Figure 1.
HAMLET lowers the methicillin MIC.
S. aureus strains 11090306 (MSSA) (left) and NRS 384 (MRSA) (right) were grown in broth for 16 hours in the presence of 2 µg/ml methicillin (5 µM) with and without the addition of 100 µg/mL (6 µM) HAMLET. The figure shows representative growth curves for the lowest concentration of antibiotic and HAMLET that inhibited bacterial growth by combination treatment without either agent alone affecting growth.
Table 1.
MIC and MBC values for S. aureus strains exposed to various antibiotics in the absence and presence of HAMLET.
Figure 2.
The effect of HAMLET/antibiotic combination treatment on in vitro biofilm viability.
(A) The activity of methicillin (250 μg/mL or 660 µM), HAMLET (200 μg/mL or 12 µM), or the combination of both agents were tested on in vitro biofilms of the methicillin-resistant strain NRS 70 (MRSA) or the methicillin-sensitive strain 11090306 (MSSA) by determining the bacterial death (in log10) after culturing dilutions overnight on blood agar. (B) The activity of vancomycin (32 μg/mL or 21 µM), HAMLET (500 μg/mL or 30 µM), or the combination of both agents on in vitro biofilms of the vancomycin-resistant strain NRS 1 (VISA) and the vancomycin-sensitive strain NRS384 (VSSA) were tested in a similar fashion as was the activity of (C) 50 µg/ml (105 µM) gentamicin alone or in combination with 500 µg/ml (30 µM) HAMLET for the gentamicin-resistant strain. The results are based on three independent experiments with duplicate samples. Statistics was performed using the paired Student t-test. Significance was indicated as follows: ns = not significant, * = P<0.05, ** = P<0.01.
Figure 3.
HAMLET/Methicillin combination treatment reduces Staphylococcal nasopharyngeal colonization.
Mice were colonized with S. aureus NRS 70 for 24 hours, treated intranasally with various doses of gentamicin in the presence (blue) or absence (black) of HAMLET (100 µg) for 12 hours, and the bacterial burden associated with the nasal wash (A) and the nasopharyngeal tissue (B) was determined. The graph shows colonization data for individual mice, with the mean recovered bacteria depicted with a line. The results are based on experiments using groups of 6 mice. Statistics was performed using the unpaired Student t-test. Significance was indicated as follows: * = P<0.05.
Figure 4.
Effect of HAMLET on membrane potential.
(A) Representative growth curves for S. aureus strain NRS 123 (MRSA) grown in broth for 16 hours (960 min) in the presence of methicillin with and without the addition of HAMLET and the inhibitors Ruthenium Red (RuR) or Amiloride (Amil). (B) Mid-log phase grown NRS 123 Staphylococci re-suspended in PBS alone or PBS plus Amiloride or Ruthenium Red, were incubated with the fluorescent indicator dye DiBAC4(3) and membrane depolarization was detected by measuring fluorescence over time. HAMLET was added at twenty minutes (arrow). The detergent Triton X-100 (0.1%) was included as a positive control. The results presented are from one representative experiment. (C) Mid-log phase grown NRS 384 Staphylococci were incubated with the radioisotope 45Ca2+ (2.5 µCi/mL) in PBS or PBS + Ruthenium Red (30 µM). After recording baseline readings, PBS (untreated), or HAMLET was added (Time = 0 min) to the bacteria and radioactivity was measured over time. Results from a representative experiment are shown. (D) NRS 384 Staphylococci were loaded with the pH sensitive dye BCECF-AM, and were washed and resuspended in PBS + 25 mM glucose. After recording baseline readings, at the first arrow, PBS (untreated), the protonophore CCCP (100 µM), HAMLET (100 µg/mL or 6 µM), HAMLET + RuR (30 µM), or HAMLET + Amiloride (1 mM) were added to the bacteria and fluorescence was measured over time. At the second arrow 20 µM each of nigericin and valinomycin was added to completely dissipate the transmembrane proton gradient.
Figure 5.
Impact of HAMLET on uptake and binding of Bocillin FL and vancomycin FL.
(A) Staphylococci were incubated with Bocillin FL or (B) with vancomycin FL in the presence or absence of 100 µg/mL (6 µM) HAMLET. The results are based on three individual experiments with duplicate samples. Statistics was performed using the unpaired Student's t-test. Significance was indicated as follows: ** = P<0.01, *** = P<0.001, ns = not significant.
Figure 6.
HAMLET and resistance development.
Methicillin adaptation of the MRSA strain NRS 384 after exposure to stepwise increasing concentrations of methicillin alone (1–512 µg/mL or 2.5–1,350 µM) or methicillin in combination with 100 µg/mL (6 µM) HAMLET. The filled blue circles show the methicillin MICs after each cycle when no HAMLET was used. The addition of 100 µg/mL of HAMLET reduced methicillin-induced resistance (blue unfilled circles). The unfilled green circle represents the MIC of methicillin of the bacteria grown in presence of HAMLET, when HAMLET was also present during the MIC assay to potentiate the effect of the antibiotic. Reintroduction of HAMLET to these isolates again returned the methicillin MIC back to the levels denoted by the green unfilled circle.