No abstract

Myeloperoxidase (MPO), a microbicidal haloperoxidase of neutrophil leukocytes, was observed to selectively bind to bacteria. Binding was quantified by dithionite-reduced minus oxidized (R-O) difference spectral analysis. Escherichia coli and Pseudomonas aeruginosa showed large MPO binding by R-O difference spectral analysis, whereas Streptococcus sanguinis did not. For increased sensitivity, free and microbe-bound MPO and chloroperoxidase (CPO) activities were quantified by acid-optimum haloperoxidase-dependent chemiluminescence (CL) measurements, and these data were used for Scatchard analysis. The MPO bound/free (B/F) CL ratio was 49.5 for P. aeruginosa, 14.6 for Staphylococcus aureus, 2.8 for E. coli, 0.7 for Candida albicans and 0.4 for S. sanguinis. By comparison, the CPO B/F CL ratio was 0.03 for P. aeruginosa, 0.09 for S. aureus, 0.31 for E. coli, 0.18 for C. albicans and 0.16 for S. sanguinis. As a member of the lactic acid family of bacteria and a viridans streptococcus, S. sanguinis does not synthesize cytochromes and is catalase-negative. The metabolic products of S. sanguinis, i.e. lactic acid and hydrogen peroxide, provide optimal acidity and substrate for MPO oxidation of chloride to hypochlorite. Hypochlorite can react with organic substrates to yield dehydrogenated or chlorinated products, but when peroxide is not limiting, hypochlorite reacts with peroxide yielding singlet oxygen. The reactivity of hypochlorite is dependent on substrate availability. The microsecond half-life of electronically excited singlet oxygen restricts reactivity to within a radius of <0.25 mm; i.e. the reactivity of singlet oxygen is both substrate and half-life dependent. Poor MPO binding provides protection and possibly competitive advantage to viridans streptococci.

E-101 showed MIC(90) values of 0.03, 0.5 and 0.5 mg pMPO/L for staphylococci (n = 140), streptococci (n = 95) and enterococci (n = 55), respectively. MIC(90) values ranged between 0.03-0.5 and ≤ 0.004-0.12 mg pMPO/L for Enterobacteriaceae (n = 148) and Gram-negative non-Enterobacteriaceae (n = 92) strains, respectively. There was no antimicrobial tolerance to E-101 for Staphylococcus aureus, Streptococcus agalactiae or Streptococcus pyogenes. Time-kill studies demonstrated a rapid (<30 min) bactericidal effect against S. aureus, Enterococcus faecalis, Escherichia coli and Pseudomonas aeruginosa in a concentration-dependent and time-dependent manner. There was no evidence of stable resistance to E-101 among staphylococci, enterococci, E. coli or P. aeruginosa strains and no evidence of E-101 interaction with antibiotics commonly used in clinical medicine. Conclusions E-101 shows potent and broad-spectrum in vitro activity against bacteria that are the causative pathogens of SSIs, thereby providing the impetus to test its clinical utility in the prevention of SSIs.

Lactic acid bacteria (LAB) are incapable of cytochrome synthesis and lack the heme electron transport mechanisms required for efficient oxygen-based metabolism. Consequently, LAB redox activity is flavoenzyme-based and metabolism is fermentative, producing lactic acid, and in many cases, hydrogen peroxide (H 2 O 2). Despite this seeming metabolic limitation, LAB dominate in the normal flora of the mouth, vagina and lower gastrointestinal tract in man. Myeloperoxidase (MPO) is produced by the neutrophil leukocytes and monocytes that provide the innate phagocyte defense against infecting pathogens. MPO is unique in its ability to catalyze the H 2 O 2-dependent oxidation of chloride (Cl −) to hypochlorite (OCl −). In turn, this OCl − directly reacts with a second H 2 O 2 to produce singlet molecular oxygen (1 2 O ), a metastable electronic excitation state of oxygen with a microsecond lifetime that restricts its combustive reactivity within a submicron radius of its point of generation. Each day a healthy human adult produces about a hundred billion neutrophils containing about 4 femtograms MPO per neutrophil. Inflammatory states and G-CSF treatment increase both neutrophil production and the quantity of MPO per neutrophil. After a short circulating lifetime, neutrophils leave the blood and migrate into body spaces including the mouth, vagina, urinary tract, and gastrointestinal tract. Greater than a hundred thousand neutrophils are lavaged from the mouths of healthy humans; the quantity lavaged is proportional to the blood neutrophil count. MPO selectively and avidly binds to most Gram-positive and all Gram-negative bacteria tested, but LAB do not show significant MPO binding. Neutrophils migrating to normal flora sites release MPO into the LAB-conditioned milieu containing adequate acidity and H 2 O 2 to support extra-phagocyte MPO microbicidal action. In combination, LAB plus MPO exert a potent synergistic microbicidal action against high MPO-binding microbes. This LAB-MPO synergy provides a mechanism for the establishment and maintenance of LAB in the normal flora of man.

E-101 solution is a first-in-class myeloperoxidase-mediated antimicrobial developed for topical application. It is composed of porcine myeloperoxidase (pMPO), glucose oxidase (GO), glucose, sodium chloride, and specific amino acids in an aqueous solution. Once activated, the reactive species hydrogen peroxide (H2O2), hypochlorous acid, and singlet oxygen are generated. We evaluated the treatment effects of E-101 solution and its oxidative products on ultrastructure changes and microbicidal activity against methicillin-resistantStaphylococcus aureus(MRSA) andEscherichia coli. Time-kill and transmission electron microscopy studies were also performed using formulations with pMPO or GO omitted. The glutathione membrane protection assay was used to study the neutralization of reactive oxygen species. The potency of E-101 solution was also measured in the presence of serum and whole blood by MIC and minimal bactericidal concentration (MBC) determinations. E-101 solution demonstrated rapid bactericidal activity and ultracellular changes in MRSA andE. colicells. When pMPO was omitted, high levels of H2O2generated from GO and glucose demonstrated slow microbicidal activity with minimal cellular damage. When GO was omitted from the formulation, no antimicrobial activity or cellular damage was observed. Protection from exposure to E-101 solution reactive oxygen species in the glutathione protection assay was competitive and temporary. E-101 solution maintained its antimicrobial activity in the presence of inhibitory substances, such as serum and whole blood. E-101 solution is a potent myeloperoxidase enzyme system with multiple oxidative mechanisms of action. Our findings suggest that the primary site where E-101 solution exerts microbicidal action is the cell membrane, by inactivation of essential cell membrane components.