Rapid Species Identification and Partial Strain Differentiation of Clostridium butyricum by PCR Using 16S‐23S rDNA Intergenic Spacer Regions

Abstract

Some Clostridium butyricum strains have been used as probiotics for both humans and animals. Strain‐specific identification is necessary for the manufacturing process of probiotics. The aim of this study was to determine whether there are sufficient genetic variations in 16S‐23S intergenic spacer regions (ISRs) to discriminate C. butyricum at the biovar level. We amplified ISRs from five reference strains, a probiotic strain (MIYAIRI 588) and 22 isolates, and we classified them into four groups on the basis of amplification patterns (type A through D). However, amplification of ISRs is not sufficient for discriminating strains. Moreover, we compared genetic structures of these ISRs. Sequence analysis revealed that the size variations of ISRs were generated by the insertion of tRNA genes and unique sequences into the internal portion, while the external portions were highly conserved. On the basis of the highly conserved nucleotide sequences within the ISRs, we developed a PCR primer set specific to C. butyricum. In addition, the PCR primer designed from the unique inserted sequence in type B strain was useful to differentiate probiotic strains at the biovar level.

Safety assessment of the Clostridium butyricum MIYAIRI 588® probiotic strain including evaluation of antimicrobial sensitivity and presence of Clostridium toxin genes in vitro and teratogenicity in vivo

“…The biochemical and genetic characteristics were well identified and reported previously. 26,27 Antimicrobial powders were obtained from the following sources: benzyl penicillin, lincomycin, colistin, bacitracin, chlortetracycline, oxytetracycline, doxycycline, amikacin and fosfomycin from Wako Pure Chemical Industries Ltd (Osaka, Japan); ampicillin, chloramphenicol, clindamycin hydrochloride, erythromycin, gentamicin sulfate, kanamycin sulfate, metronidazole, streptomycin sulfate, tetracycline hydrochloride, vancomycin hydrochloride, carbenicillin, cefmetazole, cinoxacin, norfloxacin, ofloxacin, ciprofloxacin, lomefloxacin, cephalothin, cefazolin, and cefoperazone from Sigma-Aldrich Co. (St Louis, Missouri, USA); nalidixic acid, enoxacin, tosufloxacin, ceftizoxime, ceftazidime, minocycline, neomycin, and spiramycin from Tokyo Chemical Industry Co. Ltd (Tokyo, Japan); and imipenem and paromomycin from LKT Laboratories, Inc. (St Paul, Minnesota, USA). Antimicrobial powders were diluted as recommended and prepared fresh for each experiment.…”

“…,27 Standard culturing conditions for CBM 588 are anaerobic conditions (nitrogen 80%, carbon dioxide 10%, and hydrogen 10%) at 37 C for 24 h in brain heart infusion (BHI) broth (Oxoid, Hampshire, UK). Evaluation of minimum inhibitory concentration included incubation with various antibiotics.ATCC strains (Bacteroides fragilis ATCC 25285, C. butyricum ATCC 19398 T ) were obtained directly from the American Type Culture Collection (ATCC.…”

Analysis of the 16S–23S rRNA Gene Internal Transcribed Spacer Region in Klebsiella Species

“…The 16S-23S rRNA gene inter-nal transcribed spacer (ITS) sequence, which is not subject to the same selective pressure as the rRNA genes and consequently has a 10-times-greater evolution rate, appears to be able to overcome the apparent limitation of rRNA genes (2,14). Sequence and length polymorphisms found in the ITS are increasingly being used as tools for bacterial species and subspecies identification (17,27,29,37), typing (25,37), and evolutionary studies (1,12,33). Gürtler et al reported that ITS sequence analysis is complementary to the 16S rRNA gene for phylogenetic analysis (13).…”

Use of a DNA Microarray for Detection and Identification of Bacterial Pathogens Associated with Fishery Products

“…In comparison with the 16S rRNA gene, the sequence of the ITS region is considered to be under more evolutionary pressure and is therefore prone to more genetic variation. Sequence and length polymorphisms of ITS regions have been used increasingly as tools for bacterial species and/or subspecies identification (19,21) and typing (16,25), as well as for evolutionary studies (1,7,23,29). Primers wl-5793 and wl-5794 were designed based on highly conserved regions of the 16S and 23S rRNA genes and were used successfully to amplify the ITS regions of all strains.…”