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

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2005, p Vol. 71, No /05/$ doi: /aem Copyright 2005, American Society for Microbiology. All Rights Reserved. Evaluation of New Broth Media for Microdilution Antibiotic Susceptibility Testing of Lactobacilli, Pediococci, Lactococci, and Bifidobacteria Ingo Klare, 1 * Carola Konstabel, 1 Sibylle Müller-Bertling, 1 Rolf Reissbrodt, 1 Geert Huys, 2 Marc Vancanneyt, 3 Jean Swings, 2,3 Herman Goossens, 4 and Wolfgang Witte 1 Robert Koch Institute, Wernigerode Branch, Wernigerode, Germany 1 ; Laboratory of Microbiology 2 and BCCM/LMG Bacteria Collection, 3 Ghent University, Ghent, Belgium; and Medical Microbiology, University of Antwerp, Wilrijk, Belgium 4 Received 17 December 2004/Accepted 9 August 2005 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. * Corresponding author. Mailing address: Robert Koch Institute, Wernigerode Branch, Burgstraße 37, D Wernigerode, Germany. Phone: Fax: i.klare@rki.de. 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: // 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). 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 (; Oxoid), with the growth enrichment supplement Vitox (supplementation according to the instructions of the manufacturer; Oxoid), supplemented with lysed horse blood (LHB; and 2.; Oxoid), mixtures of with various portions of MRS broth (50%, 2, 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 gml 1 noted in parentheses) for a set of international control strains (Table 3): penicillin G (PEN; to 64), ampicillin (AMP; to 64), ampicillin/sulbactam (ASU [sulbactam was tested as fixed concentration of 8 g ml 1 ]; to 64), gentamicin (GEN; 1 to 2,048), streptomycin (STR; 2 to 4,096), vancomycin (VAN; to 256), teicoplanin (TPL; to 256), erythromycin (ERY; to 32), clindamycin (CLI; to 32), quinupristin-dalfopristin (Q/D [30:70 ratio]; to 64), oxytetracycline (OTE; to 128), chloramphenicol (CMP; to 256), fusidic acid (FUS; to 128), trimethoprim (TMP; 0.25 to 512), sulfamethoxazole/trimethoprim (SXT [19:1 ratio]; 0.25 to 512), ciprofloxacin (CIP; to 16), moxifloxacin (MFL; to 16), linezolid (LIN; to 32), and cefazolin (CEZ; 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 8982

2 VOL. 71, 2005 BROTH MICRODILUTION TEST OF LACTIC ACID BACTERIA 8983 TABLE 1. Growth of type and reference strains of Lactobacillus, Lactococcus, and Pediococcus species in different nutrient broth media Growth in indicated medium after incubation a Type or reference strain MRS broth Vitox c LHB d (50%) MRS broth (50%) e (90%) MRS broth (10%) LSM broth: IST broth (90%) MRS broth (10%) b 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. The determined MICs of these antibiotics were compared b b Lactobacillus strains L. acidophilus LMG ( ) ( ) ( ) L. acidophilus LMG 9433 T ( ) ( ) ( ) L. johnsonii LMG ( ) ( ) L. johnsonii LMG 9436 T ( ) ( ) ( ) ( ) L. paracasei subsp. paracasei LMG T ( ) ( ) L. paracasei subsp. paracasei LMG ( ) ( ) ( ) L. plantarum LMG 6907 T ( ) ( ) L. plantarum LMG 9212 ( ) ( ) L. reuteri LMG ( ) ( ) L. reuteri LMG 9213 T ( ) ( ) L. rhamnosus LMG ( ) ( ) L. rhamnosus LMG 6400 T ( ) ( ) ( ) Lactococcus strains L. lactis subsp. lactis LMG ( ) L. lactis subsp. lactis LMG 6890 T ( ) Pediococcus strains P. acidilactici LMG T ( ) ( ) P. acidilactici LMG ( ) ( ) P. pentosaceus LMG T ( ) P. pentosaceus LMG ( ) ( ) a 37 C for 24 h., growth; ( ), weak growth;, no growth. b Identical results after incubation in atmosphere. c Vitox supplement (supplementation according to the instructions of the manufacturer; Oxoid). d Identical growth results with 2. LHB. e Identical growth results with (7) MRS broth (2). TABLE 2. Growth of type and reference strains of Bifidobacterium species in different nutrient broth media under anaerobic atmosphere Type or reference Bifidobacterium strain TPY broth Growth in a : LSM C broth B. adolescentis LMG T ( ) B. adolescentis LMG ( ) B. animalis subsp. animalis LMG T B. animalis subsp. lactis LMG ( ) B. animalis subsp. lactis LMG T B. bifidum LMG T ( ) B. bifidum LMG ( ) B. breve LMG ( ) ( ) B. breve LMG T ( ) B. longum biotype infantis LMG ( ) B. longum biotype infantis LMG 8811 B. longum LMG ( ) B. longum LMG T ( ) ( ) B. longum LMG ( ) a Anaerobic atmosphere; 37 C; 48 h., growth; ( ), weak growth;, no growth. with those received from parallel determinations in (for Streptococcus pneumoniae, supplemented with 2% to LHB was used [8]; Table 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 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- l volume of LSM broth per well) with the standardized strain suspensions was performed by use of a 96-needle multipoint inoculator ( 1 l of inoculum per needle was transferred in each well resulting in a final LAB inoculum of 10 5 bacteria ml 1 ). The inoculated plates were subsequently incubated aerobically and in a 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- l portion served as the inoculum for each well of the manually prepared MIC microtiter plates with 50 l of LSM C broth (final in-

3 8984 KLARE ET AL. APPL. ENVIRON. MICROBIOL. TABLE 3. Influence of different LAB nutrient broth media on the MICs of international control strains determined by broth microdilution test a Control strain Broth medium and incubation type Susceptibilities to the following antibiotic (MIC [ g ml 1 ] b ): PEN AMP ASU GEN STR VAN TPL Streptococcus pneumoniae LHB n.d. n.d. n.d n.d. ATCC LSM c Staphylococcus aureus n.d n.d ATCC LSM c Enterococcus faecalis n.d n.d ATCC LSM c Escherichia coli n.d n.d. n.d. n.d. ATCC LSM c a incubation at 37 C for 24 h. b Upper MICs; acceptable MIC limits according to CLSI (8) determined in (or in with 2% to LHB in the case of Streptococcus pneumoniae ATCC 49619). Other MICs were determined in LSM. n.d., no data. c incubation was performed in LSM broth; anaerobic incubation was performed in LSM C broth; however, these resulted in identical MICs for only one value noted or in MICs with a difference of only 1 log 2 dilution step (standard deviation of this microbiological test) in the majority. d MICs of CLSI (8) for tetracycline. TABLE 4. MICs of LAB reference strains to different antibiotics determined by broth microdilution in LSM broth and LSM C broth a Reference/control strain Broth medium incubation type Susceptibilities to the following antibiotic (MICs [ g ml 1 ]) c : PEN AMP ASU GEN STR VAN TPL L. johnsonii ATCC LSM broth aerobic b L. lactis subsp. lactis LMG LSM broth aerobic b P. acidilactici LMG LSM broth aerobic b B. bifidum ATCC LSM C broth, anaerobic a Incubation: aerobically at 37 C for 24 h; in the case of B. bifidum ATCC 29521, anaerobically at 37 C for 48 h. LSM broth was used for lactobacilli, pediococci, and lactococci; LSM C broth was used for bifidobacteria. b Anaerobic incubation in LSM resulted in MICs identical to those obtained under aerobic conditions (only one value noted) or in MICs with 1 log 2 dilution step difference (standard deviation of this microbiological test). c n.t., not tested. Downloaded from oculum, 10 5 bacteria ml 1 ). The inoculated plates were incubated at 37 C for 48 h in an anaerobic atmosphere (Anaero- Gen; 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 ) 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. The addition of various percentages of MRS broth to improved the situation, but these modifications were still inefficient in supporting the growth of all tested LAB type and reference strains (Table 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 atmosphere (Table 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 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 [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 log 2 steps lower in LSM broth than in and supplemented with LHB. Changes of 1 log 2 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 log 2 step, 4 test pairs showed differences of 2 MIC log 2 steps, and 3 test pairs differed by 3 MIC log 2 steps. Overall, these MICs were in good agreement with those determined with and on January 21, 2019 by guest

4 VOL. 71, 2005 BROTH MICRODILUTION TEST OF LACTIC ACID BACTERIA 8985 TABLE 3 Continued Susceptibilities to the following antibiotic (MIC [ g ml 1 ] b ): Q/D ERY CLI OTE CMP FUS TMP SXT CIP MFL LIN CEZ d 2 8 n.d. n.d n.d n.d d 2 16 n.d d 4 16 n.d n.d n.d. n.d. n.d d 2 8 n.d n.d with LHB supplementation according to the data of the CLSI (formerly NCCLS) (8) (Table 3). Minimal quantitative differences were found when MICs were determined in LSM broth for three LAB reference strains incubated aerobically or anaerobically (Table 4): 31 test pairs showed identical MICs, 19 test pairs differed by 1 MIC log 2 step, and 4 pairs displayed differences of 2 MIC log 2 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. 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). This work was supported by a grant from the European Commission (QLRT , 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 Susceptibility of 40 lactobacilli to six antimicrobial agents with broad gram-positive anaerobic spectra. Antimicrob. Agents Chemother. 14: Biavati, B., B. Sgorbati, and V. Scardovi The genus Bifidobacterium, p 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 In-vitro activity of RP 59500, a new semisynthetic streptogramin antibiotic, against gram-positive bacteria. J. Antimicrob. Chemother. 30(Suppl. A): TABLE 4 Continued Susceptibilities to the following antibiotic (MICs [ g ml 1 ]) c : Q/D ERY CLI OTE CMP FUS TMP SXT CIP MFL LIN CEZ n.t n.t n.t n.t Charteris, W. P., P. M. Kelly, L. Morelli, and J. K. Collins Antibiotic susceptibility of potentially probiotic Bifidobacterium isolates from the human gastrointestinal tract. Lett. Appl. Microbiol. 26: Charteris, W. P., P. M. Kelly, L. Morelli, and J. K. Collins Antibiotic susceptibility of potentially probiotic Lactobacillus species. J. Food Prot. 61: Charteris, W. P., P. M. Kelly, L. Morelli, and J. K. Collins Gradient diffusion antibiotic susceptibility testing of potentially probiotic lactobacilli. J. Food Prot. 64: Chow, A. W., and N. Cheng In vitro activities of daptomycin (LY146032) and paldimycin (U-70,138F) against anaerobic gram-positive bacteria. Antimicrob. Agents Chemother. 32: Clinical and Laboratory Standards Institute 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 Evaluation of the E-test for antimicrobial spectrum and potency determinations of anaerobes associated with bacterial vaginosis and peritonitis. Diagn. Microbiol. Infect. Dis. 20: Danielsen, M., and A. Wind Susceptibility of Lactobacillus spp. to antimicrobial agents. Int. J. Food Microbiol. 82: de la Maza, L., K. L. Ruoff, and M. J. Ferraro In vitro activities of daptomycin and other antimicrobial agents against vancomycin-resistant gram-positive bacteria. Antimicrob. Agents Chemother. 33: Dubreuil, L., I. Houcke, and E. Singer Susceptibility testing of anaerobic bacteria: evaluation of the redesigned (version 96) biomérieux ATB ANA device. J. Clin. Microbiol. 37: Eliopoulos, G. M., C. B. Wennersten, G. Cole, and R. C. Moellering In vitro activities of two glycylcyclines against gram-positive bacteria. Antimicrob. Agents Chemother. 38: Elliott, J. A., and R. R. Facklam Antimicrobial susceptibilities of Lactococcus lactis and Lactococcus garvieae and a proposed method to discriminate between them. J. Clin. Microbiol. 34: Felten, A., C. Barreau, C. Bizet, P. H. Lagrange, and A. Philippon Lactobacillus species identification, H 2 O 2 production, and antibiotic resistance and correlation with human clinical status. J. Clin. Microbiol. 37: Frei, A., D. Goldenberger, and M. Teubner Antimicrobial susceptibility of intestinal bacteria from Swiss poultry flocks before the ban of antimicrobial growth promoters. Syst. Appl. Microbiol. 24: Goldstein, E. J. C., D. M. Citron, C. V. Merriam, Y. A. Warren, K. L. Tyrrell, and H. A. T. Fernandez In vitro activities of daptomycin, vancomycin,

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