Effect of Bacteriophages on Viability and Growth of Co-cultivated Weissella and Leuconostoc in Kimchi Fermentation.

This study aimed to understand the survival and growth patterns of bacteriophage-sensitive Weissella and Leuconostoc strains involved in kimchi fermentation. Dongchimi kimchi was prepared, and Weissella and Leuconostoc were co-cultivated in the dongchimi broth. Weissella cibaria KCTC 3807 growth was accompanied by rapid lysis with an increase in the bacteriophage quantity. Leuconostoc citreum KCCM 12030 followed the same pattern. The bacteriophage-insensitive strains W. cibaria KCTC 3499 and Leuconostoc mesenteroides KCCM 11325 survived longer under low pH as their growth was not accompanied by bacteriophages. The bacteriophage lysate of W. cibaria KCTC 3807 accelerated and promoted the growth of Leuconostoc. Overall, our results show that bacteriophages might affect the viability and population dynamics of lactic acid bacteria during kimchi fermentation.

A lytic bacteriophage of the newly emerging rainbow trout pathogen Weissella ceti.

This study was conducted to isolate and characterize a bacteriophage of a newly emerging pathogen, Weissella ceti, which causes weissellosis outbreaks of intensively farmed rainbow trout worldwide. The phage appeared together with the cultured Weissella ceti during isolation of pathogen from kidney of diseased rainbow trout. The morphological, physiological, proteomic and lytic spectrum were characterized. This phage, named PWc, belonged to the family Siphoviridae and possessed an isometric head (approximately 65 nm in diameter) and a flexible, non-contractile tail of 170-180 nm in length. The latent time and burst size of PWc were approximately 25 min and 16 PFU/infected cells, respectively. The PWc was relatively stable over a wide range of temperatures and pH values and possessed a broad lytic spectrum, lysing all 36 tested W. ceti strains isolated from diseased rainbow trout in Japan. The protein profile of the phage was obtained using SDS-PAGE analysis, and the potential packaging strategy was determined based on terminase large subunit sequence analysis. This is the first study to investigate a lytic bacteriophage of a newly emerging pathogen W. ceti that causes infectious disease in rainbow trout.

Genomics of Weissella cibaria with an examination of its metabolic traits.

Weissella is a genus of lactic acid bacteria (LAB) consisting of species formerly included in the Leuconostoc paramesenteroides group. Similar to other LAB, they are commonly found in fermented foods but have also been isolated from environmental and human samples. Currently there are 20 recognized species. Herein, three Weissella cibaria genomes were sequenced using Illumia Mi-Seq and Roche 454 technologies. Annotation was performed using the Prokka and JGI IMG pipelines. A thorough analysis of the genomics of the W. cibaria strains was performed, in addition to brief comparative analyses of the genus Weissella as a whole. Genomic sequence data from the newly sequenced W. cibaria strains and data available in GenBank for other Weissella strains was used (n = 10; four Weissella cibaria, one Weissella ceti, one Weissella confusa, one Weissella halotolerans, two Weissella koreensis and one Weissella paramesenteroides). The genomes had sizes varying from 1.3 to 2.4 Mb. DNA G+C contents ranged from 35 to 45 mol%. The core- and pan-proteome at genus and species levels were determined. The genus pan-proteome was found to comprise 4712 proteins. Analysis of the four W. cibaria genomes indicated that the core-proteome, consisting of 729 proteins, constitutes 69 % of the species pan-proteome. This large core-set may explain the divergent niches in which this species has been found. In W. cibaria, in addition to a number of phosphotransferase systems conferring the ability to assimilate plant-associated polysaccharides, an extensive proteolytic system was identified.

Bacteriophage ecology in a commercial cucumber fermentation.

To reduce high-salt waste from cucumber fermentations, low-salt fermentations are under development. These fermentations may require the use of starter cultures to ensure normal fermentations. Because potential phage infection can cause starter culture failure, it is important to understand phage ecology in the fermentations. This study investigated the phage ecology in a commercial cucumber fermentation. Brine samples taken from a fermentation tank over a 90-day period were plated onto deMan-Rogosa-Sharpe agar plates. A total of 576 lactic acid bacterial isolates were randomly selected to serve as potential hosts for phage isolation. Filtered brine served as a phage source. Fifty-seven independent phage isolates were obtained, indicating that 10% of the bacterial isolates were sensitive to phage attack. Phage hosts include Lactobacillus brevis (67% of all hosts), Lactobacillus plantarum (21%), Weissella paramesenteroides, Weissella cibaria, and Pediococcus ethanolidurans. Nearly 50% of phages were isolated on day 14, and the majority of them attacked L. brevis. Some phages had a broad host range and were capable of infecting multiple hosts in two genera. Other phages were species specific or strain specific. About 30% of phage isolates produced turbid pinpoint plaques or only caused reduced cell growth on the bacterial lawns. Six phages with distinct host ranges were characterized. The data from this study showed that abundant and diverse phages were present in the commercial cucumber fermentation, which could cause significant mortality to the lactic acid bacteria population. Therefore, a phage control strategy may be needed in low-salt cucumber fermentations.

Novel Podoviridae family bacteriophage infecting Weissella cibaria isolated from Kimchi.

The first complete genome sequence of a phage infecting Weissella cibaria (Weissella kimchii) is presented. The bacteriophage YS61 was isolated from kimchi, a Korean fermented vegetable dish. Bacteriophages are recognized as a serious problem in industrial fermentations; however, YS61 differed from many virulent phages associated with food fermentations since it was difficult to propagate and was very susceptible to resistance development. Sequence analysis revealed that YS61 resembles Podoviridae of the subfamily Picovirinae. Within the subfamily Picovirinae, the 29-like phages have been extensively studied, and their terminal protein-primed DNA replication is well characterized. Our data strongly suggest that YS61 also replicates by a protein-primed mechanism. Weissella phage YS61 is, however, markedly different from members of the Picovirinae with respect to genome size and morphology. Picovirinae are characterized by small (approximately 20-kb) genomes which contrasts with the 33,594-bp genome of YS61. Based on electron microscopy analysis, YS61 was classified as a member of the Podoviridae of morphotype C2, similar to the 29-like phages, but its capsid dimensions are significantly larger than those reported for these phages. The novelty of YS61 was also emphasized by the low number of open reading frames (ORFs) showing significant similarity to database sequences. We propose that the bacteriophage YS61 should represent a new subfamily within the family Podoviridae.

Isolation and characterisation of a novel Podoviridae-phage infecting Weissella cibaria N 22 from Nham, a Thai fermented pork sausage.

A novel Podoviridae lactic acid bacteria (LAB) phage from Nham, a Thai fermented pork sausage, is reported. From a total of 36 samples, 41 isolates of LAB were obtained and employed as hosts for the isolation of phages. From these LAB, only one phage, designated Φ 22, was isolated. The lactic acid bacterial isolate named N 22, sensitive to phage Φ 22 infection was identified by an API 50 CHL kit and N 22's complete sequence of the 16S rDNA sequence. BLASTN analysis of the 16S rDNA sequence revealed a 99% similarity to the 16S rDNA sequence of Weissella cibaria in the GenBank database. Electron micrographs indicated that the phage head was icosahedral with head size and tail length of 92 × 50 nm and 27 nm, respectively. On the basis of the morphology, this phage belongs to the family Podoviridae. Host-range determination revealed that the phage Φ 22 was not capable of infecting the other 40 isolates of LAB and referenced Weissella strains used. A one-step growth experiment showed that the latent period and burst size were estimated at 110 min and 55 phage particles/infected cell, respectively. Furthermore, the phage was infective over a wide range of pH (pH 5.0-8.0) and the D time of Φ 22 was calculated as 88 s at 70 °C and 15s at 80 °C. Phage titers decreased below the detection limit (20 PFU/ml) after heating for more than 60s at 80 °C, or 20s at 90 °C or less than 10s at 100 °C. The results from the study of Nham revealed that Φ 22 was active against the potential starter culture (W. cibaria N 22) for Nham fermentation. Phage infection could adversely affect the fermentation process of Nham by delaying acidification when using W. cibaria N 22 as a starter. However, the results from a sensory test revealed that the panelists did not detect any defects in the final products. This is the first report on the isolation of W. cibaria phage.