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The isolation of two diarylnonanoids from Dioscorea cotinifolia possessing antibiotic-potentiating activity against resistant strains of S. aureus are reported. The diarylnonanoids are a class of natural products similar in structure to the diarylheptanoids, which have a wide spectrum of reported biological activities. One of the diarylnonanoids ( 1 ) isolated possesses a chiral center, and to deduce its configuration, the modified Mosher ester method was used. Using both 1D and 2D NMR data, as many protons as possible were assigned to both the R - and S -MTPA esters, and the configuration of the chiral center in 1 was determined to be R . Both the chiral and achiral diarylnonanoid ( 2 ) exhibited potent antibiotic-potentiating activity with the chiral natural product showing a greater tetracycline-potentiating activity than 2 . Interestingly, 2 gave a higher norfloxacin-potentiating activity with a resultant higher efflux pump inhibitory activity. Manipulation of the structure of the diarylnonanoids through synthesis could lead to improved biological activity.

Ethnopharmacological Importance: Capsicum and ginger are used widely in human diets and in folklore medicines. Chemically, gingerol is a relative of capsaicin and both classes of compounds are notable for their spiciness and characteristic pungent aroma. Previous studies have demonstrated that these compounds contain antimicrobial compounds with robust pharmacological importance., Aim: The present study evaluated the in vitro antibacterial activities of capsaicinoids and gingerols against a panel of clinical MRSA strains and their inhibitory effect on the conjugal transfer of R-plasmids harboured in E. coli., Materials and Methods: Crude methanol extract of C. annum was fractionated using solid phase extraction (SPE) and screened for R-plasmid transfer inhibition: TP114, PUB 307, PKM 101, R6K and R7K. The bio-guided assay led to the isolation of bioactive compounds with strong R-plasmid transfer inhibition. The compounds were identified using Nuclear Magnetic resonance (NMR) and Mass spectroscopy (MS). Capsaicin analogues nonivamide, 6-gingerol, 6-shogaol, capsaicin and dihydrocapsaicin were screened for antimicrobial activity against a panel of methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative bacteria strains using microdilution method while the plasmid transfer inhibition assay of the compounds was determined by broth mating method., Results: The bioactive fraction Ca-11 showed good inhibition rates (8.57-25.52%) against three R-plasmids PUB307, PKM 101, TP114 followed by the crude extract of C. annum (8.59%) respectively leading to the bioassay-guided isolation of capsaicin and dihydrocapsaicin as the bioactive principles. The antiplasmid effect of pure capsaicin and dihydrocapsaicin were broad and within active ranges (5.03-31.76%) against the various antibiotic resistance-conferring plasmids including R6K, R7K. Capsaicin, 6-gingerol and 6-shogaol had good broad antibacterial activity with MIC values ranging from 8 to 256 mg/L against effluxing MRSA strains SA1199B (NorA), XU212 (TetK) and RN4220 (MsrA). While they exhibited moderate antibacterial activity (128-512 mg/L) against the Gram-negative bacteria. The effect of 6-gingerol, 6-shogaol and nonivamide on the plasmids were very active on PKM 101 (6.24-22.16%), PUB 307 (1.22-45.63%) and TP114 (0.1-7.19%) comparative to the positive control plumbagin (5.70-31.76%)., Conclusion: These results are suggestive that the R-plasmids could possess substrate for capsaicinoids-like compounds and for their ability to inhibit the plasmid conjugation processes. Plant natural products possess the potential value of antibacterial and mechanistic antiplasmid activity as demonstrated by the compounds and should be evaluated in developing antimicrobial leads to novel mechanism against multidrug-resistant bacteria., (Copyright © 2019. Published by Elsevier B.V.)

Bioactivity-guided fractionation of the EtOH extract of the branches of Kielmeyera variabilis led to the isolation of a new acylphoroglucinol (1), which was active against all the MRSA strains tested herein, with pronounced activity against strain EMRSA-16. Compound 1 displayed an MIC of 0.5 mg/L as compared with an MIC of 128 mg/L for the control antibiotic norfloxacin. The structure of the new compound was elucidated by 1D and 2D NMR spectroscopic analysis and mass spectrometry, and experimental and calculated ECD were used to determine the absolute configurations. The compounds β-sitosterol (2), stigmasterol (3), ergost-5-en-3-ol (4), and osajaxanthone (5) also occurred in the n-hexane fraction. The EtOAc fraction contained nine known xanthones: 3,6-dihydroxy-1,4,8-trimethoxyxanthone (6), 3,5-dihydroxy-4-methoxyxanthone (7), 3,4-dihydroxy-6,8-dimethoxyxanthone (8), 3,4-dihydroxy-2-methoxyxanthone (9), 5-hydroxy-1,3-dimethoxyxanthone (10), 4-hydroxy-2,3-dimethoxyxanthone (11), kielcorin (12), 3-hydroxy-2-methoxyxanthone (13), and 2-hydroxy-1-methoxyxanthone (14), which showed moderate to low activity against the tested MRSA strains.

Isothermal microcalorimetry (IMC) is particularly suited to the study of microbiological samples in complex or heterogeneous environments because it does not require optical clarity of the sample and can detect metabolic activity from as few as 10(4) CFU/mL cells. While the use of IMC for studying planktonic cultures is well established, in the clinical environment bacteria are most likely to be present as biofilms. Biofilm prevention and eradication present a number of challenges to designers and users of medical devices and implants, since bacteria in biofilm colonies are usually more resistant to antimicrobial agents. Analytical tools that facilitate investigation of biofilm formation are therefore extremely useful. While it is possible to study pre-prepared biofilms in closed ampoules, better correlation with in vivo behaviour can be achieved using a system in which the bacterial suspension is flowing. Here, we discuss the potential of flow microcalorimetry for studying biofilms and report the development of a simple flow system that can be housed in a microcalorimeter. The use of the flow system is demonstrated with biofilms of Staphylococcus aureus., (Copyright © 2014 Elsevier Inc. All rights reserved.)

An isothermal microcalorimetric assay was used to quantify the efficacy of a silver-containing wound dressing against two common wound pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. The growth patterns of the two species were unique and varied depending on the environment in which the organisms were grown. Addition of non-silver-containing dressing altered the growth kinetics while addition of silver (contained either in a dressing or as AgNO3 solution) was seen to elicit inhibition and/or kill depending on concentration. Tests were conducted in nutrient broth and simulated wound fluid. It was found that minimum inhibitory and minimum bactericidal concentration values were higher in simulated wound fluid and under anaerobic conditions. Bioavailability of silver from the wound dressing was 35% against S. aureus in nutrient broth and 68% against both species in simulated wound fluid. The data highlight the importance of developing and conducting in vitro assays in biorelevant media., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

(-)-Epicatechin gallate (ECg), a component of green tea, sensitizes meticillin-resistant Staphylococcus aureus (MRSA) to beta-lactam antibiotics, promotes staphylococcal cell aggregation and increases cell-wall thickness. The potentiation of beta-lactam activity against MRSA by ECg was not due to decreased bacterial penicillin-binding protein (PBP) 2a expression or ECg binding to peptidoglycan. A 5-10 % reduction in peptidoglycan cross-linking was observed. Reduced cross-linking was insufficient to compromise the integrity of the cell wall and no evidence of PBP2a activity was detected in the muropeptide composition of ECg-grown cells. ECg increased the quantity of autolysins associated with the cell wall, even though the cells were less susceptible to Triton X-100-induced autolysis than cells grown in the absence of ECg. ECg promoted increased lysostaphin resistance that was not due to alteration of the pentaglycine cross-bridge configuration or inhibition of lysostaphin activity. Rather, decreased lysostaphin susceptibility was associated with structural changes to wall teichoic acid (WTA), an acid-labile component of peptidoglycan. ECg also promoted lipoteichoic acid (LTA) release from the cytoplasmic membrane. It is proposed that ECg reduces beta-lactam resistance in MRSA either by binding to PBPs at sites distinct from the penicillin-binding site or by intercalation into the cytoplasmic membrane, displacing LTA from the phospholipid palisade. Thus, ECg-mediated alterations to the physical nature of the bilayer will elicit structural changes to WTA that result in modulation of the cell-surface properties necessary to maintain the beta-lactam-resistant phenotype.

Binding of two bactericidal pyrrolobenzodiazepine (PBD) dimers, SJG-136 and ELB-21, to genomic DNA from Staphylococcus aureus EMRSA-16 was investigated. Both agents cross-linked purified EMRSA-16 DNA. The more potent agent, ELB-21, had a greater capacity to cross-link DNA after incubation with intact cells than SJG-136. Extensive interstrand cross-linking at multiple sites on the EMRSA-16 genome was demonstrated by probing EcoRI-restricted DNA with mecA and 16S rDNA. Cross-linking was again greater in DNA extracted from ELB-21-treated cells and was compatible with frequency analysis of preferred binding sequences in EMRSA-16 DNA. These studies support the premise that the potency of ELB-21 is due to efficient cell penetration and provide evidence that the antibacterial activity of PBD dimers results from cross-linking at specific genomic sites.

Staphylococcus aureus grows in the presence of high-salt concentrations. The green tea polyphenolic compound epicatechin gallate (ECg) extended the staphylococcal lag phase to values greater than 6 h in the presence of NaCl, KCl and LiCl; this effect was not observed with epicatechin. Osmoprotectants glycine betaine and l-proline were unable to relieve the effect of ECg on halotolerance. The capacity of ECg to suppress staphylococcal growth in the presence of salt suggests that this molecule could be used to aid the preservation of salt-containing foods.

(-)-Epicatechin gallate (ECg) and (-)-epigallocatechin gallate (EGCg) reduce oxacillin resistance in mecA-containing strains of Staphylococcus aureus. Their binding to staphylococcal cells is enhanced by the nongalloyl analogues (-)-epicatechin (EC) and (-)-epigallocatechin (EGC). EC and EGC significantly increased the capacity of ECg and EGCg to reduce levels of staphylococcal oxacillin resistance.

Epicatechin gallate (ECg), a component of green tea with weak activity against Staphylococcus aureus, reduces oxacillin resistance in methicillin-resistant S. aureus at concentrations below the MIC. Because catechins bind to artificial lipid bilayers, we investigated whether the anti-staphylococcal activity of catechins could be improved by increasing their capacity to interact with the cytoplasmic membrane. Substitution of the gallate group of ECg with 3-O-acyl chains of varying lengths (C(4)-C(18)) led to enhanced anti-staphylococcal activity with chain lengths of C(8) (octanoyl) and C(10) (decanoyl). 3-O-octanoyl catechin was bactericidal against MRSA as the result of membrane damage. 3-O-acyl catechins tested at a 1/4 MIC did not reduce the oxacillin MIC greater than two-fold. 3-O-acyl catechins warrant further investigation as anti-staphylococcal agents.

Aqueous extracts of Japanese green tea (Camellia sinensis) are able to reverse beta-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). We have attributed the capacity to reverse oxacillin resistance in the homogeneous PBP2a producer BB568 and in EMRSA-16 to (-)-epicatechin gallate (ECG) and (-)-catechin gallate (CG). Minimum inhibitory concentration (MIC) values for oxacillin were reduced from 256 and 512 to 1-4 mg/l, respectively, in the presence of these polyphenols. In addition, (-)-epigallocatechin gallate (EGCG) had a moderate capacity to modulate oxacillin activity against S. aureus BB568, but none against EMRSA-16. ECG, CG and EGCG increased the sensitivity of EMRSA-15 to oxacillin. The gallate moiety was essential for the oxacillin-modulating activity of ECG, as both (-)-epicatechin and (-)-epicatechin-3-cyclohexylcarboxylate were unable to reverse resistance to oxacillin. Gallic acid and three alkyl gallates (methyl gallate, propyl gallate, and octyl gallate) did not modulate beta-lactam resistance in MRSA. Octyl gallate exhibited direct antibacterial activity against S. aureus BB568 (16 mg/l). Modulation of beta-lactam resistance by ECG significantly enhanced the activities of flucloxacillin and the carbapenem antibiotics imipenem and meropenem against 40 MRSA isolates, with MIC(90) values for the antibiotics reduced to the susceptibility breakpoint or below. Consequently, EGCG, CG and, particularly, ECG warrant further investigation as agents to combat beta-lactam resistance in S. aureus.

Staphylococcus aureus is a major pathogen both within hospitals and in the community. Methicillin, a beta-lactam antibiotic, acts by inhibiting penicillin-binding proteins (PBPs) that are involved in the synthesis of peptidoglycan, an essential mesh-like polymer that surrounds the cell. S. aureus can become resistant to methicillin and other beta-lactam antibiotics through the expression of a foreign PBP, PBP2a, that is resistant to the action of methicillin but which can perform the functions of the host PBPs. Methicillin-resistant S. aureus isolates are often resistant to other classes of antibiotics (through different mechanisms) making treatment options limited, and this has led to the search for new compounds active against these strains. An understanding of the mechanism of methicillin resistance has led to the discovery of accessory factors that influence the level and nature of methicillin resistance. Accessory factors, such as Fem factors, provide possible new targets, while compounds that modulate methicillin resistance such as epicatechin gallate, derived from green tea, and corilagin, provide possible lead compounds for development of inhibitors.

Graphical abstract Co-inoculation of MRSA with the probiotic suspension Symprove resulted in eradication of the pathogen after 48 h. Abstract This work reports the anti-pathogenic effect of a commercially available water-based probiotic suspension, Symprove™, against three commonly encountered infectious organisms; Escherichia coli , methicillin-resistant Staphylococcus aureus (MRSA) and Shigella sonnei. An isothermal calorimetric assay was used to the monitor growth of the species individually and in binary combinations, while colony plate counting was used to enumerate viable cell numbers. It was observed that all pathogenic species were faster growing than the probiotic bacteria in Symprove™ after inoculation into growth medium yet in all instances bacterial enumeration at the end of the experiments revealed a significant reduction in the pathogen population compared with the controls. A control population between 108 and 109 CFU/ml was obtained for E. coli and S. sonnei whilst approximately 106 CFU/ml was obtained for MRSA. Upon co-incubation for 48 h, no viable counts were obtained for E. coli ; a 4-log reduction was obtained for S. sonnei whilst MRSA numbers were down to less than 10 cells/ml. The results show that Symprove™ has antipathogenic activity against E. coli , S. sonnei and MRSA. [ABSTRACT FROM AUTHOR]

Abstract: Two enantiomerically pure B-ring modified analogues of (−)-epicatechin gallate were synthesised and their modulation of β-lactam resistance using three strains of methicillin resistant Staphylococcus aureus (BB 568, EMRSA-15 and EMRSA-16) evaluated. Sub-inhibitory concentrations (12.5 and 25mg/L) of the two analogues fully sensitised each of the three MRSA strains to oxacillin, reducing the MIC to less than 0.5mg/L, identical to levels achieved with ECg. Lower concentrations demonstrated that the position and degree of hydroxylation of the B-ring is important for activity. [Copyright &y& Elsevier]

Abstract: Hydrolytically more stable analogues of (−)-epicatechin gallate (ECg) have been synthesised from ECg where an amine or amide function has been substituted for the ester linkage that joins the C-ring with the galloyl D-ring. Sub-inhibitory concentrations (25mg/L) of the amide analogue 7, possessing the natural C-3 stereochemistry, were able to reduce the resistance to oxacillin of three strains of methicillin resistant Staphylococcus aureus (BB 568, EMRSA-15 and EMRSA-16) comparable to levels achieved with ECg. [Copyright &y& Elsevier]

• The herbal extracts showed weak antibacterial activity against the MRSA strains having MIC > 512 µg/mL. • The combined extracts with standard antibiotics was synergistic (2–64 fold potentiation) and bactericidal (≤ 3log10 CFU/mL). • LILE inhibited PKM 101 only by 30% but strongly against TP114 (74%), whilst LLFE was inactive to both plasmids. • The chromatogram peaks showed that both extracts contain rutin and gallic acid at 330 nm. • LLFE displayed a toxic effect towards HepG2 cells whilst LILE acted non-toxic. • Molecular docking of lobelanidine was the most safer and potent inhibitor of DNA‐gyrase A of Staphylococcus aureus. The present study evaluated antibacterial, plasmid conjugal transfer inhibition, growth kinetics and bacterial resistance modifying activities of methanol crude extracts from the fruits of Ligustrum lucidum (LLFE) and the leaves of Lobelia inflata (LILE) against a panel of methicillin-resistant Staphylococcus aureus (MRSA) expressing active efflux pumps. The results showed that both extracts had weak antibacterial activity against the MRSA strains to have MIC > 512 mg/L. The combination of extracts with standard antibiotics was synergistic (2–64 fold potentiation) and bactericidal (≤ 3log10 CFU/mL) on more than 70% of the bacteria. LLFE displayed a toxic effect on HepG2 cells having IC 50 27.61 mg/L while LILE demonstrated moderate toxicity (IC 50 125.06 mg/L). LILE extract exhibited a weak inhibition of transfer conjugation mediated by PKM 101 (30%) and promising inhibition against TP114 (74%), while LLFE enhanced conjugal transfer to both plasmids. The chromatogram peaks showed that both extracts contain rutin and gallic acid with a retention time corresponding to the standards at 330 nm. Molecular docking of lobelanidine showed that it was the safest and most potent inhibitor of DNA-gyrase A of Staphylococcus aureus when compared with the standard norfloxacin. Our findings suggest that Ligustrum lucidum and Lobelia inflata are potential pharmacological agents in the future design of combination anti-infective therapies against multidrug-resistant strains. [ABSTRACT FROM AUTHOR]

Abstract: An in-depth evaluation was undertaken of a new antibacterial natural product (1) recently isolated and characterised from the plant Hypericum olympicum L. cf. uniflorum. Minimum inhibitory concentrations (MICs) were determined for a panel of bacteria, including: meticillin-resistant and -susceptible strains of Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus haemolyticus; vancomycin-resistant and -susceptible Enterococcus faecalis and Enterococcus faecium; penicillin-resistant and -susceptible Streptococcus pneumoniae; group A streptococci (Streptococcus pyogenes); and Clostridium difficile. MICs were 2–8mg/L for most staphylococci and all enterococci, but were ≥16mg/L for S. haemolyticus and were >32mg/L for all species in the presence of blood. Compound 1 was also tested against Gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa and Salmonella enterica serovar Typhimurium but was inactive. The MIC for Mycobacterium bovis BCG was 60mg/L, and compound 1 inhibited the ATP-dependent Mycobacterium tuberculosis MurE ligase [50% inhibitory concentration (IC50)=75μM]. In a radiometric accumulation assay with a strain of S. aureus overexpressing the NorA multidrug efflux pump, the presence of compound 1 increased accumulation of 14C-enoxacin in a concentration-dependent manner, implying inhibition of efflux. Only moderate cytotoxicity was observed, with IC50 values of 12.5, 10.5 and 8.9μM against human breast, lung and fibroblast cell lines, respectively, highlighting the potential value of this chemotype as a new antibacterial agent and efflux pump inhibitor. [Copyright &y& Elsevier]