Comunication icar phage therapy in larvae

Efficiency of phage cocktails in the inactivation of Vibrio in aquaculture

Adelaide Almeida Department of Biology and CESAM University of Aveiro, Portugal

Antibiotherapy has shown to be a rapid and effective method to treat or prevent bacterial infections but: the use of large amounts of a wide variety of antibiotics, including non-biodegradable ones, results in: the emergence of antibiotic-resistant bacteria in the environment the increase of antibiotic resistance in animal pathogenic bacteria the transfer of these resistance determinants to human pathogenic bacteria alterations of the bacterial flora both in sediments and in water column

New approaches

New approaches Phage therapy Non-antibiotic approach that use: litic phages

Advantages of phage therapy specific target (natural non-target bacteria are not affected) limited resistance development self-replicating (one dose) legislative approval (phages are naturally occurring) high resistance of phages to environmental conditions technology flexible, fast and cost effective

Desvantages of phage therapy Specificity of phages can be a disadvantage when it is not known the bacteria that cause the infection Difficulty overcomed when phage therapy is applied to specific cases (pathogenic bacteria known)

Aquaculture (Vibrio, Photobacterium)

Resistance development is possible Phages can outcompete the adaptation of the bacteria It is easy to find new phages phage co-evolve with their host, rapid isolation of new phages

Why phage therapy in aquaculture Important economic activity around the world Cultured fish are subject to many bacterial infection (high mortality and financial losses) High incidence of drug-resistant strains Few antibiotics licensed to aquaculture use Vaccination not likely in fish larvae is practically unfeasible to handle these small animals fish larvae do not have the ability to develop specific immunity

Objective Evaluate the efficiency of cocktails of two and three phages to control Vibrio in aquaculture phage characteristics(VP-1, VP-2 and VP-3) phage survival in seawater effect of multiplicity of infection (MOI) efficiency of cocktails of two and three phages impact of phage addition on natural community

Approach

Phage characterization

Host range, burst size, explosion time and survival

Vibrio parahaemolyticus

Bacteria isolated from aquaculture water Phages produced on pathogenic bacteria Phage host range (cross infection) Burst size and explosion time (one step growth curves) Survival in seawater Vibrio parahaemolyticus

phages

Results Phage host range - Efficacy of plating (%) FISH PATHOGENIC BACTERIA

V. parahemolyticus V. anguillarum A. salmonicida A. hydrophilla V.fisheri P. damselae subsp. damselae E. coli P. aeruginosa P. fluorescens P. putida P. segetis P. gingeri

VP-1 100 83.27 64.75 0 0 0 0 0 0 0 0 0

PHAGES VP-2 100 93.39 92.03 0 0 0 0 0 0 0 0 0

VP-3 100 51.21 73.78 0 0 0 0 0 0 0 0 0

Results One step growth curve of Vibrio phages Log (PFU mL-1)

4

Explosion time /burst size

3

VP-1: 120 min/ 44

2

VP-2: 90 min/ 66

1 0

20

40

60

80

100

120

Time (minutes) VP-1

VP-2

VP-3

140

160

VP-3: 40 min/ 135

Results

Log (PFU mL-1)

Phage survival in seawater 8

VP-1

6

VP-3

VP-2

4 2 0 0

40

80

120

160

200

240

280

Time (days)

VP-1 more than 7 month; VP-2 more than 9 months; VP-3 more than 9 months.

Phage therapy

Approach

Selection of multiplicity of infection (MOI) MOI tested: 1, 10, 100 and 1000 Phage used: VP-2 phage: 105-9 CFP mL-1 Bacterial host concentration: 105 CFU mL-1 Phage therapy at 25째C during 36 hours Samples collection: 0, 2, 4, 6, 8, 10, 12, 18, 24 and 36 hour Phage quantification: double agar layer method Bacteria quantification: pour plating technique Three independent assays

Phage therapy at different MOI

Results

Inactivation of V.

parahaemolyticus by

10

Log (CFU mL-1)

the VP-2 phage.

BC 1000

8

BP 1000 BC 100

6

BP 100

4

MOI 1 ≠ MOI 10, 100, 1000

BC 10

MOI 10 ≠ MOI 100, 1000

BP 10

MOI 100 = 1000

BC 1 BP 1

2 0

10

20

30

40

BC – Bacteria control, BP – Bacteria plus phage.

Values represent the mean of three independent experiments; error bars indicate the standard deviation.

Time (h) Maximum inactivation: MOI 1: 3 log, MOI 10: 3.4 log, MOI 100: 4.1 log, MOI 1000: 4.7 log

Phage therapy

Approach

Cocktails of two and three phages MOI tested: 100 Phages used: VP-1, VP-2 and VP-3 (107 CFP mL-1) Bacterial host concentration: 105 CFU mL-1 Phage therapy at 25째C during 24 hours Samples collection: 0, 2, 4, 6, 8, 10, 12, 18 and 24 hour Phage quantification: double agar layer method Bacteria quantification: pour plating Three independent assays

Results

Phage therapy with phage cocktails

Inactivation of V.

parahaemolyticus BC – Bacteria control, BP – Bacteria plus phage Values represent the mean of 3 independent experiments.

Approach Impact of phage addition on natural bacterial community Water samples from Aquaculture Corte das Freiras Phages of Vibrio parahemoliticus (107 PFU mL-1) Impact determined by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments after ten hour of phage addition.

Negative controls (water without incubation, water incubated 10 h, TSB with 1% chloroform – phage preservation solution)

Impact on bacterial community Total community

A

Vibrio community

Relationship between the bioluminescence signal (RLU) and viable counts (CFU mL-1) of an overnight culture of a transformed bioluminescent E. coli

DGGE profile of PCR-amplified Vibrio genus fragments after phages addition to bacterial community of the aquaculture system.

M – molecular weights marker T0 – Water samples at time 0 h T8 - Water samples incubated without phages TSB-CL – Water samples incubated with TSB and 1% of chloroform without phages ELY-1 100 – Water samples with phage ELY-1 VP-2 100 – Water samples with phage VP-2 Inactivation of samples bioluminescent E.VA-1 coli by the Dendogram phage at different MOI values. generated from the pattern VA-1 100 – Water with phage Bacterial bioluminescence along the 72 – Bacteria AS-2 100 – Water samples withvariation phage AS-2 ofhours bandsexperiment. obtained byBC DGGE of VP-2.

control, BP – Bacteria plus phage.

Conclusions Vibrio phages showed good survival time in marine water, no

significant impact on the bacterial community structure and specificity for host pathogenic bacteria.

V. parahaemolyticus phages with high burst sizes and short lytic

cycles increase the efficiency of phage therapy.

The phage VP-3 that present the high burst size and the short lytic cycle was more

efficient to inactivate V. parahaemolyticus than VP-1 and VP-2 phages (more 2 log of inactivation).

They are potential candidates for therapy of fish infectious diseases and phage characterization is an important aspect to select phages for phage therapy.

Conclusions The efficiency of phage therapy increased with the MOI, but

the increase in MOI from 100 to 1000 did not promote a significant increase in the efficiency of phage therapy.

Precise initial doses may not be essential, because of the self-perpetuating nature of phages, phage titers increasing along with bacteria in aquaculture water.

Conclusions The utilization of phage cocktails increases the efficiency of phage therapy against Vibrio.

The bacterial inactivation with phage cocktails occurred sonner and with higher efficiencies of inactivation than when phages are used alone.

As the use of phage cocktails avoid the development of bacterial resistance, the use of more than one phage, beside increase the effectiveness of phage therapy, avoid also resistance development.

Department of Biology Prof Dra Ângela Cunha Dr Newton Gomes PhD Liliana Costa MSc Yolanda Silva MSc Carla Pereira MSc Cristiana Mateus

Acknowledgements

AZTI Dr Igor Hernandez

Corte das Freiras fish farming staff

Universidade de Aveiro CESAM/Biology Department Fundação para a Ciência e Tecnologia FEDER through COMPETE- Programa Operacional Factores de Competitividade, and

by National funding through FCT-Fundação para a Ciência e Tecnologia, within the research project FCOMP-01-0124-FEDER-013934 (Project Phage Therapy Life+, within the research project LIFE13-ENV/ES/001048 Grant of Yolanda Silva, Carla Pereira and Liliana Costa

Thanks for your kind attention