Wan-Kyu Lee

Real-time PCR for differentiation of F4 (K88) variants (F4ab, F4ac, F4ad) of enterotoxigenic Escherichia coli from diarrhoeic piglets

A one-step real-time PCR using one set of oligonucleotide primers and three probes was developed for differentiation of F4 (K88) variants (F4ab, F4ac, F4ad) of enterotoxigenic Escherichiacoli (ETEC) from diarrhoeic pigs. The limits of detection of F4ab, F4ac and F4ad in broth dilution were 10(6), 10(5) and 10(4)colony forming units (CFU)/mL, respectively. In faecal samples spiked with E.coli, the limits of detection of F4ab, F4ac and F4ad were 10(6), 10(6) and 10(4)CFU/g faeces, respectively, without enrichment and 10(3), 10(2) and 10(2)CFU/g faeces following enrichment. In 42 ETEC field isolates from pigs in Korea encoding the F4 gene, all were identified as the F4ac variant.

Potential use of lactic acid bacteria Leuconostoc mesenteroides as a probiotic for the removal of Pb(II) toxicity

It has been demonstrated that certain lactic acid bacteria (LAB) can sequester metal ions by binding them to their surfaces. In the present study, lead (Pb)-resistant LAB were isolated from kimchi, a Korean fermented food. A total of 96 different LAB strains were isolated, and 52 strains showed lead resistance. Among them, an LAB strain-96 (L-96) identified as Leuconostoc mesenteroides showed remarkable Pb resistance and removal capacity. The maximum adsorption capacity of this strain calculated using the Langmuir isotherm was 60.6 mg Pb/g. In an in vivo experiment, young male mice were provided with water (A), Pb-water (B), or Pb-water+ L-96 (C) during puberty. Lower glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels in Pb-exposed male mice that received strain L-96 as a probiotic were suggestive of reduced hepatotoxicity. Moreover, feces from mice treated with L-96 contained more Pb than feces from untreated mice. Increased Pb elimination likely reduced internal accumulation, and this hypothesis was supported by significantly lower Pb concentrations in kidneys and testes of the mice treated with strain L-96. The motility and ATP content of epididymal spermatozoa were partially restored if strain L-96 was administered. In conclusion, isolated L-96 LAB had lead-biosorption activity and efficiently detoxified lead-poisoned male mice, resulting in recovering male reproductive function. These results suggest the potential use of LAB as a probiotic to protect humans from the adverse effects of Pb exposure.

Immunomodulatory Potential of Weissella cibaria in Aged C57BL/6J Mice

Aging is associated with distinct changes in immune cells and a decline in immune function, leading to increased susceptibility to infection and reduced responses to vaccination. Certain strains of lactic acid bacteria exert beneficial effects on the immune system. Previously, we reported that Weissella cibaria JW15 isolated from kimchi possesses immune stimulatory activity in vitro. In the present study, we further investigated whether oral administration of JW15 improves immune function in aged mice. Eighteen-month-old female mice were administered JW15 daily at low (JW15-L; 1 × 10⁸ CFU/mouse) or high dosage (JW15-H; 1 × 10⁹ CFU/mouse), or with Lactobacillus rhamnosus GG (LGG) using oral gavage. Twomonth- old female mice were included as healthy young mice. After 4 weeks, the mice were euthanized and immune profiles were analyzed using whole blood and spleen. In complete blood count analysis, the numbers of white and red blood cells were significantly increased in the JW15-L group compared with those in the old mouse (OM) control group. In addition, administration of either JW15 of LGG resulted in higher numbers of splenocytes in comparison with the OM group. Furthermore, proliferative potentials were higher in all probiotic groups than OM. Cytokines such as IFN-γ and IL-6 were secreted at higher levels in splenocytes isolated from JW15-fed mice than in OM control mice. Similarly, mRNA expression of various cytokines was altered in the JW15 groups. Collectively, these results suggest that JW15 supplementation induces immunomodulatory effects in aged mice and indicate JW15 as a potential probiotic strain to improve immune function in aged animals.

Real-time PCR for differentiation of F18 variants among enterotoxigenic and Shiga toxin-producing Escherichia coli from piglets with diarrhoea and oedema disease.

One-step real-time PCR using one set of primers and four probes was developed for differentiation of F18 variants (F18 common, F18ab, F18ac, F18new variant) of enterotoxigenic (ETEC) and Shiga toxin-producing (STEC) Escherichia coli from piglets with diarrhoea and oedema disease. The limits of detection for F18common, F18ab, F18ac, and F18new variant were 10(7), 10(7), 10(5) and 10(7)colony forming units/g faeces, respectively. Of 94 Korean isolates of E. coli encoding F18, 70 were F18ac (43 STEC/ETEC, 4 STEC and 23 ETEC), 15 were F18ab (all STEC) and nine were F18new variant (1 STEC/ETEC, 7 STEC, 1 ETEC).

Screening of Bacteriocin-producing Enterococcus faecalis Strains for Antagonistic Activities against Clostridium perfringens

This study was conducted to isolate and characterize bacteriocin-producing bacteria against Clostridium perfringens (C. perfringens) from domestic animals to determine their usefulness as probiotics. Bacteriocin-producing bacteria were isolated from pig feces by the spot-on-lawn method. A total of 1,370 bacterial stains were isolated, and six were tentatively selected after identifying the inhibitory activity against the pathogenic indicator C. perfringens KCTC 3269 and KCTC 5100. The selected strains were identified as Enterococcus faecalis (E. faecalis) by 16s rRNA sequencing. Most of the isolated bacterial strains were resistant to 0.5% bile salts for 48 h and remained viable after 2 h at pH 3.0. Some E. faecalis also showed strong inhibitory activity against Listeria monocytogenes KCTC 3569, KCTC 3586 and KCTC 3710. In the present study, we finally selected E. faecalis AP 216 and AP 45 strain based on probiotic selection criteria such as antimicrobial activity against C. perfringens and tolerance to acid and bile salts. The bacteriocins of E. faecalis AP 216 and AP 45 strains were highly thermostable, showing anticlostridial activities even after incubation at 121℃ for 15 min. These bacteriocinproducing bacteria and/or bacteriocins could be used in feed manufacturing as probiotics as an alternative to antibiotics in the livestock industry.

Immune enhancing effects of Weissella cibaria JW15 in C57BL/6J mice aged 28 months

Aging is a growing concern of age-associated aberrations in immune system or immunosenescence. Probiotics contain not only these general health advantages but also other the regulation of host immune function. Among those probiotics, the immunomodulatory effects of Weissella cibaria JW15 (JW15) strain has already been reported in our previous studies. The objective of this study is to assess improved immunity of JW15 strain in aged mice. In experiment, mice were divided into five groups. Twenty eight–month-old C57BL/6J mice were given a daily dose of 1×10 CFU/ mouse (JW15-H group) and 1×10 CFU/mouse (JW15L group) of viable JW15. The young mice group (YM) and old mice group (OM) were given PBS. After four weeks of administration, mice were euthanized. Mice blood was collected and analyzed for complete blood cell count (CBC). Then, mice spleen was weighed and analyzed for splenocyte. Finally, we measured the concentration of cytokine secreted by splenocyte and serum. The results showed splenocyte proliferation of JW15-L group by the addition of LPS and concanavalin A was significantly higher than that of OM group. Splenocyte TNF-α production was significantly increased by JW15 intake. In particular, splenocyte TNF-α production of JW15-L group by the addition of LPS (5 μg/mL) was significantly greater than that other group. Splenocyte IL-6 production was also the highest in JW15-L group. Serum IFN-γ production of JW15-L (59.13 ± 16.13 pg/mL) group was significantly higher than that of OM group (39.71 ± 2.51 pg/ mL). When compared to the OM group (32.22 ± 1.92 pg/mL), Serum TNF-α production was significantly increased by JW15-H intake (69.01± 26.51 pg/mL). In conclusion, JW15 enhanced immunomodulative effects. In particular, the effect was significantly excellent in JW15-L group (1 × 10 CFU/mouse). Therefore, Original Article