Adjuvant Compounds Research Articles

Evaluation of humoral and cellular immune responses against Vibrio cholerae using oral immunization by multi-epitope-phage-based vaccine

IntroductionCholera is a severe gastrointestinal disease that manifests with rapid onset of diarrhea, vomiting, and high mortality rates. Due to its widespread occurrence in impoverished communities with poor water sanitation, there is an urgent demand for a cost-effective and highly efficient vaccine. Multi-epitope vaccines containing dominant immunological epitopes and adjuvant compounds have demonstrated potential in boosting the immune response. Material and methodsB and T epitopes of OMPU, OMPW, TCPA, CTXA, and CTXB proteins were predicted using bioinformatics methods. Subsequently, highly antigenic multi-epitopes that are non-allergenic and non-toxic were synthesized. These multi-epitopes were then cloned into the pCOMB phagemid. A plasmid M13KO7ΔpIII containing all helper phage proteins except pIII was created to produce the recombinant phage. Female Balb/c mice were divided into three groups and immunized accordingly. The mice received the helper phage, recombinant phage or PBS via gavage feeding thrice within two weeks. Serum samples were collected before and after immunization for the ELISA test as well as evaluating immune system induction through ELISpot testing of spleen lymphocytes. ResultsThe titer of the recombinant phage was determined to be 1011 PFU/ml. The presence of the recombinant phage was confirmed through differences in optical density between sample and control groups in the ELISA phage technique, as well as by observing transduction activity, which demonstrated successful production of a recombinant phage displaying the Vibrio multi-epitope on M13 phage pIII. ELISA results revealed significant differences in phage antibodies before and after inoculation, particularly notable in the negative control mice. Mice treated with multi-epitope phages exhibited antibodies against Vibrio cholerae lysate. Additionally, ELISpot results indicated activation of cellular immunity in mice receiving both Vibrio and helper phage. ConclusionThis study emphasizes the potential of multi-epitope on phage to enhance both cellular and humoral immunity in mice, demonstrating how phages can be used as adjuvants to stimulate mucosal immunity and act as promising candidates for oral vaccination.

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery.

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

The role of membrane transporters in the absorption of atrazine following nasal exposure

Objective The purpose of these studies was to investigate the uptake of atrazine across the nasal mucosa to determine whether direct transport to the brain through the olfactory epithelium is likely to occur. These studies were undertaken to provide important new information about the potential for the enhanced neurotoxicity of herbicides following nasal inhalation. Materials and Methods Transport of atrazine from aqueous solution and from commercial atrazine-containing herbicide products was assessed using excised nasal mucosal tissues. The permeation rate and the role of membrane transporters in the uptake of atrazine across the nasal mucosa were also investigated. Histological examination of the nasal tissues was conducted to assess the effects of commercial atrazine-containing products on nasal tissue morphology. Results Atrazine showed high flux across both nasal respiratory and olfactory tissues, and efflux transporters were found to play an essential role in limiting its uptake at low exposure concentrations. Commercial atrazine-containing herbicide products showed remarkably high transfer across the nasal tissues, and histological evaluation showed significant changes in the morphology of the nasal epithelium following exposure to the herbicide products. Discussion Lipophilic herbicides such as atrazine can freely permeate across the nasal mucosa despite the activity of efflux transporters. The adjuvant compounds in commercial herbicide products disrupt the nasal mucosa’s epithelial barrier, resulting in even greater atrazine permeation across the tissues. The properties of the herbicide itself and those of the formulated products play crucial roles in the potential for the enhanced neurotoxicity of herbicides following nasal inhalation.

Saffron the “Red Gold” and Its CNS Activity: A Challenge for Future Applications in Nutraceuticals

Saffron, indicated as “the red gold,” is an extraordinary spice with a unique aroma and organoleptic properties that is largely diffused in food preparation as well as a traditional medicine in many countries of the world. In the last years, several studies have considered this spice for CNS-based disorders showing its potential usefulness. In this review, we considered the role of saffron as a nutraceutical for the management of the most diffused CNS diseases considering the important role of oxidative stress on the pathogenesis of such diseases. In fact, recent findings support a crucial role of oxidative stress in different CNS diseases suggesting an important role of antioxidants. Preclinical and clinical evidence of its efficacy in different physiopathological pathways involved in several CNS diseases were discussed showing evidence of pharmacological activities and beneficial effects in pathological models or in small trials. Due to low toxicity and significant activities on oxidative stress and inflammation as well as the ability to modulate mitochondrial function, the saffron extracts and their constituents appear to be promising nutraceutical active compounds in this area. Further investigations are in progress to assess the efficacy and safety of preventive agents as nutraceuticals or as adjuvant compounds to be used in combinations with other therapeutic approaches. Saffron nutraceuticals with significant antioxidant activity can be useful in improving the quality of life of patients suffering from several different pathological conditions related to CNS. In this review, we summarized the more recent studies showing that standardized saffron products can be a valuable instrument of well-being due to their effects on multiple targets that support the health of the brain and related tissues.

Pharmacological Stimulation of Soluble Guanylate Cyclase Counteracts the Profibrotic Activation of Human Conjunctival Fibroblasts.

Conjunctival fibrosis is a serious clinical concern implicated in a wide spectrum of eye diseases, including outcomes of surgery for pterygium and glaucoma. It is mainly driven by chronic inflammation that stimulates conjunctival fibroblasts to differentiate into myofibroblasts over time, leading to abnormal wound healing and scar formation. Soluble guanylate cyclase (sGC) stimulation was found to suppress transforming growth factor β (TGFβ)-induced myofibroblastic differentiation in various stromal cells such as skin and pulmonary fibroblasts, as well as corneal keratocytes. Here, we evaluated the in vitro effects of stimulation of the sGC enzyme with the cell-permeable pyrazolopyridinylpyrimidine compound BAY 41-2272 in modulating the TGFβ1-mediated profibrotic activation of human conjunctival fibroblasts. Cells were pretreated with the sGC stimulator before challenging with recombinant human TGFβ1, and subsequently assayed for viability, proliferation, migration, invasiveness, myofibroblast marker expression, and contractile properties. Stimulation of sGC significantly counteracted TGFβ1-induced cell proliferation, migration, invasiveness, and acquisition of a myofibroblast-like phenotype, as shown by a significant downregulation of FAP, ACTA2, COL1A1, COL1A2, FN1, MMP2, TIMP1, and TIMP2 mRNA levels, as well as by a significant reduction in α-smooth muscle actin, N-cadherin, COL1A1, and FN-EDA protein expression. In addition, pretreatment with the sGC stimulator was capable of significantly dampening TGFβ1-induced acquisition of a contractile phenotype by conjunctival fibroblasts, as well as phosphorylation of Smad3 and release of the proinflammatory cytokines IL-1β and IL-6. Taken together, our findings are the first to demonstrate the effectiveness of pharmacological sGC stimulation in counteracting conjunctival fibroblast-to-myofibroblast transition, thus providing a promising scientific background to further explore the feasibility of sGC stimulators as potential new adjuvant therapeutic compounds to treat conjunctival fibrotic conditions.

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.

Significance of Vitamin Supplementation in Reducing the Severity of COVID-19.

Coronavirus disease-19 (COVID-19), a serious pandemic due to the SARS-CoV-2 virus infection, caused significant lockdowns, healthcare shortages, and deaths worldwide. The infection leads to an uncontrolled systemic inflammatory response causing severe respiratory distress and multiple-organ failure. Quick development of several vaccines efficiently controlled the spread of COVID-19. However, the rise of various new subvariants of COVID-19 demonstrated some concerns over the efficacy of existing vaccines. Currently, better vaccines to control these variants are still under development as several new subvariants of COVID-19, such as omicron BA-4, BA-5, and BF-7 are still impacting the world. Few antiviral treatments have been shown to control COVID-19 symptoms. Further, control of COVID-19 symptoms has been explored with many natural and synthetic adjuvant compounds in hopes of treating the deadly and contagious disease. Vitamins have been shown to modulate the immune system, function as antioxidants, and reduce the inflammatory response. Recent studies have investigated the potential role of vitamins, specifically vitamins A, B, C, D, and E, in reducing the immune and inflammatory responses and severity of the complication. In this brief article, we discussed our current understanding of the role of vitamins in controlling COVID-19 symptoms and their potential use as adjuvant therapy.

An aluminium adjuvant compound with ginseng stem leaf saponins enhances the potency of inactivated Pseudomonas plecoglossicida vaccine in large yellow croaker (Larimichthys crocea)

Large yellow croaker (Larimichthys crocea) farm industry in China suffered from huge economic loss caused by Pseudomonas plecoglossicida infection. Due to multi-antibiotic resistance, efficient vaccines are urgent to be developed to combat this pathogen. In this study, an inactivated vaccine was developed with an aluminium adjuvant (Alum) plus ginseng stem and leaf saponins (GSLS). As a result, the relative percentage survival (RPS) against P. plecoglossicida was up to 67.8 %. Comparatively, RPS of groups that vaccinated with only inactivated vaccine and vaccine containing Alum or Montanide™ 763A as adjuvant were 21.8 %, 32.2 % and 62.1 %, respectively. Assays for total serum protein and serum lysozyme activity in group vaccinated with inactivated vaccine plus Alum + GSLS adjuvant were significantly higher than that in control group. Moreover, specific antibody in serum elicited a rapid and persistent level. According to the expression of some immune related genes, inactivated vaccine plus Alum + GSLS adjuvant induced a stronger cellular immune response which was vital to defend against P. plecoglossicida. In conclusion, our study demonstrated that the compound Alum and GSLS adjuvant is a potential adjuvant system to develop LYC vaccine.

Antibacterial and antibiofilm effects of Camellia oleifera seed dreg extract and its application in cosmetics.

Cosmetic care products contain a high proportion of water and nutrients. Therefore, preventing bacterial growth is an important issue to ensure product quality and safety. The application of antibacterial natural ingredients derived from plants is considered to have the potential to maintain product quality and reduce the use of chemicals in formulations. Additionally, chemically synthesized antiseptic and antibacterial agents are widely used in the industry at present. However, some preservative ingredients have been reported that may cause skin irritation, redness, allergies, and even dermatitis. This study aimed to prepare extract from Camellia oleifera tea seed dregs (CTSD), investigate the antibacterial effects on two pathogenic bacteria and evaluate the product preservative ability. Ethanol extraction was prepared and subjected to characterize their triterpenoid contents. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC) were determined for Pseudomonas aeruginosa and Staphylococcus aureus. The product's stability and preservative qualities, along with its ability to scavenge free radicals through antioxidant activity, were also assessed. The gram-positive S. aureus showed greater susceptibility to the treatment. In additional, CTSD possessed significant free radical scavenging activity invitro and cultured normal human skin fibroblast CCD-966SK cells under nontoxic concentration. The challenge test and accelerated storage test confirmed the CTSD containing formulated emulsion is eligible for commercialization. CTSD has the potential to be developed as an alternative agent to control microbial biofilm formation, or can be used as an adjuvant compound for infectious disease control.

Emulsion and liposome-based adjuvanted R21 vaccine formulations mediate protection against malaria through distinct immune mechanisms

Adjuvanted protein vaccines offer high efficacy, yet most potent adjuvants remain proprietary. Several adjuvant compounds are being developed by the Vaccine Formulation Institute in Switzerland for global open access clinical use. In the context of the R21 malaria vaccine, in a mouse challenge model, we characterize the efficacy and mechanism of action of four Vaccine Formulation Institute adjuvants: two liposomal (LQ and LMQ) and two squalene emulsion-based adjuvants (SQ and SMQ), containing QS-21 saponin (Q)and optionally a synthetic TLR4 agonist (M). Two R21 vaccine formulations, R21/LMQ and R21/SQ, offer the highest protection (81%-100%), yet they trigger different innate sensing mechanisms in macrophages with LMQ, but not SQ, activating the NLRP3 inflammasome. The resulting invivo adaptive responses have a different TH1/TH2 balance and engage divergent innate pathways while retaining high protective efficacy. We describe how modular changes in vaccine formulation allow for the dissection of the underlying immune pathways, enabling future mechanistically informed vaccine design.

Antimicrobial Peptidomimetics Prevent the Development of Resistance against Gentamicin and Ciprofloxacin in Staphylococcus and Pseudomonas Bacteria.

Bacteria readily acquire resistance to traditional antibiotics, resulting in pan-resistant strains with no available treatment. Antimicrobial resistance is a global challenge and without the development of effective antimicrobials, the foundation of modern medicine is at risk. Combination therapies such as antibiotic-antibiotic and antibiotic-adjuvant combinations are strategies used to combat antibiotic resistance. Current research focuses on antimicrobial peptidomimetics as adjuvant compounds, due to their promising activity against antibiotic-resistant bacteria. Here, for the first time we demonstrate that antibiotic-peptidomimetic combinations mitigate the development of antibiotic resistance in Staphylococcus aureus and Pseudomonas aeruginosa. When ciprofloxacin and gentamicin were passaged individually at sub-inhibitory concentrations for 10 days, the minimum inhibitory concentrations (MICs) increased up to 32-fold and 128-fold for S. aureus and P. aeruginosa, respectively. In contrast, when antibiotics were passaged in combination with peptidomimetics (Melimine, Mel4, RK758), the MICs of both antibiotics and peptidomimetics remained constant, indicating these combinations were able to mitigate the development of antibiotic-resistance. Furthermore, antibiotic-peptidomimetic combinations demonstrated synergistic activity against both Gram-positive and Gram-negative bacteria, reducing the concentration needed for bactericidal activity. This has significant potential clinical applications-including preventing the spread of antibiotic-resistant strains in hospitals and communities, reviving ineffective antibiotics, and lowering the toxicity of antimicrobial chemotherapy.

Biological evaluation of [4-(4-aminophenyl)-1-(4-fluorophenyl)-1H-pyrrol-3-yl](3,4,5-trimethoxyphenyl)methanone as potential antineoplastic agent in 2D and 3D breast cancer models.

Targeting tubulin polymerization and depolymerization represents a promising approach to treat solid tumors. In this study, we investigated the molecular mechanisms underlying the anticancer effects of a structurally novel tubulin inhibitor, [4-(4-aminophenyl)-1-(4-fluorophenyl)-1H-pyrrol-3-yl](3,4,5-trimethoxyphenyl)methanone (ARDAP), in two- and three-dimensional MCF-7 breast cancer models. At sub-cytotoxic concentrations, ARDAP showed a marked decrease in cell proliferation, colony formation, and ATP intracellular content in MCF-7 cells, by acting through a cytostatic mechanism. Additionally, drug exposure caused blockage of the epithelial-to-mesenchymal transition (EMT). In 3D cell culture, ARDAP negatively affected tumor spheroid growth, with inhibition of spheroid formation and reduction of ATP concentration levels. Notably, ARDAP exposure promoted the differentiation of MCF-7 cells by inducing: (i) expression decrease of Oct4 and Sox2 stemness markers, both in 2D and 3D models, and (ii) downregulation of the stem cell surface marker CD133 in 2D cell cultures. Interestingly, treated MCF7 cells displayed a major sensitivity to cytotoxic effects of the conventional chemotherapeutic drug doxorubicin. In addition, although exhibiting growth inhibitory effects against breast cancer cells, ARDAP showed insignificant harm to MCF10A healthy cells. Collectively, our results highlight the potential of ARDAP to emerge as a new chemotherapeutic agent or adjuvant compound in chemotherapeutic treatments.

Exploring Mangifera indica (Anacardiaceae) leaf and bark methanol extracts as potential adjuvant therapy in the management of multidrug-resistant Staphylococcus aureus

Background: After several decades of antibiotic use, pathogenic bacteria have reached alarming levels of resistance. Staphylococcus aureus is the most common cause of nosocomial infections, and treatment is difficult owing to the advent of multidrug-resistant (MDR) strains. This motivates the search for more potent drugs. Foremost, adjuvant compounds that increase the effectiveness of conventional antibiotics are also being researched extensively. The current study examined the anti-staphylococcal and antibiotic-resistance reversal effects of the methanol extracts of Mangifera indica (leaves and bark). Methods: Botanicals were tested alone, in the presence of reserpine (efflux pump inhibitor), and in association with commonly prescribed antibiotics, using a 96-well broth microdilution method against a panel of seventeen MDR strains and clinical isolates of S. aureus, including methicillin-resistant strains (MRSA). The ability of the leaf extract to inhibit H+-ATPase-mediated proton pumping was determined by controlling the acidification of the bacterial solution, whereas the influence on bacterial kinetic growth was determined by measuring absorbance (OD600 nm) after exposure to various concentrations of the test extract. Results: M. indica leaf and bark extracts exhibited exceptional anti-staphylococcal capabilities, inhibiting 100% of the S. aureus strains tested. The minimal inhibitory concentrations (MICs) recorded varied from 256 to 2048 µg/mL; the effects were bactericidal (MBC/MIC ≤ 4) in most cases. The bark extract demonstrated an outstanding potential to improve the efficacy of conventional antibiotics. The activity of chloramphenicol, doxycycline, tetracycline, levofloxacin, and ampicillin was enhanced against 100% of studied MDR S. aureus in association with the bark extract at sub-inhibitory concentrations of MIC/2 and MIC/4. The leaf extract of M. indica induced a concentration-dependent inhibition of S. aureus MRSA4 growth over 20 hours of exposure at 0.5×MIC, MIC, and 2×MIC. The latent phase has been extended up to 6 hours after treatment with the extract. Similarly, M. indica leaf extract significantly reduced the acidity of the bacterial solution in a concentration-dependent manner, indicating a possible target of its antibacterial effect. Conclusion: The present study revealed the remarkable activity of M. indica leaf and bark extracts against MDR strains and clinical isolates of S. aureus, including MRSA. The bark extract might be used as an adjuvant to antibiotic therapy, as indicated by its notable potentiation activity when combined with conventional antibiotics. Keywords: Antibacterial activity; Mangifera indica; multidrug resistance; potentiation; Staphylococcus aureus.

Antibiotic-Potentiating Effect of Some Bioactive Natural Products against Planktonic Cells, Biofilms, and Virulence Factors of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections that are mediated by both virulence factor production and biofilm formation. In addition, many antibiotics are increasingly losing their efficacy due to the development of resistance. The screening of potentially bioactive natural compounds that have both antivirulence and antibiofilm activities to enhance antibiotic efficacy and reverse antibiotic resistance is a good strategy to overcome these issues. In this study, the antibacterial, antibiofilm, and antivirulence factor activities of some bioactive natural products in combination with conventional antibiotics were evaluated against clinical isolates of P. aeruginosa. The broth microdilution method was used to determine the antibacterial and antibiofilm activities. The checkerboard method was used to evaluate the combination interactions. Spectrophotometric and agar plate techniques were used to assess the effect of the combination on the pyocyanin production and the motility in P. aeruginosa ATCC 27853 strain. Out of the eighteen combinations tested, ten exhibited synergistic effects against planktonic cells, seven against biofilm inhibition, and five against the eradication of mature biofilm of P. aeruginosa biofilm. The best synergistic effect was the association of amikacin and sinapic acid against planktonic cells (FICI = 0.08) with a 70-fold reduction in the MIC value of amikacin. The same combination showed significant synergistic inhibition of biofilm formation (FICI = 0.1) and biofilm eradication (FICI = 0.15) reducing the MBIC and MBEC of amikacin by 32-fold. Some selected synergistic combinations showed statistically significant differences (p < 0.01 or p < 0.001) in the inhibition of virulence factors compared to the antimicrobials alone. In summary, this study revealed sinapic acid as an antibiotic adjuvant and antivirulence compound to overcome P. aeruginosa infections. This finding indicates that the combinations of amikacin plus sinapic acid, ceftazidime plus thymol, and norfloxacin plus curcumin could be considered promising candidates for the development of combination therapies targeting virulence factors against P. aeruginosa infections.

Effects of Laparoscopic Hyperthermic Perfusion Therapy Combined with Adjuvant Treatment of Compound Yew Capsule on Ovarian Blood Flow Parameters and Immune Function in Patients with Ovarian Cancer.

Objective To determine the effects of laparoscopic hyperthermic perfusion therapy combined with adjuvant compound yew capsules on ovarian blood flow parameters and immune function in patients with ovarian cancer (OC). Methods A total of 90 OC patients enrolled in our hospital between January 2019 and January 2020 were randomly distributed into the control (Con group) and experimental group (Exp group) based on the sealed envelope method. The Con group was administered laparoscopic hyperthermic perfusion therapy. On this basis, the Exp group was subjected to compound yew capsules; the ovarian blood flow parameters and immune function indexes were compared between the two groups. Results The Exp group was reported to perform better than the Con group regarding ovarian blood flow parameters and immune indexes after treatment (p < 0.001). Conclusion Laparoscopic hyperthermic perfusion therapy combined with adjuvant compound yew capsules for patients with OC can substantially improve the clinical indexes and immune function. Furthermore, research and adequate promotion are needed to elicit the evidence beyond preclinical studies to understand the intricacies of its implementation.

Identification of an anti-virulence drug that reverses antibiotic resistance in multidrug resistant bacteria

The persistent incidence of high levels of multidrug-resistant (MDR) bacteria seriously endangers global public health. In response to MDR-associated infections, new antibacterial drugs and strategies are particularly needed. Screening to evaluate a potential compound to reverse antibiotic resistance is a good strategy to alleviate this crisis. In this paper, using high-throughput screening methods, we identified that oxyclozanide potentiated tetracycline antibiotics act against MDR bacterial pathogens by promoting intracellular accumulation of tetracycline in resistant bacteria. Furthermore, mechanistic studies demonstrated that oxyclozanide could directly kill bacteria by disrupting bacterial membrane and inducing the overproduction of bacterial reactive oxygen species. Oxyclozanide effectively reduced the production of virulence proteins in S. aureus and neutralized the produced α-hemolysin, thereby effectively alleviating the inflammatory response caused by bacteria. Finally, oxyclozanide significantly reversed tetracycline resistance in animal infection assays. In summary, these results demonstrated the capacity of oxyclozanide as a novel antibiotic adjuvant, antibacterial and anti-virulence multifunctional compound to circumvent MDR bacteria and improve the therapeutic effect of persistent infections caused by MDR bacteria worldwide.

Brominated Carbazole with Antibiotic Adjuvant Activity Displays Pleiotropic Effects in MRSA's Transcriptome.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major threat to human health, as the US mortality rate outweighs those from HIV, tuberculosis, and viral hepatitis combined. In the wake of the COVID-19 pandemic, antibiotic-resistant bacterial infections acquired during hospital stays have increased. Antibiotic adjuvants are a key strategy to combat these bacteria. We have evaluated several small molecule antibiotic adjuvants that have strong potentiation with β-lactam antibiotics and are likely inhibiting a master regulatory kinase, Stk1. Here, we investigated how the lead adjuvant (compound 8) exerts its effects in a more comprehensive manner. We hypothesized that the expression levels of key resistance genes would decrease once cotreated with oxacillin and the adjuvant. Furthermore, bioinformatic analyses would reveal biochemical pathways enriched in differentially expressed genes. RNA-seq analysis showed 176 and 233 genes significantly up- and downregulated, respectively, in response to cotreatment. Gene ontology categories and biochemical pathways that were significantly enriched with downregulated genes involved carbohydrate utilization, such as the citrate cycle and the phosphotransferase system. One of the most populated pathways was S. aureus infection. Results from an interaction network constructed with affected gene products supported the hypothesis that Stk1 is a target of compound 8. This study revealed a dramatic impact of our lead adjuvant on the transcriptome that is consistent with a pleiotropic effect due to Stk1 inhibition. These results point to this antibiotic adjuvant having potential broad therapeutic use in combatting MRSA.

Esterases From Bifidobacteria Exhibit the Conversion of Albiflorin in Gut Microbiota.

Bifidobacteria is an important microbe that inhabits the human gut. It is capable of metabolizing complex compounds in the human diet. Albiflorin, an antidepressant natural product from Radix Paeoniae Alba in China, is difficult to absorb after oral administration, and its metabolism has been proven to be closely related to the gut microbiota. In this study, we demonstrated in vitro that several Bifidobacteria species were able to convert albiflorin to benzoic acid, and four esterases (B2, B3, B4, and BL) from Bifidobacterium breve and Bifidobacterium longum were found through genome mining and modeled by SWISS-MODEL. B2 and B3 presented the strongest albiflorin metabolism ability. The optimal conditions, including temperature, buffer, and pH, for the conversion of albiflorin by the four esterases were investigated. Furthermore, the effect of esterase on the metabolism of albiflorin in vivo was confirmed by transplanting bacteria containing esterase B2. This study demonstrated the vital role of esterases from Bifidobacteria in the metabolism of natural compounds containing ester bonds, which could contribute to the development of new enzymes, microbial evolution, and probiotic adjuvant compounds for treatment.

Genetic Dissection of Antibiotic Adjuvant Activity.

ABSTRACTSmall molecule adjuvants that enhance the activity of established antibiotics represent promising agents in the battle against antibiotic resistance. Adjuvants generally act by inhibiting antibiotic resistance processes, and specifying the process acted on is a critical step in defining an adjuvant’s mechanism of action. This step is typically carried out biochemically by identifying molecules that bind adjuvants and then inferring their roles in resistance. Here, we present a complementary genetic strategy based on identifying mutations that both sensitize cells to antibiotic and make them “adjuvant blind.” We tested the approach in Acinetobacter baumannii AB5075 using two adjuvants: a well-characterized β-lactamase inhibitor (avibactam) and a compound enhancing outer membrane permeability (aryl 2-aminoimidazole AI-1). The avibactam studies showed that the adjuvant potentiated one β-lactam (ceftazidime) through action on a single β-lactamase (GES-14) and a second (meropenem) by targeting two different enzymes (GES-14 and OXA-23). Mutations impairing disulfide bond formation (DsbAB) also reduced potentiation, possibly by impairing β-lactamase folding. Mutations reducing AI-1 potentiation of canonical Gram-positive antibiotics (vancomycin and clarithromycin) blocked lipooligosaccharide (LOS/LPS) synthesis or its acyl modification. The results indicate that LOS-mediated outer membrane impermeability is targeted by the adjuvant and show the importance of acylation in the resistance. As part of the study, we employed Acinetobacter baylyi as a model to verify the generality of the A. baumannii results and identified the principal resistance genes for ceftazidime, meropenem, vancomycin, and clarithromycin in A. baumannii AB5075. Overall, the work provides a foundation for analyzing adjuvant action using a comprehensive genetic approach.

β-Glucans Could Be Adjuvants for SARS-CoV-2 Virus Vaccines (COVID-19).

Waiting for an effective treatment against the SARS-CoV-2 virus (the cause of COVID-19), the current alternatives include prevention and the use of vaccines. At the moment, vaccination is the most effective strategy in the fight against pandemic. Vaccines can be administered with different natural biological products (adjuvants) with immunomodulating properties. Adjuvants can be taken orally, complementing vaccine action. Adjuvant compounds could play a key role in alleviating the symptoms of the disease, as well as in enhancing vaccine action. Adjuvants also contribute to an effective immune response and can enhance the protective effect of vaccines in immunocompromised individuals such as the elderly. Adjuvants must not produce adverse effects, toxicity, or any other symptoms that could alter immune system function. Vaccine adjuvants are substances of wide varying chemical structure that are used to boost the immune response against a simultaneously administered antigen. Glucans could work as adjuvants due to their immunomodulatory biological activity. In this respect, β-(1,3)-(1,6) glucans are considered the most effective and safe according to the list issued by the European Commission. Only glucans with a β-(1,3) bond linked to a β-(1,6) are considered modulators of certain biological responses. The aim of this review is to present the possible effects of β-glucans as adjuvants in the efficacy of vaccines against SARS-CoV-2 virus.