Phellinstatin, a new inhibitor of enoyl-ACP reductase produced by the medicinal fungus Phellinus linteus

A new trimeric hispidin derivative, phellinstatin, was isolated from a culture broth of the medicinal fungus Phellinus linteus and its structure was established by various spectral analysis. Phellinstatin strongly inhibited Staphylococcus aureus enoyl-ACP reductase with an IC₅₀ of 6 μM and also showed antibacterial activity against S. aureus and MRSA.     

An antibacterial hydroxy fusidic acid analogue from Acremonium crotocinigenum

A fusidane triterpene, 16-deacetoxy-7-beta-hydroxy-fusidic acid (1), was isolated from a fermentation of the mitosporic fungus Acremonium crotocinigenum. Full unambiguous assignment of all (1)H and (13)C data of 1 was carried out by extensive one- and two-dimensional NMR studies employing HMQC and HMBC spectra. Compound 1 was tested against a panel of multidrug-resistant (MDR) and methicillin-resistant Staphylococcus aureus (MRSA) strains and showed minimum inhibitory concentration values of 16 microg/ml.     

The antibacterial activity of phospholipase A2 type IIA is regulated by the cooperative lipid chain melting behavior in Staphylococcus aureus

As one of its primary physiological functions, sPLA₂-IIA appears to act as an antibacterial agent. In particular, sPLA₂-IIA shows high activity towards Gram-positive bacteria such as Staphylococcus aureus (S. aureus). This antibacterial activity results from the preference of the enzyme towards membranes enriched in anionic lipids, which is a common feature of bacterial membranes. An intriguing aspect observed in a variety of bacterial membranes is the presence of a broad but cooperative lipid chain melting event where the lipids in the membrane transition from a solid-ordered (so) into a liquid-disordered (ld) state close to physiological temperatures. It is known that the enzyme is sensitive to the level of lipid packing, which changes sharply between the so and the ld states. Therefore, it would be expected that the enzyme activity is regulated by the bacterial membrane thermotropic behavior. We determine by FTIR the thermotropic lipid chain melting behavior of S. aureus and find that the activity of sPLA₂-IIA drops sharply in the so state. The activity of the enzyme is also evaluated in terms of its effects on cell viability, showing that cell survival increases when the bacterial membrane is in the so state during enzyme exposure. These results point to a mechanism by which bacteria can develop increased resistance towards antibacterial agents that act on the membrane through a cooperative increase in the order of the lipid chains. These results show that the physical behavior of the bacterial membrane can play an important role in regulating physiological function in an in vivo system.     

Evidence for a new post-translational modification in Staphylococcus aureus: hydroxymethylation of asparagine and glutamine

Staphylococcus aureus is an opportunistic pathogen whose infectious capacity depends on surface proteins, which enable bacteria to colonize and invade host tissues and cells. We analyzed “trypsin-shaved” surface proteins of S. aureus cultures by high resolution LC-MS/MS at different growth stages and culture conditions. Some modified peptides were identified, with a mass shift corresponding to the addition of a CH₂O group (+ 30.0106 u). We present evidence that this shift corresponds to a hyxdroxymethylation of asparagine and glutamine residues. This known but poorly documented post-translational modification was only found in a few proteins of S. aureus grown under specific conditions. This specificity seemed to exclude the hypothesis of an artifact due to sample preparation. Altogether hydroxymethylation was observed in 35 peptides from 15 proteins in our dataset, which corresponded to 41 modified sites, 35 of them being univocally localized. While no function can currently be assigned to this post-translational modification, we hypothesize that it could be linked to modulation of virulence factors, since it was mostly found on some surface proteins of S. aureus.     

Molecular weight and pH aspects of the efficacy of oligochitosan against methicillin-resistant Staphylococcus aureus (MRSA)

Oligochitosan samples varying in molecular weight (M_w) and having narrow polydispersities were prepared by means of depolymerization of chitosan in hydrochloric acid, and their antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) was measured at pH values 5.5-8.0. The antibacterial testing of oligochitosans obtained showed that oligochitosans having M_w in the range of 0.73-20.0 kDa could be used both at slightly acidic and neutral pH values, and that the activity against MRSA remained moderate for oligochitosan samples having M_w about 3-5 kDa even at slightly basic pH values. The self-assembling behavior of oligochitosan macromolecules in the dilute solution at various pH values as a function of chain length was investigated. At first it was shown that oligochitosans formed supramolecular aggregates in dilute solutions below the critical pH value 6.5. Despite the aggregation phenomenon, the formation of nano-sized aggregates did not prevent oligochitosan from demonstrating the bactiostatic activity.     

Oxidative stress involved in the antibacterial action of different antibiotics

Staphylococcus aureus and Escherichia coli sensitive to chloramphenicol incubated with this antibiotic suffered oxidative stress with increase of anion superoxide (O2-). This reactive species of oxygen was detected by chemiluminescence with lucigenin. S. aureus, E. coli, and Enterococcus faecalis sensitive to ciprofloxacin exhibited oxidative stress when they were incubated with this antibiotic while resistant strains did not show stimuli of O2-. Other bacteria investigated was Pseudomonas aeruginosa, strains sensitive to ceftazidime and piperacillin presented oxidative stress in presence of these antibiotics while resistant strains were not stressed. Higher antibiotic concentration was necessary to augment O2- in P. aeruginosa biofilm than in suspension, moreover old biofilms were resistant to oxidative stress caused by antibiotics. A ceftazidime-sensitive mutant of P. aeruginosa, coming from a resistant strain, exhibited higher production of O2- than wild type in presence of this antibiotic. There was relation between antibiotic susceptibility and production of oxidative stress.     

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.     

Bacterial resistance modifying agents from Lycopus europaeus

As part of an ongoing project to identify plant natural products which modulate bacterial multidrug resistance (MDR), bioassay-guided isolation of an extract of Lycopus europaeus yielded two new isopimarane diterpenes, namely methyl-1alpha-acetoxy-7alpha 14alpha-dihydroxy-8,15-isopimaradien-18-oate (1) and methyl-1alpha,14alpha-diacetoxy-7alpha-hydroxy-8,15-isopimaradien-18-oate (2). The structures were established by spectroscopic methods. These compounds and several known diterpenes were tested for in vitro antibacterial and resistance modifying activity against strains of Staphylococcus aureus possessing the Tet(K), Msr(A), and Nor(A) multidrug resistance efflux mechanisms. At 512 microg/ml none of the compounds displayed any antibacterial activity but individually in combination with tetracycline and erythromycin, a two-fold potentiation of the activities of these antibiotics was observed against two strains of S. aureus that were highly resistant to these agents due to the presence of the multidrug efflux mechanisms Tet(K) (tetracycline resistance) and Msr(A) (macrolide resistance).     

Biofunctionalization of cellulosic fibres with l-cysteine: Assessment of antibacterial properties and mechanism of action against Staphylococcus aureus and Klebsiella pneumoniae

The main purpose of this work is to obtain a cotton-based textile material functionalized with l-cysteine (l-cys) to achieve an antimicrobial effect with potential application in biomedical, geriatric or pediatric textiles. The binding capacity of l-cys to cotton fibres was assessed through different functionalization strategies—surface activation and exhaustion processes. A subsequent analysis of the possible antibacterial action against Staphylococcus aureus and Klebsiella pneumoniae was performed according with the Japanese International standard ( JISL, 2008). To determine the mechanism of action of l-cys on the selected strains, flow cytometry was used.     

Synthesis and antimicrobial activity of N¹-benzyl or N¹-benzyloxy-1,6-dihydro-1,3,5-triazine-2,4-diamines

The emergence and spread of multidrug-resistant strains of Staphylococcus aureus and Mycobacterium tuberculosis are generating a threat to public health worldwide. In the current study, a series of N(1)-benzyl and N(1)-benzyloxy-1,6-dihydro-1,3,5-triazine-2,4-diamine derivatives were synthesized and investigated for their antimicrobial activity against S. aureus, and Mycobacterium smegmatis which is taxonomically related to M. tuberculosis. Most of the compounds exhibited good activity against M. smegmatis as determined by comparison of diameters of the zone of inhibition of test compounds and standard antibiotics. Compound 7o showed potent antimycobacterial activity against M. smegmatis without mammalian DHFR inhibition liability. The results from this study indicate that 1-benzyl derivatives of 1,6-dihydro-1,3,5-triazine-2,4-diamines may be used as lead compounds for the discovery of antimycobacterial agents.     

Development of pooled suppression subtractive hybridization to analyze the pangenome of Staphylococcus aureus

We describe the development and application of a Pooled Suppression Subtractive Hybridization (PSSH) method to describe differences between the genomic content of a pool of clinical Staphylococcus aureus isolates and a sequenced reference strain. In comparative bacterial genomics, Suppression Subtractive Hybridization (SSH) is normally utilized to compare genomic features or expression profiles of one strain versus another, which limits its ability to analyze communities of isolates. However, a PSSH approach theoretically enables the user to characterize the entirety of gene content unique to a related group of isolates in a single reaction. These unique fragments may then be linked to individual isolates through standard PCR. This method was applied to examine the genomic diversity found in pools of S.aureus isolates associated with complicated bacteremia infections leading to endocarditis and osteomyelitis. Across four pools of 10 isolates each, four hundred and twenty seven fragments not found in or significantly divergent from the S. aureus NCTC 8325 reference genome were detected. These fragments could be linked to individual strains within its pool by PCR. This is the first use of PSSH to examine the S. aureus pangenome. We propose that PSSH is a powerful tool for researchers interested in rapidly comparing the genomic content of multiple unstudied isolates.     

Structural elucidation of the extracellular and cell-wall teichoic acids of Staphylococcus aureus MN8m, a biofilm forming strain

Extracellular teichoic acid, an essential constituent of the biofilm produced by Staphylococcus epidermidis strain RP62A, is also an important constituent of the extracellular matrix of another biofilm producing strain, Staphylococcus aureus MN8m. The structure of the extracellular and cell wall teichoic acids of the latter strain was studied by NMR spectroscopy and capillary electrophoresis-mass spectrometry. Both teichoic acids were found to be a mixture of two polymers, a (1-->5)-linked poly(ribitol phosphate), substituted at the 4-position of ribitol residues with beta-GlcNAc, and a (1-->3)-linked poly(glycerol phosphate), partially substituted with the D-Ala at 2-position of glycerol residue. Such mixture is unusual for S. aureus.     

Biochemical characterisation and genetic analysis of aureocin A53, a new,atypical bacteriocin from Staphylococcus aureus

Aureocin A53 is produced by Staphylococcus aureus A53. It is encoded on a 10.4 kb plasmid, pRJ9, and is active against Listeria monocytogenes. Aureocin A53 is a highly cationic 51-residue peptide containing ten lysine and five tryptophan residues. Aureocin A53 was purified to homogeneity by hydrophobic-interaction, cation-exchange, and reverse-phase chromatography. MALDI-TOF mass spectrometry yielded a molecular mass of 6012.5 Da, which was 28 Da higher than predicted from the structural gene sequence of the bacteriocin. The mass increment resulted from an N-formylmethionine residue, indicating that the aureocin A53 is synthesised and secreted without a typical bacteriocin leader sequence or sec-dependent signal peptide. The structural identity of aureocin A53 was verified by Edman sequencing after de-blocking with cyanogen bromide and extensive mass spectrometry analysis of enzymatically and laser-generated fragments. The complete sequence of pRJ9 was determined and none of the open reading frames identified in the vicinity of the structural gene aucA showed similarity to genes that are typically found in bacteriocin gene clusters. Thus, neither a dedicated protease or transporter, nor modifying enzymes and regulatory elements seemed to be involved in the production of aureocin A53. Further unique features that distinguish aureocin A53 from other peptide bacteriocins include remarkable protease stability and a defined, rigid structure in aqueous solution.     

Bacterial resistance modifying tetrasaccharide agents from Ipomoea murucoides

As part of an ongoing project to identify oligosaccharides which modulate bacterial multidrug resistance, the CHCl₃-soluble extract from flowers of a Mexican arborescent morning glory, Ipomoea murucoides, through preparative-scale recycling HPLC, yielded five lipophilic tetrasaccharide inhibitors of Staphylococcusaureus multidrug efflux pumps, murucoidins XII-XVI (1-5). The macrocyclic lactone-type structures for these linear hetero-tetraglycoside derivatives of jalapinolic acid were established by spectroscopic methods. These compounds were tested for in vitro antibacterial and resistance modifying activity against strains of Staphylococcus aureus possessing multidrug resistance efflux mechanisms. Only murucoidin XIV (3) displayed antimicrobial activity against SA-1199B (MIC 32 μg/ml), a norfloxacin-resistant strain that over-expresses the NorA MDR efflux pump. The four microbiologically inactive (MIC > 512 μg/ml) tetrasaccharides increased norfloxacin susceptibility of this strain by 4-fold (8 μg/ml from 32 μg/ml) at concentrations of 25 μg/ml, while murucoidin XIV (3) exerted the same potentiation effect at a concentration of 5 μg/ml.     

Cardioactive and antibacterial terpenoids from some Salvia species

Seven Salvia species were investigated recently for their chemical and biological activities. Some terpenoidal compounds exhibited cardiovascular and antibacterial activities. A number of new diterpenoids were obtained and their structures were established through extensive spectral analysis.     

Antibacterial and resistance modifying activity of Rosmarinus officinalis

As part of a project to characterise plant-derived natural products that modulate bacterial multidrug resistance (MDR), bioassay-guided fractionation of a chloroform extract of the aerial parts of Rosmarinus officinalis led to the characterisation of the known abietane diterpenes carnosic acid (1), carnosol (2) and 12-methoxy-trans-carnosic acid. Additionally, a new diterpene, the cis A/B ring junction isomer of 12-methoxy-trans-carnosic acid, 12-methoxy-cis-carnosic acid (5), was isolated. The major components were assessed for their antibacterial activities against strains of Staphylococcus aureus possessing efflux mechanisms of resistance. Minimum inhibitory concentrations ranged from 16 to 64 microg/ml. Incorporation of 1 and 2 into the growth medium at 10 microg/ml caused a 32- and 16-fold potentiation of the activity of erythromycin against an erythromycin effluxing strain, respectively. Compound 1 was evaluated against a strain of S. aureus possessing the NorA multidrug efflux pump and was shown to inhibit ethidium bromide efflux with an IC50 of 50 microM, but this activity is likely to be related to the inhibition of a pump(s) other than NorA. The antibacterial and efflux inhibitory activities of these natural products make them interesting potential targets for synthesis.     

An anti-staphylococcal acylphloroglucinol from Hypericum foliosum

An investigation into the antibacterial properties of Hypericum foliosum Aiton. (Guttiferae) has led to the isolation of a new bioactive acylphloroglucinol natural product which by NMR spectroscopy and mass spectrometry was characterised as 1,3,5-trihydroxy-6-[2',3'-epoxy-3'-methyl-butyl]-2-[2'-methyl-butanoyl]-4-[3'-methyl-2'-butenyl]-benzene and is described here for the first time. This metabolite was evaluated against a panel of multidrug-resistant strains of Staphylococcus aureus and minimum inhibitory values ranged from 16 to 32 microg/ml.     

Anti-staphylococcal acylphloroglucinols from Hypericum beanii

As part of an ongoing project to investigate the anti-staphylococcal properties of the Hypericum genus, an acylphloroglucinol, 1,5-dihydroxy-2-(2'-methylpropionyl)-3-methoxy-6-methylbenzene (1), was isolated from the dichloromethane extract of the aerial parts of H. beanii (Guttiferae), together with a minor related acylphloroglucinol 1,5-dihydroxy-2-(2'-methylbutanoyl)-3-methoxy-6-methylbenzene (2) as a mixture in a 5:2 ratio. The known compounds 1,7-dihydroxyxanthone (3), stigmasterol, catechin and shikimic acid were also isolated from this plant. The structures of the compounds were characterized by extensive 1- and 2D NMR spectroscopy and mass spectrometry. The minimum inhibitory concentration (MIC) values the acylphloroglucinol mixture and (3) against a panel of multidrug-resistant strains of Staphylococcus aureus ranged from 16-32 microg/ml to 128-256 microg/ml, respectively.     

SsrA (tmRNA) acts as an antisense RNA to regulate Staphylococcus aureus pigment synthesis by base pairing with crtMN mRNA

SsrA RNA (small stable RNA A), also known as tmRNA and 10Sa RNA, functions both as tRNA and mRNA through its unique structure. The carotenoid pigment is the eponymous feature of human pathogen Staphylococcus aureus. Here, we found that the pigment of the mutant strain with ssrA deletion was increased. Furthermore, it was demonstrated that ssrA could act as an antisense RNA aside from its well-known biological function, and crtMN, encoding two essential enzymes for the pigment synthesis, was identified as target of ssrA. Further investigation showed ssrA could specifically base pair with the RBS (ribosomal binding site) region of the crtMN mRNA. Our results revealed a new mechanism by which ssrA regulated the biosynthesis of pigment in S. aureus.     

Assessment of the antibacterial activity of galangin against 4-quinolone resistant strains of Staphylococcus aureus

The flavonol galangin is present in numerous plants and is a major constituent of Helichrysum aureonitens, a perennial herb used by South African indigenes to treat infection. In the present study, the antibacterial activity of galangin was assessed against 17 strains of 4-quinolone resistant S. aureus using an agar dilution assay. It was determined that the flavonol had a minimum inhibitory concentration (MIC) of approximately 50 microg/ml against 16 of these strains, including those which exhibited 250- and 500-fold increases in norfloxacin resistance. The remaining one strain, which possessed an amino acid alteration in the GrlB subunit of topoisomerase IV, had increased susceptibility to galangin. Control strains of 4-quinolone sensitive S. aureus were also found to have MICs of 50 microg/ml. The topoisomerase IV enzyme may therefore be implicated in the antibacterial mechanism of action of galangin. Clearly however, there is no cross-resistance between galangin and the 4-quinolones, and the flavonol therefore warrants further investigation as an antibacterial agent.