Isolation and Characterization of Two Lytic Bacteriophages, φSt2 and φGrn1; Phage Therapy Application for Biological Control of Vibrio alginolyticus in Aquaculture Live Feeds

Abstract

Bacterial infections are a serious problem in aquaculture since they can result in massive mortalities in farmed fish and invertebrates. Vibriosis is one of the most common diseases in marine aquaculture hatcheries and its causative agents are bacteria of the genus Vibrio mostly entering larval rearing water through live feeds, such as Artemia and rotifers. The pathogenic Vibrio alginolyticus strain V1, isolated during a vibriosis outbreak in cultured seabream, Sparus aurata, was used as host to isolate and characterize the two novel bacteriophages φSt2 and φGrn1 for phage therapy application. In vitro cell lysis experiments were performed against the bacterial host V. alginolyticus strain V1 but also against 12 presumptive Vibrio strains originating from live prey Artemia salina cultures indicating the strong lytic efficacy of the 2 phages. In vivo administration of the phage cocktail, φSt2 and φGrn1, at MOI = 100 directly on live prey A. salina cultures, led to a 93% decrease of presumptive Vibrio population after 4 h of treatment. Current study suggests that administration of φSt2 and φGrn1 to live preys could selectively reduce Vibrio load in fish hatcheries. Innovative and environmental friendly solutions against bacterial diseases are more than necessary and phage therapy is one of them.

Aislamiento y caracterización del bacteriófago Va1 específico a Vibrio alginolyticus

“…Además, otros autores han reportado el aislamiento de bacteriófagos líticos contra V. alginolyticus a partir de muestras de ambiente acuático (Lin et al 2012, Kalatzis et al 2016). …”

“…Sin embargo, la curva de un paso depende del índice de multiplicidad de infección (MOI), la cual se sugiere que debe utilizarse en un rango de 0.1 y 0.01 debido a que una MOI más alta incrementa la probabilidad de que una bacteria sea infectada por más de un fago incrementado así el número total de fagos por centro infectivo (Middelboe et al 2010). Así, el fago Va1 tiene un tamaño de explosión de 192 unidades víricas por centro infectivo utilizando un índice de multiplicidad de infección (MOI) de 0.1 comparado con otros fagos específicos a V. alginolyticus cuyo tamaño de explosión fue de 97 y 44 fagos por centro infectivo utilizando un MOI de 0.01 (Kalatzis et al 2016). Se ha evidenciado que mientras más corto es el periodo de latencia y mayor el tamaño de explosión, el bacteriófago puede ser aplicado con mayores probabilidades de éxito en fagoterapia (Middelboe et al 2010, Abedon et al 2001, ya que al producirse más partículas virales a partir de un centro infectivo, aumentan las posibilidades de controlar mejor las poblaciones bacterianas.…”

Comparative Functional Genomic Analysis of Two Vibrio Phages Reveals Complex Metabolic Interactions with the Host Cell

“…Both phages φ Grn1 and φ St2 belong to Myoviridae family and have been isolated from coastal seawater in Crete, Greece (Kalatzis et al, 2016). The bacterial host was a clinical V. alginolyticus strain isolated from sick gilthead seabream ( Sparus aurata ) and has been fully sequenced (Castillo et al, 2015).…”

“…Cells were immediately centrifuged at 4°C and washed with 150 mM NaCl prior to RNA extraction. The duration of the experiment was set based on the latency time which is 30 min for both phages (Kalatzis et al, 2016). …”

“…These Vibrio alginolyticus lytic phages have also been proposed as promising agents for disinfecting live feeds in aquaculture (Kalatzis et al, 2016), but information over genomic features was lacking. In this study, the phages were sequenced, annotated, and compared, revealing the largest known double stranded DNA Vibrio phage until now with various notable features.…”