Molecular Weights of Polyethyleneimine-Dependent Physicochemical Tuning of Gold Nanoparticles and FRET-Based Turn-On Sensing of Polymyxin B.

Environmental monitoring and the detection of antibiotic contaminants require expensive and time-consuming techniques. To overcome these challenges, gold nanoparticle-mediated fluorometric "turn-on" detection of Polymyxin B (PMB) in an aqueous medium was undertaken. The molecular weight of polyethyleneimine (PEI)-dependent physicochemical tuning of gold nanoparticles (PEI@AuNPs) was achieved and employed for the same. The three variable molecular weights of branched polyethyleneimine (MW 750, 60, and 1.3 kDa) molecules controlled the nano-geometry of the gold nanoparticles along with enhanced stabilization at room temperature. The synthesized gold nanoparticles were characterized through various advanced techniques. The results revealed that polyethyleneimine-stabilized gold nanoparticles (PEI@AuNP-1-3) were 4.5, 7.0, and 52.5 nm in size with spherical shapes, and the zeta potential values were 29.9, 22.5, and 16.6 mV, respectively. Accordingly, the PEI@AuNPs probes demonstrated high sensitivity and selectivity, with a linear relationship curve over a concentration range of 1-6 µM for polymyxin B. The limit of detection (LOD) was calculated as 8.5 nM. This is the first unique report of gold nanoparticle nano-geometry-dependent FRET-based turn-on detection of PMB in an aqueous medium. We believe that this approach would offer a complementary strategy for the development of a highly sophisticated and advanced sensing system for PMB and act as a template for the development of new nanomaterial-based engineered sensors for rapid antibiotic detection in environmental as well as biological samples.

Comparison of Antibacterial Efficacy of Triple Antibiotic-Loaded Hydrogel Versus Modified Triple Antibiotic-Loaded Hydrogel as Intracanal Medicament Against Enterococcus faecalis: An In vitro Study.

OBJECTIVE: Triple antibiotic paste (TAP) is known to have an essential role in the success of endodontic treatment by eliminating pathogens from the root canal system. Unfortunately, it causes discolouration and cytotoxicity at high concentrations. The objective of this research was to assess and compare the antimicrobial effectiveness of various concentrations (1 mg, 5 mg, 10 mg) of TAP, TAP hydrogel (TAPH), M-TAP, and M-TAP hydrogel (MTAPH) against Enterococcus faecalis. METHODS: The agar well diffusion method was used to assess the antibiotic sensitivity of the following intracanal medicaments: TAP (ciprofloxacin, metronidazole, and minocycline) mixed in a ratio of 1: 1: 1; TAPH, M-TAP (ciprofloxacin, metronidazole, and amoxicillin), M-TAPH and plain hydrogel. Each tested medicament was individually evaluated for its antimicrobial activity against Enterococcus faecalis. Structural and topographical characterisation were analysed using a Scanning Electron Microscope (SEM) and interpreted using ImageJ software. A microdilution broth test was performed to examine the minimum inhibitory concentration and minimum bactericidal concentration (MBC) of M-TAP and TAP. RESULTS: Except for the plain hydrogel, M-TAP and hydrogel and TAP and hydrogel showed significantly varied inhibitory zones at different concentrations. M-TAPH showed the highest mean zone of inhibition of 21.6, 33.33 and 38.0 mm at a concentration of 1, 5, and 10 mg/mL when compared to TAPH, which showed a mean zone of inhibition of 3.3 mm,12.3 mm, 21.3 mm at the respective concentrations. The MIC study shows that more than 75% of Enterococcus faecalis growth was inhibited by M-TAP at a concentration of 5 µg/mL, whereas TAP showed inhibition at a concentration of 35 µg/mL. MBC results indicate that almost 99.9% of the bacterial population was killed at a concentration of 100 µg/mL (10-1) for TAP and 10 µg/mL (10-2) for M-TAP. CONCLUSION: The antibacterial efficacy of M-TAP was significantly higher than TAP. Application of M-TAP at lower doses is advised to overcome the disadvantages seen with TAP.

Polymyxin B-Enriched Exogenous Lung Surfactant: Thermodynamics and Structure.

The use of an exogenous pulmonary surfactant (EPS) to deliver other relevant drugs to the lungs is a promising strategy for combined therapy. We evaluated the interaction of polymyxin B (PxB) with a clinically used EPS, the poractant alfa Curosurf (PSUR). The effect of PxB on the protein-free model system (MS) composed of four phospholipids (diC16:0PC/16:0-18:1PC/16:0-18:2PC/16:0-18:1PG) was examined in parallel to distinguish the specificity of the composition of PSUR. We used several experimental techniques (differential scanning calorimetry, small- and wide-angle X-ray scattering, small-angle neutron scattering, fluorescence spectroscopy, and electrophoretic light scattering) to characterize the binding of PxB to both EPS. Electrostatic interactions PxB-EPS are dominant. The results obtained support the concept of cationic PxB molecules lying on the surface of the PSUR bilayer, strengthening the multilamellar structure of PSUR as derived from SAXS and SANS. A protein-free MS mimics a natural EPS well but was found to be less resistant to penetration of PxB into the lipid bilayer. PxB does not affect the gel-to-fluid phase transition temperature, Tm, of PSUR, while Tm increased by ∼+ 2 °C in MS. The decrease of the thickness of the lipid bilayer (dL) of PSUR upon PxB binding is negligible. The hydrophobic tail of the PxB molecule does not penetrate the bilayer as derived from SANS data analysis and changes in lateral pressure monitored by excimer fluorescence at two depths of the hydrophobic region of the bilayer. Changes in dL of protein-free MS show a biphasic dependence on the adsorbed amount of PxB with a minimum close to the point of electroneutrality of the mixture. Our results do not discourage the concept of a combined treatment with PxB-enriched Curosurf. However, the amount of PxB must be carefully assessed (less than 5 wt % relative to the mass of the surfactant) to avoid inversion of the surface charge of the membrane.

Optimization of an in vitro Pseudomonas aeruginosa Biofilm Model to Examine Antibiotic Pharmacodynamics at the Air-Liquid Interface.

Pseudomonas aeruginosa is an important cause of lower respiratory tract infections, such as ventilator-associated bacterial pneumonia (VABP). Using inhaled antibiotics to treat VABP can achieve high drug concentrations at the infection site while minimizing systemic toxicities. Despite the theoretical advantages, clinical trials have failed to show a benefit for inhaled antibiotic therapy in treating VABP. A potential reason for this discordance is the presence of biofilm-embedded bacteria in lower respiratory tract infections. Drug selection and dosing are often based on data from bacteria grown planktonically. In the present study, an in vitro air-liquid interface pharmacokinetic/pharmacodynamic biofilm model was optimized to evaluate the activity of simulated epithelial lining fluid exposures of inhaled and intravenous doses of polymyxin B and tobramycin against two P. aeruginosa strains. Antibiotic activity was also determined against the P. aeruginosa strains grown planktonically. Our study revealed that inhaled antibiotic exposures were more active than their intravenous counterparts across biofilm and planktonic populations. Inhaled exposures of polymyxin B and tobramycin exhibited comparable activity against planktonic P. aeruginosa. Although inhaled polymyxin B exposures were initially more active against P. aeruginosa biofilms (through 6 h), tobramycin was more active by the end of the experiment (48 h). Together, these data slightly favor the use of inhaled tobramycin for VABP caused by biofilm-forming P. aeruginosa that are not resistant to either antibiotic. The optimized in vitro air-liquid interface pharmacokinetic/pharmacodynamic biofilm model may be beneficial for the development of novel anti-biofilm agents or to optimize antibiotic dosing for infections such as VABP.

Suppression of planktonic and biofilm of Escherichia coli by the synergistic lantibiotics-polymyxins combinations.

Escherichia coli are generally resistant to the lantibiotic's action (nisin and warnerin), but we have shown increased sensitivity of E. coli to lantibiotics in the presence of subinhibitory concentrations of polymyxins. Synergistic lantibiotic-polymyxin combinations were found for polymyxins B and M. The killing of cells at the planktonic and biofilm levels was observed for two collection and four clinical multidrug-resistant E. coli strains after treatment with lantibiotic-polymyxin B combinations. Thus, 24-h treatment of E. coli mature biofilms with warnerin-polymyxin B or nisin-polymyxin B leads to five to tenfold decrease in the number of viable cells, depending on the strain. AFM revealed that the warnerin and polymyxin B combination caused the loss of the structural integrity of biofilm and the destruction of cells within the biofilm. It has been shown that pretreatment of cells with polymyxin B leads to an increase of Ca2+ and Mg2+ ions in the culture medium, as detected by atomic absorption spectroscopy. The subsequent exposure to warnerin caused cell death with the loss of K+ ions and cell destruction with DNA and protein release. Thus, polymyxins display synergy with lantibiotics against planktonic and biofilm cells of E. coli, and can be used to overcome the resistance of Gram-negative bacteria to lantibiotics.

Synergistic interaction of polymyxin B with carvacrol: antimicrobial strategy against polymyxin-resistant Klebsiella pneumoniae.

Objective: The antimicrobial activities of the synergistic combination of carvacrol and polymyxin B against polymyxin-resistant Klebsiella pneumoniae were evaluated. Methods: The methods employed checkerboard assays to investigate synergism, biofilm inhibition assessment and membrane integrity assay. In addition, the study included in vivo evaluation using a mouse infection model. Results: The checkerboard method evaluated 48 combinations, with 23 indicating synergistic action. Among these, carvacrol 10 mg/kg plus polymyxin B 2 mg/kg exhibited in vivo antimicrobial activity in a mouse model of infection, resulting in increased survival and a significant decrease in bacterial load in the blood. Conclusion: Polymyxin in synergy with carvacrol represents a promising alternative to be explored in the development of new antimicrobials.

In this study, we wanted to find a new way to fight a bacteria called Klebsiella pneumoniae, which is not easily killed by medication. We mixed two drugs, carvacrol and polymyxin B, to see if they would work together to fight the bacteria. We found that the mixed treatment helped to kill the bacteria. We also tried this mixed treatment in sick mice, and they got better. Our study shows that this mixed treatment might be a new way to fight bacteria that are hard to kill with regular drugs. Next, we hope to learn more about how it works.

Dronedarone Enhances the Antibacterial Activity of Polymyxin B and Inhibits the Quorum Sensing System by Interacting with LuxS.

Quorum sensing (QS) is considered an appealing target for interference with bacterial infections. ß-Adrenergic blockers are promising anti-QS agents but do not have antibacterial activity. We assessed the potential ability of adrenergic receptor inhibitors to enhance the antibacterial activity of polymyxin B (PB) against Klebsiella pneumoniae and determined that dronedarone has the most potent activity both in vitro and in vivo. We found that dronedarone increases the thermal stability of LuxS, decreases the production of AI-2, and affects the biofilm formation of K. pneumoniae. We also identified the direct binding of dronedarone to LuxS. However, the mechanism by which dronedarone enhances the antibacterial activity of PB has not been elucidated and is worthy of further exploration. Our study provides a basis for the future development of drug combination regimens.

Synergistic antimicrobial photodynamic therapy using gated mesoporous silica nanoparticles containing curcumin and polymyxin B.

Photodynamic Therapy is a therapy based on combining a non-toxic compound, known as photosensitizer (PS), and irradiation with light of the appropriate wavelength to excite the PS molecule. The photon absorption by the PS leads to reactive oxygen species generation and a subsequent oxidative burst that causes cell damage and death. In this work, we report an antimicrobial nanodevice that uses the activity of curcumin (Cur) as a PS for antimicrobial Photodynamic Therapy (aPDT), based on mesoporous silica nanoparticles in which the action of the classical antibiotic PMB is synergistically combined with the aPDT properties of curcumin to combat bacteria. The synergistic effect of the designed gated device in combination with irradiation with blue LED light (470 nm) is evaluated against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus epidermidis. The results show that the nanodevice exhibits a noteworthy antibacterial activity against these microorganisms, a much more significant effect than free Cur and PMB at equivalent concentrations. Thus, 0.1 µg/mL of MSNs-Cur-PMB eliminates a bacterial concentration of about 105 CFU/mL of E. coli, while 1 µg/mL of MSNs-Cur-PMB is required for P. aeruginosa and S. epidermidis. In addition, antibiofilm activity against the selected bacteria was also tested. We found that 0.1 mg/mL of MSNs-Cur-PMB inhibited 99 % biofilm formation for E. coli, and 1 mg/mL of MSNs-Cur-PMB achieved 90 % and 100 % inhibition of biofilm formation for S. epidermidis and P. aeruginosa, respectively.

Polymyxin B nonapeptide potentiates the eradication of Gram-negative bacterial persisters.

Antibiotic-resistant Gram-negative bacteria remain a globally leading cause of bacterial infection-associated mortality, and it is imperative to identify novel therapeutic strategies. Recently, the advantage of using antibacterials selective against Gram-negative bacteria has been demonstrated with polymyxins that specifically target the lipopolysaccharides of Gram-negative bacteria. However, the severe cytotoxicity of polymyxins limits their clinical use. Here, we demonstrate that polymyxin B nonapeptide (PMBN), a polymyxin B derivative without the terminal amino acyl residue, can significantly enhance the effectiveness of commonly used antibiotics against only Gram-negative bacteria and their persister cells. We show that although PMBN itself does not exhibit antibacterial activity or cytotoxicity well above the 100-fold minimum inhibitory concentration of polymyxin B, PMBN can increase the potency of co-treated antibiotics. We also demonstrate that using PMBN in combination with other antibiotics significantly reduces the frequency of resistant mutant formation. Together, this work provides evidence of the utilities of PMBN as a novel potentiator for antibiotics against Gram-negative bacteria and insights for the eradication of bacterial persister cells during antibiotic treatment. IMPORTANCE: The significance of our study lies in addressing the problem of antibiotic-resistant Gram-negative bacteria, which continue to be a global cause of mortality associated with bacterial infections. Therefore, identifying innovative therapeutic approaches is an urgent need. Recent research has highlighted the potential of selective antibacterials like polymyxins, which specifically target the lipopolysaccharides of Gram-negative bacteria. However, the clinical use of polymyxins is limited by their severe cytotoxicity. This study unveils the effectiveness of polymyxin B nonapeptide (PMBN) in significantly enhancing the eradication of persister cells in Gram-negative bacteria. Although PMBN itself does not exhibit antibacterial activity or cytotoxicity, it remarkably reduces persister cells during the treatment of antibiotics. Moreover, combining PMBN with other antibiotics reduces the emergence of resistant mutants. Our research emphasizes the utility of PMBN as a novel potentiator to decrease persister cells during antibiotic treatments for Gram-negative bacteria.

Easy and versatile cellulosic support inhibiting broad spectrum strains: synergy between photodynamic antimicrobial therapy and polymyxin B.

Despite advances achieved in the health field over the last decade, infections caused by resistant bacterial strains are an increasingly important societal issue that needs to be addressed. New approaches have already been developed to overcome this problem. Photodynamic antimicrobial chemotherapy (PACT) could provide a promising alternative method to eradicate microbes. This approach has already inspired the development of innovative surfaces. Interesting results were achieved against Gram-positive bacteria, but it also appeared that Gram-negative strains, especially Pseudomonas aeruginosa, were less sensitive to PACT. However, materials coated with cationic porphyrins have already proven their wide-spectrum activity, but these materials were not suitable for industrial-scale production. The main aim of this work was the design of a large-scale evolutionary material based on PACT and antibiotic prophylaxis. Transparent regenerated cellulose has been simply impregnated with a usual cationic porphyrin (N-methylpyridyl) and an antimicrobial peptide (polymyxin B). In addition to its photophysical properties, this film exhibited a wide-spectrum bactericidal activity over 4 days despite daily application of fresh bacterial inoculums. The efficiency of PACT and polymyxin B combination could help to reduce the emergence of bacterial multi-resistant strains and we believe that this kind of material would provide an excellent opportunity to prevent bacterial contamination of bandages or packaging.

Comparison of in vitro synergy between polymyxin B or colistin in combination with 16 antimicrobial agents against multidrug-resistant Acinetobacter baumannii isolates.

PURPOSES: This study determined the synergy of polymyxin B (POLB) and colistin (COL) with 16 other tested antimicrobial agents in the inhibition of multidrug-resistant Acinetobacter baumannii (MDR-AB). METHODS: We used chequerboard assays to determine synergy between the drugs against 50 clinical MDR-AB from a tertiary hospital in the Zhejiang province in 2019, classifying combinations as either antagonistic, independent, additive, or synergistic. The efficacy of hit combinations which showed highest synergistic rate were confirmed using time-kill assays. RESULTS: Both POLB and COL displayed similar bactericidal effects when used in combination with these 16 tested drugs. Antagonism was only observed for a few strains (2%) exposed to a combination of POLB and cefoperazone/sulbactam (CSL). A higher percentage of synergistic combinations with POLB and COL were observed with rifabutin (RFB; 90%/96%), rifampicin (RIF; 60%/78%) and rifapentine (RFP; 56%/76%). Time-kill assays also confirmed the synergistic effect of POLB and rifamycin class combinations. 1/2 MIC rifamycin exposure can achieve bacterial clearance when combined with 1/2 MIC POLB or COL. CONCLUSION: Nearly no antagonism was observed when combining polymyxins with other drugs by both chequerboard and time-kill assays, suggesting that polymyxins may be effective in combination therapy. The combinations of POLB/COL with RFB, RIF, and RFP displayed neat synergy, with RFB showing the greatest effect.

Pseudomonas aeruginosa MipA-MipB envelope proteins act as new sensors of polymyxins.

Due to the rising incidence of antibiotic-resistant infections, the last-line antibiotics, polymyxins, have resurged in the clinics in parallel with new bacterial strategies of escape. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa develops resistance to colistin/polymyxin B by distinct molecular mechanisms, mostly through modification of the lipid A component of the LPS by proteins encoded within the arnBCDATEF-ugd (arn) operon. In this work, we characterized a polymyxin-induced operon named mipBA, present in P. aeruginosa strains devoid of the arn operon. We showed that mipBA is activated by the ParR/ParS two-component regulatory system in response to polymyxins. Structural modeling revealed that MipA folds as an outer-membrane ß-barrel, harboring an internal negatively charged channel, able to host a polymyxin molecule, while the lipoprotein MipB adopts a ß-lactamase fold with two additional C-terminal domains. Experimental work confirmed that MipA and MipB localize to the bacterial envelope, and they co-purify in vitro. Nano differential scanning fluorimetry showed that polymyxins stabilized MipA in a specific and dose-dependent manner. Mass spectrometry-based quantitative proteomics on P. aeruginosa membranes demonstrated that ∆mipBA synthesized fourfold less MexXY-OprA proteins in response to polymyxin B compared to the wild-type strain. The decrease was a direct consequence of impaired transcriptional activation of the mex operon operated by ParR/ParS. We propose MipA/MipB to act as membrane (co)sensors working in concert to activate ParS histidine kinase and help the bacterium to cope with polymyxin-mediated envelope stress through synthesis of the efflux pump, MexXY-OprA.IMPORTANCEDue to the emergence of multidrug-resistant isolates, antibiotic options may be limited to polymyxins to eradicate Gram-negative infections. Pseudomonas aeruginosa, a leading opportunistic pathogen, has the ability to develop resistance to these cationic lipopeptides by modifying its lipopolysaccharide through proteins encoded within the arn operon. Herein, we describe a sub-group of P. aeruginosa strains lacking the arn operon yet exhibiting adaptability to polymyxins. Exposition to sub-lethal polymyxin concentrations induced the expression and production of two envelope-associated proteins. Among those, MipA, an outer-membrane barrel, is able to specifically bind polymyxins with an affinity in the 10-µM range. Using membrane proteomics and phenotypic assays, we showed that MipA and MipB participate in the adaptive response to polymyxins via ParR/ParS regulatory signaling. We propose a new model wherein the MipA-MipB module functions as a novel polymyxin sensing mechanism.

In vitro conversion of ellagic acid to urolithin A by different gut microbiota of urolithin metabotype A.

The metabolite urolithin A, a metabolite of the dietary polyphenol ellagic acid (EA), has significant health benefits for humans. However, studies on the gut microbiota involved in ellagic acid metabolism are limited. In this study, we conducted in vitro fermentation of EA using human intestinal microbiome combined with antibiotics (vancomycin, polymyxin B sulfate, and amphotericin B). Liquid chromatography-mass spectrometry (LC-MS/MS) analysis demonstrated that the production capacity of urolithin A by gut microbiota co-treated with polymyxin B sulfate and amphotericin B (22.39 µM) was similar to that of untreated gut microbiota (24.26 µM). Macrogenomics (high-throughput sequencing) was used to analyze the composition and structure of the gut microbiota. The results showed that the abundance of Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium bifidum in the gut microbiota without antibiotic treatment or co-treated with polymyxin B sulfate and amphotericin B during EA fermentation was higher than that in other antibiotic treatment gut microbiota. Therefore, B. longum, B. adolescentis, and B. bifidum may be new genera involved in the conversion of EA to urolithin A. In conclusion, the study revealed unique interactions between polyphenols and gut microbiota, deepening our understanding of the relationship between phenolic compounds like EA and the gut microbiota. These findings may contribute to the development of gut bacteria as potential probiotics for further development. KEY POINTS: • Intestinal microbiome involved in ellagic acid metabolism. • Gram-positive bacteria in the intestinal microbiome are crucial for ellagic acid metabolism. • Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium bifidum participate in ellagic acid metabolism.

Model-based learn and confirm: designing effective treatment regimens against multidrug resistant Gram-negative pathogens.

Over the last decade, there has been a growing appreciation for the use of in vitro and in vivo infection models to generate robust and informative nonclinical PK/PD data to accelerate the clinical translation of treatment regimens. The objective of this study was to develop a model-based "learn and confirm" approach to help with the design of combination regimens using in vitro infection models to optimise the clinical utility of existing antibiotics. Static concentration time-kill studies were used to evaluate the PD activity of polymyxin B (PMB) and meropenem against two carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates; BAA2146 (PMB-susceptible) and BRKP67 (PMB-resistant). A mechanism-based model (MBM) was developed to quantify the joint activity of PMB and meropenem. In silico simulations were used to predict the time-course of bacterial killing using clinically-relevant PK exposure profiles. The predictive accuracy of the model was further evaluated by validating the model predictions using a one-compartment PK/PD in vitro dynamic infection model (IVDIM). The MBM captured the reduction in bacterial burden and regrowth well in both the BAA2146 and BRKP67 isolate (R2 = 0.900 and 0.940, respectively). The bacterial killing and regrowth predicted by the MBM were consistent with observations in the IVDIM: sustained activity against BAA2146 and complete regrowth of the BRKP67 isolate. Differences observed in PD activity suggest that additional dose optimisation might be beneficial in PMB-resistant isolates. The model-based approach presented here demonstrates the utility of the MBM as a translational tool from static to dynamic in vitro systems to effectively perform model-informed drug optimisation.

Polymyxin B and ethylenediaminetetraacetic acid act synergistically against Pseudomonas aeruginosa and Staphylococcus aureus.

Polymyxin B and ethylenediaminetetraacetic acid are antimicrobials possessing antibiofilm activity. They act by displacement and chelation, respectively, of divalent cations in bacterial membranes and may therefore act synergistically when applied in combination. If so, this combination of agents may be useful for the treatment of diseases like cystic fibrosis (CF), in which biofilms are present on the respiratory epithelium. We used checkerboard assays to investigate the synergy between these agents using reference strains Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 6538 in planktonic form. We then determined the efficacy of each agent against biofilms of both species grown on 96-pin lids and proceeded to combination testing against the P. aeruginosa reference strain and 10 clinical isolates from patients with CF. Synergism was observed for planktonic forms of both species and for biofilms of P. aeruginosa. The susceptibility of biofilms of P. aeruginosa clinical isolates to these agents was variable compared to the laboratory reference strain. This combination of agents may be useful in the management of biofilm-associated conditions, particularly those amenable to topical therapies. These results provide a basis upon which the antimicrobial and antibiofilm efficacy of preparations containing these agents may be enhanced.IMPORTANCEBacteria living in biofilms produce a protective matrix which makes them difficult to kill. Patients with severe respiratory disease often have biofilms. Polymyxin B is an antibiotic commonly used in topical medications, such as eye drops and nasal sprays. Ethylenediaminetetraacetic acid (EDTA) is used widely as a preservative in medication but also has antimicrobial properties. It has been hypothesized that Polymyxin B and EDTA could have a synergistic relationship: when used in combination their antimicrobial effect is enhanced. Here, we evaluated the levels at which Polymyxin B and EDTA work together to kill common pathogens Pseudomonas aeruginosa and Staphylococcus aureus. We found that Polymyxin B and EDTA were synergistic. This synergy may be useful in the management of planktonic infection with P. aeruginosa and S. aureus, or biofilm infection with P. aeruginosa. This synergy may be beneficial in the treatment of respiratory biofilms, in which P. aeruginosa biofilms are common.

Robust and Multifunctional Therapeutic Nanoparticles against Peritonitis-Induced Sepsis.

Apart from bacterial growth and endotoxin generation, the excessive production of reactive radicals linked with sepsis also has a substantial impact on triggering an inflammatory response and further treatment failure. Hence, the rational design and fabrication of robust and multifunctional nanoparticles (NPs) present a viable means of overcoming this dilemma. In this study, we used antibiotic polymyxin B (PMB) and antioxidant natural polyphenolic protocatechualdehyde (PCA) to construct robust and multifunctional NPs for sepsis treatment, leveraging the rich chemistries of PCA. The PMB release profile from the NPs demonstrated pH-responsive behavior, which allowed the NPs to exhibit effective bacterial killing and radical scavenging properties. Data from in vitro cells stimulated with H2O2 and lipopolysaccharide (LPS) showed the multifunctionalities of NPs, including intracellular reactive oxygen species (ROS) scavenging, elimination of the bacterial toxin LPS, inhibiting macrophage M1 polarization, and anti-inflammation capabilities. Additionally, in vivo studies further demonstrated that NPs could increase the effectiveness of sepsis treatment by lowering the bacterial survival ratio, the expression of the oxidative marker malondialdehyde (MDA), and the expression of inflammatory cytokine TNF-α. Overall, this work provides ideas of using those robust and multifunctional therapeutic NPs toward enhanced sepsis therapy efficiency.

Clinical relevance, mechanisms, and evolution of polymyxin B heteroresistance carbapenem-resistant Klebsiella pneumoniae: A genomic, retrospective cohort study.

OBJECTIVES: To study the clinical relevance, mechanisms, and evolution of polymyxin B (POLB) heteroresistance (PHR) in carbapenem-resistant Klebsiella pneumoniae (CRKP), potentially leading to a significant rise in POLB full resistant (FR) CRKP. METHODS: Total of 544 CRKP isolates from 154 patients treated with POLB were categorized into PHR and POLB non-heteroresistance (NHR) groups. We performed statistical analysis to compare clinical implications and treatment responses. We employed whole-genome sequencing, bioinformatics, and PCR to study the molecular epidemiology, mechanisms behind PHR, and its evolution into FR. RESULTS: We observed a considerable proportion (118 of 154, 76.62%) of clinically undetected PHR strains before POLB exposure, with a significant subset of them (33 of 118, 27.97%) evolving into FR after POLB treatment. We investigated the clinical implications, epidemiological characteristics, mechanisms, and evolutionary patterns of PHR strains in the context of POLB treatment. About 92.86% (39 of 42) of patients had PHR isolates before FR, highlighting the clinical importance of PHR. the ST15 exhibited a notably lower PHR rate (1 of 8, 12.5% vs. 117 of 144, 81.25%; p < 0.01). The ST11 PHR strains showing significantly higher rate of mgrB mutations by endogenous insertion sequences in their resistant subpopulation (RS) compared with other STs (78 of 106, 73.58% vs. 4 of 12, 33.33%; p < 0.01). The mgrB insertional inactivation rate was lower in FR isolates than in the RS of PHR isolates (15 of 42, 35.71% vs. 84 of 112, 75%; p < 0.01), whereas the pmrAB mutation rate was higher in FR isolates than in the RS of PHR isolates (8 of 42, 19.05% vs. 2 of 112, 1.79%; p < 0.01). The evolution from PHR to FR was influenced by subpopulation dynamics and genetic adaptability because of hypermutability. DISCUSSION: We highlight significant genetic changes as the primary driver of PHR to FR in CRKP, underscoring polymyxin complexity.

Daily fluid intake as a novel covariate affecting the population pharmacokinetics of polymyxin B in patients with sepsis.

BACKGROUND: Polymyxin B dosing in patients with sepsis is difficult because pathophysiological changes and supportive therapies alter drug pharmacokinetics (PK). This study aimed to investigate the impact of fluid management and renal function on the PK of polymyxin B and to propose alternative dosing regimens. METHODS: Patients (aged ≥ 18 y) with sepsis and receiving intravenous polymyxin B for ≥ 96 h were enrolled. Blood samples were collected at steady state. Plasma concentrations were measured by liquid chromatography-tandem mass spectrometry and subjected to population PK modelling. Monte Carlo simulations were used to optimise dosage regimens. RESULTS: Eighty-three patients with a median (range) daily fluid intake of 4.2 (1.3-8.4) L and a creatinine clearance (CrCL) of 87.5 (17.3-309.7) mL/min were included. Polymyxin B PK was adequately characterised by a two-compartment model. The PK covariate analysis revealed daily fluid intake statistically significantly affected central volume of distribution and central compartment clearance (CL), and CrCL influenced CL. Simulation indicated that a decreased dosing would be suitable for patients with renal dysfunction (CrCL < 40 mL/min), and therapeutic drug monitoring is recommended to avoid exposure fluctuation when patients have fluid overload. CONCLUSIONS: Fluid management as well as renal function are essential factors affecting polymyxin B PK for patients with sepsis, which can help optimise dosage regimens.

Carbapenem-resistant Klebsiella pneumoniae bloodstream infections in haematological malignances and hematopoietic stem cell transplantation: Clinical impact of combination therapy in a 10-year Brazilian cohort.

Bacterial bloodstream infections (BSI) are a common threat among patients with haematological malignancies (HM) and hematopoietic stem cell transplant recipients (HSCT). The purpose of this research was to describe clinical and microbiological aspects of BSI caused by carbapenem-resistant Klebsiella pneumoniae (CRKp) and assess risk factors associated with 30-day mortality in a 10-year cohort of haematological patients. A total of 65 CRKp-BSI episodes occurring in HM patients and HSCT recipients and CRKp-BSI between January 2010 and December 2019 were retrospectively studied. Acute leukemias were the most frequently observed underlying disease (87.7%) and 18 patients (27.7%) received HSCT. Mucosal barrier injury in the gastrointestinal tract was the primary cause of bacteremia (86.1%). Also, 14 individuals (21.6%) had an Invasive Fungal Disease (IFD) throughout the episode. Regarding treatment, in 31 patients (47.7%) empirical therapy was deemed appropriate, whereas 33 (50.8%) patients received a combination therapy. Microbiological data revealed that the majority of isolates (53-58%) had the Polymyxin B co-resistance phenotype, while amikacin resistance was less common (16 samples, or 24.7%). The mortality rates at 14 and 30 days were 32.3% and 36.9%, respectively. In a multivariate Cox regression analysis, prompt appropriate antibiotic administration within three days was associated with a better outcome (Adjusted Hazard Ratio [aHR]: 0.33; 95% Confidence Interval [CI]: 0.14-0.76; p = 0.01), whereas hypotension at presentation (aHR: 3.88; 95% CI: 1.40-10.74; p = 0.01) and concurrent IFD (aHR: 2.97; 95% CI: 1.20-7.37; p = 0.02) were independently associated with death within 30 days. Additionally, a favorable correlation between combination therapy and overall survival was found (aHR: 0.18; 95%CI: 0.06-0.56; p = 0.002). In conclusion, 30-day mortality CRKp-BSI was elevated and most of the isolates were polymyxin B resistant. Early appropriate antimicrobial treatment and the use of combination therapy were linked to a better outcome.

Response of Paenibacillus polymyxa SC2 to the stress of polymyxin B and a key ABC transporter YwjA involved.

Polymyxins are cationic peptide antibiotics and regarded as the "final line of defense" against multidrug-resistant bacterial infections. Meanwhile, some polymyxin-resistant strains and the corresponding resistance mechanisms have also been reported. However, the response of the polymyxin-producing strain Paenibacillus polymyxa to polymyxin stress remains unclear. The purpose of this study was to investigate the stress response of gram-positive P. polymyxa SC2 to polymyxin B and to identify functional genes involved in the stress response process. Polymyxin B treatment upregulated the expression of genes related to basal metabolism, transcriptional regulation, transport, and flagella formation and increased intracellular ROS levels, flagellar motility, and biofilm formation in P. polymyxa SC2. Adding magnesium, calcium, and iron alleviated the stress of polymyxin B on P. polymyxa SC2, furthermore, magnesium and calcium could improve the resistance of P. polymyxa SC2 to polymyxin B by promoting biofilm formation. Meanwhile, functional identification of differentially expressed genes indicated that an ABC superfamily transporter YwjA was involved in the stress response to polymyxin B of P. polymyxa SC2. This study provides an important reference for improving the resistance of P. polymyxa to polymyxins and increasing the yield of polymyxins. KEY POINTS: • Phenotypic responses of P. polymyxa to polymyxin B was performed and indicated by RNA-seq • Forming biofilm was a key strategy of P. polymyxa to alleviate polymyxin stress • ABC transporter YwjA was involved in the stress resistance of P. polymyxa to polymyxin B.