A Dual-Mechanism Antibiotic Kills Gram-Negative Bacteria and Avoids Drug Resistance.

The rise of antibiotic resistance and declining discovery of new antibiotics has created a global health crisis. Of particular concern, no new antibiotic classes have been approved for treating Gram-negative pathogens in decades. Here, we characterize a compound, SCH-79797, that kills both Gram-negative and Gram-positive bacteria through a unique dual-targeting mechanism of action (MoA) with undetectably low resistance frequencies. To characterize its MoA, we combined quantitative imaging, proteomic, genetic, metabolomic, and cell-based assays. This pipeline demonstrates that SCH-79797 has two independent cellular targets, folate metabolism and bacterial membrane integrity, and outperforms combination treatments in killing methicillin-resistant Staphylococcus aureus (MRSA) persisters. Building on the molecular core of SCH-79797, we developed a derivative, Irresistin-16, with increased potency and showed its efficacy against Neisseria gonorrhoeae in a mouse vaginal infection model. This promising antibiotic lead suggests that combining multiple MoAs onto a single chemical scaffold may be an underappreciated approach to targeting challenging bacterial pathogens.

Two broadly conserved families of polyprenyl-phosphate transporters.

Peptidoglycan and almost all surface glycopolymers in bacteria are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP)1-4. These UndP-linked precursors are transported across the membrane and polymerized or directly transferred to surface polymers, lipids or proteins. UndP is then flipped to regenerate the pool of cytoplasmic-facing UndP. The identity of the flippase that catalyses transport has remained unknown. Here, using the antibiotic amphomycin that targets UndP5-7, we identified two broadly conserved protein families that affect UndP recycling. One (UptA) is a member of the DedA superfamily8; the other (PopT) contains the domain DUF368. Genetic, cytological and syntenic analyses indicate that these proteins are UndP transporters. Notably, homologues from Gram-positive and Gram-negative bacteria promote UndP transport in Bacillus subtilis, indicating that recycling activity is broadly conserved among family members. Inhibitors of these flippases could potentiate the activity of antibiotics targeting the cell envelope.

Xenobiotics shape the physiology and gene expression of the active human gut microbiome.

The human gut contains trillions of microorganisms that influence our health by metabolizing xenobiotics, including host-targeted drugs and antibiotics. Recent efforts have characterized the diversity of this host-associated community, but it remains unclear which microorganisms are active and what perturbations influence this activity. Here, we combine flow cytometry, 16S rRNA gene sequencing, and metatranscriptomics to demonstrate that the gut contains a distinctive set of active microorganisms, primarily Firmicutes. Short-term exposure to a panel of xenobiotics significantly affected the physiology, structure, and gene expression of this active gut microbiome. Xenobiotic-responsive genes were found across multiple bacterial phyla, encoding antibiotic resistance, drug metabolism, and stress response pathways. These results demonstrate the power of moving beyond surveys of microbial diversity to better understand metabolic activity, highlight the unintended consequences of xenobiotics, and suggest that attempts at personalized medicine should consider interindividual variations in the active human gut microbiome.

Microbiota-derived lantibiotic restores resistance against vancomycin-resistant Enterococcus.

Intestinal commensal bacteria can inhibit dense colonization of the gut by vancomycin-resistant Enterococcus faecium (VRE), a leading cause of hospital-acquired infections1,2. A four-strained consortium of commensal bacteria that contains Blautia producta BPSCSK can reverse antibiotic-induced susceptibility to VRE infection3. Here we show that BPSCSK reduces growth of VRE by secreting a lantibiotic that is similar to the nisin-A produced by Lactococcus lactis. Although the growth of VRE is inhibited by BPSCSK and L. lactis in vitro, only BPSCSK colonizes the colon and reduces VRE density in vivo. In comparison to nisin-A, the BPSCSK lantibiotic has reduced activity against intestinal commensal bacteria. In patients at high risk of VRE infection, high abundance of the lantibiotic gene is associated with reduced density of E. faecium. In germ-free mice transplanted with patient-derived faeces, resistance to VRE colonization correlates with abundance of the lantibiotic gene. Lantibiotic-producing commensal strains of the gastrointestinal tract reduce colonization by VRE and represent potential probiotic agents to re-establish resistance to VRE.

Insights into the molecular mechanism of translation inhibition by the ribosome-targeting antibiotic thermorubin.

Thermorubin (THR) is an aromatic anthracenopyranone antibiotic active against both Gram-positive and Gram-negative bacteria. It is known to bind to the 70S ribosome at the intersubunit bridge B2a and was thought to inhibit factor-dependent initiation of translation and obstruct the accommodation of tRNAs into the A site. Here, we show that thermorubin causes ribosomes to stall in vivo and in vitro at internal and termination codons, thereby allowing the ribosome to initiate protein synthesis and translate at least a few codons before stalling. Our biochemical data show that THR affects multiple steps of translation elongation with a significant impact on the binding stability of the tRNA in the A site, explaining premature cessation of translation. Our high-resolution crystal and cryo-EM structures of the 70S-THR complex show that THR can co-exist with P- and A-site tRNAs, explaining how ribosomes can elongate in the presence of the drug. Remarkable is the ability of THR to arrest ribosomes at the stop codons. Our data suggest that by causing structural re-arrangements in the decoding center, THR interferes with the accommodation of tRNAs or release factors into the ribosomal A site.

Genome-encoded ABCF factors implicated in intrinsic antibiotic resistance in Gram-positive bacteria: VmlR2, Ard1 and CplR.

Genome-encoded antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F subfamily (ARE-ABCFs) mediate intrinsic resistance in diverse Gram-positive bacteria. The diversity of chromosomally-encoded ARE-ABCFs is far from being fully experimentally explored. Here we characterise phylogenetically diverse genome-encoded ABCFs from Actinomycetia (Ard1 from Streptomyces capreolus, producer of the nucleoside antibiotic A201A), Bacilli (VmlR2 from soil bacterium Neobacillus vireti) and Clostridia (CplR from Clostridium perfringens, Clostridium sporogenes and Clostridioides difficile). We demonstrate that Ard1 is a narrow spectrum ARE-ABCF that specifically mediates self-resistance against nucleoside antibiotics. The single-particle cryo-EM structure of a VmlR2-ribosome complex allows us to rationalise the resistance spectrum of this ARE-ABCF that is equipped with an unusually long antibiotic resistance determinant (ARD) subdomain. We show that CplR contributes to intrinsic pleuromutilin, lincosamide and streptogramin A resistance in Clostridioides, and demonstrate that C. difficile CplR (CDIF630_02847) synergises with the transposon-encoded 23S ribosomal RNA methyltransferase Erm to grant high levels of antibiotic resistance to the C. difficile 630 clinical isolate. Finally, assisted by uORF4u, our novel tool for detection of upstream open reading frames, we dissect the translational attenuation mechanism that controls the induction of cplR expression upon an antibiotic challenge.

Ring-fused 2-pyridones effective against multidrug-resistant Gram-positive pathogens and synergistic with standard-of-care antibiotics.

The alarming rise of multidrug-resistant Gram-positive bacteria has precipitated a healthcare crisis, necessitating the development of new antimicrobial therapies. Here we describe a new class of antibiotics based on a ring-fused 2-pyridone backbone, which are active against vancomycin-resistant enterococci (VRE), a serious threat as classified by the Centers for Disease Control and Prevention, and other multidrug-resistant Gram-positive bacteria. Ring-fused 2-pyridone antibiotics have bacteriostatic activity against actively dividing exponential phase enterococcal cells and bactericidal activity against nondividing stationary phase enterococcal cells. The molecular mechanism of drug-induced killing of stationary phase cells mimics aspects of fratricide observed in enterococcal biofilms, where both are mediated by the Atn autolysin and the GelE protease. In addition, combinations of sublethal concentrations of ring-fused 2-pyridones and standard-of-care antibiotics, such as vancomycin, were found to synergize to kill clinical strains of VRE. Furthermore, a broad range of antibiotic resistant Gram-positive pathogens, including those responsible for the increasing incidence of antibiotic resistant healthcare-associated infections, are susceptible to this new class of 2-pyridone antibiotics. Given the broad antibacterial activities of ring-fused 2-pyridone compounds against Gram-positive (GmP) bacteria we term these compounds GmPcides, which hold promise in combating the rising tide of antibiotic resistant Gram-positive pathogens.

Design of Ultrasound-Driven Charge Interference Therapy for Wound Infection.

Wound infections, especially those caused by pathogenic bacteria, present a considerable public health concern due to associated complications and poor therapeutic outcomes. Herein, we developed antibacterial nanoparticles, namely, PGTP, by coordinating guanidine derivatives with a porphyrin-based sonosensitizer. The synthesized PGTP nanoparticles, characterized by their strong positive charge, effectively disrupted the bacterial biosynthesis process through charge interference, demonstrating efficacy against both Gram-negative and Gram-positive bacteria. Additionally, PGTP nanoparticles generated reactive oxygen species under ultrasound stimulation, resulting in the disruption of biofilm integrity and efficient elimination of pathogens. RNA-seq analysis unveiled the detailed mechanism of wound healing, revealing that PGTP nanoparticles, when coupled with ultrasound, impair bacterial metabolism by interfering with the synthesis and transcription of amino acids. This study presents a novel approach to combatting wound infections through ultrasound-driven charge-interfering therapy, facilitated by advanced antibacterial nanomaterials.

Biguanide-Vancomycin Conjugates are Effective Broad-Spectrum Antibiotics against Actively Growing and Biofilm-Associated Gram-Positive and Gram-Negative ESKAPE Pathogens and Mycobacteria.

Strategies to increase the efficacy and/or expand the spectrum of activity of existing antibiotics provide a potentially fast path to clinically address the growing crisis of antibiotic-resistant infections. Here, we report the synthesis, antibacterial efficacy, and mechanistic activity of an unprecedented class of biguanide-antibiotic conjugates. Our lead biguanide-vancomycin conjugate, V-C6-Bg-PhCl (5e), induces highly effective cell killing with up to a 2 orders-of-magnitude improvement over its parent compound, vancomycin (V), against vancomycin-resistant enterococcus. V-C6-Bg-PhCl (5e) also exhibits improved activity against mycobacteria and each of the ESKAPE pathogens, including the Gram-negative organisms. Furthermore, we uncover broad-spectrum killing activity against biofilm-associated Gram-positive and Gram-negative bacteria as well as mycobacteria not observed for clinically used antibiotics such as oritavancin. Mode-of-action studies reveal that vancomycin-like cell wall synthesis inhibition with improved efficacy attributed to enhanced engagement at vancomycin binding sites through biguanide association with relevant cell-surface anions for Gram-positive and Gram-negative bacteria. Due to its potency, remarkably broad activity, and lack of acute mammalian cell toxicity, V-C6-Bg-PhCl (5e) is a promising candidate for treating antibiotic-resistant infections and notoriously difficult-to-treat slowly growing and antibiotic-tolerant bacteria associated with chronic and often incurable infections. More generally, this study offers a new strategy (biguanidinylation) to enhance antibiotic activity and facilitate clinical entry.

IMT-P8 potentiates Gram-positive specific antibiotics in intrinsically resistant Gram-negative bacteria.

Gram-negative bacteria (GNB) pose a major global public health challenge as they exhibit a remarkable level of resistance to antibiotics. One of the factors responsible for promoting resistance against a wide range of antibiotics is the outer membrane (OM) of Gram-negative bacteria. The OM acts as a barrier that prevents the entry of numerous antibiotics by reducing their influx (due to membrane impermeability) and enhancing their efflux (with the help of efflux pumps). Our study focuses on analyzing the effect of IMT-P8, a cell-penetrating peptide (CPP), to enhance the influx of various Gram-positive specific antibiotics in multi-drug resistant Gram-negative pathogens. In the mechanistic experiments, IMT-P8 permeabilizes the OM at the same concentrations at which it enhances the activity of various antibiotics against GNB. Cytoplasmic membrane permeabilization was also observed at these concentrations, indicating that IMT-P8 acts on both the outer and cytoplasmic membranes. IMT-P8 interferes with the intrinsic resistance mechanism of GNB and has the potential to make Gram-positive specific antibiotics effective against GNB. IMT-P8 extends the post-antibiotic effect and in combination with antibiotics shows anti-persister activity. The IMT-P8/fusidic acid combination is effective in eliminating intracellular pathogens. IMT-P8 with negligible toxicity displayed good efficacy in murine lung and thigh infection models. Based on these findings, IMT-P8 is a potential antibiotic adjuvant to treat Gram-negative bacterial infections that pose a health hazard.

Optimization, characterization and biosafety of carotenoids produced from whey using Micrococcus luteus.

This study aimed to optimize the production of carotenoid pigments from Micrococcus luteus (ATCC 9341) through the statistical screening of media components and the characterization of antimicrobial, antioxidant, cytogenetic and cytotoxic activities. A BOX-Behnken design was used to assess the effects of whey concentration, inoculum size, pH, temperature, and agitation speed on carotenoid yield. The optimum combination increased production to 2.19 g/L, with a productivity of 0.045 g L-1 h-1 and a productivity yield of 0.644 g/g, as confirmed by an observed carotene production of 2.19 g/L. The final response surface model fitting the data had an R2 of 0.9461. High-performance liquid chromatography (HPLC) analysis identified 12 carotenoid pigment compounds produced by M. luteus. The extracts displayed moderate antimicrobial efficacy against Gram-positive bacteria such as Bacillus cereus (ATCC 11778), Staphylococcus aureus (ATCC 6538), and E. faecalis (ATCC 19433), with inhibition zone diameters (IZD) of 29.0, 14.0, and 37.0 mm, respectively, at 1000 µg/mL. However, its effectiveness against Gram-negative bacteria is limited. In comparison, tetracycline exhibited greater antimicrobial potency. The IC50 value of carotenoids was used to indicate the antioxidant activity. IC50 value from the DPPH assay was 152.80 mg/100mL. An IC50 cytotoxicity value greater than 300 µg/mL was found against normal mouse liver cells, with over 68% cell viability even at 300 µg/mL, indicating low toxicity. Histological structure studies revealed normal myocardial muscle tissue, lung tissue, and kidney tissue sections, whereas liver tissue sections revealed ballooning degeneration of hepatocytes and disorganization of hepatic cords. Cytogenetic parameters revealed that the carotene treatment group had a mitotic index (70%) lower than that of the control but higher than that of the positive control, mitomycin, and did not substantially increase numerical (1.2%) or structural aberrations compared with those of the control, suggesting a lack of genotoxic effects under the experimental conditions. In conclusion, optimized culture conditions enhanced carotenoid yields from M. luteus, and the extracts displayed promising bioactivity as moderate antibiotics against certain gram-positive bacteria and as antioxidants. The high IC50 values demonstrate biosafety. Overall, this bioprocess for enhanced carotenoid production coupled with bioactivity profiling and low cytotoxicity support the application of M. luteus carotenoids.

Ferula latisecta gels for synthesis of zinc/silver binary nanoparticles: antibacterial effects against gram-negative and gram-positive bacteria and physicochemical characteristics.

This study explores the potential antibacterial applications of zinc oxide nanoparticles (ZnO NPs) enhanced with silver (Ag) using plant gel (ZnO-AgO NPs). The problem addressed is the increasing prevalence of pathogenic bacteria and the need for new, effective antimicrobial agents. ZnO NPs possess distinctive physicochemical properties that enable them to selectively target bacterial cells. Their small size and high surface area-to-volume ratio allow efficient cellular uptake and interaction with bacterial cells. In this study, the average size of the synthesized ZnO-Ag nanoparticles was 77.1 nm, with a significant standard deviation of 33.7 nm, indicating a wide size distribution. The nanoparticles demonstrated remarkable antibacterial efficacy against gram-negative and gram-positive bacteria, with inhibition zones of 14.33 mm for E. coli and 15.66 mm for B. subtilis at a concentration of 300 µg/ml. Minimum inhibitory concentrations (MIC) were determined to be 100 µg/ml for E. coli and 75 µg/ml for S. saprophyticus. Additionally, ZnO-Ag NPs exhibited excellent biocompatibility, making them appropriate for various pharmacological uses. This study utilizes Ferula latisecta gels, offering a sustainable and eco-friendly approach to nanoparticle synthesis. Incorporating of Ag into ZnO NPs significantly enhances their antimicrobial properties, with the combined results showing great inhibition effects on pathogenic microbes. The findings suggest that ZnO-Ag NPs could be a promising candidate for addressing the challenges posed by drug-resistant bacterial infections and enhancing antimicrobial treatments.

A New Theranostic Platform Against Gram-Positive Bacteria Based on Near-Infrared-Emissive Aggregation-Induced Emission Nanoparticles.

Infections induced by Gram-positive bacteria pose a great threat to public health. Antibiotic therapy, as the first chosen strategy against Gram-positive bacteria, is inevitably associated with antibiotic resistance selection. Novel therapeutic strategies for the discrimination and inactivation of Gram-positive bacteria are thus needed. Here, a specific type of aggregation-induced emission luminogen (AIEgen) with near-infrared fluorescence emission as a novel antibiotic-free therapeutic strategy against Gram-positive bacteria is proposed. With the combination of a positively charged group into a highly twisted architecture, self-assembled AIEgens (AIE nanoparticles (NPs)) at a relatively low concentration (5 µm) exhibited specific binding and photothermal effect against living Gram-positive bacteria both in vitro and in vivo. Moreover, toxicity assays demonstrated excellent biocompatibility of AIE NPs at this concentration. All these properties make the AIE NPs as a novel generation of theranostic platform for combating Gram-positive bacteria and highlight their promising potential for in vivo tracing of such bacteria.

Exploration of Rhenium Bisquinoline Tricarbonyl Complexes for their Antibacterial Properties.

Metal complexes have emerged as a promising source for novel classes of antibacterial agents to combat the rise of antimicrobial resistance around the world. In the exploration of the transition metal chemical space for novel metalloantibiotics, the rhenium tricarbonyl moiety has been identified as a promising scaffold. Here we have prepared eight novel rhenium bisquinoline tricarbonyl complexes and explored their antibacterial properties. Significant activity against both Gram-positive and Gram-negative bacteria was observed. However, all complexes also showed significant toxicity against human cells, putting into question the prospects of this specific rhenium compound class as metalloantibiotics. To better understand their biological effects, we conduct the first mode of action studies on rhenium bisquinoline complexes and show that they are able to form pores through bacterial membranes. Their straight-forward synthesis and tuneability suggests that further optimisation of this compound class could lead to compounds with enhanced bacterial specificity.

Raffinocyclicin is a novel plasmid-encoded circular bacteriocin produced by Lactococcus raffinolactis with broad-spectrum activity against many gram-positive food pathogens.

This study describes the discovery and characterization of raffinocyclicin, a novel plasmid-encoded circular bacteriocin, produced by the raw milk isolate Lactococcus raffinolactis APC 3967. This bacteriocin has a molecular mass of 6,092 Da and contains 61 amino acids with a three-amino acid leader peptide. It shows the highest identity to the circular bacteriocins bacicyclicin XIN-1 (42.62%), aureocyclicin 4185 (42.62%), and garvicin ML (41.53%). A broad inhibitory spectrum includes strains from Staphylococcus, Enterococcus, Streptococcus, Micrococcus, Lactobacillus, Leuconostoc, and Listeria, in addition to a pronounced inhibitory effect against Lactococcus and Clostridium. It displays low sensitivity to trypsin, most likely as a result of its circular nature. The raffinocyclicin gene cluster is composed of 10 genes: 6 core genes, genes encoding an accessory three-component ABC transporter (rafCDE), and a putative transcriptional regulator related to the MutR family. A lack of inhibitory activity in the cell-free supernatant combined with the pronounced activity of cell extracts suggests that the majority of raffinocyclicin is associated with the cell rather than being released to the extracellular environment. This is the first report of a bacteriocin produced by the L. raffinolactis species.IMPORTANCEThe present study aimed to characterize raffinocyclicin, a novel circular bacteriocin produced by the lactic acid bacteria Lactococcus raffinolactis APC 3967. Bacteriocins are generally cationic and hydrophobic peptides with antimicrobial activity, which present diverse biotechnological properties of interest for the food industry. Raffinocyclicin inhibits a wide range of bacteria, including foodborne pathogens, and is stable against different treatments which suggest its potential as a natural biopreservative. Whole-genome sequencing and the genetic analysis of the raffinocyclicin gene cluster showed that it is encoded by plasmid that could be used in the future to transfer the ability to produce the bacteriocin to other lactic acid bacteria for industrial applications. These results together highlight the potential of this novel antimicrobial as a biopreservative to be used by the food industry.

A multisite validation of a two hours antibiotic susceptibility flow cytometry assay directly from positive blood cultures.

BACKGROUND: Rapid antimicrobial susceptibility testing (AST) is urgently needed to provide safer treatment to counteract antimicrobial resistance. This is critical in septic patients, because resistance increases empiric therapy uncertainty and the risk of a poor outcome. We validate a novel 2h flow cytometry AST assay directly from positive blood cultures (PBC) by using a room temperature stable FASTgramneg and FASTgrampos kits (FASTinov® Porto, Portugal) in three sites: FASTinov (site-1), Hospital Ramon y Cajal, Madrid, Spain (site-2) and Centro Hospitalar S. João, Porto, Portugal (site-3). A total of 670 PBC were included: 333 spiked (site-1) and 337 clinical PBC (151 site-2 and 186 site-3): 367 gram-negative and 303 gram-positive. Manufacturer instructions were followed for sample preparation, panel inoculation, incubation (1h/37ºC) and flow cytometry analysis using CytoFlex (Site-1 and -2) or DxFlex (site-3) both instruments from Beckman-Coulter, USA. RESULTS: A proprietary software (bioFAST) was used to immediately generate a susceptibility report in less than 2 h. In parallel, samples were processed according to reference AST methods (disk diffusion and/or microdilution) and interpreted with EUCAST and CLSI criteria. Additionally, ten samples were spiked in all sites for inter-laboratory reproducibility. Sensitivity and specificity were >95% for all antimicrobials. Reproducibility was 96.8%/95.0% for FASTgramneg and 95.1%/95.1% for FASTgrampos regarding EUCAST/CLSI criteria, respectively. CONCLUSION: FASTinov® kits consistently provide ultra-rapid AST in 2h with high accuracy and reproducibility on both Gram-negative and Gram-positive bacteria. This technology creates a new paradigm in bacterial infection management and holds the potential to significantly impact septic patient outcomes and antimicrobial stewardship.

Sorbate metal complexes as newer antibacterial, antibiofilm, and anticancer compounds.

BACKGROUND: The ineffectiveness of treatments for infections caused by biofilm-producing pathogens and human carcinoma presents considerable challenges for global public health organizations. To tackle this issue, our study focused on exploring the potential of synthesizing new complexes of Co(II), Cu(II), Ni(II), and Zn(II) with sorbic acid to enhance its antibacterial, antibiofilm, and anticancer properties. METHODS: Four novel complexes were synthesized as solid phases by reacting sorbic acid with Co(II), Cu(II), Ni(II), and Zn(II). These complexes were characterized by various technique, including infrared spectra, UV-Visible spectroscopy, proton nuclear magnetic resonance (1H NMR), and thermal analysis techniques, including thermogravimetry (TG). RESULTS: The data acquired from all investigated chemical characterization methods confirmed the chemical structure of the sorbate metal complexes. These complexes exhibited antibacterial and antibiofilm properties against both Gram-positive and Gram-negative bacteria. Furthermore, these complexes enhanced the antibacterial effects of commonly used antibiotics, such as gentamicin and imipenem, with fractional inhibitory concentration (FIC) indices ≤ 0.5. Notably, the Cu(II) complex displayed the most potent antibacterial and antibiofilm activities, with minimum inhibitory concentration (MIC) values of 312.5 µg/mL and 625.0 µg/mL for Bacillus cereus and Escherichia coli, respectively. Additionally, in vitro assays using the methyl thiazolyl tetrazolium (MTT) method showed inhibitory effects on the growth of the human colon carcinoma cell line (HCT-116 cells) following treatment with the investigated metal complexes. The IC50 values for Co(II), Cu(II), Zn(II), and Ni(II) were 3230 µg/mL, 2110 µg/mL, 3730 µg/mL, and 2240 µg/mL, respectively. CONCLUSION: Our findings offer potential for pharmaceutical companies to explore the development of novel combinations involving traditional antibiotics or anticancer drugs with sorbate copper complex.

Antimicrobial lipids loaded on lectin display reduced MIC, curtail pathogenesis and protect zebrafish from reinfection by immunomodulation.

Antibiotic resistance and re-emergence of highly resistant pathogens is a grave concern everywhere and this has consequences for all kinds of human activities. Herein, we showed that N-palmitoylethanolamine-derived cationic lipid (cN16E) had a lower minimum inhibitory concentration (MIC) against both Gram-positive and Gram-negative bacteria when it was loaded with Butea monosperma seed lectin (BMSL). The analysis using lectin-FITC conjugate labelling indicated that the improved antibacterial activity of BMSL conjugation was due to bacterial cell surface glycan recognition. Live and dead staining experiments revealed that the BMSL-cN16E conjugate (BcN16E) exerts antibacterial activity by damaging the bacterial membrane. BcN16E antimicrobial activity was demonstrated using an infected zebrafish animal model because humans have 70 % genetic similarity to zebrafish. BcN16E therapeutic potential was established successfully by rescuing fish infected with uropathogenic Escherichia coli (UPEC). Remarkably, the rescued infected fish treated with BcN16E prevented reinfection without further therapy, indicating BcN16E immunomodulatory potential. Thus, the study examined the expression of immune-related genes, including tnfα, ifnγ, il-1ß, il-4, il-10, tlr-2, etc. There was a significant elevation in the expression of all these genes compared to control and fish treated with BMSL or cN16E alone. Interestingly, when the rescued zebrafish were reinfected with the same pathogen, the levels of expression of these genes were many folds higher than seen earlier. Radial immune diffusion analyses (RIA) using zebrafish serum revealed antibody production during the initial infection and treatment. Interestingly, reinfected fish had significant immunoprecipitation in RIA, a feature absent in the groups treated with cN16E, BMSL, and control. These results clearly show that the BcN16E complex not only rescued infected zebrafish but also conferred long-lasting protection in terms of immunomodulation that protects against multiple reinfections. The findings support that BcN16E has immense potential as a novel immunostimulant for various biomedical applications.

The Complete Assessment of Small Molecule and Peptidomimetic Inhibitors of Sortase A Towards Antivirulence Treatment.

This review covers the most recent advances in the development of inhibitors for the bacterial enzyme sortase A (SrtA). Sortase A (SrtA) is a critical virulence factor, present ubiquitously in Gram-positive bacteria of which many are pathogenic. Sortases are key enzymes regulating bacterial adherence to host cells, by anchoring extracellular matrix-binding proteins to the bacterial outer cell wall. By targeting virulence factors, effective treatment can be achieved, without inducing antibiotic resistance to the treatment. This is a potentially more sustainable, long-term approach to treating bacterial infections, including ones that display multiple resistance to current therapeutics. There are many promising approaches available for SrtA inhibition, some of which have the potential to advance into further clinical development, with peptidomimetic and in vivo active small molecules being among the most promising. There are currently no approved drugs on the market targeting SrtA, despite its promise, adding to the relevance of this review article, as it extends to the pharmaceutical industry additionally to academic researchers.

Vicia ervilia lectin (VEA) has an antibiofilm effect on both Gram-positive and Gram-negative pathogenic bacteria.

Bacterial growing resistance to antibiotics poses a critical threat to global health. This study investigates, for the first time, the antibiofilm properties of Vicia ervilia agglutinin (VEA) from six different V. ervilia accessions against pathogenic bacteria, and the yeast Candida albicans. In the absence of antimicrobial properties, purified VEA significantly inhibited biofilm formation, both in Gram-positive and Gram-negative bacteria, but not in C. albicans. With an inhibitory concentration ranging from 100 to 500 µg/ml, the VEA antibiofilm activity was more relevant against the Gram-positive bacteria Streptococcus aureus and Staphylococcus epidermidis, whose biofilm was reduced up to 50% by VEA purified from accessions #5 and #36. VEA antibiofilm variability between accessions was observed, likely due to co-purified small molecules rather than differences in VEA protein sequences. In conclusion, VEA seed extracts from the accessions with the highest antibiofilm activity could represent a valid approach for the development of an effective antibiofilm agent.