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Assessment of Antibiotic Resistance in Probiotic Strain Lactobacillus Brevis KB290

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Assessment of Antibiotic Resistance in Probiotic Strain Lactobacillus Brevis KB290 Name /food/72-07-44 08/24/2009 07:39AM Plate # 0-Composite pg 1923 # 1 1923 Journal of Food Protection, Vol. 72, No. 9, 2009, Pages 1923–1929 Copyright ᮊ, International Association for Food Protection Assessment of Antibiotic Resistance in Probiotic Strain Lactobacillus brevis KB290 MASANORI FUKAO,1* HARUYOSHI TOMITA,2 TAKAFUMI YAKABE,1 TAKAHIRO NOMURA,2 YASUYOSHI IKE,2 AND NOBUHIRO YAJIMA1 1Probiotics Research Department, Research Institute, Kagome Company, Limited, 17 Nishitomiyama, Nasushiobara, Tochigi, 329-2762, Japan; and 2Department of Bacteriology, Gunma University Graduate School of Medicine, 3-39-22 Showa, Maebashi, Gunma, 371-8511, Japan Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/9/1923/1681776/0362-028x-72_9_1923.pdf by guest on 25 September 2021 MS 09-060: Received 6 February 2009/Accepted 14 March 2009 ABSTRACT Our purpose was to investigate the safety of the probiotic strain Lactobacillus brevis KB290. The European Qualified Presumption of Safety (QPS) evaluation approach was applied to the strain. We determined the strain’s antibiotic resistance, verified it at the genetic level, and determined whether it could be transferred to intestinal microflora. Of 14 antibiotics tested, 11 showed MICs within the limits of the QPS criteria. However, the L. brevis KB290 MICs of ciprofloxacin (a fluoroquinolone), tetracycline, and vancomycin were two, four, and eight times, respectively, the breakpoint MICs suggested by the European Scientific Committee on Animal Nutrition, and the MIC of tetracycline was eight times the breakpoint MIC suggested by the European Scientific Panel on Additives and Products or Substances Used in Animal Feed. Using analysis of gapped-genome sequences, we found no known transferable determinants for tetracycline or vancomycin resistance, and we found no mutations in the quinolone resistance–determining regions of the genes encoding GyrA or ParC for ciprofloxacin resistance associated with insertion sequences, integrons, or transposons. These data were confirmed by using PCR primers specific for the respective genes. We assessed the transferability of the resistance traits in conjugation experiments with enterococci and obtained no transconjugants, strongly suggesting that the resistance traits were not transferable. This study demonstrated that the antibiotic resistance observed in L. brevis KB290 was due not to dedicated mechanisms but to intrinsic resistance. According to the QPS criteria, these results provide safety assurance for the ongoing use of L. brevis KB290 as a probiotic. Many microbial species have probiotic properties, but has been used since 1993 in fermented food products and those most commonly used are lactic acid bacteria (LAB). as a freeze-dried powder in Japan. L. brevis KB290 displays LAB have a long history of safe use in the production and unique properties. After oral administration, it enhances consumption of fermented foods and beverages (8, 30, 40, alpha interferon production and natural killer cell activity 47). Over recent decades, as awareness of the beneficial (27). effects of probiotics in promoting gut and general health L. brevis KB290 was isolated from suguki, a traditional has grown, the development and consumption of probiotic Japanese pickle. Plant-derived LAB are genetically similar foods has increased worldwide (38). Thus, it is essential to to milk-derived strains, but they show additional useful investigate thoroughly the safety of LAB strains used in characteristics. For example, plant-derived streptococci probiotic products (7, 40). demonstrate a greater tolerance to stress than do milk-de- When probiotic LAB enter the intestines, they interact rived strains (45) and can ferment a wider range of carbo- with the native microbiota, and gene transfer can occur (4, hydrate sources (35). 32, 42). Recent reports have described the prevalence and In this study, we determined the MICs for L. brevis mechanisms of antibiotic resistance transfer in commensal KB290 of a range of antibiotics, investigated the genes re- bacteria, such as LAB isolated from food (9, 19, 21, 22, sponsible for low-level antibiotic resistance, and examined 49). In the European Union, any probiotic added to food- the conjugative transferability of the low-level resistance. stuffs must comply with the Qualified Presumption of Safe- MATERIALS AND METHODS ty (QPS) concept (16). The European Scientific Committee on Animal Nutrition (SCAN) and the European Scientific Bacterial strains and growth medium. Table 1 lists the Panel on Additives and Products or Substances Used in strains used in this study. We cultured lactobacilli on deMan Ro- Animal Feed (FEEDAP) recommended that the absence of gosa Sharpe medium (Oxoid, Ltd., Basingstoke, UK) or Mueller- Hinton (MH) medium (Difco, Becton Dickinson, Sparks, MD) transferable resistance genes should be a prerequisite for supplemented with 1.0% horse blood, and cultured enterococci approval. SCAN and FEEDAP provide explicit details for and staphylococci on brain heart infusion (Difco, Becton Dick- the screening of strains for the absence of transferable re- inson) or MH medium, also supplemented with 1.0% horse blood. sistance genes (17, 18). We grew L. brevis and Lactobacillus plantarum at 30ЊC and all Lactobacillus brevis KB290 is a probiotic strain that other strains at 37ЊC. * Author for correspondence. Tel: ϩ81-287-36-2935; Fax: ϩ81-287-39- Antibiotic susceptibility testing and MIC determination. 1038; E-mail: Masanori࿞[email protected]. We determined antibiotic MICs according to NCCLS (now CLSI) Name /food/72-07-44 08/24/2009 07:39AM Plate # 0-Composite pg 1924 # 2 1924 FUKAO ET AL. J. Food Prot., Vol. 72, No. 9 TABLE 1. Bacterial strains used in this study Straina Relative properties (plasmid)b Reference Lactobacillus brevis KB290 This study L. plantarum LMG21684 tet(M), erm (B) 20 L. plantarum LMG21687 tet(M) 20 L. rhamnosus GG (ATCC 53103) 44 Enterococcus faecalis ATCC 29212 34 E. faecalis JH2SS Derivative of JH2; Emr, Strr, Spcr, (pIP501) 23 E. faecalis FA2-2 Derivative of JH2; Fusr, Rifr, plasmid free 23 E. faecalis BM4147 vanA (pIP816) 29 E. faecalis V583 vanB 39 Staphylococcus aureus ATCC 29213 34 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/72/9/1923/1681776/0362-028x-72_9_1923.pdf by guest on 25 September 2021 a LMG, Bacteria Collection of the Laboratory of Microbiology, University of Ghent; ATCC, American Type Culture Collection. b Emr, erythromycin resistant; Strr, streptomycin resistant; Spcr, spectinomycin resistant; Fusr, fusidic acid resistant; Rifr, rifampin resistant. Approved Standard M7-A4 (34), which defines MIC as the min- L. brevis KB290 genome, which contained several plasmids, was imum concentration of antibiotic needed for total inhibition of sequenced to a depth of 35ϫ and assembled by the 454 Newbler bacterial growth after incubation for 48 h. Overnight cultures of de novo assembler to 109 contigs per genome. We compared nu- the strains grown on MH broth supplemented with 1.0% horse cleotide sequences by using the Basic Local Alignment Search blood were diluted 100 times with fresh broth. Five microliters Tool (BLAST) (2), and determined the nucleotide sequence of the (about 5 ϫ 103 to 5 ϫ 104 cells) of each dilution was transferred highly conserved quinolone resistance-determining region to MH agar plates supplemented with 1.0% horse blood contain- (QRDR) of the gyrA and parC genes as well as the vancomycin ing ampicillin, streptomycin, kanamycin, gentamicin, chloram- resistance–related genes. All the sequences reported here have phenicol, tetracycline, erythromycin, quinupristin-dalfopristin, been deposited in the DNA Data Bank of Japan, the European vancomycin, trimethoprim, linezolid, rifampin, clindamycin (Sig- Molecular Biology Laboratory, and the GenBank nucleotide se- ma, St. Louis, MO), or ciprofloxacin (MP Biomedicals, Illkirch, quence databases under accession numbers AB462548, France). Table 2 lists the MIC breakpoints suggested for Lacto- AB462549, AB462550, AB462551, and AB462552. bacillus by SCAN (17) and FEEDAP (18). We considered MICs higher than both of the suggested breakpoints as evidence of re- Detection of antibiotic resistance genes. We isolated ge- sistance (17, 18). nomic DNA from overnight Lactobacillus cultures by using IS- OPLANT (Nippon Gene, Tokyo, Japan) according to the manu- DNA sequencing, genome assembly, and coding sequence facturer’s protocol. We determined the presence of the following prediction. The Dragon Genomics Center, Takara Bio, Inc., using types of antibiotic resistance genes by PCR: tetracycline genes a 454 Life Sciences GS-20 sequencer, carried out the DNA se- encoding ribosomal protection proteins [tet(M), tet(O), tet(S), and quencing, genome assembly, and coding sequence prediction pro- tet(W)], genes encoding tetracycline efflux pumps [tet(K) and cedures necessary for draft sequencing of the whole genome. The tet(L)], and genes encoding glycopeptide resistance proteins (vanA, vanB, vanD, vanE, and vanG). We performed PCR by us- ing TaKaRa Taq polymerase (Takara Bio, Shiga, Japan), a Ther- TABLE 2. MICs for Lactobacillus brevis KB290 and breakpoint mal Cycler Model 9600 (PerkinElmer, Waltham, MA), and the MICs for Lactobacillus primer sets listed in Table 3. MICs for Conjugation experiments. We examined the transferability Lactobacillus brevis Breakpoint MICs for of antibiotic resistance from L. brevis KB290 to Enterococcus Antibiotic KB290 (␮g/ml) Lactobacillusa faecalis by filter mating, as follows. We grew donor and recipient Ampicillin
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