Preparation of Metabolically Active Staphylococcus aureus Protoplasts by Use of the Aeromonas hydrophila Lytic Enzyme

Stable, metabolically active protoplasts of Staphylococcus aureus have been prepared by the use of a staphylolytic enzyme produced by Aeromonas hydrophila. Respiratory and glycolytic rates and synthesis of nucleic acids, protein, and lipid in these protoplasts, stabilized variously in 1.1 M sucrose, 0.37 M sodium succinate, or 0.37 M sodium sulfate, have been shown to be comparable with the same parameters in intact cells under the same conditions.     

Lysis of Staphylococcus aureus Cell Walls by a Soluble Staphylococcal Enzyme

Enzyme preparations of Staphylococcus aureus were examined for their ability to solubilize (32)P-labeled cell walls of the parent organism. Enzymatic activity was observed in the growth medium, in soluble fractions, and associated with native cell walls. Enzyme associated with isolated cell walls could be inactivated with formaldehyde without reducing the susceptibility of the walls to the action of added enzyme. When cells are frozen and thawed, 50 to 75% of the intracellular enzyme is released along with 2% of the intracellular protein. This freeze-thaw extracted enzyme has little, if any, activity on intact S. aureus cells. It appears that the enzyme resides near the cell wall and acts on the cell-wall inner surface.     

Staphylococcal polysaccharide products inhibitory to Staphylococcus aureus bacteriophage.

Four products derived from Staphylococcus aureus cultures were partially purified and tested for inhibitory activity against staphylococcal phage. Phage inhibition, a specific stable phenomenon, was concentration dependent. All inhibitory products contained carbohydrate and amino acids, the most active (phage 73 lysate product) having a high carbohydrate content. Galactose, glucosamine, five or six amino acids, and possibly 3-O-methylglucose and a uronic acid were found as components in all active preparations. However, the exact nature of the active material remains undetermined.     

Subcellular Location of the Soluble Lytic Transglycosylase Homologue in Staphylococcus aureus

The immunodominant antigen A, IsaA, of Staphylococcus aureus was found to include a putative soluble lytic transglycosylase domain in its C-terminal region. Since the presence of this distinctive domain suggested that the protein might participate in peptidoglycan turnover, as indicated in Gram-negative bacteria, its cellular location was investigated. The protein was found not only in the culture supernatant but also in the cell wall fraction. To estimate its physiological role for the bacterium, its cell surface distribution was studied by immunoelectron microscopy. Protein A-gold particles binding to the immune complex were mainly located on the septal region of the bacterial cell surface. These data suggested that IsaA might be involved in bacterial cell separation through a preferential interaction with peptidoglycan chain.     

Electron Microscopic Studies on the Outer Layers of Staphylococcus aureus Using a Lytic Enzyme from Flavobacterium

The structure of the outer layers (cell wall and membrane) of Staphylococcus aureus was studied by electron microscope using a bacteriolytic enzyme from Flavobacterium sp. called the L-11 enzyme. Comparative studies on the morphology of bacteria before and after treatment with this enzyme and cell wall and membrane fractions obtained from bacteria after the enzyme treatment led to the following conclusions. (1) The cell wall of S. aureus is composed of morphologically distinct two layers which are both susceptible to the L-11 enzyme. (2) Between the cell wall and membrane, there is an electron opaque region which could not be stained using any of the methods tested. (3) Before treatment of bacteria with the enzyme the cell membrane could not be seen clearly. However, after enzyme treatment the membrane was clearly seen. (4) The infolding of the inner layer of the cell wall, forming a structure like a mesosome, was liberated by extensive enzyme treatment.     

Electron Microscopy of the Lysis of Staphylococcus aureus Cell Walls by Aeromonas Lytic Factor

Isolated and purified cell walls of Staphylococcus aureus were treated with a purified fraction of the culture supernatant fluid of a species of Aeromonas. The course of lysis of the cell walls was followed over a period of time by examination of samples under an electron microscope. The undifferentiated cell wall was rapidly digested, but the equatorial rings were more resistant. The undifferentiated cell wall became a very thin sheet before completely dissolving, leaving a series of equatorial rings of various widths. As digestion proceeded, solubilization of the entire cell wall occurred. Analogous findings were obtained with purified S. aureus mucopeptide. It is concluded that the Aeromonas lytic principle is an enzyme, and that susceptible bonds are more concentrated in the undifferentiated cell wall mucopeptide.     

Simple Procedure for Production by Group C Streptococci of Phage-Associated Lysin Active Against Group A Streptococci

Phage-associated lysin of high potency was prepared by growing the host group C streptococcal strain 26RP66 in a semisynthetic medium. The lysin was stabilized by adding dithiothreitol and neutralized ethylenediaminetetraacetic acid (EDTA) to facilitate further concentration and partial purification. The lysin remained active when stored at -65 C for 1 year. Lysin was active against all strains of group A streptococci tested and was more active against living cells than heat-killed cells. The procedure outlined is practicable for most bacteriological research laboratories and does not require column purification or other complex biochemical procedures. It should be useful to any laboratory which requires small amounts of lysin to produce L-forms and protoplasts or to release streptococcal antigens.     

Novel Light-Activated Antimicrobial Coatings Are Effective Against Surface-Deposited Staphylococcus aureus

Aerosols constitute a major route of transmission for a wide range of infectious diseases in the hospital setting. The aim of this study was to determine the survival of Staphylococcus aureus on a light-activated antimicrobial coating. S. aureus suspended in phosphate-buffered saline (PBS), saliva, or horse serum was sprayed onto cellulose acetate coatings containing toluidine blue O and rose bengal and the survival of the organism on these surfaces was determined following 6 h of exposure to a 28-W domestic fluorescent lamp (light intensity = 3700 +/- 20 lux). Kills ranging from 78.9% (in horse serum) to 99.8% (in PBS) were obtained when the bacterial density on the coatings was approximately 10(5) colony-forming units/m(2). The results of this study have shown that a coating containing toluidine blue and rose bengal can achieve significant kills of S. aureus when illuminated by a domestic light source. Light-activated coatings could provide a simple, low-cost means of reducing the microbial load in hospitals and other facilities.     

Staphylococcal lipase affects phagocytosis of Staphylococcus aureus by human granulocytes and monocytes.

The rate of immunological and non-immunological phagocytosis of staphylococci by lipase pre-treated human granulocytes and monocytes was compared. It was found that the effect of this enzyme on two types of cells is opposite. Lipase decreases phagocytosis by granulocytes and increases by monocytes. The revealed differences between phagocytosing cells studied prompted us to investigate the influence of lipase on Fc receptors on these cells (rosette EA test). The different susceptibility of Fc receptors on non-activated phagocytes to lipase was found. This could be at least partially responsible for the difference observed between phagocytic activity of granulocytes (decreased) and monocytes (increased) pretreated with staphylococcal lipase. Inactivated enzyme showed a similar effect as active enzyme in the case of granulocytes. However, inactivated enzyme had no effect on rosette formation by lipase pretreated monocytes, indicating an enzymatic effect.     

A New Isolation Method of Plasmid Deoxyribonucleic Acid from Staphylococcus aureus Using a Lytic Enzyme of Achromobacter lyticus

An enzymatic procedure for the differential determination of polyamines, spermine and spermidine, has been established using beef plasma amine oxidase. This method was specific for these polyamines and required only one reaction system. Small amounts of polyamines (10µm to 80 µm of spermine and 10 µm to 100 µm of spermidine) were assayed by solving two simultaneous equations obtained from the rate assay method and the end point assay method. The calculated values were in good agreement with those obtained by other method.     

Active Immunization with Extracellular Vesicles Derived from Staphylococcus aureus Effectively Protects against Staphylococcal Lung Infections,Mainly via Th1 Cell-Mediated Immunity

Staphylococcus aureus is an important pathogenic bacterium that causes various infectious diseases. Extracellular vesicles (EVs) released from S. aureus contain bacterial proteins, nucleic acids, and lipids. These EVs can induce immune responses leading to similar symptoms as during staphylococcal infection condition and have the potential as vaccination agent. Here, we show that active immunization (vaccination) with S. aureus-derived EVs induce adaptive immunity of antibody and T cell responses. In addition, these EVs have the vaccine adjuvant ability to induce protective immunity such as the up-regulation of co-stimulatory molecules and the expression of T cell polarizing cytokines in antigen-presenting cells. Moreover, vaccination with S. aureus EVs conferred protection against lethality induced by airway challenge with lethal dose of S. aureus and also pneumonia induced by the administration of sub-lethal dose of S. aureus. These protective effects were also found in mice that were adoptively transferred with splenic T cells isolated from S. aureus EV-immunized mice, but not in serum transferred mice. Furthermore, this protective effect of S. aureus EVs was significantly reduced by the absence of interferon-gamma, but not by the absence of interleukin-17. Together, the study herein suggests that S. aureus EVs are a novel vaccine candidate against S. aureus infections, mainly via Th1 cellular response.     

In-Silico Repurposing of Anticancer Drug (5-FU) as an Antimicrobial Agent Against Methicillin-Resistant Staphylococcus aureus (MRSA)

International Journal of Peptide Research and Therapeutics - The Centers for Disease Control and Prevention (CDC) reported earlier that more than 11,000 people died from a methicillin-resistant...     

Purified Recombinant Phage Lysin LySMP: An Extensive Spectrum of Lytic Activity for Swine Streptococci

Bacteriophage lysin has attracted considerable attentions as possible antimicrobial agents for solution of antibiotic resistance. SMP was a Streptococcus suis serotype 2 bacteriophage isolated from nasal swabs of healthy Bama minipigs. The putative SMP bacteriophage lysin, designated LySMP, was recombinantly expressed in Escherichia coli BL21, and chromatographically purified. Treated with 0.8% of β-mercaptoethanol, LySMP exhibited an extensive lysin spectrum than those of whole phage against bacteria investigated. S. suis serotype 2, S. suis serotype 7 and S. suis serotype 9 strains were recovered from diseased pigs between 1998 and 2005 in China. Fifteen of seventeen strains of S. suis serotype 2 could be lysed, as well as S. suis serotype 7 and 9, Streptococcus equi ssp. zooepidemicus and Staphylococcus aureus. But E. coli and Salmonella enterica were not affected. Purified LySMP showed high degrading efficiency against PMSF or lysozyme treated cells comparing to PBS washed cells. Optimum pH and temperature conditions for the lysin were investigated by turbidity reduction assay. The lysin exerted efficient lysis activity at 37°C, pH 5.2. The turbidity of bacterium investigated was observed to decrease by 1.2–68% in 30 min. Result indicated that putative LySMP could be a candidate antimicrobial agent in controlling S. suis infection.     

Monoclonal Antibody Targeting Staphylococcus aureus Surface Protein A (SasA) Protect Against Staphylococcus aureus Sepsis and Peritonitis in Mice

Epidemic methicillin-resistant Staphylococcus aureus (MRSA) imposes an increasing impact on public health. Due to multi-antibiotics resistance in MRSA strains, there is an urgent need to develop novel therapeutics such as effective monoclonal antibodies (mAbs) against MRSA infections. Staphylococcus aureus surface protein A (SasA), a large surface-located protein (~240 kDa), is one of MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) and a potential target for immunotherapeutic approaches against S. aureus infections. In the present study, we analyzed the sequence of SasA with bioinformatics tools and generated a protective monoclonal antibody (2H7) targeting the conserved domain of SasA. 2H7 was shown to recognize wild-type S. aureus and promote opsonophagocytic killing of S. aureus. In both sepsis and peritoneal infection models, prophylactic administration of 2H7 improved the survival of BALB/c mice challenged by S. aureus strain USA300 and ST239 (prevalent MRSA clones in North America and Asian countries, respectively) and enhanced bacterial clearance in kidneys. Additionally, 2H7 prophylaxis prevented the formation of intraperitoneal abscess in a murine model of peritoneal infection and therapeutic administration of 2H7 showed protective efficacy in a murine sepsis model. Our results presented here provide supporting evidences that an anti-SasA mAb might be a potential component in an antibody-based immunotherapeutic treatment of MRSA infections.