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.
Highlights
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the principal multi-drug resistant bacterial pathogens causing serious community and hospital-acquired infections [1,2,3], such as skin and soft tissue infections, bone, joint and implant infections, ventilator-associated pneumonia, and sepsis [4]
Sensitizing activity of HAMLET on antibiotics in vitro A standard checkerboard broth microdilution assay was used to test whether HAMLET interfered with the susceptibility of bacteria to antibiotics that target cell wall synthesis as well antibiotics that target protein synthesis
The purpose of this study was to demonstrate that even though HAMLET showed no antimicrobial activity against any of the S. aureus strains used, HAMLET acted as an effective antimicrobial adjuvant with the ability to increase the efficacy of a broad range of commonly used antibiotics including methicillin, vancomycin, erythromycin, and gentamicin, to the degree that drug-resistant S. aureus could again become sensitive to these antibiotics both in in vitro assays determining minimal concentration that inhibited growth (MIC) and minimal bactericidal concentration (MBC), as well as for eradication of biofilms and nasopharyngeal colonization in vivo
Summary
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the principal multi-drug resistant bacterial pathogens causing serious community and hospital-acquired infections [1,2,3], such as skin and soft tissue infections, bone, joint and implant infections, ventilator-associated pneumonia, and sepsis [4]. It is estimated that multi-drug resistant Staphylococcus aureus infections leads to 19,000 deaths per year in the United States, with an associated 3-4 billion US dollars in annual health care costs [5,6] Despite this high mortality rate, there are relatively few new antibacterial agents in the pharmaceutical pipeline [7]. One attractive strategy would be the reintroduction of current and previously used antibiotics to which MRSA strains are resistant, when used in combination with other sensitizing agents These ‘antimicrobial adjuvants’ may not have significant antibiotic activity alone, would improve the biologic activity of antibiotics when used in combination [9,10,11,12]. Introduction of such antimicrobial adjuvants would provide a new dimension of safe and widely available treatment possibilities
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