Rutin, A Natural Inhibitor of IGPD Protein, Partially Inhibits Biofilm Formation in Staphylococcus xylosus ATCC700404 in vitro and in vivo.

Staphylococcus xylosus (S. xylosus) has become an emerging opportunistic pathogen due to its strong biofilm formation ability. Simultaneously, the biofilm of bacteria plays an important role in antibiotic resistance and chronic infection. Here, we confirmed that rutin can effectively inhibit biofilm formation in S. xylosus, of which the inhibition mechanism involves its ability to interact with imidazole glycerol phosphate dehydratase (IGPD), a key enzyme in the process of biofilm formation. We designed experiments to target IGPD and inhibited its activities against S. xylosus. Our results indicated that the activity of IGPD and the amount of histidine decreased significantly under the condition of 0.8 mg/ml rutin. Moreover, the expression of IGPD mRNA (hisB) and IGPD protein was significantly down-regulated. Meanwhile, the results from molecular dynamic simulation and Bio-layer interferometry (BLI) technique showed that rutin could bind to IGPD strongly. Additionally, in vivo studies demonstrated that rutin treatment reduced inflammation and protect mice from acute mastitis caused by S. xylosus. In summary, our findings provide new insights into the treatment of biofilm mediated persistent infections and chronic bacterial infections. It could be helpful to design next generation antibiotics to against resistant bacteria.

Effect of Syringopicroside Extracted from Syringa oblata Lindl on the Biofilm Formation of Streptococcus suis.

Syringopicroside is a natural drug with antibacterial activity, which is the main ingredient of Syringa oblata Lindl (S. oblata). In order to further develop the application of S. oblata and evaluate the ability of syringopicroside against Streptococcus suis (S. suis), this investigation first applied an ultrasonic-assisted method to extract syringopicroside, and then response surface methodology (RSM) was performed to get the optimum condition. Based on RSM analysis, a second-order polynomial equation about the syringopicroside yield and four variables, including ultrasonic power, time, temperature, and liquid-to-solid ratio, was purposed. Through RSM prediction and model verification experiments, the optimum conditions were determined, as follows: ultrasonic time was 63 min, temperature was 60 °C, a liquid-to-solid ratio was set to 63 mL/g, and ultrasonic power was 835 W. Under this condition, a high syringopicroside yield was obtained (3.07 ± 0.13 mg/g), which was not significantly different with a predicated value. After separation and purification by HPD 500 microporous resin, then mass spectrum was applied to identify the main ingredient in aqueous extract. A minimal inhibitory concentration (MIC) assay revealed the value against S. suis of syringopicroside was 2.56 µg/µL and syringopicroside with sub-inhibitory concentrations that could effectively inhibit biofilm formation of S. suis. Besides, scanning electron microscopy analysis indicated syringopicroside could destroy the multi-layered aggregation structure of S. suis. Finally, molecular docking analysis confirmed that syringopicroside was combined with Orfy protein of S. suis through hydrogen bonds, hydrophobic interaction, and π-π stacking.

Antiviral Strategies of Chinese Herbal Medicine Against PRRSV Infection.

Bioactive compounds from Traditional Chinese Medicines (TCMs) are gradually becoming an effective alternative in the control of porcine reproductive and respiratory syndrome virus (PRRSV) because most of the commercially available PRRSV vaccines cannot provide full protection against the genetically diverse strains isolated from farms. Besides, the incomplete attenuation procedure involved in the production of modified live vaccines (MLV) may cause them to revert to the more virulence forms. TCMs have shown some promising potentials in bridging this gap. Several investigations have revealed that herbal extracts from TCMs contain molecules with significant antiviral activities against the various stages of the life cycle of PRRSV, and they do this through different mechanisms. They either block PRRSV attachment and entry into cells or inhibits the replication of viral RNA or viral particles assembly and release or act as immunomodulators and pathogenic pathway inhibitors through cytokines regulations. Here, we summarized the various antiviral strategies employed by some TCMs against the different stages of the life cycle of PRRSV under two major classes, including direct-acting antivirals (DAAs) and indirect-acting antivirals (IAAs). We highlighted their mechanisms of action. In conclusion, we recommended that in making plans for the use of TCMs to control PRRSV, the pathway forward must be built on a real understanding of the mechanisms by which bioactive compounds exert their effects. This will provide a template that will guide the focus of collaborative studies among researchers in the areas of bioinformatics, chemistry, and proteomics. Furthermore, available data and procedures to support the efficacy, safety, and quality control levels of TCMs should be well documented without any breach of data integrity and good manufacturing practices.

1-Hydroxyanthraquinone exhibited antibacterial activity by regulating glutamine synthetase of Staphylococcus xylosus as a virulence factor.

Staphylococcus xylosus (S. xylosus) is one of the emerging pathogens causing bovine mastitis with high rate of isolation in most of the reported clinical and field cases. To verify the role of glutamine synthetase (GS) in the pathogenesis of S. xylosus, we evaluated the virulence level of the wild-type strain and its glnA mutant strain in biofilm assays in vitro and murine infection model in vivo. From the results, it was observed that the glnA mutant strain was attenuated and could reduce tissue damage. 1-Hydroxyanthraquinone (1-HAQ) is a kind of anthraquinones, it exhibited a significant inhibitory effect on the growth of S. xylosus and biofilm formation in vitro and provided anti-inflammatory effects in vivo. In addition, the rate at which it inhibits the biofilm, inflammatory factors, and CFU of wild-type strains were significantly higher than that of the mutant strains, indicating that 1-hAQ might have pharmacological effects against S. xylosus through the regulation of GS protein. The effect of 1-hAQ on GS was further confirmed by the down-regulation of glnA expression, reduced GS activity, Gln content and the results of molecular docking. Taken together, these findings suggest that 1-hAQ facilitated a significant attenuation of S. xylosus pathogenicity by regulating the GS protein: a vital virulence factor. Therefore, it can be inferred that 1-hAQ may serve as a potential source of organic compound for the development of novel alternative drugs in mitigating the menace of bovine mastitis.

In vitro activity and In vivo efficacy of Isoliquiritigenin against Staphylococcus xylosus ATCC 700404 by IGPD target.

Staphylococcus xylosus (S. xylosus) is a type of coagulase-negative Staphylococcus, which was previously considered as non-pathogenic. However, recent studies have linked it with cases of mastitis in cows. Isoliquiritigenin (ISL) is a bioactive compound with pharmacological functions including antibacterial activity. In this study, we evaluated the effect of ISL on S. xylosus in vitro and in vivo. The MIC of ISL against S. xylosus was 80 µg/mL. It was observed that sub-MICs of ISL (1/2MIC, 1/4MIC, 1/8MIC) significantly inhibited the formation of S. xylosus biofilm in vitro. Previous studies have observed that inhibiting imidazole glycerol phosphate dehydratase (IGPD) concomitantly inhibited biofilm formation in S. xylosus. So, we designed experiments to target the formation of IGPD or inhibits its activities in S. xylosus ATCC 700404. The results indicated that the activity of IGPD and its histidine content decreased significantly under 1/2 MIC (40 µg/mL) ISL, and the expression of IGPD gene (hisB) and IGPD protein was significantly down-regulated. Furthermore, Bio-layer interferometry experiments showed that ISL directly interacted with IGPD protein (with strong affinity; KD = 234 µM). In addition, molecular docking was used to predict the binding mode of ISL and IGPD. In vivo tests revealed that, ISL significantly reduced TNF-α and IL-6 levels, mitigated the destruction of the mammary glands and reversed the production of inflammatory cells in mice. The results of the study suggest that, ISL may inhibit S. xylosus growth by acting on IGPD, which can be used as a target protein to treat infections caused by S. xylosus.

Discovery of Potential Anti-infective Therapy Targeting Glutamine Synthetase in Staphylococcus xylosus.

Glutamine synthetase (GS), which catalyzes the production of glutamine, plays essential roles in most biological growth and biofilm formation, suggesting that GS may be used as a promising target for antibacterial therapy. We asked whether a GS inhibitor could be found as an anti-infective agent of Staphylococcus xylosus (S. xylosus). Here, computational prediction followed by experimental testing was used to characterize GS. Sorafenib was finally determined through computational prediction. In vitro experiments showed that sorafenib has an inhibitory effect on the growth of S. xylosus by competitively occupying the active site of GS, and the minimum inhibitory concentration was 4 mg/L. In vivo experiments also proved that treatment with sorafenib significantly reduced the levels of TNF-α and IL-6 in breast tissue from mice mastitis, which was further confirmed by histopathology examination. These findings indicated that sorafenib could be utilized as an anti-infective agent for the treatment of infections caused by S. xylosus.

Histidine Metabolism and IGPD Play a Key Role in Cefquinome Inhibiting Biofilm Formation of Staphylococcus xylosus.

Staphylococcus xylosus (S. xylosus) is an AT-rich and coagulase-negative Staphylococcus (CNS). It is normally regarded as non-pathogenic, however, recent studies have demonstrated that it is related to human opportunistic infections and bovine mastitis. In addition, S. xylosus strains have the ability to form biofilm. Biofilms are also involved in chronic infections and antibiotic resistance, there are only a few reports about cefquinome inhibiting S. xylosus biofilm formation and the protein targets of cefquinome. In our study, we found that sub-MICs of cefquinome were sufficient to inhibit biofilm formation. To investigate the potential protein targets of cefquinome, we used iTRAQ for the analyses of cells at two different conditions: 1/2-MIC (0.125 µg/mL) cefquinome treatment and no treatment. Using iTRAQ technique and KEGG database analysis, we found that proteins differently expression in histidine metabolism pathway may play a role in the process by which 1/2-MIC (0.125 µg/mL) cefquinome inhibits S. xylosus biofilm formation. Interestingly, we found a sharply down-regulated enzyme [A0A068E9J3 imidazoleglycerol-phosphate dehydratase (IGPD)] involved in histidine metabolism pathway in cefquinome-treated cells. We demonstrated the important role of IGPD in sub-MICs cefquinome inhibiting biofilm formation of S. xylosus by gene (hisB) knockout, IGPD enzyme activity and histidine content assays. Thus, our data sheds light on important role of histidine metabolism in S. xylosus biofilm formation; especially, IGPD involved in histidine metabolism might play a crucial role in sub-MICs cefquinome inhibition of biofilm formation of S. xylosus, and we propose IGPD as an attractive protein target of cefquinome.

Chitosan Grafted With ß-Cyclodextrin: Synthesis, Characterization, Antimicrobial Activity, and Role as Absorbefacient and Solubilizer.

We synthesized chitosan grafted with ß-cyclodextrin (CD-g-CS) from mono-6-deoxy-6-(p-toluenesulfonyl)-ß-cyclodextrin and chitosan. Two different amounts of immobilized ß-cyclodextrin (ß-CD) on CD-g-CS (QCD: 0.643 × 103 and 0.6 × 102 µmol/g) were investigated. The results showed that the amino contents of CD-g-CS with QCD = 0.643 × 103 and 0.6 × 102 µmol/g were 6.34 ± 0.072 and 9.41 ± 0.055%, respectively. Agar diffusion bioassay revealed that CD-g-CS (QCD = 0.6 × 102 µmol/g) was more active against Staphylococcus xylosus and Escherichia coli than CD-g-CS (QCD = 0.643 × 103 µmol/g). Cell membrane integrity tests and scanning electron microscopy observation revealed that the antimicrobial activity of CD-g-CS was attributed to membrane disruption and cell lysis. Uptake tests showed that CD-g-CS promoted the uptake of doxorubicin hydrochloride by S. xylosus, particularly for CD-g-CS with QCD = 0.6 × 102 µmol/g, and the effect was concentration dependent. CD-g-CS (QCD = 0.6 × 102 and 0.643 × 103 µmol/g) also improved the aqueous solubilities of sulfadiazine, sulfamonomethoxine, and sulfamethoxazole. These findings provide a clear understanding of CD-g-CS and are of great importance for reducing the dosage of antibiotics and antibiotic residues in animal-derived foods. The results also provide a reliable, direct, and scientific theoretical basis for its wide application in the livestock industry.