Bacterial Membrane Permeability Research Articles

Rhein against Staphylococcus xylosus by interfering with respiratory metabolism and inducing oxidative stress

Currently, dairy mastitis caused by Staphylococcus xylosus poses a serious challenge for dairy farming. In this study, we explored the role and mechanism of rhein against S. xylosus with the hope of providing new research ideas to solve mastitis in dairy cows and ensure the source safety of dairy products. Through in vitro antimicrobial studies, we found that the minimum inhibitory concentration (MIC) of rhein was 64 μg/mL, and it significantly interfered with the formation of S. xylosus biofilm at sub-MIC. In experiments on mastitis in mice, rhein alleviated inflammation in mammary tissue, reduced the levels of TNF-α and IL-6, and decreased the number of S. xylosus. To explore the anti-S. xylosus mechanism of rhein, we identified the relevant proteins involved in carbon metabolism (Glycolysis/gluconeogenesis, TCA cycle, Fatty acid degradation) through proteomics. Additionally, proteins associated with the respiratory chain, oxidative stress (proteins of antioxidant and DNA repair), and nitrate respiration were also found to be upregulated. Thus, rhein may act as an antibacterial agent by interfering with the respiratory metabolism of S. xylosus and inducing the production of ROS, high levels of which alter the permeability of bacterial cell membranes and cause damage to them. We measured the concentrations of extracellular β-galactosidase and nucleic acids. Additionally, SEM observation of S. xylosus morphology showed elevated membrane permeability and damage to the cell membrane. Finally, RT-PCR experiments showed that mRNAs of key proteins of the TCA cycle (odhA, mqo) and nitrate respiration (nreB, nreC, narG) were significantly up-regulated, consistent with proteomic results. In conclusion, rhein has good anti-S. xylosus effects in vitro and in vivo, by interfering with bacterial energy metabolism, inducing ROS production, and causing cell membrane and DNA damage, which may be one of the important mechanisms of its antimicrobial activity.

Antibacterial, bacteriolytic, antibiofilm, and synergistic effects of Curcuma species ethanol extracts with antibiotic against multidrug resistant Pseudomonas aeruginosa

Introduction: Curcuma species have shown antibacterial activity against Pseudomonas aeruginosa. The current study was conducted to analyze the antibacterial activity of ethanol extracts of Curcuma species rhizomes, including Curcuma domestica, C. xanthorrhiza, C. mangga, C. zedoaria, and C. aeruginosa against multidrug-resistant Pseudomonas aeruginosa (MDR P. aeruginosa). Furthermore, the mechanism action of Curcuma species in combination with antibiotic against MDR P. aeruginosa and its chemical component were also investigated. Methods: Determination of minimum inhibitory concentration (MIC) was carried out by the microdilution method. The synergistic effects of the extract and tetracycline were determined by the checkerboard method. The effect of the combination of Curcuma species and tetracycline to prevent bacterial resistance was investigated using inhibition of biofilm formation, permeability of bacterial cell membrane, and EtBr accumulation methods. Gas chromatography-mass spectrometry (GC-MS) analysis was also performed. Results: MIC of C. domestica, C. xanthorrhiza, and C. mangga against MDR P. aeruginosa were 125, 250, and 125 µg/mL, respectively. C. xanthorrhiza ethanol extract (7.8 µg/mL) in combination with tetracycline (1.9 µg/mL) revealed a synergistic activity with Fractional Inhibitory Concentration Index (FICI) value of 0.06. The combination of C. xanthorrhiza ethanol extract and tetracycline showed inhibitory effects on biofilm formation and efflux pump of MDR P. aeruginosa. This combination also had bacteriolytic activity. GC-MS analysis led to the identification of ar-turmerone (11.63%) and xanthorrhizol (11.36%) as the major compounds. Conclusion: Combination of C. xanthorrhiza ethanol extract and tetracycline might be developed as an alternative treatment against MDR P. aeruginosa.

S-Se-S type molecule: A bactericidal promoter against H<sub>2</sub>S-induced antibiotic resistance

<p>The hydrogen sulfide (H<sub>2</sub>S)-induced defense system is a crucial bacterial pathway that leads to antibiotic resistance. Herein, a unique S-Se-S molecule, namely, 2,2’-(selenobis(sulfanediyl))diacetic acid (Se-Acid), is first reported to relieve H<sub>2</sub>S-induced antibiotic resistance by acting as a hydrogen selenide (H<sub>2</sub>Se) donor. The S-Se-S molecular structure was formed using the carboxyl terminal as an electron acceptor. After being endocytosed by cells, Se-Acid effectively released H<sub>2</sub>Se molecules by reacting with glutathione (GSH). The released H<sub>2</sub>Se increased the endocytosis of antibiotics by promoting bacterial membrane permeability. Moreover, H<sub>2</sub>Se effectively reactivated the bacterial respiratory flux by functioning as an H<sub>2</sub>S disguiser. The synergistic effect of Se-Acid and Gentamicin (Gm) on H<sub>2</sub>S-induced antibiotic-resistant MRSA was proven on MRSA<sup>S+</sup> wound infection model. Our results establish S-Se-S type molecules as potential tools for addressing the challenge of H<sub>2</sub>S-induced antibiotic resistance and reducing the risk of antibiotic resistance.</p>

PdMo bimetallene nanozymes for photothermally enhanced antibacterial therapy and accelerated wound healing.

Although antibacterial platforms involving nanozymes have been extensively investigated, there are still problems of poor reactive oxygen species generation efficiency and obstinate bacterial biofilms. Developing a nanozyme-photothermal therapy nanoplatform with superior sterilization effects and minimal side effects would be a good alternative for completely eliminating bacteria and biofilms. Herein, an ultrathin PdMo bimetallene nanozyme with a planar topology and boosted metal utilization, exhibiting excellent photothermal and peroxidase-like activity, is designed for synergistic nanozyme-photothermal sterilization applications and accelerated wound healing. The superior catalytic activity of PdMo bimetallene nanozymes could convert a biosafe concentration of hydrogen peroxide (H2O2) into large quantities of toxic hydroxyl radicals (•OH) under laser irradiation, enhancing bacterial membrane permeability and thermal sensitivity for efficient removal of bacteria and biofilms. In addition, PdMo bimetallene presents a good wound-healing ability according to the results of fibroblast proliferation and collagen deposition with minor side effects. This work would provide an innovative avenue for developing metallene-based nanozymes for biomedical applications.

Combining with domiphen bromide restores colistin efficacy against colistin-resistant Gram-negative bacteria in vitro and in vivo

Today, colistin is considered a last-resort antibiotic for treating multidrug-resistant (MDR) Gram-negative bacteria (GNB). However, the increased and improper use of colistin has led to the emergence of colistin-resistant (Col-R) GNB. Thus, it is urgent to develop new drugs and therapies in response to the ongoing emergence of colistin resistance. In this study, we investigated the antibacterial and antibiofilm activities of the quaternary ammonium compound domiphen bromide (DB) in combination with colistin against clinical Col-R GNB both in vitro and in vivo. Checkerboard assay and time-kill analysis demonstrated significant synergistic antibacterial effects of the colistin/DB combination. The synergistic antibiofilm activity was confirmed through crystal violet staining and scanning electron microscopy (SEM). Furthermore, the colistin/DB combination exhibited increased survival rates in infected larvae and reduced bacterial loads in a mouse thigh infection model. The cytotoxicity measurement and hemolysis test showed that the combination did not adversely affect cell viability at synergistic concentrations. The alkaline phosphatase (ALP) leak test and propidium iodide (PI) staining analysis further revealed that the colistin/DB combination enhanced the therapeutic effect of colistin by altering bacterial membrane permeability. The ROS assays revealed that the combination induced the accumulation of bacterial ROS, leading to bacterial death. In conclusion, our study is the first to identify DB as a colistin potentiator, effectively restoring the sensitivity of bacteria to colistin. It provides a promising alternative approach for combating Col-R GNB infections.

A new variant of the colistin resistance gene MCR-1 with co-resistance to β-lactam antibiotics reveals a potential novel antimicrobial peptide.

The emerging and global spread of a novel plasmid-mediated colistin resistance gene, mcr-1, threatens human health. Expression of the MCR-1 protein affects bacterial fitness and this cost correlates with lipid A perturbation. However, the exact molecular mechanism remains unclear. Here, we identified the MCR-1 M6 variant carrying two-point mutations that conferred co-resistance to β-lactam antibiotics. Compared to wild-type (WT) MCR-1, this variant caused severe disturbance in lipid A, resulting in up-regulation of L, D-transpeptidases (LDTs) pathway, which explains co-resistance to β-lactams. Moreover, we show that a lipid A loading pocket is localized at the linker domain of MCR-1 where these 2 mutations are located. This pocket governs colistin resistance and bacterial membrane permeability, and the mutated pocket in M6 enhances the binding affinity towards lipid A. Based on this new information, we also designed synthetic peptides derived from M6 that exhibit broad-spectrum antimicrobial activity, exposing a potential vulnerability that could be exploited for future antimicrobial drug design.

Antibacterial, antioxidant and fruit packaging ability of biochar-based silver nanoparticles-polyvinyl alcohol-chitosan composite film

Silver nanoparticles were prepared by loading Ag+ into biochar of waste barley distillers' grains shell by reduction with trisodium citrate, and this silver-loaded biochar was introduced into polyvinyl alcohol-chitosan. Various analysis with Fourier Transform Infrared spectroscopy, X-ray diffraction, Thermogravimetric analysis, and water contact angle revealed that biochar-based silver nanoparticle was incorporated into the polyvinyl alcohol-chitosan film, the biochar-based silver nanoparticles-polyvinyl alcohol-chitosan (C-Ag-loaded PVA/CS) composite film had good thermostability and hydrophobicity. Through the analysis via disk diffusion method, the composite containing 3 % of biochar-based silver nanoparticles-polyvinyl alcohol-chitosan had high antibacterial activity (inhibition zone: 18 mm against E. coli and 15 mm against S. aureus), and the bacterial membrane permeability was measured, indicating that C-Ag-loaded PVA/CS composite film could destroy the cell membrane, release intracellular substances, and have high antioxidant activity. During the storage, the weight loss rate of the biochar-based silver nanoparticles-polyvinyl alcohol-chitosan plastic wrap group was 0.14 %, and the titratable acid content only decreased by 0.061 %, which had a good effect on extending the shelf life of blueberries. The C-Ag-loaded PVA/CS composite film could also delay deterioration of blueberries and prolong storage time. Overall, this composite film had potential in food packaging and extending food shelf-life aspects.

New broad-spectrum and potent antibacterial agents with dual-targeting mechanism: Promoting FtsZ polymerization and disrupting bacterial membranes

The emergence of multidrug-resistant bacteria and the slow development of new antibacterial agents have led to a growing global health crisis. Here, we identified an antibacterial agent possessing 1-methyl-2,5-diphenylpyridin-1-ium core, MA220607, with a dual-targeting mechanism of action (MOA), which exhibited effective killing activity against both Gram-positive (MIC = 0.062–2 μg/mL) and Gram-negative bacteria (MIC = 0.5–4 μg/mL). Moreover, our study revealed that MA220607 could block the formation of bacterial biofilm, which might be the reason for low frequency of resistance. MOA studies showed that MA220607 not only promoted FtsZ protein polymerization, but also increased the permeability of bacterial membranes and altered their proton gradients. In addition, MA220607 had low hemolytic toxicity and could significantly inhibit the growth of bacteria in mice. Molecular dynamics simulations demonstrated that MA220607 could block the transition from the tense (T) to relaxed (R) state of FtsZ protein, thereby perturbing the stepping mechanism of FtsZ protein. Overall, our findings suggest that integrating the dual mechanisms targeting FtsZ protein and cell membranes of bacteria into a single scaffold represents a promising direction for the development of new antibacterial agents.

Preparation, characterization, bacteriostatic efficacy, and mechanism of zinc/selenium-loaded sodium humate.

In recent years, metal-based complexes including selenium (Se) and zinc (Zn)-containing compounds have been widely explored for their therapeutic properties due to their roles in biological processes and modulation of diverse molecular targets. Humic acid, as a metal complexing agent, is also widely used in biomedical field. In this work, three kinds of modified sodium humate (HNa), including Zn-HNA, Se-HNa, and Zn/Se-HNa, were prepared by ion exchange reaction method. The modified HNa was characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and elemental mapping. The bacteriostatic activity and mechanism of modified HNa against gram-positive and gram-negative bacteria were investigated by testing bacterial inhibition zone, minimum inhibitory concentration, and capacity to destroy integrity of the bacterial membrane, promoting ROS generation level and prevention of biofilms. FTIR results showed that HNa could combine with zinc ions and selenite ions. The main XRD peaks did not change significantly. In the modified HNa, the particle shape was irregular. Compared to HNa, Zn-HNA, and Se-HNa, Zn/Se-HNa showed the strongest bacteriostatic activity. Zn/Se-HNa exhibited high bacteriostatic activity against gram-negative bacteria (Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae) and gram-positive bacteria (Staphylococcus aureus), but showed weak antibacterial activity against another gram-positive bacteria, Bacillus subtilis. The bacteriostasis was achieved by altering the permeability of bacterial cell membranes, generating ROS, and preventing the formation of biofilms. In conclusion, Zn/Se-HNa has high bacteriostatic activity, making it a suitable alternative to antibiotics in fields like the treatment of trauma infections and animal husbandry. KEY POINTS: • Preparate and characterize zinc- and selenium-loaded sodium humate (Zn/Se-HNa). • The combination of Zn and Se enhanced the bacteriostatic activity of HNa. • Zn/Se-HNa alters the permeability of bacterial cell membranes and promotes generation of ROS.

Silver Nanoparticles Prepared Using Magnolia officinalis Are an Effective Antimicrobial Agent on Candida albicans, Escherichia coli, and Staphylococcus aureus.

Silver nanoparticles (AgNPs) prepared by plants are simple, eco-friendly, and economical. In this study, Magnolia officinalis (MO) extract was applied to synthesize MO@AgNPs. Ultraviolet-visible (UV-vis) spectrum analysis indicated a peak at 440nm. Most of the particles were spherical with sizes from 1 to approximately 60nm based on transmission electron microscopy (TEM). X-ray diffraction (XRD) patterns showed a face-centered cubic crystal structure. The zeta value of MO@AgNPs was - 36.5 ± 0.6mV, which was stable at 25°C and 4°C. Growth kinetic studies and the Kirby-Bauer diffusion method showed significant inhibitory activity on Candida albicans (ATCC 10231), Escherichia coli (ATCC BAA-2340), and Staphylococcus aureus (ATCC 25923); the minimum inhibitory concentrations (MIC) were 3, 9, and 9μg/mL, and corresponding minimum bactericidal concentrations (MBC) were 5, 11, and 9μg/mL, respectively. MO@AgNPs exhibited better antifungal activity compared to AgNPs prepared using sodium citrate. Further research revealed that MO@AgNPs increased the permeability of bacterial cell membranes. Moreover, the effect of MO@AgNPs on Candida albicans was significantly enhanced by blocking autophagy. The reactive oxygen species (ROS) induced by MO@AgNPs in Candida albicans was limited and may be related to its good antioxidant activity. Finally, MO@AgNPs have no significant cytotoxicity to the human liver LO2 cell line under 20μg/mL.

Fluoride releasing photothermal responsive TiO2 matrices for antibiosis, biosealing and bone regeneration

Peri-implantitis induced by infection leads to gingival recession, alveolar resorption and eventual dental implant failure. So, antibiosis and biosealing of abutments as well as osseointegration of roots need to be projected seriously during the whole service lifespan of dental implants. In this work, a multipurpose photothermal therapy strategy based on Si/P/F doped TiO2 matrix is proposed to address the above issues. This TiO2 matrix not only has outstanding photothermal response, but also triggers the release of F ions under near-infrared (NIR) light irradiation. Local hyperthermia assisted with the released F ions reduces adenosine triphosphate (ATP) synthesis of staphylococcus aureus (S. aureus), increases bacterial membrane permeability, and induces abundant of reactive oxygen species, resulting in the oxidation of cellular components and eventual death of bacteria. Furthermore, the synergic action of mild photothermal stimulation and Si/P/F ions of TiO2 matrix up-regulates gingival epithelial cells behavior (e.g., hemidesmosome formation) and osteoblasts response in vitro. In an infected model, this TiO2 matrix obviously eliminates bacteria, reduces inflammatory response, improves epithelial sealing and osseointegration, and reduces alveolar resorption by regulating NIR irradiation.

High-efficiency expression of a novel antimicrobial peptide I20 with superior bactericidal ability and biocompatibility in Pichia pastoris and its efficiency enhancement to aquaculture

One of the major constraints on aquaculture is the susceptibility of farmed fish to diseases or slow growing. Antimicrobial peptide I20, as a novel feed additive, is used to cope with these limiting factors. In the current study, the Pichia pastoris expression system was used to produce I20 in large quantities, thereby reducing its application cost. In terms of molecular strategy, the I20 expression codon was optimized according to the preference of P. pastoris codon. Through traditional strategy optimization (expression strain, time, methanol concentration, temperature, and pH), the expression level of I20 in shaking bottles was 21.09 ± 1.10 mg/L. Surprisingly, the expression level of I20 in a 20 L high-cell-density cultivation was 514.78 ± 22.07 mg/L after methanol induction of 72 h. Comprehensive analyses using a series of fluorescence and microscopy revealed that I20 killed bacteria by increasing bacterial membrane permeability and binding bacterial DNA. I20 effectively killed a variety of pathogenic bacteria (Aeromonas hydrophila, Aeromonas veronii, Elizabethkingia miricola, Flavobacterium columnare, Edwardsiella ictaluri, Vibrio parahaemolyticus, Streptococcus agalactiae, Nocardia seriolae, Escherichia coli, and Staphylococcus aureus). I20 was found to have excellent biocompatibility after both feeding and injection studies. Grass carp were fed I20 diet supplemented with 200 mg/Kg I20 for 8 weeks. I20 can effectively promote intestinal villus length, secretion of digestive enzymes, and expression of growth genes to improve growth performance. I20 significantly promotes immune response to reduce tissue damage and bacterial load, enhancing survival after Aeromonas hydrophila infection. In conclusion, we systematically optimized the expression of antimicrobial peptide I20 in P. pastoris, and achieved a satisfactory efficiency for producing antimicrobial peptide I20 by integrating several strategies. The results herein showed that I20-feed promoted growth performance and disease resistance in fish, suggesting a feed supplement with great potential applications for aquaculture.

Hydrophobic moment drives penetration of bacterial membranes by transmembrane peptides

With antimicrobial resistance (AMR) remaining a persistent and growing threat to human health worldwide, membrane-active peptides are gaining traction as an alternative strategy to overcome the issue. Membrane-embedded multi-drug resistant (MDR) efflux pumps are a prime target for membrane-active peptides, as they are a well-established contributor to clinically relevant AMR infections. Here, we describe a series of transmembrane peptides (TMs) to target the oligomerization motif of the AcrB component of the AcrAB-TolC MDR efflux pump from Escherichia coli. These peptides contain an N-terminal acetyl-A-(Sar)3 (sarcosine; N-methylglycine) tag and a C-terminal lysine tag-a design strategy our lab has utilized to improve the solubility and specificity of targeting for TMs previously. While these peptides have proven useful in preventing AcrB-mediated substrate efflux, the mechanisms by which these peptides associate with and penetrate the bacterial membrane remained unknown. In this study, we have shown peptide hydrophobic moment (μH)-the measure of concentrated hydrophobicity on one face of a lipopathic α-helix-drives bacterial membrane permeabilization and depolarization, likely through lateral-phase separation of negatively-charged POPG lipids and the disruption of lipid packing. Our results show peptide μH is an important consideration when designing membrane-active peptides and may be the determining factor in whether a TM will function in a permeabilizing or non-permeabilizing manner when embedded in the bacterial membrane.

Low‐Dose Cu Ions Assisted by Mild Thermal Stimulus Inducing Bacterial Cuproptosis‐Like Death for Antibiosis and Biointegration

AbstractMethicillin‐resistant Staphylococcus aureus (MRSA) is threatening human health due to its resistance to multiple antibiotics. Excessive copper (Cu) ions target the lipoylated proteins of tricarboxylic acid cycle of cancer cells, inducing proteotoxic stress and their cuproptosis death. Whether cuproptosis plays a part in killing MRSA by low‐dose supplement of Cu ions remains to be explored. Herein, Cu‐doped hydroxyapatite nanorods (PC) are prepared on polyetheretherketone (PEEK) to resist infection and improve PEEK performance in tissue integration with the assistance of near‐infrared (NIR) irradiation, and the mechanism against MRSA is elucidated. Mild photothermal stimulation increases bacterial membrane permeability, accelerating Cu ions’ intake and consequently inducing cuproptosis‐like death of MRSA. It is confirmed by aggregation of dihydrolipoamide S‐acetyltrans‐ferase (DLAT), deactivation of glutathione peroxides 4 (GPX4), and destabilization of Fe─S cluster proteins ferredoxin (FDX1) and lipoyl synthase (LIAS). Fortunately, fibroblast behaviors are upregulated on NIR‐irradiated PC. In vivo, PC with NIR irradiation exhibits outstanding MRSA elimination, reduced inflammation response, and improved biointegration. Overall, it is demonstrated that bacterial cuproptosis‐like death can be induced by Cu ions at a non‐cytotoxic dose when cooperated with mild heat stimulus, and this photothermal strategy of PC has greatly promising application in improving PEEK performance in clinic.

In Vitro, In Vivo, and In Silico Activities of Ginkgolic Acid C15:1 against Streptococcus agalactiae Clinical Isolates.

Streptococcus agalactiae is the major cause of invasive neonatal infections and is a recognized pathogen associated with various diseases in nonpregnant adults. The emergence and spread of antibiotic-resistant S. agalactiae necessitate the development of a novel antibacterial agent. Here, the potential antibacterial activities and mechanisms of ginkgolic acid C15:1 (GA (15:1)) from Ginkgo biloba against clinical S. agalactiae are characterized. The MIC50 and MIC90 values for GA (15:1) against 72 clinical S. agalactiae isolates were 6.25 and 12.5 μM, respectively. GA (15:1) showed a strong bactericidal effect against both planktonic bacteria and bacteria embedded in biofilms as well as significant effectiveness in suppressing the growth of S. agalactiae biofilms. Moreover, GA (15:1) possesses intracellular antibacterial activity and could significantly decrease the bacterial burden in the intraperitoneal infection model of S. agalactiae. Mechanistic studies showed that GA (15:1) triggers membrane damage of S. agalactiae through a unique dual-targeting mechanism of action (MoA). First, GA (15:1) targets phospholipids in the bacterial cytoplasmic membrane. Second, by using mass-spectrometry-based drug affinity responsive target stability (DARTS) and molecular docking, lipoprotein signaling peptidase II (lspA) was identified as a target protein of GA (15:1), whose role is crucial for maintaining bacterial membrane depolarization and permeabilization. Our findings suggest a potential therapeutic strategy for developing GA (15:1) to combat S. agalactiae infections.

Murepavadin induces envelope stress response and enhances the killing efficacies of β-lactam antibiotics by impairing the outer membrane integrity of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen that can cause a variety of acute and chronic infections. The bacterium is highly resistant to numerous antibiotics. Murepavadin is a peptidomimetic antibiotic that blocks the function of P. aeruginosa lipopolysaccharide (LPS) transport protein D (LptD), thus inhibiting the insertion of LPS into the outer membrane. In this study, we demonstrated that sublethal concentrations of murepavadin enhance the bacterial outer membrane permeability. Proteomic analyses revealed the alteration of protein composition in bacterial inner and outer membranes following murepavadin treatment. The antisigma factor MucA was upregulated by murepavadin. In addition, the expression of the sigma E factor gene algU and the alginate synthesis gene algD was induced by murepavadin. Deletion of the algU gene reduces bacterial survival following murepavadin treatment, indicating a role of the envelope stress response in bacterial tolerance. We further demonstrated that murepavadin enhances the bactericidal activities of β-lactam antibiotics by promoting drug influx across the outer membrane. In a mouse model of acute pneumonia, the murepavadin-ceftazidime/avibactam combination showed synergistic therapeutic effect against P. aeruginosa infection. In addition, the combination of murepavadin with ceftazidime/avibactam slowed down the resistance development. In conclusion, our results reveal the response mechanism of P. aeruginosa to murepavadin and provide a promising antibiotic combination for the treatment of P. aeruginosa infections.IMPORTANCEThe ever increasing resistance of bacteria to antibiotics poses a serious threat to global public health. Novel antibiotics and treatment strategies are urgently needed. Murepavadin is a novel antibiotic that blocks the assembly of lipopolysaccharide (LPS) into the Pseudomonas aeruginosa outer membrane by inhibiting LPS transport protein D (LptD). Here, we demonstrated that murepavadin impairs bacterial outer membrane integrity, which induces the envelope stress response. We further found that the impaired outer membrane integrity increases the influx of β-lactam antibiotics, resulting in enhanced bactericidal effects. In addition, the combination of murepavadin and a β-lactam/β-lactamase inhibitor mixture (ceftazidime/avibactam) slowed down the resistance development of P. aeruginosa. Overall, this study demonstrates the bacterial response to murepavadin and provides a new combination strategy for effective treatment.

Effect of hydrophobic property on antibacterial activities of green tea polysaccharide conjugates against Escherichia coli

We previously found that green tea polysaccharide conjugates (gTPCs) have antibacterial activity against Escherichia coli. In this study, the effect of hydrophobic property on the antibacterial activities of gTPCs was evaluated to elucidate their property-activity relationship. Three gTPCs (gTPCs-5 h, gTPCs-12 h and gTPCs-24 h) were extracted from green tea with the ethanol precipitation time of 5 h, 12 h and 24 h, respectively. These three gTPCs did not differ significantly in terms of molecular weight distribution, amino acids composition and zeta potentials. Fourier transform infrared spectroscopy results revealed that gTPCs-5 h and gTPCs-12 h processed more hydrogen bonds than gTPCs-24 h. The surface hydrophobicity and contact angle of gTPCs-5 h were larger than that of gTPCs-12 h and gTPCs-24 h. The antibacterial activity of gTPCs against E. coli decreased in the order of gTPCs-5 h > gTPCs-12 h > gTPCs-24 h. There wasn't significant difference among the zeta potentials of E. coli treated by gTPCs-5 h, gTPCs-12 h and gTPCs-24 h, but the bacterial contact angles of E. coli treated by gTPCs-5 h were higher compared with those of the other two gTPCs. Furthermore, gTPCs-5 h exhibited higher activity to decrease bacterial membrane proteins, and increase bacterial membrane permeability than the other two gTPCs. In conclusion, gTPCs with higher hydrophobicity property exhibited stronger antibacterial activity against E. coli.

A near-infrared light-triggered nano-domino system for efficient biofilm eradication: Activation of dispersing and killing functions by generating nitric oxide and peroxynitrite via cascade reactions

One of the serious threats to global public health is the bacterial biofilm, which results in numerous persistent and recurrent infections. Herein, we proposed a near-infrared (NIR) light-triggered “nano-domino” system with “dispersing and killing” functionality for biofilm eradication. The nanoplatform was fabricated by the self-assembly of chitosan conjugated with L-arginine (L-Arg, a natural nitric oxide (NO) donor) and indocyanine green (ICG, a phototherapy agent). Using an NIR irradiation “trigger”, a series of reactive oxygen species (ROS) including singlet oxygen (1O2), hydrogen peroxide (H2O2), and superoxide anions (·O2−), as well as heat were generated from ICG aggregates. Subsequently, 1O2 and H2O2 catalyzed L-Arg to produce NO, which dispersed the biofilm and reacted with ·O2− to form peroxynitrite to kill bacteria with ROS collaboratively. Meanwhile, the generated heat increased the permeability of bacterial membranes, aggravating the damage to biofilm bacteria. The experiments on biofilm eradication demonstrated that this “nano-domino” system was capable to eradicate over 99.99% of biofilms formed by Methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa under 5-min NIR irradiation. Notably, these integrated benefits allowed the system to promote the healing of MRSA biofilm-infected wounds in vivo with negligible toxicity. Overall, this reported NIR-triggered “nano-domino” system holds great promise for addressing the difficulties associated with bacterial biofilm eradication. Statement of significanceNovel agents for biofilm eradication are urgently needed due to the alarming rise in antimicrobial resistance to conventional antibiotics and the critical shortage of new drugs. In this study, we created a nano-domino system that uses near-infrared (NIR) light as a trigger to eradicate mature biofilms. In response to a short-term NIR irradiation, the proposed nanoplatform could generate nitric oxide and peroxynitrite to disperse the biofilm and kill the bacteria inside, respectively, leading to efficient eradication of Methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa biofilms with minimal cytotoxicity. The findings, therefore, indicate that this nanoplatform with enhanced antibiofilm performance might provide a reliable and promising solution to biofilm-related problems.

A quaternary ammonium salt grafted tannin-based flocculant boosts the conjugative transfer of plasmid-born antibiotic resistance genes: The nonnegligible side of their flocculation-sterilization properties

This study developed dual-function tannin-based flocculants, namely tannin-graft-acrylamide-diallyl dimethyl ammonium chloride (TGCC-A/TGCC-C), endowed with enhanced flocculation-sterilization properties. The impacts of these flocculants on proliferation and transformation of antibiotic resistance genes (ARGs) among bacteria during the flocculation-deposition process were examined. TGCC-A/TGCC-C exhibited remarkable flocculation capacities towards both Escherichia coli and Staphylococcus aureus, encompassing a logarithmic range of initial cell density (108–109 CFU/mL) and a broad pH spectrum (pH 2–11). The grafted quaternary ammonium salt groups played pivotal parts in flocculation through charge neutralization and bridging mechanisms, concurrently contributing to sterilization by disrupting cellular membranes. The correlation between flocculation and sterilization entails a sequential progression, where an excess of TGCC, initially employed for flocculation, is subsequently consumed for sterilization purposes. The frequencies of ARGs conjugative transfer were enhanced in bacterial flocs across all TGCC treatments, stemming from augmented bacterial aggregation and cell membrane permeability, elicited stress response, and up-regulated genes encoding plasmid transfer. These findings underscore the indispensable role of flocculation-sterilization effects in mediating the propagation of ARGs, consequently providing substantial support for the scientific evaluation of the environmental risks associated with flocculants in the context of ARGs dissemination during the treatment of raw water featuring high bacterial density.

A Mildly Acidic Environment Alters Pseudomonas aeruginosa Virulence and Causes Remodeling of the Bacterial Surface.

Pseudomonas aeruginosa is a versatile pathogen that resists environmental stress, such as suboptimal pH. As a result of exposure to environmental stress, P. aeruginosa shows an altered virulence-related phenotype. This study investigated the modifications that P. aeruginosa undertakes at a mildly low pH (pH 5.0) compared with the bacteria grown in a neutral medium (pH 7.2). Results indicated that in a mildly acidic environment, expression of two-component system genes (phoP/phoQ and pmrA/pmrB), lipid A remodeling genes such as arnT and pagP and virulence genes, i.e., pqsE and rhlA, were induced. Moreover, lipid A of the bacteria grown at a mildly low pH is modified by adding 4-amino-arabinose (l-Ara4N). Additionally, the production of virulence factors such as rhamnolipid, alginate, and membrane vesicles is significantly higher in a mildly low-pH environment than in a neutral medium. Interestingly, at a mildly low pH, P. aeruginosa produces a thicker biofilm with higher biofilm biomass. Furthermore, studies on inner membrane viscosity and permeability showed that a mildly low pH causes a decrease in the inner membrane permeability and increases its viscosity. Besides, despite the importance of PhoP, PhoQ, PmrA, and PmrB in Gram-negative bacteria for responding to low pH stress, we observed that the absence of each of these two-component systems does not meaningfully impact the remodeling of the P. aeruginosa envelope. Given that P. aeruginosa is likely to encounter mildly acidic environments during infection in its host, the alterations that the bacterium undertakes under such conditions must be considered in designing antibacterial strategies against P. aeruginosa. IMPORTANCE P. aeruginosa encounters environments with acidic pH when establishing infections in hosts. The bacterium develops an altered phenotype to tolerate a moderate decrease in the environmental pH. At the level of the bacterial envelope, modified lipid A composition and a reduction of the bacterial inner membrane permeability and fluidity are among the changes P. aeruginosa undergoes at a mildly low pH. Also, the bacterium is more likely to form biofilm in a mildly acidic environment. Overall, these alterations in the P. aeruginosa phenotype put obstacles in the way of antibacterial activities. Thus, considering physiological changes in the bacterium at low pH helps design and implement antimicrobial approaches against this hostile microorganism.