to MSSA. The murine pharmacokinetic profile of each compound was determined following i.v. bolus injection. Interestingly, those compounds with poor efficacy in vivo displayed a significantly lower maximum concentration of the drug in serum and a higher volume of distribution at steady state than compounds with good therapeutic properties. S. aureus was unable to easily develop spontaneous resistance upon prolonged exposure to the peptides at sublethal concentrations, in agreement with the proposed interaction with multiple components of the bacterial membrane canopy. Although additional structure-activity relationship studies are required to improve the therapeutic window of this class of antimicrobial peptides, our results suggest that these amphipathic cyclic d,l-α-peptides have potential for systemic administration and treatment of otherwise antibiotic-resistant infections.

scholarly journals Comparative Efficacies of Human Simulated Exposures of Telavancin and Vancomycin against Methicillin-Resistant Staphylococcus aureus with a Range of Vancomycin MICs in a Murine Pneumonia Model

2010 ◽  
Vol 54 (12) ◽  
pp. 5115-5119 ◽  
Author(s):  
Jared L. Crandon ◽  
Joseph L. Kuti ◽  
David P. Nicolau
Keyword(s):  
Staphylococcus Aureus ◽  
Bacterial Load ◽  
Infection Model ◽  
Time Curve ◽  
Unbound Fraction ◽  
Methicillin Resistant ◽  
Vancomycin Mics ◽  
Mrsa Strains

ABSTRACT Telavancin displays potent in vitro and in vivo activity against methicillin-resistant Staphylococcus aureus (MRSA), including strains with reduced susceptibility to vancomycin. We compared the efficacies of telavancin and vancomycin against MRSA strains with vancomycin MICs of ≥1 μg/ml in a neutropenic murine lung infection model. Thirteen clinical MRSA isolates (7 vancomycin-susceptible, 2 vancomycin-heteroresistant [hVISA], and 4 vancomycin-intermediate [VISA] isolates) were tested after 24 h, and 7 isolates (1 hVISA and 4 VISA isolates) were tested after 48 h of exposure. Mice were administered subcutaneous doses of telavancin at 40 mg/kg of body weight every 12 h (q12h) or of vancomycin at 110 mg/kg q12h; doses were designed to simulate the area under the concentration-time curve for the free, unbound fraction of drug (fAUC) observed for humans given telavancin at 10 mg/kg q24h or vancomycin at 1 g q12h. Efficacy was expressed as the 24- or 48-h change in lung bacterial density from pretreatment counts. At dose initiation, the mean bacterial load was 6.16 ± 0.26 log10 CFU/ml, which increased by averages of 1.26 ± 0.55 and 1.74 ± 0.68 log in untreated mice after 24 and 48 h, respectively. At both time points, similar CFU reductions were noted for telavancin and vancomycin against MRSA, with vancomycin MICs of ≤2 μg/ml. Both drugs were similarly efficacious after 24 and 48 h of treatment against the hVISA strains tested. Against VISA isolates, telavancin reduced bacterial burdens significantly more than vancomycin for 1 of 4 isolates after 24 h and for 3 of 4 isolates after 48 h. These data support the potential utility of telavancin for the treatment of MRSA pneumonia caused by pathogens with reduced susceptibility to vancomycin.

scholarly journals 1244. Assessment of the In Vivo Activity of Human-Simulated Exposure of WCK 4282 (High Dose Cefepime [FEP]-Tazobactam [TZB]) against Enterobacterales (EB) and Pseudomonas aeruginosa (PA) in the Neutropenic Murine Thigh Infection Model

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S640-S641
Author(s):  
Christian M Gill ◽  
Kamilia Abdelraouf ◽  
David P Nicolau
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Carbapenem Resistance ◽  
Bacterial Suspension ◽  
Infection Model ◽  
High Dose ◽  
Comparative Efficacy ◽  
Research Support ◽  
Variable Activity

Abstract Background Carbapenems are often used for infections due to extended-spectrum-β-lactamase (ESBL) and cephalosporinase (CSase)-producers. As increased carbapenem utilization is associated with the development of carbapenem resistance, antimicrobial stewardship has targeted non-carbapenem options. WCK 4282 (FEP 2 g-TZB 2 g) offers pharmacodynamically optimized TZB exposure and demonstrated potent activity in vitro against ESBL-phenotype isolates. We describe the pharmacodynamics of a WCK 4282 human-simulated regimen (HSR) in the neutropenic murine thigh model. Methods 19 clinical strains harboring ESBLs or CSase (EB; n=8 and PA; n=4) or serine-carbapenemases (EB; KPC n=4 or OXA-48-like n=3) were tested in vivo. Per CLSI, 19, 18, and 17 isolates were cefepime, ceftolozane/tazobactam, and piperacillin/tazobactam (TZP) non-susceptible, respectively. Thighs of neutropenic, female, CD-1 mice (3 per group) were inoculated with ~107 CFU/mL of bacterial suspension 2 h prior to dosing. Mice received WCK 4282 HSR, FEP HSR, or saline (controls) for 24 h. WCK 4282 HSR and FEP HSR provided plasma exposures in mice that were similar in f%T > MIC and fAUC to FEP-TZB 2 g-2 g and FEP 2 g, respectively, as IV infusions over 1.5 h q8h in humans. Bacterial densities and their changes at 24 h relative to 0 h controls were determined to assess efficacy and reported as mean±SD log10 CFU/thigh. Results Bacterial burdens were 5.81±0.36 at 0 h and 9.29±0.88 at 24 h in untreated controls. WCK 4282 produced potent activity against ESBL/CSase producing EB and PA with WCK 4282 MIC ≤ 16 mg/L; mean change in log10 CFU from 0 h was -1.70±0.77, while growth was observed with FEP alone. WCK 4282 produced variable activity against OXA-48-like harboring EB. Against KPC-harboring EB, WCK 4282 produced stasis to growth. Mean Log10 CFU changes are reported in Table 1 and Figure 1. Table 1. Comparative efficacy of FEP HSR and WCK 4282 HSR by genotypic β-lactamase Figure 1. Mean Change in log10CFU/thigh for 24 h controls, FEP HSR, and WCK 4282 HSR across the tested MIC distribution. Conclusion WCK 4282, a novel TZB containing regimen, resulted in enhance in vitro potency against ESBL/CSase and OXA-48-like producers. Humanized exposures of WCK 4282 produced substantial kill in vivo against ESBL/CSase producers with MICs ≤ 16 mg/L including FEP resistant/TZP non-susceptible PA. These data support further evaluations of WCK 4282 as a carbapenem-sparing regimen for ESBL/cephalosporinase harboring strains. Disclosures David P. Nicolau, PharmD, Cepheid (Other Financial or Material Support, Consultant, speaker bureau member or has received research support.)Merck & Co., Inc. (Consultant, Grant/Research Support, Speaker’s Bureau)Wockhardt (Grant/Research Support)

scholarly journals In vivo activity of human-simulated regimens of imipenem alone and in combination with relebactam against Pseudomonas aeruginosa in the murine thigh infection model

2020 ◽  
Author(s):  
Sergio Reyes ◽  
Kamilia Abdelraouf ◽  
David P Nicolau
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Clinical Utility ◽  
Infection Model ◽  
Treatment Groups ◽  
Log Reduction ◽  
Wide Range ◽  
To Receive ◽  
Inhibitor Combination

Abstract Background Imipenem/relebactam is a carbapenem/β-lactamase inhibitor combination with in vitro activity against Pseudomonas aeruginosa and Enterobacterales, including KPC producers. Objectives To provide translational data to support the clinical utility of the imipenem/relebactam 500/250 mg q6h regimen using a human-simulated regimen (HSR) of imipenem/relebactam, compared with imipenem alone, against a phenotypically and genotypically diverse population of P. aeruginosa. Methods Twenty-nine P. aeruginosa isolates, including KPC (n = 6), PDC (n = 9), PAO (n = 4), GES (n = 5) and VIM (n = 1) producers, were used for the in vivo efficacy studies. Neutropenic mice were thigh-inoculated and randomized to receive HSRs of either imipenem 500 mg q6h, imipenem 1 g q8h, imipenem/relebactam 500/250 mg q6h or saline. Results Twenty-seven of the 29 isolates examined were imipenem resistant, with 24/29 isolates showing imipenem MICs of ≥32 mg/L. The addition of relebactam decreased the MICs up to 64-fold; imipenem/relebactam MICs ranged from 0.25 to >32 mg/L. Efficacies of the imipenem monotherapies and the imipenem/relebactam therapy were comparable for the two imipenem-susceptible organisms. Among the imipenem-resistant isolates, an increased mean growth was observed in the imipenem 500 mg q6h HSR and 1 g q8h HSR treatment groups of 1.31 ± 1.01 and 0.18 ± 1.67 log10 cfu/thigh, respectively. In contrast, a ≥2 log reduction in bacterial density was observed in 27/29 (93%) of the imipenem-resistant isolates subjected to imipenem/relebactam 500/250 mg q6h HSR. Conclusions The imipenem/relebactam 500/250 mg q6h HSR demonstrated superior in vivo activity compared with the conventionally employed imipenem regimens against MDR P. aeruginosa over a wide range of imipenem/relebactam MICs.

scholarly journals Synergistic Activity of Berberine with Azithromycin against Pseudomonas Aeruginosa Isolated from Patients with Cystic Fibrosis of Lung In Vitro and In Vivo

2017 ◽  
Vol 42 (4) ◽  
pp. 1657-1669 ◽  
Author(s):  
YongTao Li ◽  
JianRong Huang ◽  
LanJuan Li ◽  
LinSheng Liu
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Lung Infection ◽  
Extracellular Dna ◽  
Pcr Analysis ◽  
Infection Model ◽  
Synergistic Activity ◽  
Time Kill

Background/Aims: Pseudomonas aeruginosa (PA) is one of the major opportunistic pathogens which can cause chronic lung infection of cystic fibrosis (CF). The formation of PA biofilm promotes CF development and restricts the antimicrobial efficacies of current antibiotics. Methods: The antimicrobial effects of azithromycin (AZM) and berberine (BER) alone and in combination were evaluated using microdilution method, checkerboard assay, time-kill test, qRT-PCR analysis and absorption method. The treatments of AZM and/or BER were further evaluated in an animal lung infection model via observing survival rate, bacterial burden and histopathology of lung, the levels of pro-/anti-inflammatory cytokines. Results: AZM-BER were demonstrated to be synergistic against ten clinical PA isolates as well as the standard reference PA ATCC27853, in which PA03 was the most susceptible isolate to AZM-BER with FICI of 0.13 and chosen for subsequent experiments. The synergism of AZM-BER was further confirmed against PA03 in time-kill test and scanning electron microscope (SEM) at their concentrations showing synergism. In PA03, we found that AZM-BER could significantly attenuate productions of a series of virulence factors including alginate, LasA protease, LasB protease, pyoverdin, pyocyanin, chitinase as well as extracellular DNA, and remarkably inhibit the levels of quorum sensing (QS) molecules and the expressions of lasI, lasR, rhlI, rhlR at 1/2×MIC, 1×MIC and 2×MIC. In the infection model, the mice survival were increased markedly, the inflammations of infected lungs were improved greatly along with reduced IL-6, IL-8 and ascended IL-10 at 0.8 mg/kg of AZM combined with 3.2 mg/kg of BER. Conclusion: BER might be a promising synergist to enhance the antimicrobial activity of AZM in vitro and in vivo.

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