Comunication Aquaculture

Phage therapy as an approach to prevent Aeromonas salmonicida infections in aquaculture

Adelaide Almeida

5

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) 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, Aeromonas)

Resistance development is possible

After three streak-plating steps on solid medium, the bacteria regained sensitivity to phages

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 subjected to many bacterial infection (high mortality and financial losses) High incidence of drug-resistant strains Few antibiotics licensed to aquaculture use Efficient vaccines are available but: are expensive and often associated with undesirable side effects is practically unfeasible to handle small animal size

small animals do not have the ability to develop specific immunity

Objective

Causative agent of furunculosis, responsible for significant losses worldwide. High mortality and morbility of fish species: turbot, Atlantic cod, rockfish, seabream, wolffish

The main goals of this work were to isolate, characterize and evaluate the safety and effectiveness of an Aeromonas salmonicida phage to control furunculosis infection in Senegalese sole (Solea senegalensis) juveniles.

Furunculosis severe in the absence of vacina, which are expensive and often associated to undesirable side effects

phage characteristics efficiency of inactivation in vitro efficiency to control infection in fish development of resistances impact of phage addition on natural community

Approach

Phage characterization Host range, burst size and

Aeromonas salmonicida phage lysis plaques

explosion time

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

Aeromonas salmonicida phage

Results

Phage host range - Efficacy of plating (%) Species A. salmonicida CECT 894 A. caviae A. hydrophilla ATCC 7966 V. parahaemolyticus DSM 27657 V. anguillarum DSM 21597 V. fischeri ATCC 49387 P. damselae damselae DSM 7482 P. aeruginosa P. fluorescens P. putida P. segetis P. gingeri S. enterica serovar Typhimurium ATCC 13311 S. E. E. E. E. E. E. E. E. E. E. E.

enterica serovar Typhimurium ATCC 14028 coli ATCC 25922 coli ATCC 13706 coli BC30 coli AE11 coli AD6 coli AF15 coli AN19 coli AC5 coli AJ23 coli BN65 coli BM62

Infectivity of phage AS-A + -

Efficacy of plating (%) AS-A 100 0 0 0 0 0 0 0 0 0 0 0 0

-

0 0 0 0 0 0 0 0 0 0 0 0

Results One step growth curve of Vibrio phages

Log (PFU.mL-1)

3

Explosion time / burst size 2

VP-1: 20 min/22 1 0

10

20

30 40 Time (minutes)

50

60

70

In vitro experiments

Approach

MOI tested 100 Phage used: AS-A phage: 107 PFU mL-1 Bacterial host concentration: 105 CFU mL-1 Phage therapy at 25째C during 24 h Samples collection: 0, 2, 4, 6, 8, 10, 12, 18 and 24 h Phage quantification: double agar layer method Bacteria quantification: pour plating technique Three independent assays Determination of the frequency of emergence of phage-resistant mutants

Results

In vitro experiments

Phage concentration

Bacterial concentration A

10

60

B

↗ 0.6 Log PFU mL-1

Log CFU mL -1

6

BC

4

PFU mL-1 x 107

50 8

40 PC 30 PB 100 20

BP 100

↘ 4 Log CFU mL-1 after 8 h

10 0

2 0

5

10

15 Time (h)

20

25

0

4

8

12

16

20

24

Time (h)

Inactivation of A. salmonicida by the AS-A phage at MOI 100, during 24 h. (A) Bacterial concentration (Log CFU mL−1): BC – Bacterial control; BP – Bacteria plus phage.

(B) Phage concentration (PFU mL−1): PC – Phage control, and PB – Phage plus bacteria

Approach

Phage-resistant mutants A

Colonies that grew within the lysis plaque after 24 h of incubation

Spontaneous mutants were isolated by plating on double-layer agar with the phage (10 colonies)

Isolated resistant colonies were inoculated in liquid medium, diluted and inoculated on solid medium

Aliquots of dilutions were plated on solid medium without phage for determination of CFU concentration

Mutants in 1 ml of samples prepared from the culture with phages were divided by live bacterial cells prepared from the culture without phages

Results

Phage-resistant mutants:

susceptible to phages after 3 streak-plating steps on solid medium were

smaller than colonies formed by the non-phage added control and were visible only after 2 days of incubation.

Control sample (CFU mL-1)

Sample treated with phages

Frequency of mutants

1.72 Âą 0.12 x109

2.81 Âą 0.15 x105

2.24 x10-4

Approach Phage therapy in vivo, Solea senegalensis juveniles

Bacterial concentration: 108 CFU/mL (tested 107 Nยบ of dead juveniles 8 CFU/mL and 10 CFU/mL ) Nยบ of dead juveniles Mortality (%) Phage concentration: 1010 48 h 72 h 96 h 72 h 96 h PFU/mL Control Fish (1 x 10 CFU mL ) Time period: 72 h 0 0 1 0.0 3.3 4 groups of fish Fish + AS (1 x 10 CFU mL ) 1 9 10 30.0 33.3 Number of samples per Control Fish (1 x 10 CFU mL ) 0 0 2 0.0 6.7 condition:3 Fish + AS (1 x 10 CFU mL ) 5 12 14 40.0 46.7 Number of fish per Determination of fish mortality by Aeromonas salmonicida sample:10 7

7

-1

8

8

-1

-1

-1

Results Phage therapy in vivo with in Solea senegalensis Fish mortality

(n = 30)

Sum of all assays (n = 90)

Average percentage of all assays (%)

2

1

3

3.3

0

0

0

0

0

Fish+Aeromonas

11

10

11

32

35.6

Fish+Aeromonas+Phage

0

0

1

1

1.1

Assay 1

Assay 2

Assay 3

(n = 30)

(n = 30)

Control Fish

0

Fish+Phage

Sample

Phage therapy in vivo

9

↘ 2.2 Log CFU mL-1 after 6 h Bacteria already present in the juveniles

Log UFC/mL

8

Aeromonas in seawater (control)

7

Fish + Aeromonas 6

Fish + Aeromonas + phage

5

Fish + phage (control)

4

Fish (control)

3 0

12

24

36 Time (h)

48

60

72

Approach

Impact of phage addition on natural bacterial community and on fish bacteriome

Water samples from Aquaculture Corte das Freiras and fish juvenilles of Solea senegalensis Phages of Aeromonas salmonicida Impact determined by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments after 8 h of phage addition to seawater after 72 h of phage addition to fish

Negative controls water without incubation, water incubated 8 h, TSB with 1% chloroform (phage preservation solution) and fish juveniles without phages

Impact on bacterial community of aquaculture water A

Relationship between the bioluminescence signal (RLU) and viable counts (CFU mL-1) of an overnight culture of a DGGE profile cDNA fragments of the 16S gene transformed bioluminescent amplified by PCRE. following addition of phage. coli DGGE profile cDNA fragments of the 16S gene amplified by PCR following addition of phage.

(T0) Control without incubation, (T8) water incubated without phage (T8-TSBCL) phage preservative solution (TSB with 1 % chloroform) (T8-Phage AS-A) water incubated with phage.

Groups T0, T8 T0, T8-TSBCl T0, T8-Phage T8, T8-TSBCl

R 0.148 0.536 0.602 0.426

T8, T8-Phage

0.633

T8-TSBCL, T8-Phage

0.356

Analysis of similarities (ANOSIM)

Range 0-1, R > 0.75 well separated, 0.50 ≤ R ≤ 0.75 moderately separated but overlapping, 0.25 < R < 0.50 separated but strongly overlapping groups, R ≤ 0.25, barely separated.

Separated but strongly overlapping groups of water incubated with phage in phage preservative solution and water incubated without phage (T8 –TSBCL vs T8- Phage) (R=0.356).

Higher similarity between water samples without incubation (T0) and water incubation during 8 hours (T8) (R=0.148)

Impact on fish bacteriome A

Relationship between the bioluminescence signal (RLU) and viable counts (CFU mL-1) of an overnight culture of a transformed bioluminescent E. Moderate separation coli of Fish control group DGGE profile of 16S cDNA fragments.

and

Fish+Phage (R=0.669).

Control Fish - fish sample (gastrointestinal tract) without phage; Fish+Phage AS-A - fish sample after incubation with phage (72 h); Fish + AS - sample fish after incubation with bacteria; Fish + AS + Phage â&#x20AC;&#x201C; fish sample after incubation with bacteria and phage.

Group Fish+AS similar to the group exposed to bacteria AS and phage AS-A (R=0.269).

Groups

R

Control Fish, Fish+Phage AS-A

0.669

Control Fish, Fish+AS

0.792

Control Fish, Fish+AS+Phage AS-A

0.825

Fish+Phage AS-A, Fish+AS

0.569

Fish+Phage AS-A, Fish+AS+Phage AS-A

0.666

Analysis of similarities (ANOSIM) Fish+AS, Fish+AS+Phage AS-A

0.269

Conclusions Aeromonas salmonicida phage showed high burst size, short lytic

cycle, specificity for host Aeromonas salmonicida, no significant impact on the bacterial community structure of aquaculture water and only a moderate impact on fish bacteriome.

Aeromonas salmonicida was effectively inactivated in vitro by the

phage

It is a potential candidate for inactivation of Aeromonas salmonicida.

Conclusions The phage treatment of challenged juveniles with Aeromonas salmonicida reduced the fish mortality After 3 days of incubation fish mortality was similar to that observed normally in aquaculture and was significantly lower in phage-treated juveniles than in non-treated ones No increase in bacterial number as observed in vitro (influence of fish immune system and/or less fit bacteria after treatment)

Phage therapy can be applied as a preventive approach against bacterial infections in fish juveniles.

Conclusions A low rate of phage-resistant mutants (2.24 x 10-4) after phage therapy and these mutants after 3 streak-plating steps on solid medium were susceptible to phages.

Phage-resistant bacteria seems to be less fit (smaller than control colonies formed and visible only after two days), so they can be expected to be eliminated from the environment faster than their wild-type relatives

These results suggest that the emergence of phage-resistant mutants should not be a major problem to the application of phages to control bacterial infections in aquaculture.

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