Ginkgo biloba L. exocarp petroleum ether extract inhibits methicillin-resistant Staphylococcus aureus by modulating ion transport, virulence, and biofilm formation in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: As reported in the Ancient Chinese Medicinal Books, Ginkgo biloba L. fruit has been used as a traditional Chinese medicine for the treatment asthma and cough or as a disinfectant. Our previous study demonstrated that G. biloba exocarp extract (GBEE), an extract of a traditional Chinese herb, inhibits the formation of methicillin-resistant Staphylococcus aureus (MRSA) biofilms. However, GBEE is a crude extract that contains many components, and the underlying mechanisms of purified GBEE fractions extracted with solvents of different polarities are unknown. AIM OF THE STUDY: This study aimed to investigate the different components in GBEE fractions extracted with solvents of different polarities and their antibacterial effects and mechanisms against MRSA and Staphylococcus haemolyticus biofilms both in vitro and in vivo. METHODS: The components in different fractions were detected by high-performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS). Microbroth dilution assays and time growth curves were used to determine the antibacterial effects of the fractions on 15 clinical bacterial isolates. Crystal violet staining, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were utilized to identify the fractions that affected bacterial biofilm formation. The potential MRSA targets of the GBEE fraction obtained with petroleum ether (PE), denoted GBEE-PE, were screened by transcriptome sequencing, and the gene expression profile was verified by quantitative polymerase chain reaction (qPCR). RESULTS: HPLC-HRMS analysis revealed that the four GBEE fractions (extracted with petroleum ether, ethyl acetate, n-butanol, and water) contained different ginkgo components, and the antibacterial effects decreased as the polarity of the extraction solvent increased. The antibacterial activity of GBEE-PE was greater than that of the GBEE fraction extracted with ethyl acetate (EA). GBEE-PE improved H. illucens survival and reduced MRSA colonization in model mouse organs. Crystal violet staining and SEM and TEM analyses revealed that GBEE-PE inhibited MRSA and S. haemolyticus biofilm formation. Transcriptional analysis revealed that GBEE-PE inhibits MRSA biofilms by altering ion transport, cell wall metabolism and virulence-related gene expression. In addition, the LO2 cell viability and H. illucens toxicity assay data showed that GBEE-PE at 20 mg/kg was nontoxic. CONCLUSION: The GBEE fractions contained different components, and their antibacterial effects decreased with increases in the polarity of the extraction solvent. GBEE-PE limited MRSA growth and biofilm formation by affecting ion transport, cell wall synthesis, and virulence-related pathways. This research provides a more detailed overview of the mechanism by which GBEE-PE inhibits MRSA both in vitro and in vivo and suggests that GBEE-PE is a new prospective antimicrobial with the potential to be used in MRSA therapeutics in the future.

Evaluation of the antimicrobial function of Ginkgo biloba exocarp extract against clinical bacteria and its effect on Staphylococcus haemolyticus by disrupting biofilms.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Ginkgo biloba L. (Ginkgo nuts) has been used for a long time as a critical Chinese medicine material to treat cough and asthma, as well as a disinfectant. Similar records were written in the Compendium of Materia Medica (Ben Cao Gang Mu, pinyin in Chinese) and Sheng Nong's herbal classic (Shen Nong Ben Cao Jing, pinyin in Chinese). Recent research has shown that Ginkgo biloba exocarp extract (GBEE) has the functions of unblocking blood vessels and improving brain function, as well as antitumour activity and antibacterial activity. GBEE was shown to inhibit methicillin-resistant Staphylococcus aureus (MRSA) biofilm formation as a traditional Chinese herb in our previous report in this journal. AIM OF THE STUD: yThe antibiotic resistance of clinical bacteria has recently become increasingly serious. Thus, this study aimed to investigate the Ginkgo biloba exocarp extract (GBEE) antibacterial lineage, as well as its effect and mechanism on S. haemolyticus biofilms. This study will provide a new perspective on clinical multidrug resistant (MDR) treatment with ethnopharmacology herbs. METHODS: The microbroth dilution assay was carried out to measure the antibacterial effect of GBEE on 13 types of clinical bacteria. Bacterial growth curves with or without GBEE treatment were drawn at different time points. The potential targets of GBEE against S. haemolyticus were screened by transcriptome sequencing. The effects of GBEE on bacterial biofilm formation and mature biofilm disruption were determined by crystal violet staining and scanning electron microscopy. The metabolic activity of bacteria inside the biofilm was assessed by colony-forming unit (CFU) counting and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2HY-tetrazolium bromide (MTT) assay. Quantitative polymerase chain reaction (qPCR) was used to measure the gene expression profile of GBEE on S. haemolyticus biofilm-related factors. RESULTS: The results showed that GBEE has bacteriostatic effects on 3 g-positive (G+) and 2 g-negative (G-) bacteria among 13 species of clinical bacteria. The antibacterial effect of GBEE supernatant liquid was stronger than the antibacterial effect of GBEE supernviaould-like liquid. GBEE supernatant liquid inhibited the growth of S. epidermidis, S. haemolyticus, and E. faecium at shallow concentrations with minimum inhibitory concentrations (MICs) of 2 µg/ml, 4 µg/ml and 8 µg/ml, respectively. Genes involved in quorum sensing, two-component systems, folate biosynthesis, and ATP-binding cassette (ABC) transporters were differentially expressed in GBEE-treated groups compared with controls. Crystal violet, scanning electron microscopy (SEM) and MTT assays showed that GBEE suppressed S. haemolyticus biofilm formation in a dose-dependent manner. Moreover, GBEE supernatant liquid downregulated cidA, cidB and atl, which are involved in cell lysis and extracellular DNA (eDNA) release, as well as downregulated the cbp, ebp and fbp participation in encoding cell-surface binding proteins. CONCLUSIONS: GBEE has an excellent antibacterial effect on gram-positive bacteria and also inhibits the growth of gram-negative bacteria, such as A. baumannii (carbapenem-resistant Acinetobacter baumannii) CRABA and S. maltophilia. GBEE inhibits the biofilm formation of S. haemolyticus by altering the regulation and biofilm material-related genes, including the release of eDNA and cell-surface binding proteins.

Ginkgo biloba exocarp extracts inhibit S. aureus and MRSA by disrupting biofilms and affecting gene expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba L. fruit, also known as Bai Guo, Ya Jiao Zi (in pinyin Chinese), and ginkgo nut (in English), has been used for many years as an important material in Chinese traditional medicine to treat coughs and asthma and as a disinfectant, as described in the Compendium of Materia Medica (Ben Cao Gang Mu, pinyin in Chinese), an old herbal book. Ginkgo nuts are used to treat phlegm-associated asthma, astringent gasp, frequent urination, gonorrhoea and turgidity; consumed raw to reduce phlegm and treat hangovers; and used as a disinfectant and insecticide. A similar record was also found in Sheng Nong's herbal classic (Shen Nong Ben Cao Jing, pinyin in Chinese). Recent research has shown that Ginkgo biloba L. exocarp extract (GBEE) can unblock blood vessels and improve brain function and exhibits antitumour and antibacterial activities. AIM OF STUDY: To investigate the inhibitory effect of Ginkgo biloba L. exocarp extract (GBEE) on methicillin-resistant S. aureus (MRSA) biofilms and assess its associated molecular mechanism. MATERIALS AND METHODS: The antibacterial effects of GBEE on S. aureus and MRSA were determined using the broth microdilution method. The growth curves of bacteria treated with or without GBEE were generated by measuring the CFU (colony forming unit) of cultures at different time points. The effects of GBEE on bacterial biofilm formation and mature biofilm disruption were determined by crystal violet staining. Quantitative polymerase chain reaction (qPCR) was used to measure the effects of GBEE on the gene expression profiles of MRSA biofilm-related factors at 6, 8, 12, 16 and 24 h. RESULTS: The minimum inhibitory concentration (MIC) of GBEE on S. aureus and MRSA was 4 µg/mL, and the minimum bactericidal concentration (MBC) was 8 µg/ml. Moreover, GBEE (4-12 µg/mL) inhibited S. aureus and MRSA biofilm formation in a dose-dependent manner. Interestingly, GBEE also destroyed mature biofilms of S. aureus and MRSA at 12 µg/ml. The expression of the MRSA biofilm-associated factor icaA and sarA were downregulated after 6 h of treatment with GBEE, while sigB was downregulated after 12 h. MeanwhileMeanwhile, icaR was upregulated at 12 h. In addition, GBEE also downregulated the virulence gene hld and inhibited the synthesis of staphyloxanthin. CONCLUSIONS: GBEE has excellent antibacterial effects against S. aureus and MRSA and inhibits their biofilm-forming ability by altering related gene expression.