Effect of Calcium and Magnesium Ions on the Susceptibility of Pseudomonas Species to Tetracycline, Gentamicin Polymyxin B, and Carbenicillin

Abstract

The effect of calcium and magnesium on the susceptibility of 13 species of Pseudomonas to tetracycline, gentamicin, polymyxin B, and carbenicillin was measured. The majority of the minimum inhibitory concentrations (MICs) of these antibiotics was increased if these cations were added to the test media. The increases in MICs caused by calcium or magnesium were similar, but the combination of both -ions generally caused a greater change than either alone. Although the MIC of polymyxin B was most affected by calcium and magnesium, its interpretive susceptibilities (i.e., whether susceptible or resistant) were least changed. Susceptibility tests on Pseudomonas species probably should be done with Muller-Hinton broth supplemented with physiological concentrations of calcium and magnesium to better approximate the in vivo activity of these antibiotics. When the susceptibility tests were performed with Mueller-Hinton agar, the MICs were slightly less than those obtained with Mueller-Hinton broth supplemented with both cations but greater than those obtained with MuellerHinton broth supplemented with individual cations.

Cation Components of Mueller-Hinton Agar Affecting Testing of Pseudomonas aeruginosa Susceptibility to Gentamicin

“…Calculations assuming that 100% of the medium volume was occupied by fluid overestimate total cation content. The fluid volume of each batch was determined by mixing water and Mueller-Hinton medium powders in a graduated cylinder, allowing 1 over the top of the tube, and the adapted screwcap was replaced. In the resulting chamber, the insoluble medium fractions were washed with at least 500 volumes of deionized water to measure the freely soluble cation then washed with 0.2 M ethylenediaminetetraacetate (disodium salt) to measure agar-bound cation.…”

“…The susceptibility of Pseudomonas aeruginosa to aminoglycoside antibiotics is affected by the ionic strength of the medium, especially and uniquely by the cations magnesium and calcium (1,(3)(4)(5)(6)12) and perhaps zinc (13). As the cation content of the medium is increased, this species becomes more resistant to these drugs.…”

“…As the cation content of the medium is increased, this species becomes more resistant to these drugs. This phenomenon has been described with antibiotic dilution tests in broth and agar (1,4,6,(11)(12)(13) and with the disk diffusion test (3,5,7,11,12). Garrod and Waterworth and other investigators (3,4) suggested adjustment of calcium and magnesium in Mueller-Hinton media to physiological levels to improve reliability of these media for testing aminoglycosides with pseudomonads.…”

High Concentrations of Manganese in Mueller-Hinton Agar Increase MICs of Tigecycline Determined by Etest

“…When the activity of tigecycline was determined in agar medium from Difco supplemented with either manganese chloride (Sigma, Madrid, Spain) or sulfate chloride (Sigma) to concentrations of 630 g/ml of free Mn 2ϩ (similar to those observed in the medium from Merck), MICs increased against all tested organisms except P. aeruginosa ATCC 27853 (Table 3). It has been reported that calcium and magnesium interfere with the activity of tetracycline, aminoglycosides, and colistin against P. aeruginosa and other gram-negative bacteria (7,18). Magnesium also decreases the in vitro activity of fluoroquinolones by chelating these agents (1), and zinc interferes with the action of carbapenems against P. aeruginosa by decreasing the expression of the OprD porin (6).…”

Influence of Media and Testing Methodology on Susceptibility to Tigecycline of Enterobacteriaceae with Reported High Tigecycline MIC

“…The content in other ions could have some influence. Media from Merck and Difco have lower Mg content than Oxoid media (around 195 versus 409 ppm) (9), and it has been reported that magnesium and calcium interfere with the activity of tetracyclines, aminoglycosides, and colistin against Pseudomonas aeruginosa and other Gram-negative bacteria (8,15).…”