Bacterial Assay Research Articles

Comparative Analysis of the Antibacterial Properties of Various Periodontal Bone Graft Materials In Vitro.

Periodontal bone grafting is a common procedure in dentistry to restore bone loss resulting from periodontal disease. Various graft materials are available, each with different antibacterial properties. Understanding these properties is crucial for successful grafting outcomes. In this study, we conducted a comparative analysis of the antibacterial properties of four commonly used periodontal bone graft materials: The graft used were A: Hydroxyapatite, B: B TCP, C: Bioactive glass, D: Calcium Phosphate materials. Each material was subjected to in vitro testing using a standardized bacterial culture assay. Antibacterial activity was assessed by measuring the zone of inhibition around each material after exposure to bacterial cultures. Material A exhibited the highest antibacterial activity with an average zone of inhibition of 12.3 mm, followed by Material C with 9.8 mm, Material D with 8.5 mm, and Material B with 6.2 mm. These arbitrary values reflect the relative efficacy of each material in inhibiting bacterial growth. Our findings suggest that Material A possesses the strongest antibacterial properties among the tested periodontal bone graft materials. This may contribute to improved clinical outcomes in periodontal bone grafting procedures by reducing the risk of postoperative infections. Further research is warranted to elucidate the mechanisms underlying the antibacterial effects of these materials and to validate our in vitro findings in clinical settings.

Toxicity Evaluation of a Non-Pain Pharmacopuncture Extract Using a Bacterial Reverse Mutation Test.

The objective of this study was to assess the genotoxicity of a no-pain pharmacopuncture (NPP) extract developed in 2022 using a bacterial reverse mutation assay, aiming to further substantiate the safety profile of NPP. The genotoxicity evaluation involved a bacterial reverse mutation assay to assess the mutagenic potential of NPP extracts with and without metabolic activation. Histidine-requiring Salmonella typhimurium strains (TA98, TA100, TA1535, and TA1537) and tryptophan-requiring Escherichia coli strains (WP2uvrA) were used in the assay. The NPP extract did not induce a revertant colony count exceeding two times that of the negative control at any dose level in any of the tested strains, both with and without metabolic activation. Additionally, no growth inhibition or precipitation was observed in the presence of NPP. Based on the findings, it can be concluded that the NPP extract exhibited no mutagenic potential in the in vitro genotoxicity tests conducted.

Assessment of Mutans Streptococci Adhesion on Different Esthetic Coatings of Nickel-Titanium (Niti) Archwires: An In Vitro Study

Plaque and enamel demineralization can occur when orthodontic archwires are present in the oral cavity because they provide a unique surface for bacterial adhesion. Bacterial biofilm plays a major role in the development of complications such dental caries and periodontal disease. The purpose of the study was to examine the effect that an esthetic surface coating of NiTi archwires would have on the adherence of mutans streptococci. Material and method: three types of coated NiTi archwires (rhodium, gold and flexy blue) and one uncoated NiTi archwire with round cross section 0.016 inch were tested in the current study. Five pieces of each types of archwire were incubated in phosphate-buffered saline for two hours. Then, these wire segments were immersed for 5, 90, and 180 minutes with mutans streptococci suspension to conduct the bacterial adhesion assay. Result: there were statistically significant differences in the degree to which bacteria adhered to the various archwires at each time interval, with the level of adhesion being statistically highest on the uncoated NiTi wire and the level being statistically lowest on the gold-coated NiTi wire (p < 0.05). The result showed that the highest bacterial adhesion in 180 min followed by 90 min and the lowest in 5 min. A positive correlation was detected between bacterial adhesion and incubation time. Conclusion: the esthetic surface coating were influence the biofilm adhesion on the archwires, gold coated significantly decrease mutans streptococci adhesion when compare with uncoated NiTi wire. Higher incubation time increased the number of bacterial adherence for all types of archwires.

Deciphering the Antibiofilm, Antibacterial, and Antioxidant Potential of Essential Oil from Indian Garlic and its Phytocompounds Against Foodborne Pathogens.

Garlic (Allium sativum L.), particularly its volatile essential oil, is widely recognized for medicinal properties. We have evaluated the efficacy of Indian Garlic Essential Oil (GEO) for antimicrobial and antibiofilm activity and its bioactive constituents. Allyl sulfur-rich compounds were identified as predominant phytochemicals in GEO, constituting 96.51% of total volatile oils, with 38% Diallyl trisulphide (DTS) as most abundant. GEO exhibited significant antibacterial activity against eleven bacteria, including three drug-resistant strains with minimum inhibitory concentrations (MICs) ranging from 78 to 1250µg/mL. In bacterial growth kinetic assay GEO effectively inhibited growth of all tested strains at its ½ MIC. Antibiofilm activity was evident against two important human pathogens, S. aureus and P. aeruginosa. Mechanistic studies demonstrated that GEO disrupts bacterial cell membranes, leading to the release of nucleic acids, proteins, and reactive oxygen species. Additionally, GEO demonstrated potent antioxidant activity at IC50 31.18mg/mL, while its isolated constituents, Diallyl disulphide (DDS) and Diallyl trisulphide (DTS), showed effective antibacterial activity ranging from 125 to 500µg/mL and 250-1000µg/mL respectively. Overall, GEO displayed promising antimicrobial and antibiofilm activity against enteric bacteria, suggesting its potential application in the food industry.

Generic viability qPCR for monitoring shelf life of microbial biological control agents coated on seeds based on the nucleic acid intercalating dyes EMA and PMAxx

Biological control of seedborne pathogens and soilborne seedling pathogens through antagonists applied on seeds is an alternative to chemical seed treatments. Information on the viability of inocula on treated seeds is essential for any development and use of beneficial fungi or bacteria on seeds. Generic fungal and bacterial qPCR assays were combined with the nucleic acid intercalating dyes ethidium monoazide (EMA) and propidium monoazide (PMAxx) for the quantification of viable cells of fungi and bacteria on seeds. The applied protocols for generic fungal viability qPCR (v-qPCR) in combination with EMA and PMAxx and for generic bacterial v-qPCR in combination with PMAxx allowed the viability quantification of fungal and bacterial isolates representing a broad range of species with the exception of fungal species with highly melanized conidia. A first application of v-qPCR to coated seeds of onion and spinach indicated a differential plant species effect on survival of a coated fungus and a yeast with a generally better survival on seeds of spinach compared to seeds of onions and a similar good survival of the bacterium L. enzymogenes 3.1T8 on both seed types. The v-qPCR protocols can be applied in screening assays aiming at the selection of new antagonists with higher survival potentials and the development of new seed processing technologies compatible with coated antagonists.

Phage-Mediated Digestive Decolonization in a Gut-On-A-Chip Model: A Tale of Gut-Specific Bacterial Prosperity.

Infections due to antimicrobial-resistant bacteria have become a major threat to global health. Some patients may carry resistant bacteria in their gut microbiota. Specific risk factors may trigger the conversion of these carriages into infections in hospitalized patients. Preventively eradicating these carriages has been postulated as a promising preventive intervention. However, previous attempts at such eradication using oral antibiotics or probiotics have led to discouraging results. Phage therapy, the therapeutic use of bacteriophage viruses, might represent a worthy alternative in this context. Taking inspiration from this clinical challenge, we built Gut-On-A-Chip (GOAC) models, which are tridimensional cell culture models mimicking a simplified gut section. These were used to better understand bacterial dynamics under phage pressure using two relevant species: Pseudomonas aeruginosa and Escherichia coli. Model mucus secretion was documented by ELISA assays. Bacterial dynamics assays were performed in GOAC triplicates monitored for 72 h under numerous conditions, such as pre-, per-, or post-bacterial timing of phage introduction, punctual versus continuous phage administration, and phage expression of mucus-binding properties. The potential genomic basis of bacterial phage resistance acquired in the model was investigated by variant sequencing. The bacterial "escape growth" rates under phage pressure were compared to static in vitro conditions. Our results suggest that there is specific bacterial prosperity in this model compared to other in vitro conditions. In E. coli assays, the introduction of a phage harboring unique mucus-binding properties could not shift this balance of power, contradicting previous findings in an in vivo mouse model and highlighting the key differences between these models. Genomic modifications were correlated with bacterial phage resistance acquisition in some but not all instances, suggesting that alternate ways are needed to evade phage predation, which warrants further investigation.

Heparin mimicking sulfonated polyester synergistically promotes bacterial Infected-Wound healing with ROS responsive cinnamaldehyde based prodrugs

Excessive antibiotic usage has resulted in antibiotic resistance. This work introduces a novel ROS-responsive, cinnamaldehyde (CA)-based sulfonated all-polyester prodrug (RCSAP) to combat antibiotic resistance. RCSAP is a unique amphiphilic pseudo triblock copolymer comprising of two hydrophilic, heparin-mimicking sulfonated polyester (HSP) blocks and a hydrophobic polymeric block containing ROS-responsive CA thioacetal. RCSAP nanoparticles (NPs) not only achieve self-amplifying degradation and release, but orchestrate a “dual wielding” synergistic antibacterial mechanism. HSP shell competes with bacteria for host cell surface binding, hindering its internalization and infection. Simultaneously, ROS-responsive core liberates CA, boosting bacterial membrane permeability. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined. RCSAP NPs display exceptional antibacterial efficacy against both E. coli and S. aureus, as confirmed by survival analysis, colony-forming unit (CFU) quantification, microbial growth curve analysis, and live/dead bacterial viability assays. Importantly, RCSAP NPs effectively eradicated methicillin-resistant Staphylococcus aureus (MRSA). In addition, RCSAP NPs possess the unique ability to bind to S. aureus, aggregate and precipitate it. They competitively hinder S. aureus infection in both ARPE-19 and 293 T cells. Molecular simulation further verified the electrostatic interactions between HSP and the S. aureus SpA protein, which may mediate the binding of RCSAP NPs to S. aureus. In vivo assessments demonstrated that RCSAP NPs significantly accelerates wound healing in S. aureus-infected models, with excellent biocompatibility and minimal systemic toxicity. This work paves the way for all-polyester-based nanomedicines harnessing the power of natural antibacterial and heparin-mimicking agents synergistically for enhanced disinfection and wound healing, curbing drug resistance.

Nanobody-enhanced split-luciferase technology for innovative detection of the liver cancer biomarker alpha-fetoprotein

Liver cancer is one of the most common cancers and the third leading cause of cancer deaths worldwide. Diagnosis and screening for liver cancer rely on the alpha-fetoprotein (AFP) biomarker. This study aimed to pioneer a novel assay for AFP detection utilizing a tri-part split-luciferase system in conjunction with nanobodies targeting AFP. The strategy involved fusing anti-AFP nanobodies P5 and P15 to the split-nanoluciferase components β9 and β10, respectively. Upon binding to AFP and in the presence of the third nanoluciferase component Δ11S, the proximity-induced reassembly of split-nanoluciferase components triggers luciferase activation and luminescence emission. Following expression in a bacterial system, purification, and assay implementation, the developed assay exhibited high sensitivity in detecting AFP within a linear range of 1–20 ng/ml, with a Limit of Detection (LOD) of 0.5 ng/ml. The assay results were in line with those obtained from ELISA, indicating its efficiency. This study highlights the specificity of the homogenous assay developed with nanobodies for AFP, offering a reliable and user-friendly test for AFP detection.

The mannose-binding lectin (MBL) gene cloned from Exopalaemon carinicauda plays a key role in resisting infection by Vibrio parahaemolyticus

Mannose-binding lectin (MBL) is a vital member of the lectin family, crucial for mediating functions within the complement lectin pathway. In this study, following the cloning of the mannose-binding lectin (MBL) gene in the ridgetail white prawn, Exopalaemon carinicauda, we examined its expression patterns across various tissues and its role in combating challenges posed by Vibrio parahaemolyticus. The results revealed that the MBL gene spans 1342 bp, featuring an open reading frame of 972 bp. It encodes a protein comprising 323 amino acids, with a predicted relative molecular weight of 36 kDa and a theoretical isoelectric point of 6.18. The gene exhibited expression across various tissues including the eyestalk, heart, gill, hepatopancreas, stomach, intestine, ventral nerve cord, muscle, and hemolymph, with the highest expression detected in the hepatopancreas. Upon challenge with V. parahaemolyticus, RT-PCR analysis revealed a trend of MBL expression in hepatopancreatic tissues, characterized by an initial increase followed by a subsequent decrease, peaking at 24 h post-infection. Employing RNA interference to disrupt MBL gene expression resulted in a significant increase in mortality rates among individuals challenged with V. parahaemolyticus. Furthermore, we successfully generated the Pet32a-MBL recombinant protein through the construction of a prokaryotic expression vector for conducting in vitro bacterial inhibition assays, which demonstrated the inhibitory effect of the recombinant protein on V. parahaemolyticus, laying a foundation for further exploration into its immune mechanism in response to V. parahaemolyticus challenges.

Reducing baseline toxicity in fishery product-related sediments from land to sea: Region-specific solutions are required

Eastern China is a major producer of fishery products (including inland aquaculture, coastal mariculture, and coastal fishing products). The quality of the products is affected by hydrophobic organic contaminants (HOCs) in the sediments. Based on in-vitro luminescent bacterial assay, the baseline toxicity (BEQBio) of 56 common HOCs were assessed in the present study. Specifically, the BEQBio of sediments declined from land (31–400 mg/kg) to sea (9.1–270 mg/kg). However, the toxicity contribution explained by the HOCs increased gradually from land (0.70 %) to sea (10 %) using Iceberg Modeling. In the inland pond, current use HOCs (pyrethroid pesticide (PEs), organic tin (OTCs), and antibiotic) exhibited considerable concentrations, although their toxicity contribution was very small (0.076 %), thus more regulations on the use of HOCs should be proposed and further screening is needed to confirm the major toxicants. In coastal mariculture area, the toxicity contribution of current use HOCs further declined (0.010 %), whereas environmental background HOCs, such as polycyclic aromatic hydrocarbons (PAHs), became increasingly significant, with the contribution ratio increasing from 0.37 % to 2.4 %. To minimize the negative impacts of PAHs, optimization of energy structure in transportation and coastal industry is required. In the coastal fishing area, the phased-out persistent organic pollutants (POPs) remained a major concern, in terms of both concentration and toxicity contribution. The phased-out POPs explained 7.0 % of the toxic effects of the sediments from the coastal fishing area, due to historical residue, industrial emissions, and their high toxicities. For this reason, it is critical to improve the relevant emission regulations and standards, so as to eventually reduce the unintentional discharges of POPs.

In vivo efficacy of phage cocktails against carbapenem resistance Acinetobacter baumannii in the rat pneumonia model.

Acinetobacter baumannii, an opportunistic pathogen, poses a significant threat in intensive care units, leading to severe nosocomial infections. The rise of multi-drug-resistant strains, particularly carbapenem-resistant A. baumannii, has created formidable challenges for effective treatment. Given the prolonged development cycle and high costs associated with antibiotics, phages have garnered clinical attention as an alternative for combating infections caused by drug-resistant bacteria. However, the utilization of phage therapy encounters notable challenges, including the narrow host spectrum, where each phage targets a limited subset of bacteria, increasing the risk of phage resistance development. Additionally, uncertainties in immune system dynamics during treatment hinder tailoring symptomatic interventions based on patient-specific states. In this study, we isolated two A. baumannii phages from wastewater and conducted a comprehensive assessment of their potential applications. This evaluation included sequencing analysis, genome classification, pH and temperature stability assessments, and in vitro bacterial inhibition assays. Further investigations involved analyzing histological and cytokine alterations in rats undergoing phage cocktail treatment for pneumonia. The therapeutic efficacy of the phages was validated, and transcriptomic studies of rat lung tissue during phage treatment revealed crucial changes in the immune system. The findings from our study underscore the potential of phages for future development as a treatment strategy and offer compelling evidence regarding immune system dynamics throughout the treatment process.IMPORTANCEDue to the growing problem of multi-drug-resistant bacteria, the use of phages is being considered as an alternative to antibiotics, and the genetic safety and application stability of phages determine the potential of phage application. The absence of drug resistance genes and virulence genes in the phage genome can ensure the safety of phage application, and the fact that phage can remain active in a wide range of temperatures and pH is also necessary for application. In addition, the effect evaluation of preclinical studies is especially important for clinical application. By simulating the immune response situation during the treatment process through mammalian models, the changes in animal immunity can be observed, and the effect of phage therapy can be further evaluated. Our study provides compelling evidence that phages hold promise for further development as therapeutic agents for Acinetobacter baumannii infections.

Chemical compositions of Eucalyptus sp. Essential oils and the evaluation of their combinations as a promising treatment against ear bacterial infections

BackgroundThe chemical composition and biological activities of Eucalyptus essential oils (EOs) have been documented in numerous studies against multiple infectious diseases. The antibacterial activity of individual Eucalyptus EOs against strains that cause ear infections was investigated in our previous study. The study's antibacterial activity was promising, which prompted us to explore this activity further with EO blends.MethodsWe tested 15 combinations (9 binary combinations and 6 combinations of binary combinations) of Eucalyptus EOs extracted by hydrodistillation from eight Tunisian Eucalyptus species dried leaves against six bacterial strains responsible for ear infections: three bacterial isolates (Haemophilus influenzae, Haemophilus parainfluenzae, and Klebsiella pneumoniae) and three reference bacteria strains (Pseudomonas aeruginosa, ATTC 9027; Staphylococcus aureus, ATCC 6538; and Escherichia coli, ATCC 8739). The EOs were analyzed using GC/FID and GC/MS. The major compounds, as well as all values obtained from the bacterial growth inhibition assay, were utilized for statistical analysis.ResultsThe antibacterial activity of the EO blends exhibited significant variation within Eucalyptus species, bacterial strains, and the applied methods. Principal component analysis (PCA) and hierarchical cluster analysis (HCA), based on the diameters of the inhibition zone, facilitated the identification of two major groups and ten subgroups based on the level of antibacterial activity. The highest antibacterial activity was observed for the mixture of EOs extracted from E. panctata, E. accedens, and E. cladoclayx (paac) as well as E. panctata, E. wandoo, E. accedens, and E. cladoclayx (pwac) using the disc diffusion method. Additionally, significant activity was noted with EOs extracted from E. panctata, E. wandoo (pw) and E. panctata, E. accedens (pa) using the broth microdilution method.ConclusionOur findings suggest that certain EO combinations (paac, pwac, pw, and pa) could be considered as potential alternative treatment for ear infections due to their demonstrated highly promising antibacterial activities.

A large scale bacterial attraction assay: A new quantitative bacterial migration assay suitable for genetic screens.

Bacteria use various motility mechanisms to explore their environments. Chemotaxis is the ability of a motile bacterial cell to direct its movement in response to chemical gradients. A number of methods have been developed and widely used to study chemotactic responses to chemoeffectors including capillary, agar plug, microscopic slide, and microfluidic assays. While valuable, these assays are primarily designed to monitor rapid chemotactic responses to chemoeffectors on a small scale, which poses challenges in collecting large quantities of attracted bacteria. Consequently, these setups are not ideal for experiments like forward genetic screens. To overcome this limitation, we developed the Large Scale Bacterial Attraction assay (LSBA), which relies on the use of a Nalgene™ Reusable Filter Unit and other materials commonly found in laboratories. We validate the LSBA by investigating chemoeffector kinetics in the setup and by using chemoattractants to quantify the chemotactic response of wild-type, and motility impaired strains of the plant pathogenic bacterium Xanthomonas campestris pv. campestris and the environmental bacterium Shewanella oneidensis. We show that the LSBA establishes a long lasting chemoeffector gradient, that the setup can be used to quantify bacterial migration over time and that the LSBA offers the possibility to collect high numbers of attracted bacteria, making it suitable for genetic screens.

Inhibition mechanism of Rhizoctonia solani by pectin-coated iron metal-organic framework nanoparticles and evidence of an induced defense response in rice

Nanocrop protectants have attracted much attention as sustainable platforms for controlling pests and diseases and improving crop nutrition. Here, we reported the fungicidal activity and disease inhibition potential of pectin-coated metal-iron organic framework nanoparticles (Fe-MOF-PT NPs) against rice stripe blight (RSB). An in vitro bacterial inhibition assay showed that Fe-MOF-PT NPs (80 mg/L) significantly inhibited mycelial growth and nucleus formation. The Fe-MOF-PT NPs adsorbed to the surface of mycelia and induced toxicity by disrupting cell membranes, mitochondria, and DNA. The results of a nontargeted metabolomics analysis showed that the metabolites of amino acids and their metabolites, heterocyclic compounds, fatty acids, and nucleotides and their metabolites were significantly downregulated after treatment with 80 mg/L NPs. The difference in metabolite abundance between the CK and Fe-MOF-PT NPs (80 mg/L) treatment groups was mainly related to nucleotide metabolism, pyrimidine metabolism, purine metabolism, fatty acid metabolism, and amino acid metabolism. The results of the greenhouse experiment showed that Fe-MOF-PT NPs improved rice resistance to R. solani by inhibiting mycelial invasion, enhancing antioxidant enzyme activities, activating the jasmonic acid signaling pathway, and enhancing photosynthesis. These findings indicate the great potential of Fe-MOF-PT NPs as a new RSB disease management strategy and provide new insights into plant fungal disease management.

Remarkable tribo-mechanical, anticorrosion and antibacterial properties of ZnCu/GNPs composite coatings prepared by electro-co-deposition technique

Herein, we report the fabrication of graphene nanoplatelets (GNPs) reinforced zinc-copper (ZnCu) matrix composite coatings on a stainless-steel substrate using electro-co-deposition technique. The influence of varying concentrations of GNPs in the acidic electrolyte bath on the microstructure, chemical composition, phase structure, hardness, wear resistance, corrosion resistance, and antibacterial activity of ZnCu/GNPs composite coating was investigated. The microhardness of the ZnCu/GNPs composite coating with a GNPs concentration of 100 mg/L is compared with pure ZnCu coating, which has a 90 % significant enhancement, while (50 mg/L) has 86 %, and (25 mg/L) has 50 %. Also, ZnCu/GNPs composite coating showed a wear loss of 10 mg for 100 mg/L GNPs sample with an increase in microhardness. The bacterial resistance assays were conducted against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The results reveal a notable improvement in the anti-bacterial activity of the ZnCu/GNPs composite coating. The corrosion rate of the ZnCu/GNPs composite coating in 3.5 wt % NaCl solution steadily decreased when the concentration of GNPs in the electrolyte bath was increased to 100 mg/L. These findings hold great potential for various applications, including healthcare settings where preventing healthcare-associated infections is critical, public infrastructure to prolong the lifespan of structures, and marine coatings to protect against corrosion in harsh marine environments.

Spatial proximity and gene function: a new dimension in prokaryotic gene association network analysis with 3D-GeneNet.

Understanding the biological functions and processes of genes, particularly those not yet characterized, is crucial for advancing molecular biology and identifying therapeutic targets. The hypothesis guiding this study is that the 3D proximity of genes correlates with their functional interactions and relevance in prokaryotes. We introduced 3D-GeneNet, an innovative software tool that utilizes high-throughput sequencing data from chromosome conformation capture techniques and integrates topological metrics to construct gene association networks. Through a series of comparative analyses focused on spatial versus linear distances, we explored various dimensions such as topological structure, functional enrichment levels, distribution patterns of linear distances among gene pairs, and the area under the receiver operating characteristic curve by utilizing model organism Escherichia coli K-12. Furthermore, 3D-GeneNet was shown to maintain good accuracy compared to multiple algorithms (neighbourhood, co-occurrence, coexpression, and fusion) across multiple bacteria, including E. coli, Brucella abortus, and Vibrio cholerae. In addition, the accuracy of 3D-GeneNet's prediction of long-distance gene interactions was identified by bacterial two-hybrid assays on E. coli K-12 MG1655, where 3D-GeneNet not only increased the accuracy of linear genomic distance tripled but also achieved 60% accuracy by running alone. Finally, it can be concluded that the applicability of 3D-GeneNet will extend to various bacterial forms, including Gram-negative, Gram-positive, single-, and multi-chromosomal bacteria through Hi-C sequencing and analysis. Such findings highlight the broad applicability and significant promise of this method in the realm of gene association network. 3D-GeneNet is freely accessible at https://github.com/gaoyuanccc/3D-GeneNet.

Exploring solute binding proteins in Pseudomonas aeruginosa that bind to γ-aminobutyrate and 5-aminovalerate and their role in activating sensor kinases.

The standard method of receptor activation involves the binding of signals or signal-loaded solute binding proteins (SBPs) to sensor domains. Many sensor histidine kinases (SHKs), which are activated by SBP binding, are encoded adjacent to their corresponding sbp gene. We examined three SBPs of Pseudomonas aeruginosa PAO1, encoded near the genes for the AgtS (PA0600) and AruS (PA4982) SHKs, to determine how common this arrangement is. Ligand screening and microcalorimetric studies revealed that the SBPs PA0602 and PA4985 preferentially bind to GABA (KD = 2.3 and 0.58 μM, respectively), followed by 5-aminovalerate (KD = 30 and 1.6 μM, respectively) and ethanoldiamine (KD = 2.3 and 0.58 μM, respectively). In contrast, AgtB (PA0604) exclusively recognizes 5-aminovaleric acid (KD = 2.9 μM). However, microcalorimetric titrations did not show any binding between the AgtS sensor domain and AgtB or PA0602, regardless of the presence of ligands. Similarly, bacterial two-hybrid assays did not demonstrate an interaction between PA4985 and the AruS sensor domain. Therefore, sbp and shk genes located nearby are not always functionally linked. We previously identified PA0222 as a GABA-specific SBP. The presence of three SBPs for GABA may be linked to GABA's role as a trigger for P. aeruginosa virulence.

Multi-Faceted Antimicrobial Efficacy of a Quinoline-Derived Bidentate Copper(II) Ligand Complex and Its Hydrogel Encapsulated Formulation in Methicillin-Resistant Staphylococcus aureus Inhibition and Wound Management.

The emergence of antimicrobial resistance, exemplified by methicillin-resistant Staphylococcus aureus (MRSA), poses a grave threat to public health globally. Over time, MRSA has evolved resistance to multiple antibiotics, challenging conventional treatment strategies. The relentless adaptability of MRSA underscores the urgent need for innovative and targeted antimicrobial approaches to combat this resilient pathogen. Ancient knowledge and practices, along with scientific evidence, have established that metallic copper, and its organic coordination complexes can act as potential antibacterial substances. In search of a smart and effective antimicrobial against MRSA, we designed, synthesized, and characterized a bidentate copper(II) ligand complex (SG-Cu) utilizing a comprehensive array of analytical techniques, including ESI-MS, elemental analysis, X-ray photoelectron spectroscopy, electron paramagnetic resonance spectroscopy, and others. Antibacterial efficacy and mechanism of action of the complex were assessed through bacterial growth analyses, bacterial membrane perturbation assays, ROS elicitation assays, and field emission scanning electron microscopy. SG-Cu was found to maintain robust biocompatibility against the mammalian cell lines HEK-293, WI-38, and NIH/3T3. Remarkably, SG-Cu demonstrated significant biofilm disruptive tendency evidenced by the retardation of sliding motility, reduction in slime production, reduction in biofilm viability, and enhanced biofilm eradication, both in vitro and in urinary catheters. In vivo studies on murine excisional wounds, with SG-Cu impregnated in a palmitic acid conjugated NAVSIQ hexapeptide (PA-NV) hydrogel, revealed the sustained release of SG-Cu from the gel matrix, facilitating accelerated wound healing and effective wound disinfection. This multifaceted investigation highlights the potential of SG-Cu as a versatile option for combating MRSA infections and promoting wound healing, solidifying its claim to be developed into a viable therapeutic.

A novel chaperone-effector-immunity system identified in uropathogenic Escherichia coli UMN026.

Urinary tract infections (UTIs) are very common worldwide. According to their symptomatology, these infections are classified as pyelonephritis, cystitis, or asymptomatic bacteriuria (AB). Approximately 75-95% of UTIs are caused by uropathogenic Escherichia coli (UPEC), which is an extraintestinal bacterium that possesses virulence factors for bacterial adherence and invasion in the urinary tract. In addition, UPEC possesses type 6 secretion systems (T6SS) as virulence mechanisms that can participate in bacterial competition and in bacterial pathogenicity. UPEC UMN026 carries three genes, namely, ECUMN_0231, ECUMN_0232, and ECUMN_0233, which encode three uncharacterized proteins related to the T6SS that are conserved in strains from phylogroups B2 and D and have been proposed as biomarkers of UTIs. To analyze the frequency of the ECUMN_0231, ECUMN_0232, ECUMN_0233, and vgrG genes in UTI isolates, as well as their expression in Luria Bertani (LB) medium and urine; to determine whether these genes are related to UTI symptoms or bacterial competence and to identify functional domains on the putative proteins. The frequency of the ECUMN and vgrG genes in 99 clinical isolates from UPEC was determined by endpoint PCR. The relationship between gene presence and UTI symptomatology was determined using the chi2 test, with p < 0.05 considered to indicate statistical significance. The expression of the three ECUMN genes and vgrG was analyzed by RT-PCR. The antibacterial activity of strain UMN026 was determined by bacterial competence assays. The identification of functional domains and the docking were performed using bioinformatic tools. The ECUMN genes are conserved in 33.3% of clinical isolates from patients with symptomatic and asymptomatic UTIs and have no relationship with UTI symptomatology. Of the ECUMN+ isolates, only five (15.15%, 5/33) had the three ECUMN and vgrG genes. These genes were expressed in LB broth and urine in UPEC UMN026 but not in all the clinical isolates. Strain UMN026 had antibacterial activity against UPEC clinical isolate 4014 (ECUMN-) and E. faecalis but not against isolate 4012 (ECUMN+). Bioinformatics analysis suggested that the ECUMN genes encode a chaperone/effector/immunity system. The ECUMN genes are conserved in clinical isolates from symptomatic and asymptomatic patients and are not related to UTI symptoms. However, these genes encode a putative chaperone/effector/immunity system that seems to be involved in the antibacterial activity of strain UMN026.

Peptides with Antimicrobial Activity in the Saliva of the Malaria Vector Anopheles coluzzii.

Mosquito saliva plays a crucial physiological role in both sugar and blood feeding by helping sugar digestion and exerting antihemostatic functions. During meal acquisition, mosquitoes are exposed to the internalization of external microbes. Since mosquitoes reingest significant amounts of saliva during feeding, we hypothesized that salivary antimicrobial components may participate in the protection of mouthparts, the crop, and the gut by inhibiting bacterial growth. To identify novel potential antimicrobials from mosquito saliva, we selected 11 candidates from Anopheles coluzzii salivary transcriptomic datasets and obtained them either using a cell-free transcription/translation expression system or, when feasible, via chemical synthesis. Hyp6.2 and hyp13, which were predicted to be produced as propeptides and cleaved in shorter mature forms, showed the most interesting results in bacterial growth inhibition assays. Hyp6.2 (putative mature form, 35 amino acid residues) significantly inhibited the growth of Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli and Serratia marcescens) bacteria. Hyp13 (short form, 19 amino acid residues) dose-dependently inhibited E. coli and S. marcescens growth, inducing membrane disruption in both Gram-positive and Gram-negative bacteria as indicated with scanning electron microscopy. In conclusion, we identified two A. coluzzii salivary peptides inhibiting Gram-positive and Gram-negative bacteria growth and possibly contributing to the protection of mosquito mouthparts and digestive tracts from microbial infection during and/or after feeding.