Phage therapy as an approach to prevent Aeromonas salmonicida infections in juvenile flounder fish Yolanda J. Silva, Carla Pereira, Luísa Santos, Ângela Cunha, Newton Gomes, Ricardo Calado, Adelaide Almeida1* 1Department
of Biology and CESAM, University of Aveiro, Campus Universitário de Santiago, 3810-193 Aveiro, Portugal
* Corresponding author: aalmeida@ua.pt Four fish groups:
Introduction Fish intensive culture is an important economic activity. Bacterial infections in fishfarming plants, however, cause high mortality rates in larvae, juveniles and adult fish . Aeromonas salmonicida is the causative agent of furunculosis, a systemic disease characterized by high mortality and morbidity. The worldwide rising of antibiotic resistance in common pathogenic bacteria and the concern about the spreading of antibiotics in the environment, enhance the need for new methods to control fish pathogens. Phage therapy appears to represent a useful and flexible tool for the inactivation of bacterial pathogens in aquaculture. The aim of the present study was to test the efficacy of phage therapy applied during the production of juvenile fish.
- Aeromonas + Phage: fish infected with A . salmonicida (108 CFUmL-1) and treated with AS-A phage (104 PFU mL-1). - Control Aeromonas: fish infected with the bacterium (108 CFUmL-1) but not treated with the phage. - Control Phage: fish not infected with the bacterium but added of phages (104 PFU mL1).
Study area
Samples were incubated at 25 °C and water samples were collected at 0, 6, 12, 24, 48, and 72 h after infection for determine bacteria and phage concentration . Fish mortality in each group was determined by visual inspection (inspecting for dead fish) at 48, 60, 72 and 96 h. was
Effect of AS-A phage on the structure of the bacterial community of aquaculture water Fig.1 Ria de Aveiro with the aquaculture system (A).
This study was conducted in the semiintensive aquaculture system Corte das Freiras located in the estuarine system Ria de Aveiro (Fig.1). The aquaculture is divided in ten earthen ponds of approximately 2500 m2 each
Phage therapy in juvenile sole fish
- Control Fish: fish without phage and without bacteria
Bacterial and phage concentration determined as described before.
Material and Methods
Fig. 3 Inactivation of A. salmonicida by the AS-A phage at a MOI of 100 during the 24 h. CB – Bacteria control; Phage AS-A – Bacteria plus phage. Values represent the mean of three experiments; error bars represent the standard deviation.
Three negative controls [T0, T8, T8 (TSB-Cl)liquid medium TSB with 1% of chloroform, suspension where the phages were maintained in the laboratory] and water samples added AS-A was incubated at 25 ºC during T8 (AS-A phage).
Fig. 4 Inactivation of A. salmonicida by the AS-A phage in juvenile sole fish during the 72 h. Aeromonas + phage Bacteria plus phage; Control bacteria: fish infected with bacteria but not treated with phages; Control phage: fish with phages; Control Fish: fish without phages and without bacteria. Values represent the mean of three experiments; error bars represent the standard deviation. Table1:Mortality of juvenile fish Mortality (n=90)
The results showed that AS-A phage inhibited the growth of the A. salmonicida, causing a decrease in bacterial abundance of ≈ 3.5, 3 and 3 log after 6, 12 and 24 h of treatment (Fig. 4), respectively. After 72 h, the mortality of juvenile fish was higher (34%) in the control groups than in the infected and treated groups (0%), indicating that phage treatment was effective (Table 1).
Time (hours) 48h
60h
72h
96h
Average percentage of all assays (%)
0
0
0
3
0,033
0
0
0
0
0
Control Aermonas
11
7
14
-
35,55
Aeromonas + Phage
0
0
0
1
0,01
Sample
Control Fish
Control Phage
Effect AS-A phage on the structure of the bacterial community of aquaculture water and in fish microbiota
After incubation, each sample was filtered through 0,22 µm pore-size-filters. The RNA was extracted from the sample [2] and reverse transcription of mRNA was done. The cDNA was amplified and Denaturing Gradient Gel Electrophoresis (DGGE) was done.
Bacteria and growth conditions A. salmonicida (CECT 894) grew at 30 ºC, pH 7.3, in tryptic soy broth (TSB).
Effect of AS-A phage on the structure of the bacterial community of fish Incubated at 25 ºC during 72 h:
Bacteriophage
-
AS-A phage was isolated from sewage water, according to the procedure described by Mateus et al, 2014 [1].
Control Fish - fish without phages and bacteria
-
Fish + Phage– fish after incubation with phages
-
Fish + AS - fish after incubation with bacteria
Phage therapy assays Phage therapy was performed using AS-A (107 PFU mL-1) phage using the bacterium A. salmonicida (105 CFU mL-1) as host, at a multiplicity of infection (MOI) of 100. Bacteria and phage were inoculated in TSB and incubated at 25 °C. Two control samples were included: the bacterial control and the phage control, without phages and without bacteria, respectively. For host quantification, aliquots were serially diluted, pour-plated in duplicate in tryptic Soy agar medium (TSA) and incubated at 30 °C for 48 hours.
-
Fish + AS + Phage – fish after incubation with bacteria and phages
After incubation, fish was homogenized in extraction buffer, DNA was extraction [2] and reverse transcription of mRNA was done. The cDNA was amplified and Denaturing Gradient Gel Electrophoresis (DGGE) was done.
Fig. 5 DGGE profile cDNA fragments of the 16S gene amplified by PCR following addition of phage of A. salmonicida to the natural bacterial community of aquaculture water. M - molecular weight marker; T0 water sample without phage at time 0; T8 - water sample after 8 h incubation without phage; T8 (TSB-Cl) - water sample with TSB culture medium and chloroform at 1% after 8 h incubation without phage; T8 (Phage AS) water sample with TSB culture medium and chloroform at 1% with phage AS-A after 8 h incubation.
Fig. 6 DGGE profile of the 16S gene fragments amplified by PCR following addition of phage to the fish. M molecular weight marker; Control Fish - Fish sample (gastrointestinal area) without phage; Phage + fish – fish sample (gastrointestinal area) after incubation with phage (72 h); Fish + AS - sample fish (gastrointestinal area) after incubation with bacteria (72 hours); Fish + AS + Phage – fish sample (gastrointestinal area) after incubation with bacteria and phage (72 h).
The results showed that the natural bacterial community of aquaculture water was not significantly affected by the addition of A. salmonicida phages. However, fish microbiota was significantly affected by the addition of phages of A. salmonicida. Nevertheless, when assays were done in the presence of the host A. salmonicida, the differences were not statistically different from the control.
Conclusion This study provides evidence that phage therapy is a feasible alternative approach against furunculosis during fish juvenile production in aquaculture systems.
Results and Discussion Phage isolation and enrichment AS-A formed clear plaques on the host strain with a diameter of 0.5- 2 mm (Fig. 2).
References [1] Mateus C, Costa L, Pereira C, Silva Y, Almeida A (2014) Efficiency of phage cocktails in the inactivation of Vibrio in aquaculture. Aquaculture 424-425, 167-173. [2] Griffiths RI, Whiteley AS, O’Donnell AG, Bailey MJ (2000) Rapid method for coextraction of DNA and RNA from natural environments for analysis of ribosomal DNA and rRNA-based microbial community composition. Appl. Environ. Microbiol. 66, 5488–5491.
The phage titre was determined by the double-layer method and incubated at 30 °C for 24 hours.
Acknowledgement
Phage therapy in juvenile flounder fish
The authors also thank the University of Aveiro and the Center for Environmental and Marine Studies (CESAM) for funding (Project Pest-C/MAR/LA0017/2013).
Each set of 10 specimens of the 4 groups was treated separately, corresponding to 3 independent samples per condition.
Fig.2. Phage plaques of the AS-A phage.
Phage therapy assays The maximum of bacterial inactivation with AS-A phage was 3.9 log achieved after 8 h of phage therapy. After 12 h of treatment, the rate of inactivation was still considerably high (2.4 log) (Fig. 3).
This work was financed by LIFE+ (project LIFE13-ENV/ES/001048) and PROMAR 31-03-05-FEP0028. Financial support to Y. J. Silva, C. Pereira and L. Santos was provided by FCT in the form of PhD grants (SFRH/BD/65147/2009 and SFRH/BD/76414/2011) and a Postdoctoral grant (CESAM/PTDC/MAR-EST/2314/2012), respectively .