Exploring the Effect of Phage Therapy in Preventing Vibrio anguillarum Infections in Cod and Turbot Larvae.

The aquaculture industry is suffering from losses associated with bacterial infections by opportunistic pathogens. Vibrio anguillarum is one of the most important pathogens, causing vibriosis in fish and shellfish cultures leading to high mortalities and economic losses. Bacterial resistance to antibiotics and inefficient vaccination at the larval stage of fish emphasizes the need for novel approaches, and phage therapy for controlling Vibrio pathogens has gained interest in the past few years. In this study, we examined the potential of the broad-host-range phage KVP40 to control four different V. anguillarum strains in Atlantic cod (Gadus morhua L.) and turbot (Scophthalmus maximus L.) larvae. We examined larval mortality and abundance of bacteria and phages. Phage KVP40 was able to reduce and/or delay the mortality of the cod and turbot larvae challenged with V. anguillarum. However, growth of other pathogenic bacteria naturally occurring on the fish eggs prior to our experiment caused mortality of the larvae in the unchallenged control groups. Interestingly, the broad-spectrum phage KVP40 was able to reduce mortality in these groups, compared to the nonchallenge control groups not treated with phage KVP40, demonstrating that the phage could also reduce mortality imposed by the background population of pathogens. Overall, phage-mediated reduction in mortality of cod and turbot larvae in experimental challenge assays with V. anguillarum pathogens suggested that application of broad-host-range phages can reduce Vibrio-induced mortality in turbot and cod larvae, emphasizing that phage therapy is a promising alternative to traditional treatment of vibriosis in marine aquaculture.

Lactococcus lactis Diversity in Undefined Mixed Dairy Starter Cultures as Revealed by Comparative Genome Analyses and Targeted Amplicon Sequencing of epsD.

Undefined mesophilic mixed (DL) starter cultures are used in the production of continental cheeses and contain unknown strain mixtures of Lactococcus lactis and leuconostocs. The choice of starter culture affects the taste, aroma, and quality of the final product. To gain insight into the diversity of Lactococcus lactis strains in starter cultures, we whole-genome sequenced 95 isolates from three different starter cultures. Pan-genomic analyses, which included 30 publically available complete genomes, grouped the strains into 21 L. lactis subsp. lactis and 28 L. lactis subsp. cremoris lineages. Only one of the 95 isolates grouped with previously sequenced strains, and the three starter cultures showed no overlap in lineage distributions. The culture diversity was assessed by targeted amplicon sequencing using purR, a core gene, and epsD, present in 93 of the 95 starter culture isolates but absent in most of the reference strains. This enabled an unprecedented discrimination of starter culture Lactococcus lactis and revealed substantial differences between the three starter cultures and compositional shifts during the cultivation of cultures in milk.IMPORTANCE In contemporary cheese production, standardized frozen seed stock starter cultures are used to ensure production stability, reproducibility, and quality control of the product. The dairy industry experiences significant disruptions of cheese production due to phage attacks, and one commonly used countermeasure to phage attack is to employ a starter rotation strategy, in which two or more starters with minimal overlap in phage sensitivity are used alternately. A culture-independent analysis of the lactococcal diversity in complex undefined starter cultures revealed large differences between the three starter cultures and temporal shifts in lactococcal composition during the production of bulk starters. A better understanding of the lactococcal diversity in starter cultures will enable the development of more robust starter cultures and assist in maintaining the efficiency and stability of the production process by ensuring the presence of key bacteria that are important to the characteristics of the product.

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.

Characterization of a Leuconostoc bacteriophage infecting flavor producers of cheese starter cultures.

Dairy siphovirus φLmd1, which infects starter culture isolate Leuconostoc mesenteroides subsp. dextranicum A1, showed resistance to pasteurization and was able to grow on 3 of the 4 commercial starter cultures tested. Its 26,201-bp genome was similar to that of Leuconostoc phage of vegetable origin but not to those of dairy phages infecting Lactococcus.