Finding of acquired resistance genes in isolates intended for probiotic or nutritional use highlights the importance of antimicrobial susceptibility testing in documenting the safety of commercial LAB.

Nine pure or mixed broth media were evaluated for their suitabilities to determine MICs in a microdilution test of 19 antibacterial agents for lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Lactococcus, and Bifidobacterium. A mixed formulation of Iso-Sensitest broth (90%) and deMan-Rogosa-Sharpe broth (10%) with or without supplementation with L-cysteine, referred to as the LAB susceptibility test medium, provided the most optimal medium basis in terms of growth support of nonenterococcal LAB and correct indication of MICs of international control strains.A large variety of methods to determine antibiotic susceptibilities of nonenterococcal lactic acid bacteria (LAB) belonging to the genera Lactobacillus, Pediococcus, Lactococcus, and Bifidobacterium based on either agar disk diffusion (4, 5, 15, 20, 26, 29, 32, 33, 35), E-test (6, 9, 10, 13, 15, 16, 19, 21), agar dilution (3,7,11,17,19,22) or broth dilution (1,12,14,18,23,24,25,27,29,30,31, 34) has been described. Due to the fact that many of these organisms require special growth conditions in terms of medium acidity and nutrient supplementation, conventional media such as Mueller-Hinton and Iso-Sensitest (IST) agar or broth are not uniformly suitable for susceptibility testing of nonenterococcal LAB. In this study, we describe the evaluation of two variants of a newly developed broth formula referred to as the LAB susceptibility test medium (LSM) with or without supplementation with L-cysteine for the determination of MICs for Lactobacillus, Pediococcus, Lactococcus, and Bifidobacterium species for a range of 19 antibacterial agents representing all major antibiotic classes.Type and reference strains of relevant nonenterococcal LAB species (Tables 1 and 2) were obtained from BCCM/LMG Bacteria Collection, Ghent University (Ghent, Belgium; http: //www.belspo.be/bccm/db/bacteria_search.htm). Lactobacilli, pediococci, and lactococci were routinely cultured on deManRogosa-Sharpe (MRS) agar (Oxoid) aerobically or under microaerophilic conditions, whereas bifidobacteria were grown anaerobically (AnaeroGen; Oxoid) on modified Columbia agar (CM331 [Oxoid] supplemented with 0.3 g liter Ϫ1 L-cysteine hydrochloride and 5 g liter Ϫ1 glucose). A series of nine broth media was evaluated for the abilities of the media to support growth of lactobacilli, pediococci, and lactococci: MRS broth (Oxoid), cation-adjusted Mueller-Hinton (CAMHB; Oxoid), CAMHB with the growth enrichment supplement Vitox (supplementation according to the instructions of the manufacturer; Oxoid), CAMHB supplemented with lysed horse blood (LHB; 5% and 2.5%; Oxoid), mixtures of CAMHB with various portions of MRS broth (50%, 25%, and 10%), and, finally, a mixture of IST broth (90%; Oxoid) and MRS broth (10%) adjusted to pH 6.7. Growth of bifidobacteria was tested in trypticase-phytone-yeast extract (TPY; Becton-Dickinson) broth (2) and in a mixture of IST broth (90%) and MRS broth (10%) adjusted to pH 6.7 and supplemented with L-cysteine hydrochloride (0.3 g liter Ϫ1 and 0.5 g liter ...