Ethanolic Extract Propolis-Loaded Niosomes Diminish Phospholipase B1, Biofilm Formation, and Intracellular Replication of Cryptococcus neoformans in Macrophages.

Secretory phospholipase B1 (PLB1) and biofilms act as microbial virulence factors and play an important role in pulmonary cryptococcosis. This study aims to formulate the ethanolic extract of propolis-loaded niosomes (Nio-EEP) and evaluate the biological activities occurring during PLB1 production and biofilm formation of Cryptococcus neoformans. Some physicochemical characterizations of niosomes include a mean diameter of 270 nm in a spherical shape, a zeta-potential of -10.54 ± 1.37 mV, and 88.13 ± 0.01% entrapment efficiency. Nio-EEP can release EEP in a sustained manner and retains consistent physicochemical properties for a month. Nio-EEP has the capability to permeate the cellular membranes of C. neoformans, causing a significant decrease in the mRNA expression level of PLB1. Interestingly, biofilm formation, biofilm thickness, and the expression level of biofilm-related genes (UGD1 and UXS1) were also significantly reduced. Pre-treating with Nio-EEP prior to yeast infection reduced the intracellular replication of C. neoformans in alveolar macrophages by 47%. In conclusion, Nio-EEP mediates as an anti-virulence agent to inhibit PLB1 and biofilm production for preventing fungal colonization on lung epithelial cells and also decreases the intracellular replication of phagocytosed cryptococci. This nano-based EEP delivery might be a potential therapeutic strategy in the prophylaxis and treatment of pulmonary cryptococcosis in the future.

Study on the Interaction of Algal Peptides on Virulence Factors of Helicobacter pylori: In Silico Approach.

In the Asian region, Helicobacter pylori infects about 80% populations, which is most leading cause of peptic ulcers, and it is an asymptomatic infection. Studies reported that the particular bacteria carry specific virulence factors that leads to severe complications. These virulence factors can be used as a drug targets to inhibit their growth and pathogenicity. Chronic infection with H. pylori virulence factors are CagA, VacA and HtrA positive strains the risk factor of gastric cancer. In this study, we aimed to study the antagonistic interaction pattern between the potential eight algal peptides against the virulence factors of H. pylori through in silico analysis intended to treat peptic ulcer and prevent the further complications such as cancer. The proteins of virulent factors are docked using C-Docker algorithm and calculated the bind energy of the complexes. The results showed that the peptide derived from a green alga, Tetradesmus sp. are active against the three virulent factors such as cag-A, vac-A, and Htr-A with multiple hydrogen, vdW, electrostatic interactions, and mild π-hydrophobic bindings with the libdock energy score for CagA, VacA and HtrA are 175.625, 158.603 and 89.397 kcal/mol. These primes and the peptide lead to develop a better and potential inhibitors against H. pylori infection.

Inhibition of Virulence Factors and Biofilm Formation by Wogonin Attenuates Pathogenicity of Pseudomonas aeruginosa PAO1 via Targeting pqs Quorum-Sensing System.

Pseudomonas aeruginosa, an important opportunistic pathogen, is capable of producing various virulence factors and forming biofilm that are regulated by quorum sensing (QS). It is known that targeting virulence factor production and biofilm formation instead of exerting selective pressure on growth such as conventional antibiotics can reduce multidrug resistance in bacteria. Therefore, many quorum-sensing inhibitors (QSIs) have been developed to prevent or treat this bacterial infection. In this study, wogonin, as an active ingredient from Agrimonia pilosa, was found to be able to inhibit QS system of P. aeruginosa PAO1. Wogonin downregulated the expression of QS-related genes and reduced the production of many virulence factors, such as elastase, pyocyanin, and proteolytic enzyme. In addition, wogonin decreased the extracellular polysaccharide synthesis and inhibited twitching, swimming, and swarming motilities and biofilm formation. The attenuation of pathogenicity in P. aeruginosa PAO1 by wogonin application was further validated in vivo by cabbage infection and fruit fly and nematode survival experiments. Further molecular docking analysis, pathogenicity examination of various QS-related mutants, and PQS signal molecule detection revealed that wogonin could interfere with PQS signal molecular synthesis by affecting pqsA and pqsR. Taken together, the results indicated that wogonin might be used as an anti-QS candidate drug to attenuate the infection caused by P. aeruginosa.

Molecular Insights into Binding Mode and Interactions of Structure-Based Virtually Screened Inhibitors for Pseudomonas aeruginosa Multiple Virulence Factor Regulator (MvfR).

Multi-drug resistance (MDR) bacterial pathogens pose a threat to global health and warrant the discovery of new therapeutic molecules, particularly those that can neutralize their virulence and stop the evolution of new resistant mechanisms. The superbug nosocomial pathogen, Pseudomonas aeruginosa, uses a multiple virulence factor regulator (MvfR) to regulate the expression of multiple virulence proteins during acute and persistent infections. The present study targeted MvfR with the intention of designing novel anti-virulent compounds, which will function in two ways: first, they will block the virulence and pathogenesis P. aeruginosa by disrupting the quorum-sensing network of the bacteria, and second, they will stop the evolution of new resistant mechanisms. A structure-based virtual screening (SBVS) method was used to screen druglike compounds from the Asinex antibacterial library (~5968 molecules) and the comprehensive marine natural products database (CMNPD) (~32 thousand compounds), against the ligand-binding domain (LBD) of MvfR, to identify molecules that show high binding potential for the relevant pocket. In this way, two compounds were identified: Top-1 (4-((carbamoyloxy)methyl)-10,10-dihydroxy-2,6-diiminiodecahydropyrrolo[1,2-c]purin-9-yl sulfate) and Top-2 (10,10-dihydroxy-2,6-diiminio-4-(((sulfonatocarbamoyl)oxy)methyl)decahydropyrrolo[1,2-c]purin-9-yl sulfate), in contrast to the co-crystallized M64 control. Both of the screened leads were found to show deep pocket binding and interactions with several key residues through a network of hydrophobic and hydrophilic interactions. The docking results were validated by a long run of 200 ns of molecular dynamics simulation and MM-PB/GBSA binding free energies. All of these analyses confirmed the presence of strong complex formation and rigorous intermolecular interactions. An additional analysis of normal mode entropy and a WaterSwap assay were also performed to complement the aforementioned studies. Lastly, the compounds were found to show an acceptable range of pharmacokinetic properties, making both compounds potential candidates for further experimental studies to decipher their real biological potency.

Staphylococcus aureus Toxins: An Update on Their Pathogenic Properties and Potential Treatments.

Staphylococcus aureus is a clinically important pathogen that causes a wide range of human infections, from minor skin infections to severe tissue infection and sepsis. S. aureus has a high level of antibiotic resistance and is a common cause of infections in hospitals and the community. The rising prevalence of community-acquired methicillin-resistant S. aureus (CA-MRSA), combined with the important severity of S. aureus infections in general, has resulted in the frequent use of anti-staphylococcal antibiotics, leading to increasing resistance rates. Antibiotic-resistant S. aureus continues to be a major health concern, necessitating the development of novel therapeutic strategies. S. aureus uses a wide range of virulence factors, such as toxins, to develop an infection in the host. Recently, anti-virulence treatments that directly or indirectly neutralize S. aureus toxins have showed promise. In this review, we provide an update on toxin pathogenic characteristics, as well as anti-toxin therapeutical strategies.

Citral modulates virulence factors in methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is responsible for high morbidity and mortality rates. Citral has been studied in the pharmaceutical industry and has shown antimicrobial activity. This study aimed to analyze the antimicrobial activity of citral in inhibiting biofilm formation and modulating virulence genes, with the ultimate goal of finding a strategy for treating infections caused by MRSA strains. Citral showed antimicrobial activity against MRSA isolates with minimum inhibitory concentration (MIC) values between 5 mg/mL (0.5%) and 40 mg/mL (4%), and minimum bactericidal concentration (MBC) values between 10 mg/mL (1%) and 40 mg/mL (4%). The sub-inhibitory dose was 2.5 mg/mL (0.25%). Citral, in an antibiogram, modulated synergistically, antagonistically, or indifferent to the different antibiotics tested. Prior to evaluating the antibiofilm effects of citral, we classified the bacteria according to their biofilm production capacity. Citral showed greater efficacy in the initial stage, and there was a significant reduction in biofilm formation compared to the mature biofilm. qPCR was used to assess the modulation of virulence factor genes, and icaA underexpression was observed in isolates 20 and 48. For icaD, seg, and sei, an increase was observed in the expression of ATCC 33,591. No significant differences were found for eta and etb. Citral could be used as a supplement to conventional antibiotics for MRSA infections.

Inhibiting Type VI Secretion System Activity with a Biomimetic Peptide Designed To Target the Baseplate Wedge Complex.

Human health is threatened by bacterial infections that are increasingly resistant to multiple drugs. A recently emerged strategy consists of disarming pathogenic bacteria by targeting and blocking their virulence factors. The type VI secretion system (T6SS) is a widespread secretion nanomachine encoded and employed by pathogenic strains to establish their virulence process during host invasion. Given the conservation of T6SS in several human bacterial pathogens, the discovery of an effective broad-spectrum T6SS virulence blocker represents an attractive target for development of antivirulence therapies. Here, we identified and validated a protein-protein interaction interface, TssK-TssG, as a key factor in the assembly of the T6SS baseplate (BP) complex in the pathogen enteroaggregative Escherichia coli (EAEC). In silico and biochemical studies revealed that the determinants of the interface are broadly conserved among pathogenic species, suggesting a role for this interface as a target for T6SS inhibition. Based on the high-resolution structure of the TssKFGE wedge complex, we rationally designed a biomimetic cyclic peptide (BCP) that blocks the assembly of the EAEC BP complex and inhibits the function of T6SS in bacterial cultures. Our BCP is the first compound completely designed from prior structural knowledge with anti-T6SS activity that can be used as a model to target human pathogens. IMPORTANCE New therapeutic options are urgently needed to fight drug-resistant and life-threatening infections. In contrast to antibiotics that inhibit the growth pathways of bacteria, the antivirulence strategy is a promising approach to disarm pathogens by interfering with bacterial virulence factors without exerting evolutionary pressure. The type VI secretion system (T6SS) is used by many pathogens, including members of the antibiotic-resistant ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), to establish their virulence during the invasion of the human host. Although the T6SS is undoubtedly involved in pathogenesis, strategies targeting this virulence factor are crucially lacking. Here, we used a combination of genetics, microbiology, biochemical, biophysics, and bioinformatics approaches to rationally design a biomimetic peptide that interferes with T6SS assembly and functioning. This study represents a novel proof of concept for an antivirulence strategy which aims to interfere with the assembly of the T6SS.

Phloretin inhibited the pathogenicity and virulence factors against Candida albicans.

Oral candidiasis is one of the most common types of fungal infection caused by Candida albicans (C. albicans). The present study aims to investigate the antifungal effects of phloretin (a dihydrochalcone flavonoid) against the C. albicans pathogenicity. In this work, we treated C. albicans SC5314 with 37.28, 74.55, or 149.10 µg/mL (equivalent to 0.5×, 1× or 2× MIC) phloretin in vitro. Besides, we established a mice model of oral candidiasis by a sublingual infection of C. albicans suspension (1 × 107 colony-forming unit/mL), and mice were treated with phloretin (3.73 or 7.46 mg/mL, which were equivalent to 50× or 100× MIC) twice a day starting on day one post-infection. The results showed that the MIC of phloretin against C. albicans was 74.55 µg/mL. Phloretin exerted antifungal activity by inhibiting the biofilm formation and suppressing the yeast-to-hyphae transition upon the downregulation of hypha-associated genes including enhanced adherence to polystyrene 1, the extent of cell elongation gene 1, hyphal wall protein 1 gene, and agglutinin-like sequence gene 3. Next, phloretin repressed the secretion of proteases and phospholipases via reducing the expression of protease-encoding genes secreted aspartyl proteases (SAP)1 and SAP2, as well as phospholipase B1. Subsequently, the in vivo antifungal activity of phloretin was testified by the reverse of the enhanced lesion severity, inflammatory infiltration, and the increased colony-forming unit counts caused by C. albicans of tongue tissues in oral candidiasis mice. In conclusion, phloretin suppressed the pathogenicity and virulence factors against C. albicans both in vivo and in vitro.

Discovery of uncompetitive inhibitors of SapM that compromise intracellular survival of Mycobacterium tuberculosis.

SapM is a secreted virulence factor from Mycobacterium tuberculosis critical for pathogen survival and persistence inside the host. Its full potential as a target for tuberculosis treatment has not yet been exploited because of the lack of potent inhibitors available. By screening over 1500 small molecules, we have identified new potent and selective inhibitors of SapM with an uncompetitive mechanism of inhibition. The best inhibitors share a trihydroxy-benzene moiety essential for activity. Importantly, the inhibitors significantly reduce mycobacterial burden in infected human macrophages at 1 µM, and they are selective with respect to other mycobacterial and human phosphatases. The best inhibitor also reduces intracellular burden of Francisella tularensis, which secretes the virulence factor AcpA, a homologue of SapM, with the same mechanism of catalysis and inhibition. Our findings demonstrate that inhibition of SapM with small molecule inhibitors is efficient in reducing intracellular mycobacterial survival in host macrophages and confirm SapM as a potential therapeutic target. These initial compounds have favourable physico-chemical properties and provide a basis for exploration towards the development of new tuberculosis treatments. The efficacy of a SapM inhibitor in reducing Francisella tularensis intracellular burden suggests the potential for developing broad-spectrum antivirulence agents to treat microbial infections.

Morpholinium-based ionic liquids show antimicrobial activity against clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a multi-drug resistant (MDR) pathogen. It is classified by WHO as one of the most life-threatening pathogens causing nosocomial infections. Some of its clinical isolates and their subpopulations show high persistence to many antibiotics that are recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Thus, there is a need for non-traditional classes of antibiotics to fight the increasing threat of MDR P. aeruginosa. Ionic liquids (IL) are one such promising class of novel antibiotics. We selected four strains of P. aeruginosa and studied the growth inhibition and other effects of 12 different ILs. We used the well-characterized P. aeruginosa PAO1 (ATCC 15692) as model strain and compared it to three other isolates from chronic lung infection (LES B58), skin burn infection (UCBPP-PA14) and keratitis infection (39016), respectively. The ILs consisted of either 4,4-didecylmorpholinium [Dec2Mor]+ or 4-decyl-4-ethylmorpholinium [DecEtMor]+ cations combined with different anions. We found that the ILs with 4,4-didecylmorpholinium [Dec2Mor]+ cations most effectively inhibited bacterial growth as well as reduced strain fitness and virulence factor production. Our results indicate that these ILs could be used to treat P. aeruginosa infections.

High-Throughput Screen for Inhibitors of the Type IV Pilus Assembly ATPase PilB.

The bacterial type IV pilus (T4P) is a prominent virulence factor in many significant human pathogens, some of which have become increasingly antibiotic resistant. Antivirulence chemotherapeutics are considered a promising alternative to antibiotics because they target the disease process instead of bacterial viability. However, a roadblock to the discovery of anti-T4P compounds is the lack of a high-throughput screen (HTS) that can be implemented relatively easily and economically. Here, we describe the first HTS for the identification of inhibitors specifically against the T4P assembly ATPase PilB in vitroChloracidobacterium thermophilum PilB (CtPilB) had been demonstrated to have robust ATPase activity and the ability to bind its expected ligands in vitro. We utilized CtPilB and MANT-ATP, a fluorescent ATP analog, to develop a binding assay and adapted it for an HTS. As a proof of principle, we performed a pilot screen with a small compound library of kinase inhibitors and identified quercetin as a PilB inhibitor in vitro Using Myxococcus xanthus as a model bacterium, we found quercetin to reduce its T4P-dependent motility and T4P assembly in vivo. These results validated our HTS as effective in identifying PilB inhibitors. This assay may prove valuable in seeking leads for the development of antivirulence chemotherapeutics against PilB, an essential and universal component of all bacterial T4P systems.IMPORTANCE Many bacterial pathogens use their type IV pili (T4P) to facilitate and maintain infection of a human host. Small chemical compounds that inhibit the production or assembly of T4P hold promise in the treatment and prevention of infections, especially in the era of increasing threats from antibiotic-resistant bacteria. However, few chemicals are known to have inhibitory or anti-T4P activity. Their identification has not been easy due to the lack of a method for the screening of compound collections or libraries on a large scale. Here, we report the development of an assay that can be scaled up to screen compound libraries for inhibitors of a critical T4P assembly protein. We further demonstrate that it is feasible to use whole cells to examine potential inhibitors for their activity against T4P assembly in a bacterium.

Betulin attenuates pneumolysin-induced cell injury and DNA damage.

AIMS: Pneumolysin, a pore-forming toxin, is an important virulence factor of Streptococcus pneumoniae with multiple biological activity, such as cell lysis and DNA damage. Thus, targeting this toxin is alternative strategy for the treatment of S. pneumoniae infection. METHODS AND RESULTS: Haemolysin assay was performed to identify the potential PLY inhibitor. The mechanism by which betulin, a natural compound from birch bark, against PLY was determined via MICs determination, western blot analysis and oligomerization analysis. Cytotoxicity and Immunofluorescence assays were further used to evaluate the protection of betulin against PLY-induced cell injury and DNA damage. Here, betulin, a natural compound from birch bark, was indentified as an effective inhibitor of PLY. Importantly, at the concentrations required for such inhibition, betulin has no influence on S. pneumoniae viability or PLY production. The interaction of betulin with PLY restrict the olgomerizaiton of this toxin and, thus, directly neutralizing the activity of PLY. Additionally, betulin treatment alleviate PLY induced cells injury and DNA damage in the co-culture system of PLY and A549 cells. CONCLUSIONS: Betulin could be used as a promising leading compound against S. pneumoniae virulence by directly targeting PLY without antibacterial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The results presented in this work provided a novel strategy and candidate for S. pneumoniae infection.

In silico prediction of host-pathogen protein interactions in melioidosis pathogen Burkholderia pseudomallei and human reveals novel virulence factors and their targets.

The aerobic, Gram-negative motile bacillus, Burkholderia pseudomallei is a facultative intracellular bacterium causing melioidosis, a critical disease of public health importance, which is widely endemic in the tropics and subtropical regions of the world. Melioidosis is associated with high case fatality rates in animals and humans; even with treatment, its mortality is 20-50%. It also infects plants and is designated as a biothreat agent. B. pseudomallei is pathogenic due to its ability to invade, resist factors in serum and survive intracellularly. Despite its importance, to date only a few effector proteins have been functionally characterized, and there is not much information regarding the host-pathogen protein-protein interactions (PPI) of this system, which are important to studying infection mechanisms and thereby develop prevention measures. We explored two computational approaches, the homology-based interolog and the domain-based method, to predict genome-scale host-pathogen interactions (HPIs) between two different strains of B. pseudomallei (prototypical, and highly virulent) and human. In total, 76 335 common HPIs (between the two strains) were predicted involving 8264 human and 1753 B. pseudomallei proteins. Among the unique PPIs, 14 131 non-redundant HPIs were found to be unique between the prototypical strain and human, compared to 3043 non-redundant HPIs between the highly virulent strain and human. The protein hubs analysis showed that most B. pseudomallei proteins formed a hub with human dnaK complex proteins associated with tuberculosis, a disease similar in symptoms to melioidosis. In addition, drug-binding and carbohydrate-binding mechanisms were found overrepresented within the host-pathogen network, and metabolic pathways were frequently activated according to the pathway enrichment. Subcellular localization analysis showed that most of the pathogen proteins are targeting human proteins inside cytoplasm and nucleus. We also discovered the host targets of the drug-related pathogen proteins and proteins that form T3SS and T6SS in B. pseudomallei. Additionally, a comparison between the unique PPI patterns present in the prototypical and highly virulent strains was performed. The current study is the first report on developing a genome-scale host-pathogen protein interaction networks between the human and B. pseudomallei, a critical biothreat agent. We have identified novel virulence factors and their interacting partners in the human proteome. These PPIs can be further validated by high-throughput experiments and may give new insights on how B. pseudomallei interacts with its host, which will help medical researchers in developing better prevention measures.

Identifying the Molecular Targets of an Anti-pathogenic Hydroalcoholic Extract of Punica granatum Peel Against Multidrug-resistant Serratia marcescens.

BACKGROUND: Antibiotic-resistant members of the family Enterobacteriaceae are among the serious threats to human health globally. This study reports the anti-pathogenic activity of Punica granatum peel extract (PGPE) against a multi-drug resistant, beta-lactamase producing member of this family i.e. Serratia marcescens. OBJECTIVE: This study aimed at assessing the anti-pathogenic activity of PGPE against the gramnegative bacterial pathogen S. marcescens and identifying the molecular targets of this extract in the test bacterium. METHODS: Effect of PGPE on S. marcescens growth and quorum sensing (QS)-regulated pigment production was assessed through broth dilution assay. In vivo anti-infective and prophylactic activity of PGPE was assessed employing the nematode worm Caenorhabditis elegans as a model host. Differential gene expression in PGPE-exposed S. marcescens was studied through a whole transcriptome approach. RESULTS: PGPE was able to modulate QS-regulated pigment production in S. marcescens without exerting any heavy growth-inhibitory effect at concentrations as low as ≥2.5 µg/mL. It could attenuate the virulence of the test bacterium towards the worm host by 22-42% (p≤0.01) at even lower concentrations (≥0.5 µg/mL). PGPE also exerted a post-extract effect on S. marcescens. This extract was found to offer prophylactic benefit too, to the host worm, as PGPE-pre-fed worms scored better (34-51%; p≤0.001) survival in face of subsequent bacterial attack. Differential gene expression analysis revealed that PGPE affected the expression of a total of 66 genes in S. marcescens by ≥1.5 fold. CONCLUSION: The anti-virulence effect of PGPE against S. marcescens is multifaceted, affecting stress-response machinery, efflux activity, iron homeostasis, and cellular energetics of this bacterium notably. Among the major molecular targets identified in this study are LPS export transporter permease (LptF), t-RNA pseudouridine synthase (TruB), etc.

Inhibition of Virulence Factors and Biofilm Formation of Acinetobacter Baumannii by Naturally-derived and Synthetic Drugs.

Acinetobacter baumannii is a gram-negative, aerobic, non-motile, and pleomorphic bacillus. A. baumannii is also a highly-infectious pathogen causing high mortality and morbidity rates in intensive care units. The discovery of novel agents against A. baumannii infections is urgently needed due to the emergence of drug-resistant A. baumannii strains and the limited number of efficacious antibiotics available for treatment. In addition to the production of several virulence factors, A. baumannii forms biofilms on the host cell surface as well. Formation of biofilms occurs through initial surface attachment, microcolony formation, biofilm maturation, and detachment stages, and is one of the major drug resistance mechanisms employed by A. baumannii. Several studies have previously reported the efficacy of naturally-derived and synthetic compounds as anti- biofilm and anti-virulence agents against A. baumannii. Here, inhibition of biofilm formation and virulence factors of A. baumannii using naturally-derived and synthetic compounds are reviewed.

Possible drugs for the treatment of bacterial infections in the future: anti-virulence drugs.

Antibiotic resistance is a global threat that should be urgently resolved. Finding a new antibiotic is one way, whereas the repression of the dissemination of virulent pathogenic bacteria is another. From this point of view, this paper summarizes first the mechanisms of conjugation and transformation, two important processes of horizontal gene transfer, and then discusses the approaches for disarming virulent pathogenic bacteria, that is, virulence factor inhibitors. In contrast to antibiotics, anti-virulence drugs do not impose a high selective pressure on a bacterial population, and repress the dissemination of antibiotic resistance and virulence genes. Disarmed virulence factors make virulent pathogens avirulent bacteria or pathobionts, so that we human will be able to coexist with these disarmed bacteria peacefully.

Inhibition of biofilm and quorum sensing-regulated virulence factors in Pseudomonas aeruginosa by Cuphea carthagenensis (Jacq.) J. F. Macbr. Leaf extract: An in vitro study.

ETHNOPHARMCOLOGICAL RELEVANCE: Microbial biofilm formation, a quorum sensing (QS) regulated process, is one of the major causes of nosocomial and chronic infections, foodborne diseases, and associated deaths. Various approaches have been used to eradicate the menace of biofilm. Ethnomedicinal plants as potent antibiofilm agents are gaining a lot of interest in an era where the drug resistance is increasing and the availability of potent antibiotics is no longer promised. In this context, the methanol extract of Cuphea carthagenensis (CCMD), an ethno-medicinal and culinary herb, was evaluated as an antibiofilm and anti-QS agent against Pseudomonas aeruginosa. AIM OF THE STUDY: The aim of the study is to evaluate the antibiofilm and anti-QS activity of an ethnomedicinal plant against a strong biofilm forming microorganism, P. aeruginosa. METHODS: Antibiofilm activity of CCMD was demonstrated at different concentrations by Tissue Culture Plate, Test Tube method and other microscopic techniques. The effect of CCMD on QS and QS-related virulence factors viz. Pyocyanin, exopolymeric substance matrix (EPS), total protease, elastase, pyoverdin and swimming motility in P. aeruginosa were also evaluated. Antioxidant activity (DPPH & FRAP), total phenolic and flavonoid content were also checked. In order to determine the composition of the extract HPLC analysis was also performed. RESULTS: In vitro study demonstrated a significant inhibition of biofilm formation (81.88 ± 2.57%) as well as production of QS-dependent virulence factors in P. aeruginosa. The extract also inhibited violacein production (83.31 ± 2.77%) in Chromobacterium violaceum which correlates with the reduction in QS-mediated virulence factors. The extract showed 64.79% ± 0.83% DPPH scavenging activity and reduction of ferricyanide complex (Fe3+) to the ferrous form (Fe2+) in DPPH and FRAP assay, respectively. Furthermore, the extract showed thermal stability and does not have any growth inhibitory effect on P. aeruginosa. The HPLC analysis demonstrated the presence of ellagic acid, ascorbic acid and hippuric acid in the extract. CONCLUSION: This work is the first to demonstrate that C. carthagenensis can attenuate biofilm formation and QS-mediated virulence factors of P. aeruginosa. Further investigation is required to use this ethnomedicinal plant (CCMD) as an important source of antibiofilm agents.

Development of Anti-Virulence Therapeutics against Mono-ADP-Ribosyltransferase Toxins.

Mono-ADP-ribosyltransferase toxins are often key virulence factors produced by pathogenic bacteria as tools to compromise the target host cell. These toxins are enzymes that use host cellular NAD+ as the substrate to modify a critical macromolecule target in the host cell machinery. This post-translational modification of the target macromolecule (usually protein or DNA) acts like a switch to turn the target activity on or off resulting in impairment of a critical process or pathway in the host. One approach to stymie bacterial pathogens is to curtail the toxic action of these factors by designing small molecules that bind tightly to the enzyme active site and prevent catalytic function. The inactivation of these toxins/enzymes is targeted for the site of action within the host cell and small molecule therapeutics can function as anti-virulence agents by disarming the pathogen. This represents an alternative strategy to antibiotic therapy with the potential as a paradigm shift that may circumvent multi-drug resistance in the offending microbe. In this review, work that has been accomplished during the past two decades on this approach to develop anti-virulence compounds against mono-ADP-ribosyltransferase toxins will be discussed.

New Agents in Development for Sepsis: Any Reason for Hope?

Sepsis is a syndrome which is defined as a dysregulated host response to infection leading to organ failure. Since it remains one of the leading causes of mortality worldwide, numerous drug candidates have already been tested, and continue to be developed, as potential adjunct therapies. Despite convincing mechanisms of action and robust pre-clinical data, almost all drug candidates in the field of sepsis have failed to demonstrate clinical efficacy in the past two decades. Accordingly, the development of new sepsis drugs has markedly decreased in the past few years. Nevertheless, thanks to a better understanding of sepsis pathophysiology and pathways, new promising drug candidates are currently being developed. Instead of a unique sepsis profile as initially suspected, various phenotypes have been characterised. This has  resulted in the identification of multiple targets for new drugs together with relevant biomarkers, and a better understanding of the most appropriate time to intervention. Within the entire sepsis drugs portfolio, those targeting the immune response are probably the most promising. Monoclonal antibodies targeting either cytokines or infectious agents are undoubtedly part of the potential successful therapeutic classes to come.