Discovery of highly neutralizing human antibodies targeting Pseudomonas aeruginosa.

Drug-resistant Pseudomonas aeruginosa (PA) poses an emerging threat to human health with urgent need for alternative therapeutic approaches. Here, we deciphered the B cell and antibody response to the virulence-associated type III secretion system (T3SS) in a cohort of patients chronically infected with PA. Single-cell analytics revealed a diverse B cell receptor repertoire directed against the T3SS needle-tip protein PcrV, enabling the production of monoclonal antibodies (mAbs) abrogating T3SS-mediated cytotoxicity. Mechanistic studies involving cryoelectron microscopy identified a surface-exposed C-terminal PcrV epitope as the target of highly neutralizing mAbs with broad activity against drug-resistant PA isolates. These anti-PcrV mAbs were as effective as treatment with conventional antibiotics in vivo. Our study reveals that chronically infected patients represent a source of neutralizing antibodies, which can be exploited as therapeutics against PA.

Structure-based design of small molecule inhibitors of the cagT4SS ATPase Cagα of Helicobacter pylori.

We here describe the structure-based design of small molecule inhibitors of the type IV secretion system of Helicobacter pylori. The secretion system is encoded by the cag pathogenicity island, and we chose Cagα, a hexameric ATPase and member of the family of VirB11-like proteins, as target for inhibitor design. We first solved the crystal structure of Cagα in a complex with the previously identified small molecule inhibitor 1G2. The molecule binds at the interface between two Cagα subunits and mutagenesis of the binding site identified Cagα residues F39 and R73 as critical for 1G2 binding. Based on the inhibitor binding site we synthesized 98 small molecule derivates of 1G2 to improve binding of the inhibitor. We used the production of interleukin-8 of gastric cancer cells during H. pylori infection to screen the potency of inhibitors and we identified five molecules (1G2_1313, 1G2_1338, 1G2_2886, 1G2_2889, and 1G2_2902) that have similar or higher potency than 1G2. Differential scanning fluorimetry suggested that these five molecules bind Cagα, and enzyme assays demonstrated that some are more potent ATPase inhibitors than 1G2. Finally, scanning electron microscopy revealed that 1G2 and its derivatives inhibit the assembly of T4SS-determined extracellular pili suggesting a mechanism for their anti-virulence effect.

Piceatannol: a renaissance in antibacterial innovation unveiling synergistic potency and virulence disruption against serious pathogens.

In the relentless battle against multi-drug resistant Gram-negative bacteria, piceatannol emerges as a beacon of hope, showcasing unparalleled antibacterial efficacy and a unique ability to disrupt virulence factors. Our study illuminates the multifaceted prowess of piceatannol against prominent pathogens-Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. Notably, piceatannol demonstrated a remarkable ability to inhibit biofilm formation, reduce bacterial mobility, and diminish extracellular enzyme synthesis.Mechanistic insights into piceatannol's activity unraveled its impact on membrane potential, proton motive force, and ATP production. Furthermore, our study delved into piceatannol's anti-quorum sensing (QS) activity, showcasing its potential to downregulate QS-encoding genes and affirming its affinity to critical QS receptors through molecular docking. Crucially, piceatannol exhibited a low propensity for resistance development, positioning it as a promising candidate for combating antibiotic-resistant strains. Its mild effect on red blood cells (RBCs) suggests safety even at higher concentrations, reinforcing its potential translational value. In an in vivo setting, piceatannol demonstrated protective capabilities, significantly reducing pathogenesis in mice infected with P. aeruginosa and P. mirabilis. This comprehensive analysis positions piceatannol as a renaissance in antibacterial innovation, offering a versatile and effective strategy to confront the evolving challenges posed by resilient Gram-negative pathogens.

Boosting the Anti-Helicobacter Efficacy of Azithromycin through Natural Compounds: Insights From In Vitro, In Vivo, Histopathological, and Molecular Docking Investigations.

BACKGROUND: Antimicrobial-resistant Helicobacter pylori (H. pylori) poses a significant public health concern, especially given the limited therapeutic options for azithromycin-resistant strains. Hence, there is a necessity for new studies to reconsider the use of azithromycin, which has diminished in effectiveness against numerous strains. Thus, we aimed to augment azithromycin's anti-Helicobacter properties by combining it with curcumin in different formulations, including curcumin in clove oil, curcumin nano-gold emulsion, and curcumin nanoemulsion. METHODS: The antimicrobial activities of the investigated compounds, both individually and in combination with other anti-Helicobacter drugs, were evaluated. Their antibiofilm and anti-virulence properties were assessed using both phenotypic and genotypic methods, alongside molecular docking studies. Our findings were further validated through mouse protection assays and histopathological analysis. RESULTS: We observed high anti-Helicobacter activities of curcumin, especially curcumin nanoemulsion. A synergistic effect was detected between curcumin nanoemulsion and azithromycin with fraction inhibitory concentration index (FICI) values <0.5. The curcumin nanoemulsion was the most active anti-biofilm and anti-virulence compound among the examined substances. The biofilm-correlated virulence genes (babA and hopQ) and ureA genes were downregulated (fold change <1) post-treatment with curcumin nanoemulsion. On the protein level, the anti-virulence activities of curcumin nanoemulsion were documented based on molecular docking studies. These findings aligned with histopathological scoring of challenge mice, affirming the superior efficacy of curcumin nanoemulsion/azithromycin combination. CONCLUSION: The anti-Helicobacter activities of all curcumin physical forms pose significant challenges due to their higher  minimum inhibitory concentration (MIC) values exceeding the maximum permissible level. However, using curcumin nanoemulsion at sub-MIC levels could enhance the anti-Helicobacter activity of azithromycin and exhibit anti-virulence properties, thereby improving patient outcomes and addressing resistant pathogens. Therefore, more extensive studies are necessary to assess the safety of incorporating curcumin nanoemulsion into H. pylori treatment.

Inhibition of the type IV secretion system from antibiotic-resistant Helicobacter pylori clinical isolates supports the potential of Cagα as an anti-virulence target.

Helicobacter pylori resistance to antibiotics is a growing problem and it increasingly leads to treatment failure. While the bacterium is present worldwide, the severity of clinical outcomes is highly dependent on the geographical origin and genetic characteristics of the strains. One of the major virulence factors identified in H. pylori is the cag pathogenicity island (cagPAI), which encodes a type IV secretion system (T4SS) used to translocate effectors into human cells. Here, we investigated the genetic variability of the cagPAI among 13 antibiotic-resistant H. pylori strains that were isolated from patient biopsies in Québec. Seven of the clinical strains carried the cagPAI, but only four could be readily cultivated under laboratory conditions. We observed variability of the sequences of CagA and CagL proteins that are encoded by the cagPAI. All clinical isolates induce interleukin-8 secretion and morphological changes upon co-incubation with gastric cancer cells and two of them produce extracellular T4SS pili. Finally, we demonstrate that molecule 1G2, a small molecule inhibitor of the Cagα protein from the model strain H. pylori 26695, reduces interleukin-8 secretion in one of the clinical isolates. Co-incubation with 1G2 also inhibits the assembly of T4SS pili, suggesting a mechanism for its action on T4SS function.

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 3 h of growth. However, an increase in bauE, abaI, and pgAB expression was observed after 6 h 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.

Myricetin Acts as an Inhibitor of Type II NADH Dehydrogenase from Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is a common pathogenic microorganism in humans and animals. Type II NADH oxidoreductase (NDH-2) is the only NADH:quinone oxidoreductase present in this organism and represents a promising target for the development of anti-staphylococcal drugs. Recently, myricetin, a natural flavonoid from vegetables and fruits, was found to be a potential inhibitor of NDH-2 of S. aureus. The objective of this study was to evaluate the inhibitory properties of myricetin against NDH-2 and its impact on the growth and expression of virulence factors in S. aureus. RESULTS: A screening method was established to identify effective inhibitors of NDH-2, based on heterologously expressed S. aureus NDH-2. Myricetin was found to be an effective inhibitor of NDH-2 with a half maximal inhibitory concentration (IC50) of 2 µM. In silico predictions and enzyme inhibition kinetics further characterized myricetin as a competitive inhibitor of NDH-2 with respect to the substrate menadione (MK). The minimum inhibitory concentrations (MICs) of myricetin against S. aureus strains ranged from 64 to 128 µg/mL. Time-kill assays showed that myricetin was a bactericidal agent against S. aureus. In line with being a competitive inhibitor of the NDH-2 substrate MK, the anti-staphylococcal activity of myricetin was antagonized by MK-4. In addition, myricetin was found to inhibit the gene expression of enterotoxin SeA and reduce the hemolytic activity induced by S. aureus culture on rabbit erythrocytes in a dose-dependent manner. CONCLUSIONS: Myricetin was newly discovered to be a competitive inhibitor of S. aureus NDH-2 in relation to the substrate MK. This discovery offers a fresh perspective on the anti-staphylococcal activity of myricetin.

Anti-quorum sensing activity of poly-amidoamine dendrimer generation 5 dendrimer loaded kinase inhibitor peptide against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant concern in both healthcare and community settings, as it causes numerous infections worldwide with high morbidity and mortality rates. One promising strategy is to target the quorum sensing (QS) system of MRSA using a dendrimer loaded with kinase inhibitor peptide. The present investigation has formulated a poly-amidoamine dendrimer (PAMAM) G5 dendrimer that is loaded with Quorum Quencher (QQ) peptide, which functions as a histidine kinase inhibitor. The particle average size of the formulated G5-QQ3 complex was determined to be 276 nm, and polydispersity index values of 0.33. The MIC50 for the formulated nanoparticles was 18 µM as demonstrated by a growth assay. Furthermore, the G5-QQ3 complex was able to inhibit the hemolysis activity of the MRSA with a concentration of 10 µM, and for Staphylococcus aureus was 3 µM. The G5-QQ3 complex possesses the ability to inhibit, penetrate, and eradicate biofilm in MRSA, Staphylococcus aureus, and different agr mutants with inhibition percentages ranging from 60 to 72%. Furthermore, live/dead viability assay confirmed the ability of the formulated nanoparticles to effectively kill all strains within the biofilm structure as evidenced by a confocal microscope, and the cytotoxicity of the G5-QQ3 complex was dose-dependent (p < 0.05). against RAW 264.7 cells. In general, the study confirmed that encapsulating QQ3 peptide within PAMAM G5 dendrimer results in a potent anti-virulence and anti-bacterial action and suggests a synergistic effect. The findings of this study have significant implications for the development of new treatments for MRSA infections, which are a major public health concern.

Structure of Staphylococcus aureus ClpP Bound to the Covalent Active-Site Inhibitor Cystargolide A.

The caseinolytic protease is a highly conserved serine protease, crucial to prokaryotic and eukaryotic protein homeostasis, and a promising antibacterial and anticancer drug target. Herein, we describe the potent cystargolides as the first natural ß-lactone inhibitors of the proteolytic core ClpP. Based on the discovery of two clpP genes next to the cystargolide biosynthetic gene cluster in Kitasatospora cystarginea, we explored ClpP as a potential cystargolide target. We show the inhibition of Staphylococcus aureus ClpP by cystargolide A and B by different biochemical methods in vitro. Synthesis of semisynthetic derivatives and probes with improved cell penetration allowed us to confirm ClpP as a specific target in S. aureus cells and to demonstrate the anti-virulence activity of this natural product class. Crystal structures show cystargolide A covalently bound to all 14 active sites of ClpP from S. aureus, Aquifex aeolicus, and Photorhabdus laumondii, and reveal the molecular mechanism of ClpP inhibition by ß-lactones, the predominant class of ClpP inhibitors.

Pseudomonas aeruginosa strains belonging to phylogroup 3 frequently exhibit an atypical quorum sensing response: the case of MAZ105, a tomato rhizosphere isolate.

Pseudomonas aeruginosa is a widespread γ-proteobacterium and an important opportunistic pathogen. The genetically diverse P. aeruginosa phylogroup 3 strains are characterized by producing the pore-forming ExlA toxin and by their lack of a type III secretion system. However, like all strains of this species, they produce several virulence-associated traits, such as elastase, rhamnolipids and pyocyanin, which are regulated by quorum sensing (QS). The P. aeruginosa QS response comprises three systems (Las, Rhl and Pqs, respectively) that hierarchically regulate these virulence factors. The Pqs QS system is composed of the PqsR transcriptional factor, which, coupled with the alkyl-quinolones HHQ or PQS, activates the transcription of the pqsABCDE operon. The products of the first four genes of this operon produce HHQ, which is then converted to PQS by PqsH, while PqsE forms a complex with RhlR and stabilizes it. In this study we report that mutations affecting the Pqs system are particularly common in phylogroup 3 strains. To better understand QS in phylogroup 3 strains we studied strain MAZ105 isolated from tomato rhizosphere and showed that it contains mutations in the central QS transcriptional regulator, LasR, and in the gene encoding the PqsA enzyme involved in the synthesis of PQS. However, it can still produce QS-regulated virulence factors and is virulent in Galleria mellonella and mildly pathogenic in the mouse abscess/necrosis model; our results show that this may be due to the expression of pqsE from a different PqsR-independent promoter than the pqsA promoter. Our results indicate that using anti-virulence therapy based on targeting the PQS system will not be effective against infections by P. aeruginosa phylogroup 3 strains.

Emerging strategies to target virulence in Pseudomonas aeruginosa respiratory infections.

Pseudomonas aeruginosa is an opportunistic pathogen that is responsible for infections in people living with chronic respiratory conditions, such as cystic fibrosis (CF) and non-CF bronchiectasis (NCFB). Traditionally, in people with chronic respiratory disorders, P. aeruginosa infection has been managed with a combination of inhaled and intravenous antibiotic therapies. However, due in part to the prolonged use of antibiotics in these people, the emergence of multi-drug resistant P. aeruginosa strains is a growing concern. The development of anti-virulence therapeutics may provide a new means of treating P. aeruginosa lung infections whilst also combatting the AMR crisis, as these agents are presumed to exert reduced pressure for the emergence of drug resistance as compared to antibiotics. However, the pipeline for developing anti-virulence therapeutics is poorly defined, and it is currently unclear as to whether in vivo and in vitro models effectively replicate the complex pulmonary environment sufficiently to enable development and testing of such therapies for future clinical use. Here, we discuss potential targets for P. aeruginosa anti-virulence therapeutics and the effectiveness of the current models used to study them. Focus is given to the difficulty of replicating the virulence gene expression patterns of P. aeruginosa in the CF and NCFB lung under laboratory conditions and to the challenges this poses for anti-virulence therapeutic development.

Inhibitory effect of resveratrol on swimming motility and adhesion ability against Salmonella enterica serovar Typhimurium infection.

Salmonella enterica serovar Typhimurium (S. typhimurium) is a common Gram-negative foodborne pathogen that threatens public health and hinders the development of livestock industry. Resveratrol, an important component in grape fruits and seeds, has been shown to possess multiple biological activities, but its potential effects on S. typhimurium-mediated virulence have been rarely reported. In this study, we investigated the effect of resveratrol on S. typhimurium flagella -mediated virulence. The results showed that resveratrol significantly reduced the transcription of flagella genes and swimming motility of S. typhimurium, and also inhibited the transcription of T3SS-related virulence genes with varying degrees inhibiting bacterial growth. Simultaneously, resveratrol significantly reduced the adhesion of S. typhimurium to HeLa cells. Unfortunately, resveratrol does not improve the survival rate of S. typhimurium-infected mice, but it reduces the bacterial load in the liver and spleen of infected mice, and it also has a certain degree of anti-inflammatory activity. In summary, these results indicated that resveratrol has the potential to be developed as an alternative drug or antibacterial agent to prevent Salmonella infection.

Isorhamnetin as a novel inhibitor of pneumolysin against Streptococcus pneumoniae infection in vivo/in vitro.

The increasing incidence of Streptococcus pneumoniae (S. pneumoniae) infection severely threatened the global public heath, causing a significant fatality in immunocompromised hosts. Notably, pneumolysin (PLY) as a pore-forming cytolysin plays a crucial role in the pathogenesis of pneumococcal pneumonia and lung injury. In this study, a natural flavonoid isorhamnetin was identified as a PLY inhibition to suppress PLY-induced hemolysis by engaging the predicted residues and attenuate cytolysin PLY-mediated A549 cells injury. Underlying mechanisms revealed that PLY inhibitor isorhamnetin further contributed to decrease the formation of bacterial biofilms without affecting the expression of PLY. In vivo S. pneumoniae infection confirmed that the pathological injury of lung tissue evoked by S. pneumoniae was ameliorated by isorhamnetin treatment. Collectively, these results presented that isorhamnetin could inhibit the biological activity of PLY, thus reducing the pathogenicity of S. pneumoniae. In summary, our study laid a foundation for the feasible anti-virulence strategy targeting PLY, and provided a promising PLY inhibitor for the treatment of S. pneumoniae infection.

Anti-virulence prospects of Metformin against Pseudomonas aeruginosa: A new dimension to a multifaceted drug.

Metformin (MeT) is an FDA-approved drug with a myriad of health benefits. Besides being used as an anti-diabetic drug, MeT is also effective against various cancers, liver-, cardiovascular-, and renal diseases. This study was undertaken to examine its unique potential as an anti-virulence drug against an opportunistic bacterial pathogen, Pseudomonas aeruginosa. Due to the menace of multidrug resistance in pathogenic microorganisms, many novel or repurposed drugs with anti-virulence prospects are emerging as next-generation therapies with the aim to overshadow the application of existing antimicrobial regimens. The quorum sensing (QS) mechanisms of P. aeruginosa are an attractive drug target for attenuating bacterial virulence. In this context, the anti-QS potential of MeT was scrutinized using biosensor assays. MeT was comprehensively evaluated for its effects on different motility phenotypes, virulence factor production (phenotypic and genotypic expression) along with biofilm development in P. aeruginosa in vitro. At sub-lethal concentrations, MeT displayed prolific quorum quenching (QQ) ability and remarkably inhibited AHL biosynthesis in P. aeruginosa. Moreover, MeT (1/8 MIC) effectively downregulated the expression levels of various QS- and virulence genes in P. aeruginosa, which coincided with a notable reduction in the levels of alginate, hemolysin, pyocyanin, pyochelin, elastase, and protease production. In silico analysis through molecular docking also predicted strong associations between MeT and QS receptors of P. aeruginosa. MeT also compromised the motility phenotypes and successfully abrogated biofilm formation by inhibiting EPS production in P. aeruginosa. Hence, MeT may be repurposed as an anti-virulence drug against P. aeruginosa in clinical settings.

Hesperidin methyl chalcone, a citrus flavonoid, inhibits Aeromonas hydrophila infection mediated by quorum sensing.

Plant-derived phytocompounds are effective in treating a variety of ailments and disorders, the most common of which are bacterial infections in humans, which are a major public health concern. Flavonoids, one of the groups of phytocompounds, are known to have significant antimicrobial and anti-infective properties. Hence, the current study investigates the efficacy of the citrus flavonoid hesperidin methylchalcone (HMC) in addressing this major issue. The results of this study indicate that the anti-quorum sensing (anti-QS) action against Aeromonas hydrophila infections is exhibited with a decrease in biofilm development and virulence factors production through in vitro and in silico analyses. In addition, the qPCR findings indicate that HMC has antivirulence action on A. hydrophila by reducing the expression of QS-related virulence genes, including ahyR, ahyB, ahh1, aerA, and lip. Interestingly, HMC significantly rescued the A. hydrophila-infected zebrafish by reducing the internal colonization, demonstrating the in vivo anti-infective potential of HMC against A. hydrophila infection. Based on these results, this study recommends that HMC could be employed as a possible therapeutic agent to treat A. hydrophila-related infections in humans.

Sponge-derived fatty acids inhibit biofilm formation of MRSA and MSSA by down-regulating biofilm-related genes specific to each pathogen.

AIM: A promising approach for the development of next-generation antimicrobials is to shift their target from causing bacterial death to inhibiting virulence. Marine sponges are an excellent potential source of bioactive anti-virulence molecules (AVM). We screened fractions prepared from 26 samples of Irish coastal sponges for anti-biofilm activity against clinically relevant pathogens. METHODS AND RESULTS: Fifteen fractions from eight sponge species inhibited biofilm of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), and/or Listeria monocytogenes without causing growth inhibition. Gas chromatograph/mass spectroscopy analyses of Mycale contarenii fractions revealed the presence of myristic acid and oleic acid. These fatty acids repressed transcription of the fibronectin-binding protein fnbA and fnbB genes and the polysaccharide intercellular adhesin icaADBC operon, which are required for MRSA and MSSA biofilm formation, respectively. CONCLUSIONS: This study illustrates the potential of AVM from Irish coastal sponges to specifically target bacterial virulence phenotypes, in this case, repression of biofilm formation via decreased transcription of biofilm-associated genes in MSSA and MRSA.

Multivalent calix[4]arene-based mannosylated dendrons as new FimH ligands and inhibitors.

We report the synthesis and biological characterization of a novel class of multivalent glycoconjugates as hit compounds for the design of new antiadhesive therapies against urogenital tract infections (UTIs) caused by uropathogenic E. coli strains (UPEC). The first step of UTIs is the molecular recognition of high mannose N-glycan expressed on the surface of urothelial cells by the bacterial lectin FimH, allowing the pathogen adhesion required for mammalian cell invasion. The inhibition of FimH-mediated interactions is thus a validated strategy for the treatment of UTIs. To this purpose, we designed and synthesized d-mannose multivalent dendrons supported on a calixarene core introducing a significant structural change from a previously described family of dendrimers bearing the same dendrons units on a flexible pentaerythritol scaffold core. The new molecular architecture increased the inhibitory potency against FimH-mediated adhesion processes by about 16 times, as assessed by yeast agglutination assay. Moreover, the direct molecular interaction of the new compounds with FimH protein was assessed by on-cell NMR experiments acquired in the presence of UPEC cells.

Drug repositioning: doxazosin attenuates the virulence factors and biofilm formation in Gram-negative bacteria.

The resistance development is an increasing global health risk that needs innovative solutions. Repurposing drugs to serve as anti-virulence agents is suggested as an advantageous strategy to diminish bacterial resistance development. Bacterial virulence is controlled by quorum sensing (QS) system that orchestrates the expression of biofilm formation, motility, and virulence factors production as enzymes and virulent pigments. Interfering with QS could lead to bacterial virulence mitigation without affecting bacterial growth that does not result in bacterial resistance development. This study investigated the probable anti-virulence and anti-QS activities of α-adrenoreceptor blocker doxazosin against Proteus mirabilis and Pseudomonas aeruginosa. Besides in silico study, in vitro and in vivo investigations were conducted to assess the doxazosin anti-virulence actions. Doxazosin significantly diminished the biofilm formation and release of QS-controlled Chromobacterium violaceum pigment and virulence factors in P. aeruginosa and P. mirabilis, and downregulated the QS encoding genes in P. aeruginosa. Virtually, doxazosin interfered with QS proteins, and in vivo protected mice against P. mirabilis and P. aeruginosa. The role of the membranal sensors as QseC and PmrA was recognized in enhancing the Gram-negative virulence. Doxazosin downregulated the membranal sensors PmR and QseC encoding genes and could in silico interfere with them. In conclusion, this study preliminary documents the probable anti-QS and anti-virulence activities of doxazosin, which indicate its possible application as an alternative or in addition to antibiotics. However, extended toxicological and pharmacological investigations are essential to approve the feasible clinical application of doxazosin as novel efficient anti-virulence agent. KEY POINTS: • Anti-hypertensive doxazosin acquires anti-quorum sensing activities • Doxazosin diminishes the virulence of Proteus mirabilis and Pseudomonas aeruginosa • Doxazosin could dimmish the bacterial espionage.

Neem oil against Aeromonas hydrophila infection by disrupting quorum sensing and biofilm formation.

Aeromonas hydrophila is an opportunistic pathogen that can cause a number of infectious diseases in fish and is widely distributed in aquatic environments. Antibiotics are the main approach against A. hydrophila infections, while the emergence of resistant bacteria limits the application of antibiotics. Here, quorum-sensing (QS) was defined as the target and the inhibitory effects of neem oil against QS of A. hydrophila was studied. The results showed that neem oil could dose-dependently reduce aerolysin, protease, lipase, acyl-homoserine lactones (AHLs), biofilm and swarming motility at sub-inhibitory concentrations. Results of real-time PCR demonstrated that neem oil could down-regulate the transcription of aerA, ahyI and ahyR. Moreover, neem oil showed significant protections to A549 cells and a fish infection model. Taken together, these results indicated that neem oil could be chosen as a promising candidate for the treatment of A. hydrophila infections.

Evaluating the Antibacterial and Antivirulence Activities of Floxuridine against Streptococcus suis.

Streptococcus suis is an emerging zoonotic pathogen that can cause fatal diseases such as meningitis and sepsis in pigs and human beings. The overuse of antibiotics is leading to an increased level of resistance in S. suis, and novel antimicrobial agents or anti-virulence agents for the treatment of infections caused by S. suis are urgently needed. In the present study, we investigated the antibacterial activity, mode of action and anti-virulence effects of floxuridine against S. suis. Floxuridine showed excessive antibacterial activity against S. suis both in vivo and in vitro; 4 × MIC of floxuridine could kill S. suis within 8 h in a time-kill assay. Meanwhile, floxuridine disrupted the membrane structure and permeability of the cytoplasmic membrane. Molecular docking revealed that floxuridine and SLY can be directly bind to each other. Moreover, floxuridine effectively inhibited the hemolytic capacity and expression levels of the virulence-related genes of S. suis. Collectively, these results indicate that the FDA-approved anticancer drug floxuridine is a promising agent and a potential virulence inhibitor against S. suis.