Publications

Histones possess significant antimicrobial potential, yet their activity against biofilms remains underexplored. Moreover, concerns regarding adverse effects limit their clinical implementation. We investigated the antibacterial efficacy of human recombi nant histone H1 subtypes against Pseudomonas aeruginosa PAO1, both planktonic and in biofilms. After the in vitro tests, toxicity and efficacy were assessed in a P. aeruginosa PAO1 infection model using Galleria mellonella larvae. Histones were also evaluated in combination with ciprofloxacin (Cpx) and gentamicin (Gm). Our results demonstrate antimicrobial activity of all three histones against P. aeruginosa PAO1, with H1.0 and H1.4 showing efficacy at lower concentrations. The bactericidal effect was associated with a mechanism of membrane disruption. In vitro studies using static and dynamic models showed that H1.4 had antibiofilm potential by reducing cell biomass. Neither H1.0 nor H1.4 showed toxicity in G. mellonella larvae, and both increased larvae survival when infected with P. aeruginosa PAO1. Although in vitro synergism was observed between ciprofloxacin and H1.0, no improvement over the antibiotic alone was noted in vivo. Differences in antibacterial and antibiofilm activity were attributed to sequence and structural variations among histone subtypes. Moreover, the efficacy of H1.0 and H1.4 was influenced by the presence and strength of the extracellular matrix. These findings suggest histones hold promise for combating acute and chronic infections caused by pathogens such as P. aeruginosa.

JTD Keywords: Antimicrobial peptide, Antimicrobial peptides, Binding, Biofilm, Galleria mellonella, Host-defense, Peptid, Protein, Proteins, Toxicity, Treatment

Hydrogels with cell adhesive moieties stand out as promising materials to enhance tissue healing and regeneration. Nonetheless, bacterial infections of the implants represent an unmet major concern. In the present work, we developed an alginate hydrogel modified with a multifunctional peptide containing the RGD cell adhesive motif in combination with an antibacterial peptide derived from the 1-11 region of lactoferrin (LF). The RGD-LF branched peptide was successfully anchored to the alginate backbone by carbodiimide chemistry, as demonstrated by 1H NMR and fluorescence measurements. The functionalized hydrogel presented desirable physicochemical properties (porosity, swelling and rheological behavior) to develop biomaterials for tissue engineering. The viability of mesenchymal stem cells (MSCs) on the peptide-functionalized hydrogels was excellent, with values higher than 85 % at day 1, and higher than 95 % after 14 days in culture. Moreover, the biological characterization demonstrated the ability of the hydrogels to significantly enhance ALP activity of MSCs as well as to decrease bacterial colonization of both Gram-positive and Gram-negative models. Such results prove the potential of the functionalized hydrogels as novel biomaterials for tissue engineering, simultaneously displaying cell adhesive activity and the capacity to prevent bacterial contamination, a dual bioactivity commonly not found for these types of hydrogels. In this work we report on the functionalization of an alginate hydrogel with a tailor-made multifunctional peptide containing the cell adhesive RGD motif and the LF1-11 antibacterial peptide. Such novel multifunctional biomaterial ensures the viability of human mesenchymal stem cells, enhances ALP activity and decreases bacterial infections of both Gram-positive and Gram-negative models. image

JTD Keywords: Alginate hydrogel, Alginates, Anti-bacterial agents, Antimicrobial peptid, Antimicrobial peptide, Antimicrobial peptides, Arginyl-glycyl-aspartic acid, Biocompatible materials, Biofunctionalization, Bone, Cell adhesion, Cell survival, Composite hydrogels, Cross-linking, Hlf1-11 peptide, Human lactoferrin, Humans, Hydrogels, Immobilization, Mesenchymal stem cells, Multifunctional peptide, Oligopeptides, Peptides, Physical-properties, Scaffolds, Surfac, Tissue engineering

Microbial biofilms are complex three-dimensional structures where sessile microbes are embedded in a polymeric extracellular matrix. Their resistance toward the host immune system as well as to a diverse range of antimicrobial treatments poses a serious health and development threat, being in the top 10 global public health threats declared by the World Health Organization. In an effort to combat biofilm-related microbial infections, several strategies have been developed to independently eliminate biofilms or to complement conventional antibiotic therapies. However, their limitations leave room for other treatment alternatives, where the application of nanotechnology to biofilm eradication has gained significant relevance in recent years. Their small size, penetration efficiency, and the design flexibility that they present makes them a promising alternative for biofilm infection treatment, although they also present set-backs. This review aims to describe the main possibilities and limitations of nanomedicine against biofilms, while covering the main aspects of biofilm formation and study, and the current therapies for biofilm treatment. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

JTD Keywords: Anti-bacterial agents, Anti-infective agents, Antiinfective agent, Antimicrobial, Antimicrobials, Antimicrobials,bacteria,biofilm,infectious diseases,microorganism, Bacteria, Biofilm, Biofilm infections, Biofilms, Complex three dimensional structures, Diseases, Diverse range, Drug-delivery systems,in-vitro,cellular toxicity,nanoparticles,penetration,model,biocompatibility,perspectives,hyperthermia,diagnosi, Extracellular matrices, Global public health, Health risks, Infectious disease, Infectious diseases, Medical nanotechnology, Microbial biofilm, Microorganisms, Nanomedicine, Polymer, Polymers, Regulatory issues, Roadmap

Dental implant-associated infection is a clinical challenge which poses a significant healthcare and socio-economic burden. To overcome this issue, developing antimicrobial surfaces, including antimicrobial peptide coatings, has gained great attention. Different physical and chemical routes have been used to obtain these biofunctional coatings, which in turn might have a direct influence on their bioactivity and functionality. In this study, we present a silane-based, fast, and efficient chemoselective conjugation of antimicrobial peptides (Cys-GL13K) to coat titanium implant surfaces. Comprehensive surface analysis was performed to confirm the surface functionalization of as-prepared and mechanically challenged coatings. The antibacterial potency of the evaluated surfaces was confirmed against both Streptococcus gordonii and Streptococcus mutans, the primary colonizers and pathogens of dental surfaces, as demonstrated by reduced bacteria viability. Additionally, human dental pulp stem cells demonstrated long-term viability when cultured on Cys-GL13K-grafted titanium surfaces. Cell functionality and antimicrobial capability against multi-species need to be studied further; however, our results confirmed that the proposed chemistry for chemoselective peptide anchoring is a valid alternative to traditional site-unspecific anchoring methods and offers opportunities to modify varying biomaterial surfaces to form potent bioactive coatings with multiple functionalities to prevent infection.

JTD Keywords: biocompatibility, cytotoxicity, delivery, dental implants, prevention, release, stability, surface coating, titanium, zirconia, Antimicrobial peptide, Biocompatibility, Dental implants, Peri-implantitis, Surface coating, Titanium

Titanium implantation success may be compromised by Staphylococcus aureus surface colonization and posterior infection. To avoid this issue, different strategies have been investigated to promote an antibacterial character to titanium. In this work, two antibacterial agents (silver nanoparticles and a multifunctional antimicrobial peptide) were used to coat titanium surfaces. The modulation of the nanoparticle (≈32.1 ± 9.4 nm) density on titanium could be optimized, and a sequential functionalization with both agents was achieved through a two-step functionalization method by means of surface silanization. The antibacterial character of the coating agents was assessed individually as well as combined. The results have shown that a reduction in bacteria after 4 h of incubation can be achieved on all the coated surfaces. After 24 h of incubation, however, the individual antimicrobial peptide coating was more effective than the silver nanoparticles or their combination against Staphylococcus aureus. All tested coatings were non-cytotoxic for eukaryotic cells.

JTD Keywords: antimicrobial peptide, biomaterials, bone, coatings, performance, ph, resistance, silanization, silver nanoparticles, staphylococcus aureus, Anti-bacterial agents, Antimicrobial peptide, Coated materials, biocompatible, Metal nanoparticles, Reduces bacterial adhesion, Silanization, Silver, Silver nanoparticles, Staphylococcus aureus, Surface properties, Titanium, Titanium functionalization

Mycobacterium brumae is a rapid-growing, non-pathogenic Mycobacterium species, originally isolated from environmental and human samples in Barcelona, Spain. Mycobacterium brumae is not pathogenic and it's in vitro phenotype and immunogenic properties have been well characterized. However, the knowledge of its underlying genetic composition is still incomplete. In this study, we first describe the 4 Mb genome of the M. brumae type strain ATCC 51384T assembling PacBio reads, and second, we assess the low intraspecies variability by comparing the type strain with Illumina reads from three additional strains. Mycobacterium brumae genome is composed of a circular chromosome with a high GC content of 69.2% and containing 3,791 CDSs, 97 pseudogenes, one prophage and no CRISPR loci. Mycobacterium brumae has shown no pathogenic potential in in vivo experiments, and our genomic analysis confirms its phylogenetic position with other non-pathogenic and rapid growing mycobacteria. Accordingly, we determined the absence of virulence-related genes, such as ESX-1 locus and most PE/PPE genes, among others. Although the immunogenic potential of M. brumae was proved to be as high as Mycobacterium bovis BCG, the only mycobacteria licensed to treat cancer, the genomic content of M. tuberculosis T cell and B cell antigens in M. brumae genome is considerably lower than those antigens present in M. bovis BCG genome. Overall, this work provides relevant genomic data on one of the species of the mycobacterial genus with high therapeutic potential.Copyright © 2023 Renau-Mínguez, Herrero-Abadía, Ruiz-Rodriguez, Sentandreu, Torrents, Chiner-Oms, Torres-Puente, Comas, Julián and Coscolla.

JTD Keywords: antimicrobial susceptibility, bcg, identification, immunogenic, non-pathogenic, nontuberculous mycobacteria, resistance mechanisms, strain, therapeutic, Diversity, Immunogenic, Non-pathogenic, Nontuberculous mycobacteria, Therapeutic

Chronic wounds infected by Pseudomonas aeruginosa and Staphylococcus aureus are a relevant health problem worldwide because these pathogens grow embedded in a network of polysaccharides, proteins, lipids, and extracellular DNA, named biofilm, that hinders the transport of antibiotics and increases their antimicrobial tolerance. It is necessary to investigate therapies that improve the penetrability and efficacy of antibiotics. In this context, our main objectives were to study the relationship between P. aeruginosa and S. aureus and how their relationship can affect the antimicrobial treatment and investigate whether functionalized silver nanoparticles can improve the antibiotic therapy. We used an optimized in vitro wound model that mimics an in vivo wound to co-culture P. aeruginosa and S. aureus biofilm. The in vitro wound biofilm was treated with antimicrobial combinatory therapies composed of antibiotics (gentamycin and ciprofloxacin) and biofilm-dispersing free or silver nanoparticles functionalized with enzymes (alpha-amylase, cellulase, DNase I, or proteinase K) to study their antibiofilm efficacy. The interaction and colocalization of P. aeruginosa and S. aureus in a wound-like biofilm were examined and detailed characterized by confocal and electronic microscopy. We demonstrated that antibiotic monotherapy is inefficient as it differentially affects the two bacterial species in the mixed biofilm, driving P. aeruginosa to overcome S. aureus when using ciprofloxacin and the contrary when using gentamicin. In contrast, dual-antibiotic therapy efficiently reduces both species while maintaining a balanced population. In addition, DNase I nanoparticle treatment had a potent antibiofilm effect, decreasing P. aeruginosa and S. aureus viability to 0.017 and 7.7%, respectively, in combined antibiotics. The results showed that using nanoparticles functionalized with DNase I enhanced the antimicrobial treatment, decreasing the bacterial viability more than using the antibiotics alone. The enzymes alpha-amylase and cellulase showed some antibiofilm effect but were less effective compared to the DNase I treatment. Proteinase K showed insignificant antibiofilm effect. Finally, we proposed a three-dimensional colocalization model consisting of S. aureus aggregates within the biofilm structure, which could be associated with the low efficacy of antibiofilm treatments on bacteria. Thus, designing a clinical treatment that combines antibiofilm enzymes and antibiotics may be essential to eliminating chronic wound infections.

JTD Keywords: antimicrobial therapies, biofilm, chronic infection, nanoparticle, Antimicrobial therapies, Biofilm, Chronic infection, In-vitro, Matrix, Model, Nanoparticle, Wound healing

The increasing resistance of bacteria to existing antibiotics constitutes a major public health threat globally. Most current antibiotic treatments are hindered by poor delivery to the infection site, leading to undesired off-target effects and drug resistance development and spread. Here, we describe micro- and nanomotors that effectively and autonomously deliver antibiotic payloads to the target area. The active motion and antimicrobial activity of the silica-based robots are driven by catalysis of the enzyme urease and antimicrobial peptides, respectively. These antimicrobial motors show micromolar bactericidal activity in vitro against different Gram-positive and Gram-negative pathogenic bacterial strains and act by rapidly depolarizing their membrane. Finally, they demonstrated autonomous anti-infective efficacy in vivo in a clinically relevant abscess infection mouse model. In summary, our motors combine navigation, catalytic conversion, and bactericidal capacity to deliver antimicrobial payloads to specific infection sites. This technology represents a much-needed tool to direct therapeutics to their target to help combat drug-resistant infections.

JTD Keywords: antibiotic-resistance, antimicrobial peptides, autonomous treatment, bacterial infection, delivery, ll-37, nanomotors, nanoparticles, peptide, self-propulsion, tissue, vitro, wasp venom, Antibiotic-resistance, Antimicrobial peptides, Autonomous treatment, Bacterial infection, Delivery, Ll-37, Mesoporous silica nanoparticles, Nanomotors, Nanoparticles, Peptide, Self-propulsion, Tissue, Vitro, Wasp venom

Transmission of a plasmid from one bacterial cell to another, in several instances, underlies the dissemination of antimicrobial resistance (AMR) genes. The process requires well-characterized enzymatic machinery that facilitates cell-to-cell contact and the transfer of the plasmid.

JTD Keywords: antimicrobial resistance, bacterial ig-like proteins, bacterial lg-like proteins, chromosomal genes, identification, inca/c, mutational analysis, plasmid conjugation, products, r-factors, resistance plasmids, salmonella-enterica, sequence, Antimicrobial resistance, Bacterial ig-like proteins, Escherichia-coli, Plasmid conjugation

The preparation, characterization, and controlled release of hydroxyapatite (HAp) nanopar-ticles loaded with streptomycin (STR) was studied. These nanoparticles are highly appropriate for the treatment of bacterial infections and are also promising for the treatment of cancer cells. The analyses involved scanning electron microscopy, dynamic light scattering (DLS) and Z-potential measurements, as well as infrared spectroscopy and X-ray diffraction. Both amorphous (ACP) and crystalline (cHAp) hydroxyapatite nanoparticles were considered since they differ in their release behavior (faster and slower for amorphous and crystalline particles, respectively). The encapsulated nanoparticles were finally incorporated into biodegradable and biocompatible polylactide (PLA) scaf-folds. The STR load was carried out following different pathways during the synthesis/precipitation of the nanoparticles (i.e., nucleation steps) and also by simple adsorption once the nanoparticles were formed. The loaded nanoparticles were biocompatible according to the study of the cytotoxicity of extracts using different cell lines. FTIR microspectroscopy was also employed to evaluate the cytotoxic effect on cancer cell lines of nanoparticles internalized by endocytosis. The results were promising when amorphous nanoparticles were employed. The nanoparticles loaded with STR increased their size and changed their superficial negative charge to positive. The nanoparticles’ crystallinity decreased, with the consequence that their crystal sizes reduced, when STR was incorporated into their structure. STR maintained its antibacterial activity, although it was reduced during the adsorption into the nanoparticles formed. The STR release was faster from the amorphous ACP nanoparticles and slower from the crystalline cHAp nanoparticles. However, in both cases, the STR release was slower when incorporated in calcium and phosphate during the synthesis. The biocompatibility of these nanoparticles was assayed by two approximations. When extracts from the nanoparticles were evaluated in cultures of cell lines, no cytotoxic damage was observed at concen-trations of less than 10 mg/mL. This demonstrated their biocompatibility. Another experiment using FTIR microspectroscopy evaluated the cytotoxic effect of nanoparticles internalized by endocytosis in cancer cells. The results demonstrated slight damage to the biomacromolecules when the cells were treated with ACP nanoparticles. Both ACP and cHAp nanoparticles were efficiently encapsulated in PLA electrospun matrices, providing functionality and bioactive properties. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: antibiotics, antimicrobial activity, behavior, cytotoxicity, delivery, drug, drug delivery, hydroxyapatite nanoparticles, in-vitro, mechanisms, mitochondria, polylactide, release, streptomycin, Antimicrobial activity, Cancer stem-cells, Cytotoxicity, Drug delivery, Hydroxyapatite nanoparticles, Polylactide, Streptomycin

Contact-based antimicrobials, as antibiotic-free technologies that use non-specific interactions with bacterial cells to exert antimicrobial activity, are a prospective solution in fighting the global issue of bacterial resistance. A very simplified approach to their design considers the direct bonding of cationic guanidine-containing amino acids to the surface of nano-gold carriers. The structure enables antimicrobial activity due to a high density of cationic surface charges. This opens a set of novel questions that are important for their effective engineering, particularly regarding (i) chemistry and events that take place at the interface between NPs and cells, (ii) the direct influence of a charge (and its change) on interactions with bacterial and mammalian cells, and (iii) the stability of structures (and their antimicrobial activity) in the presence of enzymes, which are addressed in this paper. Because of the ability of amino acid-functionalized nano-gold to retain structural and functional activity, even after exposure to a range of physicochemical stimuli, they provide an excellent nanotechnological platform for designing highly effective contact-based antimicrobials and their applications.

JTD Keywords: agents, antibiotic-free technology, arginine, charged amino acids, contact-based antimicrobials, discovery, enzyme-resistant antimicrobials, functionalized gold, peptides, polymers, resistant, Antibiotic-free technology, Charged amino acids, Contact-based antimicrobials, Enzyme-resistant antimicrobials, Functionalized gold, Nanoparticles

Pseudomonas aeruginosa biofilms and the capacity of the bacterium to coexist and interact with a broad range of microorganisms have a substantial clinical impact. This review focuses on the main traits of P. aeruginosa biofilms, such as the structural composition and regulatory networks involved, placing particular emphasis on the clinical challenges they represent in terms of antimicrobial susceptibility and biofilm infection clearance. Furthermore, the ability of P. aeruginosa to grow together with other microorganisms is a significant pathogenic attribute with clinical relevance; hence, the main microbial interactions of Pseudomonas are especially highlighted and detailed throughout this review. This article also explores the infections caused by single and polymicrobial biofilms of P. aeruginosa and the current models used to recreate them under laboratory conditions. Finally, the antimicrobial and antibiofilm strategies developed against P. aeruginosa mono and multispecies biofilms are detailed at the end of this review.

JTD Keywords: aeruginosa models, antibiotic-resistance, antimicrobials, bacterial biofilms, biofilms, c-di-gmp, chronic infections, enterococcus-faecalis, extracellular dna, in-vitro, lectin pa-iil, p, p. aeruginosa models, polymicrobial, polymicrobial interactions, staphylococcus-aureus, Antimicrobials, Biofilms, Chronic infections, P. aeruginosa models, Polymicrobial, Pseudomonas aeruginosa, Urinary-tract-infection

Background: Emerging concepts for designing innovative drugs (i.e., novel generations of antimicrobials) frequently include nanostructures, new materials, and nanoparticles (NPs). Along with numerous advantages, NPs bring limitations, partly because they can limit the analytical techniques used for their biological and in vivo validation. From that standpoint, designing innovative drug delivery systems requires advancements in the methods used for their testing and investigations. Considering the well-known ability of resazurin-based methods for rapid detection of bacterial metabolisms with very high sensitivity, in this work we report a novel optimization for tracking bacterial growth kinetics in the presence of NPs with specific characteristics, such as specific optical properties. Results: Arginine-functionalized gold composite (HAp/Au/arginine) NPs, used as the NP model for validation of the method, possess plasmonic properties and are characterized by intensive absorption in the UV/vis region with a surface plasmon resonance maximum at 540 nm. Due to the specific optical properties, the NP absorption intensively interferes with the light absorption measured during the evaluation of bacterial growth (optical density; OD600). The results confirm substantial nonspecific interference by NPs in the signal detected during a regular turbidity study used for tracking bacterial growth. Instead, during application of a resazurin-based method (Presto Blue), when a combination of absorption and fluorescence detection is applied, a substantial increase in the signal-to-noise ratio is obtained that leads to the improvement of the accuracy of the measurements as verified in three bacterial strains tested with different growth rates (E. coli, P. aeruginosa, and S. aureus). Conclusions: Here, we described a novel procedure that enables the kinetics of bacterial growth in the presence of NPs to be followed with high time resolution, high sensitivity, and without sampling during the kinetic study. We showed the applicability of the Presto Blue method for the case of HAp/Au/arginine NPs, which can be extended to various types of metallic NPs with similar characteristics. The method is a very easy, economical, and reliable option for testing NPs designed as novel antimicrobials.

JTD Keywords: Antimicrobial nanoparticles, Arginine-functionalized gold, Bacterial growth kinetics, Plasmonic nanoparticles, Presto Blue

Oleanolic acid (OA) and maslinic acid (MA) are pentacyclic triterpenic compounds that abound in industrial olive oil waste. These compounds have renowned antimicrobial properties and lack cytotoxicity in eukaryotic cells as well as resistance mechanisms in bacteria. Despite these advantages, their antimicrobial activity has only been tested in vitro, and derivatives improving this activity have not been reported. In this work, a set of 14 OA and MA C-28 amide derivatives have been synthesized. Two of these derivatives, MA-HDA and OA-HDA, increase the in vitro antimicrobial activity of the parent compounds while reducing their toxicity in most of the Gram-positive bacteria tested, including a methicillin-resistant Staphylococcus aureus-MRSA. MA-HDA also shows an enhanced in vivo efficacy in a Galleria mellonella invertebrate animal model of infection. A preliminary attempt to elucidate their mechanism of action revealed that these compounds are able to penetrate and damage the bacterial cell membrane. More significantly, their capacity to reduce antibiofilm formation in catheters has also been demonstrated in two sets of conditions: a static and a more challenged continuous-flow S. aureus biofilm.

JTD Keywords: Antibiofilm, Galleria mellonella, In vitro and in vivo antimicrobials, Maslinic and oleanolic acids, Natural products, Staphylococcus aureus

In dentistry and orthopedics, it is well accepted that implant fixation is a major goal. However, an emerging concern is bacterial infection. Infection of metallic implants can be catastrophic and significantly reduce patient quality of life. Accordingly, in this work, we focus on multifunctional coatings to simultaneously address and mitigate both these problems. We have developed a tailor-made peptide-based chemical platform that integrates the well-known RGD cell adhesive sequence and the lactoferrin-derived LF1-11 antimicrobial peptide. The platform was covalently grafted on titanium via silanization and the functionalization process characterized by contact angle, XPS, and QCM-D. The presence of the platform statistically improved the adhesion, proliferation and mineralization of osteoblast-like cells compared to control surfaces. At the same time, colonization by representative bacterial strains was significantly reduced on the surfaces. Furthermore, the biological potency of the multifunctional platform was verified in a co-culture in vitro model. Our findings demonstrate that this multifunctional approach can be useful to functionalize biomaterials to both improve cell integration and reduce the risk of bacterial infection.

JTD Keywords: Antimicrobial peptides, Cell adhesive peptides, Multifunctionality, Osseointegration, Surface functionalization

IncHI plasmids account for multiple antimicrobial resistance in Salmonella and other enterobacterial genera. These plasmids are generally very stable in their bacterial hosts. R27 is the archetype of IncHI1 plasmids. A high percentage of the R27-encoded open reading frames (ORFs) (66.7%) do not show similarity to any known ORFs. We performed a deletion analysis of all non-essential R27 DNA sequences to search for hitherto non-identified plasmid functions that might be required for plasmid stability. We report the identification of a short DNA sequence (incC) that is essential for R27 stability. That region contains several repeats (incC repeats), belongs to one of the three-plasmid replicons (R27 FIA-like) and is targeted by the R27 E protein. Deletion of the incC sequence drastically reduces R27 stability both in Escherichia coli and in Salmonella, the effect being more pronounced in this latter species. Interfering with incC-E protein interaction must lead to a reduced IncHI1 plasmid stability, and may represent a new approach to combat antimicrobial resistance.

JTD Keywords: Antimicrobial resistance, E protein, IncC, IncHI1 plasmids, Plasmid R27, Plasmid stability