Simple screening method for gram-positive bacterial beta-lactam antibiotic tolerance on routine laboratory...

A simple screening method served to detect beta-lactam antibiotic-tolerant variants of clinical isolates and laboratory control strains of staphylococcus aureus, S. epidermis, group B beta-hemolytic streptococci, and Listeria monocytogenes. The beta-lactamase(s) of a multiple drug-resistant strain of Enterobacter cloacae (isolate No. 19) yielded most consistent results as compared with several other beta-lactamase producers; the E. cloacae beta-lactamase(s) was neutralized by clavulanic acid. Spot inocula of E. cloacae isolate No. 19, following overnight "induction" with 1 microgram/ml of ampicillin and 3 microgram/ml of cephalothin in tryptic soya broth, were applied centrally to beta-lactam antibiotic inhibition zones of Bauer-Kirby antibiogram plates (Mueller-Hinton agar, MHA, and diagnostic sensitivity test agar, DSTA) following removal of the appropriate disks. The spot-inoculated plates were incubated overnight at 35 degrees C and inspected for satellite growths of tolerant variants around the E. cloacae spot inocula. Satellite growths of less than or equal to 10 colonies were interpreted to indicate tolerance of the relevant cell wall synthesis inhibitor. The method readily permitted detection of variants tolerant for ampicillin, cephalothin, penicillin G, piperacillin, azlocillin, and mezlocillin. However, strains documented by minimal inhibitory and minimal bactericidal concentrations to be tolerant for cefotaxime, cefoxitin, fosfomycin, and vancomycin only rarely gave rise to respective satellite growths. DSTA proved superior to MHA with respect to "rescue" of inhibited tolerant staphylococcal variants; furthermore, the diameters of inhibition zones obtained on DSTA correlated well with those on MHA. Therefore, DSTA was adopted as the routine test medium for clinical staphylococcal isolates.