In this study, we isolated and characterized a bacteriocin-producing strain and two bacteriophages (P4, A3), showing antimicrobial effects against Clostridium perfringens, from chicken and swine feces by the spot-on-the lawn antagonism method. The selected strain was identified as Streptococcus hyoi...
In this study, we isolated and characterized a bacteriocin-producing strain and two bacteriophages (P4, A3), showing antimicrobial effects against Clostridium perfringens, from chicken and swine feces by the spot-on-the lawn antagonism method. The selected strain was identified as Streptococcus hyointestinalis by 16S rRNA gene sequencing. The bacteriocin from the isolated strain exhibited strong inhibitory activity against four strains of C. perfringens and all the tested strains of Listeria monocytogenes, and the bacteriocin were highly heat- and pH-stable even at pH 2, pH 10 and $121^{\circ}C$ for 15 min. We also evaluated the combined effects of the isolated bacteriocin and phages. Combining the phage treatments and bacteriocin resulted in a synergetic effect compared with the phage or the bacteriocin alone. In addition, during the probiotic test, the bacteriocin-producing S. hyointestinalis B19 strain reduced the population of C. perfringens significantly. Treatment with S. hyointestinalis B19 and a cocktail of lytic bacteriophages eradicated the C. perfringens KCTC $3269^T$, completely. Consequently, the isolated bacteriocin and bacteriophages represent candidates for effective biocontrol of C. perfringens, and bacteriocin-producing S. hyointestinalis B19 is a potential probiotic candidate for use in domestic animals.
In this study, we isolated and characterized a bacteriocin-producing strain and two bacteriophages (P4, A3), showing antimicrobial effects against Clostridium perfringens, from chicken and swine feces by the spot-on-the lawn antagonism method. The selected strain was identified as Streptococcus hyointestinalis by 16S rRNA gene sequencing. The bacteriocin from the isolated strain exhibited strong inhibitory activity against four strains of C. perfringens and all the tested strains of Listeria monocytogenes, and the bacteriocin were highly heat- and pH-stable even at pH 2, pH 10 and $121^{\circ}C$ for 15 min. We also evaluated the combined effects of the isolated bacteriocin and phages. Combining the phage treatments and bacteriocin resulted in a synergetic effect compared with the phage or the bacteriocin alone. In addition, during the probiotic test, the bacteriocin-producing S. hyointestinalis B19 strain reduced the population of C. perfringens significantly. Treatment with S. hyointestinalis B19 and a cocktail of lytic bacteriophages eradicated the C. perfringens KCTC $3269^T$, completely. Consequently, the isolated bacteriocin and bacteriophages represent candidates for effective biocontrol of C. perfringens, and bacteriocin-producing S. hyointestinalis B19 is a potential probiotic candidate for use in domestic animals.
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제안 방법
Lytic activities of the four phages were evaluated by measuring the increase in optical density (O.D.) of C. perfringens KCTC 3269T using a 96-well microplate reader. All the treatments showed inhibition of Clostridium perfringens KCTC 3269T growth compared with control (indicator only), however, the degrees of inhibition varied.
Lytic activities of the phages were evaluated by measuring the optical density (O.D.) during incubation of the mixtures containing C. perfringens and the phage. We incubated 3 mL of BHI broth with 1% (v/v) overnight cultured C.
The bile tolerance test was conducted by spreading the overnight cultures of S. hyointestinalis B19 on GM17 agar containing bovine bile (Sigma, USA) at 0%, 0.5%, 1%, 2%, 3%, 4%, and 5%, respectively. The plates were incubated at 37°C for 48 h (Dunne et al.
The combined effect of the isolated bacteriocin and phages was evaluated using the P4 and A3 phages in this experiment. The viable cell counts and O.
To identify the bacteriocin-producing bacteria, their DNA was isolated using a Powerfecal DNA isolation kit (MOBIO., USA) and the 16S rRNA gene was PCR-amplified using universal primers (27F and 1492R) followed by purification and sequencing (Solgent, Republic of Korea). The identified strains were confirmed and compared with BLAST in NCBI (Basic Local Aliments Search Tools in National Center for Biotechnology Information, USA).
대상 데이터
The pig fecal samples were collected from a local farm in Anseong, Gyeonggi-do, Republic of Korea. Fecal samples were isolated from 35-day-old Ross 308 broiler chickens, 38-week-old Hyline Brown laying hens, and 22-week-old Landrace pigs. Samples were used immediately for the screening of bacteriocin-producing bacteria and lytic bacteriophages.
These isolated phages were propagated by double-layer methods to reach 109 PFU/mL. In this process, four phages, A1, A3, P2, and P4 were selected for further experiments (data not shown).
Indicators used in this study were obtained from the Korean Collection for Type Culture (KCTC). Bacteriocinproducing bacteria were isolated from the feces of chickens and maintained at -80°C in stock solution (skim milk and glycerol mixture).
The fecal samples of chickens were collected from a poultry farm at Chung-Ang University. The pig fecal samples were collected from a local farm in Anseong, Gyeonggi-do, Republic of Korea.
The fecal samples of chickens were collected from a poultry farm at Chung-Ang University. The pig fecal samples were collected from a local farm in Anseong, Gyeonggi-do, Republic of Korea. Fecal samples were isolated from 35-day-old Ross 308 broiler chickens, 38-week-old Hyline Brown laying hens, and 22-week-old Landrace pigs.
이론/모형
, 2015). Any residual activity of the bacteriocin against C. perfringens KCTC 3269T was determined by the spot-on-the-lawn method (Teo and Tan, 2005).
Isolated phage titer was determined by spot-on-the-lawn method as described above. The propagated phages were serially diluted in SM buffer followed by the addition of 10 μL on the surface of soft BHI agar containing C.
The bacteriocin-producing strain was incubated in BHI broth (1% v/v) at 37°C, and the samples were removed at 2-h intervals to measure the antagonistic activity of bacteriocin. The bacteriocin activities were conducted by the spot-on-the-lawn method (Teo and Tan, 2005). Bacteriocin activity was expressed in terms of arbitrary units per mL (AU/mL), defined as the highest two-fold dilution showing definite inhibition of the indicator (Han et al.
The buffer was centrifuged at 10,000 g for 10 min and the supernatant was filtered to eliminate the indicator. The filtered supernatant was propagated by the double-layer method (Carvalho et al., 2010; Salama et al., 1989).
The pre-enriched samples were processed using the double-layer method for phage propagation (Zinno et al., 2010). Each double-layered plate contained 100 μL of samples and 100 μL of overnight incubated C.
성능/효과
All the treatments showed inhibition of Clostridium perfringens KCTC 3269T growth compared with control (indicator only), however, the degrees of inhibition varied. After 12 h of incubation, P2 at MOI 1, P4 at MOI 1 and 0.1, and A3 at MOI 1 and 0.1 treatments completely (rate of O.D. increasing values less than 5%) inhibited the growth of C. perfringens compared with control. The P2 at MOI 0.
perfringens KCTC 3269T using a 96-well microplate reader. All the treatments showed inhibition of Clostridium perfringens KCTC 3269T growth compared with control (indicator only), however, the degrees of inhibition varied. After 12 h of incubation, P2 at MOI 1, P4 at MOI 1 and 0.
In conclusion, the isolated bacteriocin and bacteriophages represent potential options for the biocontrol of C. perfringens. The bacteriocin-producing S.
In this study, the bacteriocin-producing strain, S. hyointestinalis B19 and two lytic phages, P4 and A3 phages, were isolated from feces of chickens and pigs, and were found to exhibit antagonistic effects against C. perfringens.
The strain was identified by 16S rDNA sequencing and compared with BLAST in NCBI. The 16S rDNA sequence showed 99.09% similarity with that of Streptococcus hyointestinalis ATCC 49169T. Therefore this strain was identified as Streptococcus hyointestinalis B19.
6). The antagonistic effect of S. hyointestinalis B19 strain alone and in combination with the bacteria and the lytic phages was evaluated. Treatment with S.
perfringens was also observed (data not shown), which was in accordance with previous reports (Dykes and Moorhead, 2002). The most important finding in this study was that a synergistic effect was observed when the phages and bacteriocin were combined. In addition, co-culturing with the bacteriocin-producing S.
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