The Application of Cinnamon Twig Extract as an Inhibitor of Listeriolysin O against Listeria monocytogenes Infection.

As a major virulence factor of Listeria monocytogenes (L. monocytogenes), listeriolysin O (LLO) can assist in the immune escape of L. monocytogenes, which is critical for the pathogen to evade host immune recognition, leading to various infectious diseases. Cinnamon twig (CT), as a traditional medicine, has been widely used in clinics for multiple functions and it has exhibited excellent safety, efficacy and stability. There are few reports on the effects of the extracts of traditional medicine on bacterial virulence factors. CT has not been reported to be effective in the treatment of L. monocytogenes infection. Therefore, this study aims to explore the preventive effect of CT against L. monocytogenes infection in vivo and in vitro by targeting LLO. Firstly, a hemolysis assay and a cell viability determination are used to detect the effect of CT extract on the inhibition of the cytolytic activity of LLO. The potential mechanism through which CT extract inhibits LLO activity is predicted through network pharmacology, molecular docking assay, real-time polymerase chain reaction (RT-PCR), Western blotting and circular dichroism (CD) analysis. The experimental therapeutic effect of CT extract is examined in a mouse model infected with L. monocytogenes. Then, the ingredients are identified through a high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC) analysis. Here we find that CT extract, containing mainly cinnamic acid, cinnamaldehyde, ß-sitosterol, taxifolin, catechin and epicatechin, shows a potential inhibition of LLO-mediated hemolysis without any antimicrobial activity. The results of the mechanism research show that CT extract treatment can simultaneously inhibit LLO expression and oligomerization. Furthermore, the addition of CT extract led to a remarkable alleviation of LLO-induced cytotoxicity. After treatment with CT extract, the mortality, bacterial load, pathological damage and inflammatory responses of infected mice are significantly reduced when compared with the untreated group. This study suggests that CT extract can be a novel and multicomponent inhibitor of LLO with multiple strategies against L. monocytogenes infection, which could be further developed into a novel treatment for infections caused by L. monocytogenes.

Shionone-Targeted Pneumolysin to Ameliorate Acute Lung Injury Induced by Streptococcus pneumoniae In Vivo and In Vitro.

Streptococcus pneumoniae (S. pneumoniae), as a Gram-positive bacterium, can cause severe bacterial pneumonia, and result in high morbidity and mortality in infected people. Meanwhile, isolated drug-resistant S. pneumoniae is growing, which raises concerns about strategies for combatting S. pneumoniae infection. To disturb S. pneumoniae pathogenicity and its drug-resistance, developing novel anti-infective strategies or compounds is urgent. In this study, the anti-infective effect of shionone was explored. A minimum inhibitory concentration (MIC) assay and growth curve determination were performed to evaluate the effect of the tetracyclic triterpenoid compound shionone against S. pneumoniae. Hemolysis tests, western blotting, oligomerization inhibition assays, and molecular docking were carried out to explore the anti-infective mechanism of shionone. Moreover, the protective effect of shionone was also confirmed in a mousepneumonia model. The results showed that the excellent hemolytic inhibitory activity of shionone was observed at less than 8 µg/mL. Meanwhile, shionone could disturb the oligomerization of pneumolysin (PLY) but did not interfere with PLY expression at less than 4 µg/mL. Molecular docking suggested that shionone targeted the ASP-59, ILE-60, THR-57, PHE-344, and ASN-346 amino acid sites to reduce S. pneumoniae pathogenicity. Furthermore, shionone alleviated lung histopathologic injury and decreased lung bacterial colonization in vivo. The above results showed that shionone could bind to the PLY active pocket under the concentrations of 8 µg/mL and neutralize PLY hemolysis activity to reduce S. pneumoniae pathogenicity in vitro and in vivo.

NMPA-approved traditional Chinese medicine-Pingwei Pill: new indication for colistin recovery against MCR-positive bacteria infection.

BACKGROUND: The wide spread of plasmid-mediated colistin resistance by mobile colistin resistance (MCR) in Enterobacteriaceae severely limits the clinical application of colistin as a last-line drug against bacterial infection. The identification of colistin potentiator from natural plants or their compound preparation as antibiotic adjuncts is a new promising strategy to meet this challenge. METHODS: Herein, the synergistic activity, as well as the potential mechanism, of Pingwei pill plus antibiotics against MCR-positive Gram-negative pathogens was examined using checkerboard assay, time-killing curves, combined disk test, western blot assay, and microscope analysis. Additionally, the Salmonella sp. HYM2 infection models of mouse and chick were employed to examine the in vivo efficacy of Pingwei pill in combination with colistin against bacteria infection. Finally, network pharmacology and molecular docking assay were used to predicate other actions of Pingwei pill for Salmonella infection. RESULTS: Our results revealed that Pingwei Pill synergistically potentiated the antibacterial activity of colistin against MCR-1-positive bacteria by accelerating the damage and permeability of the bacterial outer membrane with an FIC (Fractional Inhibitory Concentration) index less than 0.5. The treatment of Pingwei Pill neither inhibited bacterial growth nor affected MCR production. Notably, Pingwei Pill in combination with colistin significantly prolonged the median survival in mouse and chick models of infection using the Salmonella sp. strain HYM2, decreased bacteria burden and organ index of infected animal, alleviated pathological damage of cecum, which suggest that Pingwei Pill recovered the therapeutic performance of colistin for MCR-1- positive Salmonella infection in mice and the naturally infected host chick. Pharmacological network topological analysis, molecular docking, bacterial adhesion, and invasion pathway verification assays were performed to identify the other molecular mechanisms of Pingwei Pill as a colistin potentiator against Gram-negative bacteria infection. CONCLUSION: Taken together, NMPA (National Medical Products Administration)-approved Pingwei Pill is a promising adjuvant with colistin for MCR-positive bacterial infection with a shortened R&D (research and development) cycle and affordable R&D cost and risk.

Phloretin potentiates polymyxin E activity against gram-negative bacteria.

AIMS: The spread of plasmid-mediated polymyxin resistance has jeopardized the use of polymyxin, the last defender that combats infections caused by multidrug-resistant (MDR) gram-negative pathogens. MAIN METHODS: In this study, phloretin, as a monomeric compound extracted from natural plants, showed a good synergistic effect with polymyxin E against gram-negative bacteria, as evaluated by minimal inhibit concentration (MIC) assay and a series of assays, including growth curve, time-killing, and Western blot assays. A model of mice infected by Salmonella sp. stain HYM2 was established to further identify the synergistic effect of phloretin with polymyxin E. KEY FINDINGS: The results suggested that phloretin had the potential ability to recover the antibacterial sensitivity of polymyxin E from 64 µg/mL to no more than 2 µg/mL in E. coli ZJ478 or in Salmonella sp. stain HYM2 with a 32-fold decrease. A series of strains, including mcr-1-positive and mcr-1-negative strains, were treated with a combination of phloretin and polymyxin E, and the fractional inhibitory concentration (FIC) values were all found to be below 0.5. However, the combination of phloretin and polymyxin E did not lead to bacterial resistance. In vivo, the survival rate of infected mice reached nearly 80% with the combination treatment, and the cecal colony value also decreased significantly. SIGNIFICANCE: All the above results indicated that phloretin is a potential polymyxin potentiator to combat gram-negative stains.