Silver nanoparticles biosynthesis using mixture of Lactobacillus sp. and Bacillus sp. growth and their antibacterial activity.

The biosynthesis of nanoparticles offers numerous advantages, including ease of production, cost-effectiveness, and environmental friendliness. In our research, we focused on the bioformation of silver nanoparticles (AgNPs) using a combination of Lactobacillus sp. and Bacillus sp. growth. These AgNPs were then evaluated for their biological activities against multidrug-resistant bacteria. Our study involved the isolation of Bacillus sp. from soil samples and Lactobacillus sp. from raw milk in Dhamar Governorate, Yemen. The synthesized AgNPs were characterized using various techniques such as UV-visible spectroscopy, X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). The antibacterial properties of the AgNPs were assessed using the modified Kirby Bauer disk diffusion method against multidrug-resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa. Our results demonstrated that the use of a bacterial mixture for biosynthesis led to faster and more effective production of AgNPs compared to using a single bacterium. The UV-visible spectra showed characteristic peaks indicative of silver nanoparticles, while XRD analysis confirmed the crystalline nature of the synthesized particles. FTIR results suggested the presence of capping proteins that contribute to the synthesis and stability of AgNPs. Furthermore, TEM images revealed the size and morphology of the AgNPs, which exhibited spherical shapes with sizes ranging from 4.65 to 22.8 nm. Notably, the antibacterial activity of the AgNPs was found to be more pronounced against Staphylococcus aureus than Pseudomonas aeruginosa, indicating the potential of these nanoparticles as effective antimicrobial agents. Overall, our study highlights the promising antibacterial properties of AgNPs synthesized by a mixture of Lactobacillus sp. and Bacillus sp. growth. Further research is warranted to explore the potential of utilizing different bacterial combinations for enhanced nanoparticle synthesis.

Antibacterial activity of lysozyme after association with carboxymethyl konjac glucomannan.

The unique high isoelectric point of lysozyme (LYZ) restricts its application in composite antibacterial coating due to the unfavorable liability to electrostatic interaction with other components. In this work, the antibacterial activity of a dispersible LYZ-carboxymethyl konjac glucomannan (CMKGM) polyelectrolyte complex was evaluated. Kinetic analysis revealed that, compared with free LYZ, the complexed enzyme exhibited decreased affinity (Km) but markedly increased Vmax against Micrococcus lysodeikticus, and QCM and dynamic light scattering analysis confirmed that the complex could bind with the substrate but in a much lower ratio. The complexation with CMKGM did not alter the antibacterial spectrum of LYZ, and the complex exerted antibacterial function by delaying the logarithmic growth phase and impairing the cell integrity of Staphylococcus aureus. Since the LYZ-CMKGM complex is dispersible in water and could be assembled easily, it has great potential as an edible coating in food preservation.

The Staphylococcus aureus regulatory program in a human skin-like environment.

Staphylococcus aureus is a Gram-positive pathogen responsible for the majority of skin and soft tissue infections (SSTIs). S. aureus colonizes the anterior nares of approximately 20%-30% of the population and transiently colonizes the skin, thereby increasing the risk of developing SSTIs and more serious infections. Current laboratory models that mimic the skin surface environment are expensive, require substantial infrastructure, and limit the scope of bacterial physiology studies under human skin conditions. To overcome these limitations, we developed a cost-effective, open-source, chemically defined media recipe termed skin-like medium (SLM) that incorporates key aspects of the human skin surface environment and supports growth of several staphylococcal species. We utilized SLM to investigate the transcriptional response of methicillin-resistant Staphylococcus aureus (MRSA) following growth in SLM compared to a commonly used laboratory media. Through RNA-seq analysis, we observed the upregulation of several virulence factors, including genes encoding functions involved in adhesion, proteolysis, and cytotoxicity. To further explore these findings, we conducted quantitative reverse transcription-PCR (qRT-PCR) experiments to determine the influence of media composition, pH, and temperature on the transcriptional response of key factors involved in adhesion and virulence. We also demonstrated that MRSA primed in SLM adhered better to human corneocytes and demonstrated adhesin-specific phenotypes that previously required genetic manipulation. This improved adherence to corneocytes was dependent on both acidic pH and growth in SLM. These results support the potential utility of SLM as an in vitro model for assessing staphylococcal physiology and metabolism on human skin. IMPORTANCE: Staphylococcus aureus is the major cause of skin diseases, and its increased prevalence in skin colonization and infections present a need to understand its physiology in this environment. The work presented here outlines S. aureus upregulation of colonization and virulence factors using a newly developed medium that strives to replicate the human skin surface environment and demonstrates roles for adhesins clumping factor A (ClfA), serine-rich repeat glycoprotein adhesin (SraP), and the fibronectin binding proteins (Fnbps) in human corneocyte adherence.

In Vitro and In Vivo Testing of Microbe Growth on Antimicrobial Nursing Scrubs.

Around 5% to 10% of hospitalized patients develop a hospital-acquired infection (HAI). Scrubs are a potential vector of HAIs. To compare the antimicrobial characteristics of scrubs with and without an antimicrobial fabric coating, as tested in the laboratory (in vitro) and hospital (in vivo) environments. Two protocols were conducted to address the purpose. The in vitro protocol was a laboratory study that involved observing the microbe growth after inoculating coated and uncoated scrub fabric swatches with S. aureus and then processing them in moist and dry environments. The in vivo protocol was a clinical trial that measured microbe growth on coated and uncoated scrubs prior to and following nursing staff completing a 12-hr shift on an acute care unit, as measured by colony forming units (CFUs). For high-humidity environments, the in vitro study indicated that swatches treated with an antimicrobial coating exhibited minimal microbe growth, while untreated swatches exhibited significant microbe growth. For low-humidity environments, coated and uncoated swatches were all found to exhibit minimal microbe growth. In the in vivo study, the CFUs increased on scrubs worn by nurses over a 12-hr shift with no significant difference in CFUs for coated and uncoated scrubs. For bacteria in a warm and moist environment, the antimicrobial coating was found to be important for inhibiting growth. For bacteria in a warm and dry environment, both coated and uncoated fabrics performed similarly as measured at 24 hr, with minimal bacterial growth observed. In a hospital environment, microbe growth was observed, but no significant difference was detected when comparing coated and uncoated scrubs. This may have been due to the short time between exposure and culturing the scrubs for analysis immediately at the end of the shift not allowing for enough time to kill or inhibit growth. Contact time between the bacteria and scrub fabric (coated or uncoated) in the in vivo study more directly correlated with the 0-hr observations for the in vitro study, suggesting that the ineffectiveness of the treated scrubs in the clinical results may be due in part to short residence times before collection.

Application of interaction models in predicting the simultaneous growth of Staphylococcus aureus and different concentrations of background microbiota in Chinese-style braised beef.

This study aimed to investigate the growth kinetics of S. aureus and different concentrations of background microbiota in Chinese-style braised beef (CBB). A one-step analysis method was applied to develop predictive model to describe the simultaneous growth and interaction of S. aureus with different concentrations of background microbiota in CBB. The results show that a one-step method successfully models the growth of S. aureus and background microbiota in CBB and the competing interactions between the two. In sterile CBB, the estimated minimum growth temperatures (Tmin,S) and the maximum growth concentrations (Ymax,S) were 8.76 °C and 9.58 log CFU/g for S. aureus. Under competition, the growth of background microbiota was not affected by S. aureus, the estimated Tmin,B and Ymax,B was 4.46 °C and 9.94 log CFU/g. The background microbiota in CBB did not affect the growth rate of S. aureus (α1 = 1.04), but had an inhibitory effect on the number of S. aureus (α2 = 0.69) at the later growth stage. The Root Mean Square Error (RMSE) of the modeling data was 0.34 log CFU/g, with 85.5% of the residual errors within ±0.5 log CFU/g of experimental observations. The one-step analysis and dynamic temperatures (8 °C-32 °C) verification indicated that the RMSE of prediction was <0.5 log CFU/g for both S. aureus and background microbiota. This study demonstrates that microbial interaction models are a useful and promising tool for predicting and evaluating the spatiotemporal population dynamics of S. aureus and background microbiota in CBB products.

The YAP1-TEAD axis promotes autophagy against intracellular Staphylococcus aureus in vitro.

Previously considered as an exclusive extracellular bacterium, Staphylococcus aureus has been shown to be able to invade many cells in vitro and in humans. Once inside the host cell, both cytosolic and endosome-associated S. aureus strongly induce macroautophagy/autophagy. Whether autophagy fosters S. aureus intracellular survival or clearance remains unclear. The YAP1-TEAD axis regulates the expression of target genes controlling the cell fate (e.g., proliferation, migration, cell cycle …). Growing evidence indicates that YAP1-TEAD also regulates autophagy and lysosomal pathways. Recently we showed that the YAP1-TEAD axis promotes autophagy and lysosome biogenesis to restrict S. aureus intracellular replication. We also discovered that the C3 exoenzyme-like EDIN-B toxin produced by the pathogenic S. aureus ST80 strain inhibits YAP1 nuclear translocation resulting in a strong increase of intracellular S. aureus burden.

Chemical Composition and In Vitro Antioxidant and Antimicrobial Activities of the Marine Cyanolichen Lichina pygmaea Volatile Compounds.

Volatile compounds from the marine cyanolichen Lichina pygmaea, collected from the Moroccan Atlantic coast, were extracted by hydrodistillation and their putative chemical composition was investigated by gas chromatography coupled to mass spectrometry (GC/MS). Based on the obtained results, Lichina pygmaea volatile compounds (LPVCs) were mainly dominated by sesquiterpenes compounds, where γ-himachalene, ß-himachalene, (2E,4E)-2,4 decadienal and α-himachalene were assumed to be the most abundant constituents, with percentage of 37.51%, 11.71%, 8.59% and 7.62%, respectively. LPVCs depicted significant antimicrobial activity against all tested strains (Staphylococcus aureus CCMM B3, Pseudomonas aeruginosa DSM 50090, Escherichia coli ATCC 8739 and Candida albicans CCMM-L4) with minimum inhibitory concentration (MIC) values within the range of 1.69-13.5 mg/mL. Moreover, this LPVC showed interesting scavenging effects on the 2,2-diphenyl-1-picrylhydrazyl radical with an IC50 of 0.21 mg/mL. LPVCs could be an approving resource with moderate antimicrobial potential and interesting antioxidant activity for cosmetics and pharmaceutical applications.

Leucine-Rich, Potent Anti-Bacterial Protein against Vibrio cholerae, Staphylococcus aureus from Solanum trilobatum Leaves.

A 24 kDa leucine-rich protein from ion exchange fractions of Solanum trilobatum, which has anti-bacterial activity against both the Gram-negative Vibrio cholerae and Gram-positive Staphylococcus aureus bacteria has been purified. In this study, mass spectrometry analysis identified the leucine richness and found a luminal binding protein (LBP). Circular dichroism suggests that the protein was predominantly composed of α- helical contents of its secondary structure. Scanning electron microscopy visualized the characteristics and morphological and structural changes in LBP-treated bacterium. Further in vitro studies confirmed that mannose-, trehalose- and raffinose-treated LBP completely inhibited the hemagglutination ability towards rat red blood cells. Altogether, these studies suggest that LBP could bind to sugar moieties which are abundantly distributed on bacterial surface which are essential for maintaining the structural integrity of bacteria. Considering that Solanum triolbatum is a well-known medicinal and edible plant, in order to shed light on its ancient usage in this work, an efficient anti-microbial protein was isolated, characterized and its in vitro functional study against human pathogenic bacteria was evaluated.

Multilayered Curcumin-Loaded Hydrogel Microcarriers with Antimicrobial Function.

The design of multifunctional microcarriers has attracted significant attention because they combine various functions within a single system. In this study, we developed a set of multilayered hydrogel microcarriers, which were first loaded with chemotherapeutic curcumin (CUR), then, using the layer-by-layer (LbL) technique, coated through a polyelectrolyte shell consisting of chitosan (CHIT) or poly(allylamine hydrochloride) (PAH). As an outer layer with antimicrobial function, newly synthesised alkylene quaternary ammonium salt functionalised polyelectrolytes (A-QAS-PEs) were applied. For this purpose, poly(acrylic acid) (PAA) was decorated with different hydrophobic side chains (n-hexane and n-dodecane side entities) and different degrees of substitution (m) of quaternary ammonium groups (abbreviated as PAA-C(O)O-(CH2)n-N+(CH3)3(m); n = 6, 12; m = 8-14%). The grafting approach of PAA with the alkylene quaternary ammonium salt moiety was performed under mild reaction conditions using Steglich esterification followed by quaternisation. The structure of antimicrobial decorated PAA was confirmed by 1H NMR and FTIR, and the mean diameter of all multifunctional microparticles was characterised by SEM. The viscoelastic properties of the functional layers were studied using quartz crystal microbalance with a dissipation (QCM-D). The release of CUR from the microcarriers was described using a hybrid model, i.e., a combination of first-order kinetics and the Korsmeyer-Peppas model. The antimicrobial activity of functionalised PAA and multilayered CUR-loaded hydrogel microcarriers with quaternary ammonium function was assessed against Staphylococcus aureus and Serratia marcescens by the agar diffusion assay method. Only a limited inhibition zone of PAA was observed, but in the case of both antimicrobial decorated PAA and the corresponding multilayered nanocarriers, the inhibitory activity increase was achieved against both strains of bacteria.

Host Cell Oxidative Stress Induces Dormant Staphylococcus aureus Persisters.

Persisters are transiently nongrowing and antibiotic-tolerant phenotypic variants identified in major human pathogens, including intracellular Staphylococcus aureus. Due to their capacity to regrow once the environmental stress is relieved and to promote resistance, persisters possibly contribute to therapeutic failures. While persistence and its related quiescence have been mostly studied under starvation, little is known within host cell environments. Here, we examined how the level of reactive oxygen species (ROS) in different host cells affects dormancy depth of intracellular S. aureus. Using single-cell approaches, we found that host ROS induce variable dormant states in S. aureus persisters, displaying heterogeneous and increased lag times for resuscitation in liquid medium. Dormant persisters displayed decreased translation and energy metabolism, but remained infectious, exiting from dormancy and resuming growth when reinoculated in low-oxidative-stress cells. In high-oxidative-stress cells, ROS-induced ATP depletion was associated with the formation of visible dark foci similar to those induced by the protein aggregation inducer CCCP (carbonyl cyanide m-chlorophenylhydrazone) and with the recruitment of the DnaK-ClpB chaperone system involved in the clearance of protein aggregates. ATP depletion led to higher fractions of dormant persisters than ROS, due to a counterbalancing effect of ROS-induced translational repression, suggesting a pivotal role of translation in the dormant phenotype. Consistently, protein synthesis inhibition limited dormancy to levels similar to those observed in low-oxidative-stress cells. This study supports the hypothesis that intracellular S. aureus persisters can reach heterogeneous dormancy depths and highlights the link between ROS, ATP depletion, dark focus formation, and subsequent dormancy state. IMPORTANCE By their capacity to survive to antibiotic pressure and to regrow and give rise to a susceptible population once this pressure is relieved, intracellular persisters of S. aureus may contribute to explain therapeutic failures and recurrent infections. Here, we show that the level of dormancy and the subsequent capacity to resuscitate from this resting state are dependent on the level of oxidative stress in the host cells where bacteria survive. This observation nourishes the debate as whether the most appropriate strategy to cope with S. aureus intracellular infections would consist of trying to push persisters to a deep dormancy state from which wakening is improbable or, on the contrary, to prevent ROS-induced dormancy and force bacteria to maintain regular metabolism in order to restore their responsiveness to antibiotics. Importantly also, our data highlight the interest in single-cell analyses with conventional enumeration of CFU to quantify persisters and study host-pathogen interactions.

Comprehensive properties of photodynamic antibacterial film based on κ-Carrageenan and curcumin-ß-cyclodextrin complex.

In this study, a biodegradable photodynamic antibacterial film (Car-Cur) was prepared using casting method with κ-Carrageenan (κ-Car) as film-forming substrate and curcumin-ß-cyclodextrin (Cur-ß-CD) complex as photosensitizer. The comprehensive performance of this Car-Cur film was investigated. The obtained results showed that the concentration of Cur-ß-CD was an important factor determining the properties of film including tensile strength (TS) elongation at break (EB), water vapor permeability (WVP), water content (WC) and thermal stability. When the concentration of Cur-ß-CD is 1%, the film demonstrated the maximum TS and EB, increased thermal stability, with desirable WVP and WC. Furthermore, this film also showed good photodynamic antibacterial potential against Staphylococcus aureus and Escherichia coli upon irradiation of blue LED light. Moreover, the film can be degraded in the soil in one week. In conclusion, our results suggested Car-Cur photodynamic film could be developed as biodegradable antimicrobial packaging material for food preservation.

Development of a novel reinforced scaffold based on chitosan/cellulose nanocrystals/halloysite nanotubes for curcumin delivery.

Chitosan, cellulose nanocrystals, and halloysite nanotubes in the presence of calcium cations were used to fabricate a three-dimensional nanocomposite scaffold. The FTIR and XRD analyses revealed that formation of the network and incorporation of halloysite nanotubes into it were successful. FESEM images showed that the addition of higher amounts of halloysite nanotubes into the scaffold's matrix leads to more and smaller pores. The addition of halloysite nanotubes enhanced the thermal stability, mechanical characteristics, water uptake, and degradation rate of the nanocomposite scaffold. The nanocomposite scaffold represented good biomineralization, great cell proliferation, and acceptable cell attachment. Furthermore, the capability of the nanocomposite scaffold for curcumin delivery was approved through cell proliferation, cumulative release, and antibacterial studies. Cell proliferation of the nanocomposite with 10 wt% curcumin-loaded halloysite nanotubes reached around 175% after 72 h. Considering the results, the prepared nanocomposite scaffold holds great potential for being used in bone tissue engineering applications.

Chitosan and polyhexamethylene guanidine dual-functionalized cotton gauze as a versatile bandage for the management of chronic wounds.

Development of versatile medical dressing with good immediate and long-lasting antibacterial, hygroscopic and moisturizing abilities is of great significance for management of chronic wounds. Cotton gauze (CG) can protect wounds and promote scabbing, but can cause wound dehydration and loss of biologically active substances, thereby greatly delays wound healing. Herein, a bi-functional CG dressing (CPCG) was developed by chemically grafting polyhexamethylene guanidine (PHMG) and physically adsorbing chitosan (CS) onto the CG surface. Due to the powerful microbicidal activity of PHMG, CPCG exhibited excellent immediate and long-lasting antibacterial activity against gram-positive and gram-negative bacteria. Moreover, the abundant hydroxyl and amino groups in CS endowed CPCG with good biocompatibility, moisture absorption, moisturizing and cell scratch healing performances. Importantly, CPCG can be easily fabricated into a bandage to conveniently manage infected full-skin wounds. Together, this study suggests that CPCG is a versatile wound dressing, having enormous application potential for management chronic wounds.

Electrospun ZnO-loaded chitosan/PCL bilayer membranes with spatially designed structure for accelerated wound healing.

A multifunctional bilayer membrane with electrospinning chitosan (CS) and active ZnO nanoparticles was designed. The outer-layer was constructed with ZnO-encapsulated poly(ε-caprolactone) (PCL) ultrafine fibers in a randomly-orientated structure, which could impart the bilayer membrane with great antibacterial activity. The inner-layer was composed with CS fibers with aligned core-shell structure, which could provide anti-inflammatory and effective cell contact guide function. The structure, morphology and crystallization behavior of the bilayer membrane was investigated by FTIR, TEM, SEM and XRD. Importantly, the bi-layered CS/PCL electrospun membrane loading 1.2 wt% ZnO nanoparticles exhibited an enhanced tensile strength and an obvious inhibitory zone against E. coli and S. aureus, and also presented a non-cytotoxic behavior to fibroblasts. Moreover, the as-prepared bi-layered membrane enabled the maintenance of high bioavailability of ZnO nanoparticles and synchronization with the aligned structural feature of CS fibers, which alleviated inflammation, stimulated cellular migration and re-epithelialization in vivo.

Deep Functional Profiling of Wild Animal Microbiomes Reveals Probiotic Bacillus pumilus Strains with a Common Biosynthetic Fingerprint.

The biodiversity of microorganisms is maintained by intricate nets of interactions between competing species. Impaired functionality of human microbiomes correlates with their reduced biodiversity originating from aseptic environmental conditions and antibiotic use. Microbiomes of wild animals are free of these selective pressures. Microbiota provides a protecting shield from invasion by pathogens in the wild, outcompeting their growth in specific ecological niches. We applied ultrahigh-throughput microfluidic technologies for functional profiling of microbiomes of wild animals, including the skin beetle, Siberian lynx, common raccoon dog, and East Siberian brown bear. Single-cell screening of the most efficient killers of the common human pathogen Staphylococcus aureus resulted in repeated isolation of Bacillus pumilus strains. While isolated strains had different phenotypes, all of them displayed a similar set of biosynthetic gene clusters (BGCs) encoding antibiotic amicoumacin, siderophore bacillibactin, and putative analogs of antimicrobials including bacilysin, surfactin, desferrioxamine, and class IId cyclical bacteriocin. Amicoumacin A (Ami) was identified as a major antibacterial metabolite of these strains mediating their antagonistic activity. Genome mining indicates that Ami BGCs with this architecture subdivide into three distinct families, characteristic of the B. pumilus, B. subtilis, and Paenibacillus species. While Ami itself displays mediocre activity against the majority of Gram-negative bacteria, isolated B. pumilus strains efficiently inhibit the growth of both Gram-positive S. aureus and Gram-negative E. coli in coculture. We believe that the expanded antagonistic activity spectrum of Ami-producing B. pumilus can be attributed to the metabolomic profile predetermined by their biosynthetic fingerprint. Ultrahigh-throughput isolation of natural probiotic strains from wild animal microbiomes, as well as their metabolic reprogramming, opens up a new avenue for pathogen control and microbiome remodeling in the food industry, agriculture, and healthcare.

Comparative Assessment of Bacteriophage and Antibiotic Activity against Multidrug-Resistant Staphylococcus aureus Biofilms.

Problems connected with biofilm-related infections and antibiotic resistance necessitate the investigation and development of novel treatment strategies. Given their unique characteristics, one of the most promising alternatives to conventional antibiotics are bacteriophages. In the in vitro and in vivo larva model study, we demonstrate that phages vB_SauM-A, vB_SauM-C, and vB_SauM-D are effective antibiofilm agents. The exposure of biofilm to phages vB_SauM-A and vB_SauM-D led to 2-3 log reductions in the colony-forming unit number in most of the multidrug-resistant S. aureus strains. It was found that phage application reduced the formed biofilms independently of the used titer. Moreover, the study demonstrated that bacteriophages are more efficient in biofilm biomass removal and reduction in staphylococci count when compared to the antibiotics used. The scanning electron microscopy analysis results are in line with colony forming unit (CFU) counting but not entirely consistent with crystal violet (CV) staining. Additionally, phages vB_SauM-A, vB_SauM-C, and vB_SauM-D can significantly increase the survival rate and extend the survival time of Galleria mellonella larvae.

Synergistic Effect of Co-Delivering Ciprofloxacin and Tetracycline Hydrochloride for Promoted Wound Healing by Utilizing Coaxial PCL/Gelatin Nanofiber Membrane.

Combining multiple drugs or biologically active substances for wound healing could not only resist the formation of multidrug resistant pathogens, but also achieve better therapeutic effects. Herein, the hydrophobic fluoroquinolone antibiotic ciprofloxacin (CIP) and the hydrophilic broad-spectrum antibiotic tetracycline hydrochloride (TH) were introduced into the coaxial polycaprolactone/gelatin (PCL/GEL) nanofiber mat with CIP loaded into the PCL (core layer) and TH loaded into the GEL (shell layer), developing antibacterial wound dressing with the co-delivering of the two antibiotics (PCL-CIP/GEL-TH). The nanostructure, physical properties, drug release, antibacterial property, and in vitro cytotoxicity were investigated accordingly. The results revealed that the CIP shows a long-lasting release of five days, reaching the releasing rate of 80.71%, while the cumulative drug release of TH reached 83.51% with a rapid release behavior of 12 h. The in vitro antibacterial activity demonstrated that the coaxial nanofiber mesh possesses strong antibacterial activity against E. coli and S. aureus. In addition, the coaxial mats showed superior biocompatibility toward human skin fibroblast cells (hSFCs). This study indicates that the developed PCL-CIP/GEL-TH nanofiber membranes hold enormous potential as wound dressing materials.

Exposure to b-LED Light While Exerting Antimicrobial Activity on Gram-Negative and -Positive Bacteria Promotes Transient EMT-like Changes and Growth Arrest in Keratinocytes.

While blue LED (b-LED) light is increasingly being studied for its cytotoxic activity towards bacteria in therapy of skin-related infections, its effects on eukaryotic cells plasticity are less well characterized. Moreover, since different protocols are often used, comparing the effect of b-LED towards both microorganisms and epithelial surfaces may be difficult. The aim of this study was to analyze, in the same experimental setting, both the bactericidal activity and the effects on human keratinocytes. Exposure to b-LED induced an intense cytocidal activity against Gram-positive (i.e, Staphylococcus aureus) and Gram-negative (i.e., Pseudomonas aeruginosa) bacteria associated with catheter-related infections. Treatment with b-LED of a human keratinocyte cell line induced a transient cell cycle arrest. At the molecular level, exposure to b-LED induced a transient downregulation of Cyclin D1 and an upregulation of p21, but not signs of apoptosis. Interestingly, a transient induction of phosphor-histone γ-H2Ax, which is associated with genotoxic damages, was observed. At the same time, keratinocytes underwent a transient epithelial to mesenchymal transition (EMT)-like phenotype, characterized by E-cadherin downregulation and SNAIL/SLUG induction. As a functional readout of EMT induction, a scratch assay was performed. Surprisingly, b-LED treatment provoked a delay in the scratch closure. In conclusion, we demonstrated that b-LED microbicidal activity is associated with complex responses in keratinocytes that certainly deserve further analysis.

Recombinant HNP-1 Produced by Escherichia coli Triggers Bacterial Apoptosis and Exhibits Antibacterial Activity against Drug-Resistant Bacteria.

Human neutrophil peptide-1 (HNP-1) is a promising antibiotic candidate, but its clinical applications have been hampered by challenges during mass production and an inadequate understanding of its bactericidal mechanisms. In this study, we demonstrated that Escherichia coli expressing full-length preproHNP-1 secretes a soluble form of HNP-1, which can be recovered from the total cell lysate after isopropyl thio-ß-d-galactoside (IPTG) induction and ultrafiltration. Label-free quantitative proteomics and co-immunoprecipitation experiments revealed that HNP-1 induces cell apoptosis in bacteria by causing DNA and membrane damage. Notably, we found that HNP-1 disrupts the DNA damage response pathway by interfering with the binding of RecA to single-stranded DNA (ssDNA). Further experiments demonstrated that HNP-1 encapsulated in liposomes inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA) and meropenem-resistant Pseudomonas aeruginosa (MRPA). These results indicated that recombinant protein expression may be a simple and cost-effective solution to produce HNP-1 and that RecA inhibition via HNP-1 may serve as an alternative strategy to counteract antibiotic resistance. IMPORTANCE Human neutrophil peptide-1 (HNP-1) is a promising antibiotic candidate, but its clinical application has been hampered by the difficulty of mass production and an inadequate understanding of its bactericidal mechanisms. In this study, we demonstrated that recombinant protein expression combined with ultrafiltration may be a simple and cost-effective solution to HNP-1 production. We further found that HNP-1 induces bacterial apoptosis and prevents its SOS repair pathway from binding to the RecA protein, which may be a new antibacterial mechanism. In addition, we showed that HNP-1 encapsulated in liposomes inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA) and meropenem-resistant Pseudomonas aeruginosa (MRPA). These results provide new insights into the production and antibacterial mechanism of HNP-1, both of which may promote its clinical application.

Hydrophilic nanoparticles that kill bacteria while sparing mammalian cells reveal the antibiotic role of nanostructures.

To dissect the antibiotic role of nanostructures from chemical moieties belligerent to both bacterial and mammalian cells, here we show the antimicrobial activity and cytotoxicity of nanoparticle-pinched polymer brushes (NPPBs) consisting of chemically inert silica nanospheres of systematically varied diameters covalently grafted with hydrophilic polymer brushes that are non-toxic and non-bactericidal. Assembly of the hydrophilic polymers into nanostructured NPPBs doesn't alter their amicability with mammalian cells, but it incurs a transformation of their antimicrobial potential against bacteria, including clinical multidrug-resistant strains, that depends critically on the nanoparticle sizes. The acquired antimicrobial potency intensifies with small nanoparticles but subsides quickly with large ones. We identify a threshold size (dsilica ~ 50 nm) only beneath which NPPBs remodel bacteria-mimicking membrane into 2D columnar phase, the epitome of membrane pore formation. This study illuminates nanoengineering as a viable approach to develop nanoantibiotics that kill bacteria upon contact yet remain nontoxic when engulfed by mammalian cells.