Evaluation of Bacteriophage ϕ11 host recognition protein and its host-binding peptides for diagnosing/targeting of Saphylococcus aureus infections.

Evaluating the potential of using both synthetic and biological products as targeting agents for the diagnosis, imaging, and treatment of infections due to particularly antibiotic-resistant pathogens is important for controlling infections. We examined the interaction between Gp45, a receptor-binding protein of the ϕ11 lysogenic phage, and its host S. aureus, a common cause of nosocomial infections. Using molecular dynamics and docking simulations, we identified the peptides that bind to S. aureus wall teichoic acids via Gp45. We compared the binding affinity of Gp45 and the two highest-scoring peptide sequences (P1 and P3) and their scrambled forms using microscopy, spectroscopy, and ELISA. Our results revealed that rGp45 (recombinant Gp45) and chemically synthesized P1 had a higher binding affinity for S. aureus compared with all other peptides, with the exception of E. coli. Furthermore, rGp45 had a capture efficiency of over 86%; P1 had a capture efficiency of over 64%. Overall, our findings suggest that receptor-binding proteins such as rGp45, which provide a critical initiation of the phage life cycle for host adsorption, might play an important role in the diagnosis, imaging, and targeting of bacterial infections. Studying such proteins could accordingly enable the development of effective strategies for controlling infections.

Tannic Acid Incorporated Antibacterial Polyethylene Glycol Based Hydrogel Sponges for Management of Wound Infections.

Conventional wound dressings fail to provide features that can assist the healing process of chronic wounds. Multifunctional wound dressings address this issue by incorporating attributes including antibacterial and antioxidant activity, and the ability to enhance wound healing. Herein, polyethylene glycol (PEG)-based antibacterial hydrogel sponge dressings are prepared by a rapid and facile gas foaming method based on an acid chloride/alcohol reaction where tannic acid (TA) is included as a reactant to impart antibacterial efficacy as well as to enhance the mechanical properties of the samples. The results reveal that the TA-integrated sponges possess excellent antibacterial properties against both Escherichia coli and Staphylococcus aureus with approximately 6-8 log reduction in the microbial colony count after 6 h, indicating their high potential for management of infection-prone wounds. Compared to the control sample, TA incorporation increases the elastic modulus by twofold. As the samples also exhibit biocompatibility, antioxidant activity, and wound healing capacity, the novel TA-incorporated hydrogels can be an alternative to traditional wound dressings for wounds with low-to-moderate exudate.

Targeted delivery of rifaximin using P6.2-decorated bifunctional PLGA nanoparticles for combating Staphylococcus aureus infections.

The emergence of antibiotic resistance makes the treatment of bacterial infections difficult and necessitates the development of alternative strategies. Targeted drug delivery systems are attracting great interest in overcoming the limitations of traditional antibiotics. Here, we aimed for targeted delivery of rifaximin (RFX) by decorating RFX-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) with synthetic P6.2 peptide, which was used as a targeting agent for the first time. Our results showed that encapsulation of RFX into NPs increased its antibacterial activity by improving its solubility and providing controlled release, while P6.2 modification allowed targeting of NPs to S. aureus bacterial cells. A promising therapeutic approach for bacterial infections, these P6.2-conjugated RFX-loaded PLGA NPs (TR-NP) demonstrated potent antibacterial activity against both strains of S. aureus. The antibacterial activity of RFX-loaded PLGA NPs (R-NP) showed significant results with an increase of 8 and 16-fold compared to free RFX against S. aureus and MRSA, respectively. Moreover, the activity of targeted nanoparticles was found to be increased 32 or 16-fold with an MBC value of 0.0078 µg/mL. All nanoparticles were found to be biocompatible at doses where they showed antimicrobial activity. Finally, it revealed that P6.2-conjugated targeted nanoparticles extremely accumulated in S. aureus rather than E. coli.

Propolis-Loaded Poly(lactic-co-glycolic Acid) Nanofibers: An In Vitro Study.

Nanofibers have high potential through their high porosity, small pore sizes, lightweight materials, and their ability to mimic the extracellular matrix structure for use in the manufacture of wound dressings for wound treatment. In this study, poly(lactic-co-glycolic acid) (PLGA) nanofibers were produced by electrospinning. Propolis was loaded into the PLGA nanofibers by the dropping method. The average diameters and effects of propolis loading on the morphology of 37.5, 50, and 100% propolis-loaded PLGA nanofibers (PLGA-P37.5, PLGA-P50, and PLGA-P100) were evaluated by scanning electron microscopy (SEM). The successful loading of propolis into PLGA nanofibers was confirmed with Fourier transform infrared spectroscopy (FTIR) analysis. In vitro propolis release was examined at physiological pH. The antioxidant activity of propolis-loaded nanofibers was studied with 2,2-diphenyl-1-picrylhydrazyl (DPPH). Antimicrobial activities of the nanofibers against Escherichia coli, Staphylococcus aureus and Candida albicans strains were determined by the disk diffusion method. Consequently, PLGA-P50 and PLGA-P100 showed high antimicrobial activity on S. aureus and C. albicans. Cell viability was tested by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and propolis-loaded PLGA nanofibers were found to be biocompatible with human fibroblast cells. In the wound scratch assay, propolis-loaded nanofibers supported wound closure with cell migration and proliferation. Thus, in vitro wound closure properties of propolis-loaded PLGA nanofibers were evaluated for the first time in the literature.

Genome editing in Acinetobacter baumannii through enhanced natural transformation.

Acinetobacter baumannii, a multidrug-resistant bacterium has become a significant cause of life-threatening infections acquired in hospitals worldwide. The existing drugs used to treat A. baumannii infections are rapidly losing efficacy, and the increasing antimicrobial resistance, which is expected to turn into a global health crisis, underscores the urgency to develop novel prevention and treatment strategies. We reasoned that the discovery of novel virulence targets for vaccine and therapy interventions requires a more enhanced method for the introduction of multiple elements of foreign DNA for genome editing than the current methods of natural transformation techniques. Herein, we employed a novel and a much-improved enhanced technique for the natural transformation of elements of the genome editing system CRISPR-Cas9 to suppress specific genomic regions linked to selectively suppress bacterial virulence. We modified the genome of the laboratory-adapted strain of A. baumannii BAA-747 by targeting the AmpC, as a marker gene, for disruption by three different genomic manipulation strategies, and created mutant strains of A. baumannii that are, at least, fourfold susceptible to ampicillin. This work has established an optimized enhanced natural transformation system that enables efficient genome editing of pathogenic bacteria in a laboratory setting, providing a valuable future tool for exploring the function of unidentified virulence genes in bacterial genomes.

Recombinant proteins production in Escherichia coli BL21 for vaccine applications: a cost estimation of potential industrial-scale production scenarios.

Recent SARS-CoV-2 pandemic elevated research interest in microorganism-related diseases, and protective health application importance such as vaccination and immune promoter agents emerged. Among the production methods for proteins, recombinant technology is an efficient alternative and frequently preferred method. However, since the production and purification processes vary due to the protein nature, the effect of these differences on the cost remains ambiguous. In this study, brucellosis and its two important vaccine candidate proteins (rOmp25 and rEipB) with different properties were selected as models, and industrial-scale production processes were compared with the SuperPro Designer® for estimating the unit production cost. Simulation study showed raw material cost by roughly 60% was one of the barriers to lower-cost production and 52.5 and 559.8 $/g were estimated for rEipB and rOmp25, respectively.

HighlightsTechno-economic evaluation of recombinant protein produced for vaccine purposesRecombinant proteins rOmp25 and rEipB production process using E.coli BL21Effect of outer membrane and periplasmic space proteins on purification costSimulated cost estimation of rEipB and rOmp25 were 52.5 and 559.8 $/g, respectively.

Host-Guest Interactions of Caffeic Acid Phenethyl Ester with ß-Cyclodextrins: Preparation, Characterization, and In Vitro Antioxidant and Antibacterial Activity.

The aim of this study is to improve the solubility, chemical stability, and in vitro biological activity of caffeic acid phenethyl ester (CAPE) by forming inclusion complexes with ß-cyclodextrin (ß-CD) and hydroxypropyl-ß-cyclodextrin (Hß-CD) using the solvent evaporation method. The CAPE contents of the produced complexes were determined, and the complexes with the highest CAPE contents were selected for further characterization. Detailed characterization of inclusion complexes was performed by using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and electrospray ionization-mass spectrometry (ESI-MS). pH and thermal stability studies showed that both selected inclusion complexes exhibited better stability compared to free CAPE. Moreover, their antimicrobial activities were evaluated against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) for the first time. According to the broth dilution assay, complexes with the highest CAPE content (10C/ß-CD and 10C/Hß-CD) exhibited considerable growth inhibition effects against both bacteria, 31.25 µg/mL and 62.5 µg/mL, respectively; contrarily, this value for free CAPE was 500 µg/mL. Furthermore, it was determined that the in vitro antioxidant activity of the complexes increased by about two times compared to free CAPE.

Evaluating the Stability of Lytic and Lysogenic Bacteriophages in Various Protectants.

Phage therapy has regained value as a potential alternative and a complementary anti-infective approach to antibiotics in the fight against bacterial pathogens. Due to their host specificity, non-pathogenic nature for humans, and low production cost, phages offer an effective opportunity for utilization in healthcare, agriculture, and food preservation. Well-defined storage conditions are essential for commercialization and dissemination of phage usage. For this purpose, in our study, after the isolation and characterization of two different phages, one lytic and the other lysogenic; storage and shelf-life studies of phages were evaluated in a presence of various protectants (glycerol, sodium azide, DMSO with chloroform) and without any protectant during 8-month period at four different temperatures. The short-time stability of the lytic P. syringae phage and lysogenic MRSA phage, which were determined by STEM analysis to belong to the Straboviridae and Siphoviridae families, respectively were also examined for the different temperatures and the pH levels ranging from 1.0 to 14.0. This study revealed the storage-model of phages that exhibit distinct lifecycles, for the first time and provided a theoretical basis for development and application of phages, has yielded valuable findings contributing to understanding of phage biology.

Time-dependent change in the microbiota structure of seminal stains exposed to indoor environmental.

Seminal stains acquired from fabric surfaces stand as pivotal biological evidence of utmost significance for elucidating sexual assault cases. The ability to determine the temporal aspect of a forensic incident via the analysis of a biological specimen found at the crime scene is crucial in resolving most cases. This study aimed to investigate the time-dependent change in the microbiota structure of human seminal stains exposed to indoor environmental conditions. Stains on polyester fabric generated using semen samples from five male volunteers were kept indoors for varying durations of up to 20 days, followed by sequencing of the V1-V9 regions of the 16S rRNA gene of the microbial DNA extracted from the stains. The acquired data provided the taxonomic composition, and microbial alterations across different days were examined. The most abundantly detected phyla in all samples were Firmicutes, Proteobacteria, and Bacteroidetes, and the relative abundances of bacteria were observed to change over time. Statistically significant changes at the species level were found for Treponema medium, Corynebacterium tuberculostearicum, Faecalibacterium prausnitzii, and Anaerostipes hadrus. Alterations observed in the samples between the analyzed time periods were investigated. The changes during the specified time periods were examined, identifying rare bacterial species that were initially present on certain days but later ceased to exist in the environment. Conversely, bacterial species that were absent before exposure but emerged at a later stage were also identified. The findings of this study demonstrate that species-level evaluations, in particular, can provide crucial insights into semen stain age.

Fabrication of poly(ß-amino ester) and hyaluronic acid based pH responsive nanocomplex as an antibiotic release system.

World Health Organization (WHO) warns about antimicrobial resistance (AMR) considered as the most serious threats to global health, food security, and development. There are various efforts for elimination of this serious issue. These efforts include education of individuals, new policies, development of new antimicrobials and new materials for effective delivery. Novel drug delivery systems with ability of local and on-demand delivery are one of the promising approaches for prevention of AMR. In this regard, a pH-responsive antibiotic delivery system based on pH-responsive poly(ß-amino ester) (PBAE) and enzyme responsive hyaluronic acid (HA). The polymeric nanocomplexes were obtained via electrostatic complexation of PBAE and HA in the presence of a model antibiotics, colistin and vancomycin. The particle sizes at pH 7.4 were determined in the range of 131-730 nm and 120-400 nm by DLS and STEM, respectively. When pH was switched from 7.4 to 5.5, the hydrodynamic diameter increased 2.5-32 fold. The drug release performances were tested using FITC-labeled antibiotics via fluorescence spectroscopy. The nanocomplexes released the drugs more at pH 5.5 compared to pH 7.4. Antibacterial activity of the system was evaluated on various bacteria. The nanocomplex loaded with the antibiotics exhibited significantly greater efficacy against E. coli and S. aureus.

Ketoconazole-loading strategy to improve antifungal activity and overcome cytotoxicity on human renal proximal tubular epithelial cells.

Ketoconazole (KTZ), an antifungal agent used to treat localized or systemic fungal infections by inhibiting ergosterol synthesis, exhibits restricted efficacy within eukaryotic cells owing to its elevated toxicity and limited solubility in water. This study aims to improve the biological activity and overcome cytotoxic effects in the renal system of the hydrophobic KTZ by incorporating it into poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) utilizing biomaterial nano-engineering techniques. KTZ-loaded PLGA NPs (KTZ-NPs) were prepared by single emulsion solvent evaporation method and characterized by using dynamic light scattering (DLS), electrophoretic light scattering (ELS), Fourier transform-infrared (FT-IR) spectroscopy and scanning light microscopy (SEM). Particle size and zeta potential of KTZ-NPs were determined as 182.0 ± 3.27 nm and -27.4 ± 0.56 mV, respectively. Antifungal activity was analyzed with the time-kill and top agar dilution methods onCandida albicans(C. albicans) andAspergillus flavus(A. flavus). Both KTZ and KTZ-NPs caused a significant decrease inA. flavuscell growth; however, the same effect was only observed in time-killing analysis onC. albicans, indicating a methodological difference in the antifungal analysis. According to the top agar method, the MIC value of KTZ-NPs againstA. flavuswas 9.1µg ml-1, while the minimum inhibition concentration (MIC) value of KTZ was 18.2µg ml-1. The twofold increased antifungal activity indicates that nanoparticular drug delivery systems enhance the water solubility of hydrophobic drugs. In addition, KTZ-NPs were not cytotoxic on human renal proximal tubular epithelial cells (HRPTEpCs) at fungistatic concentration, thus reducing fungal colonization without cytotoxic on renal excretion system cells.

The uselessness of using salivary microbiota in forensic identification purposes of a person with recent antibiotic use.

The detection of microbial flora changes in saliva samples because of antibiotic use through advanced molecular genetic analysis is important for forensic and clinical applications. This study aims to reveal the variability in the microbial structure of human saliva after antibiotic use with metagenomic analysis techniques from a forensic point of view. Within the scope of the study, saliva samples were collected from patients who were under the effect of regional anesthesia to be administered a standardized course of antibiotic therapy that lasted for a week. The analysis was conducted on 56 saliva samples from 14 individuals over four different time intervals. Isolation of the 16S rRNA region and PCR analysis were performed prior to sequence analysis to determine the microbiome structure of the samples at phylum, genus, and species levels. As expected, changes were observed in bacterial species found in saliva samples after administration of antibiotics and this was linked to the specific type of antibiotics that were administered. This change was statistically significant for Firmicutes, Spirochetes, and Verrucomicrobiota. Furthermore, although the oral microbiome tends to return to its former state at the phylum and genus level within a 4-week period after the start of antibiotic use, it is observed that the change, especially in some bacterial species, still continues. The findings of this study show that because of the inability of stabilization at species-level in a period of 4 weeks from the start of antibiotic use, it is not suitable to assess saliva samples at species-level for forensic identification.

The evaluation of biotechnological potential of Gp144, the key molecule of natural predator bacteriophage K in Staphylococcus aureus hunting mechanism.

Bacteriophages, which selectively infect bacteria, and phage-derived structures are considered promising agents for the diagnosis and treatment of bacterial infections due to the increasing antibiotic resistance. The binding of phages to their specific receptors on host bacteria is highly specific and irreversible, and therefore, the characterization of receptor-binding proteins(RBPs), which are key determinants of phage specificity, is crucial for the development of new diagnostic and therapeutic products. This study highlights the biotechnological potential of Gp144, an RBP located in the tail baseplate of bacteriophage K and responsible for adsorption of phageK to S. aureus. Once it was established that recombinant Gp144 (rGp144)is biocompatible and does not exhibit lytic effects on bacteria, its interaction with the host, the binding efficiency and performance were assessed in vitro using microscopic and serological methods. Results showed that rGp144 has a capture efficiency (CE) of over 87% and the best CE score is %96 which captured 9 CFU mL-1 out of 10 CFU mL-1 bacteria, indicating that very low number of bacteria could be detected. Additionally, it was shown for the first time in the literature that rGp144 binds to both S. aureus and methicillin-resistant S. aureus (MRSA) cells in vitro, while its affinity to different Gram-positive bacteria (E. faecalis and B. cereus) was not observed. The findings suggest that rGp144 can be effectively used for the diagnosis of S. aureus and MRSA, and that the use of RBPs in host-phage interaction can be a novel and effective strategy for imaging and diagnosing the site of infection.

A new strategy to achieve high antimicrobial activity: green synthesised silver nanoparticle formulations with Galium aparine and Helichrysum arenarium.

Silver nanoparticles (AgNPs), which have recently gained attention due to their antimicrobial activity, can also be produced by green synthesis. The aims of this study were to (i) characterise green synthesized AgNPs using microwave-assisted aqueous extracts of Galium aparine (G-AgNPs) and Helichrysum arenarium (H-AgNPs) and (ii) investigate the combined antimicrobial effects of the G- and H-AgNPs in different ratios. Nanoparticle formation and reactions were determined with UV-Vis spectroscopy. The G-AgNPs were 52.0±10.9 nm in size, with a 0.285±0.034 polydispersity index (PDI), and a -17.9±0.9 mV zeta potential. For H-AgNPs these characteristics were 23.9±1.0 nm, 0.280±0.032, and -21.3±2.7 mV, respectively. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) confirmed that the particles were monodisperse and spherical. The Fourier transform-infrared spectroscopy (FT-IR) results showed the presence of reducing agents that stabilised the AgNPs. Three different nanoformulations (NF-1, NF-2, and NF-3) were prepared by combining these two synthesised nanoparticles in different ratios and their antimicrobial activity was tested against E. coli, S. aureus, C. albicans, and A. flavus. Our study is the first to show that combining AgNPs from two different biological sources can produce effective nanoformulations with improved antibacterial activity against E. coli and S. aureus. These nanoformulations showed lower minimum inhibitory concentrations (31.25 µg/mL against E. coli with all NFs; 62.5 µg/mL for NF-1 and 125 µg/mL for NF-2/3 against S. aureus) than G-AgNPs (62.5 µg/mL for E. coli) or H-AgNPs (125 µg/mL for S. aureus) alone. Their high combined inhibitory effect against E. coli (NF-1-3) was synergistic and against S. aureus (NF-2 and NF-3) potentially additive. Considering such promising results, we believe our study provides some direction for new research and strategies in antimicrobial therapeutics.

Design of a bactericidal hydrogel scaffold containing genipin crosslinked HF-18 peptide.

Wound healing is a process getting affected by internal and external factors and might be interrupted by infections. To overcome infections during wound healing, novel antibacterial agents such as antimicrobial peptides have gained popularity because of the rising antibiotic resistance. Therefore, in this study, a three-dimensional polymeric scaffold was designed for the controlled release of HF-18 peptide, with the contribution of hyaluronic acid, chondroitin sulfate, and chitosan polymers with the crosslinker genipin. The obtained scaffold structure (OPT) was found to have interconnected pores, was pH-responsive and swelled more in acidic conditions (5446.5% at pH: 5.0). It was observed that HF-18-loaded OPT (P-OPT) was able to release HF-18 peptide both in acidic and neutral conditions in a controlled release manner. This study also demonstrated that both OPT and P-OPT were biocompatible and promoted L929 cell attachment and migration. Antimicrobial activity assessments demonstrated that P-OPT was effectively bactericidal on Staphylococcus aureus and methicillin-resistant S. aureus. Moreover, OPT produced a synergistic effect on the antimicrobial activity of HF-18 peptide, as P-OPT showed activity below the reported MIC value. As a result, OPT is considered a promising scaffold as a carrier for HF-18 for wound healing.

Synthesis of silane- and quaternary ammonium-bearing copolymers and application as a coating resin on cotton fabric.

In this study, silane and quaternary ammonium functional methacrylate monomers were synthesized and used to construct a copolymer using an emulsion polymerization technique to control the reaction rate. The copolymer was then designed using different ratios of silane and quaternary ammonium groups to investigate the relationship between the structure and properties. The presence of the ethoxy silane group in the copolymer series provided covalent bonding through the silanol group onto cotton fabric. The presence of cationic groups also helped to cover the fabric surface. After coating the cotton textile fabric, the resistance of the dye on the fabric surface to friction was assessed and tests were conducted on washing, rubbing, water, and light fastness. Finally, the textile surfaces were investigated for their antibacterial activity against Staphylococcus aureus and Escherichia coli. It was observed that the copolymer series showed >99% killing efficiency against S. aureus but had no effect on E. coli.

Investigating changes in salivary microbiota due to dental treatment: A metagenomic analysis study for forensic purposes.

The advent of next generation sequencing techniques as well as the existing traditional culture methods has enabled metagenomic studies on the usability of microbiomes for the forensic identification of individuals to gain momentum. However, before the utilization of microbiomes as a potential technique for real forensic case resolutions, it is necessary to understand the stability of the microbiota compositions in an individual's biological samples and the factors responsible for their variations. In the present study, we compared the microbiota compositions present in the saliva of individuals with active dental caries before and after treatment from a forensic and clinical perspective using an approach based on the sequencing of all the variable regions (V1-V9) of the bacterial 16 S rRNA gene. For this purpose, 10 individuals were included in the study comprising of 8 individuals between the ages of 18-50 years with at least 3 deep dentin caries as patients and 2 healthy individuals without any dental or gingival diseases as controls. Saliva samples were collected from the patients at two timepoints, before and after treatment, as well as from the healthy individuals (before and after control) at an interval of 1 month. The collected 20 saliva samples were subjected to metagenomic analysis using the MinION device, which was developed by Oxford Nanopore Technologies (ONT Oxford, UK). Bioinformatic analyses were performed on the obtained data and the results were evaluated using statistical comparison methods and alpha/beta diversity analyses within the scope of the study objective. On evaluation using the distance metrics, it was observed that the microbial compositions in the saliva of individuals with active caries remained relatively stable after treatment. However, the relative abundance levels of bacteria of 28 genera and species showed statistically significant differences before and after treatment (p < 0.05). As a result, although the composition of salivary microbiome remained relatively stable after caries treatment, there were significant changes in many types of bacteria, especially at the species level, between the BT and AT samples. Our results provide a framework for further forensic and clinical investigations regarding the factors that affect human salivary microbiome diversity.