In vitro activity of allicin against Staphylococcus epidermidis and influence of subinhibitory concentrations on biofilm formation

AIMS: The aim of this study is to determine the in vitro activity of allicin against Staphylococcus epidermidis and to evaluate the influence of allicin on biofilm formation. METHODS AND RESULTS: In vitro activity of allicin (diallyl thiosulphinate) against 38 strains of S. epidermidis was investigated. The activity of allicin was similar against S. epidermidis methicillin susceptible and methicillin resistant strains [minimum inhibitory concentration (MIC)90=8 mg l(-1)]. In general, subinhibitory concentrations (sub-MIC) of allicin diminished biofilm formation in the five strains analysed. CONCLUSION: The results confirm the antibacterial effect of allicin. Sub-MICs of allicin also diminished the biofilm formations by S. epidermidis. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study shows that allicin is active in vitro against S. epidermidis and that sub-MICs of allicin may play a role in the prevention of adherence of this bacteria to medical devices.     

Detection and recovery of sublethally-injured enterotoxigenic Staphylococcus aureus

AIMS: To determine whether sublethally-injured (acid- or heat-shocked) Staphylococcus aureus cells are recoverable using selective agar overlays. METHODS AND RESULTS: Brain Heart Infusion (BHI) Agar overlaid with either Baird-Parker Agar (BPA) or Gram-Positive Agar (GPA) was compared in the ability to resuscitate heat- and acid-shocked enterotoxigenic Staph. aureus. BHI/BPA overlays allowed for greater recovery of both heat- and acid-shocked cells than BHI/GPA, although the former was not selective and allowed growth of bacteria other than Staph. aureus. No significant difference existed in percent recovery of heat- and acid-shocked cells between the two overlay approaches. Significant differences were noted in counts on BHI/GPA plates and straight selective GPA/GPA plates, however. Viability of heat- and acid-shocked Staph. aureus was also examined using fluorescence microscopy, the relative counts of which correlated well to the calculated percent recovery on selective agar overlays. CONCLUSIONS: This work has shown that an improved agar overlay technique increases the sensitivity of the standard plate count while enumerating sublethally-injured enterotoxigenic Staph. aureus compared with direct plating onto selective media. SIGNIFICANCE AND IMPACT OF THE STUDY: These data emphasize the need to develop practical and cost-effective methods that reliably detect and enumerate sublethally-injured pathogens such as Staph. aureus.     

Discrimination of Staphylococcus aureus biotypes by pulsed-field gel electrophoresis of DNA macro-restriction fragments

AIMS: To examine whether pulsed-field gel electrophoresis (PFGE) of DNA macro-restriction fragments could provide better discrimination among the different biotypes previously described within the species Staphylococcus aureus than the traditional biochemical approach. METHODS AND RESULTS: Seventy three Staph. aureus strains from various sources (human, animal or food origin) and belonging to eight biotypes, including the poultry-like biotype, tentatively designated as an 'abattoir' biotype, were genotyped by PFGE after SmaI digestion of DNA. The PFGE patterns were compared using the average linkage matching method (UPGMA) with the Dice coefficient. A total of 61 PFGE patterns were observed, showing between 31 and 100% similarity. In most cases, strains with the same biotype were grouped specifically into one, two or three separate sub-clusters. Strains from the 'abattoir' biotype were clustered in one separate sub-cluster. CONCLUSIONS: The PFGE typing is useful to distinguish the traditional biotypes of Staph. aureus and has a more discriminatory power than the biochemical typing. SIGNIFICANCE AND IMPACT OF THE STUDY: The PFGE typing confirms the 'abattoir' biotype as a separate group on a genetic level and is well suited to investigate modes of staphylococcal contamination of food.     

Purification and characterization of nitric oxide synthase from Staphylococcus aureus

We previously reported the presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P whose activity was induced by methanol. In the present study, the methanol-induced NOS was purified 900-fold from S. aureus by means of Mono Q ion exchange column, 2',5'-ADP-agarose affinity column, and Superdex 200HR gel permeation column chromatography. The purified bacterial NOS showed two protein bands with 67 and 64 kDa molecular mass on SDS-PAGE. However, the molecular mass of the NOS was 135 kDa on Superdex 200HR gel permeation column chromatography, indicating that the native enzyme exists as a heterodimer. This bacterial NOS had K(m) value of 13.4x10(-6) M for L-arginine and V(max) of 35.3 nmol min(-1) mg(-1) protein. In addition, reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, calmodulin and Ca(2+) were required as cofactors in the conversion of L-arginine to L-citrulline, and NOS inhibitors selectively inhibited the activity of the purified NOS.     

Understanding the mechanism of action of the exfoliative toxins of Staphylococcus aureus

The exfoliative toxins of Staphylococcus aureus are responsible for the staphylococcal scalded skin syndrome, a blistering skin disorder that particularly affects infants and young children, as well as adults with underlying disease. Their three-dimensional structure is similar to other glutamate-specific trypsin-like serine proteases with two substrate-binding domains and a serine-histidine-aspartate catalytic triad that forms the active site. However, unlike other serine proteases, the exfoliative toxins possess a highly charged N-terminal alpha-helix and a unique orientation of a critical peptide bond, which blocks the active site of the toxins so that, in their native state, they do not possess any significant enzymatic activity. The target for the toxins has recently been identified as desmoglein-1, a desmosomal glycoprotein which plays an important role in maintaining cell-to-cell adhesion in the superficial epidermis. It is speculated that binding of the N-terminal alpha-helix to desmoglein-1 results in a conformation change that opens the active site of the toxin to cleave the extracellular domain of desmoglein-1 between the third and fourth domains, resulting in disruption of intercellular adhesion and formation of superficial blisters. Elucidating the mechanism of action of the toxins and identifying desmoglein-1 as their specific epidermal substrate has not only given us an insight into the pathogenesis of the staphylococcal scalded skin syndrome, but also provided us with useful information on normal skin physiology and the pathogenesis of other toxin-mediated diseases. It is hoped that this knowledge will lead to development of rapid screening and diagnostic tests, and new antitoxin strategies for the treatment and prevention of the staphylococcal scalded skin syndrome in the near future.     

Relationship between haemolytic and sphingomyelinase activities in a partially purified beta-like toxin from Staphylococcus schleiferi

beta-Toxins of staphylococcal species possess dual activity in that they can both lyse erythrocytes (by 'hot-cold' lysis) and catalyse hydrolysis of membrane-associated sphingomyelin. However, the precise relationship between these two activities has not been extensively studied. We have partially purified a beta-like toxin from culture supernatants of Staphylococcus schleiferi N860375 which exhibits both 'hot-cold' lysis of erythrocytes and neutral sphingomyelinase activities. This toxin has a strong preference for sheep erythrocytes, the membranes of which are rich in sphingomyelin. Kinetic analysis suggests that haemolysis and sphingomyelinase activities are very closely associated obeying identical Michaelis-Menten kinetics. However, pre-treatment with antibodies to Staphylococcus aureus beta-toxin, Ca(2+), dithiothreitol and phenylmethylsulfonyl fluoride appear to inhibit sphingomyelinase activity significantly more strongly than haemolysis while Mg(2+) activates sphingomyelinase activity more strongly than haemolysis. We attribute these effects to differences in binding properties in the two assays. Micropurification by both sphingosylphosphocholine-agarose affinity chromatography and preparative electrophoresis revealed that the 34-kDa toxin associates non-covalently with individual proteins.     

Staphylococcus aureus alpha-toxin induces apoptosis in endothelial cells

The internalization of Staphylococcus aureus by cultured human umbilical vein endothelial cells was recently shown to induce apoptosis. We examined the role of alpha-toxin, a major pore-forming toxin secreted by S. aureus, in causing apoptosis in vitro. Purified alpha-toxin, at sublytic concentrations, induced apoptosis in endothelial cell monolayers. Comparisons of two alpha-toxin (hla)-positive S. aureus strains and their isogenic hla-deficient mutants in the invasion assay of endothelial cells demonstrated that the capacity to produce alpha-toxin was associated with a greater propensity for apoptosis in endothelial cells. These results demonstrate for the first time that expression of alpha-toxin during endothelial cell invasion by S. aureus enhances apoptosis.     

Identification of a fmtA-like gene that has similarity to other PBPs and beta-lactamases in Staphylococcus aureus

We identified a gene from Staphylococcus aureus, flp (fmtA-like protein), encoding a protein of 489 amino acid residues with a molecular mass of 56.4 kDa. The deduced amino acid sequence shows similarity to previously characterized penicillin binding proteins (PBPs) and FmtA of S. aureus (one of the factors which affect methicillin resistance). FLP protein has three motifs, which are conserved in PBPs and beta-lactamases, suggesting that it might be associated with cell wall synthesis. Recombinant FLP protein, however, lacks penicillin binding activity, and the inactivation of flp in two methicillin-resistant S. aureus strains did not cause a reduction in the methicillin resistance.     

Room temperature sterilization of surfaces and fabrics with a One Atmosphere Uniform Glow Discharge Plasma

We report the results of an interdisciplinary collaboration formed to assess the sterilizing capabilities of the One Atmosphere Uniform Glow Discharge Plasma (OAUGDP). This newly-invented source of glow discharge plasma (the fourth state of matter) is capable of operating at atmospheric pressure in air and other gases, and of providing antimicrobial active species to surfaces and workpieces at room temperature as judged by viable plate counts. OAUGDP exposures have reduced log numbers of bacteria, Staphylococcus aureus and Escherichia coli, and endospores from Bacillus stearothermophilus and Bacillus subtilis on seeded solid surfaces, fabrics, filter paper, and powdered culture media at room temperature. Initial experimental data showed a two-log₁₀ CFU reduction of bacteria when 2 × 10² cells were seeded on filter paper. Results showed ≥3 log₁₀ CFU reduction when polypropylene samples seeded with E. coli (5 × 10⁴) were exposed, while a 30 s exposure time was required for similar killing with S. aureus-seeded polypropylene samples. The exposure times required to effect ≥6 log₁₀ CFU reduction of E. coli and S. aureus on polypropylene samples were no longer than 30 s. Experiments with seeded samples in sealed commercial sterilization bags showed little or no differences in exposure times compared to unwrapped samples. Plasma exposure times of less than 5 min generated ≥5 log₁₀ CFU reduction of commercially prepared Bacillus subtilis spores (1 × 10⁶); 7 min OAUGDP exposures were required to generate a ≥3 log₁₀ CFU reduction for Bacillus stearothermophilus spores. For all microorganisms tested, a biphasic curve was generated when the number of survivors vs time was plotted in dose-response cures. Several proposed mechanisms of killing at room temperature by the OAUGDP are discussed. Received 06 June 1997/ Accepted in revised form 01 November 1997     

Improved high-performance liquid chromatographic separation of peptidoglycan isolated from various Staphylococcus aureus strains for mass spectrometric characterization

Reversed-phase high-performance liquid chromatography (RP-HPLC) of muropeptides, obtained by muramidase digestion of peptidoglycan in combination with amino acid analysis and plasma desorption time-of-flight mass spectrometry is today by far the best tool to analyze the fine structure of the peptidoglycans. Here we report further improvements of the RP-HPLC separation of muropeptides for analyzing the peptidoglycans of various methicillin-resistant strains of Staphylococcus aureus, with emphasis on a more detailed characterization of the interpeptide bridge of the peptidoglycans of this species.