Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5.

Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 µg ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 µg ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 µg ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 µg ml-1) and P. aeruginosa P2307 (65.00 µg ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.

Catalase-templated nanozyme-loaded microneedles integrated with polymyxin B for immunoregulation and antibacterial activity in diabetic wounds.

Diabetic wounds are characterized by chronic trauma, with long-term non-healing attributed to persistent inflammation and recurrent bacterial infections. Exacerbation of the inflammatory response is largely due to increased levels of reactive oxygen species (ROS). In this study, catalase (CAT) was used as a biological template to synthesize nanozyme-supported natural enzymes (CAT-Mn(SH)x) using a biomimetic mineralization method. Subsequently, polymyxin B (CAT-Mn(SH)x@PMB) was immobilized on its surface through electrostatic assembly. CAT-Mn(SH)x@PMB demonstrates the ability for slow and sustained release of hydrogen sulfide (H2S). Finally, CAT-Mn(SH)x@PMB loaded microneedles (MNs) substrate were synthesized using polyvinyl alcohol (PVA) and hydroxyethyl methacrylate (HEMA), and named CAT-(MnSH)x@PMB-MNs. It exhibited enhanced enzyme and antioxidant activities, along with effective antibacterial properties. Validation findings indicate that it can up-regulate the level of M2 macrophages and reduce the level of pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Additionally, it promotes angiogenesis and rapid nerve regeneration, thereby facilitating wound healing through its dual anti-inflammatory and antibacterial effects. Hence,this study introduces a time-space tissue-penetrating and soluble microneedle patch with dual anti-inflammatory and antibacterial effects for the treatment of diabetic wounds.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

An efflux pump in genomic island GI-M202a mediates the transfer of polymyxin B resistance in Pandoraea pnomenusa M202.

BACKGROUND: Polymyxin B is considered a last-line therapeutic option against multidrug-resistant gram-negative bacteria, especially in COVID-19 coinfections or other serious infections. However, the risk of antimicrobial resistance and its spread to the environment should be brought to the forefront. METHODS: Pandoraea pnomenusa M202 was isolated under selection with 8 mg/L polymyxin B from hospital sewage and then was sequenced by the PacBio RS II and Illumina HiSeq 4000 platforms. Mating experiments were performed to evaluate the transfer of the major facilitator superfamily (MFS) transporter in genomic islands (GIs) to Escherichia coli 25DN. The recombinant E. coli strain Mrc-3 harboring MFS transporter encoding gene FKQ53_RS21695 was also constructed. The influence of efflux pump inhibitors (EPIs) on MICs was determined. The mechanism of polymyxin B excretion mediated by FKQ53_RS21695 was investigated by Discovery Studio 2.0 based on homology modeling. RESULTS: The MIC of polymyxin B for the multidrug-resistant bacterial strain P. pnomenusa M202, isolated from hospital sewage, was 96 mg/L. GI-M202a, harboring an MFS transporter-encoding gene and conjugative transfer protein-encoding genes of the type IV secretion system, was identified in P. pnomenusa M202. The mating experiment between M202 and E. coli 25DN reflected the transferability of polymyxin B resistance via GI-M202a. EPI and heterogeneous expression assays also suggested that the MFS transporter gene FKQ53_RS21695 in GI-M202a was responsible for polymyxin B resistance. Molecular docking revealed that the polymyxin B fatty acyl group inserts into the hydrophobic region of the transmembrane core with Pi-alkyl and unfavorable bump interactions, and then polymyxin B rotates around Tyr43 to externally display the peptide group during the efflux process, accompanied by an inward-to-outward conformational change in the MFS transporter. Additionally, verapamil and CCCP exhibited significant inhibition via competition for binding sites. CONCLUSIONS: These findings demonstrated that GI-M202a along with the MFS transporter FKQ53_RS21695 in P. pnomenusa M202 could mediate the transmission of polymyxin B resistance.

Comparative evaluation of colistin broth disc elution (CBDE) and broth microdilution (BMD) in clinical isolates of Pseudomonas aeruginosa with special reference to heteroresistance.

PURPOSE: Antimicrobial resistance (AMR) in Pseudomonas aeruginosa has been ever-increasing. Among other reasons, colistin resistance might be attributed to limited routine testing by approved methods. Both broth microdilution (BMD) and colistin broth disc elution (CBDE) methods have been advocated, with limited data on the performance of these methods in the Indian settings. This prospective study was conducted to determine colistin resistance in P. aeruginosa, compare the BMD and CBDE methods with special reference to heteroresistance. MATERIALS AND METHODS: A total of 100 isolates of P. aeruginosa from admitted patients were included. Antimicrobial susceptibility testing was done against standard antibiotics by disc diffusion test. Minimum inhibitory concentration (MIC) against polymyxins was studied by BMD and CBDE (for colistin only). Heteroresistance to colistin was studied by population analysis profile (PAP). CBDE and BMD were compared by performance calculations. Discrepancy in results were analyzed based on heteroresistance. RESULTS: Majority of the P. aeruginosa isolates were from pus samples (62, 62 â€‹%). Disc diffusion method revealed maximum susceptibility towards aztreonam (74, 74 â€‹%) followed by meropenem (68, 68 â€‹%) and piperacillin-tazobactam (65, 65 â€‹%). Polymyxin B resistance was seen in 6 â€‹% (6) while colistin resistance was seen in 9 â€‹% (9) isolates by BMD. CBDE revealed 8 â€‹% (8) resistance to colistin, having 97 â€‹% essential agreement and 95 â€‹% categorical agreement with BMD. Further, by PAP analysis, 9 isolates were resistant to colistin which included 9 resistant isolates by BMD. On discrepancy analysis, 1 isolate was found to be heteroresistant to colistin. No heteroresistance was seen in the isolates that were susceptible by all the methods. CONCLUSIONS: Heteroresistance to colistin in P. aeruginosa accounted for the discrepancy in results where CBDE method failed to detect heteroresistant isolate. As heteroresistance is a least studied phenotype, it's exact prevalence should be studied so that challenges in susceptibility testing could be addressed.

Polysaccharide-Targeting Lipid Nanoparticles to Kill Gram-Negative Bacteria.

The rapid increase and spread of Gram-negative bacteria resistant to many or all existing treatments threaten a return to the preantibiotic era. The presence of bacterial polysaccharides that impede the penetration of many antimicrobials and protect them from the innate immune system contributes to resistance and pathogenicity. No currently approved antibiotics target the polysaccharide regions of microbes. Here, describe monolaurin-based niosomes, the first lipid nanoparticles that can eliminate bacterial polysaccharides from hypervirulent Klebsiella pneumoniae, are described. Their combination with polymyxin B shows no cytotoxicity in vitro and is highly effective in combating K. pneumoniae infection in vivo. Comprehensive mechanistic studies have revealed that antimicrobial activity proceeds via a multimodal mechanism. Initially, lipid nanoparticles disrupt polysaccharides, then outer and inner membranes are destabilized and destroyed by polymyxin B, resulting in synergistic cell lysis. This novel lipidic nanoparticle system shows tremendous promise as a highly effective antimicrobial treatment targeting multidrug-resistant Gram-negative pathogens.

Polymyxin B-targeted liposomal photosensitizer cures MDR A. baumannii burn infections and accelerates wound healing via M1/M2 macrophage polarization.

Multidrug-resistant (MDR) Acinetobacter baumannii infections pose a significant challenge in burn wound management, necessitating the development of innovative therapeutic strategies. In this work, we introduced a novel polymyxin B (PMB)-targeted liposomal photosensitizer, HMME@Lipo-PMB, for precise and potent antimicrobial photodynamic therapy (aPDT) against burn infections induced by MDR A. baumanni. HMME@Lipo-PMB-mediated aPDT exhibited enhanced antibacterial efficacy by specifically targeting and disrupting bacterial cell membranes, and generating increased intracellular ROS. Remarkably, even at low concentrations, this targeted approach significantly reduced bacterial viability in vitro and completely eradicated burn infections induced by MDR A. baumannii in vivo. Additionally, HMME@Lipo-PMB-mediated aPDT facilitated burn infection wound healing by modulating M1/M2 macrophage polarization. It also effectively promoted acute inflammation in the early stage, while attenuated chronic inflammation in the later stage of wound healing. This dynamic modulation promoted the formation of granulation tissue, angiogenesis, and collagen regeneration. These findings demonstrate the tremendous potential of HMME@Lipo-PMB-mediated aPDT as a promising alternative for the treatment of burn infections caused by MDR A. baumannii.