Gradient diffusion antibiotic susceptibility testing of potentially probiotic lactobacilli

Minimum inhibitory contentrations (MICs) of selected inhibitors of cell wall synthesis (benzylpenicillin, ampicillin, and vancomycin), protein synthesis (gentamicin, streptomycin, tetracycline, chloramphenicol, and erythromycin), and nucleic acid synthesis (co-trimoxazole, rifampicin, and metronidazole) were determined by gradient diffusion (E test; AB Biodisk, Solna, Sweden) on deMan, Rogosa, Sharpe (MRS) agar for Lactobacillus strain GG and 11 closely related, rapidly growing, facultatively anaerobic, potentially probiotic Lactobacillus rhamnosus strains. All strains were resistant to vancomycin (MIC90 greater than or equal to 256 mug/ml), co-trimoxazole (MIC90 greater than or equal to 32 mug/ml), metronidazole (MIC90 greater than or equal to 32 mug/ml), gentamicin (MIC90 greater than or equal to 128 mug/ml), and streptomycin (MIC90 greater than or equal to 256 mug/ml), and sensitive to pencillin G (MIC90 > 0.375 mug/ml), ampicillin (MIC90 > 0.750 mug/ml), rifampicin (MIC90 > 0.375 mug/ml), tetracycline (MIC90 > 1.5 mug/ml), chloramphenicol (MIC90 > 8 mug/ml), and erythromycin (MIC90 > 2 mug/ml). E test MICs were also determined for L. acidophilus National Collection of Food Bacteria (NCFB) 1748 and L. reuteri Deutsche Sammlung von Mikroorganismen 20016T by the inoculum application method recommended by the manufacturer (swabbing), with and without antibiotic prediffusion for 1 h at room temperature, and by an alternative inoculum application (agar overlay) method, without antibiotic prediffusion. Antibiotic prediffusion increased the MICs for penicillin G, ampicillin, tetracycline, and chloramphenicol by up to 2 log(2) MIC dilutions without changing antibiotic susceptibility category. Agar overlay application also increased the MICs for these antibiotics as well as for,gentamicin by up to 3 log(2) MIC dilutions without changing antibiotic susceptibility category. Exact agreement between MICs determined by swab and agar overlay application without antibiotic prediffusion was strain dependent: 54.5% for strain DSM 20016T and 72.7% for strain NCFB 1748. The swab and agar overlay gradient diffusion methods provide a reliable basis for antibiotic susceptibility testing of rapidly growing, facultatively anaerobic lactobacilli, using MRS agar as test medium and are readily applicable for testing individual isolates as needed.