Enhancement of antibacterial effects of epigallocatechin gallate, using ascorbic acid

Although plant polyphenols such as (−)-epigallocatechin gallate (EGCG) have antibacterial activity towards methicillin-resistant Staphylococcus aureus (MRSA), such polyphenols are unstable in solution. Because the instability of polyphenols is attributable to their oxidation, we examined the effects of antioxidants and inhibitors of polyphenol oxidation on the maintenance of polyphenol antibacterial activity. The antibacterial activity of EGCG was enhanced in the presence of ascorbic acid, and ascorbic acid was the most effective for retaining the concentration of stable EGCG. On the other hand, the antibacterial activity of EGCG was lowered in the presence of casein in spite of its suppressing effect on the EGCG decrease. The effect of EGCG on the antibiotic resistance of MRSA was also enhanced in the presence of ascorbic acid. The addition of an antioxidant may affect other pharmacological effects of polyphenols in analogous ways, although this does not mean the clinical usefulness of the addition directly.     

Variation in antimicrobial action of proanthocyanidins from Dorycnium rectum against rumen bacteria

The proanthocyanidin polymer fractions of the leaves of the forage legume Dorycnium rectum were analysed by acid catalysis with benzyl mercaptan, NMR and ES-MS. The results showed that D. rectum differs from other temperate proanthocyanidin-containing forage legumes in that the range of polymers extends up to very high degrees of polymerisation. Three fractions were characterised as low, medium, and high molecular weight proanthocyanidin fractions with mean degree of polymerisations of 10.3, 41 and 127, respectively. Epigallocatechin was the most abundant extension unit and the terminating flavan-3-ols comprised largely catechin and gallocatechin units in equal proportions. Formation of thiolyated dimer products showed the interflavan-linkages of the lower molecular weight proanthocyanidins to be predominantly C4-->C8 with a small amount of C4-->C6. ES-MS spectra distinguished lower from higher polymeric proanthocyanidins from M2- to M8(2)-. The antibacterial activity of proanthocyanidin fractions against pure cultures of microbes selected from the ruminal population to represent fibre degrading, proteolytic and hyper ammonia producing bacteria in broth culture was evaluated. The activity of proanthocyanidin fractions against Clostridium aminophilum, Butyrivibrio fibrisolvens and Clostridium proteoclasticum was significantly dependent on their structure but not so against Ruminococcus albus and Peptostreptococcus anaerobius. The latter observation was unique in that they were sensitive to all proanthocyanidin fractions evaluated, even at the lowest concentration (100 microg/ml). The results suggest the effects of the extractable proanthocyanidins on rumen microbes should be considered when evaluating an alternative proanthocyanidin-containing forage source for ruminants, such as D. rectum.     

The anti-staphylococcal activity of Angelica dahurica (Bai Zhi)

Bioassay-guided fractionation of a hexane extract prepared from the roots of the Chinese drug Angelica dahurica (Bai Zhi) led to the isolation of the polyacetylenic natural product falcarindiol (1). The absolute stereochemistry of this compound was confirmed by careful 1H NMR analysis of its (R)- and (S)-Mosher ester derivatives as the 3(R), 8(S) isomer. Activity was tracked using a Mycobacterium fortuitum screening assay and the purified product was evaluated against multidrug-resistant and methicillin-resistant strains of Staphylococcus aureus (MRSA). The minimum inhibitory concentrations (MIC) of this metabolite ranged from 8 to 32 microg/ml highlighting the potential of the acetylene natural product class as antibiotic-lead compounds. These MIC values compare favourably with some of the newest agents in development for the treatment of MRSA infection and indicate that further evaluation of the antibiotic activity of acetylenes is warranted.     

Loop-mediated isothermal amplification assays for identification of antiseptic- and methicillin-resistant Staphylococcus aureus

A method for rapid identification of antiseptic- and methicillin-resistant Staphylococcus aureus (MRSA) based on 3 loop-mediated isothermal amplification (LAMP) assays was developed. LAMP targeting the femB gene identified S. aureus with 100% specificity, and LAMP targeting the mecA gene associated with methicillin resistance identified methicillin-resistant staphylococci with 100% specificity. LAMP targeting the qacA/B gene encoding an efflux pump responsible for antiseptic resistance identified high-acriflavine-resistant (MIC ≥ 100 mg/L) MRSA (92.5% positive) and acriflavine-susceptible (MIC < 25 mg/L) MRSA (100% negative). They were performed under the same reaction conditions within 60 min at 63 °C. The combined LAMP assays will be useful for rapid identification of S. aureus isolates and determination of their antibiotic and antiseptic resistance patterns with regard to methicillin and organic cationic substrates.     

Gene sequences and specific detection for Panton-Valentine leukocidin

A new category of methicillin-resistant Staphylococcus aureus (MRSA), called community-acquired MRSA (CA-MRSA), has emerged worldwide. In contrast to previous MRSA, most CA-MRSA carries the Panton-Valentine leukocidin (PVL) genes (lukPVSF) as a virulence genetic trait. Sequence analysis of the lukPVSF gene of a Japanese isolate demonstrated that the gene has more similarity to methicillin-susceptible S. aureus from France than MRSA from the United States. Based on the sequences, we developed a real-time PCR assay for the three key genes of CA-MRSA; that is, lukPVSF, mecA (for methicillin resistance), and spa (for S. aureus). Dual or triple assay for lukPVSF, mecA, and spa in one test tube became possible. The detection limit of the assay with probe and SYBR Green methods was between 2.7 and 2.7 x 10(1) CFU/ml. The assay detected PVL-positive MRSA in clinical (blood) isolates.     

Electron paramagnetic resonance studies of radical species of proanthocyanidins and gallate esters

The polyphenols present in green tea or red wine comprise both regular flavon(ol)s and proanthocyanidins, i.e., derivatives of flavan-3-ols, whose distinct antioxidative potential is of great importance for explaining the beneficial effects of these nutrient beverages. Using EPR spectroscopy, we investigated radical structures obtained after oxidation of the parent compounds either by horseradish peroxidase/hydrogen peroxide or after autoxidation in moderately alkaline solutions. Both proanthocyanidins (monomers of condensed tannins, e.g., (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin, (-)-epigallocatechin gallate, Pycnogenol) and gallate esters (hydrolyzable tannins, e.g., propylgallate, beta-glucogallin, pentagalloyl glucose and tannic acid) yielded predominantly semiquinone structures derived from the parent catechol or pyrogallol moieties. Evidence for a time-dependent oligomerization was obtained for (-)-epigallocatechin gallate, supporting our hypothesis that o-quinones formed from the initial semiquinone disproportionation are prone to nucleophilic addition reactions. Such phenolic coupling reactions would retain the numbers of reactive catechol/pyrogallol structures and thus the antioxidative potential. We therefore propose that proanthocyanidins are superior antioxidants as compared to flavon(ol)s proper, whose quinones are more likely to redox-cycle and act as prooxidants.     

Synergistic effect between dieckol from <Emphasis Type="Italic">Ecklonia stolonifera</Emphasis> and β-lactams against methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

We have been attempting for some time to discover a compound evidencing antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). The dieckol isolated from Ecklonia stolonifera has been shown to exhibit antibacterial activity against methicillin-susceptible S. aureus (MSSA) and MRSA. The minimum inhibitory concentrations (MICs) of dieckol were determined in a range of 32 to 64 μg/mL against standard MSSA and MRSA strains. Furthermore, dieckol clearly reversed the high-level ampicillin and penicillin resistance of MRSA. The MICs of ampicillin against two standard strains of MRSA were dramatically reduced from 512 to 0.5 μg/mL in combination with 1/4 MIC of dieckol (16 μg/mL). The fractional inhibitory concentration (FIC) indices of ampicillin and penicillin were measured from 0.066 to 0.266 in combination with 8 or 16 μg/mL of dieckol against all tested MRSA strains, thereby suggesting that dieckol-ampicillin or dieckol-penicillin combinations exert a synergistic effect against MRSA. The results of this study indicate that dieckol, administered in combination with β-lactams, may prove effective in the treatment of MRSA infections. Our finding may also contribute to the development of an alternative phytotherapeutic anti-MRSA agent.     

Proteome of a methicillin-resistant Staphylococcus aureus clinical strain of sequence type ST398

Proteomics is a powerful tool to analyze the differences in gene expression of bacterial strains. Staphylococcus aureus has long been recognized as an important pathogen in human disease. In order to investigate this pathogen, the proteome of a clinical methicillin-resistant S. aureus (MRSA) strain of the sequence type ST398 was determined using 2-DE. Using 2-DE we obtained a total of 105 spots the MRSA strain. Furthermore in correlation with bioinformatic databases, they allowed accurate identification and characterization of proteins, resulting in 227 identified proteins. There were found proteins related to basic function of the cell, but also proteins related to virulence like catalase, specific of S. aureus species, and proteins related to antibiotic resistance. Proteins associated with antibiotic resistance or virulence factors are related to genomic databases. The most abundant classes identified involved glycolysis, energy production, one-carbon metabolism, and oxidation-reduction process, all of which reflect an active metabolism. These results highlight the importance of proteomics to deepen in the knowledge of protein expression of MRSA strain of the lineage ST398, microorganism with diverse and important resistance mechanisms. With this proteome map we have an essential tool for a better understanding of this pathogen and providing new data for protein databases. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Characterization and structure identification of an antimicrobial peptide, hominicin, produced by Staphylococcus hominis MBBL 2-9

Hominicin, antimicrobial peptide displaying potent activity against Staphylococcus aureus ATCC 25923, methicillin-resistant S. aureus (MRSA) ATCC 11435 and vancomycin-intermediate S. aureus (VISA) CCARM 3501, was purified by chloroform extraction, ion-exchange column chromatography and reverse-phase HPLC from culture supernatant of Staphylococcushominis MBBL 2-9. Hominicin exhibited heat stability up to 121 °C for 15 min and activity under both acidic and basic conditions (from pH 2.0 to 10.0). Hominicin was cleaved into two fragments after treatment with proteinase K, resulting in the loss of its antibacterial activity, while it was resistant to trypsin, α-chymotrypsin, pepsin and lipase. The molecular mass of hominicin determined by mass spectrometry was 2038.4 Da. LC-mass spectrometry and NMR spectroscopy analyses of the two fragments revealed the sequence of hominicin as DmIle-Dhb-Pro-Ala-Dhb-Pro-Phe-Dhb-Pro-Ala-Ile-Thr-Glu-Ile-Dhb-Ala-Ala-Val-Ile-Ala-Dmp, which had no similarity with other antimicrobial peptides previously reported. The present study is the first report of this novel antimicrobial peptide, which has uncommon amino acid residues like the ones in Class I group and shows potent activity against clinically relevant S. aureus, MRSA and VISA.     

Salt resistance and synergistic effect with vancomycin of alpha-helical antimicrobial peptide P18.

P18 (KWKLFKKIPKFLHLAKKF-NH(2)) is an alpha-helical antimicrobial peptide designed from a cecropin A-magainin 2 hybrid. In this study, P18 was found to show strong antimicrobial activity against several antibiotic-resistant bacterial and fungal strains. Both the salt resistance on antimicrobial activity and the synergistic effect with clinically used antibiotic agents are critical factors in developing effective peptide antibiotic drugs. For this reason, we investigated the salt resistance of P18 to antagonism by NaCl, CaCl(2), and MgCl(2) on antimicrobial activity and the synergistic effect of P18 with vancomycin against vancomycin-resistant Enterococcus faecium (VREF). Compared to magainin 2, P18 showed strong resistance on antimicrobial activity against bacterial strains and C. albicans under high NaCl concentrations of 100-200 mM. In addition, P18 displayed much greater salt resistance on antibacterial activity against Gram-negative bacteria at the physiological or elevated concentrations of CaCl(2) and MgCl(2) than magainin 2. Furthermore, the combination study revealed that P18 has a relatively effective synergistic effect with vancomycin against VREF. Thus, these results support that P18 may prove to be a salt-resistant antibiotic peptide potentially useful in the treatment of cystic fibrosis patients as well as a valuable adjuvant for antimicrobial chemotherapy.     

Evaluation of a duplex real-time PCR assay to detect MRSA from broth culture,human sera seeded with MRSA and from patient's serum

The need for rapid methods in order to precisely detect methicillin-resistant Staphylococcus aureus (MRSA) is extensively acknowledged. This study evaluated a quantitative real-time PCR assay targeting mecA (encoding high level resistance to methicillin) and femB (a specific genomic marker for S. aureus) genes to detect MRSA from broth culture, from serum seeded with MRSA and straight from the patient''s serum. One hundred and thirty-five clinical isolates of MRSA strains and different species were utilised in this study. In addition, a pilot study with 9 patients'' serum samples was performed. The sensitivity and specificity values for this assay were 99% and 100% respectively. The detection limit for this method was 1.23×10² CFU/ml from the serum seeded with MRSA cells and the limiting concentration of DNA for detection was 18 fg, which equates to 5.14 genomic DNA copies. In addition, this assay detected MRSA from patient''s serum (7 out of 9) with sensitivity of 77.8%. Overall, the assay was rapid, efficient, sensitive and easy to perform.     

Tryptoline-based benzothiazoles re-sensitize MRSA to β-lactam antibiotics

Resistance-modifying agents (RMAs) offer a promising solution to combat bacterial antibiotic resistance. Here we report the discovery and structure–activity relationships of a new class of RMAs with a novel tryptoline-based benzothiazole scaffold. Our most potent compound in this series (4ad) re-sensitizes multiple MRSA strains to cephalosporins at low concentrations (2 μg/mL) and has low mammalian cytotoxicity with a half growth inhibitory concentration (GI₅₀) > 100 μg/mL in human cervical carcinoma (HeLa) cells. In addition, the same core scaffold with different substitutions also gives good antibacterial activity against MRSA.     

Anti-MRSA activity of alkyl gallates.

A series of alkyl gallates (3,4,5-trihydroxybenzoates) was found to show antibacterial activity against Gram-positive bacteria including methicillin resistant Staphylococcus aureus (MRSA) strains. For example, dodecyl (C(12)) gallate (1) exhibited bactericidal activity against MRSA ATCC 33591 strain with the minimum bactericidal concentration (MBC) of 25 microg/mL (74 microM). The time-kill curve study showed that dodecyl gallate is bactericidal against this MRSA strain. This bactericidal activity comes in part from its ability to inhibit respiratory electron transport systems. The length of the alkyl chain is not a major contributor but plays an important role in eliciting the activity.     

Green peach aphid infestation induces Arabidopsis PHYTOALEXIN-DEFICIENT4 expression at site of insect feeding

The Arabidopsis thaliana PHYTOALEXIN-DEFICIENT4 (PAD4) protein, which has homology to lipases, is required for phloem-based resistance against the green peach aphid (GPA; Myzus persicae Sülzer). PAD4 modulates antibiotic and antixenotic defenses against GPA. PAD4 in conjunction with its interacting partner ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) also functions in basal resistance to bacterial and oomycete pathogens by promoting salicylic acid (SA)-dependent and SA-independent defenses. By contrast, neither EDS1 nor SA is required for PAD4-controlled defense against GPA. Distinct molecular activities of PAD4 are involved in different aspects of Arabidopsis defense against GPA and pathogens. Histochemical analysis of plants containing a PAD4_p:GUS chimera, which expresses the GUS reporter from the PAD4 promoter, indicated strong PAD4 promoter activity at the site of penetration of the vasculature by the insect stylet. GUS activity was also observed in non-vascular tissues of GPA-infested leaves, thus raising the possibility that a combination of distinct PAD4 activities in vascular and non-vascular tissues contribute to Arabidopsis defense against GPA.     

Multiple antibiotic resistances in metal tolerant E. coli from hospital waste water

Study of antibiotic resistance was done among the metal tolerant E. coli isolates from hospital wastewater at Lucknow city. Metal tolerance was determined in terms of visible growth on metal amended plates at their varying concentrations. MICs were also determined among all metal tolerant E. coli isolates. All the isolates showed their MIC in between 100-2000 µg/ml while maximum isolates demonstrated their MICs at 400, 800 and 1600 µg/ml against all the metal tested. 23.07% of the isolates showed their MIC at 2000 µg/ml against Ni³⁺. Multiple antibiotic resistances were recorded among all the metal resistant E.coli isolates. A high level of resistance was observed against Methicillin (86.53%) followed by penicillin (73.07%), Cephradin (57.69%), Rifampicin (34.61%), Erythromycin (26.92%), Nalidixic acids (25%), Chloramphenicol (3.84%) and least to Gentamycine (1.92%). Streptomycin was recorded most effective against E.coli isolates among the entire antibiotic tested. Antimicrobial resistance observed among the bacteria from the aquatic system contaminated with hospital wastes may be threatful for the environment and public health both.     

Single-molecule sequencing reveals the molecular basis of multidrug-resistance in ST772 methicillin-resistant Staphylococcus aureus

Background

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infection, but there is growing awareness of the emergence of multidrug-resistant lineages in community settings around the world. One such lineage is ST772-MRSA-V, which has disseminated globally and is increasingly prevalent in India. Here, we present the complete genome sequence of DAR4145, a strain of the ST772-MRSA-V lineage from India, and investigate its genomic characteristics in regards to antibiotic resistance and virulence factors.

Results

Sequencing using single-molecule real-time technology resulted in the assembly of a single continuous chromosomal sequence, which was error-corrected, annotated and compared to nine draft genome assemblies of ST772-MRSA-V from Australia, Malaysia and India. We discovered numerous and redundant resistance genes associated with mobile genetic elements (MGEs) and known core genome mutations that explain the highly antibiotic resistant phenotype of DAR4145. Staphylococcal toxins and superantigens, including the leukotoxin Panton-Valentinin Leukocidin, were predominantly associated with genomic islands and the phage φ-IND772PVL. Some of these mobile resistance and virulence factors were variably present in other strains of the ST772-MRSA-V lineage.

Conclusions

The genomic characteristics presented here emphasize the contribution of MGEs to the emergence of multidrug-resistant and highly virulent strains of community-associated MRSA. Antibiotic resistance was further augmented by chromosomal mutations and redundancy of resistance genes. The complete genome of DAR4145 provides a valuable resource for future investigations into the global dissemination and phylogeography of ST772-MRSA-V.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1599-9) contains supplementary material, which is available to authorized users.     

Effect of new antioxidant cysteinyl-flavanol conjugates on skin cancer cells

Novel catechin derivatives obtained from grape procyanidins and l-cysteine scavenge free radicals by hydrogen atom donation, rather than electron transfer, and reduce cell viability in A375 and M21 melanoma cells. In particular, 4beta-(S-cysteinyl)epicatechin 3-O-gallate has a free radical scavenging capacity as strong as that of tea (-)-epigallocatechin gallate and causes a significant S-phase cell-cycle arrest in both cell lines at doses higher than 100 microM. The other cysteinyl compounds do not affect normal cell cycle distribution. The gallate derivative also induces apoptosis in melanoma cells more strongly than the other derivatives and the parent (-)-epicatechin do. The gallate compound seems to trigger nuclear condensation and fragmentation, which is confirmed by DNA laddering. Interestingly, they do not induce apoptosis in keratinocytes (HaCaT).     

A stochastic mathematical model of methicillin resistant Staphylococcus aureus transmission in an intensive care unit: predicting the impact of interventions

OBJECTIVES: To estimate the transmission rate of MRSA in an intensive care unit (ICU) in an 800 bed Australian teaching hospital and predict the impact of infection control interventions. METHODS: A mathematical model was developed which consisted of four compartments: colonised and uncolonised patients and contaminated and uncontaminated health-care workers (HCWs). Patient movements, MRSA acquisition and daily prevalence data were collected from an ICU over 939 days. Hand hygiene compliance and the probability of MRSA transmission from patient to HCW per discordant contact were measured during the study. Attack rate and reproduction ratio were estimated using Bayesian methods. The impact of a number of interventions on attack rate was estimated using both stochastic and deterministic versions of the model. RESULTS: The mean number of secondary cases arising from the ICU admission of colonised patients, also called the ward reproduction ratio, R(w), was estimated to be 0.50 (95% CI 0.39-0.62). The attack rate was one MRSA transmission per 160 (95% CI 130-210) uncolonised-patient days. Results were not sensitive to uncertainty in measured model parameters (hand hygiene rate and transmission probability per contact). Hand hygiene was predicted to be the most effective intervention. Decolonisation was predicted to be relatively ineffective. Increasing HCW numbers was predicted to increase MRSA transmission, in the absence of patient cohorting. The predictions of the stochastic model differed from those of the deterministic model, with lower levels of colonisation predicted by the stochastic model. CONCLUSIONS: The number of secondary cases of MRSA colonisation within the ICU in this study was below unity. Transmission of MRSA was sustained through admission of colonised patients. Stochastic model simulations give more realistic predictions in hospital ward settings than deterministic models. Increasing staff does not necessarily lead to reduced transmission of nosocomial pathogens.     

Effects of antibiotic resistance alleles on bacterial evolutionary responses to viral parasites

Antibiotic resistance has wide-ranging effects on bacterial phenotypes and evolution. However, the influence of antibiotic resistance on bacterial responses to parasitic viruses remains unclear, despite the ubiquity of such viruses in nature and current interest in therapeutic applications. We experimentally investigated this by exposing various Escherichia coli genotypes, including eight antibiotic-resistant genotypes and a mutator, to different viruses (lytic bacteriophages). Across 960 populations, we measured changes in population density and sensitivity to viruses, and tested whether variation among bacterial genotypes was explained by their relative growth in the absence of parasites, or mutation rate towards phage resistance measured by fluctuation tests for each phage. We found that antibiotic resistance had relatively weak effects on adaptation to phages, although some antibiotic-resistance alleles impeded the evolution of resistance to phages via growth costs. By contrast, a mutator allele, often found in antibiotic-resistant lineages in pathogenic populations, had a relatively large positive effect on phage-resistance evolution and population density under parasitism. This suggests costs of antibiotic resistance may modify the outcome of phage therapy against pathogenic populations previously exposed to antibiotics, but the effects of any co-occurring mutator alleles are likely to be stronger.