Sensitization of Staphylococcus aureus to Methicillin and Other Antibiotics In Vitro and In Vivo in the Presence of HAMLET

Abstract

HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a protein-lipid complex from human milk with both tumoricidal and bactericidal activities. HAMLET exerts a rather specific bactericidal activity against some respiratory pathogens, with highest activity against Streptococcus pneumoniae, but lacks activity against most other bacterial pathogens, including Staphylococci. Still, ion transport associated with death in S. pneumoniae is also detected to a lower degree in insensitive organisms. In this study we demonstrate that HAMLET acts as an antimicrobial adjuvant that can increase the activity of a broad spectrum of antibiotics (methicillin, vancomycin, gentamicin and erythromycin) against multi-drug resistant Staphylococcus aureus, to a degree where they become sensitive to those same antibiotics, both in antimicrobial assays against planktonic and biofilm bacteria and in an in vivo model of nasopharyngeal colonization. We show that HAMLET exerts these effects specifically by dissipating the proton gradient and inducing a sodium-dependent calcium influx that partially depolarizes the plasma membrane, the same mechanism induced during pneumococcal death. These effects results in an increased cell associated binding and/or uptake of penicillin, gentamicin and vancomycin, especially in resistant stains. Finally, HAMLET inhibits the increased resistance of methicillin seen under antibiotic pressure and the bacteria do not become resistant to the adjuvant, which is a major advantageous feature of the molecule. These results highlight HAMLET as a novel antimicrobial adjuvant with the potential to increase the clinical usefulness of antibiotics against drug resistant strains of S. aureus.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in <em>Streptococcus pneumoniae</em> Using Fluorescent Dyes

“…2+ in the pneumococcus 19,20 , which we have also used in our recent studies 18,21 . However, working with these radioactive ions has several drawbacks.…”

Protein–lipid complexes: molecular structure, current scenarios and mechanisms of cytotoxicity

“…5). 63,[129][130][131][132][133] HAMLET was found to kill certain bacterial strains, such as S. pneumonia, Haemophilus inuenzae, antibiotic-susceptible and -resistant Streptococcus pyogenes (GAS) and Streptococcus agalactiae (GBS), and multidrugresistant Mycobacterium tuberculosis (MD-TB), 128,133,134 and subtoxic doses of HAMLET not only stimulate the bactericidal effect for treatment of pneumococci, 135 but potentiate a remarkably broad array of TB drugs and antibiotics against M. tuberculosis as well as S. pneumoniae, GAS and GBS. The synergistic action of HAMLET on the viability of TB decreased the minimal inhibitory concentrations of rifampin, bedaquiline, delamanid, and clarithromycin by 8-to 16-fold.…”

Norfloxacin salts of carboxylic acids curtail planktonic and biofilm mode of growth in ESKAPE pathogens

“…The degree of cell membrane damage due to treatment with norfloxacin and its carboxylic acid salts was determined as reported earlier (Marks et al 2013). Antibiotictreated cells of both P. aeruginosa and S. aureus were collected at 0, 1, 2, 3 and 4 h pelleted at 13 000 rev min À1 for 5 min.…”