In vitro activity of gallium maltolate against Staphylococci in logarithmic, stationary, and biofilm growth phases: comparison of conventional and calorimetric susceptibility testing methods

Abstract

Ga(3+) is a semimetal element that competes for the iron-binding sites of transporters and enzymes. We investigated the activity of gallium maltolate (GaM), an organic gallium salt with high solubility, against laboratory and clinical strains of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), methicillin-susceptible Staphylococcus epidermidis (MSSE), and methicillin-resistant S. epidermidis (MRSE) in logarithmic or stationary phase and in biofilms. The MICs of GaM were higher for S. aureus (375 to 2000 microg/ml) than S. epidermidis (94 to 200 microg/ml). Minimal biofilm inhibitory concentrations were 3,000 to >or=6,000 microg/ml (S. aureus) and 94 to 3,000 microg/ml (S. epidermidis). In time-kill studies, GaM exhibited a slow and dose-dependent killing, with maximal action at 24 h against S. aureus of 1.9 log(10) CFU/ml (MSSA) and 3.3 log(10) CFU/ml (MRSA) at 3x MIC and 2.9 log(10) CFU/ml (MSSE) and 4.0 log(10) CFU/ml (MRSE) against S. epidermidis at 10x MIC. In calorimetric studies, growth-related heat production was inhibited by GaM at subinhibitory concentrations; and the minimal heat inhibition concentrations were 188 to 4,500 microg/ml (MSSA), 94 to 1,500 microg/ml (MRSA), and 94 to 375 microg/ml (MSSE and MRSE), which correlated well with the MICs. Thus, calorimetry was a fast, accurate, and simple method useful for investigation of antimicrobial activity at subinhibitory concentrations. In conclusion, GaM exhibited activity against staphylococci in different growth phases, including in stationary phase and biofilms, but high concentrations were required. These data support the potential topical use of GaM, including its use for the treatment of wound infections, MRSA decolonization, and coating of implants.

FIG. 1.

Time-kill curves of the activity of GaM in RPMI against MSSA (A), MRSA (B), MSSE (C), and MRSE (D). Values are the mean numbers of CFU/ml ± SDs of three measurements. Continuous lines represent kill curves (at inhibitory concentrations) and dashed lines represent growth curves (at subinhibitory concentrations) over 24 h (S. aureus) or 48 h (S. epidermidis) of incubation. GaM concentrations are reported as multiples of the MIC for the corresponding test strain (MICs are listed in Table 1). GC, growth controls tested in RPMI without GaM; horizontal dotted line, 3-log₁₀-CFU/ml reduction. The scales on the x and y axes are adapted for S. aureus and S. epidermidis.

FIG. 2.

Calorimetry curves representing the total heat generated by MSSA (A), MRSA (B), MSSE (C), and MRSE (D) at 37°C for 24 h in the presence of twofold dilutions of GaM in RPMI. Dashed lines, growth controls without GaM; continuous lines, GaM concentrations below the MIC; dashed-dot lines, GaM concentrations above the MIC for the corresponding test strain. The scales on the y axes are adapted for S. aureus and S. epidermidis. GC, growth control; curve 1, 23 μg/ml GaM; curve 2, 46 μg/ml GaM; curve 3, 94 μg/ml GaM; curve 4, 188 μg/ml GaM; curve 5, 375 μg/ml GaM; curve 6, 750 μg/ml GaM; curve 7, 1,500 μg/ml GaM; curve 8, 3,000 μg/ml GaM; curve 9, 6,000 μg/ml GaM.