Analysis of a single reference strain for determination of gentamicin regression line constants and inhibition zone diameter breakpoints in quality control of disk diffusion antibiotic susceptibility testing

Abstract

An equation was derived from known formulas to express the size of the inhibition zone diameter in the disk diffusion antibiotic susceptibility test as a function of the disk content of antibiotic. The equation permitted a calculation of regression line constants for the correlation between zone diameter and the minimum inhibitory concentration (MIC) with a single reference strain. Analysis of reference strains Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853, as well as 12 clinical isolates belonging to these species, showed a linearity between zone size squared and the logarithm of disk content in tests with 10-, 30-, and 100-micrograms gentamicin disks. All three species, however, gave regression line constants which were characteristic for the individual bacterial species. Calculations of zone diameter breakpoints corresponding to recommended MIC limits with E. coli and P. aeruginosa reference strains gave an accurate prediction of gentamicin susceptibility. Histogram analysis of 48 strains of Streptococcus faecalis from clinical specimens showed a distribution of zone diameter values which would result in false classification of susceptibility with breakpoints calculated for the other bacterial species studied. Single reference strain analysis of S. faecalis ATCC 29212 (gentamicin MIC, 8 micrograms/ml) permitted the calculation of breakpoints which accurately assigned the strains tested to the intermediate category of susceptibility. Single reference strain analysis offers a quality control method for individual laboratories that allows the determination of inhibition zone diameter breakpoints corresponding to recommended MIC limits with no MIC determinations required.