Antivirulence Compounds Research Articles

VFDB 2025: an integrated resource for exploring anti-virulence compounds.

With the escalating crisis of bacterial multidrug resistance, anti-virulence therapeutic strategies have emerged as a highly promising alternative to conventional antibiotic treatments. Anti-virulence compounds are specifically designed to target virulence factors (VFs), disarming pathogens without affecting bacterial growth and thus reduce the selective pressure for resistance development. However, due to the complexity of bacterial pathogenesis, no anti-virulence small molecules have been approved for clinical use thus far, despite the documentation of hundreds of potential candidates. To provide valuable reference resources for drug design, repurposing, and target selection, the virulence factor database (VFDB, http://www.mgc.ac.cn/VFs/) has systematically collected public data on anti-virulence compounds through extensive literature mining, and further integrated this information with its existing knowledge of bacterial VFs. To date, the VFDB has curated a comprehensive dataset of 902 anti-virulence compounds across 17 superclasses reported by 262 studies worldwide. By cross-linking the current knowledge of bacterial VFs with information on relevant compounds (e.g. classification, chemical structure, molecular targets and mechanisms of action), the VFDB aims to bridge the gap between chemists and microbiologists, providing crucial insights for the development of innovative and effective antibacterial therapies to combat bacterial infections and address antibiotic resistance.

Harmine acts as a quorum sensing inhibitor decreasing the virulence and antibiotic resistance of Pseudomonas aeruginosa

BackgroundAs antimicrobial resistance (AMR) has become a global health crisis, new strategies against AMR infection are urgently needed. Quorum sensing (QS), responsible for bacterial communication and pathogenicity, is among the targets for anti-virulence drugs that thrive as one of the promising treatments against AMR infection.MethodsWe identified a natural compound, Harmine, through virtual screening based on three QS receptors of Pseudomonas aeruginosa (P. aeruginosa) and explored the effect of Harmine on QS-controlled and pathogenicity-related phenotypes including pyocyanin production, exocellular protease excretion, biofilm formation, and twitching motility of P. aeruginosa PA14. The protective effect of Harmine on Caenorhabditis elegans (C. elegans) and mice infection models was determined and the synergistic effect of Harmine combined with common antibiotics was explored. The underlaying mechanism of Harmine’s QS inhibitory effect was illustrated by molecular docking analysis, transcriptomic analysis, and target verification assay.ResultsIn vitro results suggested that Harmine possessed QS inhibitory effects on pyocyanin production, exocellular protease excretion, biofilm formation, and twitching motility of P. aeruginosa PA14, and in vivo results displayed Harmine’s protective effect on C. elegans and mice infection models. Intriguingly, Harmine increased susceptibility of both PA14 and clinical isolates of P. aeruginosa to polymyxin B and kanamycin when used in combination. Moreover, Harmine down-regulated a series of QS controlled genes associated with pathogenicity and the underlying mechanism may have involved competitively antagonizing autoinducers’ receptors LasR, RhlR, and PqsR.ConclusionsThis study shed light on the anti-virulence potential of Harmine against QS targets, suggesting the possible use of Harmine and its derivates as anti-virulence compounds.

Evaluating virulence features of Acinetobacter baumannii resistant to polymyxin B.

The increasing resistance to polymyxins in Acinetobacter baumannii has made it even more urgent to develop new treatments. Anti-virulence compounds have been researched as a new solution. Here, we evaluated the modification of virulence features of A. baumannii after acquiring resistance to polymyxin B. The results showed lineages attaining unstable resistance to polymyxin B, except for Ab7 (A. baumannii polymyxin B resistant lineage), which showed stable resistance without an associated fitness cost. Analysis of virulence by a murine sepsis model indicated diminished virulence in Ab7 (A. baumannii polymyxin B resistant lineage) compared with Ab0 (A. baumannii polymyxin B susceptible lineage). Similarly, downregulation of virulence genes was observed by qPCR at 1 and 3h of growth. However, an increase in bauE, abaI, and pgAB expression was observed after 6h of growth. Comparison analysis of Ab0, Ab7, and Pseudomonas aeruginosa suggested no biofilm formation by Ab7. In general, although a decrease in virulence was observed in Ab7 when compared with Ab0, some virulence feature that enables infection could be maintained. In light of this, virulence genes bauE, abaI, and pgAB showed a potential relevance in the maintenance of virulence in polymyxin B-resistant strains, making them promising anti-virulence targets.

Chemical-mediated virulence: the effects of host chemicals on microbial virulence and potential new antivirulence strategies.

The rising antimicrobial resistance rates and declining antimicrobial discovery necessitate alternative strategies to combat multidrug-resistant pathogens. Targeting microbial virulence is an emerging area of interest. Traditionally, virulence factors were largely restricted to bacteria-derived toxins, adhesins, capsules, quorum sensing systems, secretion systems, factors required to sense, respond to, acquire, or synthesize, and utilize trace elements (such as iron and other metals) and micronutrients (such as vitamins), and other factors bacteria use to establish infection, form biofilms, or damage the host tissues and regulatory elements thereof. However, this traditional definition overlooks bacterial virulence that may be induced or influenced by host-produced metabolites or other chemicals that bacteria may encounter at the infection site. This review will discuss virulence from a non-traditional perspective, shedding light on chemical-mediated host-pathogen interactions and outlining currently available mechanistic insight into increased bacterial virulence in response to host factors. This review aims to define a possibly underestimated theme of chemically mediated host-pathogen interactions and encourage future validation and characterization of the contribution of host chemicals to microbial virulence in vivo. From this perspective, we discuss proposed antivirulence compounds and suggest new potential targets for antimicrobials that prevent chemical-mediated virulence. We also explore proposed host-targeting therapeutics reducing the level of host chemicals that induce microbial virulence, serving as virulence attenuators. Understanding the host chemical-mediated virulence may enable new antimicrobial solutions to fight multidrug-resistant pathogens.

Linoleic acid acts as a potential anti-virulence agent in Klebsiella pneumoniae.

The rise in antimicrobial resistance among bacterial pathogens is a global concern, and anti-virulence therapy may be an alternative strategy to address the issue. Multidrug resistant (MDR) hypervirulent Klebsiella pneumoniae (HvKp) is known to be associated with healthcare associated infections. These are often challenging to treat and here anti-virulence therapy may be a treatment option. The study of anti-virulence compounds against HvKp by in-silico prediction, in-vitro experiments and in-vivo assay enables to determine which anti-virulence compounds are suitable for an alternative approach MDR HvKp. Modeling of the proteins, ligand binding and molecular docking were performed targeting different hypervirulence genes viz., rmpA, rmpA2 and, iroC by in-silico analysis using different bioinformatics tool and software. Minimum inhibitory concentration (MIC) was determined for six anti-virulence compounds; curcumin, eugenol, reserpine, linoleic acid, ε-anethole, and α-thujone by standard protocol. Quantitative real-time PCR was performed selecting two isolates harboring rmpA, rmpA2 and iroC genes. Galleria mellonella larva killing assay was used for in-vivo experiment. In-silico analysis observed that linoleic acid could be the best fit in comparison with the other compounds. None of the anti-virulence compounds showed any inhibitory activity and upon transcriptional expression analysis of the hypervirulence genes; rmpA was marginally increased for both the isolates when linoleic acid exposure was given. In-vivo study revealed that linoleic acid and reserpine showed anti-virulence activity.

Evaluation of novel compounds as anti-bacterial or anti-virulence agents.

Antimicrobial resistance is a global threat, leading to an alarming increase in the prevalence of bacterial infections that can no longer be treated with available antibiotics. The World Health Organization estimates that by 2050 up to 10 million deaths per year could be associated with antimicrobial resistance, which would equal the annual number of cancer deaths worldwide. To overcome this emerging crisis, novel anti-bacterial compounds are urgently needed. There are two possible approaches in the fight against bacterial infections: a) targeting structures within bacterial cells, similar to existing antibiotics; and/or b) targeting virulence factors rather than bacterial growth. Here, for the first time, we provide a comprehensive overview of the key steps in the evaluation of potential new anti-bacterial and/or anti-virulence compounds. The methods described in this review include: a) in silico methods for the evaluation of novel compounds; b) anti-bacterial assays (MIC, MBC, Time-kill); b) anti-virulence assays (anti-biofilm, anti-quorum sensing, anti-adhesion); and c) evaluation of safety aspects (cytotoxicity assay and Ames test). Overall, we provide a detailed description of the methods that are an essential tool for chemists, computational chemists, microbiologists, and toxicologists in the evaluation of potential novel antimicrobial compounds. These methods are cost-effective and have high predictive value. They are widely used in preclinical studies to identify new molecular candidates, for further investigation in animal and human trials.

Characterization of the dispirotripiperazine derivative PDSTP as antibiotic adjuvant and antivirulence compound against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major human pathogen, able to establish difficult-to-treat infections in immunocompromised and people with cystic fibrosis (CF). The high rate of antibiotic treatment failure is due to its notorious drug resistance, often mediated by the formation of persistent biofilms. Alternative strategies, capable of overcoming P. aeruginosa resistance, include antivirulence compounds which impair bacterial pathogenesis without exerting a strong selective pressure, and the use of antimicrobial adjuvants that can resensitize drug-resistant bacteria to specific antibiotics. In this work, the dispirotripiperazine derivative PDSTP, already studied as antiviral, was characterized for its activity against P. aeruginosa adhesion to epithelial cells, its antibiotic adjuvant ability and its biofilm inhibitory potential. PDSTP was effective in impairing the adhesion of P. aeruginosa to various immortalized cell lines. Moreover, the combination of clinically relevant antibiotics with the compound led to a remarkable enhancement of the antibiotic efficacy towards multidrug-resistant CF clinical strains. PDSTP-ceftazidime combination maintained its efficacy in vivo in a Galleria mellonella infection model. Finally, the compound showed a promising biofilm inhibitory activity at low concentrations when tested both in vitro and using an ex vivo pig lung model. Altogether, these results validate PDSTP as a promising compound, combining the ability to decrease P. aeruginosa virulence by impairing its adhesion and biofilm formation, with the capability to increase antibiotic efficacy against antibiotic resistant strains.

Extraction, determination and evaluation the anti-virulence compounds of Citrus aurantium against multi-drug resistant Staphylococcus aureus

Extraction, determination and evaluation the anti-virulence compounds of Citrus aurantium against multi-drug resistant Staphylococcus aureus

New Antimicrobial Strategies to Treat Multi-Drug Resistant Infections Caused by Gram-Negatives in Cystic Fibrosis.

People with cystic fibrosis (CF) suffer from recurrent bacterial infections which induce inflammation, lung tissue damage and failure of the respiratory system. Prolonged exposure to combinatorial antibiotic therapies triggers the appearance of multi-drug resistant (MDR) bacteria. The development of alternative antimicrobial strategies may provide a way to mitigate antimicrobial resistance. Here we discuss different alternative approaches to the use of classic antibiotics: anti-virulence and anti-biofilm compounds which exert a low selective pressure; phage therapies that represent an alternative strategy with a high therapeutic potential; new methods helping antibiotics activity such as adjuvants; and antimicrobial peptides and nanoparticle formulations. Their mechanisms and in vitro and in vivo efficacy are described, in order to figure out a complete landscape of new alternative approaches to fight MDR Gram-negative CF pathogens.

Discovery of dual-active ethionamide boosters inhibiting the Mycobacterium tuberculosis ESX-1 secretion system

Drug-resistant Mycobacterium tuberculosis (Mtb) remains a major public health concern requiring complementary approaches to standard anti-tuberculous regimens. Anti-virulence molecules or compounds that enhance the activity of antimicrobial prodrugs are promising alternatives to conventional antibiotics. Exploiting host cell-based drug discovery, we identified an oxadiazole compound (S3) that blocks the ESX-1 secretion system, a major virulence factor of Mtb. S3-treated mycobacteria showed impaired intracellular growth and a reduced ability to lyse macrophages. RNA sequencing experiments of drug-exposed bacteria revealed strong upregulation of a distinct set of genes including ethA, encoding a monooxygenase activating the anti-tuberculous prodrug ethionamide. Accordingly, we found a strong ethionamide boosting effect in S3-treated Mtb. Extensive structure-activity relationship experiments revealed that anti-virulence and ethionamide-boosting activity can be uncoupled by chemical modification of the primary hit molecule. To conclude, this series of dual-active oxadiazole compounds targets Mtb via two distinct mechanisms of action.

Bioprospecting of inhibitors of EPEC virulence from metabolites of marine actinobacteria from the Arctic Sea.

A considerable number of antibacterial agents are derived from bacterial metabolites. Similarly, numerous known compounds that impede bacterial virulence stem from bacterial metabolites. Enteropathogenic Escherichia coli (EPEC) is a notable human pathogen causing intestinal infections, particularly affecting infant mortality in developing regions. These infections are characterized by microvilli effacement and intestinal epithelial lesions linked with aberrant actin polymerization. This study aimed to identify potential antivirulence compounds for EPEC infections among bacterial metabolites harvested from marine actinobacteria (Kocuria sp. and Rhodococcus spp.) from the Arctic Sea by the application of virulence-based screening assays. Moreover, we demonstrate the suitability of these antivirulence assays to screen actinobacteria extract fractions for the bioassay-guided identification of metabolites. We discovered a compound in the fifth fraction of a Kocuria strain that interferes with EPEC-induced actin polymerization without affecting growth. Furthermore, a growth-inhibiting compound was identified in the fifth fraction of a Rhodococcus strain. Our findings include the bioassay-guided identification, HPLC-MS-based dereplication, and isolation of a large phospholipid and a likely antimicrobial peptide, demonstrating the usefulness of this approach in screening for compounds capable of inhibiting EPEC virulence.

Development of Quinazolinone Derivatives as Modulators of Virulence Factors of Pseudomonas aeruginosa Cystic Fibrosis Strains.

Pseudomonas aeruginosa (PA), one of the ESKAPE pathogens, is an opportunistic Gram-negative bacterium responsible for nosocomial infections in humans but also for infections in patients affected by AIDS, cancer, or cystic fibrosis (CF). Treatment of PA infections in CF patients is a global healthcare problem due to the ability of PA to gain antibiotic tolerance through biofilm formation. Anti-virulence compounds represent a promising approach as adjuvant therapy, which could reduce or eliminate the pathogenicity of PA without impacting its growth. Pyocyanin is one of the virulence factors whose production is modulated by the Pseudomonas quinolone signal (PQS) through its receptor PqsR. Different PqsR modulators have been synthesized over the years, highlighting this new powerful therapeutic strategy. Based on the promising structure of quinazolin-4(3H)-one, we developed compounds 7a-d, 8a,b, 9, 10, and 11a-f able to reduce biofilm formation and the production of virulence factors (pyocyanin and pyoverdine) at 50 µM in two PA strains responsible for CF acute and chronic infections. The developed compounds did not reduce the cell viability of IB3-1 bronchial CF cells, and computational studies confirmed the potential ability of novel compounds to act as potential Pqs system modulators.

Fatty Acids Abolish Shigella Virulence by Inhibiting Its Master Regulator, VirF.

The pathogenicity of Shigella, the intracellular pathogen responsible for human bacillary dysentery, depends on a coordinated and tightly regulated expression of its virulence determinants. This is the result of a cascade organization of its positive regulators, with VirF, a transcriptional activator belonging to the AraC-XylS family, in a pivotal position. VirF itself is submitted to several well-known regulations at the transcriptional level. In this work, we present evidence for a novel posttranslational regulatory mechanism of VirF mediated by the inhibitory interaction with specific fatty acids. By homology modeling and molecular docking analyses, we identify a jelly roll motif in the structure of ViF capable of interacting with medium-chain saturated and long-chain unsaturated fatty acids. In vitro and in vivo assays show that capric, lauric, myristoleic, palmitoleic, and sapienic acids interact effectively with the VirF protein, abolishing its transcription-promoting activity. This silences the virulence system of Shigella, leading to a drastic reduction in its ability to invade epithelial cells and proliferate in their cytoplasm. IMPORTANCE In the absence of a valid vaccine, the main therapeutic approach currently used to treat shigellosis is based on the use of antibiotics. The emergence of antibiotic resistance jeopardizes the future effectiveness of this approach. The importance of the present work resides both in the identification of a new level of posttranslational regulation of the Shigella virulence system and in the characterization of a mechanism offering new opportunities for the design of antivirulence compounds, which may change the treatment paradigm of Shigella infections by limiting the emergence of antibiotic-resistant bacteria.

Expanding therapeutic potential of Bdellovibrio bacteriovorus against multidrug-resistant pathogens

Novel treatments toward Gram-negative bacteria are urgently needed to prevent even higher mortality levels associated with resistant bacterial infections. Predatory bacteria have been studied as a new type of treatment against pathogenic bacteria, including resistant species. However, because of limitations related to eradication efficacy, combination therapy using predatory bacteria with other agents has also been tested. Here, we discuss recent advances in the use of predatory bacteria to treat infections and propose novel combinatory strategies with antivirulence compounds.

Effect of traditional Chinese medicine monomers interfering with quorum-sensing on virulence factors of extensively drug-resistant Acinetobacter baumannii.

The antimicrobial resistance of Acinetobacter baumannii (A. baumannii) clinical isolates has emerged as a great threat to public health. Quorum sensing (QS) is one of the resistance mechanisms for drug-resistant A. baumannii. Interfering with QS is a promising strategy to combat infections caused by drug-resistant bacteria. This study explored the QS inhibition ability of thirty-four traditional Chinese medicine monomers (TCMMs) and assessed the effect of QS inhibitors (QSIs) on the virulence factors of twelve extensively drug-resistant A. baumannii (XDRAB) strains. Nine traditional Chinese medicine monomers, such as caffeic acid, cinnamic acid, and myricetin, were found to be able to inhibit the bacterial QS. Then, at 1/8 of the minimal inhibitory concentration, we found that these QSIs inhibited extensively drug-resistant A. baumannii adhesion and biofilm formation and downregulated the expression levels of virulence-associated genes (abaI, abaR, csuE, pgaA, and bap). In conclusion, nine traditional Chinese medicine monomers have QS inhibitory activity and may downregulate QS genes to interfere with the QS system, which could inhibit the expression of extensively drug-resistant A. baumannii virulence factors. These results suggest that traditional Chinese medicine monomers could develop as novel anti-virulence compounds to control extensively drug-resistant A. baumannii infections.

Antiquorum Sensing Activity of Methanolic Seed (Nutmeg) Extract of Myristica Fragrans Against a Gram-Positive Bacterium

The increase in multidrug-resistant pathogens has created new anti-pathogenic and anti-virulence compounds. This is because of the behavioural changes bacteria acquire, such as increased antibiotic resistance and virulence capacity. This social behaviour is maintained through a signalling transduction pathway called Quorum sensing. The present study aims to evaluate the inhibition of quorum sensing in Bacillus subtilis with Myristica fragrans seed extract. The seed of M. fragrans is called nutmeg. Methanolic nutmeg extract was used to test the anti-quorum sensing activity of B. subtilis. Total flavonoid and phenolic concentrations of nutmeg were estimated. 10 μg/ml, 30 μg/ml, 50 μg/ml, 70 μg/ml and 90 μg/ml concentrations of nutmeg extract were used to check against bacterial motilities and biofilm formations. Inhibition of biofilm formation was observed under a fluorescence microscope, and inhibition of swimming and swarming motilities were observed on 0.3 % and 0.5 % agar plates, respectively. These observations suggest that the seed of M. fragrans showed anti-quorum sensing activity against B. subtilis. This research work helps to study and isolate natural quorum-sensing inhibitors from medicinal plants. These inhibitors can synthesize novel anti-pathogenic or anti-virulence drugs that combat bacterial infections by interrupting with quorum-sensing controlled phenotypes and decreasing bacterial virulence.

Staphylococcus aureus Strains Isolated from Cattle Livestock and Possible Use of Anti-Virulence Strategies

Staphylococcus aureus is known for its ability to acquire resistance to commonly used antibiotics and causes mastitis in cows. Bacterial strains belonging to this species can spread in humans and animals, through diffusion in different contexts, including workplaces and the environment. The antibiotic-resistance in S. aureus strains evidenced the need for novel therapeutic approaches that do not exert selective pressure on the evolutionary adaptation of the bacteria. Alternative approaches can be represented by anti-virulence therapies that interfere with virulence factors, or relative pathways that regulate the production of toxins. Various S. aureus toxins and regulatory systems involved in secreting these toxins can be investigated. The potential of targeting S. aureus toxins and virulence-mediated pathways as anti-virulence strategies can be a substantial and important alternative, in contrast to traditional antibiotics directed at pathogen viability but triggering the mechanisms of antibiotic-resistance. Thus, the antivirulence approach must be aimed to reduce the production of virulence factors without affecting bacterial growth. Strategies to reduce bacterial virulence include compounds able to inhibit quorum sensing, disassemble bacterial membranes, disrupt biofilm formation, or neutralize the bacterial toxin, thus reducing the spread of the infection. Virulence factors eventually related to the infectiousness of S. aureus strains can offer new insights into vaccine development and possible identification of new vaccine targets. The benefits of the antivirulence-antibiotic combination during the treatment against S. aureus infections have been enhanced by virtue of the synergistic action between antibiotics and the antivirulence compounds. The characteristics of the spread of antibiotic resistant S. aureus and its virulence characteristics for anti-virulence strategies have been described.

Auranofin inhibits virulence pathways in Pseudomonas aeruginosa

Pseudomonas aeruginosa is widely attributed as the leading cause of hospital-acquired infections. Due to intrinsic antibiotic resistance mechanisms and the ability to form biofilms, P. aeruginosa infections are challenging to treat. P. aeruginosa employs multiple virulence mechanisms to establish infections, many of which are controlled by the global virulence regulator Vfr. An attractive strategy to combat P. aeruginosa infections is thus the use of anti-virulence compounds. Here, we report the discovery that FDA-approved drug auranofin attenuates virulence pathways in P. aeruginosa, including quorum sensing (QS) and Type IV pili (TFP). We show that auranofin acts via multiple targets, one of which being Vfr. Consistent with inhibition of QS and TFP expression, we show that auranofin attenuates biofilm maturation, and when used in combination with colistin, displays strong synergy in eradicating P. aeruginosa biofilms. Auranofin may have immediate applications as an anti-virulence drug against P. aeruginosa infections.

Structural Insights into Regulation of Vibrio Virulence Gene Networks.

One of the best studied aspects of pathogenic Vibrios are the virulence cascades that lead to the production of virulence factors and, ultimately, clinical outcomes. In this chapter, we will examine the regulation of Vibrio virulence gene networks from a structural and biochemical perspective. We will discuss the recent research into the numerous proteins that contribute to regulating virulence in Vibrio spp such as quorum sensing regulator HapR, the transcription factors AphA and AphB, or the virulence regulators ToxR and ToxT. We highlight how insights gained from these studies are already illuminating the basic molecular mechanisms by which the virulence cascade of pathogenic Vibrios unfold and contend that understanding how protein interactions contribute to the host-pathogen communications will enable the development of new antivirulence compounds that can effectively target these pathogens.

Exploring Oceans for Curative Compounds: Potential New Antimicrobial and Anti-Virulence Molecules against Pseudomonas aeruginosa.

Although several antibiotics are already widely used against a large number of pathogens, the discovery of new antimicrobial compounds with new mechanisms of action is critical today in order to overcome the spreading of antimicrobial resistance among pathogen bacteria. In this regard, marine organisms represent a potential source of a wide diversity of unique secondary metabolites produced as an adaptation strategy to survive in competitive and hostile environments. Among the multidrug-resistant Gram-negative bacteria, Pseudomonas aeruginosa is undoubtedly one of the most important species due to its high intrinsic resistance to different classes of antibiotics on the market and its ability to cause serious therapeutic problems. In the present review, we first discuss the general mechanisms involved in the antibiotic resistance of P. aeruginosa. Subsequently, we list the marine molecules identified up until now showing activity against P. aeruginosa, dividing them according to whether they act as antimicrobial or anti-virulence compounds.