The induced and intrinsic resistance of Escherichia coli to sanguinarine is mediated by AcrB efflux pump.

IMPORTANCE: The use of plant extracts is increasing as an alternative to synthetic compounds, especially antibiotics. However, there is no sufficient knowledge on the mechanisms and potential risks of antibiotic resistance induced by these phytochemicals. In the present study, we found that stable drug resistant mutants of E. coli emerged after repetitive exposure to sanguinarine and demonstrated that the AcrB efflux pump contributed to the emerging of induced and intrinsic resistance of E. coli to this phytochemical. Our results offered some insights into comprehending and preventing the onset of drug-resistant strains when utilizing products containing sanguinarine.

Hinokitiol Selectively Enhances the Antibacterial Activity of Tetracyclines against Staphylococcus aureus.

The increasing prevalence of antibiotic resistance causes an urgent need for alternative agents to combat drug-resistant bacterial pathogens. Plant-derived compounds are promising candidates for the treatment of infections caused by antibiotic-resistant bacteria. Hinokitiol (ß-thujaplicin), a natural tropolone derivative found in the heartwood of cupressaceous plants, has been widely used in oral and skin care products as an antimicrobial agent. The aim of this work was to study the synergy potential of hinokitiol with antibiotics against Staphylococcus aureus, which is an extremely successful opportunistic pathogen capable of causing nosocomial and community-acquired infections worldwide. The MIC was determined by the broth microdilution method, and the effect of combinations was evaluated through fractional inhibitory concentration indices (FICI). The mechanism behind this synergy was also investigated by using fluorescence spectroscopy and high-performance liquid chromatography (HPLC). The MICs of hinokitiol alone against most S. aureus strains were 32 µg/mL. Selectively synergistic activities (FICIs of ≤0.5) were observed for combinations of this phytochemical with tetracyclines against all tested strains of S. aureus. Importantly, hinokitiol at 1 µg/mL completely or partially reversed tetracycline resistance in staphylococcal isolates. The increased accumulation of tetracycline inside S. aureus in the presence of hinokitiol was observed. In addition, hinokitiol promoted the uptake of ethidium bromide (EB) in bacterial cells without membrane depolarization, suggesting that it may be an efflux pump inhibitor. IMPORTANCE The disease caused by S. aureus is a public health issue due to the continuing emergence of drug-resistant strains, particularly methicillin-resistant S. aureus (MRSA). Tetracyclines, one of the old classes of antimicrobials, have been used for the treatment of infections caused by S. aureus. However, the increased resistance to tetracyclines together with their toxicity have limited their use in the clinic. Here, we demonstrated that the combination of hinokitiol and tetracyclines displayed synergistic antibacterial activity against S. aureus, including tetracycline-resistant strains and MRSA, offering a potential alternative approach for the treatment of infections caused by this bacterium.

New Insights Into the Antibacterial Mechanism of Cryptotanshinone, a Representative Diterpenoid Quinone From Salvia miltiorrhiza Bunge.

The rapid rise of antibiotic resistance causes an urgent need for new antimicrobial agents with unique and different mechanisms of action. The respiratory chain is one such target involved in the redox balance and energy metabolism. As a natural quinone compound isolated from the root of Salvia miltiorrhiza Bunge, cryptotanshinone (CT) has been previously demonstrated against a wide range of Gram-positive bacteria including multidrug-resistant pathogens. Although superoxide radicals induced by CT are proposed to play an important role in the antibacterial effect of this agent, its mechanism of action is still unclear. In this study, we have shown that CT is a bacteriostatic agent rather than a bactericidal agent. Metabolome analysis suggested that CT might act as an antibacterial agent targeting the cell membrane. CT did not cause severe damage to the bacterial membrane but rapidly dissipated membrane potential, implying that this compound could be a respiratory chain inhibitor. Oxygen consumption analysis in staphylococcal membrane vesicles implied that CT acted as respiratory chain inhibitor probably by targeting type II NADH:quinone dehydrogenase (NDH-2). Molecular docking study suggested that the compound would competitively inhibit the binding of quinone to NDH-2. Consistent with the hypothesis, the antimicrobial activity of CT was blocked by menaquinone, and the combination of CT with thioridazine but not 2-n-heptyl-4-hydroxyquinoline-N-oxide exerted synergistic activity against Staphylococcus aureus. Additionally, combinations of CT with other inhibitors targeting different components of the bacterial respiratory chain exhibit potent synergistic activities against S. aureus, suggesting a promising role in combination therapies.

Phenanthrene Antibiotic Targets Bacterial Membranes and Kills Staphylococcus aureus With a Low Propensity for Resistance Development.

New classes of antibiotics with different mechanisms of action are urgently required for combating antimicrobial resistance. Blestriacin, a dihydro-biphenanthrene with significant antibacterial activity, was recently isolated from the fibrous roots of Bletilla striata. Here, we report the further characterization of the antimicrobial potential and mode of action of blestriacin. The phenanthrene compound inhibited the growth of all tested clinical isolates of Staphylococcus aureus including methicillin-resistant S. aureus (MRSA). The minimum inhibitory concentrations (MICs) of blestriacin against these pathogens ranged from 2 to 8 µg/mL. Minimum bactericidal concentration (MBC) tests were conducted, and the results demonstrated that blestriacin was bactericidal against S. aureus. This effect was confirmed by the time-kill assays. At bactericidal concentrations, blestriacin caused loss of membrane potential in B. subtilis and S. aureus and disrupted the bacterial membrane integrity of the two strains. The spontaneous mutation frequency of S. aureus to blestriacin was determined to be lower than 10-9. The selection and whole genome sequencing of the blestriacin -resistant mutants of S. aureus indicated that the development of blestriacin resistance in S. aureus involves mutations in multi-genes. All these observations can be rationalized by the suggestion that membrane is a biological target of blestriacin.

The anti-Staphylococcus aureus activity of the phenanthrene fraction from fibrous roots of Bletilla striata.

BACKGROUND: Bletillae Rhizoma, the tuber of Bletilla striata, has been used in Chinese traditional medicine to treat infectious diseases. Chemical studies indicated that phenanthrene was one of the most important components of the herb, with a broad spectrum of antibiotic activity against Gram-positive bacteria. The objective of this study was to further characterize the antibacterial activity of the phenanthrene fraction from the fibrous root of the pseudobulb of B. striata. METHODS: The phenanthrene fraction (EF60) from the ethanol extract of fibrous roots of Bletilla striata pseudobulbs was isolated using polyamide column chromatography. The antibacterial activity of the fraction was evaluated in vitro using a 96-well microtiter plate and microbroth dilution method. The cytotoxicity of EF60 against mammalian cells was tested by hemolysis and MTT assays. RESULTS: EF60 was obtained using alcohol extraction and polyamide column chromatography, with a yield of 14.9 g per 1 kg of the fibrous roots of B. striata. In vitro tests indicated that EF60 was active against all tested strains of Staphylococcus aureus, including clinical isolates and methicillin-resistant S. aureus (MRSA). The minimum inhibitory concentration (MIC) values of EF60 against these pathogens ranged from 8 to 64 µg/mL. Minimum bactericidal concentration tests demonstrated that EF60 was bactericidal against S. aureus 3304 and ATCC 29213 and was bacteriostatic against S. aureus 3211, ATCC 25923, and ATCC 43300. Consistently, the time-kill assay indicated that EF60 could completely kill S. aureus ATCC 29213 at 2× the MIC within 3 h but could kill less than two logarithmic units of ATCC 43300, even at 4× the MIC within 24 h. The postantibiotic effects (PAE) of EF60 (4× MIC) against strains 29213 and 43300 were 2.0 and 0.38 h, respectively. Further studies indicated that EF60 (160 µg/mL) showed no cytotoxicity against human erythrocytes, and was minimally toxic to Human Umbilical Vein Endothelial Cells with an IC50 of 75 µg/mL. CONCLUSIONS: Our studies indicated that EF60 is worthy of further investigation as a potential phytotherapeutic agent for treating infections caused by S. aureus and MRSA.

[Protoplasts isolation, purification and plant regeneration of Pinellia cordata].

The main factors which affected the isolation, purification and cultivation of Pinellia cordata protoplasts from leaves were studied. The results indicated that the optimum enzyme solution for P. cordata leaves was 13% CPW + 1.0% Cellulose +0.1% Pectolase, at pH 6.0, temperature (25-28 degrees C ) for 4 h. The sucrose density gradient centrifugation was adopted to purificate the protoplasts collected, when 25% sucrose was used as mediator, centrifugating at 500 rpm for 10 min. When the protoplasts were shallow liquid and liquid-solid double layer cultured on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA + 13% mannitol at the density of 2.5 x 104 protoplasts/mL, or fed and nursed cultured at the density of 100-500 protoplasts/mL, cell division could be observed for 3 days; granular calli appeared for 30 days. Calli was proliferated on the medium of MS + 0.5 mg x L(-1) 6-BA + 0.25 mg x L(-1) NAA solidified by 0.55% agar, and differentiated and regenerated after 5-6 months. Plant generation of P. cordata is successfully established.

[Study function of endophytic fungus in parasitism process of mistletoe].

OBJECTIVE: To research the function of endophytes of mistletoe in parasitism process of mistletoe in Pterocarya stenoptera. METHOD: Endophytes from eight different parts of the mistletoe were separated by explant culture, and further screened by different CMC plates culture and DNS method to get cellulase high productive strains. The distribution of the endophytic fungus parasitized in mistletoe were prepared and stained to demonstrate by histological section of the intumescentia part of the P. stenoptera. RESULT: The histological section indicated that aboundent of hyphasma were distributed around the haustorium of the mistletoe. Eighty three strains of endophytic fungus were separated, 38 of them were able to degrade cellulose, 19 strains showed high cellulase activity and 10 of which were separated from the parasitic position. CONCLUSION: Endophytic fungus of mistletoe can secrete cellulase and assist the haustorium of mistletoe to breakthrough the cell walls as well as intercellular space tissues of the P. stenoptera, thus, the endophytic fungus plays an important role in the parasitism process of mistletoe in P. stenoptera.

[Application of SCAR molecular marker technology in identification of Monascus strains].

OBJECTIVE: To establish an effective way for rapid identification of Monascus strains based on DNA molecular marker. METHOD: A random amplified polymorphic DNA (RAPD) marker named F421 in genomic DNA of Monascus F strain was observed during a comparison of DNA fingerprints derived from 10 cultivated strains of Monascus. F421 was cloned and sequenced. Comparing the sequence of F421 (GenBank accession number EF063107) with other relative sequences in the GenBank databases, no distinct comparability was found. A pair of sequence characterized amplified region (SCAR) primers were designed based on the sequence of the cloned fragment and tested for the specific detection of Monascus F. RESULT: The results of polymerase chain reaction showed that only a 421bp segment of Monascus F strain was amplified compared with other 9 cultivated strains of Monascus. And the acquired SCAR marker of strain F could be used as a specific DNA fingerprint to identify Monascus strain F within one day. CONCLUSION: SCAR molecular marker technology is an effective new way to identify Monascus strains more rapidly. And also is an assistant tool to identify Monascus strains more accurately when disagreements come out using traditional classification. It could be applied widely to the protection of germ plasm resources, classification and identification distinguishing false strains of pharmaceutical fungi.

Isolation and identification of an anti-tumor component from leaves of Impatiens balsamina.

We have previously shown that ethanol or chloroform extracts of the leaves of Impatiens balsamina (LIB) have anti-tumor activity against the human hepatocellular carcinoma cell line HepG2. The ethanol extracts were separated into five fractions according to polarity. An MTT assay indicated that two of the fractions had anti-tumor activity and that the petroleum ether fraction (PEF) was the most active. But the available quantities of both the PEF and chloroform fractions (CHF) were limited, precluding further study. The chloroform extract (CHE) shared almost all the same spots with the PEF and CHF and was plentiful enough to carry out further separations. Thus, the CHE was further separated into six sub-fractions (CHE 1 approximately 6) by column chromatography. A MTT assay showed that only the CHE2 fraction had a strong tumor inhibition ratio (IC(50) = 6.47+/-0.05 mg/L), which was superior to that of curcumin (IC(50) = 13.95+/-0.11 mg/L). However, TLC revealed that CHE2 was not pure and still contained two more components. After further separation and purification, followed by TLC and MTT assay confirmation, the final active component was isolated and identified as 2-methoxy-1,4-naphthoquinone by m.p., UV, MS and (13)C- and (1)H-NMR data. This is the first report demonstrating that 2-methoxy-1,4-naphthoquinone has intensive in vitro anti-tumor activity against HepG2 cells.