1. Figure 1 : Soft agar overlay technique
Efficiency of phage cocktails in the inactivation of Salmonella Typhimurium in bivalve molluscs
Pereira C1, Pereira S1, Silva Y.J1, Romalde J.L2, Nunes M.L3, Almeida A1.
1Department of Biology and CESAM, University of Aveiro, Campus Universitário de Santiago, Aveiro, Portugal
2Departamento de Microbiología e Parasitología e CIBUS e Facultad de Biologia, Universidade de Santiago de 3Compostela e Campus Universitario Sur.,15782 Santiago de Compostela (A Coruña), Spain
Portuguese Institute for Sea and Atmosphere (IPMA, IP), Av. Brasília, Lisbon, Portugal
* Corresponding author:
Introduction
Salmonella enterica subsp enterica serovar Typhimurium (Salmonella Typhimurium) is the most frequent causative agent of human gastroenteritis, after consumption of contaminated seafood.
Depuration is a useful method to eliminate microorganisms from bivalves when occur under conditions that maximize the natural filtering activity, which results in expulsion of intestinal contents. However, some pathogenic microorganisms are resistant to this process [1,2,3]
To reduce the risk of the development and transmission of infections caused by microbial pathogens, including multidrug-resistant bacteria, other technologies associated with depuration, such as phage therapy must be developed
The aim of this study was to evaluate the efficiency of cocktails of two and three phages of S. Typhimurium (SE- 1, SE-2 and SE-5) to control Salmonella in bivalve molluscs.
Material and Methods
Bacteria and growth conditions
S. Typhimurium (ATCC 13311) grew at 37ºC, pH 7.3, in tryptic soy broth
Bacteriophages
Three phages (SE-1, SE-2 and SE- 5) were isolated from sewage water, according to the procedure described by Pereira et al, 2011 [4].
One-step growth experiments
For the one-step growth experiment were determined by the double- layer method according to the procedure described by Mateus et al, 2014 [4].
Phage therapy assays
Phage therapy was performed using one phage alone (SE-1, SE-2 or SE- 5) and with phage cocktails (two or all the three phages mixed together, each phage at the same concentration) using the bacterium S. Typhimurium as host, at a MOI of 100.
The assays with two and three phages were performed with the following phage combinations: SE-1/ SE-2, SE-1/SE-5, SE-2/SE-5 and SE-1/SE-2/SE-5 phages.
For host quantification, aliquots were serially diluted, plated by pour plating method in duplicate and incubated at 37°C for 24 hours.
The phage titre was determined by the double-layer method (Figure 1) and incubation at 37°C for 18 hours.
With SE-2 and SE-5 phages, the maximum of bacterium inactivation was 2.4 and 2.5 log, respectively, after 12 h of phage therapy (Figure 4A).
The phage control, for three phages, remained almost constant during all time but when the phage was incubated in the presence of its host, a increase of ~ log (Figure 4B).
However, for the three phages, during the subsequent incubation period, the non-infected bacteria grew again and reached densities similar to those of the control.
Figure 4. Inactivation of S. Typhimurium by the three phages (SE-1, SE-2 and SE-5) at a MOI of 100 during the 24 h. A. Bacterial concentration: BC – Bacteria control; BP – Bacteria plus phage .B. Phage concentration: PC – phage control; BP – Bacteria plus phage. Values represent the mean of three experiments; error bars represent the standard deviation
Phage therapy using phage cocktails
Figure 5. Inactivation of S. Typhimurium by phage cocktails (SE-1/SE-2, SE-1/SE-5 and SE-2/SE-5) at a MOI of 100 during the 24 h. (A) bacterial concentration BC – Bacteria control; BP – Bacteria plus phage. B. Phage concentration. PC – phage control; BP – Bacteria plus phage. Values represent the mean of three experiments; error bars represent the standard deviation.
The cocktail SE-2/SE-5 was more effective (reductions of 2 log after 4 h and 2.7 log after 12 h) than the SE-2 and SE-5 phages used alone (Figure 5A).
However, the efficiency of SE-1/SE-2, and SE-1/SE-2/SE-5 cocktails was similar, respectively, to SE-2 and SE-5 phages alone.
The efficiency of SE-5 phage alone was higher than the phage cocktail SE-1/SE-5.
No decrease of the phage survival was observed during the 24 h of the experiments for the phage cocktail controls (PC) (Figure 5B).
The suspensions with the phage cocktails when incubated in the presence of the host, presented also an increase of ~ log.
Conclusions
The three phages are efficient in the inactivation of S. Typhimurium, being potential candidates, to be used individually or in cocktails, as agents for the biological control of infections transmitted to humans by consumption of bivalve molluscs.
The use of phage cocktails with two or three phages do not always increases the efficiency of phage therapy against S. Typhimurium, but as stated before can delay the development of bacterial resistance.
References
1.FAO, Bivalve depuration: fundamental and practical aspects. In FAO Fisheries technical paper, Vol Rome, Italy: FAO of the United Nations.2. Martínez, O., Rodríguez-Calleja, J.M., Santos, J.A., Otero, A., García-López, M.L., Foodborne and indicator bacteria in farmed molluscan shellfish before and after depuration. J Food Prot 72 (7), Rong, R., Lina, H.,Wang, J., Khana, M.N., Li, M., 2014 Reductions of Vibrio parahaemolyticus in oysters after bacteriophage application during depuration. 418– – Mateus, C., Costa, L., Pereira, C , Silva, Y., Almeida, A., Efficiency of phage cocktails in the inactivation of Vibrio in aquaculture. Aquaculture ,
Acknowledgement
This study was funded by FEDER COMPETE (Competitiveness Factors Operational Program) and by FCT (Portuguese Foundation for Science and Technology), under the research project FCOMP FEDER
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).
Financial support to C. Pereira, and Y. J. Silva was provided by FCT in the form of PhD grants (SFRH/BD/76414/2011 and SFRH/BD/65147/2009) , respectively.
Figure 1 : Soft agar overlay technique
Results and Discussion
Bacteriophages
Phage isolation and enrichment
SE-1, SE-2 and SE-5 formed clear plaques on the host strain with a diameter of mm, of 1- 3mm and of 2-5 mm, respectively (Figure 2).
Figure 2 : Phage plaques of the phages. (A) SE- 1, (B) SE-2 and (C) SE-5.
One-step growth experiments
The SE-5 phage presented a larger burst size and a shorter latent period (188 and 15 min, respectively) (Figure 3).
For the SE-1 and SE-2 phages were characterized by a latent period of 20 min and each infected bacteria produced 33 and 39 PFU/host cell, respectively, (Figure 3).
Figure 3. One-step growth curves of SE-1, SE-2 and SE-5 phages in the presence of S. Typhimurium as host.
Phage therapy assays
Phage therapy using single-phage suspensions
The maximum of bacterium inactivation with SE-1 phage was 1.8 log achieved after 4 h of phage therapy. After 12 h of treatment, the rate of inactivation was still considerably high (1.3 log) (Figure 4A).