• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of aemPermissionsJournals.ASM.orgJournalAEM ArticleJournal InfoAuthorsReviewers
Appl Environ Microbiol. Dec 2005; 71(12): 8982–8986.
PMCID: PMC1317481

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 (Tables11 and and2)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 deMan-Rogosa-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−1 l-cysteine hydrochloride and 5 g liter−1 glucose).

TABLE 1.
Growth of type and reference strains of Lactobacillus, Lactococcus, and Pediococcus species in different nutrient broth media
TABLE 2.
Growth of type and reference strains of Bifidobacterium species in different nutrient broth media under anaerobic atmosphere

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−1 and 0.5 g liter−1; Sigma).

Following the evaluation of these broth formulations, the two most optimal media (LSM broth and LSM broth supplemented with 0.3 g liter−1 l-cysteine hydrochloride [LSM+C broth]) were used in a microdilution test (8) to determine the MICs of the following 19 antimicrobials (test ranges in μg ml−1 noted in parentheses) for a set of international control strains (Table (Table3):3): penicillin G (PEN; 0.032 to 64), ampicillin (AMP; 0.032 to 64), ampicillin/sulbactam (ASU [sulbactam was tested as fixed concentration of 8 μg ml−1]; 0.032 to 64), gentamicin (GEN; 1 to 2,048), streptomycin (STR; 2 to 4,096), vancomycin (VAN; 0.125 to 256), teicoplanin (TPL; 0.125 to 256), erythromycin (ERY; 0.016 to 32), clindamycin (CLI; 0.032 to 32), quinupristin-dalfopristin (Q/D [30:70 ratio]; 0.032 to 64), oxytetracycline (OTE; 0.063 to 128), chloramphenicol (CMP; 0.125 to 256), fusidic acid (FUS; 0.063 to 128), trimethoprim (TMP; 0.25 to 512), sulfamethoxazole/trimethoprim (SXT [19:1 ratio]; 0.25 to 512), ciprofloxacin (CIP; 0.008 to 16), moxifloxacin (MFL; 0.008 to 16), linezolid (LIN; 0.016 to 32), and cefazolin (CEZ; 0.125 to 256). Most tested antibiotics originated from Sigma, except sulbactam and linezolid (Pfizer), TPL and Q/D (Sanofi-Aventis), ERY (Abbott), CIP and MFL (Bayer), and CEZ (Chephasaar). For preparation of stock solutions, the majority of antibiotics were dissolved in distilled water or buffer as recommended previously (8). The following antibiotics required solubility mediators used in volumes as low as possible: OTE, 0.1 N HCl; FUS, methanol; TMP, dimethyl formamide; and sulfamethoxazole, 0.1 N NaOH.

TABLE 3.
Influence of different LAB nutrient broth media on the MICs of international control strains determined by broth microdilution testa

The determined MICs of these antibiotics were compared with those received from parallel determinations in CAMHB (for Streptococcus pneumoniae, CAMHB supplemented with 2% to 5% LHB was used [8]; Table Table3).3). Inocula of Lactobacillus, Pediococcus, and Lactococcus strains were prepared by suspending single colonies (picked up from fresh cultures on MRS agar plates incubated for 48 h at 37°C in 5% CO2 atmosphere) in a tube with 5 ml of saline to an optical density of 0.5 McFarland standard and subsequently diluting them 1:10 in saline. Inoculation of manually premade MIC microtiter test plates (containing the different antibiotic test concentrations in each 50-μl volume of LSM broth per well) with the standardized strain suspensions was performed by use of a 96-needle multipoint inoculator (~1 μl of inoculum per needle was transferred in each well resulting in a final LAB inoculum of 105 bacteria ml−1). The inoculated plates were subsequently incubated aerobically and in a 5% CO2 atmosphere at 37°C for 24 h, after which the MICs were read as the lowest concentration of a given antibiotic at which no growth of the test organism was observed. Inocula for bifidobacteria were prepared from fresh cultures (anaerobically grown on modified Columbia agar at 37°C for 48 h; AnaeroGen; Oxoid) by suspending single colonies in saline up to 0.5 McFarland standard turbidity. From the subsequent 1:15 dilution in saline, a 10-μl portion served as the inoculum for each well of the manually prepared MIC microtiter plates with 50 μl of LSM+C broth (final inoculum, ~105 bacteria ml−1). The inoculated plates were incubated at 37°C for 48 h in an anaerobic atmosphere (AnaeroGen; Oxoid), and the MICs were read as described above.

The best overall growth support of the examined Lactobacillus, Pediococcus, and Lactococcus strains was obtained with MRS broth. However, there is some concern about possible antagonistic interactions between MRS components and specific antimicrobial agents (10, 20); in particular, antagonists for trimethoprim (thymidine) and sulfonamides (p-aminobenzoic acid) inhibit the antibacterial activities of these agents competitively (28). Additionally, the low pH of MRS medium (pH 6.2 ± 0.2) could be responsible for decreased activities of some antibiotics, e.g., aminoglycosides.

Furthermore, several of the tested Lactobacillus strains exhibited only weak or even no growth when testing different preparations of the conventional susceptibility test medium CAMHB. The addition of various percentages of MRS broth to CAMHB improved the situation, but these modifications were still inefficient in supporting the growth of all tested LAB type and reference strains (Table (Table1).1). Finally, a mixed formulation of 90% IST broth with 10% MRS broth (adjusted to pH 6.7) was found to be the most optimal medium yielding sufficient to strong growth for all tested Lactobacillus, Pediococcus, and Lactococcus strains when incubated under aerobic conditions at 37°C for 24 h; only minimal differences in growth were noted if these LAB were incubated in a 5% CO2 atmosphere (Table (Table1).1). The mixed IST/MRS preparation was referred to as the LSM. For tested Bifidobacterium strains, supplementation of LSM broth with 0.3 g liter−1 l-cysteine hydrochloride and anaerobic incubation (AnaeroGen; Oxoid) at 37°C for 48 h led to sufficient growth, which was better compared to that seen with TPY broth (Table (Table2;2; see also reference 4).

In the second part of the study, LSM broth (with and without l-cysteine supplementation) was tested by microdilution for a correct indication of known MICs for 19 antimicrobials (determined in CAMHB [8]) for a set of international control strains. This nutrient medium was used for two reasons: (i) both variants of LSM broth sufficiently supported the growth of all tested nonenterococcal LAB strains, and (ii) LSM broth is composed of 90% IST broth, which is the nutrient medium recommended by the British Society for Antimicrobial Chemotherapy for antibiotic susceptibility testing, and, therefore, only minimal influences on MICs for control strains were to be expected.

Of all antibacterials tested, only MICs of PEN were 1 to 2 MIC log2 steps lower in LSM broth than in CAMHB and CAMHB supplemented with LHB. Changes of ±1 log2 dilution step in MICs are the normal standard deviation of MIC dilution tests. Likewise, the MICs of agents determined for the reference strains in LSM broth without l-cysteine (aerobic incubation) or with l-cysteine (anaerobic incubation) were comparable: 35 test pairs exhibited identical MICs, 34 test pairs displayed a difference of 1 MIC log2 step, 4 test pairs showed differences of 2 MIC log2 steps, and 3 test pairs differed by 3 MIC log2 steps. Overall, these MICs were in good agreement with those determined with CAMHB and CAMHB with LHB supplementation according to the data of the CLSI (formerly NCCLS) (8) (Table (Table3).3). Minimal quantitative differences were found when MICs were determined in LSM broth for three LAB reference strains incubated aerobically or anaerobically (Table (Table4):4): 31 test pairs showed identical MICs, 19 test pairs differed by 1 MIC log2 step, and 4 pairs displayed differences of 2 MIC log2 steps. On the condition that all strains of lactobacilli, pediococci, and lactococci are able to grow in the presence of oxygen, we recommend the incubation of susceptibility tests of these genera in LSM broth aerobically for 24 h at 37°C.

TABLE 4.
MICs of LAB reference strains to different antibiotics determined by broth microdilution in LSM broth and LSM+C brotha

In summary, both variants of LSM are suitable for MIC determinations for lactobacilli, pediococci, lactococci, and bifidobacteria in a broth microdilution test. It is expected that the use of these medium formulations will minimize previously reported growth problems with nonenterococcal LAB and antagonistic effects between some antimicrobials and growth medium components (10, 20).

Acknowledgments

This work was supported by a grant from the European Commission (QLRT-2001-01273, PROSAFE). Geert Huys is a postdoctoral fellow of the Fund for Scientific Research, Flanders, Belgium (F.W.O.-Vlaanderen).

REFERENCES

1. Bayer, A. S., A. W. Chow, N. Concepcion, and L. B. Guze. 1978. Susceptibility of 40 lactobacilli to six antimicrobial agents with broad gram-positive anaerobic spectra. Antimicrob. Agents Chemother. 14:720-722. [PMC free article] [PubMed]
2. Biavati, B., B. Sgorbati, and V. Scardovi. 1992. The genus Bifidobacterium, p. 816-833. In A. Balows, H. G. Trüper, M. Dworkin, W. Harder, and K.-H. Schleifer (ed.), The prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, identification, applications. Springer, New York, N.Y.
3. Brumfitt, W., J. M. Hamilton-Miller, and S. Shah. 1992. In-vitro activity of RP 59500, a new semisynthetic streptogramin antibiotic, against gram-positive bacteria. J. Antimicrob. Chemother. 30(Suppl. A):29-37. [PubMed]
4. Charteris, W. P., P. M. Kelly, L. Morelli, and J. K. Collins. 1998. Antibiotic susceptibility of potentially probiotic Bifidobacterium isolates from the human gastrointestinal tract. Lett. Appl. Microbiol. 26:333-337. [PubMed]
5. Charteris, W. P., P. M. Kelly, L. Morelli, and J. K. Collins. 1998. Antibiotic susceptibility of potentially probiotic Lactobacillus species. J. Food Prot. 61:1636-1643. [PubMed]
6. Charteris, W. P., P. M. Kelly, L. Morelli, and J. K. Collins. 2001. Gradient diffusion antibiotic susceptibility testing of potentially probiotic lactobacilli. J. Food Prot. 64:2007-2014. [PubMed]
7. Chow, A. W., and N. Cheng. 1988. In vitro activities of daptomycin (LY146032) and paldimycin (U-70,138F) against anaerobic gram-positive bacteria. Antimicrob. Agents Chemother. 32:788-790. [PMC free article] [PubMed]
8. Clinical and Laboratory Standards Institute. 2005. Performance standards for antimicrobial susceptibility testing: fifteenth informational supplement. Document M100-S15, Vol. 25, No. 1. Clinical and Laboratory Standards Institute, Wayne, Pa.
9. Croco, J. L., M. E. Erwin, J. M. Jennings, L. R. Putnam, and R. N. Jones. 1994. Evaluation of the E-test for antimicrobial spectrum and potency determinations of anaerobes associated with bacterial vaginosis and peritonitis. Diagn. Microbiol. Infect. Dis. 20:213-219. [PubMed]
10. Danielsen, M., and A. Wind. 2003. Susceptibility of Lactobacillus spp. to antimicrobial agents. Int. J. Food Microbiol. 82:1-11. [PubMed]
11. de la Maza, L., K. L. Ruoff, and M. J. Ferraro. 1989. In vitro activities of daptomycin and other antimicrobial agents against vancomycin-resistant gram-positive bacteria. Antimicrob. Agents Chemother. 33:1383-1384. [PMC free article] [PubMed]
12. Dubreuil, L., I. Houcke, and E. Singer. 1999. Susceptibility testing of anaerobic bacteria: evaluation of the redesigned (version 96) bioMérieux ATB ANA device. J. Clin. Microbiol. 37:1824-1828. [PMC free article] [PubMed]
13. Eliopoulos, G. M., C. B. Wennersten, G. Cole, and R. C. Moellering. 1994. In vitro activities of two glycylcyclines against gram-positive bacteria. Antimicrob. Agents Chemother. 38:534-541. [PMC free article] [PubMed]
14. Elliott, J. A., and R. R. Facklam. 1996. Antimicrobial susceptibilities of Lactococcus lactis and Lactococcus garvieae and a proposed method to discriminate between them. J. Clin. Microbiol. 34:1296-1298. [PMC free article] [PubMed]
15. Felten, A., C. Barreau, C. Bizet, P. H. Lagrange, and A. Philippon. 1999. Lactobacillus species identification, H2O2 production, and antibiotic resistance and correlation with human clinical status. J. Clin. Microbiol. 37:729-733. [PMC free article] [PubMed]
16. Frei, A., D. Goldenberger, and M. Teubner. 2001. Antimicrobial susceptibility of intestinal bacteria from Swiss poultry flocks before the ban of antimicrobial growth promoters. Syst. Appl. Microbiol. 24:116-121. [PubMed]
17. Goldstein, E. J. C., D. M. Citron, C. V. Merriam, Y. A. Warren, K. L. Tyrrell, and H. A. T. Fernandez. 2003. In vitro activities of daptomycin, vancomycin, quinupristin-dalfopristin, linezolid, and five other antimicrobials against 307 gram-positive anaerobic and 31 Corynebacterium clinical isolates. Antimicrob. Agents Chemother. 47:337-341. [PMC free article] [PubMed]
18. Green, M., R. M. Wadowsky, and K. Barbadora. 1990. Recovery of vancomycin-resistant gram-positive cocci from children. J. Clin. Microbiol. 28:484-488. [PMC free article] [PubMed]
19. Herra, C. M., M. T. Cafferkey, and C. T. Keane. 1995. The in-vitro susceptibilities of vaginal lactobacilli to four broad-spectrum antibiotics, as determined by the agar dilution and E-test methods. J. Antimicrob. Chemother. 35:775-783. [PubMed]
20. Huys, G., K. D'Haene, and J. Swings. 2002. Influence of the culture medium on antibiotic susceptibility testing of food-associated lactic acid bacteria with the agar overlay disc diffusion method. Lett. Appl. Microbiol. 34:402-406. [PubMed]
21. Katla, A. K., H. Kruse, G. Johnsen, and H. Herikstad. 2001. Antimicrobial susceptibility of starter culture bacteria used in Norwegian dairy products. Int. J. Food Microbiol. 67:147-152. [PubMed]
22. King, A., and I. Phillips. 2001. The in vitro activity of daptomycin against 514 Gram-positive aerobic clinical isolates. J. Antimicrob. Chemother. 48:219-223. [PubMed]
23. Lim, K. S., C. S. Huh, and Y. J. Baek. 1993. Antimicrobial susceptibility of bifidobacteria. J. Dairy Sci. 76:2168-2174. [PubMed]
24. Matteuzzi, D., F. Crociani, and P. Brigidi. 1983. Antimicrobial susceptibility of Bifidobacterium. Ann. Microbiol. (Paris) 134A:339-349. [PubMed]
25. Nagaraja, T. G., and M. B. Taylor. 1987. Susceptibility and resistance of ruminal bacteria to antimicrobial feed additives. Appl. Environ. Microbiol. 53:1620-1625. [PMC free article] [PubMed]
26. Orberg, P. K., and W. E. Sandine. 1985. Survey of antimicrobial resistance in lactic streptococci. Appl. Environ. Microbiol. 49:538-542. [PMC free article] [PubMed]
27. Parada, J. L., and M. Pamies de Giacchi. 1986. Resistance of Streptococcus lactis mutants to beta-lactam antibiotics. J. Dairy Sci. 69:2031-2037. [PubMed]
28. Reissbrodt, R., W. Witte, and H. Rische. 1983. A new semidefined nutrient medium for bacterial susceptibility testing. J. Hyg. Epidemiol. Microbiol. Immunol. 27:465-479. [PubMed]
29. Ruoff, K. L., D. R. Kuritzkes, J. S. Wolfson, and M. J. Ferraro. 1988. Vancomycin-resistant gram-positive bacteria isolated from human sources. J. Clin. Microbiol. 26:2064-2068. [PMC free article] [PubMed]
30. Sidhu, M. S., S. Langsrud, and A. Holck. 2001. Disinfectant and antibiotic resistance of lactic acid bacteria isolated from the food industry. Microb. Drug Resist. 7:73-83. [PubMed]
31. Swenson, J. M., R. R. Facklam, and C. Thornsberry. 1990. Antimicrobial susceptibility of vancomycin-resistant Leuconostoc, Pediococcus, and Lactobacillus species. Antimicrob. Agents Chemother. 34:543-549. [PMC free article] [PubMed]
32. Tankovic, J., R. Leclercq, and J. Duval. 1993. Antimicrobial susceptibility of Pediococcus spp. and genetic basis of macrolide resistance in Pediococcus acidilactici HM3020. Antimicrob. Agents Chemother. 37:789-792. [PMC free article] [PubMed]
33. Temmerman, R., B. Pot, G. Huys, and J. Swings. 2003. Identification and antibiotic susceptibility of bacterial isolates from probiotic products. Int. J. Food Microbiol. 81:1-10. [PubMed]
34. Yamane, N., and R. N. Jones. 1991. In vitro activity of 43 antimicrobial agents tested against ampicillin-resistant enterococci and gram-positive species resistant to vancomycin. Diagn. Microbiol. Infect. Dis. 14:337-345. [PubMed]
35. Yazid, A. M., A. M. Ali, M. Shuhaimi, V. Kalaivaani, M. Y. Rokiah, and A. Reezal. 2000. Antimicrobial susceptibility of bifidobacteria. Lett. Appl. Microbiol. 31:57-62. [PubMed]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...