Increased Innate Immune Susceptibility in Hyperpigmented Bacteriophage-Resistant Mutants of Pseudomonas aeruginosa.

Bacteriophage (phage) therapy is an alternative to traditional antibiotic treatments that is particularly important for multidrug-resistant pathogens, such as Pseudomonas aeruginosa. Unfortunately, phage resistance commonly arises during treatment as bacteria evolve to survive phage predation. During in vitro phage treatment of a P. aeruginosa-type strain, we observed the emergence of phage-resistant mutants with brown pigmentation that was indicative of pyomelanin. As increased pyomelanin (due to hmgA gene mutation) was recently associated with enhanced resistance to hydrogen peroxide and persistence in experimental lung infection, we questioned if therapeutic phage applications could inadvertently select for hypervirulent populations. Pyomelanogenic phage-resistant mutants of P. aeruginosa PAO1 were selected for upon treatment with three distinct phages. Phage-resistant pyomelanogenic mutants did not possess increased survival of pyomelanogenic ΔhmgA in hydrogen peroxide. At the genomic level, large (~300 kb) deletions in the phage-resistant mutants resulted in the loss of ≥227 genes, many of which had roles in survival, virulence, and antibiotic resistance. Phage-resistant pyomelanogenic mutants were hypersusceptible to cationic peptides LL-37 and colistin and were more easily cleared in human whole blood, serum, and a murine infection model. Our findings suggest that hyperpigmented phage-resistant mutants that may arise during phage therapy are markedly less virulent than their predecessors due to large genomic deletions. Thus, their existence does not present a contraindication to using anti-pseudomonal phage therapy, especially considering that these mutants develop drug susceptibility to the familiar FDA-approved antibiotic, colistin.

(I-3,II-3)-Biacacetin-mediated cell death involves mitochondria.

Dysregulation of the dynamic balance between cell proliferation and cell death leads to several malignancies including cancer. Biflavones are known to possess anti-proliferative activity against numerous cancer cell lines. The current study was undertaken to understand the mechanism of action of the biflavonoid (I-3,II-3)-biacacetin on MDA-MB-231. Biacacetin induces dose-dependent cell death in MDA-MB-231 cells from concentrations as low as 0.5 µM, which was further confirmed by an increase in sub-G1 cells. Furthermore, the cell death induced by biacacetin was found to be mitochondria-dependent, since cells devoid of mitochondria were viable in the presence of biacacetin even at the highest concentration tested (25 µM). Fluorescence studies clearly indicated nuclear changes and apoptotic body formation that are characteristic of apoptosis. These results were further corroborated by studies that demonstrate biacacetin to regulate several key markers of apoptosis like Caspase 3, p53, Bax, and poly-ADP-ribose polymerase-1. Furthermore, biacacetin did not induce cell death in normal macrophage cell line, RAW at concentrations up to 15 µM. In addition to MDA-MB-231 cells, biacacetin also induces apoptotic cell death in the highly chemo-resistant cell line, OVISE, where the cells stained positive for annexin. Biacacetin also induces cell death in the highly malignant fibrosarcoma cell line HT1080. Furthermore, biacacetin also induces significant cell death (50%) in 3D tumor spheroids, at a concentration of 25 µM. Taken together, these results provide an understanding of biacacetin-mediated cell death and thereby provides a strong basis for the use of such compounds as novel templates for anti-cancer therapeutics.

Natural Product Anacardic Acid from Cashew Nut Shells Stimulates Neutrophil Extracellular Trap Production and Bactericidal Activity.

Emerging antibiotic resistance among pathogenic bacteria is an issue of great clinical importance, and new approaches to therapy are urgently needed. Anacardic acid, the primary active component of cashew nut shell extract, is a natural product used in the treatment of a variety of medical conditions, including infectious abscesses. Here, we investigate the effects of this natural product on the function of human neutrophils. We find that anacardic acid stimulates the production of reactive oxygen species and neutrophil extracellular traps, two mechanisms utilized by neutrophils to kill invading bacteria. Molecular modeling and pharmacological inhibitor studies suggest anacardic acid stimulation of neutrophils occurs in a PI3K-dependent manner through activation of surface-expressed G protein-coupled sphingosine-1-phosphate receptors. Neutrophil extracellular traps produced in response to anacardic acid are bactericidal and complement select direct antimicrobial activities of the compound.

Nitric Oxide and ERK mediates regulation of cellular processes by Ecdysterone.

The complex process of wound healing is a major problem associated with diabetes, venous or arterial disease, old age and infection. A wide range of pharmacological effects including anabolic, anti-diabetic and hepato-protective activities have been attributed to Ecdysterone. In earlier studies, Ecdysterone has been shown to modulate eNOS and iNOS expression in diabetic animals and activate osteogenic differentiation through the Extracellular-signal-Regulated Kinase (ERK) pathway in periodontal ligament stem cells. However, in the wound healing process, Ecdysterone has only been shown to enhance granulation tissue formation in rabbits. There have been no studies to date, which elucidate the molecular mechanism underlying the complex cellular process involved in wound healing. The present study, demonstrates a novel interaction between the phytosteroid Ecdysterone and Nitric Oxide Synthase (NOS), in an Epidermal Growth Factor Receptor (EGFR)-dependent manner, thereby promoting cell proliferation, cell spreading and cell migration. These observations were further supported by the 4-amino-5-methylamino- 2' ,7' -difluorofluorescein diacetate (DAF FM) fluorescence assay which indicated that Ecdysterone activates NOS resulting in increased Nitric Oxide (NO) production. Additionally, studies with inhibitors of both the EGFR and ERK, demonstrated that Ecdysterone activates NOS through modulation of EGFR and ERK. These results clearly demonstrate, for the first time, that Ecdysterone enhances Nitric Oxide production and modulates complex cellular processes by activating ERK1/2 through the EGF pathway.

Anacardic acid inhibits gelatinases through the regulation of Spry2, MMP-14, EMMPRIN and RECK.

Earlier studies from our laboratory have identified Anacardic acid (AA) as a potent inhibitor of gelatinases (MMP-2 and 9), which are over-expressed in a wide variety of cancers (Omanakuttan et al., 2012). Disruption of the finely tuned matrix metalloproteinase (MMP) activator/inhibitor balance plays a decisive role in determining the fate of the cell. The present study demonstrates for the first time, that in addition to regulating the expression as well as activity of gelatinases, AA also inhibits the expression of its endogenous activators like MMP-14 and Extracellular Matrix MetalloProteinase Inducer (EMMPRIN) and induces the expression of its endogenous inhibitor, REversion-inducing Cysteine-rich protein with Kazal motifs (RECK). In addition to modulating gelatinases, AA also inhibits the expression of various components of the Epidermal Growth Factor (EGF) pathway like EGF, Protein Kinase B (Akt) and Mitogen-activated protein kinases (MAPK). Furthermore, AA also activates the expression of Sprouty 2 (Spry2), a negative regulator of EGF pathway, and silencing Spry2 results in up-regulation of expression of gelatinases as well as MMP-14. The present study thus elucidates a novel mechanism of action of AA and provides a strong basis for utilizing this molecule as a template for cancer therapeutics.

Discovery of arjunolic acid as a novel non-zinc binding carbonic anhydrase II inhibitor.

Elevated levels of carbonic anhydrase II (CA II) have been shown to be associated with cardiac hypertrophy and heart failure. Although arjunolic acid (AA) has a diverse range of therapeutic applications including cardio-protection, there have been no reports on the effect of AA on CA II. The present study describes for the first time, the novel zinc independent inhibition of CA II by AA. The molecular docking studies of AA indicated that the hydroxyl group at C2 of the A-ring, which hydrogen bonds with the catalytic site residues (His64, Asn62 and Asn67), along with the gem-dimethyl group at C20 of the E-ring, greatly influences the inhibitory activity, independent of the catalytic zinc, unlike the inhibition observed with most CA II inhibitors. Among the triterpenoids tested viz. arjunolic acid, arjunic acid, asiatic acid, oleanolic acid and ursolic acid, AA was the most potent in inhibiting CA II in vitro with an IC50 of 9µM. It was interesting to note, that in spite of exhibiting very little differences in their structures, these triterpenoids exhibited vast differences in their inhibitory activities, with IC50 values ranging from 9µM to as high as 333µM. Furthermore, AA also inhibited the cytosolic activity of CA in H9c2 cardiomyocytes, as reflected by the decrease in acidification of the intracellular pH (pHi). The decreased acidification reduced the intracellular calcium levels, which further prevented the mitochondrial membrane depolarization. Thus, these studies provide a better understanding for establishing the novel molecular mechanism involved in CA II inhibition by the non-zinc binding inhibitor AA.

Independent component analysis reveals 49 independently modulated gene sets within the global transcriptional regulatory architecture of multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii causes severe infections in humans, resists multiple antibiotics, and survives in stressful environmental conditions due to modulations of its complex transcriptional regulatory network (TRN). Unfortunately, our global understanding of the TRN in this emerging opportunistic pathogen is limited. Here, we apply independent component analysis, an unsupervised machine learning method, to a compendium of 139 RNA-seq data sets of three multidrug-resistant A. baumannii international clonal complex I strains (AB5075, AYE, and AB0057). This analysis allows us to define 49 independently modulated gene sets, which we call iModulons. Analysis of the identified A. baumannii iModulons reveals validating parallels to previously defined biological operons/regulons and provides a framework for defining unknown regulons. By utilizing the iModulons, we uncover potential mechanisms for a RpoS-independent general stress response, define global stress-virulence trade-offs, and identify conditions that may induce plasmid-borne multidrug resistance. The iModulons provide a model of the TRN that emphasizes the importance of transcriptional regulation of virulence phenotypes in A. baumannii. Furthermore, they suggest the possibility of future interventions to guide gene expression toward diminished pathogenic potential.IMPORTANCEThe rise in hospital outbreaks of multidrug-resistant Acinetobacter baumannii infections underscores the urgent need for alternatives to traditional broad-spectrum antibiotic therapies. The success of A. baumannii as a significant nosocomial pathogen is largely attributed to its ability to resist antibiotics and survive environmental stressors. However, there is limited literature available on the global, complex regulatory circuitry that shapes these phenotypes. Computational tools that can assist in the elucidation of A. baumannii's transcriptional regulatory network architecture can provide much-needed context for a comprehensive understanding of pathogenesis and virulence, as well as for the development of targeted therapies that modulate these pathways.

Comparative molecular profiling of multidrug-resistant Pseudomonas aeruginosa identifies novel mutations in regional clinical isolates from South India.

Objectives: We sought to analyse the antibiotic susceptibility profiles and molecular epidemiology of MDR clinical Pseudomonas aeruginosa isolates from South India using non-MDR isolates as a reference. Methods: We established a comprehensive clinical strain library consisting of 58 isolates collected from patients across the South Indian state of Kerala from March 2017 to July 2019. The strains were subject to antibiotic susceptibility testing, modified carbapenem inactivation method assay for carbapenemase production, PCR sequencing, comparative sequence analysis and quantitative PCR of MDR determinants associated with antibiotic efflux pump systems, fluoroquinolone resistance and carbapenem resistance. We performed in silico modelling of MDR-specific SNPs. Results: Of our collection of South Indian P. aeruginosa clinical isolates, 74.1% were MDR and 55.8% were resistant to the entire panel of antibiotics tested. All MDR isolates were resistant to levofloxacin and 93% were resistant to meropenem. We identified seven distinct, MDR-specific mutations in nalD, three of which are novel. mexA was significantly overexpressed in strains that were resistant to the entire test antibiotic panel while gyrA and gyrB were overexpressed in MDR isolates. Mutations in fluoroquinolone determinants were significantly associated with MDR phenotype and a novel GyrA Y100C substitution was observed. Carbapenem resistance in MDR isolates was associated with loss-of-function mutations in oprD and high prevalence of NDM (blaNDM-1) within our sample. Conclusions: This study provides insight into MDR mechanisms adopted by P. aeruginosa clinical isolates, which may guide the potential development of therapeutic regimens to improve clinical outcomes.

Anacardic acid inhibits the catalytic activity of matrix metalloproteinase-2 and matrix metalloproteinase-9.

Cashew nut shell liquid (CNSL) has been used in traditional medicine for the treatment of a wide variety of pathophysiological conditions. To further define the mechanism of CNSL action, we investigated the effect of cashew nut shell extract (CNSE) on two matrix metalloproteinases, MMP-2/gelatinase A and MMP-9/gelatinase B, which are known to have critical roles in several disease states. We observed that the major constituent of CNSE, anacardic acid, markedly inhibited the gelatinase activity of 3T3-L1 cells. Our gelatin zymography studies on these two secreted gelatinases, present in the conditioned media from 3T3-L1 cells, established that anacardic acid directly inhibited the catalytic activities of both MMP-2 and MMP-9. Our docking studies suggested that anacardic acid binds into the MMP-2/9 active site, with the carboxylate group of anacardic acid chelating the catalytic zinc ion and forming a hydrogen bond to a key catalytic glutamate side chain and the C15 aliphatic group being accommodated within the relatively large S1' pocket of these gelatinases. In agreement with the docking results, our fluorescence-based studies on the recombinant MMP-2 catalytic core domain demonstrated that anacardic acid directly inhibits substrate peptide cleavage in a dose-dependent manner, with an IC50 of 11.11 µM. In addition, our gelatinase zymography and fluorescence data confirmed that the cardol-cardanol mixture, salicylic acid, and aspirin, all of which lack key functional groups present in anacardic acid, are much weaker MMP-2/MMP-9 inhibitors. Our results provide the first evidence for inhibition of gelatinase catalytic activity by anacardic acid, providing a novel template for drug discovery and a molecular mechanism potentially involved in CNSL therapeutic action.

Systematic understanding of anti-tumor mechanisms of Tamarixetin through network and experimental analyses.

Tamarixetin, a flavonoid derived from Quercetin, was shown to possess anti-cancer properties in various types of cancer. However, the mechanism of action of this compound is not well understood. Observations from reverse docking and network pharmacology analysis, were validated by cell based studies to analyse the chemotherapeutic potential and elucidate the molecular mechanism of action of Tamarixetin in breast cancer. In silico analysis using reverse docking and PPI analysis clearly indicated that out of 35 proteins targeted by Tamarixetin, the top 3 hub genes, namely, AKT1, ESR1 and HSP90AA1, were upregulated in breast tumor tissues and more importantly showed strong negative correlation to breast cancer patient survival. Furthermore, the KEGG pathway analysis showed enrichment of target proteins of Tamarixetin in 33 pathways which are mainly involved in neoplastic signalling. In vitro cell-based studies demonstrated that Tamarixetin could inhibit cell proliferation, induce ROS and reduce mitochondrial membrane potential, leading to cell death. Tamarixetin induced cell cycle arrest at G2/M phase and inhibited the migration as well as the invasion of breast cancer cells. Taken together, the combination of in silico and in vitro approaches used in the present study clearly provides evidence for the chemotherapeutic potential of Tamarixetin in breast cancer.

Nuclear factor-κB plays an important role in Tamarixetin-mediated inhibition of matrix metalloproteinase-9 expression.

Flavonoids possess a broad spectrum of pharmacological properties, including anti-cancer, anti-oxidant and immunomodulatory activities. The current study explored the potential of some less-studied flavonoids in inhibiting Matrix Metalloproteinase-9 (MMP-9), a prominent biomarker, upregulated in a variety of cancers and known to promote migration and invasion of cancer cells. Amongst these, Tamarixetin, a naturally occurring flavonoid derivative of Quercetin, demonstrated significant dose-dependent inhibition of MMP-9 expression. Furthermore, a substantial inhibition of migration, invasion and clonogenic potential of HT1080 cells was also observed in the presence of Tamarixetin, which further suggests its role as a potential anti-cancer agent. It is noteworthy that Tamarixetin inhibits nuclear translocation as well the activity of nuclear factor kappa B (NFκB), both of which are functions essential for the activation of MMP-9 in promoting tumorigenesis. Additionally, the endogenous regulators of MMP-9 that tightly control its activity were also modulated by Tamarixetin, as evident from the 1.9 fold increase in the expression of Tissue Inhibitor of Metalloproteinase-1 (TIMP-1), with a concomitant 2.2 fold decrease in Matrix Metalloproteinase-14 (MMP-14) expression. The results obtained were further corroborated in three dimensional (3D) tumor models, which showed significant inhibition of MMP-9 activity as well as reduced invasive potential in the presence of Tamarixetin. Taken together, our observations demonstrate for the first time, the anti-invasive potential of Tamarixetin in cancer cells, indicating its possible use as a template for novel therapeutic applications.

A Novel N4-Like Bacteriophage Isolated from a Wastewater Source in South India with Activity against Several Multidrug-Resistant Clinical Pseudomonas aeruginosa Isolates.

Multidrug-resistant community-acquired infections caused by the opportunistic human pathogen Pseudomonas aeruginosa are increasingly reported in India and other locations globally. Since this organism is ubiquitous in the environment, samples such as sewage and wastewater are rich reservoirs of P. aeruginosa bacteriophages. In this study, we report the isolation and characterization of a novel P. aeruginosa N4-like lytic bacteriophage, vB_Pae_AM.P2 (AM.P2), from wastewater in Kerala, India. AM.P2 is a double-stranded DNA podovirus that efficiently lyses the model strain, PAO1, at a multiplicity of infection as low as 0.1 phage per bacterium and resistance frequency of 6.59 × 10-4 Synergy in bactericidal activity was observed between AM.P2 and subinhibitory concentrations of the antibiotic ciprofloxacin. Genome sequencing of AM.P2 revealed features similar to those of the N4-like P. aeruginosa phages LUZ7 and KPP21. As judged by two independent assay methods, spot tests and growth inhibition, AM.P2 successfully inhibited the growth of almost 30% of strains from a contemporary collection of multidrug-resistant P. aeruginosa clinical isolates from South India. Thus, AM.P2 may represent an intriguing candidate for inclusion in bacteriophage cocktails developed for various applications, including water decontamination and clinical bacteriophage therapy.IMPORTANCE In India, multidrug resistance determinants are much more abundant in community-associated bacterial pathogens due to the improper treatment of domestic and industrial effluents. In particular, a high bacterial load of the opportunistic pathogen P. aeruginosa in sewage and water bodies in India is well documented. The isolation and characterization of bacteriophages that could target emerging P. aeruginosa strains, representing possible epicenters for community-acquired infections, could serve as a useful alternative tool for various applications, such as phage therapy and environmental treatment. Continuing to supplement the repertoire of broad-spectrum bacteriophages is an essential tool in confronting this problem.

Oxyresveratrol drives caspase-independent apoptosis-like cell death in MDA-MB-231 breast cancer cells through the induction of ROS.

Earlier studies from our laboratory have demonstrated that Oxyresveratrol (OXY), a hydroxyl-substituted stilbene, exhibits potent inhibition of human melanoma cell proliferation. The present study defines a cytotoxic effect of OXY on the highly chemo-resistant, triple-negative human breast cancer cell line MDA-MB-231. OXY-mediated cell death resulted in accumulation of cells at the sub-G1 phase of the cell cycle, induced chromatin condensation, DNA fragmentation, phosphatidylserine externalization and PARP cleavage, indicative of apoptosis. Interestingly, morphology and cell viability studies with the pan-caspase inhibitor, QVD-OPH revealed that OXY-induced cell death was caspase-independent. Docking studies also showed that OXY can bind to the S1 site of caspase-3, and could also exert an inhibitory effect on this executioner caspase. The immunoblot analysis demonstrating the absence of caspase cleavage during cell death further confirmed these findings. OXY was also observed to induce the production of reactive oxygen species, which caused the depolarization of the mitochondrial membrane resulting in translocation of Apoptosis Inducing Factor (AIF) into the nucleus. Pretreatment of the cells with N-Acetyl Cysteine antioxidant prevented cell death resulting from OXY treatment. Thus, OXY initiates ROS-mediated, apoptosis-like cell death, involving mitochondrial membrane depolarization, translocation of AIF into the nucleus, and DNA fragmentation, resulting in caspase-independent cell death in MDA-MB-231 cells. The cytotoxicity manifested by OXY was also observed in 3D cell culture models and primary cells, thereby providing a basis for the utilization of OXY as a novel template for the future design of anticancer therapeutics.

RECK and TIMP-2 mediate inhibition of MMP-2 and MMP-9 by Annona muricata.

Up-regulation of MMP-2 and MMP-9 plays a significant role in promoting cancer progression by degrading the components of the extracellular matrix, thereby enhancing the migration of tumor cells. Although the antiproliferative and apoptotic effect of Annona muricata is well established, its effect on MMP-2 and MMP-9, a major target in several types of cancers, has not been studied. Powdered samples of various parts of A. muricata like fruit, stem, seed, and twig extracted using aqueous methanol showed significant dose-dependent inhibition of MMP-2 and MMP-9 in a highly metastatic fibrosarcoma cell line, HT1080. Additionally, these extracts also up-regulated the expression of several endogenous inhibitors of MMP-2 and MMP-9 like REversion-inducing Cysteine-rich protein with Kazal motifs (RECK) and Tissue Inhibitor of Metalloproteinase- 2 (TIMP-2). Furthermore, primary cells developed from tumor tissues obtained from patients not exposed to chemotherapy, also exhibited similar results. Remarkably, the inhibition of MMP-2 and MMP-9 observed was tumor specific, with the A. muricata fruit extract showing only 2% inhibition in cells obtained from normal tissues, when compared to 60% inhibition observed in cells obtained from tumor samples. The present study elucidates a novel mechanism by which A. muricata extracts selectively exhibit their anti-cancer activity in tumor cells by down-regulating MMP-2 and MMP-9 that are important biomarkers in cancer.

Analysis of microarray data for identification of key microRNA signatures in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is one of the most malignant types of glioma known for its reduced survival rate and rapid relapse. Previous studies have shown that the expression patterns of different microRNAs (miRNA/miR) play a crucial role in the development and progression of GBM. In order to identify potential miRNA signatures of GBM for prognostic and therapeutic purposes, we downloaded and analyzed two expression data sets from Gene Expression Omnibus profiling miRNA patterns of GBM compared with normal brain tissues. Validated targets of the deregulated miRNAs were identified using MirTarBase, and were mapped to Search Tool for the Retrieval of Interacting Genes/Proteins, Database for Annotation, Visualization and Integrated Discovery and Kyoto Encyclopedia of Genes and Genomes databases in order to construct interaction networks and identify enriched pathways of target genes. A total of 6 miRNAs were found to be deregulated in both expression datasets studied. Pathway analysis demonstrated that most of the target genes were enriched in signaling cascades connected to cancer development, such as 'Pathways in cancer', 'Focal adhesion' and 'PI3K-Akt signaling pathway'. Of the five target genes that were enriched in the glioblastoma pathway, in the WikiPathway database, both HRas proto-oncogene, GTPase and MET proto-oncogene, receptor tyrosine kinase target genes of hsa-miR-139-5p, were found to be significantly associated with patient survival. The present study may thus form the basis for further exploration of hsa-miR-139-5p, not only as a therapeutic agent, but also as a diagnostic biomarker for GBM as well as a predictive marker for patient survival.

Recent insights into natural product inhibitors of matrix metalloproteinases.

Members of the matrix metalloproteinase (MMP) family have biological functions that are central to human health and disease, and MMP inhibitors have been investigated for the treatment of cardiovascular disease, cancer and neurodegenerative disorders. The outcomes of initial clinical trials with the first generation of MMP inhibitors proved disappointing. However, our growing understanding of the complexities of the MMP function in disease, and an increased understanding of MMP protein architecture and control of activity now provide new opportunities and avenues to develop MMP-focused therapies. Natural products that affect MMP activities have been of strong interest as templates for drug discovery, and for their use as chemical tools to help delineate the roles of MMPs that still remain to be defined. Herein, we highlight the most recent discoveries of structurally diverse natural product inhibitors to these proteases.

Clove Bud Oil Modulates Pathogenicity Phenotypes of the Opportunistic Human Pathogen Pseudomonas aeruginosa.

Earlier studies from our laboratory have demonstrated that clove bud oil (CBO) attenuates expression of certain virulence factors of Pseudomonas aeruginosa PAO1. Here, we probe more deeply into the effect of CBO on four pseudomonal proteases - elastase A, elastase B, protease IV and alkaline protease - each known to play key roles in disease pathogenesis. CBO inhibited the activity of these proteases present in the bacterial culture supernatant. Zymography studies indicated that these proteases can activate host matrix metalloproteases (MMPs) to establish infection, through conversion of pro-MMP-2 to active MMP-2. PAO1 is a predominant pathogen in burn wound infections and we show the modulatory effect of CBO on MMPs in an in vitro model of burn injury. Furthermore, CBO induced dose-dependent neutrophil extracellular trap formation in human neutrophils. CBO also increased the survival of C. elegans infected with PAO1, establishing an anti-infective role in a whole animal model of pathogenesis. LC-MS/MS analysis indicated that CBO treatment elicited a significant reduction of signalling molecules (Acyl-Homoserine-Lactone) involved in quorum sensing regulation. Our observations demonstrate that CBO attenuates key virulence mechanisms of this important human pathogen, while concomitantly enhancing host innate immunomodulatory functions, with potential implications for topical therapy against antibiotic-resistant infections.