Effect of medium composition on the apparent sensitivity ofPseudomonas aeruginosato gentamicin

Abstract

SYNOPSIS The diameter of inhibition zones in cultures of Ps. aeruginosa produced by discs of gentamicin varied on different media, as did the minimum inhibitory concentration (MIC) when the antibiotic was included in the medium.These variations, which were small in parallel tests of Esch. coli, were found to depend on the general composition of the medium, but were also affected by the agar used to solidify it.An agar on which the MIC was low had a low magnesium content, and two on which it was high had a high content. The addition of magnesium sulphate in increasing amounts to a medium of low content progressively increased the MIC.It thus appears that a high magnesium content, which is necessary for pyocyanin production, also increases the resistance of this organism to gentamicin.The difficulty of determining the sensitivity ofPs. aeruginosa to gentamicin as a guide to treatment is emphasized. The best method appears to be direct comparison with another strain of the same organism of known sensitivity.It is well recognized that the activity of streptomycin and other aminoglycosides in vitro is affected by medium composition, important factors being pH, peptone, and electrolyte content. That gentamicin is also affected is evident in discrepancies among previous findings on the activity of this antibiotic against Ps. aeruginosa. In an early description of gentamicin (Weinstein, Luedemann, Oden, and Wagman, 1963) the minimum inhibitory concentration (MIC) for five out of eight strains is stated as 0 1 toO3 /tg/ml,the remainder having an MIC of 0-6, 12, and 1 2 ,tg/ml. Barber and Waterworth (1966) (Oxoid).Of these diagnostic sensitivity test agar and Columbia agar were also kindly supplied to us by Oxoid Ltd without agar, in order that different agars could be added to them.The agars used were agar no. 1, lonagar no. 2, and agar no. 3 (all Oxoid), in concentrations of 1-0, 1-0, and 1-2 % respectively. In one experiment fibre agar (Southern Group Laboratory) was also used.Some determinations of MIC were also done in 'blood' and 'urine'. To prepare these whole citrated blood was lysed by freezing and thawing and diluted with an equal volume of normal saline, and pooled normal urine was adjusted to pH 6-8 and sterilized by filtration. The blood medium was subcultivated to determine whether there had been growth in it.STRAINS OF BACTERIA Most of these were recent isolates, but those of Ps. aeruginosa included NCTC 10490 and Effect of medium composition on the apparent sensitivity ofPseudomonas aeruginosa to gentamicin 535 those of Esch. coli NCTC 10418. In tests using multiple strains, significant differences in their behaviour were never observed. DISCS These were 10 jig gentamicin discs supplied by Mast Laboratories. They were shown by exhaustive tests to behave consistently.MIC DETERMINATIONS Plates containing falling concentrations of antibiotic were prepared by adding 14-5 ml melted and cooled medium to 05 ml solution and thorough mixing. These were inoculated with a multiple replicator from an approximately 1...

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

“…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.…”

Comparison of Liquid and Agar‐Solidified Defined Media Regarding the Physiological Mechanism by which β‐2‐Thienylalanine Inhibits Growth of Escherichia, Shigella, and Salmonella Cultures

“…Sands and Bennett (33) have demonstrated that some phenolic compounds are less inhibitory to Staphylococcus aureus in unwashed agar, and that inhibition can be restored by washing agar cubes in distilled water before adding the agar to growth media. Similar agar effects have been shown for fatty amines (23), antibiotics (15,24,38), and infectivity of Dengue virus (34). Greenberg (19) has studied removal of agar factors with water, methyl alcohol, ethyl alcohol, acetone, and butanoi.…”

Functions and activities of the Area Committee on Microbiology of the National Committee for Clinical Laboratory Standards

“…This subcommittee has the responsibility for testing and selecting a reference lot of Mueller-Hinton agar (the so-called golden kilo) to be used as a physical reagent standard in evaluating production lots of the dehydrated agar (M6-P) (9). Batch-tobatch and lot-to-lot variabilities in the antipseudomonal activity of aminoglycosides have been reported by a number of investigators (4,26,29) and were found, as the result of an elegant series of experiments by Kenny et al (6), to correlate with the sums of soluble calcium and magnesium and with the amount of ionized calcium in various batches or lots of the medium. Factors such as autoclaving caused shifts among soluble, ionized, and bound pools of cation which in turn affected the aminoglycoside activity (6).…”