A mild hydrophobic interaction chromatography involving polyethylene glycol immobilized to agarose media refolding recombinant Staphylococcus aureus elongation factor G

Recombinant Staphylococcus aureus elongation factor G (EF-G) is difficult to refold by dilution due to the formation of large amounts of misfolded structures. However, refolding of EF-G by adsorption to a chromatographic column packed with immobilized polyethylene glycol 20,000 (PEG 20 K) followed by pulse elution with 8 M urea resulted in 88% mass recovery and 80% of correctly refolded structure. The PEG 20 K was coupled to brominated allyl group derivatized Sepharose High Performance to construct a mild hydrophobic adsorbent. Various other hydrophobic interaction adsorbents were also attempted to refold EF-G. However, ligands with high hydrophobicity tended to misfold EF-G, resulting in irreversible adsorption. Various solvents, detergents, and low temperature as well as 8 M urea were tried to release bound EF-G. Only pulse elution with 8 M urea was efficient. Urea concentrations favorable for efficiently refolding EF-G were investigated. Low urea concentration produced more misfolded structures.     

Effect of Cd2+ on ATP synthesis coupled to electron transfer in cadmium-resistant and -sensitive Staphylococcus aureus

In the Cd2(+)-resistant Staphylococcus aureus 17810R which contains the plasmid-coded Cd2+ efflux system, accumulation of Cd2+ was highly reduced. Consequently, neither respiration nor ATP synthesis coupled to electron transfer were inhibited. The plasmidless S. aureus strain 17810S accumulated Cd2+ via the Mn2+ porter down the membrane potential (delta phi) which resulted in inhibition of respiration and of ATP synthesis.     

Toxicity mechanism of Cu2+ ion individually and in combination with Zn2+ ion in characterizing the molecular changes of Staphylococcus aureus studied using FTIR coupled with chemometric analysis

Copper and zinc have a high binding affinity with a Staphylococcus aureus bacterial community. This causes a change in the biomolecular composition of S. aureus. Our study aims at understanding the resistance mechanism of Cu and Zn either or in various combinations using FTIR and chemometric techniques. Zn toxicity resulted in a significant change in lipid content (3100-2800 cm⁻¹) compared to Cu. A significant decrease in protein content is observed for Cu treatment in the amide region. The bio-concentration factor shows a higher value for Cu compared to Zn. The increase in band area of carbohydrates moieties 1059 cm⁻¹ shows the secretion of EPS due to Cu toxicity. A significant change in nucleic acid compositions was noted in the region1200-900 cm⁻¹ due to Zn treatment. Secondary structural change in protein shows β sheet formation. The result of the finding shows Cu has greater toxicity than Zn. Further toxicity effects were greatly enhanced for metal mixtures ratio (Cu:2Zn). This shows Zn exhibits synergism effect with Cu. The obtained ROC (receiver operating characteristic) curve area gives good reliability of the experiments. The study attempts to understand the mechanism of toxicity removal of Cu and Zn metal mixtures by bacterial population using FTIR coupled with chemometric techniques. Open in a separate windowGraphical abstract     

In vitro antimicrobial activity of pannarin alone and in combination with antibiotics against methicillin-resistant Staphylococcus aureus clinical isolates

The in vitro antimicrobial activities of pannarin, a depsidone isolated from lichens, collected in several Southern regions of Chile (including Antarctica), was evaluated alone and in combination with five therapeutically available antibiotics, using checkerboard microdilution assay against methicillin-resistant clinical isolates strains of Staphylococcus aureus. MIC(90), MIC(50), as well as MBC(90) and MBC(50), were evaluated. A moderate synergistic action was observed in combination with gentamicin, whilst antagonism was observed in combination with levofloxacin. All combinations with erythromycin were indifferent, whilst variability was observed for clindamycin and oxacillin combinations. Data from checkerboard assay were analysed and interpreted using the fractional inhibitory concentration index and the response surface approach using the ΔE model. Discrepancies were found between both methods for some combinations. In order to asses cellular lysis after exposure to pannarin, cell membrane permeability assay was performed. The treatment with pannarin produces bactericidal activity without significant calcein release, consistent with lack of lysis or even significant structural damage to the cytoplasmic membrane. Furthermore, pannarin shows low hemolytic activity and moderate cytotoxic effect on peripheral blood mononuclear cells. These findings suggest that the natural compound pannarin might be a good candidate for the individualization of novel templates for the development of new antimicrobial agents or combinations of drugs for chemotherapy.     

Synthesis and anti Methicillin resistant Staphylococcus aureus activity of substituted chalcones alone and in combination with non-beta-lactam antibiotics

A total of 30 chalcone analogues was synthesized via a base catalyzed Claisen Schmidt condensation and screened for their in vitro antibacterial activity against Methicillin-sensitive Staphylococcus aureus (MSSA) and Methicillin-resistant Staphylococcus aureus (MRSA) alone or in combination with non beta-lactam antibiotics namely ciprofloxacin, chloramphenicol, erythromycin, vancomycin, doxycycline and gentamicin. In the checkerboard technique, fractional inhibitory concentration indices (FICI) show that the following combinations like ciprofloxacin with 25 (4'-bromo-2-hydroxychalcone); doxycycline with 21 (4-hydroxychalcone); doxycycline with 25; and doxycycline with 4 (2',2-dihydroxychalcone) were synergistic against MRSA. In term SAR study, the relationship between chalcone structure and their antibacterial activity against S. aureus and synergy with tested antibiotics were discussed. Possible mechanisms for antibacterial activity of chalcones alone as well as the synergistic effect in combinations were proposed by molecular modeling studies, respectively. Combinations of chalcones with conventional antibiotics could be an effective alternative in the treatment of infection caused by MRSA.     

Protection induced in mice vaccinated with recombinant collagen-binding protein (CnBP) and alpha-toxoid against intramammary infection with Staphylococcus aureus

Mice vaccinated with a combination of two Staphylococcus aureus antigens consisting of a recombinant collagen-binding protein (CnBP) and alpha-toxoid (alpha-toxoid) were significantly protected from intramammary challenge infection with S. aureus. The average number of bacteria recovered from the glands of mice vaccinated with the combination of CnBP/alpha-toxoid was significantly lower compared to the average number of bacteria recovered from the glands of mice vaccinated with only CnBP or alpha-toxoid or controls (P< or =0.01). Histopathological examination of mammary glands of mice vaccinated with CnBP together with alpha-toxoid showed no pathological changes, whereas glands of mice vaccinated with CnBP or alpha-toxoid alone developed severe mastitis and showed both focal and disseminated necrosis.     

Experimental study on the efficacy of combination of alpha-helical antimicrobial peptides and vancomycin against Staphylococcus aureus with intermediate resistance to glycopeptides

An experimental study has been performed to compare the in vitro activity and the in vivo efficacy of magainin II and cecropin A, two alpha-helical antimicrobial peptides, and vancomycin against Staphylococcus aureus with intermediate resistance to glycopeptides. In vitro experiments included MIC determination, time-kill and synergy studies. For in vivo studies, a mouse model of staphylococcal sepsis has been used. Main outcome measures were: lethality, quantitative blood cultures and detection of TNF-alpha and interleukin-6 (IL-6) plasma levels. Combinations of alpha-helical antimicrobial peptides showed in vitro synergistic interaction. Significant increase in efficacy was also observed in vivo: combined-treated groups had significant lower bacteremia when compared to single-treated groups. Magainin II combined with vancomycin exhibited the highest efficacy on all main outcome measurements. These results highlight the potential usefulness of these combinations and provide future therapeutic alternative in infections due to glycopeptides resistant staphylococci in the coming years.     

Genetic variability in beta-defensins is not associated with susceptibility to Staphylococcus aureus bacteremia

Introduction

Human beta-defensins are key components of human innate immunity to a variety of pathogens, including Staphylococcus aureus. The aim of the present study was to investigate a potential association between gene variations in DEFB1 and DEFB103/DEFB4 and the development of S. aureus bacteremia (SAB) employing a case-control design.

Methods

Cases were unique patients with documented SAB, identified with the National S. aureus Bacteremia Register, a comprehensive dataset of all episodes of community associated-SABs (CA-SAB) occurring in children (≤20 yrs) in Denmark from 1990 to 2006. Controls were age-matched healthy individuals with no history of SAB. DNA obtained from cases and controls using the Danish Newborn Screening Biobank were genotyped for functional polymorphisms of DEFB1 by Sanger sequencing and copy number variation of the DEFB103 and DEFB4 genes using Pyrosequencing-based Paralogue Ratio Test (P-PRT).

Results

193 ethnic Danish SAB cases with 382 age-matched controls were used for this study. S. aureus isolates represented a variety of bacterial (i.e., different spa types) types similar to SAB isolates in general. DEFB1 minor allele frequencies of rs11362 (cases vs. controls 0.47/0.44), rs1800972 (0.21/0.24), and rs1799946 (0.32/0.33) were not significantly different in cases compared with controls. Also, DEFB4/DEFB103 gene copy numbers (means 4.83/4.92) were not significantly different in cases compared with controls.

Conclusions

Using a large, unique cohort of pediatric CA-SAB, we found no significant association between DEFB1 genetic variation or DEFB4/DEFB103 gene copy number and susceptibility for SAB.     

In vitro pharmacokinetics of antimicrobial cationic peptides alone and in combination with antibiotics against methicillin resistant Staphylococcus aureus biofilms

Antibiotic therapy for methicillin-resistant Staphylococcus aureus (MRSA) infections is becoming more difficult in hospitals and communities because of strong biofilm-forming properties and multidrug resistance. Biofilm-associated MRSA is not affected by therapeutically achievable concentrations of antibiotics. Therefore, we investigated the in vitro pharmacokinetic activities of antimicrobial cationic peptides (AMPs; indolicidin, cecropin [1–7]-melittin A [2–9] amide [CAMA], and nisin), either alone or in combination with antibiotics (daptomycin, linezolid, teicoplanin, ciprofloxacin, and azithromycin), against standard and 2 clinically obtained MRSA biofilms. The minimum inhibitory concentrations (MIC) and minimum biofilm-eradication concentrations (MBEC) were determined by microbroth dilution technique. The time-kill curve (TKC) method was used to determine the bactericidal activities of the AMPs alone and in combination with the antibiotics against standard and clinically obtained MRSA biofilms. The MIC values of the AMPs and antibiotics ranged between 2 to 16 and 0.25 to 512 mg/L, and their MBEC values were 640 and 512 to 5120 mg/L, respectively. The TKC studies demonstrated that synergistic interactions occurred most frequently when using nisin + daptomycin/ciprofloxacin, indolicidin + teicoplanin, and CAMA + ciprofloxacin combinations. No antagonism was observed with any combination. AMPs appear to be good candidates for the treatment of MRSA biofilms, as they act as both enhancers of anti-biofilm activities and help to prevent or delay the emergence of resistance when used either alone or in combination with antibiotics.     

Antibacterial activity of anacardic acid and totarol, alone and in combination with methicillin, against methicillinresistant Staphylococcus aureus

The inhibitory and bactericidal activities of anacardic acid and totarol, alone and in combination with methicillin, were investigated against methicillin-resistant Staphylococcus aureus (MRSA). The growth of two MRSA strains was inhibited by 6·25 μg ml^-1 of anacardic acid and 0·78 μg ml^-1 of totarol. The time-kill curve study showed that these two compounds were bactericidal against MRSA. Anacardic acid killed MRSA cells more rapidly than totarol, and no viable cells were detected after being exposed to 6·25 μg ml^-1 of anacardic acid for 6 h. Anacardic acid showed bactericidal activity against MRSA at any stage of growth, and also even when cell division was inhibited by chloramphenicol. In the combination studies, the minimal inhibitory concentration (MIC) of methicillin was lowered from 800 to 1·56 μg ml^-1 for MRSA ATCC 33591, and from 800 to 6·25 μg ml^-1 for MRSA ATCC 33592, by combining with 1/2 X MIC of anacardic acid. The time-kill curves demonstrated synergistic bactericidal activities for these combinations.     

Antibody response to Staphylococcus aureus whole cell, lipase and staphylolysin in patients with S. aureus infections

Abstract Three assays to measure antibodies against Staphylococcus aureus whole cells, lipase and staphylolysin were used to try to discriminate between complicated and uncomplicated S. aureus septicaemia. Sera were examined from 8 patients with S. aureus endocarditis, 23 patients with complicated S. aureus septicaemia, 12 patients with uncomplicated S. aureus septicaemia and 93 febrile non-septicaemic controls. No single assay could distinguish between complicated and uncomplicated S. aureus septicaemia. If the criterion for a positive result is defined as positive antibody level in the anti-lipase ELISA as well as in at least 1 of the other 2 assays, 10/31 patients with S. aureus endocarditis or complicated septicaemia were positive compared to 0/93 non-septicaemic patients and 0/12 patients with uncomplicated S. aureus septicaemia. Therefore, the combined use of serological assays in the diagnosis of complicated S. aureus septicaemia, one of which is the anti-lipase ELISA, is recommended.     

Supercritical carbon dioxide in combination with silica gel to fractionate essential oils

Supercritical fluids have high selectivity thanks to the fact that their solvating power can be varied either physically or chemically. Supercritical carbon dioxide (SC CO₂) in combination with silica gel can successfully be applied to fractionate essential oils. By modifying the elution strength of SC CO₂ through its density, several essential oils were fractionated, and it was possible to separate compounds of very similar polarity into different fractions. In particular, the essential oils of Mentha × piperita L., Tagetes × lucida L. and Matricaria × chamomilla L. were processed. Copyright © 1999 John Wiley & Sons, Ltd.     

The corneal response to infection with Staphylococcus aureus in the absence of interleukin-4

Interleukin-4 (IL-4) has previously been implicated in a protective response to Staphylococcus aureus corneal infection. Consequently, the specific role of IL-4 during S. aureus corneal infection was investigated using IL-4 gene knockout mice. The eyes of IL-4-/- mice and wild-type mice were challenged topically with S. aureus and examined at 24 h post-infection. Keratitis was examined clinically and histologically. Bacterial and polymorphonuclear leucocytes (PMN) numbers were enumerated and cytokine and chemokine levels determined by enzyme-linked immunosorbent assay. Exogenous IL-4 was administered to both IL-4-/- and wild-type mice and clinical parameters were determined. A lack of IL-4 resulted in a significant increase in clinical scores, pathology, bacterial load and neutrophil numbers. The absence of IL-4 also resulted in an upregulation of interferon (IFN)-gamma and a downregulation of IL-6, IL-10 and the chemokines KC and macrophage inflammatory protein-2. Administration of exogenous IL-4 to IL-4-/- mice was protective but time-dependent. This study highlights the protective role of IL-4 during S. aureus infection and emphasizes the balance between IL-4 and IFN-gamma in achieving bacterial control and maintaining the integrity of the cornea. This information may lead to the development of novel therapeutic strategies potentially improving the prognosis for infection of this unique avascular site.     

Dynamics of staphylococcal infection in guinea pigs with delayed hypersensitivity to Staphylococcus aureus surface antigens

The relationship between delayed hypersensitivity (DH) to S. aureus surface antigens and the intensity of the infectious process induced by the sublethal infection of guinea pigs with S. aureus was studied. The protective effect, manifested by a decrease in the staphylococcal contamination of the spleen tissue and by an increase in the level of the activation of lymphocytes, was shown to correlate with DH induced by inactivated staphylococcal cells. In infected guinea pigs having DH to different staphylococcal antigens the disease either took a more severe course (in cases of DH to cell wall or peptidoglycan) than in the animals subjected only to infection, or no aggravation of the disease was observed (in cases of DH to protein A).     

Development of an in vitro colonization model to investigate Staphylococcus aureus interactions with airway epithelia

Staphylococcus aureus is a bacterial pathogen responsible for a wide range of diseases and is also a human commensal colonizing the upper respiratory tract. Strains belonging to the clonal complex group CC30 are associated with colonization, although the colonization state itself is not clearly defined. In this work, we developed a co‐culture model with S. aureus colonizing the apical surface of polarized human airway epithelial cells. The S. aureus are grown at the air–liquid interface to allow an in‐depth evaluation of a simulated colonization state. Exposure to wild‐type, S. aureus bacteria or conditioned media killed airway epithelial cells within 1 day, while mutant S. aureus strains lacking alpha‐toxin (hla) persisted on viable cells for at least 2 days. Recent S. aureus CC30 isolates are natural hla mutants, and we observed that these strains displayed reduced toxicity toward airway epithelial cells. Quantitative real‐time polymerase chain reaction of known virulence factors showed the expression profile of S. aureus grown in co‐culture correlates with results from previous human colonization studies. Microarray analysis indicated significant shifts in S. aureus physiology in the co‐culture model toward lipid and amino acid metabolism. The development of the in vitro colonization model will enable further study of specific S. aureus interactions with the host epithelia.