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Antimicrob Agents Chemother. Jan 2007; 51(1): 394–396.
Published online Oct 23, 2006. doi:  10.1128/AAC.00637-06
PMCID: PMC1797673

Effects of Inoculum Size and Incubation Time on Broth Microdilution Susceptibility Testing of Lactic Acid Bacteria[down-pointing small open triangle]

Abstract

Inoculum size and incubation time were varied during broth microdilution testing of the susceptibilities of 35 strains of lactic acid bacteria to six antibiotics. An increase in either parameter resulted in elevated MICs for all species. An inoculum of 3 × 105 CFU/ml is recommended to assess the antibiotic susceptibilities of these bacteria by using broth microdilution.

Lactic acid bacteria (LAB) are commonly used as food-processing aids and probiotics. Due to their genetic flexibility and widespread occurrence in the food chain and in the intestinal tract, LAB can act as potential reservoirs of antibiotic resistance genes that may be transferred to other bacteria, including human pathogens (4, 21). Thus, the presence of antibiotic resistance genes should be assessed before LAB are used in food applications. For this reason, standardized and reliable testing procedures are needed to define rational microbiological breakpoints for distinguishing between strains with and without acquired resistance genes (22).

There is currently no standard method for antibiotic susceptibility testing of LAB, although several broth microdilution methods have been used (5-7, 9, 16, 19, 20). At present, the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) recommends broth microdilution for bacteria that grow aerobically (3) and for anaerobic bacteria belonging to the Bacteroides fragilis group (18). The inoculum size and incubation time are important parameters to evaluate during the development of broth microdilution methods (22) and have been extensively studied for several bacterial species (1, 8, 13, 14, 17) but not for nonenterococcal LAB. Other factors that may affect the susceptibility results are the incubation temperature and the composition of the atmosphere and the growth medium (12, 22). The poor growth of many LAB on established antibiotic susceptibility testing media such as Mueller-Hinton and Iso-Sensitest media has led to the recent development of LAB susceptibility test medium (LSM) (15). The present study was performed to evaluate the effects of the inoculum size and incubation time on antibiotic MICs for LAB using broth microdilution and LSM.

Twenty-nine LAB reference strains were tested: 27 Lactobacillus species, encompassing different phylogenetic groups and fermentation pathways, and 1 strain each of Lactococcus lactis subsp. lactis and Streptococcus thermophilus, both species widely used by the food industry (Table (Table1).1). Six clinical Lactobacillus isolates, recovered from cerebrospinal fluid, blood, dental caries, breast milk, intestines, and feces, respectively, were also tested. Enterococcus faecalis LMG 8222 was included as a quality control. Strains were obtained from the BCCM/LMG Bacteria Collection, Ghent University, Ghent, Belgium (n = 32), the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany (n = 3), and the American Type Culture Collection, Middlesex, United Kingdom (n = 1). All strains were grown anaerobically (AnaeroGen; Oxoid). The incubation temperatures and growth media used for each species are shown in Table Table11.

TABLE 1.
LAB strains and growth conditions used for antibiotic susceptibility testing

Broth microdilution was performed using ACE-ART VetMIC panels (National Veterinary Institute, Uppsala, Sweden). The 96-well microtiter plates contained six antibiotics, air dried in cabinets at approximately 30°C, in serial twofold dilution steps to determine MICs in the following ranges: oxytetracycline, 0.5 to 128 μg/ml; clindamycin, 0.12 to 8 μg/ml; streptomycin, 2 to 256 μg/ml; erythromycin, 0.12 to 16 μg/ml; gentamicin, 0.5 to 32 μg/ml; ampicillin, 0.12 to 8 μg/ml. Colonies from overnight cultures (20 to 24 h) were suspended in the corresponding growth medium (Table (Table1)1) to obtain four final densities ranging from 3 × 104 to 3 × 107 CFU/ml for each strain. Bacterial density was measured spectrophotometrically at 600 nm and verified by viable cell counts. Each well was filled with 100 μl of inoculum. The panels were covered with plastic lids and incubated anaerobically for 24 and 48 h. The MIC was defined, according to the manufacturer's recommendations, as the lowest antibiotic concentration for which there was no visible bacterial growth, i.e., the first well without a pellet.

The MICs of the quality control strain determined after 24 h at 3 × 105 CFU/ml were within the range reported by Klare et al. (15) for all antibiotics tested except erythromycin, which displayed a twofold increase in the MIC (data not shown). Interassay reproducibility was evaluated by assessing the susceptibilities of five reference strains (Lactobacillus acidophilus LMG 9433T, L. amylovorus LMG 9496T, L. plantarum LMG 6907T, L. rhamnosus LMG 6400T, and Lactobacillus sakei subsp. sakei LMG 9468T) to six antibiotics on five separate occasions. MICs determined at 3 × 105 CFU/ml after both 24 and 48 h incubation were within the accuracy limit of MIC standard tests (plus or minus one twofold dilution step) (2) for all strain-antibiotic combinations tested (data not shown).

The MICs for all strains increased with inoculum size. Increasing the density from 3 × 104 to 3 × 105 CFU/ml resulted in identical MICs after 48 h of incubation for 144 (69%) of the 210 strain-antibiotic combinations, a twofold increase in MICs for 63 (30%) of the combinations, and a fourfold increase for the remaining 3 (1%) combinations (Fig. (Fig.1a).1a). A similar stepwise increase in MICs was observed when the inoculum was further increased to 3 × 106 and 3 × 107 CFU/ml, respectively. The shift in the MIC due to increased inoculum size was independent of incubation time (data not shown).

FIG. 1.
Effects of increased inoculum size (a) and prolonged incubation time (b) on the MICs of six antibiotics for 35 LAB strains. (a) MICs obtained for the 210 strain-antibiotic combinations with different inoculum sizes, compared to MICs obtained with an inoculum ...

All strains displayed similar increases in MICs when the incubation time was extended to 48 h. With an inoculum of 3 × 105 CFU/ml, 102 (49%) of the 210 strain-antibiotic combinations were unaffected by prolonged incubation, whereas MICs increased twofold for 96 (46%) of the combinations (Fig. (Fig.1b).1b). For the remaining combinations (6%), fourfold MIC increases between 24 and 48 h of incubation were seen mainly with oxytetracycline and clindamycin, but these increases were all in the lower part of the MIC range (maximum, 2 to 8 μg/ml). The MIC increase with time was independent of inoculum size (data not shown). For the clinical isolates, the elevation in MICs due to increased inoculum size or incubation time was in accordance with the results for the LAB reference strains (data not shown).

The use of the two highest inoculum densities (3 × 106 and 3 × 107 CFU/ml) was generally associated with trailing growth, which prevented clear-cut end point readings. In addition, an inoculum of 3 × 104 CFU/ml resulted in poor growth for L. casei, L. collinoides, L. brevis, L. hilgardii, and L. buchneri after 24 h of incubation, making it difficult to determine MICs accurately. Thus, an inoculum size of 3 × 105 CFU/ml is recommended for broth microdilution susceptibility testing of LAB using LSM. This is in approximate agreement with the standardized inoculum size for aerobic bacteria (3) but is about three times lower than the density recommended for anaerobic bacteria (18). Sufficient growth for MIC determination was obtained at 3 × 105 CFU/ml after both 24 and 48 h of incubation for all strains tested. However, end points were more easily read after 48 h of incubation. In addition, LAB groups that need extended incubation for sufficient growth on LSM, such as most Bifidobacterium species (15), can be tested simultaneously when 48 h is used as the standard incubation time.

In conclusion, an increased inoculum size and an extended incubation time resulted in elevated antibiotic MICs for all LAB species, underlining the importance of controlled and standardized conditions for susceptibility testing of LAB. Hopefully, the results from this study will contribute to the development of a standard method for determining the antibiotic susceptibilities of LAB and to subsequent delineation of microbiological breakpoints for individual LAB species.

Acknowledgments

This work was supported by a grant from the European Commission, Sixth Framework Program (CT-2003-506214, “ACE-ART”). G.H. is a postdoctoral fellow of the Fund for Scientific Research, Flanders, Belgium (F.W.O.—Vlaanderen).

Footnotes

[down-pointing small open triangle]Published ahead of print on 23 October 2006.

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