Evaluation of New Broth Media for Microdilution Antibiotic Susceptibility Testing of Lactobacilli, Pediococci, Lactococci, and Bifidobacteria

Abstract

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 ...

Two Different Tetracycline Resistance Mechanisms, Plasmid-Carried tet (L) and Chromosomally Located Transposon-Associated tet (M), Coexist in Lactobacillus sakei Rits 9

“…The MICs of tetracycline for the different strains were determined by microdilution. Briefly, colonies obtained after growth on solid media were picked up and incubated overnight at 30°C in LSM broth (29). The optical density at 625 nm (OD 625 ) of the cultures was adjusted to 0.2 in LSM broth, and the suspension was diluted 500-fold in the same medium.…”

Characterization and Kinetics of Growth of Bacteriocin like Substance Produced by Lactic Acid Bacteria Isolated from Ewe Milk and Traditional Sour Buttermilk in Iran

“…The antimicrobial susceptibilities of the isolates and reference strains were preliminarily assessed by the disk diffusion method on MRS [11]. Ten antibiotics used in the treatment of the most common hospital infections were chosen, belonging to different categories: ampicillin, penicillin, tetracycline, vancomycin, erythromycin, gentamicin, clindamycin, Chloramphenicol, Kanamycin and Sterptomycin.…”

Effect of Pediocin NCDC252 as Cell Free Supernatant Produced from Pediococus acidilactici NCDC252 with EDTA on Total Viable Count and Sensory Evaluation of Chicken Carcasses Stored at Refrigeration Temperature

“…Broth was supplemented with L-cysteine hydrochloride (0.3 g/lit) (Klare et al, 2005). Cultures were preserved in MRS glycerol broth (15 per cent glycerol) and stored at -20˚C.…”