Host-Pathogen interaction for Mytilus edulis

Faculteit Bio-ingenieurswetenschappen

Academiejaar 2014 – 2015

Host-Pathogen interaction for Mytilus edulis

-

Characterisation of virulence factors

and application of blue mussel pathogens

in an adult challenge test

Suzan Demuynck

Promotor: M.Sc. Christophe Wille

Promotor: Dr. ir. Tom Defoirdt

Tutor: Mieke Eggermont

Masterproef voorgedragen tot het behalen van de graad van

Master in de industriële wetenschappen: biochemie

Copyright

The author and promotors give permission to put this thesis to disposal for consultation

and to copy parts of it for personal use. Any other use falls under the limitations of

copyright, in particular the obligation to explicitly mention the source when citing parts out

of this thesis.

Ghent, July 2015

Suzan Demuynck,

Christophe Wille,

Tom Defoirdt, Mieke Eggermont,

Acknowledgements

This master dissertation and associated internship are part of the requirements to fulfil the

degree of 'Master in Science - Industrial Engineer - specialisation Biochemistry'. I learned

a lot during this internship at the (Lab for aquaculture and) Artemia Reference Center and

have been immersed to the wonderful world of aquaculture and especially the blue

mussels.

This master dissertation would not have been possible without the help and guidance of

several people who contributed their valuable assistance in the preparation and

completion of this thesis and my studies in general.

At first I want to thank both my promotors Tom Defoirdt and Christophe Wille for making

this master dissertation and internship possible. Secondly I want to express my gratitude

to my tutor Mieke Eggermont for sharing her knowledge about the topic, securing the

supply of the test animals and her enthusiasm and encouraging words when things got

complicated. I also want to thank both Mieke and Tom for their suggestions during the

experimental work and their patience during reading, re-reading and correcting my written

report. Besides them, I want to thank Tom Baelemans for his technical help. I am also

thankful to Nancy Nevejan, Jorg Desmyter, Aäron Plovie, Geert Vandewiele, Brigitte

Moffaert and the rest of the team for their help, good chats and nice moments. At last I

want to thank my lab partner Ali Rayhan for his chats and jokes during the experiments in

the ‘musselroom’. Thanks to all this I went working with a smile every day.

Last, but not least, I want to thank my parents, sister and brother, family and friends for

their help and support during my thesis and my studies in general.

Content

List of abbreviations ............................................................................................................ i

Abstract ............................................................................................................................. ii

Samenvatting .................................................................................................................... iii

Introduction ....................................................................................................................... 1

Part I. Literature study ................................................................................................... 3

1.1. Blue mussel – Mytilus edulis ................................................................................... 3

1.1.1. Phylogeny ........................................................................................................ 3

1.1.2. Anatomy .......................................................................................................... 3

1.1.3. Physiology ....................................................................................................... 4

1.2. Mussel cultivation ..................................................................................................10

1.2.1. Habitat of the Blue mussel ..............................................................................10

1.2.2. Economic value of Mytilus edulis in aquaculture .............................................11

1.2.3. Mussel culture techniques ...............................................................................12

1.2.4. Problems associated with mussel hatcheries ..................................................13

1.3. Mass mortality events ............................................................................................14

1.3.1. General information ........................................................................................14

1.3.2. Diseases and mass mortality events in bivalve aquaculture ............................18

1.3.3. Microbial origin of mass mortality events in Mytilus edulis ...............................20

Part II. Materials and Methods ......................................................................................22

2.1. Bacterial culture media ..........................................................................................22

2.1.1. Luria-Bertani35 broth ........................................................................................22

2.1.2. Luria-Bertani35 agar .........................................................................................22

2.2. Antibiotics ..............................................................................................................22

2.2.1. Rifampicin .......................................................................................................22

2.2.2. Ampicillin, kanamycin and chloramphenicol ....................................................22

2.2.3. Tetracycline and enrofloxacin ..........................................................................22

2.3. Bacteria .................................................................................................................22

2.4. Antibiotic sensitivity tests .......................................................................................23

2.5. Virulence factor tests .............................................................................................24

2.5.1. Hemolysin production......................................................................................24

2.5.2. Exopolysaccharide production and biofilm formation .......................................25

2.6. Development of an adult challenge test for Mytilus edulis ......................................25

2.6.1. Adult experiment 1: Try-out of an adult challenge test protocol .......................25

2.6.2. Adult experiment 2: Microbiological monitoring of the rearing water in the

presence of mussels .................................................................................................26


absence of mussels ..................................................................................................27

2.6.4. Adult experiment 4: Verification of the effectivity of the mussel pre-treatment .27

Part III. Results ...........................................................................................................28

3.1. Antibiotic sensitivity & virulence factor production ..................................................28

3.1.1. Antibiotic sensitivity .........................................................................................28

3.1.2. Virulence factors .............................................................................................30

3.2. Development of an adult challenge teste for Mytilus edulis ....................................34

3.2.1. Adult experiment 1: Try-out of an adult challenge test protocol .......................34


presence of mussels .................................................................................................36


absence of mussels ..................................................................................................36

3.2.4. Adult experiment 4: Verification of the effectivity of the used pre-treatment .....37

Part IV. Discussion and recommendations for the future .............................................38

4.1. Antibiotic sensitivity tests .......................................................................................38

4.1.1. Tentative comparison of antibiotic activity of the different antibiotics towards

isolates ME1-15 ........................................................................................................39

4.1.2. Comparison of antibiotic sensitivity of the different isolates .............................40

4.1.3. Recommendations for the future regarding the antibiotic sensitivity of different

antibiotics and towards the different isolates .............................................................41

4.2. Virulence factor production ....................................................................................41

4.2.1. Hemolysin production .................................................................................41

4.2.2. Exopolysaccharide production and biofilm formation ..................................42

4.2.3. Recommendations for the future regarding the virulence factor production .43

4.3. Development of an adult challenge test for Mytilus edulis ......................................43

4.2.4. Try-out of an adult challenge test protocol ..................................................44

4.2.5. Effectivity of the used pre-treatment ...........................................................44

4.2.6. Effectivity of the cleaning and disinfection protocol .....................................45

4.2.7. Recommendations for the future regarding the adult challenge test ............45

Part V. Conclusions ...................................................................................................47

5.1. Antibiotic sensitivity ...............................................................................................47

5.2. Virulence factors ....................................................................................................47

5.3. Conslusion about the adult challenge test for Mytilus edulis ..................................48

5.4. Overall conclusion .................................................................................................48

References ......................................................................................................................... i

Appendices ....................................................................................................................... 1

i

List of abbreviations

‰ Parts per thousand

Amp Antimicrobial peptide

AMP Ampicillin

ARC (Lab for aquaculture and) Artemia Reference Center

BRD Brown Ring Disease

C Chloramphenicol

DMSO Dimethyl sulfoxide

ENR Enrofloxacin

EPS Exopolysaccharide production

FAO Food and Agriculture Organization of the United Nations

JOD Juvenile Oyster Disease

K Kanamycin

kDa kilo Dalton

LB35 Luria-Bertani35 (broth)

OD Optical Density

PON Pacific Oyster Nocardiosis

RIF Rifampicin

TE Tetracycline

USD United States Dollar

ii

Abstract

Over the last years an increased interest in cultivation of mussels has been noticed. The

cultivation of mussels, such as blue mussel (Mytilus edulis), still fully depends on natural

spat collection from the wild. To solve the problem of unreliable spat supply, spat could be

produced in hatcheries. Despite the fact that hatchery technology is available and

advantageous over natural spat collection, it is not yet economically viable. The major

bottleneck is unexpected mass mortality in dense larval cultures (i.e. before the larvae

reach the spat stage). Previous research revealed that mass mortality can be caused by

several heterotrophic bacteria that are naturally associated with healthy wild blue mussel

adults. In previous research, 15 different bacterial strains (ME1-15), from which 12 belong

to the Vibrio splendidus clade, have been isolated from wild-caught blue mussel adults.

This master dissertation aimed to evaluate the host-pathogen interactions between these

15 bacterial strains and Mytilus edulis.

The first part of this study aimed to further characterize the virulence factors (i.e. gene

products involved in infection) of these bacteria and to evaluate their virulence towards

Mytilus edulis. All strains possess hemolytic activity towards sheep blood. To confirm

hemolytic activity towards mussel hemocytes, the test should be repeated with mussel

hemolymph. Indeed, hemolytic activity towards sheep blood is just an indication of the

possible pathogenicity of these bacteria towards blue mussel. Secondly, some of the

bacteria, namely ME11 and ME15 also showed exopolysaccharide production and biofilm

formation. However, the protocol first needs to be optimized before solid conclusions can

be drawn. So, in order to fully understand and verify the mechanism of how these

bacterial strains cause disease in Mytilus edulis, further research on their virulence factors

is needed.

The second part of this study aimed at optimising a challenge test procedure to evaluate

the virulence of the 15 bacterial strains in adult wild-caught Mytilus edulis. It was found

that a further optimisation of the adult challenge test procedure is needed. First, another

antibiotic mixture should be used in the pre-treatment of the mussels. The original mixture

– consisting of ampicillin, kanamycin and chloramphenicol – was not effective enough to

kill the bacteria that are naturally occurring in, on and around blue mussel adults. A new

antibiotic mixture – consisting of oxytetracycline, enrofloxacin and chloramphenicol – was

based on the results of an antibiotic sensitivity test and worked better but was still not fully

effective. Secondly, a new cleaning and disinfection method for the material used in the

test should be established as this study shows that there are still bacteria present after

thoroughly mechanically cleaning and disinfection with a strong acid. Hence, this study

indicates that further optimisation and adaptation of the adult challenge test is needed,

before the effectivity of the virulence of the isolates can be evaluated.

iii

Samenvatting

Gedurende de laatste jaren is de interesse voor het kweken van mosselen toegenomen.

Het kweken van mosselen, zoals de blauwe mossel (Mytilus edulis) is vandaag de dag

echter nog volledig afhankelijk van het opvissen van mosselzaad uit de natuur. Om het

probleem van de onbetrouwbare aanvoer van mosselzaad uit de natuur op te lossen kan

dit mosselzaad geproduceerd worden in kwekerijen. Desondanks dat dergelijke

kwekerijen reeds bestaan en voordelen bieden ten opzichte van het natuurlijke aanbod

van mosselzaad, is deze productie nog niet economisch haalbaar. Het grootste probleem

is de massale sterfte van mossellarven voor ze het stadium van mosselzaad bereiken.

Voorgaand onderzoek heeft aangetoond dat deze massale sterfte veroorzaakt kan zijn

door heterotrofe bacteriën die van nature voorkomen in, rond en op gezonde, wilde

volwassen mosselen. Enkele van deze virulente bacteriële stammen werden reeds

geïsoleerd en geïdentificeerd (ME1-15), waarvan er 12 behoren tot de Vibrio splendidus

clade. Deze masterproef richt zich op het evalueren van de gastheer-pathogeen

interacties tussen deze 15 bacteriële stammen en Mytilus edulis.

Het eerste deel van deze studie omvat het verder karakteriseren van de virulentie factoren

(genproducten die betrokken zijn bij infecties) van deze bacteriën. Al de bacteriële

stammen vertonen hemolytische activiteit tegen schapenbloed. Om de hemolytische

activiteit tegen mossel hemocyten te bevestigen zou deze test moeten herhaald worden

met mossel hemolymfe, omdat de hemolytische activiteit tegen schapenbloed enkel maar

een indicatie is voor de mogelijke pathogeniciteit van de bacteriën tegenover de blauwe

mossel. Ten tweede vertonen enkele stammen, namelijk ME11 en ME15,

exopolysacharide productie en biofilm vorming. Het gebruikte protocol dient echter eerst

verder geoptimaliseerd te worden vooraleer degelijke conclusies kunnen bevestigd

worden. Dus, om de manier waarop deze bacteriën ziekte veroorzaken bij Mytilus edulis

volledig te begrijpen is verder onderzoek naar de virulentie factoren van deze 15

bacteriële stammen aanbevolen.

Het tweede deel van deze studie omvat het optimaliseren van een 'challenge test' protocol

om de virulentie van de 15 bacteriële stammen tegen gezonde, wilde volwassen blauwe

mossels te evalueren. Het is duidelijk dat verdere optimalisatie van het uitgeteste protocol

nodig is. Ten eerste dient tijdens de voorbehandeling van de mossels een andere

antibioticummix gebruikt te worden. De oorspronkelijke mix - die bestaat uit ampicilline,

kanamycine en chlooramfenicol - is niet effectief genoeg om de bacteriën die van nature

voorkomen in, op en rond volwassen blauwe mossels te doden. Een nieuwe

antibioticummix, gebaseerd op de resultaten van een antibioticum resistentie test - en

bestaande uit oxytetracycline, enrofloxacin en chlooramfenicol - werkt veel beter maar is

nog niet volledig effectief. Ten tweede dient een andere reinigings- en desinfectiemethode

uitgetest te worden, want deze studie toont aan dat er nog steeds bacteriën aanwezig zijn

na grondig mechanisch reinigen en desinfecteren met een sterk zuur. Deze studie bewijst

dat verdere optimalisatie en aanpassingen aan het protocol voor de 'adult challenge test'

nodig is vooraleer de virulentie van de onderzochte bacteriële stammen ten opzichte van

volwassen mosselen kan geëvalueerd worden.

1

Introduction

Aquaculture, the cultivation of marine and freshwater organisms under controlled

conditions, is a fast-growing sector with an average annual increasing production rate of

6,1% in the last decade (from 2002 till 2012). Mollusc cultivation comprises 23% of the

worldwide aquaculture production. The phylum molluscs includes the class of the

bivalves, which consists of oysters, clams, scallops and mussels (Bouchet and Gofas,

2015; FAO, 2014).

Mussels are important bivalve species for both economical and ecological reasons. In

addition to their use as a food source, mussels are ideal biological markers for monitoring

aquatic environments due to their filtering capacity and worldwide distribution (Brenner et

al., 2014; Widdows et al., 2002). In Europe, the two main mussel species are Mytilus

edulis (blue mussel) and Mytilus galloprovincialis (Mediterranean mussel) (Beaumont et

al., 2006).

Unlike for many other commercial aquatic species, bivalve production is unstable. This is

because nowadays mussel cultivation entirely depends on natural spat (or mussel seed)

collected from the wild, which leads to an unreliable spat supply (Eggermont et al., 2014).

Local shortages of natural spat have been reported in the recent past and these resulted

in economic losses. Mussel seed production in hatcheries, in which adult mussels are

collected from the wild and used as broodstock, could provide a solution for this irregular

natural spat supply. Despite the fact that hatchery technology is available and

advantageous over collection of natural spat, it is not yet economically viable and only

implemented in a few hatcheries worldwide (Galley et al., 2010).

The major bottleneck in blue mussel spat hatchery development is unexpected mass

mortality events in dense larval cultures. The causative agent is currently unknown, but it

can be assumed that the underlying cause of these mortality events is of microbial origin,

as already described for other bivalve species (Beaz-Hidalgo et al., 2010; Eggermont et

al., 2014). Even more, Eggermont et al. (2014) demonstrated that mass mortality events

in Mytilus edulis can be due to pathogens associated with healthy wild-caught blue mussel

adults. In previous research at the Laboratory of aquaculture and Artemia Reference

Center (ARC) in Ghent by Tamanji (2013) 15 different bacterial strains (ME1-15), from

which 12 strains belong to the Vibrio splendidus clade and the other three strains are

Photobacterium spp., were isolated from mass mortality events.

Further previous research at the ARC by Yumo (2014) evaluated the virulence of the

above-mentioned 15 bacterial strains. This research study revealed that all the strains

produce several (putative) virulence factors, but no correlation was found between

virulence factor production and mortality of blue mussel larvae. Another master

dissertation student, Vanmeerkerken (to be published), developed an adult challenge test

for Mytilus edulis. Such a challenge test is an essential toolbox to study host-pathogen

interactions.

2

This master dissertation is divided into two main parts. In the first part, a further

characterisation of the virulence of the 15 bacterial strains, which were isolated from

previous mass mortality events of Mytilus edulis, (ME1-15) was performed by in vitro

virulence factor experiments. This part of the study continues the work by Yumo (2014),

because the results concerning some virulence factors were questionable. So, this first

part aimed to answer the following research question: ‘Do the 15 bacterial isolates (ME1-

15) produce important virulence factors like exopolysaccharide production, biofilm

formation and hemolytic activity?’. Supplementary tests investigated the sensitivity of the

bacterial isolates towards different antibiotics belonging to several antibiotic classes.

The second part of this study aimed to further develop an adult challenge test to evaluate

the virulence of the bacterial isolates as a toolbox for further research on the interactions

between Mytilus edulis and Vibrio spp. The goal of this part of the study was to find an

answer on the following research question: ‘Could these 15 bacterial isolates (ME1-15) be

applied as pathogens in an adult blue mussel challenge test?’. Regarding the results and

difficulties encountered during the try-out of this adult challenge test, additional

experiments were performed. Because this is one of the first times an adult challenge test

was performed for Mytilus edulis, it could be regarded as pioneer-work of which the

results lead to adaptations in the design of a better challenge test.

This master dissertation is constituted in different chapters. First of all, a literature study

was performed. Information about the phylogeny, anatomy and physiology of the host

organism Mytilus edulis is given, followed by the description of the cultivation techniques

for blue mussels, and finally, information regarding mass mortality events and previous

research on host-pathogen interactions for Mytilus edulis is reported. Secondly, a

description is given on how the different experiments were performed in the Materials and

Methods section. In a third section, the results of the different experiments are presented.

Finally, in the discussion section, the results are critically analysed, and conclusions and

recommendations for the future are given.

3

Part I. Literature study

An overview of the current knowledge of the host organism Mytilus edulis will be given in

this literature study to give the reader the necessary insights to understand the performed

experiments in this thesis.

The phylogeny, general anatomy and physiology of the blue mussel will be highlighted in

the first part of this literature study. In the second part the habitat of the blue mussel, the

different mussel culture techniques and the economic value of the blue mussel will be

described. Finally more information regarding mass mortality events, diseases and

pathogens of Mytilus edulis will be described in the third and last part of this literature

study.

1.1. Blue mussel – Mytilus edulis

1.1.1. Phylogeny

Mytilus edulis, commonly known as the blue mussel, belongs to the phylum Mollusca. This

phylum is one of the largest and most diverse groups in the kingdom Animalia and

encloses six different classes, including Bivalvia to which Mytilus edulis belongs (Bouchet

and Gofas, 2015; Gosling, 2003).

Early taxonomy was almost solely based on characterisation of the shell. Later research

studies identified four different species in the genus Mytilus based on their morphological

and genetic differences, namely M. californianus, M. trossulus, M. galloprovincialis and M.

edulis (Koehn, 1991).

The scientific classification of Mytilus edulis is given in figure I-1.

Figure I-1. Taxonomy of Mytilus edulis (Linnaeus, 1758) (Bouchet and Gofas, 2015).

1.1.2. Anatomy

The blue mussel is characterised by its two elongated and triangular shaped valves that

are equal in size and have a bluish to black colour. The valves are hinged together by

means of a ligament at the anterior. The opening and closing of the two valves is

4

conducted by two muscles, a large posterior adductor muscle and a much smaller anterior

adductor muscle. Another important muscle is the foot, used for moving and anchoring.

The byssus gland, that is situated in the foot, secretes continuously tanned proteins as a

bundle of tough threads, called byssus threads. These byssus threads allow the mussel to

attach itself repeatedly to the substrate (Beaumont et al., 2006; FAO, 2015; Gosling,

2003).

Figure I-2. General anatomy of Mytilus edulis (Gosling, 2003).

The internal organs of the mussel, such as the gills and the digestive gland, are enclosed

by two lobes of connective tissue that is called the mantle. The mantle is attached to the

shell at the pallial line. The mantle margins are fused together between the inhalant and

exhalant openings. The mantle does not only cover the internal organs, it also contains

most of the gonads and is the main site for the storage of nutrient reserves, in particular

glycogen. In adult mussels the mantle colours orange in females and creamy-white in

males, due to the presence of the gametes (FAO, 2015; Gosling, 2003).

1.1.3. Physiology

1.1.3.1. Feeding mechanism and digestive system

The blue mussel is a filter feeder, a feeding process that is well conserved amongst most

bivalve species. Food particles are taken up from the incoming water current, followed by

selection, transport, digestion and finally ingestion as described below (Gosling, 2003,

Jorgensen, 1990).

The water, with suspended organic particles such as unicellular phytoplankton, enters the

mussel through the inhalant opening. By the help of cilia, located on the gills, particles of

interest are trapped in mucus and transported along the labial palps towards the mouth.

The food particles are then, again by cilia, transported through the oesophagus and

5

directed towards the stomach. The stomach is surrounded by a dark digestive gland. Due

to mechanical and enzymatic processes the food particles are digested extracellular.

These digested end-products are then absorbed in the hemolymph. Via the hemolymph

this transport of nutrients could for example end up in the mantle tissue where it is stored

as the metabolic reserve glycogen. The waste materials and rejected products are

converted to faeces in the intestines and excreted through the anus. They find their way

back into the seawater through the exhalant opening (Gosling, 2003, Jorgensen, 1990).

The selection of particles of interest is a selective process. The unwanted particles are

rejected and exit the mussel, as pseudofaeces, together with the waste water through the

inhalant opening (Jorgensen, 1996).

Figure I-3. Schematic illustration of filter feeding process (Aquascope, 2000).

The blue mussel is an active filter feeder and can filter up to 20 liters of seawater a day

(Delbaere, 2005). This large filter capacity allows the mussel to unselectively ingest not

only food particles and organic matter, but also bacteria, toxic substances, parasite larvae

and even chemical pollutants. Because of this, the blue mussel is able to concentrate

several contaminants at high levels in its tissue and is thereby an ideal biological marker

for monitoring aquatic environments (Brenner et al., 2014, Widdows et al., 2002).

1.1.3.2. Reproduction and life cycle

The blue mussel is dioecious, which means that the sexes are separate. Males and

females can be distinguished from each other by a slight difference in the colour of the

ripe gonads, as described earlier (1.1.2. Anatomy) (Gosling, 2003).

The life cycle of a mussel starts with the spawning of adult animals. This is the

simultaneously release of a large amount of sperm and eggs into the water. The sperm is

released before the eggs, in fact the sperm triggers the release of the eggs (Gosling,

2003). Females can produce up to 8 million eggs per individual and for each egg there are

about 10 000 spermatozoa released by males (Newel, 1989; Zaidi et al., 2003). This

spawning is triggered by different factors and occurs from early spring (April) till the end of

summer (September) (Gabbot, 1976). The triggering factors for spawning are a

combination of (1) internal factors such as nutrient availability and hormonal cycle and (2)

6

external factors such as food availability, temperature, salinity and tidal changes. The

most influencing factors are water temperature and food availability (Newel, 1989).

Fertilization occurs in the water. At first two polar bodies are expelled in order to bring the

chromosome number back to normal. A little later the four and eight cell stage is clearly

visible under the microscope. After 24 hours the fertilized egg is developed into a

trochophore larvae by meiotic division and passing the blastula and gastrula stage (Helm

and Bourne, 2004). This trochophore larvae is a motile planktonic larvae and is not yet

capable to eat by itself, it relies on its internal yolk for energy supply (Newel, 1989; Seed,

1976). After 48 hours the larvae develops into a D-shaped larvae, having a clearly visible

transparent double shell. During the next weeks the larvae further develop and pass

through the veliger and pediveliger stages. While further developing, the larvae enter the

pediveliger stage and it can swim freely and digest phytoplankton (Zaidi et al., 2003).

When the larvae are fully developed metamorphosis starts and it settles on hard substrate

as a juvenile mussel. In this latter stage the larvae is also called spat or mussel seed. This

settlement occurs due to the fact that the larvae develop a hard shell and thereby falls

down to the bottom of the sea. Because of this the primary settlement is also called

spatfall. In total, the larval development, from trochophore larvae to spat, varies between

15 and 35 days (Helm and Bourne, 2004; Newel, 1989).

When the juveniles are about 1-1,5 mm they can easily detach themselves from the

substrate and move to another location by crawling or floating in the water column. The

water current will transport them to densely populated mussel beds where they use their

byssus threads for their final settlement (Newel, 1989; Seed, 1976).

Figure I-4. Schematic illustration of reproduction cycle (University of Waikato, 2013).

The last stage is the maturation till an adult mussel. In optimal conditions of temperature

and food availability, a mussel is fully developed and ready for reproduction after about

7

one year. The gametogenesis takes place in winter. During winter the food supply is

limited and the necessary energy comes from their glycogen reserve (Gabbot, 1976;

Newel, 1989)

1.1.3.3. Cardiovascular system

The cardiovascular system is an open circulatory system in which the organs are bathing

directly in hemolymph (Gosling, 2003; Pruzzo, 2005). This open circulatory system

consists of several separate sinuses and a network of blood vessels. The heart is the

chief blood-propelling organ in molluscs. It lies in the mid dorsal region of the body in the

pericardial cavity. This cavity is a space that surrounds the heart and is lined by the

pericardium. The heart consists of a single but muscular ventricle and two paired atria or

auricles (Gosling, 2003).

Oxygen from the incoming water diffuses to the hemolymph through the gills. This

oxygenated hemolymph flows then via arteria to the heart. It enters the heart in the

auricles and flows from there into the ventricle. Then the ventricle contracts and thereby

drives the hemolymph in the anterior aorta, from where it is divided into many arteries.

The most important arteries are the pallial arteries, which supply the mantle with

hemolymph, and the visceral arteries, which supply other organs such as the stomach, the

intestines, the muscles and the foot. The arteries break up into a network of vessels,

which join together to form veins and then end up in the sinuses. After the oxygen is used,

the hemolymph flows back to the heart via a venous system. The venous blood from the

mantle directly returns back to the auricles, the venous blood from the viscera returns via

the kidney to the auricles for purification. From the heart the deoxygenated hemolymph

flows to the gills, where the CO2 dissolves in the exhalant current and fresh oxygen can

diffuses again to the hemolymph (Gosling, 2003).

Figure I-5. Schematic illustration of cardiovascular system of bivalves (Anonymous, 2015).

Hemolymph, or ‘mussel blood’, has an important role in different physiological systems

within bivalves, such as gas exchange, nutrient distribution, osmoregulation, waste

elimination and internal defence. Hemolymph contains serum and hemocytes, the

hemocytes are not confined to the hemolymph and can move freely in the mussel tissues

(Gosling, 2003; Pruzzo, 2005). The hemolymph is a colourless plasma, because the

oxygen level in the hemolymph is similar to that in seawater, so there is no need for a

respiratory pigment (Gosling, 2003).

8

1.1.3.4. Immune defence system

As all invertebrates, bivalves are characterised by an innate, non-specific immune system.

This means that they combat foreign components without any memory regarding previous

contacts, so without the help of antibodies, components of the adaptive immune system

(Gosling, 2003; Roch, 1998). The internal defence system of bivalves can be divided into

the cellular and humoral response, two separate mechanisms, closely working together.

The cellular response or cell-mediated immunity, includes phagocytosis and cytotoxic

reactions by circulating hemocytes. The humoral response includes various reactions

mediated by humoral defence factors such as soluble lectins, hydrolytic enzymes and

antimicrobial peptides. A combined action of these two systems results in a robust and

well working immune system. (Canesi et al., 2006; Pruzzo, 2005). A pathogen or foreign

particle has to pass primarily through the first line of defence, which consist of several

chemicophysical barriers, such as het external shell and mucus (Canesi et al., 2002).

Figure I-6. Schematic overview of the immune defence system in bivalves. Cells and serum

factors involved in bacterial killing activity of the hemolymph (canesi, 2002).

1.1.3.4.1. Cellular immunity

Cellular response is carried out by hemocytes and in Mytilus edulis they can be

subdivided into two categories, namely granulocytes and hyalinocytes or agranulocytes.

The former type, granulocytes, are the most abundant hemocytes in bivalves and have a

large variability in number and type of granules and degree of phagocytic activity. The

latter group, agranulocytes, have a few or no granules and are less phagocytic then

granulocytes (Gosling, 2003; Song et al., 2010).

There are different types of cellular defence mechanisms in bivalves, namely

hemocytosis, phagocytosis and encapsulation.

Hemocytosis, or hemocyte proliferation, is the first response to an infection or presence of

a non-self particle. This involves an increase of circulating hemocytes, which move

towards the infected of injured tissue (Gosling, 2003). This movement of the cells is

activated by chemoattractant substances, such as opsonines, to which the hemocytes are

sensitive. This movement is implemented by two motile responses, namely chemotaxis

and chemokinesis. Chemotaxis is a directional movement and in Mytilus edulis for

example triggered by the presence of lipopolysaccharides from Escherichia coli and

Serratia marcescens. Chemokinesis is a random, indirectional migration of the hemocytes

and for example stimulated by N-formyl-methionyl-leucyl-phenylal-anine (N-FMLP) in the

blue mussel (Canesi et al., 2002; Song et al., 2010).

9

After this movement, non-self recognition and attachment of the hemocyte to the targeted

particle occurs and the particle is enclosed in a vesicle in the cell by endocytosis. This

vesicle is also called a primary phagosome and fuses together with a lysosome to form a

phagolysosome. In this structure, the engulfed particle is destroyed by lysosomal

enzymes, reactive oxygen intermediates, nitric oxide and antimicrobial factors (Gosling,

2003; Song et al., 2010). This mechanisms is called phagocytosis and is illustrated in

figure I-7.

Figure I-7. Schematic illustration of phagocytosis (Anonymous, 2015a).

Encapsulation is the cellular immune response for foreign particles that are too large to be

phagocytosed. In this mechanism the foreign particle is encapsulated with hemocytes and

cytotoxic products, such as degradative enzymes and free radicals, are released to

destroy the particles (Song et al., 2010).

1.1.3.4.2. Humoral immunity

Humoral response is carried out by humoral defence factors that are present in the

hemolymph, as mentioned above. These factors can cause a direct cytotoxic effect on

pathogens and a lot of soluble molecules in the hemolymph can be categorised as

humoral defence factors. It should be noted that some of these factors are present in

hemocytes and are thereby also involved in the cellular defence. The most abundant

humoral defence factors in Mytilus species are (soluble) lectins, hydrolytic and lysosomal

enzymes such as phosphatase and lysozyme and antimicrobial peptides (Canesi et al.,

2002; Pruzzo, 2005).

Lectins

Lectins are sugar binding proteins that can interact specifically and reversibly with

membrane glycoproteins or glycolipids of bacteria or other invaders (Renwrantz, 1990).

Thereby lectins are involved in several immune functions, such as self/non-self

recognition and associated effector mechanisms (Song et al., 2010).

10

Lectins can have a direct or indirect role in the phagocytosis process. Lectins can cause

agglutination and thereby potentiate the phagocytosis process by immobilisation of

bacteria. On the other hand lectins can also act as bridge molecules between bacteria and

hemocytes and thereby act as opsonisation for phagocytosis (Pruzzo, 2005).

There exist a lot of lectins and they can be distinguished from each other by differences in

molecular size, subunit structure, agglutination properties and sugar-binding specificity

(Canesi et al., 2002).

Lysozymes

Lysozyme is an ubiquitous enzyme that is characterized by cleavage activity. It catalyses

the hydrolysis of β-1,4-glycosidic linkage between N-acetylmuraci acid and N-

acetylglucosamine of the peptidoglycan structure in bacterial cell walls (Song et al., 2010).

There are a lot of lysozymes and they are subdivided according to the organism in which

they were identified the first time, such as i-type or invertebrate-type lysozyme (Song et

al., 2010). Because of their cleavage ability, lysozyme does not only play an important role

in host defence, but they also have an important digestive role (Olsen et al., 2003).

Lysozymes occur in different types of tissue - such as the crystalline style sac, soft tissue,

hemocytes and hemolymph – and their activity level depends on the type of tissue. In

Mytilus edulis four different types of lysozymes are observed. The main mussel lysozyme

is a soft-body lysozyme and has an antibacterial activity. The three remaining lysozymes

are crystalline-style associated lysozymes from the digestive gland and are solely involved

in digestion (Olsen, et al., 2003).

Antimicrobial peptides

Antimicrobial peptides (Amp) are a major component of the innate immune defence

system of bivalves. Amp’s are molecules with a mass less than 10 kDa which show

antimicrobial and antifungal properties (Tincu and Taylor, 2004).

Approximate 10 different Amp’s are identified in the blue mussel today. They can be

categorised into four different groups according to their antimicrobial and antifungal action

and to their primary structure (hydrophobic, cationic or amphipathic). Those four groups

are called defensins, mytilins, myticins and mytimycins (Canesi et al., 2002; Song et al.,

2010).

Amp’s act by disrupting the membrane integrity, for example by the formation of pores in

the microbial membrane (Tincu and Taylor, 2004).

1.2. Mussel cultivation

1.2.1. Habitat of the Blue mussel

Mytilus edulis has a wide distribution pattern, extending from the artic towards the mild

subtropical regions. In European waters, blue mussels are distributed from the White Sea

in the North (Russia), over the North Sea (The Netherlands and Belgium) and the Atlantic

Ocean (The United Kingdom and Ireland) to the Atlantic coast at the South (southern

France) (see the red circle in figure I-8) (Beaumont et al., 2006; FAO, 2015; Gosling,

2003).

11

Figure I-8. Worldwide distribution of bivalves. Mytilus edulis is situated in the Artic regions

and European waters (red circle) (Gosling, 2003).

Mytilus edulis can also be found in a wide range of surroundings, from high intertidal to

subtidal regions, as well as oceanic seawaters to estuarine areas and also on wild rocky

coastlines to sheltered areas. However this species prefers gently sloping and slow-

drained platforms. (FAO, 2015; Gosling, 2003).

Blue mussels can withstand a wide range in temperature. They are well adapted to

temperatures between 5 and 20°C. They have an upper thermal tolerance of 29°C, but

can also survive freezing temperatures. Next to this, blue mussels are also euryhaline,

which means that they survive a wide range in salinity levels. Their growth rate reduces

below 18‰, but they still survive at a salinity level of 4‰. Blue mussels can also withstand

fluctuations in other physiological factors, such as dissolved oxygen level (FAO, 2015;

Gosling, 2003).

1.2.2. Economic value of Mytilus edulis in aquaculture

Aquaculture is a fast growing sector with an average annual production rate of 6,1% in the

last decade – from 36,8 million tonnes (life weight) in 2002 to 66,6 million tonnes in 2012 –

and an estimate value of USD 137,7 billion in 2012. Approximate 23%, or 15,7 million

tonnes (life weight), from the worldwide food fish production is obtained from molluscs.

95% of the worldwide mussel production is derived from aquaculture (European

Commission, 2012; FAO, 2014).

The blue mussel Mytilus edulis belongs to the top five economically important species in

the worldwide bivalve production. It is together with Mytilus galloprovcincialis the most

important bivalve species in Europe (Beaumont et al., 2006).

12

Worldwide, the two biggest mussel producers are China and Europe, followed by Chile

and New-Zealand. In Europe Spain is by far the greatest mussel producer, followed by the

Netherlands, France and Italy (Beaumont et al., 2006; European Commission, 2012; FAO,

2015).

1.2.3. Mussel culture techniques

Mussel culture technique can generally be divided into five phases: collection of mussel

seed, optionally attaching the seed to the culture ropes, thinning, ongrowing and finally

harvesting the mussels (Gosling, 2003).

There are three popular mussel farming methods in Europe. (1) Bottom cultivation, is

widely used in The Netherlands. (2) Bouchot culture, is a traditional technique on the

Atlantic coast of France. (3) Suspended rope techniques, such as raft culture, are applied

in Spain, and longline culture which is merely applied in Sweden and Norway (Beaumont

et al., 2006; European Commission, 2012; Gosling, 2003).

Figure I-9. Production scheme of Mytilus edulis, with the different cultivation techniques

(FAO, 2015).

- Bottom cultivation:

For bottom cultivation, mussel seed is dredged from natural sea beds and

transferred to culture plots in sheltered area, such as the Wadden Sea or the

Eastern Scheldt in the Netherlands. This allows the spat to grow in optimal growth

conditions, without competition for nutrients because of lower density and without

mortality due to predation (Beaumont et al., 2006; FAO, 2015).

13

- Bouchot culture:

The Bouchot method combines mussel seed collection and ongrowing. This

method is carried out in intertidal areas in rows of wooden poles, which are

embedded with half their length in the seabed. Spat bouchots are parallel rows of

poles with horizontal ropes of for example coconut fibre on which the spat settle

down. After settlement the young mussel seed is transferred to tubular nets that

are reattached around the wooden poles for ongrowing (see figure I-10 at the right)

(Beaumont et al., 2006; FAO, 2015; Gosling, 2003).

- Suspended rope technique:

Suspended rope method can be deployed from floating ponton raft systems or

sub-surface longlines and consists of vertical ropes that are supported by

horizontal lines. While growing, mussels are several times divided onto grow-out

ropes or into stocking collectors until they reach a marketable size. The mussel

seed for this method is either caught on collector ropes from the structure or

gathered from natural spatfall (see figure I-10 at the left) (Beaumont et al., 2006;

FAO, 2015; Gosling, 2003).

Figure I-10. Schematic illustration of different mussel cultivation techniques. Right:

Bouchot technique, Left: Longline technique (Bord bia Irish food board, 2015).

After the mussels reach a marketable size they are harvested, cleaned, declumped,

debyssused and sorted by size in processing plants or directly on the decks of the boat

(FAO, 2015).

1.2.4. Problems associated with mussel hatcheries

Mussel cultivation relies nowadays mainly on natural spat collection. Because of this

dependence on nature, the supply of spat for cultivation is unreliable (Eggermont et al.,

2014). Natural spat collection also has to be implemented while paying attention to

maintaining ecological balances and sustainability. Indeed, negative impacts such as

changes in benthic communities due to seed dredging and overfishing, as a consequence

of the increase demand, have been reported (FAO, 2015).

A solution for the unreliable spat supply is the production of spat in hatcheries (Eggermont

et al., 2014). Therefore, adult mussel are collected from nature and used as broodstock.

However, hatchery production of spat is only implemented in a few hatcheries worldwide

(Galley et al., 2010).

14

The advantages of spat production in hatcheries are, for instance, the possibility to grow a

particular strain and the possibility of genetic enhancement. The most important

advantage, however, is the reliable supply of spat and the flexible timing of this supply

(Eggermont et al., 2014; Galley et al., 2010). Despite these advantages, spat production

in hatcheries is not yet cost-effective and the major bottleneck is the unexpected mass

mortality events in dense larval cultures in hatcheries (Eggermont et al., 2014). The

causative agent of this mass mortality is currently unknown, but it can be assumed that

the underlying cause is of microbial origin, such as already described for other bivalves

species (Beaz-Hidalgo et al., 2010). This will be further explained in the next paragraphs.

1.3. Mass mortality events

1.3.1. General information

1.3.1.1. Pathogens, virulence and virulence factors

A bacterium is pathogenic when it has the capacity to cause disease. The degree of

pathogenicity of a pathogen is referred to as virulence, and can be quantitatively

measured. Virulence factors are substances, secreted by the bacterium, that enable the

bacterium to be virulent (Chen et al., 2005; Paillard et al., 2004).

So, a pathogen is generally described as a microorganism that is capable to cause

disease or cause host damage, either direct due to microbial action or indirect through the

immune response of the host organism.

According to the four postulates of Koch, a bacterium is pathogenic when (1) it is present

in large quantities in the host organism, (2) it can be isolated from the host organism and

cultured again, (3) experimental reproduction of the disease in a healthy host organism is

possible and (4) it can be isolated and identified as the same pathogenic bacterium after

this experimental reproduction. It also has to be kept in mind that the susceptibility of the

host organism towards the pathogen might be dependent on its life stage and

environmental conditions (Paillard et al., 2004).

The infection cycle includes several steps. First, the pathogen enters the host, then the

establishment of the pathogen occurs followed by its multiplication. During this phase the

pathogen survives the immune defence system of the host organism and causes damage

to the host tissues and cells or causes systematic inflammation. Finally, the pathogen

leaves the host organism (Chen et al., 2005; Defoirdt, 2014).

Virulence factors are gene products that enable the pathogen to infect and damage the

host, including proteins involved in motility and adhesion of the pathogen to the host,

protection from host defence mechanism and host tissue degradation, iron acquisition and

toxins (Chen et al., 2005; Defoirdt, 2014). Lytic enzymes, exopolysaccharide production

and biofilm formation will be further discussed in more detail because of their interest for

this master dissertation. A schematic overview of different virulence factors produced by

pathogenic bacteria is given in figure I-11.

15

Figure I-11. Schematic overview of different virulence factors produced by bacteria

(Defoirdt, 2014).

- Lytic enzymes

Lytic enzymes damage host tissues, which enables the pathogens to obtain

nutrients and to spread through the host tissues. Lytic enzymes are produced by

many bacteria and the most well-known lytic enzymes in aquaculture pathogens

are hemolysins, proteases, lipases and chitinases (Defoirdt, 2014). Only the

former one, hemolysins, will be discussed further regarding its interest for this

master dissertation.

Hemolysins are extracellular proteins which are produced by many bacteria, such

as Vibrio species (Goebel et al., 1988). Hemolysins are able to lyse blood cells,

either by breaking down the cell membrane by phospholipase activity, or by

forming pores of varying diameter in the cell membrane. This results in cell death

due to the osmotic imbalance resulting from the formed pores (Defoirdt, 2014).

Different types of hemolytic activity exist, namely α-hemolysis and β-hemolysis. α-

hemolysis results in incomplete lysis of blood cells. β-hemolysis results in a

complete lysis of blood cells (Pemberton et al., 1997). Hemolytic activity can be

screened on agar plates that contain blood cells. The occurrence of α-hemolysis

results in greening of the medium, whereas β-hemolysis results in a clearing zone

around the colonies (Defoirdt, 2014).

- Exopolysaccharide production and biofilm formation

Extracellular polysaccharides are substances that are secreted around the cell by

the bacterium and act as a capsule or as loose slime. A specific group of these

extracellular polysaccharides are exopolysaccharides (EPS). They from a loose

slime outside the cell, which acts as an extracellular matrix in biofilms (Defoirdt,

2014). Bacterial EPS are considered to play a crucial role in both the initial

adhesion of the bacteria and the development of biofilms in a later stage (Jiang et

al., 2011).

16

Biofilms are cell-to-cell or cell-to-surface attached microorganisms that form dense

aggregates in a hydrated extracellular polymeric substance matrix (Jiang et al.,

2011). These biofilms enhance the growth and survival of microorganisms by

providing nutrients and protection from antibiotics and host defence mechanisms.

In aquaculture, biofilms on submerged substrates are considered as a reservoir for

potentially pathogenic bacteria (Defoirdt, 2014). EPS is a common constituent of

bacterial biofilms and in many bacteria an increase in EPS production is often

correlated with an increase in biofilm formation (Jiang et al., 2011).

1.3.1.2. Antibiotics, antibiotic-resistance and antivirulence therapy

1.3.1.2.1. Antibiotics

An antibiotic is a substance that acts against a microorganism by its capacity to kill or to

inhibit the growth of the microorganism. Thereby, an antibiotic refers to a substance with

antibacterial, antifungal or antiparasitic characteristics (Hernández Serrano, 2005;

Kummerer, 2009).

Originally the term antibiotic referred to a substance that is produced by a microorganism

and acts against another microorganism. Nowadays, antibiotics are not only produced

naturally by microorganisms, but they can also be synthetic or semi-synthetic. The former

are substances that are produced by chemical synthesis, the latter are obtained from

chemical modification of natural compounds (Kummerer, 2009).

About 250 different chemical entities can be used as antibiotics. All these antibiotics can

be divided into different groups or classes (Kummerer, 2009). At first, antibiotics can be

subdivided in two groups according to their impact on the microorganism. (1) Bactericidal

antibiotics kill the microorganisms, whereas (2) bacteriostatic antibiotics inhibit the growth

of the microorganisms. Antibiotics can also be subdivided according to their mechanisms

of action and chemical structure (Hernández Serrano, 2005; Kummerer, 2009). Typical

modes of action of the most common antibiotics include damaging the cell membrane,

inhibiting cell wall synthesis, inhibiting folic acid synthesis (thereby inhibiting DNA and

RNA synthesis), inhibiting DNA function and inhibiting protein synthesis (Hernández

Serrano, 2005) (see figure I-12). According to their chemical structure, antibiotics can be

grouped in different classes, such as β-lactam antibiotics, tetracyclines, aminoglycosides,

macrolides, glycopeptides, sulphonamides, quinolones (see table I-1). Some of these

classes can be subdivided into different subgroups, such as β-lactam antibiotics that can

be subdivided in penicillins and cephalosporins (Hernández Serrano, 2005; Kummerer,

2009).

17

Figure I-12. Schematic overview of different mechanisms of action by antibiotics (Moore,

2015).

1.3.1.2.2. Antibiotic resistance

In aquaculture, antibiotics are used for disease treatment and as prophylactic agents.

Table I-1 summarizes different classes of antibiotics that are used in aquaculture (Defoirdt

et al., 2011). The wide and frequent use of antibiotics in aquaculture – both for treatment

and prophylactic use – results in a growing resistance development in pathogens. This

means that in many cases bacterial disease can no longer be treated with antibiotics

anymore. Antibiotic resistance can occur due to the ability of bacteria to adapt and survive

changes in external conditions because of their rapid generation rate (every 20-30

minutes under optimal conditions). Another important factor is the ability of bacteria to

exchange genetic information to each other by horizontal transfer of mobile genetic

elements, such as transposable elements or plasmids (Hernández Serrano, 2005;

Kummerer, 2009a).

Table I-1. Overview of different antibiotic classes, together with some examples.

class subgroup example

β-lactam antibiotic aminopenicillin amoxicillin, ampicillin

tetracycline tetracycline, oxytetracycline

aminoglycoside streptomycin, kanamycin

macrolides erythromycin

sulphonamides sulfadiazine, sulphamethoxazole

quinolones quinolones oxolinic acid

fluorquinolones enrofloxacin

amphenicols florfenicol

1.3.1.2.3. Antivirulence therapy

Antivirulence therapy is a promising alternative approach to treat bacterial diseases.

Because bacteria use virulence factors to cause infection, preventing pathogens from

producing and using such factors is an interesting strategy for disease control. This

therapy is thus based on a thorough understanding of the mechanisms that pathogenic

bacteria use to cause diseases (Defoirdt, 2014). The major advantage of antivirulence

18

therapy is that there will be less or no interference with non-target organisms, such as the

commensal microbiota. This is because antivirulence therapy specifically targets virulence

gene expression and regulation (Defoirdt, 2013; Defoirdt, 2014).

Antivirulence therapy consists on one hand of specifically inhibiting one or more virulence

factors. On the other hand, antivirulence therapy can interfere with the mechanisms that

control the expression of virulence factors. These mechanisms are for instance cell-to-cell

communication (or quorum sensing) and host-pathogen signalling. So for example

quorum sensing disrupting agents can prevent bacteria from attaching to the host and are

in this way effective in disease control (Defoirdt, 2014).

1.3.2. Diseases and mass mortality events in bivalve aquaculture

The major disease-causing agents in bivalves are viruses, bacteria, fungi and protozoans

(Gosling, 2003; Paillard et al., 2004). However, within the scope of this master

dissertation, only bacteria will be further discussed.

The aquatic environment harbours a diverse bacterial microbiota and due to their filter

feeding habit, bivalves can accumulate microorganisms, as mentioned above. The

bacterial microbiota is composed of various species from different bacterial genera - such

as Vibrio, Pseudomonas, Acinetobacter, Photobacterium, Moraxella, Aeromoneas,

Micrococcus and Bacillus – that are all commonly found on the shell, in the pallial fluid and

digestive tract of bivalves (Beaz-Hidalgo, 2010; Gosling, 2003). Most marine bacteria are

not harmful (Gosling, 2003).

Bacterial diseases can affect bivalves in all stages of their life cycle (larvae, juveniles and

adults), but larvae are more susceptible than juveniles and adults. Moreover, cultured

larvae are the most susceptible, because the artificial environment, high densities,

elevated temperatures and the stressful conditions in hatcheries create an ideal

environment for bacteria and the spread of bacterial diseases (Gosling, 2003; Paillard et

al., 2004).

1.3.2.1. Bacterial diseases in juvenile and adult bivalves

There exist several bacterial diseases in bivalve juveniles and adults. Some of these

diseases, such as the brown ring disease, the juvenile oyster disease and nocardiosis in

the Pacific oyster, are well-studied (Gosling, 2003; Paillard et al., 2004). These disease

will be further discussed below.

Brown ring disease (BRD) is characterised by an abnormal brown deposit of organic

matter on the inside of the shell, between the pallial line and the shell margins, forming a

brown ring (Gosling, 2003; Pailliard, 2004). The BRD is caused by Vibrio tapetis and

mainly affects the manila clam R. philippinarum. This disease is indigenous in Europe and

occurs in France, Spain, Italy, Ireland and the United Kingdom (Gosling, 2003; Paillard et

al., 2004). BRD can be treated and prevented with antibiotics (Gosling, 2003).

19

Figure I-13. Picture of an adult clam affected by BRD, with the characteristic brown deposit

on the edges of the mantle (Paillard et al., 2004).

Juvenile oyster disease (JOD) is characterised by an abnormal deposit of conchiolin on

the inner surface of the valves (Paillard et al., 2004). JOD only affects cultured juvenile

oysters of the species Crassostrea virginica and occurs mainly in summer months

(Gosling, 2003). The causative agent is a newly described species of the α-

proteobacterium of the Roseobacter clade (Gosling, 2003; Paillard et al., 2004). JOD can

be avoided by for example transferring the juvenile oysters as soon as possible to

outgrowing rears (Gosling, 2003).

Figure I-14. Picture of Crassostrea virginica affected with JOD (Paillard et al., 2004).

Pacific oyster norcardiosis (PON) is characterised by local brown discolouration on the

mantle in light infections and to large internal yellow-green nodules in most tissues in

more heavy infections (Friedman et al., 1998; Paillard et al., 2004). PON affected pacific

oysters in Japan (1945) and North America (1965) where it resulted in mass summer

mortalities in these oysters. The causative agent is identified as a gram-positive bacteria

belonging to the genera Nocardia (Paillard et al., 2004).

1.3.2.2. Bacterial diseases in bivalve larvae

The bivalve larval development starts from a planktonic embryonal stage and develops to

a free-living larvae, which finally metamorphoses to spat (as mentioned earlier). All larval

stages, certainly cultured larvae, are vulnerable to vibriosis.

Vibriosis is a collective noun for all the bacterial diseases caused by species from the

genus Vibrio. Larval vibriosis results for example in bacillary necrosis, formation of

‘swarms’ of bacteria on the margins of the larvae and ‘spotting’, which is defined as an

accumulation of moribund larvae that agglutinate together. Bacillary necrosis is

characterised by the extension of the velum, motility reduction or erratic movement in

circles. The main causal agents for bacillary necrosis are Vibrio alginolyticus, Vibrio

tubiashii and Vibrio anguillarum (Beaz-Hidalgo, 2010).

20

Vibriosis in bivalve larvae can be divided into three different patterns, namely

Pathogenesis I, Pathogenesis II and Pathogenesis III. Pathogenesis I affects all larval

stages and results in sedentary larvae. This pattern also shows signs of colonisation of

the mantle and invasion of the visceral cavity. Pathogenesis II only affects the early stage

of the veliger larvae and results in velum disruption and extension and abnormal

swimming behaviour. This leads to an active larvae that shows visceral atrophy before

invasion into organs of the digestive tract occurs. Pathogenesis III affects the late veliger

stage, the pediveliger larvae. This pattern results in sedentary larvae, a progressive and

extensive visceral atrophy and lesions in the organs of the digestive tract (Beaz-Hidalgo,

2010).

1.3.2.3. Mass mortality events of bivalve adults

Only a few mass mortality events in mussels have been reported worldwide, and they are

usually ascribed to adverse environmental conditions or to be caused by algal blooms

rather than pathogenic bacteria (Eggermont et al., 2014; Vernier et al., 2011). An example

of a mass mortality event for Mytilus edulis is the summer mortality of this species that

occurred on the Magdalen Island and along the Atlantic and Pacific coast of North

America with a first event in 1975. These outbreaks caused up to 80% mortality among

cultured blue mussels. This outbreak was associated with adverse environmental

conditions, such as a non-optimal temperature (Myrand et al., 2000). Temperature is one

of the most important environmental controls on for example psychological systems from

organisms (Harley, 2008). Another example of a mass mortality event for Mytilus edulis is

the dead or weakening of blue mussels in the Ascumpeque Bay on Prince Edward Island

in Canada in January of 1977. This mass mortality is assumed to be caused by

microparasites (Li and Clyburne, 1979). The mass mortality events that have been

reported are all mortality events of adult organisms. To the best of my knowledge, no

causative (bacterial) agents for mass mortality events in larval stages of Mytilus edulis

have ever been described.

1.3.3. Microbial origin of mass mortality events in Mytilus edulis

1.3.3.1. Previous research

Previous research indicated that mass mortality events in Mytilus edulis can be caused by

pathogenic bacteria that are associated with healthy wild-caught blue mussel adults

(Eggermont et al., 2014). The growth of these bacteria was stimulated by the addition of

dissolved organic matter to the rearing water and resulted in complete mortality within a

week. A second study isolated 15 bacterial strains (ME1-15) from mass mortality events in

wild-caught blue mussel adults (Tamanji, 2013). Further research investigated the

virulence of these 15 isolates against Mytilus edulis larvae, and this revealed that 12 of

the 15 strains are pathogenic against larvae (Yumo, 2014). It should be pointed out that

these are the first pathogens towards Mytilus edulis larvae ever described. Twelve of

these 15 isolates are vibrios belonging to the Splendidus clade.

1.3.3.2. Vibrio species

As mentioned above, bivalves have a rich bacterial commensal microbiota. The two

general groups of pathogenic bacteria in coastal waters are non-indigenous and

21

indigenous bacterial pathogens and these can be entrapped by bivalves due to their filter

feeding capacity. The first group includes bacterial species such as Salmonella and

Shigella that enters the water mostly via infected sewage. The second group mainly

consists of species from the Vibrionaceae family, which are natural inhabitants of marine

environments (Pruzzo et al., 2005). This latter group will be discussed further because of

their relevance for this master dissertation.

The Vibrionaceae is a large family of gram-negative gammaproteobacteria, that can be

subdivided in eight genera, including the genus Vibrio (Ruwandeepika et al., 2012). Vibrio

species are free-living bacteria or are associated with phytoplankton. They are

widespread among aquatic environments, can tolerate a wide range of salinities and have

a temperature preference of 17°C or higher (Pruzzo et al., 2005).

Because of the filter feeding capacity of bivalves, vibrios are found in up to 100-fold higher

concentrations in bivalve tissues than in the surrounding waters. This can result in a

dangerously high concentration of vibrios in bivalves (Pruzzo et al., 2005). The Vibrio

genus includes more than 30 different species, such as V. splendidus, V. alginolyticus, V.

harveyi, V. tubashii, V. fluvialis, V. vulnificus, V. mimicus, and V. cholerae (Beaz-Hidalgo,

2010; Paillard et al., 2004; Pruzzo et al., 2005). From the more than 30 different species

of the genus Vibrio, many are pathogenic to humans and aquatic organisms (Pruzzo et

al., 2005). The human pathogens includes strains belonging to species such as Vibrio

cholerae, Vibrio parahaemolyticus and Vibrio vulnificus. Vibrio species such as for

example Vibrio harveyi, Vibrio campbellii and Vibrio alginolyticus, belong to the major

pathogens of aquatic organism (Ruwandeepika et al., 2012). Vibrio anguillarm and Vibrio

alginolyticus are reported as causative pathogens for mass mortality events in cultured

bivalve larvae and juveniles (Gosling, 2003).

22

Part II. Materials and Methods

2.1. Bacterial culture media

2.1.1. Luria-Bertani35 broth

Luria-Bertani35 (LB35) broth was prepared by dissolving 10 g tryptone, 5 g yeast extract

and 35 g Instant Ocean in 1 l demineralised water. The solution was autoclaved for 20

minutes at 121°C and stored at room temperature until further use.

2.1.2. Luria-Bertani35 agar

Luria-Bertani35 agar (LB35 agar) was prepared by adding 1,5 % agar to the LB35 broth

solution before autoclaving. Plates were poured (15 ml/plate) in the laminar flow and

stored at room temperature until further use.

2.2. Antibiotics

2.2.1. Rifampicin

A rifampicin (RIF) stock solution was prepared at a concentration of 100 g l-1 in dimethyl

sulfoxide (DMSO). After dissolving, the solution was filter sterilized (0,2 µm) and divided

into aliquots in a laminar flow hood. The aliquots were wrapped in aluminium foil to protect

them from light and stored at -20°C.

2.2.2. Ampicillin, kanamycin and chloramphenicol

For all three antibiotics, a stock solution was prepared at a concentration of 50 g l-1.

Ampicillin (AMP) and kanamycin (K) were dissolved in demineralized water,

chloramphenicol (C) was dissolved in ethanol. After dissolving, the solution was filter

sterilized (0,2 µm) and divided in aliquots in the laminar flow hood. The ampicillin stock

solution was stored at -20°C, the kanamycin and chloramphenicol stock solutions were

stored at 4°C.

2.2.3. Tetracycline and enrofloxacin

A solution was prepared at a concentration of 50 g l-1. Tetracycline (TE) was dissolved in

demineralized water and filter sterilized (0,2 µm) in the laminar flow hood. For the

enrofloxacin (ENR) the ready to use Baytrill® 10% was used.

2.3. Bacteria

Bacterial cultures were inoculated at 1% (v/v) in LB35 broth from a -80°C stock culture.

The stock culture was thawed on ice and vortexed well before use. The bacterial cultures

were incubated overnight at 18°C on a shaker.

The strains used in this thesis are shown in Table II-1.

23

Table II-1. Overview of the bacterial strains used in this study.

Strain Identity Pathogenicity a

ME 1 Vibrio belonging to the Splendidus clade Yes

ME 2 Photobacterium spp. Yes









ME 11 Photobacterium spp. No

ME 12 Photobacterium spp. No



ME 15 Vibrio belonging to the Splendidus clade Yes a Pathogenicity towards blue mussel larvae

2.4. Antibiotic sensitivity tests

50 µl of an overnight grown culture was streaked on a LB35 agar plate with a triangular

spatulum. Antibiotic discs were put on top of the inoculated plates and the plates were

incubated upside down at 18°C. After 24 hours and 48 hours, the growth inhibition zones

were measured. The size of the clearing zone around the antibiotic disc indicates the

degree of antibiotic resistance; the larger the inhibition zone, the more sensitive the

bacterium is for the tested antibiotic (see figure II-1).

Figure II-1. Antibiotic sensitivity test. LB35 agar plate inoculated with bacteria and different

antibiotic-discs (the larger the inhibition zone, how more sensitive the bacterium is for the

antibiotic).

This procedure was performed for all isolates (ME1-15). The assays were done in

triplicate and repeated in time.

24

The tested antibiotics are summarized in table II-2.

Table II-2. Overview of used antibiotics to evaluate the antibiotic sensitivity of the ME1-15

isolates.

Antibiotic Dose (µg/disk) Antibiotic class

Ampicillin 10 β-lactam AB- penicillines

Cefadroxil 30 β-lactam AB -cefalosporines

Chloramphenicol 30 -

Enrofloxacin 5 (fluor)quinolones

Erythromycin 30 macrolides

Kanamycine 30 aminoglycosides

Metronidazole 5 -

Nitrofurantoin 50 nitrofurans

Rifampicin 30 -

Streptomycin 25 aminoglycosides

Trimethoprim/Sulphamethoxazole 25 trimethoprim-sulphonamides

Tetracycline 30 tetracyclines

2.5. Virulence factor tests

2.5.1. Hemolysin production

Hemolysin production was tested on blood agar. 5% sterile defibrinated sheep blood was

added to LB35 agar before pouring plates. An overnight grown bacterial culture was diluted

to an OD550 of 0,5 and inoculated by spotting 2 µl on each blood agar plate. The plates

were incubated at 18°C. After four days, the clearing zone was measured and the ratio

was calculated. The ratio is determined by dividing the halo diameter trough the colony

diameter. The size of the clearing zone around the bacteria colony indicates the degree of

hemolytic activity; the larger the clearing zone, the stronger the hemolytic activity of the

bacteria (see figure II-2).

Figure II-2. Hemolysin production test. LB35-blood agar plate inoculated with bacteria (the

larger the clearing zone, the stronger the hemolytic activity of the bacteria).

This procedure was performed for all isolates (ME1-15). The assays were done in

triplicate and repeated in time.

25

2.5.2. Exopolysaccharide production and biofilm formation

For these tests, overnight grown bacterial cultures were diluted to an OD600 of 0,1 in fresh

LB35 broth.

Exopolysaccharide production was assessed by inoculating 200 µl/well of the diluted

overnight grown bacterial culture into a 96-well microtiter plate and the plate was

incubated at 18°C for 24 hours. After incubation, the wells were washed three times with

300 µl physiological saline to remove all non-adherent bacteria, and the plate was dried

for 30 minutes. 100 µl phosphate buffered saline containing 5 mM calcofluor white staining

was added to each well. After 60 minutes, the fluorescence (excitation 405 nm/emission

500 nm) was measured with an Infinite M200 microplate reader (Tecan).

Biofilm production was assessed by inoculating 200 µl/well of the diluted overnight grown

bacterial culture into a 96-well microtiter plate, and the plate was incubated at 18°C for 24

hours. After incubation, the wells were washed three times with 300 µl physiological saline

to remove all non-adherent bacteria, and the plate was dried for 30 minutes. The

remaining bacteria were fixed to the microtiter plate by adding 200 µl 99% methanol to

each well and incubating for 2 hours. After fixation, the microtiter plate was emptied and

dried overnight. The fixed bacteria were stained with 200 µl/well of a 0,1% crystal violet

solution for 20 minutes. After incubation, the excess of stain was rinsed off by placing the

microtiter plate under running tap water. The dye bounded to the adherent bacteria was

resolubilized by adding 200 µl 99% ethanol to each well and the absorbance was

measured at 570 nm with Infinite M200 microplate reader (Tecan).

This procedure was performed for all isolates (ME1-15). These assays were done for

three independent grown bacteria cultures, each in four replicates.

2.6. Development of an adult challenge test for Mytilus edulis

2.6.1. Adult experiment 1: Try-out of an adult challenge test protocol

Wild-caught blue mussels (M. edulis) were transported from the Netherlands (Roem van

Yerseke B.V. - Yerseke) to the lab under thermo-stable and dry conditions. Upon arrival,

debris and dead animals were removed and the healthy mussels were rinsed several

times with filtered seawater (0,2 µm) and acclimatized for 48 hours in filtered (0,2 µm) and

aerated seawater at a room temperature of 18°C. During acclimatization, the mussels

were treated with antibiotics by dissolving the antibiotics in the rearing water. The first 24

hours, the mussels were treated with an antibiotic mix consisting of ampicillin, kanamycin

and chloramphenicol, each at a concentration of 20 mg l-1. The next 24 hours the mussels

were treated with rifampicin at a concentration of 50 mg l-1, so only the bacteria that are

added to the rearing water further survive because they are natural rifampicin resistant

mutants. In between two antibiotic treatments, the mussels were rinsed thoroughly with

filtered seawater (0,2 µm).

26

Figure II-3. Experimental set-up of the adult challenge test.

The experiment was performed in covered rectangular tanks, each filled with 4 l of filtered

(0,2 µm) and aerated seawater (see figure II-3). After 48 hours of acclimatization in both

antibiotic treatments as described above, healthy mussels were randomly stocked at 20

animals per tank and the tanks were randomly placed next to each other. The mussels

were subjected to different treatments, as shown in table II-3, and every treatment was

performed in triplicate. Mortality of the mussels and optical density (OD) at 550 nm of the

rearing water was monitored daily during 14 days. Dead mussels were removed from the

tanks.

Table II-3. Overview of different treatments used for the adult challenge test (treatment name

and composition).

Treatment Blank

0%

Blank

0,1%

Blank

1%

NC

0,1%

NC

1%

MIX

0,1%

MIX

1%

Tank code

replicate

1

a, b, c

2

a, b, c

3

a, b, c

4

a, b, c

5

a, b, c

6

a, b, c

7

a, b, c

RIF 20 mg l-1 Yes Yes Yes Yes Yes Yes Yes

LB35 broth (v/v %) No 0,1 1 0,1 1 0,1 1

Bacteria No No No ME 13 ME 13

ME 7

ME 8

ME 9

ME 10

ME 15

ME 7

ME 8

ME 9

ME 10

ME 15

After two days, the water from the tanks was replaced by fresh filtered and acclimatized

seawater. After water exchange, rifampicin was added to the tanks at a concentration of

10 mg l-1, LB35 broth and bacteria were not added again.

2.6.2. Adult experiment 2: Microbiological monitoring of the rearing water in

the presence of mussels

In order to verify whether rifampicin-resistant bacteria were present in the experimental

set-up, the following experiment was performed without the addition of any isolate.

27

Acclimatisation and pre-treatment of the mussels were done according to the description

above. 1% LB35 broth and 10 mg l-1 rifampicin were added at the start of the experiment to

the tanks containing 4 l filtered (0,2 µm), aerated seawater and 20 mussels. In addition to

measuring OD550 of the culture water and mortality of the mussels, water samples were

taken at the start of the experiment and after 24 hours. This test was performed in three

replicates. A dilution series of the water samples was prepared and inoculated on LB35

agar plates with and without rifampicin (50 mg l-1) by spiral plating. The plates were

incubated for three days at 18°C. The CFU/ml was calculated.


the absence of mussels

In order to verify whether rifampicin-resistant bacteria were present in the experimental

set-up without mussels, the following experiment was performed according to the

description in 2.6.2., however no mussels were added to the tanks.

2.6.4. Adult experiment 4: Verification of the effectivity of the mussel pre-

treatment

The set-up of this experiment was done according to the description in adult experiment 2

(2.6.2.). However, during acclimatization, the mussels were treated with different

antibiotics. During the first 24 hours, half of the mussels was treated with the previously

described antibiotic mix, consisting of ampicillin, kanamycin and chloramphenicol, each at

a concentration of 20 mg l-1. The other half of the mussels was treated with a new

antibiotic mix, consisting of chloramphenicol, tetracycline and enrofloxacin, each at a

concentration of 50 mg l-1. After thoroughly rinsing with filtered (0,2 µm) seawater, the

mussels were submerged in seawater treated with 50 mg l-1 rifampicin for 24 hours.

After the acclimatization period, 20 healthy mussels were randomly stocked per tank filled

with 4 l filtered (0,2 µm) and aerated seawater. No rifampicin, LB35 or bacteria were added

to the rearing water. At the start and after 24 hours, mussels from each tank were

sampled. For each tank, the tissues from 10 mussels were pooled, homogenised and

inoculated on LB35 agar with and without 50 mg l-1 rifampicin. To sample the mussel

tissue, the mussels were opened by cutting through the anterior and posterior muscle with

a sterile scalpel. The mussel tissue was then collected in a sterile beaker and

homogenized using a mixer. After every sample (10 mussels from the same tank) the

mixer and scalpel were sterilized with 70% ethanol. Between the different antibiotic

treatments the table and mixer were both cleaned with soap and sterilized with 70%

ethanol and a new sterile scalpel was used. After homogenisation, 100 ml sterile

physiological water was added, and the mixture was homogenized again to make the

solution more fluid. Afterwards, 3 ml of the solution was centrifuged short at high speed (1

minute at 13.000 x g). The supernatants were then used to set up a dilution series, and

these were inoculated on the agar plates. The inoculated plates were then incubated for

four days at 18°C. The CFU/ml was calculated.

28

Part III. Results

The aim of this study was to evaluate the virulence of 15 bacterial strains (ME1-15), that

were originally isolated during mass mortality events from culture water, hemolymph

samples and homogenised blue mussel adults and larvae (see appendix 1).

In the first part of this master dissertation, the antibiotic sensitivity and production of

(putative) virulence factors were screened. In the second part, an adult challenge test for

Mytilus edulis that could be used to evaluate the virulence of the isolates, was further

developed.

3.1. Antibiotic sensitivity & virulence factor production

3.1.1. Antibiotic sensitivity

In a first experiment, the sensitivity of all 15 strains (ME1-15) towards a wide range of

antibiotics was determined. The respective antibiotics were selected in order to cover a

broad range of classes and modes of action (table II-2).

Tables III-1, III-2 and III-3 summarize the results. The data given below are the averages

and standard deviation of three replicates (the results are representative for two replicates

in time).

Table III-1. Growth inhibition zones of ME1-5 for 12 tested antibiotics. Average and standard

deviation of three replicates.

Antibiotic Dose

(µg/disk)

Growth inhibition zone (mm)

ME1 ME2 ME3 ME4 ME5

Ampicillin 10 11 + 3 17 + 2 13 + 4 12 + 4 9 + 2

Cefadroxil 30 8 + 0 8 + 0 8 + 1 9 + 2 8 + 0

Kanamycine 30 12 + 1 11 + 0 13 + 1 13 + 1 15 + 0

Streptomycin 25 12 + 1 12 + 2 14 + 1 12 + 1 11 + 1

Chloramphenicol 30 27 + 7 32 + 1 30 + 1 30 + 2 34 + 3

Rifampicin 30 8 + 0 8 + 0 8 + 0 8 + 1 8 + 0

Tetracycline 30 15 + 6 17 + 3 20 + 2 15 + 1 19 + 1

Enrofloxacin 5 15 + 1 14 + 1 17 + 1 15 + 2 16 + 1

Trimethoprim/

Sulphamethoxazole 25 24 + 2 23 + 2 24 + 2 17 + 2 24 + 2

Erythromycin 30 12 + 2 18 + 3 17 + 1 15 + 2 19 + 1

Nitrofurantoin 50 21 + 1 18 + 2 22 + 1 19 + 2 19 + 3

Metronidazole 5 8 + 0 8 + 0 8 + 0 8 + 0 8 + 0

29

Table III-2. Antibiotic resistance from ME6-10 towards 12 tested antibiotics. Average and

standard deviation of three replicates.

Antibiotic Dose

(µg/disk)


ME6 ME7 ME8 ME9 ME10

Ampicillin 10 8 + 0 8 + 0 9 + 1 8 + 0 8 + 0

Cefadroxil 30 8 + 0 8 + 0 8 + 0 8 + 0 8 + 0

Kanamycine 30 13 + 2 13 + 1 12 + 1 13 + 1 11 + 0

Streptomycin 25 13 + 3 11 + 1 12 + 0 13 + 2 12 + 2


Rifampicin 30 8 + 0 8 + 0 9 + 1 8 + 0 8 + 0

Tetracycline 30 19 + 1 20 + 3 18 + 2 22 + 3 15 + 3

Enrofloxacin 5 17 + 0 15 + 1 16 + 1 15 + 2 13 + 1

Trimethoprim/


Erythromycin 30 17 + 3 18 + 2 15 + 1 12 + 5 17 + 5

Nitrofurantoin 50 23 + 2 23 + 3 21 + 4 21 + 2 14 + 1

Metronidazole 5 8 + 0 8 + 0 8 + 0 8 + 0 8 + 0

Table III-3. Antibiotic resistance from ME11-15 towards 12 tested antibiotics. Average and


Antibiotic Dose

(µg/disk)


ME11 ME12 ME13 ME14 ME15

Ampicillin 10 19 + 10 8 + 0 8 + 0 11 + 5 16 + 4

Cefadroxil 30 8 + 0 8 + 0 8 + 0 10 + 4 9 + 1

Kanamycine 30 8 + 0 8 + 0 9 + 1 11 + 1 13 + 1

Streptomycin 25 8 + 0 8 + 0 12 + 2 10 + 1 11 + 1


Rifampicin 30 8 + 0 8 + 0 8 + 0 8 + 0 8 + 0

Tetracycline 30 21 + 3 8 + 0 17 + 3 17 + 1 24 + 2

Enrofloxacin 5 16 + 2 8 + 0 15 + 1 17 + 1 19 + 2

Trimethoprim/


Erythromycin 30 18 + 2 8 + 0 13 + 4 12 + 5 19 + 5

Nitrofurantoin 50 28 + 2 8 + 0 14 + 3 20 + 2 27 + 1

Metronidazole 5 8 + 0 8 + 0 8 + 0 8 + 0 8 + 0

Based on this results, the antibiotics could be divided into three groups based on the

effectiveness towards the particular isolates ME1-15 and on the applied antibiotic dosage.

Since no standardised protocol was available, it was decided to interpret the growth

inhibition as resistant (diameter of inhibition zone < 15 mm), intermediate (inhibition zone

= 15 – 25 mm) and sensitive (inhibition zone >25 mm).

The first group consists of metronidazole, ampicillin, cefadroxil, kanamycine and

streptomycin, which are not effective towards ME1-15. The second group consists of

chloramphenicol and trimethoprim/sulphamethoxazole, which are the most effective

30

antibiotics towards the isolates. Since enrofloxacin was tested at a considerably lower

dosage we could include it also in this group. The third group consists of tetracycline,

erythromycin and nitrofurantoin, which showed rather intermediate effectivity.

From the perspective of the 15 bacterial isolates (ME1-15), following can be observed

from this results. ME1 is the most resistant bacterial strains. This strain is resistant

towards nine of the twelve tested antibiotics. ME11 and ME15 are the only bacterial

strains that are sensitive towards more than one of the tested antibiotics, namely

chloramphenicol, sulphamethoxazole/trimethoprim and nitrofurantoin. At last, ME15 is the

most sensitive bacterial isolate and ME12 seems to be resistant towards all the tested

bacteria. But it should be taken in mind that ME12 grows a lot slower than the rest of the

bacterial strains and so the result for ME12 should be taken with caution.

3.1.2. Virulence factors

Virulence factor tests were performed to determine hemolytic activity, exopolysaccharide

production and biofilm formation of the bacterial isolates ME1-15.

3.1.2.1. Hemolysin production

The hemolytic activity of ME1-15 was tested on blood agar plates. The tests were

performed in triplicate and repeated in time. The results shown below are the averages

and standard deviations of the three replicates for each experiment.

Table III-4. Colony diameter and hemolytic activity zone of ME1-15. Averages and standard

deviation of three replicates.

Strain Experiment 1 Experiment 2

colony diameter (mm) zone (mm) colony diameter (mm) zone (mm)

ME1 5 + 1 15 + 0 4 + 1 12 + 1

ME2 5 + 1 16 + 0 4 + 0 11 + 1

ME3 6 + 0 15 + 1 5 + 1 13 + 1

ME4 5 + 1 16 + 0 3 + 1 11 + 1

ME5 4 + 0 17 + 1 1 + 0 13 + 1

ME6 4 + 1 16 + 0 5 + 1 11 + 1

ME7 3 + 0 17 + 0 5 + 1 13 + 0

ME8 4 + 1 16 + 1 5 + 0 12 + 1

ME9 4 + 1 17 + 1 5 + 0 13 + 1

ME10 4 + 1 16 + 0 5 + 1 12 + 0

ME11 3 + 0 14 + 2 6 + 1 12 + 1

ME12 4 + 1 11 + 1 4 + 1 14 + 1

ME13 5 + 0 15 + 1 6 + 1 13 + 1

ME14 5 + 0 16 + 1 5 + 1 11 + 0

ME15 6 + 0 16 + 1 4 + 1 11 + 1

This table (table III-4) shows the colony and respective hemolytic activity zone in mm for

all 15 bacterial strains. All strains grew well which can be observed by the colony diameter

and exhibited hemolytic activity since the halo diameter exceeds the colony diameter for

each of the 15 strains. This finding is confirmed by the time replicate.

31

Further, the ratio between clearing zone and colony diameter was calculated for each

strain. The higher this ratio, the stronger the hemolytic activity of the bacteria. The

respective hemolytic activities are shown in figure III-1.

Figure III-1. Hemolytic activity of ME1-15 for both time replicates. Error bars represent the


This graph (figure III-1) confirms that all 15 isolates (ME1-15) possess hemolytic activity

regardless of the differences in between the time replicates for some isolates.

3.1.2.2. Exopolysaccharide production and biofilm formation

The production of exopolysaccharides and biofilm formation were evaluated for the

bacterial strains (ME1-15). Different temperatures and different incubation times were

evaluated (see chapter 2.4.3 in Material and Methods), namely (1) 24 hours at 18°C, (2)

48 hours at 28°C and (3) 72 hours at 18°C.

The results for the incubation at 28°C (not shown) were all negative for exopolysaccharide

production, which indicates that the tested bacteria prefer a lower temperature and are at

their best at a temperature of 18°C. At 18°C the test was performed once during 24 hours

and once during 72 hours incubation time. The results for incubation at 18°C are shown in

figure III-2 and III-3. The results given are the averages of twelve replicates and their

standard deviations.

32

Figure III-2. Exopolysaccharide production for 24 hours and 72 hours incubation at 18°C.

Error bars represent the standard deviation of twelve replicates.

Figure III-3. Biofilm formation for 24 hours and 72 hours incubation at 18°C. Error bars

represent the standard deviation of twelve replicates for 24 hours and eight replicates for 72

hours.

The results (figure III-2) for exopolysaccharide production show that all the strains show

exopolysaccharide production.

33

The results for biofilm formation (figure III-3) show that most of the strains form biofilms.

But there is a difference between the different incubation times, namely lower biofilm

formation is observed when incubated longer.

To evaluate the correlation between exopolysaccharide production and biofilm formation

the Spearman’s rank correlation coefficient is calculated (table III-5) and is also reflected

in scatter plots showing EPS formation and biofilm formation for each isolate (figure III-4

and III-5).

Table III-5. Correlation (Spearman’s rank) between exopolysaccharide production and

biofilm formation in the 15 isolates.

incubation time Spearman’s rank p-value

24 hours 0,818 0,000

72 hours 0,785 0,001

Figure III-4. Scatter plot of EPS production and biofilm formation at 24 hours incubation.

34

Figure III-5. Scatter plot of EPS production and biofilm formation at 72 hours incubation.

This results show that there is a strong and significant correlation between

exopolysaccharide production and biofilm formation for both incubation times, with a

correlation coefficient of 0,818 and 0,785 at 24h and 72h, respectively.

3.2. Development of an adult challenge teste for Mytilus edulis

In order to facilitate future research on host-microbe interactions in Mytilus edulis, a robust

adult challenge test is needed. Both the microbiological control of such a test and the

identification of a suitable pathogen are real challenges.

In adult experiment 1, the virulence of a mixture of some of the ME1-15 isolates was

tested on adult blue mussels. Two additional experiments (adult experiment 2 and 3) were

performed to be able to further explain the results obtained in experiment 1.

3.2.1. Adult experiment 1: Try-out of an adult challenge test protocol

The protocol for an adult challenge test as described by Pim Vanmeerkerken (to be

published) was used with some modifications as described in chapter 2.5.1 of Material

and Methods. It was reasoned that a mixture of the most virulent bacterial isolates

regarding mussel larvae (ME7, ME8, ME10 and ME15) might have the highest chance of

success. It was also reasoned that ME13, which is not pathogenic towards mussel larvae,

could act as negative control. As a blank treatment, no bacteria were added to the mussel

culture water. To create an ideal environment for the administered bacteria, extra nutrients

were added to the mussel culture water, referred to as LB35. The % survival of the adult

mussels in this experiment are given figure III-6 and figure III-7.

35

Figure III-6. % Survival of the mussels in the adult challenge test with 0,1% (v/v) LB35 in the

culture water as extra nutrients for the added bacteria. Error bars represent the standard

deviation (n=3). (Blank = blank treatment, NC = treatment with addition of non-pathogenic

bacteria, MIX = treatment with mixture of pathogenic bacteria.)

Figure III-7. % Survival of the mussels in the adult challenge test with 1% (v/v) LB35 in the

culture water as extra nutrients for the added bacteria. Error bars represent the standard

deviation (n=3). (Blank = blank treatment, NC = treatment with addition of non-pathogenic

bacteria, MIX = treatment with mixture of pathogenic bacteria.)

36

These results show that there was no significant difference in survival between the

different treatments after 14 days.

To investigate whether the bacteria in the culture water are indeed the rifampicin resistant

bacteria that were added according to the different treatments the next experiment

described underneath in 3.2.2 was performed.


the presence of mussels

For the adult challenge test it is reasoned that due to the pre-treatment of the mussels

with an antibiotic mixture as described in chapter 2.6.1 of Material and Methods, the

bacterial load at the start of the test should be limited. By combining the addition of 106

CFU ml-1 of the bacterial isolate mix (all naturally resistant to the antibiotic rifampicin) with

rifampicin and extra nutrients, the remaining host bacteria should be outcompeted in

favour of the isolate mix.

To check this hypothesis, the experiment was repeated with the difference that no

bacteria were added to the rearing water and the experiment only lasted for 24 hours after

the pre-treatment that did not changed. It was reasoned that a limited amount of bacteria

might survive the pre-treatment of the mussels. However, no rifampicin resistant bacteria

are expected to be present. The results are shown in table III-6.

Table III-6. Bacterial density in the rearing water of the adult challenge test. The plates were

incubated for 48h at 18°C. The data are averages and standard deviations of 6 replicates.

Samples were spread on agar with or without rifampicin (RIF).

LB35agar without RIF

(CFU ml-1)

LB35agar with 50 mg l-1 RIF

(CFU ml-1)

start (2,17 + 0,0) x103 (4,65 + 0,0) x103

24h (4,57 + 0,0) x103 (4,56 + 0,0) x103

The results show that there still is a considerable amount of bacteria in the rearing water

after the pre-treatment of the mussels with antibiotics, and cleaning and disinfection of the

used materials. The comparable CFU ml-1 on LB35 agar-plates supplemented with 50 mg

l-1 rifampicin, indicates that the bacteria are rifampicin resistant. This finding indicated that

the used materials are not sufficiently disinfected after cleaning and disinfection and/or

that the used antibiotics for the pre-treatment of the mussels is not effective enough. In

order to clarify this, adult experiment 3 and 4 were performed.


the absence of mussels

This experiment was designed to investigate whether the used materials, such as the 4

liter tanks and the air stones, were sufficiently disinfected after mechanical cleaning and

disinfection with a strong acid. The same experimental set-up as described in 3.2.2 was

used without adding mussels to the rearing water in order to exclude the possibility that

the bacteria came in with the mussels. The results are shown in table III-7.

37

Table III-7. Bacterial density in the rearing water of the adult challenge test. The plates were

incubated during 48h at 18°C. The date given are the averages and standard deviation of 6

replicates


(CFU ml-1)


(CFU ml-1)

start (3,50 + 0,0) x 103 (0,14 + 0,0) x 103

24ha > (60,0 + 0,0) x 103 > (60,0 + 0,0) x 103

These results show that there are still bacteria present after cleaning and disinfection of

the used materials. The presence of bacteria on the LB35 agar-plates with 50 mg l-1

rifampicin indicates that the bacteria are rifampicin resistant. It can be concluded that the

cleaning and disinfection of the used material is not sufficient.

3.2.4. Adult experiment 4: Verification of the effectivity of the used pre-

treatment

This experiment investigates whether a more effective antibiotics-mixture could be found

as pre-treatment in order to limit the bacterial load of the mussels at the start of the

experiment. For this experiment, the effect of a new antibiotic mixture (new) was

compared with the previously used mixture (old). The new mix was composed according

to the results of the antibiotic resistance test (see chapter 3.1.1.). The most effective

antibiotics against the bacterial isolates ME1-15 were chosen, taking into account their

solubility in water. The new mix is composed of chloramphenicol, enrofloxacin and

tetracycline. When compared to the old mix (where the antibiotic were dosed at 20 mg l-1)

we also used a higher concentration (50 mg l-1) for the new mix.

Table III-8. Bacterial density in mussel tissue of the adult challenge test at the start and after

24 h after pre-treatment with two different antibiotic mixtures (old mix: ampicillin,

kanamycin & chloramphenicol – each 20 mg l-1

, new mix: chloramphenicol, tetracycline and

enrofloxacin – each at 50 mg l-1

). The date given as averages and standard deviation 6

replicates).


(CFU ml-1)


(CFU ml-1)

old AB mix

start 0 + 0 0 + 0

24h (1,61 + 0,0) x 103 (1,35 + 0,0) x 103

new AB mix

start 0 + 0 0 + 0

24h 0 + 0 0 + 0

The result (table III-8) shows that there are still bacteria present after the pre-treatment

with the old antibiotic-mix (ampicillin, kanamycin and chloramphenicol), and those bacteria

are rifampicin resistant bacteria. But it has to be noticed that the pre-treatment with

rifampicin lasted 48 hours instead of 24 hours due to technical problems. However after

the pre-treatment with the new antibiotic mix no bacterial growth could be detected on the

agar plates.

38

Part IV. Discussion and recommendations for the future

The aim of this master dissertation was to evaluate the virulence of 15 bacterial strains

(ME1-15) that were originally isolated during blue mussel mass mortality events. First their

antibiotic sensitivity and virulence will be discussed, followed by their possible application

in an adult challenge test.

4.1. Antibiotic sensitivity tests

There are different testing methods to evaluate the antimicrobial sensitivity of bacteria.

Disc diffusion is one of the standard procedures in which antibiotic discs, with a fixed

concentration, are placed on an agar plate inoculated with the test strain. After incubation,

the diameter of the growth inhibition zone is determined as a measure of sensitivity to the

antibiotic. After standardisation, the results can be classified in different categories from

susceptible or sensitive (S) over intermediate (I) to resistant (R) (Madigan et al., 2012).

The disc diffusion test method is well-standardized for a lot of known pathogens, such as

S. aureus and E. coli, and for different antibiotics. In this cases accurate and valuable

conclusions regarding the effectivity of the antibiotics towards the tested organism can be

obtained from the results because the used protocols and the media, the incubation time,

etc. are fully standardized. To the best of our knowledge, no standardized protocol for

vibrios is available. Therefore, the results obtained in this study cannot be interpreted in

terms of sensitive, intermediate or resistant (Madigan et al., 2012; Verhaeghen et al.,

2010).

Furthermore, it has to be taken into account that the inhibition zone depends on (1) the

growth rate of micro-organism and (2) the diffusion rate of the antibiotic through the agar.

Regarding the growth rate of the bacteria, the bacteria should be detectable within the first

24 hours after incubation. Otherwise the antibiotic will be uniformly diluted over the entire

agar plate instead of a decreasing concentration with an increasing distance from the

antibiotic discs. Next to the growth rate, also the time between the inoculation and the

placing of the antibiotic discs should be as minimal as possible. So nor the bacterial

growth, nor the antibiotic diffusion through the agar should be favoured in respect to the

other, because this will lead to incorrect interpretation of the results.

Regarding the diffusion of the antibiotic the thickness of the agar plays an important role

because the diffusion occurs in all directions. How thinner the agar, how more diffusion

will occur in the horizontal direction and how bigger the inhibition zone will be. Also the

physico-chemical properties (e.g. solubility) of the antibiotic are important, because they

will also affect the diffusion in the agar (e.g. the lower the solubility, the lower the diffusion

rate) (Verhaeghen et al., 2010).

It has to be taken into account that the conclusions regarding the effectivity of the different

antibiotics towards the 15 bacterial strains are fully based on personal chosen criteria, by

the lack of a standardized protocol. The criteria for determining the sensitivity of the

different antibiotics (resistant, intermediate and sensitive) have rather high limits - which

rapidly classify the antibiotics as ineffective (resistant and intermediate action of the

39

antibiotics). This is because this test aimed, on one hand, to find a very effective antibiotic

mixture for the pre-treatment of the adult challenge test, which will be discussed later in

this discussion chapter. So the conclusion in the next paragraphs should be regarded with

caution.

4.1.1. Tentative comparison of antibiotic activity of the different antibiotics

towards isolates ME1-15

The results from the antibiotic sensitivity tests revealed that the majority of tested

antibiotics are not effective towards the ME1-15 isolates.

Both ampicillin and cefadroxil belongs to the antibiotic class of β-lactam antibiotics and

work by inhibiting enzymes of the peptidoglycan synthesis (Verhaegen et al., 2010). Since

all the tested bacterial isolates (ME1-15) are resistant towards both this antibiotics, it can

be concluded that antibiotics that interfere with the peptidoglycan layer of cell walls are not

effective towards infection with this 15 bacterial isolates. Resistance to this kind of

antibiotics can be due to the production of β-lactamases by the microorganism. These

enzymes cleave the antibiotics by opening their β-lactam ring (Verhaegen et al., 2010). A

study by Joseph (1978) confirms that several Vibrio species produce such β-lactamase

enzymes.

Kanamycine and streptomycin both belong to the antibiotic class of the aminoglycosides,

which inhibit the protein synthesis by binding to the 30S ribosomal subunit in the mRNA

translation complex, causing misreading of the mRNA and thereby resulting in the fact

that the bacteria are unable to synthesize important proteins. Hotta et al (1981) showed

that micro-organisms that are resistant to this kind of antibiotics produce enzymes that

inactivate these antibiotics. This might indicate that the ME isolates produces this kind of

enzymes.

The intermediate effectivity of trimethoprim/sulphamethoxazole can be explained by the

fact that this antibiotic is a combined antimicrobial agent of trimethoprim and

sulphamethoxazole. A study by Bushby (1973) revealed that both antibiotics have a

synergetic effect on each other, which results in an increased bacteriostatic and

bactericidal activity. Both antibiotics inhibit the folate synthesis by interfering with the

folate co-enzymes - folate is necessary for the synthesis of nucleic acids – and thereby

absence of folate results in the impossibility of cells to divide.

Another antibiotic with an intermediate effectivity is enrofloxacin, which belongs to the

antibiotic class of the quinolones. Ruiz (2003) explains that this class of antibiotics inhibits

the action of type II topoisomerases, namely DNA-gyrase and topoisomerase IV, and

thereby inhibiting DNA replication and translation. This might indicate that antibiotics that

interfere with the DNA and RNA synthesis are effective towards the ME isolates.

Erythromycin and tetracycline are antibiotics that show intermediate effectivity - but less

than the latter antibiotics - and also affects the DNA synthesis regarding Goldberg (2004),

by respectively inhibiting the translocation and the binding of tRNA. At last, nitrofurantoin

40

also affects the DNA and RNA synthesis, besides the inhibition of the protein and cell wall

synthesis, and possess intermediate effectivity. The mode of action of these latter three

antibiotics strengthens the idea that antibiotics that interfere with the bacterial DNA and

RNA synthesis are effective antibiotics towards the ME isolates.

Finally, the antibiotic sensitivity tests suggest that chloramphenicol is a very effective

antibiotic towards the ME isolates. Goldberg (2004) shows that chloramphenicol indirectly

influences the DNA and RNA synthesis. This bacteriostatic agent blocks the protein

synthesis by binding to the 50S subunit of the bacterial ribosome and thereby blocking the

transfer of amino acids from tRNA to the ribosome.

Two conclusions can be suggested regarding the effectivity of the tested antibiotics

towards the different ME isolates. (1) Antibiotics from the classes of aminoglycosides –

such as kanamycine and streptomycin – and β-lactam antibiotics – such as ampicillin and

cefadroxil -, interfere with the bacterial protein synthesis and are the least effective

antibiotics against ME1-15. (2) Secondly, antibiotics that interfere with the DNA and RNA

synthesis such as trimethoprim/sulphamethoxazole and enrofloxacin are the most

effective antibiotics against ME1-15. Chloramphenicol is also a very effective antibiotic

towards ME1-15, despite the fact that it primarily interferes with the protein synthesis of

the bacteria.

4.1.2. Comparison of antibiotic sensitivity of the different isolates

The conclusion that the tested bacterial strains - which are Vibrio spp. or Photobacterium

spp. - are very resistant towards ampicillin and kanamycine, intermediately sensitive

towards trimethoprim/sulphamethoxazole and sensitive towards chloramphenicol are

confirmed by several other studies. Akinbowale et al. (2005) reported out that several

bacteria belonging to the Vibrio spp. can be resistant to antibiotics including ampicillin and

kanamycine and sensitive towards chloramphenicol and trimethoprim/sulphamethoxazole.

For bacteria belonging to Photobacterium spp., they concluded that this bacteria are

resistant towards antibiotics such as ampicillin and susceptible to for example

chloramphenicol, trimethoprim/sulphamethoxazole and tetracycline. Other research

studies confirm the antibiotic resistance from Vibrio spp. towards several antibiotics from

different antibiotic classes. For example, regarding Rojas et al. (2011), Vibrio

parahaemolyticus is resistant to antibiotics belonging to the aminoglycosides, β-lactam

antibiotics and quinolones.

Regarding the individual bacterial isolates, it can be concluded that ME1 is the most

resistant bacterial isolate of this group of tested bacteria. ME11 and ME15 are the only

bacterial isolates that are sensitive to more than one of the tested antibiotics, all the other

bacterial isolates are resistant till intermediate sensitive towards the antibiotics. This

results also reveal that ME15 is the most sensitive bacterial strain, in spite of the fact the it

is the most pathogenic bacterial strain against blue mussel larvae.

41

4.1.3. Recommendations for the future regarding the antibiotic sensitivity of

different antibiotics and towards the different isolates

It is recommended to standardize the protocol for the disc diffusion test for the antibiotic

sensitivity test regarding the conditions used in this study, before solid conclusions can be

drawn from the results. The protocol should be standardized for the ideal temperature and

growth media for the tested ME1-15 isolates.

4.2. Virulence factor production

The expression of (putative) virulence-related phenotypes by the isolates was determined

in vitro. Previous research on this topic has been done by Yumo (2013). She tested the

virulence factors of these bacterial isolates (ME1-15), such as swimming motility,

production of lytic enzymes, biofilm formation and the production of exopolysaccharides.

Her results concerning hemolytic activity and biofilm formation were questionable and

because both virulence factors have an important role in the pathogenesis by bacteria

(Defoirdt, 2014), this factors are evaluated again in this study. At first, the results for the

hemolytic activity will be discussed, followed by the results of the exopolysaccharide

production and biofilm formation experiment.

4.2.1. Hemolysin production

Hemolysins are extracellular proteins that are able to lyse blood cells. These lytic

enzymes are linked to the virulence of pathogenic bacteria towards both shrimp and fish

(Defoirdt, 2014).

From the results for the production of hemolysin by ME1-15 isolates in this study, it can be

concluded that all the bacterial strains possess hemolytic activity resulting in β-hemolysis.

This is in contrast to the results from the previous research by Yumo (2013), who

concluded that only ME1 possessed hemolytic activity. The protocol used in this study

was the same as the one used by Yumo (2013). The results from both studies regarding

ME1 are quite similar. Nevertheless, the results for ME2 till ME15 are positive in this study

for both independent time replicates. In my opinion, the main difference between these

two studies is the growing of the bacteria, from samples stored at -80°C, prior to the

hemolytic experiment. In this study the bacterial isolates stored at -80°C were already

rifampicin resistant. In the previous study, the samples at -80°C originated from mass

mortality events in blue mussel larvae and adults, and still needed to be made rifampicin

resistant prior to the hemolytic activity experiment. This was done by growing the bacteria

in LB35 broth with addition of rifampicin and afterwards growing the bacteria again for 24

hours in LB35 broth without rifampicin.

Previous research by Chang et al (2000) showed that hemolytic activity against sheep

blood does not strictly mean that there is hemolytic activity against hemolymph of the

studied host organism. In this regard, the obtained results on blood agar should be seen

as indicative for the pathogenic capacity of the tested bacterial strains.

42

4.2.2. Exopolysaccharide production and biofilm formation

Biofilms are surface-associated communities of bacterial cells that are enclosed in an

adhesive matrix, which is secreted by the cells and typically consists of

exopolysaccharides (Madigan et al., 2012). Biofilm formation and exopolysaccharide

production are linked to each other because exopolysaccharide production contributes to

biofilm formation (Li et al., 2014). In this study exopolysaccharide production and biofilm

formation were evaluated after different incubation times and at different incubation

temperatures.

The reason why the tests were performed at different incubation temperatures is at first

the fact that the obtained results for exopolysaccharide production and biofilm formation

for 24 hours incubation at 18°C were rather low. Secondly, mass mortality events in

Mytilus edulis were reported during summer months, when the water temperature raised

till more than 20°C (Myrand et al., 2000). The results from this study indicate that the ME

isolates prefer a lower temperature of 18°C. Several studies, for example Amanda et al.

2002 and Motes et al. 1996, indicates that the effect of water temperature is different for

different Vibrio spp. and this is also confirmed by a study by Chavez, who shows that V.

cholerae is favoured in water temperatures of approximately 31°C and V. alginolyticus in

contrast were detected in water samples during winter months, when the water

temperature was about 21,8°C. On the other hand, it is expected that the ME isolates

prefer a lower temperature since they are isolated from mass mortality events of Mytilus

edulis in the Eastern Scheldt at the Netherlands.

Regarding the incubation times, there is a difference between 24 hours and 72 hours of

incubation. Expected was that there would have been more exopolysaccharide production

and biofilm formation after 72 hours of incubation than after 24 hours. The observations

for exopolysaccharide production confirm this expectations, but for biofilm formation the

opposite is observed. This means that after 72 hours of incubation, less biofilm is present.

This is probably due to the fact that after 72 hours of incubation the bacterial cells are

more clustered to each other instead of attaching to the well plate. Regarding this latter

conclusion biofilm formation and exopolysaccharide production for 24 hours of incubation

will be discussed further on.

Li et al. (2014) evaluated biofilm formation and exopolysaccharide production of V.

anguillarium in the presence of quorum sensing signal molecules such as indole. For the

wild type V. anguillarium they obtained an absorbance of 0,23 + 0,01 for biofilm formation

and 1414 + 178 for exopolysaccharide production, using the same protocols as in this

study. Yang et al. (2014) also used the same protocols to evaluate biofilm formation and

exopolysaccharide production of V. harveyi in relation to norepinephrine and dopamine.

They obtained an absorbance of approximately 0,35 for biofilm formation and 800 for

exopolysaccharide production. The highest absorbance for biofilm formation in this study

was obtained for ME13, ME14 and ME15. The absorbance values are respectively 0,11 +

0,02, 0,13 + 0,03 and 0,12 + 0,02. This values are two times lower than the ones

observed by Li et al. (2014) and three times lower than Yang et al. (2014). This lower

values do not mean that no biofilm was formed by the tested bacterial isolates, because

biofilm formation was visible with the naked eye during the experiment. The highest

absorbance for exopolysaccharide production in this study is obtained by ME11 and

43

ME15, respectively 790 + 229 and 1049 + 511. These values are between the values

obtained by Li et al. (2014) and Yang et al. (2014).

The correlation between exopolysaccharide production and biofilm formation was

evaluated. For both incubation times, a strong correlation was observed. This indicates

that exopolysaccharide production and biofilm formation are linked to each other, as

already reported by several studies. Besides Li et al. (2014) and Yang et al. (2014), also

Watnick (2002) and Heithoff and Mahan (2004) reported a positive correlation between

exopolysaccharide production and biofilm formation. But it should be mentioned that the

strong correlation obtained for 72 hours of incubation at 18°C in this study is ambiguous,

because the results obtained for biofilm formation after 72 hours incubation are too low to

conclude that the observed absorbance is due to biofilm formation. The idea that bacterial

cells are more clustered to each other than forming a biofilm after 72 hours, is the most

obvious reason why the results for biofilm formation after 72 hours did not follow the

expectations and observations of exopolysaccharide production after 72 hours.

4.2.3. Recommendations for the future regarding the virulence factor

production

The results from the hemolytic activity test for the 15 bacterial isolates could however be

confirmed by evaluating the hemolysin production at gene level. Hemolysin production is

linked to the occurrence of different specific genes. Virulent strains possess the tdh- or

trh-gene (respectively thermostable direct hemolysin and tdh-related gene) or both genes

in their DNA. Also the tlh-gene (thermolabile hemolysin) is linked to hemolytic activity and

this gene is species specific. Regarding this information, Rojas et al. (2011) showed that

β-hemolysin on blood agar is correlated to the presence of the tdh-gene for Vibrio

parahaemolyticus isolated from oysters and mussels. In that experiment, the trh-gene was

absent in the tested bacterial strains and the tlh-gene was used to discriminate V.

parahaemolyticus from other Vibrio species.

Regarding exopolysaccharide production and biofilm formation, the used protocol first

needs to be optimized and standardized for this 15 bacterial isolates before making a real

conclusion about exopolysaccharide production and biofilm formation. It is also

recommended to use another bacterial strain, than the tested ones, that does produce

exopolysaccharides and forms biofilms and a bacterial strain that does not do this, so an

positive and negative control is implemented in the protocol.

4.3. Development of an adult challenge test for Mytilus edulis

The aim of the second part of this master dissertation was to test the suitability of the

isolated strains (ME1-15) as pathogens in an adult challenge test for Mytilus edulis. For

this study, adult mussels that are collected from the wild, were cleaned and pre-treated by

immersion in an antibiotic mixture. This was done in order to minimize interference by the

mussel-associated bacteria.

44

4.2.4. Try-out of an adult challenge test protocol

The first experiment with blue mussel adults in this study was performed to evaluate the

possibility of using a mixture of several bacterial strains from the ME1-15 isolates, that are

proven to be pathogenic towards blue mussel larvae, in an adult challenge test.

The expected results for this experiment were that (1) there would be a significant

mortality of mussels that were exposed to the mixture of pathogenic bacteria, and (2) no

mortality would occur for the ones that were exposed to a non-pathogenic strain, namely

ME13. Also no mortality was expected for the mussels from the blank treatment. However,

all treatments resulted in almost 100% survival. The most obvious reason for the failure of

the adult challenge test is probably the fact that the used bacteria-isolate mix was not

virulent enough towards adult blue mussels. But the results form additional experiments

revealed that there were problems regarding the microbiological control of the test. One

could hypothesize that the used pre-treatment with antibiotics was not effective enough

(leaving non-pathogenic bacteria associated with the mussels that outcompeted the

pathogens that were added) and/or that the used materials were not sterile after cleaning

and disinfection at the start of the experiment. So, before concluding that the bacteria-

isolate mix is not virulent towards blue mussel adults, one should exclude other affecting

factors such as the pre-treatment and the cleaning and disinfection method.

4.2.5. Effectivity of the used pre-treatment

The used antibiotics in the original pre-treatment (ampicillin, kanamycine and

chloramphenicol) were not chosen randomly, but were based on a protocol to disinfect

bivalve larvae (Situmorang et al., 2014). Due to the assumption that the used antibiotic

mixture did not work well, a new mixture was composed based on the results from the

antibiotic sensitivity test (in chapter 1.1.1. of the discussion) and the availability of them in

the lab. The new mixture consists of tetracycline (oxytetracycline), enrofloxacin (Baytrill®)

and also chloramphenicol. The assumption that this mixture would work better is

confirmed by several other studies regarding Vibrio spp. in aquaculture. Roque et al.

(2002) for example shows that Vibrio spp. collected from Mexican shrimp cultures are

sensitive towards trimethoprim/sulphamethoxazole, enrofloxacin and florfenicol, which is

similar to chloramphenicol. Regarding oxytetracycline their results are contractionary, but

this is countered by the fact that oxytetracycline is frequently used as prophylactic agent in

shrimp aquaculture. A study by Abdel-Aziz et al. (2013) on Vibrio spp. collected from

mass mortality events of gilthead sea bream and European sea bass in Egyptian coastal

regions, confirms the sensitivity of different Vibrio spp. towards chloramphenicol,

enrofloxacin, oxytetracycline and tetracycline. The pre-treatment with rifampicin did not

change, because this study wanted to test the protocol for a system in which rifampicin-

resistant bacteria could survive.

The result obtained from this new antibiotic mixture (tetracycline, enrofloxacin and

chloramphenicol) is much better than the old antibiotic mixture (ampicillin, kanamycine

and chloramphenicol). This result, together with the fact that tetracycline is a bacteriostatic

antibiotic that binds to the 30S ribosomal subunit in the mRNA translation complex and

the above mentioned working mechanisms of chloramphenicol and enrofloxacin,

45

strengthen the idea that antibiotics that interact with the DNA and RNA synthesis are

effective to threaten aquatic organisms towards Vibrio spp.

Regarding the results for the effectivity experiment of the pre-treatment, at first, the results

of the antibiotic sensitivity test prove that the old mixture was not effective enough to kill

all the bacteria that are present in/on/around wild caught blue mussel adults.

Secondly, despite the fact that the new mixture works a lot better than the old one, it is not

yet unfillable. Because no growth on the agar plates does not means that no bacteria are

present after the pre-treatment with the old mixture. At first, in my opinion, it is possible

that bacteria that are still present are under the detection limit of 30 CFU ml-1 (which was

observed during the experiment). In this case, it is just a matter of time and available

nutrients before this bacteria reach a detectable amount. Beside this, Du et al. (2007)

showed that viable Vibrio alginolyticus can shift to the viable but not detectable state,

caused by temperature shift and absence of nutrients. Also Sun et al. (2008) showed the

possibility of Vibrio harveyi to enter into the viable but not countable state with retaining

the possibility to be pathogenic and Eiler and Bertilsson (2006) used denaturant gradient

gel electrophoresis to detect and quantify Vibrio populations because they were aware of

the viable but not culturable state of Vibrios. This information raises the recommendation

that the samples should be taken at a later stage of the experiment, so the bacteria are

able to grow till a detectable level, or other detection techniques should be used. But it

has to be taken in mind that the used system for the challenge test is para-anexic. This

means that a minimal amount of surviving bacteria is possible and this amount is also

acceptable, because at the start of the effective challenge test a large amount of desired

bacteria are added. This administered bacteria should overgrow the still present bacteria,

so the later ones have no change to affect the host-organism.

At last, regarding the results for the old antibiotic mixture, it is remarkable that also

bacteria grew on the LB35-agar plates with addition of 50 mg l-1 rifampicin. This is probably

due to the fact that bacteria who survive the pre-treatment with a mixture of antibiotics can

become rifampicin resistant.

A final noteworthy observation is the fact that the concentration of the antibiotics in the

new mixture was 50 mg l-1, which is higher than the used 20 mg l-1 concentration of the old

antibiotic mixture. It is generally known that a higher concentration of antibiotics have a

higher effectivity in the treatment towards (pathogenic) bacteria.

4.2.6. Effectivity of the cleaning and disinfection protocol

Another additional experiment shows that the used materials are not sterile after

thoroughly mechanical cleaning and disinfection with a strong acid (CID 2000®, which

consists of 20% hydrogen peroxide and 5% peracetic acid). A longer incubation time than

6 hours in CID2000® is not recommendable. Sterilizing the materials by autoclaving at

121°C for 20 minutes, which is an effective procedure for killing bacterial cells, would

probably result in deformed or damaged materials because of the high temperature.

4.2.7. Recommendations for the future regarding the adult challenge test

Besides further evaluation of the antibiotic mixture for the pre-treatment and the cleaning

and disinfection method, other possible recommendations for further research regarding

the adult challenge test protocol are given in the next paragraphs.

46

At first, it needs to be evaluated if the rifampicin – that is added to the experimental 4 liter

tanks at the start of the effective adult challenge test - works and if it works properly like it

is expected to. Because, when the added rifampicin resistant bacteria (ME1-15) are not

advantaged by creating an ideal environment for them, so they are the dominant bacteria

in the system, it is possible that they would not grow and be able to infect the host

organism as expected. Even more, while performing the adult challenge test it was seen

that the rifampicin precipitated when it was added to the seawater at a temperature of

18°C. So the solubility of the rifampicin in seawater should be evaluated in further

research. It is known that rifampicin does not dissolve well in water, but is soluble in

different chemicals, such as dimethyl sulfoxide (DMSO) and hydrogen chloride (HCl). So

the degree of solubility of rifampicin in those different chemicals should be investigated,

together with attention for the fact that some of these chemicals can be harmful to the host

organism.

Another problem that has been experienced while performing the adult challenge test is

the formation of foam due to the aeration of the seawater. This aeration was carried out in

order to provide the adult mussel from just enough oxygen that it do not closes it shell to

survive and dies because of anoxia, but keeps filtering the seawater by its filter-feeding

habit, resulting in the up-take of the added bacteria to the culture water. So, it has to be

investigated if the degree of aeration has an effect on the foam formation and if so, the

ideal degree of aeration has to be defined. The ideal degree is one that provides the adult

mussel with enough oxygen the keep filter-feeding and with a minimal foam formation as a

result. An additional fact that has to be taken in mind, is that rifampicin precipitations were

found in the foam after a while. So the rifampicin was not totally available in the culture

water and in this way did not act like it was expected to do.

A last recommendation for the adult challenge test protocol, is to investigate the

concentration of LB35 broth that is added to the culture water. This concentration needs to

be high enough to provide the added bacteria with enough nutrition and low enough to not

lead to a bad water quality for the mussels. Eggermont et al. (2014) concluded after

previous research studies on Mytilus edulis that the addition of organic matter to the

culture water resulted in an increase in total bacterial counts in the culture water, which

than can result in a significant mortality of the blue mussel adults when the added bacteria

are pathogenic towards them. But it has to be taken into account that this previous study

used a higher concentration of tryptone and yeast extract as addition of organic matter to

the culture water than was implemented in this study. They added 100 mg l-1 tryptone,

compared with 10 mg l-1 for the 1% LB35 treatments and 1 mg l-1 for the 0,1% LB35

treatments in this study, and 50 mg l-1 yeast extract compared with respectively 5 and 0,5

mg l-1. This previous study also only lasted for one week instead of two weeks like it was

performed for the experiment in this master dissertation.

47

Part V. Conclusions

5.1. Antibiotic sensitivity

After evaluating the different antibiotics regarding their effectiveness towards the different

bacterial isolates, the antibiotics can be divided into different groups and the antibiotics

that interfere with the DNA and RNA synthesis, like the trimethoprim/sulphamethoxazole

and enrofloxacin, are the most effective antibiotics to treat blue mussel from infection with

the ME1-15 bacterial isolates. Despite the fact chloramphenicol interferes with the

bacterial protein synthesis, it is also a very good antibiotic towards the ME1-15 bacterial

isolates.

Regarding the individual bacterial isolates, it can be concluded that ME1 is the most

resistant strain. Only ME11 and ME15 are sensitive to more than one of the tested

antibiotics (at the indicated dosage). All the others are resistant to intermediate sensitive

strains. This results also reveal that ME15 is the most sensitive bacterial strain, in spite

the fact that it is the most pathogenic strain towards blue mussel larvae.

In general, one can conclude that (1) there is only one antibiotic that works effectively

against all the bacterial strains, namely chloramphenicol, and there is an overall

resistance from the bacterial strains towards the tested antibiotics. But these results

create the possibility of protecting mussel larvae, from mass mortality events due to

microbial origin, with a treatment consisting of several antibiotics, such as

chloramphenicol, trimethoprim/sulphamethoxazole and enrofloxacin.

Regarding this information, the pre-treatment of the adult challenge test can be adapted to

a better working pre-treatment. This will be clarified further on in the part about the adult

challenge test.

5.2. Virulence factors

The results for the hemolysin production experiment reveals that all the 15 bacterial

isolates possess hemolytic activity. But it should be taken in mind that this experiment was

performed on sheep blood agar and not on mussel hemolymph. So this results are only an

indication of the possibility from the 15 bacterial strains to possess hemolytic activity.

The results for exopolysaccharide production and biofilm formation reveal that this

experiment gives the best results at an incubation temperature of 18°C and an incubation

time of 24 hours and there is a strong correlation between exopolysaccharide production

and biofilm formation. Regarding exopolysaccharide production, more production was

observed for the incubation time of 72 hours, but for the biofilm formation lower values

were obtained with a longer incubation time. This is probably due to the fact, that after 72

hours of incubation there are so much bacterial cells present that the bacterial cells cluster

together instead of forming a biofilm. From these results, it can be concluded that ME15

and ME11 are the most virulent bacterial isolates. But the results are still low compared to

the values in literature.

48

So, some additional experiments on the virulence factors can be done to fully understand

and verify the mechanism of how pathogenic bacteria causes diseases in Mytilus edulis.

5.3. Conslusion about the adult challenge test for Mytilus edulis

The second part of this study investigated the possibility to use bacterial strains, that are

proven to be pathogenic towards mussel larvae, in an adult challenge test that eventually

can be used in further research studies on host-pathogen interactions for Mytilus edulis.

Since this study is one of the first times a protocol for such an adult challenge test is

tested, further research and testing is recommended to optimize this protocol. The results

from this study provides information that can be used for this optimization. The evaluated

solutions - (1) a new antibiotic mix for the pre-treatment and (2) a new cleaning and

disinfection method -, could result in a better working adult challenge test protocol.

Further recommendations regarding the pre-treatment with antibiotics are that the working

mechanisms of the antibiotics and some other specification such as the solubility of the

antibiotics in seawater at a temperature of 18°C and the working concentration of the

antibiotic should be taken into account. Regarding all this, a more ideal antibiotic mix

could be composed. So further research on this topic needs to be done to optimize the

protocol for the adult challenge test.

Regarding the used cleaning and disinfection method, the test revealed that additional

research and experiments regarding this is also recommended.

5.4. Overall conclusion

So, it seems too early to conclude that the ME isolate mix that is virulent towards mussel

larvae is not virulent enough towards adult mussels and thus not suitable to be used as a

pathogen in a standardised adult mussel challenge test. The results from this study

however indicate that the microbiological control of the protocol is not yet successful and

needs further optimisation and adapted before the virulence of the possible pathogens

can be fully evaluated.

A lot of research needs to be done before the protocol for the adult challenge test is fully

optimized and ready to use in further research studies on host-pathogen interactionsdate

consult with Mytilus edulis, but seen the broad application possibilities it is worth the effort.

Also, further research on the virulence factors of the 15 bacterial isolates (ME1-15) is

needed in order to fully understand and verify the mechanism of how this bacterial isolates

cause diseases in Mytilus edulis.

i

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Appendices

Appendix 1: Overview of origin of the bacteria used in this study

Table A-1. Origin of ME1-15

Strain Isolated from

ME 1 Culture water of dead mussel larvae

ME 2 Homogenized mussel tissue

ME 3 Mussel hemolymph













Host-Pathogen interaction for Mytilus edulis

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