300 W.m-2 150 W.m-2 New approaches with low...

New approaches with low environmental impact to inactivate microorganisms in aquaculture systems

Departamento de Biologia e CESAM Universidade de Aveiro

CESAM

Centro de Estudos do Ambiente e do Mar

www.cesam.ua.pt

Adelaide Almeida [email protected]

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Why new therapies in aquaculture

Important economic activity around the world

Cultured fish subjected to many microbial infections (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

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New approaches

Antimicrobial Photodynamic Therapy

Phage Therapy

Antimicrobial photodynamic therapy Non-antibiotic approach that combines:

a nontoxic photosensitizer

visible light

oxygen

Relies on the experience of the treatment of malignant tumors by PDT

To generate highly reactive oxygen species

(ROS):

singlet oxygen

superoxide and hydroxyl radical

Irreversibly oxidize microorganism vital constituents resulting in lethal damage

N HN

NNH

N

N

N

F F

F

FF N HN

NNH

N

N

N

F F

F

FF

Chemical Formula: C41H22F5N7

Molecular Weight: 707,65

CH3

H3C

CH3

I

I

I

Chemical Formula: C44H31F5I3N7-


Tri-Py-PF Tri-Py+-Me-PF

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http://www.ifsc.usp.br/~cepofusp/terapia.jpg

Advantages of PDI Non-target specificity (bacteria, viruses, fungi and protozoa)

Lack of development of resistance

multi-target process

main targets are external structures (PS does not need to enter in the microorganism)

antioxidant enzymes (superoxide dismutase, catalase, peroxidase) protect against some ROS, not against singlet oxygen (the main ROS of PDT )

singlet oxygen inactivate these enzymes

Independent of antibiotic-resistance spectrum (resistant microorganisms are equally as susceptible as their native counterparts)

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Recombinant bioluminescent Escherichia coli

strain as model

Three photosensitizer concentrations

(0.5 µM, 1.0 µM and 5.0 µM)

Two light intensities (40 and 600 Wm-2)

Light exposure time (270 minutes)

Bacteria concentration (106 CFU mL-1)

Controls (light and dark controls)

Bioluminescence measured in a luminometer

R² = 0,9838

R² = 0,9925

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-5 0 5 10L

og

CF

U m

L-1

Log RLU

Efficiency of porphyrins to inactivate bacteria

Approach

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Results Tetra-Py+-Me Tri-Py+-Me-CO2H Tri-Py+-Me-PF

600 m

Wcm

-2

40 m

Wcm

-2

Legend: x light control, dark control, 0.5µM, 1.0 µM, 5.0 µM

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Development of resistance in bacteria

PS concentrations 5.0 µM

Light intensity 40 mWcm-2

Exposure cycles of 120 minutes (modest inactivation)

After each cycle sub-samples of 1 mL were aseptically taken and plated

Three colonies were picked for produce new cultures

Repetition 10 times


Approach

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N HN

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N

N

N

F F

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FF N HN

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CH3

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Recovery of microbial viability



Exposure of 120 minutes (modest inactivation)

Samples protected from light and aliquots collected at 24, 48 and 72, 120 and 168 h after treatment, bioluminescence signal measured


Results Escherichia coli

Variation of the concentration of

microorganisms after irradiation in ten

consecutive cycles. N0 and N colony

counts before and after the irradiation,

respectively.

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4,5 28,5 52,5 76,5 100,5124,5148,5172,5196,5L

og

Bio

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ine

sc

ên

cia

(U

RL

)

Tempo de incubação (h)

Controlo claro; Tri-Py+-Me-PF 5uM; Controlo escuro

Effect of physical and chemical properties of environmental waters in PDI

Recombinant bioluminescent Vibrio fischeri as model


Light intensities 40 mWcm-2 and solar light

Exposure cycles of 270 minutes

After each cycle sub-samples of 1 mL were

aseptically taken and light was measured

Effect of temperature, salinity, pH, O2,

organic matter


Approach

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N HN

NNH

N

N

N

F F

F

FF N HN

NNH

N

N

N

F F

F

FF



CH3

H3C

CH3

I

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I




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N HN

NNH

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N

N

F F

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FF N HN

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F F

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-1

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Log

Bio

lum

ines

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ce (

RLU

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Irradiation time (min)

Photoinactivation assay of V. fischeri with and without stirring for [O2] monitoring (values in mg/L)

Light control

Dark control (5µM)

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iolu

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U)


Photoinactivation assay of V. fischeri at different temperatures

10°C

15°C

20°C

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Log

Bio

lum

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Photoinactivation assay of V. fischeri at different pH values of the suspension medium

pH=6.5

pH=7.0

pH=8.0

pH=8.5

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Photoinactivation assay of V. fischeri at different concentrations of NaCl in the suspension medium

10 g/LNaCl

20 g/LNaCl

Aquaculture water under 4 mW cm-2 irradiation. A: non-filtered

portion; B: portion filtered by 0.7 µm membrane; C: portion filtered

by 0.2 µm membrane. Light control (), dark control with 50 µM of

porphyrin (), 10 µM (), 20 µM () and 50 µM ().

Aquaculture sample under different light sources.

A: Artificial white light (4 mW cm-2). B: Solar light

(40 mW cm-2). Light control (), dark control with 20

µM of porphyrin (), 20 µM ().

Maio 2010 Junho 2010

Outubro 2010

Re-utilization of the PS after PDI.

PS was immobilized in a solid support –

nanomagnet-porphyrin hybrids



Exposure 270 minutes

A Gram negative bacteria (V. fischeri)

Controls (light, dark and material controls)

Approach

a) Water colloidal suspensions of the magnetic materials 6, 8-10 a) in the absence of a

magnetic bar; b) in the presence of a magnetic bar.

6 – PS cationic + neutral material 8 – PS cationic + cationic material 9 – neutral PS + cationic material 4 – neutral material 10 – cationic material

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-2

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015

3060

90

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Time (min)

1º ciclo

2º ciclo

3º ciclo

4º ciclo

5º ciclo

6º ciclo

Free Tri-Py+-Me-PF (3 assays of 6 cycles)

Re-utilization of free and immobilized PS (nanomagnet-

porphyrin hybrid)

Phage therapy

Non-antibiotic approach that use:

Litic phages

Specific target (natural non-target bacteria not affected)

Self-replicating (one dose)

Legislative approval (phages are naturally occurring)

Easy penetration in infectious sites (necrosis tissue)

High resistance of phages to environmental conditions

Technology flexible, fast and cost effective

Advantages of phage therapy

Specificity of phage infection (disadvantage when pathogenic

bacteria is not known)

Difficulty overcomed when phage therapy is applied to specific cases, when the main pathogenic bacteria are known

Aquaculture (Vibrio, Photobacterium)

Poultry (Salmonella)

Skin/wound infections (e.g.Pseudomonas aeruginosa)

Desvantages of phage therapy

Resistance development

Phages can outcompete the adaptation of the bacteria

Is easy to find new phages, phage co-evolve with their host, rapid isolation of new lytic phages from the environment for phage-resistant bacterial mutants

Use of phage cocktails

Phage characterization

Host range, burst size, explosion time and survival

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

Approach

Vibrio parahaemolyticus phages

Vibrio parahaemolyticus

Phage host range - efficacy of plating (%) FISH PATHOGENIC

BACTERIA

PHAGES

VP-1 VP-2 VP-3

V. parahemolyticus V. anguillarum

100 83.3

100 93.4

100 51.2

A. salmonicida 64.8 92.0 73.8

A. hydrophilla V.fisheri

0 0

0 0

0 0

P. damselae subsp. damselae

0 0 0

E. coli P. aeruginosa P. fluorescens

P. putida P. segetis P. gingeri

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

0 0

Vibrio phages

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Time (minutes)

Log

(PFU

mL-1

)

VP-1 VP-2 VP-3

One step growth curve

Phage survival in seawater

Explosion time /burst size:

VP-1: 120 min/9; VP-2: 90 min/15; VP-3: 40 min 42

Phage survival:

VP-1 more than 7 month; VP-2 more

than 9 months; VP-3 more than 9 months

Results

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Log

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Time (days)

VP-1

VP-2

VP-3

Phage therapy

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 25°C 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

Approach

Results

Phage therapy at different MOI Inactivation of V. parahaemolyticus by the VP-2 phage.

BC – Bacteria control, BP – Bacteria plus phage.

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

Maximum inactivation: MOI 1: 3 log, MOI 10: 3.4 log, MOI 100: 4.1 log, MOI 1000: 4.7 log

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Time (h)

Log

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) BC 1000

BP 1000

BC 100

BP 100

BC 10

BP 10

BC 1

BP 1

MOI 1 MOI 10, 100, 1000

MOI 10 MOI 100, 1000

MOI 100 = 1000

Results

Phage therapy with phage cocktails

Inactivation of V. parahaemolyticus .

BC – Bacteria control, BP – Bacteria plus phage.

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

A

Relationship between the bioluminescence signal (RLU) and

viable counts (CFU mL-1) of an overnight culture of a

transformed bioluminescent E. coli

Influence of environmental variables in the efficiency of phage therapy

Phage of a recombinant bioluminescent Escherichia coli strain as model

pH

Temperature

Salinity

Organic matter R² = 0,9838

R² = 0,9925

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Log RLU

Approach

Influence of pH

A

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0 10 20 30 40 50 60 70 80

Time (h)

Log

RLU

BC 6.5

BP 6.5

BC 7.0

BP 7.0

BC 7.4

BP 7.4

Results

Increase of 14% in phage efficiency.

BC – Bacterial control; BP – Bacteria and phages

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Time (h)

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BC 15ºC

BP 15ºC

BC 20ºC

BP 20ºC

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Time (h)

Log

RLU

BC 25ºC

BP 25ºC

Influence of temperature


Influence of salinity

Inactivation of bioluminescent E. coli by the phage at different salinity values. (A), bacterial bioluminescence at salinity 0 g L-1 and (B) bacterial concentration at salinities 10, 20 and 30 g L-1 along the 72 hours experiment. BC – Bacteria control, BP – Bacteria plus phage.

A

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g (

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BC 10g/L

BP 10g/L

BC 20g/L

BP 20g/L

BP 30g/L

BP 30g/L

A B

Results


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RLU

BC 0g/L

BP 0g/L

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Time (h)Lo

g R

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BC 1:1

BP 1:1

BC 1x

BP 1x

BC 2x

BP 2x

Influence of organic matter content


BC – Bacterial control; BP – Bacteria and phages

Inactivation of bioluminescent E. coli by the phage at different MOI values. Bacterial bioluminescence variation along the 72 hours experiment. BC – Bacteria control, BP – Bacteria plus phage.

A

Phage therapy in Zebra Fish larvae.

Fish larvae selected 5 days post fecundation

Three samples for each condition, 20 larvae per sample

Fish larva stored at 27ºC with slight shake up to 72 hours.

Phages added orally 1 hour post bacteria challenge (V. anguillarum, 106 PFU mL-1)

Four conditions:

• Fish+Vibrio

• Fish+Vibrio + phage

• Fish + phage

• Fish (control)

Approach

Mortality similar in phage-treated and in non-infected samples, and much lower than in infected non-phage treated samples.

A

Condition %

Average S.D.

Vibrio + phage 2 2,7

Vibrio 16,7 2,9

Phage 0 0

Control (no Vibrio and no phage)

2,5 3,5

Fish mortality after 72 hours at 27ºC

Results

PDT Conclusions

Cationic porphyrins with 4 or 3 charges efficiently photoinactivated microorganisms

PDT is effective at micromolar concentration under different light condition

The bacteria that survived to PDT treatments in the presence of 5 M of porphyrin did not develop resistance after 10 cycles of aPDT.

The tricationic porphyrin inactivated effectively bacteria without the possibility of viability recovery after one week of dark incubation

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N HN

NNH

N

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N

F F

F

FF N HN

NNH

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F F

F

FF



CH3

H3C

CH3

I

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The variations in pH (6.5-8.5), temperature (10-25ºC), salinity (20-40 g L-1) and O2 did not significantly affect the PDI of V. fischeri (in all conditions ≈7 log reduction).

The assays using aquaculture water showed that the efficiency of PDI is affected by the suspended matter.

Total PI of V. fischeri in aquaculture water was achieved under solar light in the presence of 20 µM of PS.

The new multicharged nanomagnet-porphyrin hybrids are very stable in water and highly effective in the photoinactivation of bacteria.

The hybrid was effective to photoinactivate bacteria for at least 6 cycles, inactivating > 40 log of bacteria.

PDT Conclusions

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NNH

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F F

F

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NNH

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F F

F

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Vibrio phages showed high survival time in marine water and specificity for

host pathogenic bacteria.

Phages with high burst sizes and short lytic cycles increase the efficiency of

phage therapy (more 2 log of inactivation).

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.

The utilization of phage cocktails increases the efficiency of phage therapy

against Vibrio (bacterial inactivation with phage cocktails occurred sonner and

with higher efficiencies than when phages were used alone)

Phage Therapy Conclusions

The efficiency of phage therapy was affected by the variation of the

environmental parameters, namely by salinity and organic matter content of

the waters.

As the effectiveness of phage therapy increases with water salt content, this

approach appears to be a suitable choice for marine aquaculture systems.

The phage treatment of challenged larvae with Vibrio anguillarum reduced the disease symptoms in the fish.

The disease symptoms after 3 days of incubation were similar to those that occur normally and were significantly lower in phage-treated larvae than in non-treated ones

Phage Therapy Conclusions

Ongoing Research project As Principal Investigator (Coordinator) • Project: PTDC/AAC-AMB/112934/2009: Terapia fágica como

alternativa de baixo impacto ambiental para inactivar bactérias patogénicas em pisciculturas. (Phage therapy as a low environmental impact alternative to inactive phatogenic bacteria in fishfarming plants) (2011-2014). Funds: 150 000 €.

• Project: PROMAR nº 31-03-05-FEP-0028. Terapia Fágica - Uma Nova Tecnologia para Depuração de Bivalves (Depurofago). (Phage therapy - a new technology to depurate shellfish - Depurofago). Fundo Europeu das Pescas (FEP). Programa Operacional de Pescas 2007-2013: Projectos-piloto, Portugal (2012-2015). Funds: 273 135 €.

• Project: PTDC/MAR-EST/2314/2012. Impacto da radiação solar nos processos fotoquímicos da matéria orgânica dissolvida e microbianos no ambiente estuarino. (Impact of solar radiation on dissolved organic matter photochemical and microbial processes in the estuarine environment) (2013-2015). Funds: 160 512 €.

http://www.fct.mctes.pt/fctsig/contents/c030401/publicar_2010.asp?idrx=112934&UGuid=%7b33E020DE-B24C-4E24-A37D-42C2036BC9CA%7d





























Ongoing Research project

As Investigator • Project: AQUASAFE - Development of new technologies to anticipate and

diagnose disease outbreaks in aquaculture (2011-2014). Funds: 310 156 €.

• Project: RASTREMAR - Use of molecular tools in the traceability of marine

food products (2011- 2014). Funds: 346 799 €.

• Project: Project QREN, nº 13846 Desenvolvimento de Novas Tecnologias

de Suporte à Criação de Produtos Inovadores (NOVELTEC) (2011-2014).

• Project: Project QREN, Carne fresca com prazo de validade alargado e

maior segurança microbiológica recorrendo à tecnologia de Altas Pressões

(AP) – FRESHMEAT-AP (2014-2015).

• Project: Long term monitoring in the Ria de Aveiro: towards a deeper

understanding of ecological, environmental and economic processes. 2011-

2014). Funds: 199 999 €.

Acknowledgement

Universidade de Aveiro

CESAM, QOPNA

Fundação para a Ciência e Tecnologia (Proj.POCTI/CTM/58183/2004

POCI 2010 (FEDER) (projecto POCI/CTM/58183/2004)

Prof. José Cavaleiro Prof. Graça Neves Prof. Amparo Faustino Prof. Augusto Tomé Dr. João Tomé Dra Carla Carvalho MSc Clara Gomes

Department of Chemistry

Prof. Adelaide Almeida Prof. Ângela Cunha Dr. Newton Gomes PhD Eliana Alves PhD Liliana Costa PhD Yolanda Silva PhD Carla Pereira MSc Anabela Oliveira MSc Anabela Tavares MSc Joana Almeida MSc Joana Brás MSc Cátia Arrojado

Department of Biology

Corte das Freiras Aquaculture staff

Thanks for your kind attention

300 W.m-2 150 W.m-2 New approaches with low...

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