Journal articles: 'Polymyxin B (PMB)' – Grafiati (2024)

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ABSTRACT Polymyxin B (PMB) is a cationic antibiotic that interacts with the envelopes of gram-negative bacterial cells. The therapeutic use of PMB was abandoned for a long time due to its undesirable side effects; however, the spread of resistance to currently used antibiotics has forced the reevaluation of PMB for clinical use. Previous studies have used enteric bacteria to examine the mode of PMB action, resulting in a somewhat limited understanding of this process. This study examined the effects of PMB on marine pseudoalteromonads and demonstrates that the frequently accepted view that “what is true for Escherichia coli is true for all bacteria” does not hold true. We show here that in contrast to the growth inhibition observed for enteric bacteria, PMB induces lysis of pseudoalteromonads, which is not prevented by high concentrations of divalent cations. Furthermore, we demonstrate that a high membrane voltage is required for the interaction of PMB with the cytoplasmic membranes of pseudoalteromonads, further elucidating the mechanisms by which PMB interacts with the cell envelopes of those gram-negative bacteria.

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ABSTRACT Background Pandrug-resistant Klebsiella pneumoniae ventilator associated pneumonia (VAP) is associated with high rate of mortality in intensive care unit (ICU) and has been recognized as a difficult-to-treat infection worldwide. Polymyxin B or colistin-based combination therapies are frequently used worldwide though microbial eradication rate is not promising. Aim The aim of this study is to compare the clinical outcome of intravenous with aerosolized polymyxin B versus colistin in the treatment of pandrug-resistant K. pneumoniae VAP. Methods This retrospective cohort study was conducted on 222 mechanically ventilated patients admitted from May 11, 2019 to October 19, 2020. K. pneumoniae isolates were resistant to all available antibiotics, including polymyxins in culture sensitivity tests. As treatment, polymyxin B and colistin was administered in intravenous and aerosolized form concurrently twice daily in 106 patients and 116 patients in PMB and CLN group, respectively for 14 days. Survival rate, safety, and clinical outcomes were compared among the groups. The Cox proportional-hazard model was performed to calculate hazard ratio (HR) with 95% confidence intervals (CI). Results Patients in PMB group showed more microbial eradication than the patients CLN group [68.1% (n=116)/83% (n=106), respectively; P <0.05). The median day of intubation and ICU stay in PMB group was shorter than that in CLN group [10 (IQR: 9-12.25) vs. 14 (IQR: 11-19), P <0.05; 12 (IQR: 10-14) vs. 15 (IQR: 9-18.5), P=0.072, respectively] with reduced 60-day all-cause mortality rate [15% (n=106) vs. 21.55% (n=116)]. Polymyxin B improved survival compared to colistin (multivariate HR: 0.662; 95% CI=0.359-1.222, P=0.195). Conclusions Concurrent administration of intravenous and aerosolized polymyxin B in patients with pandrug-resistant K. pneumoniae-associated VAP revealed better microbial eradication, reduced the length of intubation and ICU stay, and improved survival rate compared to colistin.

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Polymyxin-resistant Pseudomonas aeruginosa is a major threat to public health globally. We investigated the prevalence of polymyxin-resistant P. aeruginosa in a Chinese teaching hospital and determined the genetic and drug-resistant phenotypes of the resistant isolates. P. aeruginosa isolates identified by MALDI-TOF MS were collected across a 3-month period in Ruijin Hospital. Antimicrobial susceptibility was determined by a Vitek-2 Compact system with broth dilution used to determine polymyxin B (PMB) susceptibility. Polymyxin-resistant isolates were further characterized by molecular typing using PCR, multi-locus sequence typing (MLST) and whole-genome sequencing. Phylogenetic relationships were analyzed using single nucleotide polymorphism (SNP) from the whole-genome sequencing. Of 362 P. aeruginosa isolates collected, 8 (2.2%) isolates from separate patients across six wards were polymyxin-resistant (MIC range, PMB 4–16 μg/mL and colistin 4–≥16 μg/mL). Four patients received PMB treatments (intravenous, aerosolized and/or topical) and all patients survived to discharge. All polymyxin-resistant isolates were genetically related and were assigned to five different clades (Isolate 150 and Isolate 211 being the same ST823 type). Genetic variations V51I, Y345H, G68S and R155H in pmrB and L71R in pmrA were identified, which might confer polymyxin resistance in these isolates. Six of the polymyxin-resistant isolates showed reduced susceptibility to imipenem and meropenem (MIC range ≥ 16 μg/mL), while two of the eight isolates were resistant to ceftazidime. We revealed a low prevalence of polymyxin-resistant P. aeruginosa in a Chinese teaching hospital with most polymyxin-resistant isolates being multidrug-resistant. Therefore, effective infection control measures are urgently needed to prevent further spread of resistance to the last-line polymyxins.

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ABSTRACTWe aimed to investigate polymyxin B (PMB) resistance and its molecular mechanisms in 126Klebsiella pneumoniaeisolates from rectal swabs in Brazil. Ten isolates exhibited PMB resistance with interruption ofmgrBgene by insertion sequences or missense mutations. Most of the PMB-resistant isolates harboredblaKPC-2(n= 8) and belonged to clonal complex 258 (CC258) (n= 7). These results highlight the importance of monitoring the spread of polymyxin-resistant bacteria in hospitals, since few options remain to treat multidrug-resistant isolates.

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ABSTRACT The effects of polymyxin B (PMB) on the Escherichia coli outer (OM) and cytoplasmic membrane (CM) permeabilities were studied by monitoring the fluxes of tetraphenylphosphonium, phenyldicarbaundecaborane, and K+ and H+ions. At concentrations between 2 and 20 μg/ml, PMB increased the OM permeability to lipophilic compounds and induced a leakage of K+ from the cytosol and an accumulation of lipophilic anions in the cellular membranes but did not cause the depolarization of the CM. At higher concentrations, PMB depolarized the CM, forming ion-permeable pores in the cell envelope. The permeability characteristics of PMB-induced pores mimic those of bacteriophage- and/or bacteriocin-induced channels. However, the bactericidal effect of PMB took place at concentrations below 20 μg/ml, indicating that this effect is not caused by pore formation. Under conditions of increased ionic strength, PMB made the OM permeable to lipophilic compounds and decreased the K+gradient but was not able to depolarize the cells. The OM-permeabilizing effect of PMB can be diminished by increasing the concentration of Mg2+. The major new findings of this work are as follows: (i) the OM-permeabilizing action of PMB was dissected from its depolarizing effect on the CM, (ii) the PMB-induced ion-permeable pores in bacterial envelope were registered, and (iii) the pore formation and depolarization of the CM are not obligatory for the bactericidal action of PMB and dissipation of the K+gradient on the CM.

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The receptor megalin plays an important role in the accumulation of polymyxin B (PMB) in renal cells in vitro. This study aimed to examine the effects of cytochrome c (cyto c), a typical megalin ligand, on renal accumulation and nephrotoxicity of PMB in vivo. Thirty Sprague-Dawley rats were randomly divided into the vehicle control group, PMB group, PMB + cyto c 50, 100, or 200 mg/kg group, respectively, and were treated with intravenous cyto c 30 min before the administration of PMB 4.0 mg/kg once a day for consecutive 5 days. On the 4th day after administration, 24 h urine was collected to determine N-acetyl-β-D-glucosaminidase excretion. Six hours after the last injection on the 5th day, kidneys were harvested to assay PMB concentration and observe pathological alterations, and blood samples were collected to assay serum creatinine (SCr), blood urea nitrogen (BUN), and blood β2-microglobulin (β2-MG) levels. Cyto c 50, 100, and 200 mg/kg decreased the accumulation of PMB in the kidney by 18.5%, 39.1% ( p < 0.01), and 36.8% ( p < 0.01), respectively, and reduced 24 h N-acetyl-β-D- glucosaminidase excretion by 22.5% ( p < 0.05), 40.4% ( p < 0.01), and 40.4% ( p < 0.01), respectively. Kidney pathological damage induced by PMB was markedly reduced by cyto c 100 mg/kg and 200 mg/kg. However, there were no significant differences in SCr, BUN, and blood β2-MG levels among the groups. These results indicated that cyto c may inhibit the renal accumulation and nephrotoxicity of PMB in a rat model, further proving the role of megalin in the accumulation of PMB.

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Polymyxin B (PMB), a cyclic decapeptide antibiotic, inhibits the hypoglycemic effect of insulin in vivo. To elucidate the mechanism of PMB action, we have studied its effect in vitro on insulin-stimulated pathways in the mouse skeletal muscle. PMB, added to the incubation mixture, specifically inhibited insulin-stimulated 2-deoxyglucose transport and alpha-aminoisobutyric acid uptake in the isolated soleus muscle but did not affect the basal rates of transport (measured in the absence of insulin). PMB did not alter insulin binding and hexokinase activity. PMB effect was observed at all deoxyglucose concentrations tested, and PMB was also able to inhibit vanadate-stimulated glucose transport. By contrast, insulin activation of glycogen synthase was not prevented by PMB. Basal and maximally insulin-stimulated insulin receptor tyrosine kinase activity, tested in a cell-free system, was similar for both autophosphorylation and phosphorylation of exogenous substrates in the absence or in the presence of PMB. Furthermore, the insulin sensitivity of the kinase was increased in the presence of PMB. Our results suggest that the anti-insulin effect of PMB observed in vivo is due to an inhibition of insulin-stimulated glucose transport in the skeletal muscle perhaps through a specific blockade of the insulin-induced translocation of the glucose carriers.

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Drug-resistant Gram-negative bacteria pose a serious threat to public health, and polymyxin B (PMB) is clinically used as a last-line therapy for the treatment of infections caused by these pathogens. However, the appearance of PMB resistance calls for an effort to develop new approaches to improve its antibacterial performance. In this work, a new type of nanocomposite, composed of PMB molecules being chemically decorated on the surface of graphene oxide (GO) nanosheets, was designed, which showed potent antibacterial ability through synergistically and physically disturbing the bacterial membrane. The as-fabricated PMB@GO nanocomposites demonstrated an enhanced bacterial-killing efficiency, with a minimum inhibitory concentration (MIC) value half of that of free PMB (with an MIC value as low as 0.5 μg mL−1 over Escherichia coli), and a bacterial viability less than one fourth of that of PMB (with a bacterial reduction of 60% after 3 h treatment, and 90% after 6 h incubation). Furthermore, the nanocomposite displayed moderate cytotoxicity or hemolysis effect, with cellular viabilities over 85% at concentrations up to 16 times the MIC value. Studies on antibacterial mechanism revealed that the synergy between PMB molecules and GO nanosheets greatly facilitated the vertical insertion of the nanocomposite into the lipid membrane, leading to membrane disturbance and permeabilization. Our results demonstrate a physical mechanism for improving the antibacterial performance of PMB and developing advanced antibacterial agents for better clinic uses.

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ABSTRACT Polymyxin B (PMB) is a cyclic decapeptide antibiotic which also binds and neutralizes endotoxin. Unfortunately, PMB can be considerably nephrotoxic at clinically utilized doses, thereby limiting its utility as a therapeutic antiendotoxin reagent. We sought to change the pharmaco*kinetics and toxicity profile of PMB by covalently linking it to a human immunoglobulin G (IgG) carrier. Conjugates of PMB with IgG were prepared by EDAC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide]-mediated amide formation. Analysis by dot enzyme-linked immunosorbent assay with an anti-PMB monoclonal antibody showed that the purified conjugate contained bound PMB. The IgG-PMB conjugate reacted with lipid A and J5 lipopolysaccharide in Western blot assays in a manner comparable to that of whole antiserum with anti-lipid A reactivity; unconjugated IgG had no reactivity. The PMB bound in the conjugate retained its endotoxin-neutralizing activity compared to that of unbound PMB as evidenced by its dose-dependent inhibition of tumor necrosis factor release by endotoxin-stimulated human monocytes in vitro; unconjugated IgG had no activity. By this assay, the PMB-IgG conjugate was determined to have approximately 3.0 μg of bound functional PMB per 100 μg of total protein of conjugate (five molecules of PMB per IgG molecule). The PMB-IgG conjugate was also bactericidal against clinical strains of Escherichia coli,Pseudomonas aeruginosa, and Klebsiella pneumoniae relative to unconjugated IgG with MBCs of <4 μg of conjugate per ml for each of the tested strains. The conjugate appeared to be nontoxic at the highest doses deliverable and provided statistically significant protection from death to galactosamine-sensitized, lipopolysaccharide-challenged mice in a dose-dependent fashion when administered prophylactically 2 h before challenge. However, neither free PMB nor the PMB-IgG conjugate could protect mice challenged with endotoxin 2 h after administration. This suggests that these reagents can play a role in prophylaxis but not in therapy of sepsis. These experiments demonstrated that the PMB-IgG conjugate retains bound yet functional PMB as evidenced by its endotoxin-neutralizing activity both in vitro and in vivo. Further work is required to define the role that this or related conjugate compounds may play in the prophylaxis of endotoxin-mediated disease.

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ABSTRACTPolymyxin B (PMB) is a cationic polypeptide antibiotic with activity against multidrug-resistant Gram-negative bacteria. PMB-induced nephrotoxicity consists of direct toxicity to the renal tubules and the release of reactive oxygen species (ROS) with oxidative damage. This study evaluated the nephroprotective effect of heme oxygenase-1 (HO-1) against PMB-induced nephrotoxicity in rats. Adult male Wistar rats, weighing 286 ± 12 g, were treated intraperitoneally once a day for 5 days with saline, hemin (HO-1 inducer; 10 mg/kg), zinc protoporphyrin (ZnPP) (HO-1 inhibitor; 50 μmol/kg, administered before PMB on day 5), PMB (4 mg/kg), PMB plus hemin, and PMB plus ZnPP. Renal function (creatinine clearance, Jaffe method), urinary peroxides (ferrous oxidation of xylenol orange version 2 [FOX-2]), urinary thiobarbituric acid-reactive substances (TBARS), renal tissue thiols, catalase activity, and renal tissue histology were analyzed. The results showed that PMB reduced creatinine clearance (P< 0.05), with an increase in urinary peroxides and TBARS. The PMB toxicity caused a reduction in catalase activity and thiols (P< 0.05). Hemin attenuated PMB nephrotoxicity by increasing the catalase antioxidant activity (P< 0.05). The combination of PMB and ZnPP incremented the fractional interstitial area of renal tissue (P< 0.05), and acute tubular necrosis in the cortex area was also observed. This is the first study demonstrating the protective effect of HO-1 against PMB-induced nephrotoxicity.

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Heteromultivalent design of PAMAM dendrimer by conjugation with polymyxin B (PMB) ligand and excess auxiliary ethanolamine (EA) branches led to lipopolysaccharide (LPS) avidity two orders of magnitude greater than free PMB.

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Abstract Background Nephrotoxicity is a common adverse effect of polymyxin B (PMB) with reported acute kidney injury (AKI) rates of 20% to &gt;60%. Data on PMB dosing to optimize efficacy while minimizing toxicity are limited. Previous studies suggest higher doses improve outcomes but are also associated with more AKI. Data are needed to evaluate optimal dosing and contributing factors to minimize AKI and to evaluate renal recovery. Methods Retrospective study evaluating PMB in adults at NewYork-Presbyterian Hospital from 2012 to 2016. Patients who received PMB dosed twice daily for ≥2 days were included. Patients on renal replacement therapy within 48 hours prior to PMB or with AKI at time of PMB initiation were excluded. A classification and regression tree (CART) analysis was performed to identify the PMB dose most predictive of AKI which defined the breakpoint for high- vs. low-dose PMB cohorts for all subsequent comparisons. The primary outcome was to determine whether high-dose PMB independently predicted AKI. Secondary outcomes included in-hospital mortality, time to AKI, and recovery of renal function. Results 246 patients were included: majority were male (59%) with median age 41 years. Median PMB dose was 2.9 mg/kg/day or 180 mg/day for a median duration of 10 days. AKI occurred in 64% and 38% had recovery of renal function by hospital discharge. The breakpoint for high-dose PMB determined by CART was 160 mg/day, putting 104 in low-dose and 142 in high-dose groups. High-dose PMB was associated with AKI compared with low-dose PMB on univariable (75% vs.. 49%, P &lt; 0.001) and multivariable (OR 3.43; 95% CI 1.68,6.99; P = 0.001) analyses. Concomitant vancomycin (OR 3.34; 95% CI 1.74,6.41;P &lt; 0.001), history of transplant (OR 4.96; 95% CI 2.14,11.48;P &lt; 0.001), and previous PMB exposure (OR 2.37; 95% CI 1.23,4.57; P = 0.01) were also identified as independent predictors of AKI. No significant differences were found for in-hospital mortality (28% vs. 21%, P = 0.326), renal recovery (37% vs. 41%, P = 0.723), time to AKI (median 5 vs. 6 days, P = 0.125) between groups. Conclusion High-dose PMB (&gt;160 mg/day) was independently associated with AKI as well as concomitant vancomycin, history of transplant, and previous PMB exposure. High-dose PMB did not have a significant impact on in-hospital mortality, recovery of renal function, or time to development of AKI. Disclosures M. Yin, Gilead Sciences: Consultant, Consulting fee.

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Polymyxins are considered a last-line treatment against infections caused by multidrug-resistant (MDR) Gram-negative bacteria. In addition to their use as a potent antibiotic, polymyxins have also been utilized as outer membrane (OM) permeabilizers, capable of augmenting the activity of a partner antibiotic. Several polymyxin derivatives have been developed accordingly, with the objective of mitigating associated nephrotoxicity. The conversion of polymyxins to guanidinylated derivatives, whereby the L-γ-diaminobutyric acid (Dab) amines are substituted with guanidines, are described herein. The resulting guanidinylated colistin and polymyxin B (PMB) exhibited reduced antibacterial activity but preserved OM permeabilizing properties that allowed potentiation of several antibiotic classes. Rifampicin, erythromycin, ceftazidime and aztreonam were particularly potentiated against clinically relevant MDR Gram-negative bacteria. The potentiating effects of guanidinylated polymyxins with ceftazidime or aztreonam were further enhanced by adding the β-lactamase inhibitor avibactam.

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Bacterial endotoxin, also known as lipopolysaccharide (LPS), plays a major role in the initiation of sepsis, a severe inflammatory condition. Removal of the toxin from blood is one accepted method of patient treatment. Polymyxin B (PMB)-modified columns have been employed successfully for this purpose via extra-corporeal blood-flow systems that incorporate a cartridge for toxin removal. Herein we demonstrate that PMB-modified glass beads are able to reduce the presence of LPS competitively with the equivalent fiber column used in a commercial cartridge. Analysis by gas chromatography-mass spectrometry and ELISA of released fatty acids from the toxin indicates that PMB does not physically capture or significantly remove LPS from the blood samples. In reality, interaction between the surface-bound PMB and the toxin may lead to disaggregation or monomerization of LPS aggregates. As aggregates are the bioactive form of LPS, it is possible that the monomerization of these entities may be the mechanism by which their toxicity is reduced. Moreover, this work indicates that LPS monomers are stabilized subsequent to disaggregation induced by PMB.

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Polymyxins are considered as last–resort antibiotics to treat infections caused by Acinetobacter baumannii. However, there are increasing reports of resistance in A. baumannii to polymyxins. In this study, inhalable combinational dry powders consisting of ciprofloxacin (CIP) and polymyxin B (PMB) were prepared by spray–drying. The obtained powders were characterized with respect to the particle properties, solid state, in vitro dissolution and in vitro aerosol performance. The antibacterial effect of the combination dry powders against multidrug–resistant A. baumannii was assessed in a time–kill study. Mutants from the time–kill study were further investigated by population analysis profiling, minimum inhibitory concentration testing, and genomic comparisons. Inhalable dry powders consisting of CIP, PMB and their combination showed a fine particle fraction above 30%, an index of robust aerosol performance of inhaled dry powder formulations in the literature. The combination of CIP and PMB exhibited a synergistic antibacterial effect against A. baumannii and suppressed the development of CIP and PMB resistance. Genome analyses revealed only a few genetic differences of 3–6 SNPs between mutants and the progenitor isolate. This study suggests that inhalable spray–dried powders composed of the combination of CIP and PMB is promising for the treatment of respiratory infections caused by A. baumannii, and this combination can enhance the killing efficiency and suppress the development of drug resistance.

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Polymyxins (polymyxin B (PMB) and polymyxin E (colistin)) are cyclic lipodecapeptide antibiotics, highly basic due to five free amino groups, and rapidly bactericidal against Gram-negative bacteria, such as the majority of Enterobacteriaceae as well as Acinetobacter baumannii and Pseudomonas aeruginosa. Their clinical use was abandoned in the 1960s because of nephrotoxicity and because better-tolerated drugs belonging to other antibiotic classes were introduced. Now, due to the global dissemination of extremely-drug resistant Gram-negative bacterial strains, polymyxins have resurged as the last-line drugs against those strains. Novel derivatives that are less toxic and/or more effective at tolerable doses are currently under preclinical development and their properties have recently been described in several extensive reviews. Other derivatives lack any direct bactericidal activity but damage the outermost permeability barrier, the outer membrane, of the target bacteria and make it more permeable to many other antibiotics. This review describes the properties of three thus far best-characterized “permeabilizer” derivatives, i.e., the classic permeabilizer polymyxin B nonapeptide (PMBN), NAB7061, and SPR741/NAB741, a compound that recently successfully passed the clinical phase 1. Also, a few other permeabilizer compounds are brought up.

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Systemic therapy of Gram-negative sepsis remains challenging. Polymyxin B (PMB) is well suited for sepsis therapy due to the endotoxin affinity and antibacterial activity. However, the dose-limiting toxicity has limited its systemic use in sepsis patients. For safe systemic use of PMB, we have developed a nanoparticulate system, called D-TZP, which selectively reduces the toxicity to mammalian cells but retains the therapeutic activities of PMB. D-TZP consists of an iron-complexed tannic acid nanocapsule containing a vitamin D core, coated with PMB and a chitosan derivative that controls the interaction of PMB with endotoxin, bacteria, and host cells. D-TZP attenuated the membrane toxicity associated with PMB but retained the ability of PMB to inactivate endotoxin and kill Gram-negative bacteria. Upon intravenous injection, D-TZP protected animals from pre-established endotoxemia and polymicrobial sepsis, showing no systemic toxicities inherent to PMB. These results support D-TZP as a safe and effective systemic intervention of sepsis.

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Abstract Background Currently available polymyxins are limited by toxicity and poor efficacy at tolerated doses. We have developed a new series of polymyxin derivatives with improved safety profiles and in vitro potency against major MDR bacteria. The following describes studies on the in vivo antimicrobial activity and toxicity of QPX9003 in mice and rats. Methods Mouse studies. The minimum lethal dose (MLD by IV bolus) and nephrotoxicity (6 IP doses administered 2 hours apart) of QPX9003 and polymyxin B (PMB) were determined in Swiss mice. For the neutropenic mouse thigh infection using A. baumannii, Swiss mice were infected with ~106 CFU/thigh. Doses were administered IP at various intervals starting 2-hour post-infection and continued over 24 hours. Rat studies. For the rat lung infection model, Sprague-Dawley rats were infected with ~107 CFU/lung. QPX9003 and PMB were administered IV every 4 hours starting 2 hours post-infection and continued over 24 hours. Bacteria. For both infection models, animals were infected with A. baumannii AB1016 (QPX9003 MIC of 0.5 mg/L and PMB MIC of 1.0 mg/L). Untreated control groups were sacrificed at the start of treatment and both untreated and treated groups were sacrificed 24 hours after the start of treatment, infected tissues harvested, hom*ogenized, and plated to determine colony counts. Results QPX9003 had reduced acute toxicity and nephrotoxicity compared with PMB in mice. QPX9003 showed better bacterial killing of A. baumannii than PMB at similar plasma exposures in both the mouse thigh model (−0.41 vs. +0.83 log CFU/thigh) and rat lung infection model (−1.10 vs. +1.44 log CFU/lung). Conclusion QPX9003 was less acutely toxic, less nephrotoxic, and was more efficacious in mouse and rat infection models compared with PMB. QPX9003 is a promising new polymyxin. (This work was supported in part by federal funds from the National Institutes of Allergy and Infectious Diseases [R01AI098771], and the Department of Health and Human Services; Office of the Assistant Secretary for Preparedness and Response; Biomedical Advanced Research and Development Authority (BARDA), under OTA number HHSO100201600026C). Disclosures All authors: No reported disclosures.

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A new adsorbent for hemoperfusion was successfully prepared by grafting polymyxin B (PMB) on the surface of polystyrene divinylbenzene (PS-DVB) microspheres. It showed good biocompatibility and could adsorb both bilirubin and endotoxin.

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Directed, effective therapies for feline sepsis are needed to reduce the high morbidity and mortality associated with this disease. We investigated the anti-endotoxin effects of polymyxin B (PMB) in a blinded, placebo controlled fashion, both ex vivo in a feline whole blood culture system and in vivo, using a low-dose endotoxin infusion in cats. Serial measures of systemic inflammation, and hemodynamic stability, were compared between groups. Ex vivo, PMB significantly decreased lipopolysaccharide-induced tumor necrosis factor (TNF) production from whole blood. PMB (1 mg/kg over 30 min) demonstrated anti-endotoxin effects in vivo, including decreased peak plasma TNF activity ( P<0.001) and increased white blood cell count ( P=0.019), with no adverse effects. Given the apparent safety and anti-endotoxin effects of PMB in this endotoxemia model, a carefully designed, randomized, blinded, placebo controlled clinical trial evaluating the use of PMB in naturally occurring Gram-negative feline sepsis should be considered.

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Background/Aims: Polymyxin B (PMB) is a cyclic cationic polypeptide antibiotic widely used to counteract the effects of endotoxin contamination, both in vitro and in vivo. Lipopolysaccharide (LPS) is an endotoxin that acts as a radiation protection factor. In this study, we focus on the role of PMB in LPS-induced and radiation-induced mortality in mice. Methods: Mice received total-body radiation or were pretreated by LPS or PMB, and the survival of mice was recorded. Elisa were used to detect the cytokines levels. Results: PMB decreased LPS-induced, but increased radiation-induced mortality in mice. Moreover, PMB could block the LPS-induced radioprotective effect. The ELISA and gene knock-out experiments indicated that PMB reduces TNF-α level to block LPS-induced mortality in mice, and inhibits IL-6, G-CSF and IL-10 to increase radiation-induced mortality via the TLR4-Myd88-IL-6 pathway. Conclusions: Our study revealed a role of PMB in LPS-induced endotoxemia and radiation exposure. We infer that the TLR4-Myd88-IL-6 pathway may play a crucial role in the process.

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Population pharmaco*kinetic studies have suggested that high polymyxin B (PMB) doses (≥30,000 IU/kg/day) can improve bacterial kill in carbapenem-resistant Gram-negative bacteria (CR-GNB). We aim to describe the efficacy and nephrotoxicity of patients with CR-GNB infections prescribed high-dose PMB. A single-centre cohort study was conducted from 2013 to 2016 on septic patients with CR-GNB infection and prescribed high-dose PMB (~30,000 IU/kg/day) for ≥72 h. Study outcomes included 30-day mortality and acute kidney injury (AKI) development. Factors associated with AKI were identified using multivariable regression. Forty-three patients with 58 CR-GNB received high-dose PMB; 57/58 (98.3%) CR-GNB were susceptible to PMB. The median daily dose and duration of high-dose PMB were 32,051 IU/kg/day (IQR, 29,340–34,884 IU/kg/day) and 14 days (IQR, 7–28 days), respectively. Thirty-day mortality was observed in 7 (16.3%) patients. AKI was observed in 25 (58.1%) patients with a median onset of 8 days (IQR, 6–13 days). Higher daily PMB dose (aOR,1.01; 95% CI, 1.00–1.02) and higher number of concurrent nephrotoxins (aOR, 2.14; 95% CI; 1.03–4.45) were independently associated with AKI. We observed that a sizable proportion developed AKI in CR-GNB patients described high-dose PMB; hence, the potential benefits must be weighed against increased AKI risk. Concurrent nephrotoxins should be avoided to reduce nephrotoxicity.

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ABSTRACTNephrotoxicity is the main adverse effect of colistin and polymyxin B (PMB). It is not clear whether these two antibiotics are associated with different nephrotoxicity rates. We compared the incidences of renal failure (RF) in patients treated with colistimethate sodium (CMS) or PMB for ≥48 h. A multicenter prospective cohort study was performed that included patients aged ≥18 years. The primary outcome was renal failure (RF) according to Risk, Injury, Failure, Loss, and End-stage renal disease (RIFLE) criteria. Multivariate analysis with a Cox regression model was performed. A total of 491 patients were included: 81 in the CMS group and 410 in the PMB group. The mean daily doses in milligrams per kilogram of body weight were 4.2 ± 1.3 and 2.4 ± 0.73 of colistin base activity and PMB, respectively. The overall incidence of RF was 16.9% (83 patients): 38.3% and 12.7% in the CMS and PMB groups, respectively (P< 0.001). In multivariate analysis, CMS therapy was an independent risk factor for RF (hazard ratio, 3.35; 95% confidence interval, 2.05 to 5.48;P< 0.001) along with intensive care unit admission, higher weight, older age, and bloodstream and intraabdominal infections. CMS was also independently associated with a higher risk of RF in various subgroup analyses. The incidence of RF was higher in the CMS group regardless of the patient baseline creatinine clearance. The development of RF during therapy was not associated with 30-day mortality in multivariate analysis. CMS was associated with significantly higher rates of RF than those of PMB. Further studies are required to confirm our findings in other patient populations.

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Abstract. In this paper an electrochemical endotoxin biosensor consisting of an immobilized lipopolysaccharide (LPS) ligand, polymyxin B (PmB), is presented. Several parameters involved both in the device fabrication and in the detection process were analyzed to optimize the ligand immobilization and the interaction between PmB and LPS, aiming at increasing the sensitivity of the sensor. Different electrochemical pre-treatment procedures as well as the functionalization methods were studied and evaluated. The use of a SAM (self-assembled monolayer) to immobilize PmB and the quantification of the interactions via cyclic voltammetry allowed the development of a robust and simple device for in situ detection of LPS. Thus, the biosensor proposed in this work intends an approach to the demanding needs of the market for an integrated, portable and simple instrument for endotoxin detection.

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Polymyxin B (PMB) is the final option for treating multidrug-resistant Gram-negative bacterial infections. The acceptable pharmaco*kinetic/pharmacodynamic target is an area under the concentration–time curve across 24 h at a steady state (AUCss,24h) of 50–100 mg·h/L. The limited sampling strategy (LSS) is useful for predicting AUC values. However, establishing an LSS is a time-consuming process requiring a relatively dense sampling of patients. Further, given the variability among different centers, the predictability of LSSs is frequently questioned when it is extrapolated to other clinical centers. Currently, limited data are available on a reliable PMB LSS for estimating AUCss,24h. This study assessed and validated the practicability of LSSs established in the literature based on data from our center to provide reliable and ready-made PMB LSSs for laboratories performing therapeutic drug monitoring (TDM) of PMB. The influence of infusion and sampling time errors on predictability was also explored to obtain the optimal time points for routine PMB TDM. Using multiple regression analysis, PMB LSSs were generated from a model group of 20 patients. A validation group (10 patients) was used to validate the established LSSs. PMB LSSs from two published studies were validated using a dataset of 30 patients from our center. A population pharmaco*kinetic model was established to simulate the individual plasma concentration profiles for each infusion and sampling time error regimen. Pharmaco*kinetic data obtained from the 30 patients were fitted to a two-compartment model. Infusion and sampling time errors observed in real-world clinical practice could considerably affect the predictability of PMB LSSs. Moreover, we identified specific LSSs to be superior in predicting PMB AUCss,24h based on different infusion times. We also discovered that sampling time error should be controlled within −10 to 15 min to obtain better predictability. The present study provides validated PMB LSSs that can more accurately predict PMB AUCss,24h in routine clinical practice, facilitating PMB TDM in other laboratories and pharmaco*kinetics/pharmacodynamics-based clinical studies in the future.

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Understanding membrane interactions and cell-wall permeation of Gram-negative bacteria is of great importance, owing to increasing bacterial resistance to existing drugs and therapeutic treatments. Here we use biomimetic lipid vesicles to analyse membrane association and penetration by synthetic derivatives of polymyxin B (PMB), a potent naturally occurring antibacterial cyclic peptide. The PMB analogues studied were PMB nonapeptide (PMBN), in which the hydrophobic alkyl residue was cleaved, PMBN diastereomer containing d-instead of l-amino acids within the cyclic ring (dPMBN) and PMBN where the hydrophobic alkyl chain was replaced with an Ala6 repeat (Ala6-PMBN). Peptide binding measurements, colorimetric transitions induced within the vesicles, fluorescence quenching experiments and ESR spectroscopy were applied to investigate the structural parameters underlying the different membrane-permeation profiles and biological activities of the analogues. The experiments point to the role of negatively charged lipids in membrane binding and confirm the prominence of lipopolisaccharide (LPS) in promoting membrane association and penetration by the peptides. Examination of the lipid interactions of the PMB derivatives shows that the cyclic moiety of PMB is not only implicated in lipid attachment and LPS binding, but also affects penetration into the inner bilayer core. The addition of the Ala6 peptide moiety, however, does not significantly promote peptide insertion into the hydrophobic lipid environment. The data also indicate that the extent of penetration into the lipid bilayer is not related to the overall affinity of the peptides to the membrane.

Abstract:

ABSTRACTThere is significant variation in the use of polymyxin B (PMB), and optimal dosing has not been defined. The purpose of this retrospective study was to evaluate the relationship between PMB dose and clinical outcomes. We included patients with bloodstream infections (BSIs) due to carbapenem-resistant Gram-negative rods who received ≥48 h of intravenous PMB. The objective was to evaluate the association between PMB dose and 30-day mortality, clinical cure at day 7, and development of acute kidney injury (AKI). A total of 151 BSIs were included. The overall 30-day mortality was 37.8% (54 of 151), and the median PMB dosage was 1.3 mg/kg (of total body weight)/day. Receipt of PMB dosages of <1.3 mg/kg/day was significantly associated with 30-day mortality (46.5% versus 26.3%;P= 0.02), and this association persisted in multivariable analysis (odds ratio [OR] = 1.58; 95% confidence interval [CI] = 1.05 to 1.81;P= 0.04). Eighty-two percent of patients who received PMB dosages of <1.3 mg/kg/day had baseline renal impairment. Clinical cure at day 7 was not significantly different between dosing groups. AKI was more common in patients receiving PMB dosages of ≥250 mg/day (66.7% versus 32.0%;P= 0.03), and this association persisted in multivariable analysis (OR = 4.32; 95% CI = 1.15 to 16.25;P= 0.03). PMB dosages of <1.3 mg/kg/day were administered primarily to patients with renal impairment, and this dosing was independently associated with 30-day mortality. However, dosages of ≥250 mg/day were independently associated with AKI. These data support the use of PMB without dose reduction in the setting of renal impairment.

Abstract:

Lipopolysaccharide (LPS), the major cell wall constituent of Gram-negative bacteria, evokes a multitude of biological effects in mammals including pyrogenicity and toxic shock syndrome. Polymyxin B (PmB), a polycationic cyclic peptide, is known to neutralize most of its activities. The nature of the interaction of PmB with LPS and lipid A was investigated by isothermal titration calorimetry. PmB binds to LPS as well as lipid A stoichiometrically and non-co-operatively with micromolar affinity. These interactions are driven primarily by a favourable change in entropy (∆S) and are endothermic in nature. These positive changes in enthalpies decrease with increasing temperature, yielding a heat capacity change, ∆Cp, of -2385 J·mol-1·degree-1 for PmB–LPS interactions while the binding of PmB to lipid A displays a ∆Cp of -2259 J·mol-1·degree-1. The negative heat capacity changes provide strong evidence for the role of hydrophobic interactions as the driving force for the association of PmB with LPS and lipid A. A correlation of the energetics of these interactions with analyses of the molecular models of PmB suggests that a cluster of solvent-exposed non-polar amino acid side-chains that line one surface of the molecule, together with a ring of positively charged residues on its other surface, are responsible for its strong and stoichiometric binding to LPS.

Abstract:

Although detection of gram-negative bacteria (GNB) in body fluids is important for clinical purpose, traditional gram staining and other recently developed methods have inherent limitations in terms of accuracy, sensitivity, and convenience. To overcome the weakness, this study proposed a method detecting GNB based on specific binding of polymyxin B (PMB) to lipopolysaccharides (LPS) of GNB. Fluorescent microscopy demonstrated that surface immobilized PMB using a silane coupling agent was possible to detect fluorescent signal produced by a single Escherichia coli (a model GNB) cell. Furthermore, the signal was selective enough to differentiate between GNB and gram-positive bacteria. The proposed method could detect three cells per ml within one hour, indicating the method was very sensitive and the sensing was rapid. These results suggest that highly multifold PMB binding on each GNB cell occurred, as millions of LPS are present on cell wall of a GNB cell. Importantly, the principle used in this study was realized in a microfluidic chip for a sample containing E. coli cells suspended in porcine plasma, demonstrating its potential application to practical uses. In conclusion, the proposed method was accurate, sensitive, and convenient for detecting GNB, and could be applied clinically.

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Background: Infectious diseases are one of the main causes of morbidity and mortality worldwide. Nuclear molecular imaging would be of great help to non-invasively discriminate between septic and sterile inflammation through available radiopharmaceuticals, as none is currently available for clinical practice. Here, we describe the radiolabeling procedure and in vitro and in vivo studies of 99mTc-polymyxin B sulfate (PMB) as a new single photon emission imaging agent for the characterization of infections due to Gram-negative bacteria. Results: Labeling efficiency was 97 ± 2% with an average molar activity of 29.5 ± 0.6 MBq/nmol. The product was highly stable in saline and serum up to 6 h. In vitro binding assay showed significant displaceable binding to Gram-negative bacteria but not to Gram-positive controls. In mice, 99mTc-HYNIC-PMB was mainly taken up by liver and kidneys. Targeting studies confirmed the specificity of 99mTc-HYNIC-PMB obtained in vitro, showing significantly higher T/B ratios for Gram-negative bacteria than Gram-positive controls. Conclusions: In vitro and in vivo results suggest that 99mTc-HYNIC-PMB has a potential for in vivo identification of Gram-negative bacteria in patients with infections of unknown etiology. However, further investigations are needed to deeply understand the mechanism of action and behavior of 99mTc-HYNIC-PMB in other animal models and in humans.

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Abstract This clinical study investigated the effects of endodontic treatment by using different irrigants (limewater + NaOCl and polymyxin B + NaOCl) and intracanal medication on endotoxins in teeth with primary endodontic infection and radiographically visible apical periodontitis. Thirty-three teeth with necrotic pulp and periapical lesions from different patients were selected for this study. Samples were collected after the coronal opening (S1) and after instrumentation (S2). Root canals were divided in 3 groups (n = 11) according to the irrigant combination used: NaOCl + LW: 2.5% NaOCl + calcium hydroxide solution (0.14%, limewater); NaOCl + PmB: 2.5% NaOCl + 10.000 UI/mL polymyxin B; 2.5% NaOCl (control). The third sampling (S3) was performed after ethylenediaminetetraacetic acid and the fourth (S4) after samples got 14 days with intracanal medication with 2% chlorhexidine gel + calcium hydroxide. Endotoxins (lipopolysaccharide) were quantified by chromogenic Limulus amebocyte lysate (LAL). Endotoxins were detected in all root canals after the coronal opening (S1). NaOCl + PmB group presented the greatest endotoxin reduction after instrumentation (76.17%), similar to NaOCl + LW group (67.64%, p<0.05) and different from NaOCl group (42.17%, p<0.05). After intracanal medication period (S4), there was significant increase of endotoxins neutralization. It was concluded that NaOCl + PmB promoted the greatest reduction of endotoxin levels, followed by NaOCl + LW. Intracanal medications had no significant complementary role in the reduction of endotoxins at the end of the treatment

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ABSTRACTThis study evaluated the efficacy of tigecycline (TIG), polymyxin B (PMB), and meropenem (MER) in 80 rats challenged withKlebsiella pneumoniaecarbapenemase (KPC)-producingK. pneumoniaeinfection. A time-kill assay was performed with the same strain. Triple therapy and PMB+TIG were synergistic, promoted 100% survival, and produced negative peritoneal cultures, while MER+TIG showed lower survival and higher culture positivity than other regimens (P= 0.018) and was antagonistic.In vivoandin vitrostudies showed that combined regimens, except MER+TIG, were more effective than monotherapies for this KPC-producing strain.

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The rise in antimicrobial resistant bacteria is limiting the number of effective treatments for bacterial infections. Escherichia coli and Pseudomonas aeruginosa are two of the pathogens with the highest prevalence of resistance, and with the greatest need for new antimicrobial agents. Combinations of antimicrobial peptides (AMPs) and antibiotics that display synergistic effects have been shown to be an effective strategy in the development of novel therapeutic agents. In this study, we investigated the synergy between the AMP LL-37 and various classes of antibiotics against E. coli and P. aeruginosa strains. Of the six antibiotics tested (ampicillin, tetracycline, ciprofloxacin, gentamicin, aztreonam, and polymyxin B (PMB)), LL-37 displayed the strongest synergy against E. coli MG1655 and P. aeruginosa PAO1 laboratory strains when combined with PMB. Given the strong synergy, the PMB + LL-37 combination was chosen for further examination where it demonstrated synergy against multidrug-resistant and clinical E. coli isolates. Synergy of PMB + LL-37 towards clinical isolates of P. aeruginosa varied and showed synergistic, additive, or indifferent effects. The PMB + LL-37 combination treatment showed significant prevention of biofilm formation as well as eradication of pre-grown E. coli and P. aeruginosa biofilms. Using the Galleria mellonella wax worm model, we showed that the PMB + LL-37 combination treatment retained its antibacterial capacities in vivo. Flow analyses were performed to characterize the mode of action. The results of the present study provide proof of principle for the synergistic response between LL-37 and PMB and give novel insights into a promising new antimicrobial combination against gram-negative planktonic and biofilm cells.

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ABSTRACT Lipopolysaccharide (LPS) structure impacts the bactericidal action of cationic peptides, such as polymyxin B (PMB), and sensitivity to killing by normal human serum (NHS). Cultivation of different subspecies strains of Yersinia pestis isolated from unrelated geographic origins at various temperatures (mammals, 37°C; fleas, 25°C; or winter hibernation, 6°C) affects LPS composition and structure. We tested the susceptibilities of various strains of Y. pestis grown at these different temperatures to PMB and serum bactericidal killing. Both properties varied significantly in response to temperature changes. In Y. pestis subsp. pestis (the main subspecies causing human plague), high levels of resistance to PMB and NHS were detected at 25°C. However, at the same temperature, Y. pestis subsp. caucasica was highly sensitive to PMB. At both of the extreme temperatures, all strains were highly susceptible to PMB. At 25°C and 37°C, Y. pestis subsp. caucasica strain 1146 was highly susceptible to the bactericidal activity of 80% NHS. All Y. pestis strains studied were able to grow in heat-inactivated human serum or in 80% normal mouse serum. At 6°C, all strains were highly sensitive to NHS. Variations in the PMB resistance of different bacterial cultures related to both the content of cationic components (4-amino-4-deoxyarabinose in lipid A and glycine in the core) and a proper combination of terminal monosaccharides in the LPS. The NHS resistance correlated with an elevated content of N-acetylglucosamine in the LPS. Structural variation in the LPS of Y. pestis correlates with the organism's ability to resist innate immunity in both fleas and mammals.

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The lack of consensus regarding the significance of transmembrane passage of bacterial products across hemodialysis membranes can be related to several methodological differences in the various studies, including the choice of circulating fluid in the blood compartment of the model, nature and concentration of the bacterial products employed to challenge the dialysate compartment and whether cytokine production by PMBC or the limulus amebocyte lysate (LAL) assay was used as the index of transfer and the cytokine used as the read-out. In this study, we examined the production of interleukin-1 alpha (IL-1α), interieukin-1 receptor antagonist (IL-1Ra) and interleukin-8 (IL-8) by peripheral blood mononuclear cells (PBMC) incubated with a Pseudomonas aeruginosa culture filtrate. Further, the effects of 10% autologous human plasma and Polymyxin B sulfate (PmB) on cytokine production by PBMC were also characterized. The results of our study indicate that the Ps. aeruginosa culture filtrate had both PmB suppressible and PmB non-suppressible components and that the addition of 10% human plasma significantly enhanced cytokine production by both PmB suppressible and PmB non-suppressible components. The enhancing effect of plasma was most evident at low concentrations of the filtrate. The inhibitory effect of PmB was most evident in samples cultured in the presence of 10% plasma. There was a direct correlation between the production of IL-1α and IL-1Ra suggesting that both pro-inflammatory cytokines and cytokine-specific inhibitory proteins are concurrently produced. There results have direct relevance to selection of study conditions for in vitro models used to study the transmembrane passage of bacterial products across hemodialysis membranes

Abstract:

ABSTRACT Lipopolysaccharide (LPS), or endotoxin, a structural component of gram-negative bacterial outer membranes, plays a key role in the pathogenesis of septic shock, a syndrome of severe systemic inflammation which leads to multiple-system organ failure. Despite advances in antimicrobial chemotherapy, sepsis continues to be the commonest cause of death in the critically ill patient. This is attributable to the lack of therapeutic options that aim at limiting the exposure to the toxin and the prevention of subsequent downstream inflammatory processes. Polymyxin B (PMB), a peptide antibiotic, is a prototype small molecule that binds and neutralizes LPS toxicity. However, the antibiotic is too toxic for systemic use as an LPS sequestrant. Based on a nuclear magnetic resonance-derived model of polymyxin B-LPS complex, we had earlier identified the pharmacophore necessary for optimal recognition and neutralization of the toxin. Iterative cycles of pharmacophore-based ligand design and evaluation have yielded a synthetically easily accessible N 1,mono-alkyl-mono-hom*ologated spermine derivative, DS-96. We have found that DS-96 binds LPS and neutralizes its toxicity with a potency indistinguishable from that of PMB in a wide range of in vitro assays, affords complete protection in a murine model of LPS-induced lethality, and is apparently nontoxic in vertebrate animal models.

Abstract:

Abstract This study aimed to evaluate sodium hypochlorite (NaOCl), limewater (LW), and Polymyxin B (PMB) as irrigants over MMP-3, MMP-8 and MMP-9. Thirty-three patients with apical periodontitis of single-rooted teeth were treated according to three-experimental groups (n=11): group-1: 2.5% NaOCl was used as irrigant; group-2: 2.5% NaOCl for the first two files and LW: [0.14% Ca(OH)2] for the last two files; group-3: 2.5% NaOCl for the first two files and PMB for the last two files. The association of Ca(OH)2 and CHX was used as an intracanal medication in all groups. Four root canal samplings (S) were collected: S1) immediately after access cavity; S2) after biomechanical preparation; S3) after EDTA application; and S4) after removal of the intracanal medication. After quantification of MMP-3, MMP-8, and MMP-9, the data were analyzed by Friedman and Kruskal-Wallis tests and completed by Dunn test (5%). Regardless the used irrigant, there was no difference in reducing MMP-3 or MMP-8 (P=0,5273, P=0,7048 respectively). However, in reducing MMP-9 (P=0,0246) the NaOCl group was the most effective followed by NaOCl+LW group and NaOCl+PMB group respectively. The intracanal medication [Ca(OH)2 + CHX] with the NaOCl and NaOCl+LW was effective in reducing MMP-8 (P<0,0001, P=0,0025) and MMP-9 (P=0,0007, P=0,0047) respectively, but not for the group of NaOCl+PMB which was not effective in reducing MMP-8 or MMP-9 (P=0,1718, P=0,1953) respectively. NaOCl and NaOCl+LW were effective in reducing MMP-9 levels, and this effectivity could be improved by the use of the intracanal medication [Ca(OH)2 + CHX] in reducing MMP-8 and MMP-9 levels.

Abstract:

ABSTRACTMultidrug-resistantPseudomonas aeruginosais a major cause of severe hospital-acquired infections. Currently, polymyxin B (PMB) is a last-resort antibiotic for the treatment of infections caused by Gram-negative bacteria, despite its undesirable side effects. The delivery of drug combinations has been shown to reduce the required therapeutic doses of antibacterial agents and thereby their toxicity if a synergistic effect is present. In this study, we investigated the synergy between two cyclic antimicrobial peptides, PMB and gramicidin S (GS), against differentP. aeruginosaisolates, using a quantitative checkerboard assay with resazurin as a growth indicator. Among the 28 strains that we studied, 20 strains showed a distinct synergistic effect, represented by a fractional inhibitory concentration index (FICI) of ≤0.5. Remarkably, several clinicalP. aeruginosaisolates that grew as small-colony variants revealed a nonsynergistic effect, as indicated by FICIs between >0.5 and ≤0.70. In addition to inhibiting the growth of planktonic bacteria, the peptide combinations significantly decreased static biofilm growth compared with treatment with the individual peptides. There was also a faster and more prolonged effect when the combination of PMB and GS was used compared with single-peptide treatments on the metabolic activity of pregrown biofilms. The results of the present study define a synergistic interaction between two cyclic membrane-active peptides toward 17 multidrug-resistantP. aeruginosaand biofilms ofP. aeruginosastrain PAO1. Thus, the application of PMB and GS in combination is a promising option for a topical medication and in the prevention of acute and chronic infections caused by multidrug-resistant or biofilm-formingP. aeruginosa.

Abstract:

By monitoring the pressure signal changes of the platinum nanoparticles (PtNPs) catalyzed H₂O₂ breakdown, a cost-effective and rapid analysis method for the quantitative testing of PMB is established with a portable pressure meter readout.

Abstract:

ABSTRACT Commercially available pokeweed mitogen (PWM) has been reported to activate macrophages, leading to production of proinflammatory cytokines and nitric oxide (NO). However, we found that polymyxin B (PMB), a specific inhibitor of endotoxin activity, inhibited the PWM-induced expression of proinflammatory cytokines and NO and the activation of Toll-like receptor 4 (TLR4). A kinetic-turbidimetric Limulus amebocyte lysate assay demonstrated that commercial PWM contained substantial endotoxin, over 104 endotoxin units/mg of the PWM. A PWM repurified by PMB-coupled beads no longer induced the expression of proinflammatory cytokines, TLR4 activation, or dendritic cell maturation. However, the repurified PWM remained able to induce proliferation of human lymphocytes, which is a representative characteristic of PWM. These results suggest that commercial PWM might be contaminated with a large amount of endotoxin, resulting in the attribution of misleading immunological properties to PWM.

Abstract:

ABSTRACTCommunication of antibiotic resistance among bacteria via small molecules is implicated in transient reduction of bacterial susceptibility to antibiotics, which could lead to therapeutic failures aggravating the problem of antibiotic resistance. Released putrescine from the extremely antibiotic-resistant bacteriumBurkholderia cenocepaciaprotects less-resistant cells from different species against the antimicrobial peptide polymyxin B (PmB). Exposure ofB. cenocepaciato sublethal concentrations of PmB and other bactericidal antibiotics induces reactive oxygen species (ROS) production and expression of the oxidative stress response regulator OxyR. We evaluated whether putrescine alleviates antibiotic-induced oxidative stress. The accumulation of intracellular ROS, such as superoxide ion and hydrogen peroxide, was assessed fluorometrically with dichlorofluorescein diacetate, while the expression of OxyR and putrescine synthesis enzymes was determined in luciferase assays using chromosomal promoter-luxreporter system fusions. We evaluated wild-type and isogenic deletion mutant strains with defects in putrescine biosynthesis after exposure to sublethal concentrations of PmB and other bactericidal antibiotics. Exogenous putrescine protected against oxidative stress induced by PmB and other antibiotics, whereas reduced putrescine synthesis resulted in increased ROS generation and a parallel increased sensitivity to PmB. Of the 3B. cenocepaciaputrescine-synthesizing enzymes, PmB induced only BCAL2641, an ornithine decarboxylase. This study reveals BCAL2641 as a critical component of the putrescine-mediated communication of antibiotic resistance and as a plausible target for designing inhibitors that would block the communication of such resistance among different bacteria, ultimately reducing the window of therapeutic failure in treating bacterial infections.

Abstract:

Bacterial biofilms pose a serious threat to human health, as they prevent the penetration of antimicrobial agents. Developing nanocarriers that can simultaneously permeate biofilms and deliver antibacterial agents is an attractive means of treating bacterial biofilm infections. Herein, photosensitive metal–organic framework (MOF) nanoparticles were developed to promote the penetration of antibiotics into biofilms, thereby achieving the goal of eradicating bacterial biofilms through synergistic photodynamic and antibiotic therapy. First, a ligand containing benzoselenadiazole was synthesized and incorporated into MOF skeletons to construct benzoselenadiazole-doped MOFs (Se-MOFs). The growth of the Se-MOFs could be regulated to obtain nanoparticles (Se-NPs) in the presence of benzoic acid. The singlet oxygen (1O2) generation efficiencies of the Se-MOFs and Se-NPs were evaluated. The results show that the Se-NPs exhibited a higher 1O2 generation efficacy than the Se-MOF under visible-light irradiation because the small size of the Se-NPs was conducive to the diffusion of 1O2. Afterward, an antibiotic drug, polymyxin B (PMB), was conjugated onto the surface of the Se-NPs via amidation to yield PMB-modified Se-NPs (PMB-Se-NPs). PMB-Se-NPs exhibit a synergistic antibacterial effect by specifically targeting the lipopolysaccharides present in the outer membranes of Gram-negative bacteria through surface-modified PMB. Benefiting from the synergistic therapeutic effects of antibiotic and photodynamic therapy, PMB-Se-NPs can efficiently eradicate bacterial biofilms at relatively low antibiotic doses and light intensities, providing a promising nanocomposite for combating biofilm infections.

Abstract:

ABSTRACT Polymyxin B (PMB) binds to and neutralizes endotoxin, but its systemic clinical utility is limited by neuro- and nephrotoxicity. PMX622 is a covalent conjugate of PMB and Dextran-70 designed to retain the ability of PMB to neutralize endotoxin and to retain the favorable colloidal, pharmaco*kinetic, and metabolic properties of Dextran-70. PMX622 has demonstrated efficacy in a number of animal models and effectively neutralized endotoxin in phase I clinical trials. Here, we systematically evaluated the pharmacodynamic properties of PMX622 in a murine model of endotoxin-induced lethality in galactosamine-sensitized mice. PMX622 completely and dose dependently inhibited lethality in this model. A stoichiometric relationship was found between the endotoxin challenge dose and the dose of PMX622 needed for protection. PMX622 neutralized endotoxin from four different genera of gram-negative bacteria but not Neisseria meningitidis. PMX622 was significantly less toxic than PMB in the mouse, suggesting that PMX622 has a better margin of safety than PMB. The timing of PMX622 administration relative to endotoxin was crucial. PMX622 was active for several hours prior to the endotoxin challenge; however, PMX622 did not protect mice if administered ≥15 min after endotoxin challenge. This suggests that PMX622 would best be clinically used prophylactically rather than therapeutically. These studies will be crucial in designing and interpreting human clinical trials assessing PMX622 efficacy.

Abstract:

Colonization of the gastrointestinal tract by enterohaemorrhagic Escherichia coli (EHEC) is critically dependent on its ability to sense and respond to various microenvironments within the host. EHEC exposure to physiologically relevant levels of bile salts upregulates the two-component system, pmrAB, and the arnBCADTEF operon, resulting in lipopolysaccharide modification and increased resistance to the cationic antimicrobial peptide, polymyxin B (PMB). A similar pmrAB- and arn-dependent PMB resistance has been observed in Salmonella enterica in the presence of ferric iron. Limiting magnesium levels and mild acid can also induce Salmonella resistance to PMB through another two-component system, PhoPQ and the connector protein, PmrD. This study aims to evaluate the relative contributions of a bile-salt mix (BSM), iron, limiting magnesium as well as the roles of pmrAB, phoPQ and pmrD to EHEC’s resistance to PMB. Killing assays show that EHEC treatment with the BSM or iron under excess magnesium and neutral pH conditions induces a pmrAB-dependent, phoP-independent PMB resistance. By contrast, exposure to limiting magnesium triggers a pmrB-, phoP- and pmrD-dependent PMB resistance. The iron-induced PMB resistance is independent of phoP and pmrD under limiting magnesium conditions while the bile-salt-induced PMB resistance is independent of pmrD only under non-PhoP-inducing conditions. GFP-pmrD transcriptional reporter studies reveal that the limiting magnesium enhances pmrD expression, which is repressed upon additional exposure to either BSM or iron. Our results also show that exposure to mild acid enhances PMB resistance in a pmrD-independent manner and GFP reporter results confirm minimal expression of pmrD at this pH regardless of the magnesium level. This study provides novel insights into how EHEC differentially employs PmrAB, PhoPQ and PmrD to monitor and respond to bile salts, iron, acidic pH and magnesium typically encountered within the gastrointestinal tract in order to modulate its survival against cationic antimicrobial peptides.

Abstract:

Antimicrobial peptides (AMPs) contribute to an effective protection against infections. The antibacterial function of AMPs depends on their interactions with microbial membranes and lipids, such as lipopolysaccharide (LPS; endotoxin). Hyperinflammation induced by endotoxin is a key factor in bacterial sepsis and many other human diseases. Here, we provide a comprehensive profile of peptide-mediated LPS neutralization by systematic analysis of the effects of a set of AMPs and the peptide antibiotic polymyxin B (PMB) on the physicochemistry of endotoxin, macrophage activation, and lethality in mice. Mechanistic studies revealed that the host defense peptide LL-32 and PMB each reduce LPS-mediated activation also via a direct interaction of the peptides with the host cell. As a biophysical basis, we demonstrate modifications of the structure of cholesterol-rich membrane domains and the association of glycosylphosphatidylinositol (GPI)-anchored proteins. Our discovery of a host cell–directed mechanism of immune control contributes an important aspect in the development and therapeutic use of AMPs.

Abstract:

Abstract Background The scourge of MBLs among Gram negatives, such New Delhi Metallo-beta-lactamase-producing Klebsiella pneumoniae, has resulted in an overwhelming need for new treatmens. Worryingly, additional acquisition of plasmid-mediated polymyxin resistance through the mcr gene can produce strains resistant to all last line agents. The novel combination of aztreonam (ATM, which is not an MBL substrate) with avibactam (AVI, to inhibit extended spectrum beta-lactamases that inactivate ATM) has been proposed to restore ATM activity. Methods K. pneumoniae SZ04 harboring blaNDM-5, blaCTX-M-55, and mcr-1 (MICATM = 128 mg/L, MICPolymyxin B = 4 mg/L, MICAmikacin = 1 mg/L, MICceftazidime/avibactam &gt; 16/4 mg/L) was studied at two initial inoculum (106 and 108 cfu/mL) over 24h in static time kills (SCTK). Concentration arrays of 2-, 3-, and 4-drug combinations of low- and package insert-dose polymyxin B (PMB) ± low- and package insert-dose amikacin (AMI) ± package insert dosing of ATM/AVI were simulated in &gt; 200 individual arms of SCTK. Data were summarized using the Log Ratio Area (LRA) which is calculated by integrating the area under the bacterial killing curve, normalizing to the growth control, then log-transforming. A Hill-type function was fit to the data in order to determine the maximum effect (Emax) and drug concentration for 50% effect (EC50). Results When simulating the maximum free concentration of amikacin 15 mg/kg (52 mg/L) in combination with package insert concentrations of ATM/AVI, there was a marked reduction of 3.22 in the LRA compared to the growth control for the 108 cfu/mL starting inocula. Combining ATM with low amikacin (0.813 mg/L) and polymyxin B (0.125 mg/L) resulted in a reduction in LRA of 4.32 at 106 cfu/mL. Model fitting results showed a statistically significant difference in EC50 to amikacin between low and high inocula at 1.24 and 18.1 mg/L, respectively. Combination of AMI with low-concentration PMB (0.125 mg/L) resulted in an increase in the Emax of amikacin to -5.68. Conclusion The use of ATM/AVI combinations is a promising option against MBL and MCR co-producing K. pneumoniae. Low-dose strategies of polymyxin or amikacin dosing in combination with ATM/AVI is merits further testing for future translation to the clinical setting to improve efficacy and optimize treatment. Disclosures Thomas Lodise, PharmD, PhD, Paratek Pharmaceuticals, Inc. (Consultant) Robert A. Bonomo, MD, Entasis, Merck, Venatorx (Research Grant or Support)

Abstract:

The outer membrane (OM) of Gram-negative bacteria is a robust, impermeable, asymmetric bilayer of outer lipopolysaccharides (LPSs) and inner phospholipids containing selective pore proteins which confer on it the properties of a molecular sieve. This structure severely limits the variety of antibiotic molecules effective against Gram-negative pathogens and, as antibiotic resistance has increased, so has the need to solve the OM permeability problem. Polymyxin B (PmB) represents those rare antibiotics which act directly on the OM and which offer a distinct starting point for new antibiotic development. Here we investigate PmB’s interactions with in vitro OM models and show how the physical state of the lipid matrix of the OM is a critical factor in regulating the interaction with the antimicrobial peptide. Using neutron reflectometry and infrared spectroscopy, we reveal the structural and chemical changes induced by PmB on OM models of increasing complexity. In particular, only a tightly packed model reproduced the temperature-controlled disruption of the asymmetric lipid bilayer by PmB observed in vivo. By measuring the order of outer-leaflet LPS and inner-leaflet phospholipids, we show that PmB insertion is dependent on the phase transition of LPS from the gel to the liquid crystalline state. The demonstration of a lipid phase transition in the physiological temperature range also supports the hypothesis that bacteria grown at different temperatures adapt their LPS structures to maintain a homeoviscous OM.

Abstract:

<b><i>Introduction:</i></b> Lipopolysaccharide (LPS) contamination of commercially available proteins has seriously impeded research on citrullinated fibrinogen (cit-Fb) in rheumatoid synovial cells (RSCs). <b><i>Methods:</i></b> RSCs obtained from 4 rheumatoid arthritis patients who underwent full knee arthroplasty were cultured, stimulated with cit-Fb, and cytokine expression levels were measured. We then evaluated polymyxin-B (PMB), heat inactivation, and rough (R)-type LPS mutants for rapid detection of LPS contamination. <b><i>Results:</i></b> cit-Fb induced expression of <i>CXCL10</i> and <i>IFNB</i> in RSCs via the toll-like receptor. PMB inhibited cit-Fb-mediated CXCL10 gene expression but not protein expression induced by 20 μg/mL cit-Fb. Heat inactivation did not affect LPS-mediated <i>CXCL10</i> or <i>IL-6</i> induction; however, cit-Fb-mediated <i>CXCL10</i>expression was inhibited. Wild-type LPS from <i>Escherichia coli</i> (WT-LPS) strongly induces <i>CXCL10</i> expression, but induction by Ra-LPS was weak, and induction by Rc- and Re-LPS was minimal. Re-LPS suppression of WT-LPS-mediated <i>CXCL10</i> induction in RSCs and peripheral blood monocytes (PBMs) was dose dependent. Furthermore, Re-LPS completely suppressed cit-Fb-mediated <i>CXCL10</i> induction in RSCs and PBMs. <b><i>Conclusion:</i></b> To easily identify LPS contamination during routine experiments, our results suggest that Re-LPS is a better tool for rapid detection of LPS contamination compared to PMB and heat treatment.

Abstract:

ABSTRACT Lipopolysaccharides (LPS) are proinflammatory bacterial products implicated in the pathogenesis of gram-negative sepsis and septic shock. Polymyxin B (PMB), a cyclic, cationic peptide antibiotic, inhibits biological activities of LPS through high-affinity binding to the lipid A moiety. Small synthetic peptides have been designed to mimic the primary and secondary structures of PMB to determine structural requirements for binding and detoxification of lipid A and to assess possible therapeutic potential. The purpose of this study was to compare and contrast the endotoxin-neutralizing activities of two synthetic antiendotoxin peptides (SAEP-2 and SAEP-4), PMB, and an LPS core-specific monoclonal antibody (MAb), WN1 222-5, based on their abilities to inhibit CD14-mediated target cell uptake of fluorescein isothiocyanate (FITC)-conjugated LPS, detected by flow cytometry and confocal microscopy, and LPS-induced production of the proinflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α), as measured by bioassays. PMB and SAEP-4 produced dose-dependent inhibition of FITC-LPS uptake by CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) and by human peripheral blood mononuclear cells. The anti-LPS MAb, WN1 222-5, also blocked LPS uptake by these cells and synergized with PMB and SAEP-4. LPS-induced IL-6 release was inhibited by PMB, SAEP-4, and MAb WN1 222-5, and these inhibitory activities were additive or synergistic. LPS-induced TNF-α release by PBMC was also inhibited by PMB and SAEP-4 alone and in combination with anti-LPS MAb. SAEP-2, in contrast, produced comparatively minor decrements in cellular uptake of LPS and LPS-induced cytokine responses, and did so only in the absence of serum, while a nonsense peptide exerted no discernible inhibitory effect on LPS uptake or LPS-induced cytokine expression in the presence or absence of serum. Thus, PMB and SAEP-4, like the LPS-reactive MAb, WN1 222-5, block proinflammatory activities of LPS in part by preventing LPS recognition by membrane-bound CD14-expressing target cells. Differences in peptide structure, however, like those exemplified by SAEP-2 and SAEP-4, may differentially affect the endotoxin-neutralizing potency of these peptides despite similar binding activity against lipid A, reflecting possible differences in peptide solubility or peptide regulation of intracellular signal transduction.