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Comparison of Delivery Methods in Phage Therapy against Flavobacterium columnare Infections in Rainbow Trout.

ABSTRACT: Viruses of bacteria, bacteriophages, specifically infect their bacterial hosts with minimal effects on the surrounding microbiota. They have the potential to be used in the prevention and treatment of bacterial infections, including in the field of food production. In aquaculture settings, disease-causing bacteria are often transmitted through the water body, providing several applications for phage-based targeting of pathogens, in the rearing environment, and in the fish. We tested delivery of phages by different methods (via baths, in phage-coated material, and via oral delivery in feed) to prevent and treat Flavobacterium columnare infections in rainbow trout fry using three phages (FCOV-S1, FCOV-F2, and FCL-2) and their hosts (FCO-S1, FCO-F2, and B185, respectively). Bath treatments given before bacterial infection and at the onset of the disease symptoms were the most efficient way to prevent F. columnare infections in rainbow trout, possibly due to the external nature of the disease. In a flow-through system, the presence of phage-coated plastic sheets delayed the onset of the disease. The oral administration of phages first increased disease progression, although total mortality was lower at the end of the experiment. When analysed for shelf-life, phage titers remained highest when maintained in bacterial culture media and in sterile lake water. Our results show that successful phage therapy treatment in the aquaculture setting requires optimisation of phage delivery methods in vivo.

SUBMITTER: Kunttu HMT

PROVIDER: S-EPMC8388691 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundFlavobacterium columnare is the causative agent of columnaris disease, a disease affecting many freshwater fish species. Methods for the genetic manipulation for some of the species within the Bacteroidetes, including members of the genus Flavobacterium, have been described, but these methods were not adapted to work with F. columnare.ResultsAs a first step toward developing a robust set of genetic tools for F. columnare, a protocol was developed to introduce the E. coli - Flavobacterium shuttle vector pCP29 into F. columnare strain C#2 by conjugal mating at an efficiency of 1.5 x 10(-3) antibiotic-resistant transconjugants per recipient cell. Eight of eleven F. columnare strains tested were able to receive pCP29 using the protocol. pCP29 contains the cfxA and ermF genes, conferring both cefoxitin and erythromycin resistance to recipient cells. Selection for pCP29 introduction into F. columnare was dependent on cfxA, as ermF was found not to provide strong resistance to erythromycin. This is in contrast to other Flavobacterium species where ermF-based erythromycin resistance is strong. The green fluorescent protein gene (gfp) was introduced into F. columnare strains under the control of two different native Flavobacterium promoters, demonstrating the potential of this reporter system for the study of gene expression. The transposon Tn4351 was successfully introduced into F. columnare, but the method was dependent on selecting for erythromycin resistance. To work, low concentrations of antibiotic (1 microg ml(-1)) were used, and high levels of background growth occurred. These results demonstrate that Tn4351 functions in F. columnare but that it is not an effective mutagenesis tool due to its dependence on erythromycin selection. Attempts to generate mutants via homologous recombination met with limited success, suggesting that RecA dependent homologous recombination is rare in F. columnare.ConclusionThe conjugation protocol developed as part of this study represents a significant first step towards the development of a robust set of genetic tools for the manipulation of F. columnare. The availability of this protocol will facilitate studies aimed at developing a deeper understanding of the virulence mechanisms of this important pathogen.

Project description:In the aquaculture sector, there is an increased interest in developing environmentally friendly alternatives to antibiotics in the treatment and prevention of bacterial infections. This requires an understanding of the effects of different treatments on the fish microbiota as a measure for improving the fish health status. In this study, we focused on the freshwater pathogen Flavobacterium psychrophilum and investigated the effects of antibiotics (florfenicol) and phage therapies on the gut microbiota of healthy and infected rainbow trout fry (1-2 g). Florfenicol-coated feed was administered for 10 days, starting two days after the infection procedure. A two-component mix of phage targeting F. psychrophilum (FpV4 and FPSV-D22) was continuously delivered by feed with a prophylactic period of 12 days. Samples of the distal intestine were collected over time (day -1 and 1, 8, and 33 days post-infection) and analyzed by community analysis targeting the 16S rRNA gene (V3-V4 region). Results showed the dysbiosis effect caused both by the infection and by florfenicol administration. Shifts in the overall composition were detected by β-diversity analysis, and changes in specific populations were observed during taxonomic mapping. Measures of α-diversity were only affected in infected fish (large variation observed 1 and 8 dpi). These community alterations disappeared again when fish recovered from the infection and the antibiotic treatment was terminated (33 dpi). Interestingly, phage addition altered the microbiota of the fish independently of the presence of their target bacterium. The overall gut bacterial community in fish fed phage-treated feed was different from the controls at each time point as revealed by β-diversity analysis. However, it was not possible to identify specific bacterial populations responsible for these changes except for an increase of lactic acid bacteria 33 dpi. Overall, the results indicate that the administered phages might affect the complex network of phage-bacteria interactions in the fish gut. Nevertheless, we did not observe negative effects on fish health or growth, and further studies should be directed in understanding if these changes are beneficial or not for the fish health with an additional focus on the host immune response.

Project description:Flavobacterium psychrophilum affects salmonid health worldwide and causes economic losses. The genetic diversity of the pathogen must be considered to develop control methods. However, previous studies have reported both high and low levels of genetic diversity. The present longitudinal study aimed at assessing the genetic diversity of F. psychrophilum at a small temporal and geographic scale. Four farms located on the same watershed in France were studied. Rainbow trout (Oncorhynchus mykiss) batches were monitored, and apparently healthy individuals were sampled over 1 year. A total of 288 isolates were recovered from fish organs (gills and spleen) and eggs. Pulsed field gel electrophoresis revealed high genetic diversity. Multilocus sequence typing performed on a selection of 31 isolates provided congruent results, as follows: 18 sequence types (STs) were found, of which 13 were novel. The mean gene diversity (H = 0.8413) was much higher than that previously reported for this host species, although the sampling was restricted to a single watershed and 1 year. Seven isolates out of 31 were assigned to clonal complex ST10 (CC-ST10), which is the predominant clonal complex in the main salmonid production areas. A split decomposition tree reflected a panmictic population. This finding is important for aquaculture veterinarians in their diagnostic procedure, as the choice of adequate antibiotic treatment is conditioned by the correct identification of the causative agent. Furthermore, this study expands our knowledge on genetic diversity required for the development of an effective vaccine against F. psychrophilumIMPORTANCE The bacterium Flavobacterium psychrophilum is a serious pathogen in many fish species, especially salmonids, that is responsible for considerable economic losses worldwide. In order to treat infections and to develop vaccines, the genetic diversity of this bacterium needs to be known. We assessed the genetic diversity of F. psychrophilum isolates from apparently healthy rainbow trout raised in several fish farms in the same watershed in France. Two different genotyping methods revealed high diversity. The majority of isolates were unrelated to clonal complex sequence type 10 (CC-ST10), the clonal complex that is predominant worldwide and associated with disease in rainbow trout. In addition, we found 13 novel sequence types. These results suggest that a diverse subpopulation of F. psychrophilum may be harbored by rainbow trout.

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Comparison of Delivery Methods in Phage Therapy against Flavobacterium columnare Infections in Rainbow Trout.

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