Effects of Lactiplantibacillus plantarum FBT215 and prebiotics on the gut microbiota structure of mice.

Imbalanced intestinal microbiota is associated with diseases, including inflammatory bowel disease and obesity, and diet can alter the structure of the gut microbiota. In this study, the effects of dietary treatments including the potential probiotic Lactiplantibacillus plantarum FBT215 with/without prebiotics on the intestinal microbiota composition of mice were investigated. Lactiplantibacillus plantarum FBT215 administration significantly decreased the Firmicutes/Bacteroidetes ratio and increased the abundance of Muribaculum and Duncaniella. The diversity within and between groups was measured according to α and ß diversity metrics, respectively. The Shannon index of α diversity decreased significantly in all treatment groups except the probiotic group, although this group showed an increase in the Chao1 index. Principal coordinate analysis of ß diversity showed that the groups had different species distributions. Finally, gamma-aminobutyric acid (GABA) concentration increased in groups fed L. plantarum FBT215. These findings improve our understanding of the association between the gut microbiota structure and specific probiotic/prebiotic-containing diets.

Probiotic Properties and Optimization of Gamma-Aminobutyric Acid Production by Lactiplantibacillus plantarum FBT215.

Gamma-aminobutyric acid (GABA) improves various physiological illnesses, including diabetes, hypertension, depression, memory lapse, and insomnia in humans. Therefore, interest in the commercial production of GABA is steadily increasing. Lactic acid bacteria (LAB) have widely been reported as a GABA producer and are safe for human consumption. In this study, GABA-producing LAB were preliminarily identified and quantified via GABase assay. The acid and bile tolerance of the L. plantarum FBT215 strain were evaluated. The one-factor-at-a-time (OFAT) strategy was applied to determine the optimal conditions for GABA production using HPLC. Response surface methodology (RSM) with Box-Behnken design was used to predict the optimum GABA production. The strain FBT215 was shown to be acid and bile tolerant. The optimization of GABA production via the OFAT strategy resulted in an average GABA concentration of 1688.65 ± 14.29 µg/ml, while it was 1812.16 ± 23.16 µg/ml when RSM was applied. In conclusion, this study provides the optimum culture conditions for GABA production by the strain FBT215 and indicates that L. plantarum FBT215 is potentially promising for commercial functional probiotics with health claims.

Large-Scale Production of Cronobacter sakazakii Bacteriophage Φ CS01 in Bioreactors via a Two-Stage Self-Cycling Process.

Cronobacter sakazakii is an opportunistic pathogenic bacterium found in powdered infant formula and is fatal to neonates. Antibiotic resistance has emerged owing to overuse of antibiotics. Therefore, demand for high-yield bacteriophages as an alternative to antibiotics has increased. Accordingly, we developed a modified mass-production method for bacteriophages by introducing a two-stage self-cycling (TSSC) process, which yielded high-concentration bacteriophage solutions by replenishing the nutritional medium at the beginning of each process, without additional challenge. pH of the culture medium was monitored in real-time during C. sakazakii growth and bacteriophage CS01 propagation, and the changes in various parameters were assessed. The pH of the culture medium dropped to 5.8 when the host bacteria reached the early log phase (OD540 = 0.3). After challenge, it decreased to 4.65 and then recovered to 4.94; therefore, we set the optimum pH to challenge the phage at 5.8 and that to harvest the phage at 4.94. We then compared phage production during the TSSC process in jar-type bioreactors and the batch culture process in shaker flasks. In the same volume of LB medium, the concentration of the phage titer solution obtained with the TSSC process was 24 times higher than that obtained with the batch culture process. Moreover, we stably obtained high concentrations of bacteriophage solutions for three cycles with the TSSC process. Overall, this modified TSSC process could simplify large-scale production of bacteriophage CS01 and reduce the unit cost of phage titer solution. These results could contribute to curing infants infected with antibiotic-resistant C. sakazakii.

Isolation, characterization, and genomic analysis of the novel T4-like bacteriophage ΦCJ20.

Pathogenic Escherichia coli infections have been consistently reported annually. The basic characteristics and genome of the newly isolated ΦCJ20 from swine feces was analyzed. To determine basic characteristics, dotting assays and double-layer agar assays were conducted. Bacteriophage particles were analyzed via transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed to determine the sizes of major structural proteins. The complete genome of the phage was analyzed. Bacteriophage particles were identified as Myoviridae, with a head measuring 110.57 ± 1.89 nm and a contractile tail measuring 107.97 ± 3.20 nm and were found to infect E. coli. Major structural proteins of ΦCJ20 showed two well-pronounced bands of approximately 53.6 and 70.9 kDa. The genome size of ΦCJ20 was 169,884 bp, and 118 of 307 open reading frames were annotated. This study provides a baseline for the development of E. coli infection treatment strategies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00906-y.

Genetic Analysis and Characterization of a Bacteriophage ØCJ19 Active against Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the major pathogenic E. coli that causes diarrhea and edema in post-weaning piglets. In this study, we describe the morphology and characteristics of ØCJ19, a bacteriophage that infects ETEC, and performed genetic analysis. Phage ØCJ19 belongs to the family Myoviridae. One-step growth curve showed a latent phase of 5 min and burst size of approximately 20 phage particles/infected cell. Phage infectivity was stable for 2 h between 4°C and 55°C, and the phage was stable between pH 3 and 11. Genetic analysis revealed that phage ØCJ19 has a total of 49,567 bases and 79 open reading frames (ORFs). The full genomic sequence of phage ØCJ19 showed the most similarity to an Escherichia phage, vB_EcoS_ESCO41. There were no genes encoding lysogeny, toxins, virulence factors, or antibiotic resistance in this phage, suggesting that this phage can be used safely as a biological agent to control ETEC. Comparative genomic analysis in terms of the tail fiber proteins could provide genetic insight into host recognition and the relationship with other coliphages. These results showed the possibility to improve food safety by applying phage ØCJ19 to foods of animal origin contaminated with ETEC and suggests that it could be the basis for establishing a safety management system in the animal husbandry.

Characterization and Genomic Analysis of Novel Bacteriophage ΦCS01 Targeting Cronobacter sakazakii.

Cronobacter sakazakii is an opportunistic pathogen causing serious infections in neonates. In this study, a bacteriophage ΦCS01, which infects C. sakazakii, was isolated from swine feces and its morphology, growth parameters, and genomic analysis were investigated. Transmission electron microscopy revealed that ΦCS01 has a spherical head and is 65.74 nm in diameter with a 98.75 nm contracted tail, suggesting that it belongs to the family Myoviridae. The major viral proteins are approximately 71 kDa and 64 kDa in size. The latent period of ΦCS01 was shown to be 60 min, and the burst size was 90.7 pfu (plaque-forming units)/ infected cell. Bacteriophage ΦCS01was stable at 4-60°C for 1 h and lost infectivity after 1 h of heating at 70°C. Infectivity remained unaffected at pH 4-9 for 2 h, while the bacteriophage was inactivated at pH <3 or >10. The double-stranded ΦCS01 DNA genome consists of 48,195 base pairs, with 75 predicted open reading frames. Phylogenetic analysis is closely related to that of the previously reported C. sakazakii phage ESP2949-1. The newly isolated ΦCS01 shows infectivity in the host bacterium C. sakazakii, indicating that it may be a promising alternative to antibacterial agents for the removal of C. sakazakii from powdered infant formulas.

Whole-Genome Sequencing and Genomic Analysis of a Virulent Bacteriophage Infecting Bacillus cereus.

OBJECTIVE: Infants with a weak immune system are prone to infection with Bacillus cereus, which is commonly found in natural environments. With the aim of achieving better control of this pathogenic bacterium, in the present study we characterized a new bacteriophage, ΦBC01. METHODS: Bacteriophage particles were analyzed by transmission electron microscopy. Factors influencing adsorption were identified in a double-layer plaque assay. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was conducted to determine the size of major structural proteins. The complete genome of the phage was analyzed. RESULTS: Bacteriophage particles (105.3 ± 3.1 nm icosahedral head and 208.8 ± 3.6 nm contractile tail) were identified as Myoviridae. ΦBC01 was found to have host specificity to B. cereus. Major structural proteins of ΦBC01 showed 2 well-pronounced bands of 99 and 56 kDa. The 158,385-bp genome sequence of ΦBC01 was determined: 56 of the 239 open reading frames were annotated, indicating involvement in bacteriophage DNA manipulation, cell lysis, packaging, virion structure, and other functions. CONCLUSION: Because of characterization and genotyping of a new bacteriophage from soil samples containing earthworms, this study provides a baseline for the development of alternatives to antibiotics.