Virulence properties, biofilm formation and random amplified polymorphic DNA analysis of Photobacterium damselae subsp. damselae isolates from cultured sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax)

Accepted Manuscript Virulence properties, biofilm formation and random amplified polymorphic DNA analysis of Photobacterium damselae subsp. damselae isolates from cultured sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax) Sadok Khouadja, Faouzi Lamari, Amina Bakhrouf, Kamel Gaddour PII:

S0882-4010(14)00040-0

DOI:

10.1016/j.micpath.2014.03.007

Reference:

YMPAT 1476

To appear in:

Microbial Pathogenesis

Received Date: 4 November 2013 Revised Date:

18 March 2014

Accepted Date: 20 March 2014

Please cite this article as: Khouadja S, Lamari F, Bakhrouf A, Gaddour K, Virulence properties, biofilm formation and random amplified polymorphic DNA analysis of Photobacterium damselae subsp. damselae isolates from cultured sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax), Microbial Pathogenesis (2014), doi: 10.1016/j.micpath.2014.03.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Virulence properties, biofilm formation and random amplified polymorphic DNA

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analysis of Photobacterium damselae subsp. damselae isolates from cultured sea bream

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(Sparus aurata) and sea bass (Dicentrarchus labrax)

4 Sadok Khouadja 1*, Faouzi Lamari1, Amina Bakhrouf 1, Kamel Gaddour1

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1. Laboratoire d’Analyse, Traitement et Valorisation des Polluants de l’Environnement

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et des Produits, Département de Microbiologie, Faculté de Pharmacie, Rue Avicenne,

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5000, Monastir, Tunisie

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Abstract

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Photobacterium damselae subsp. damselae has been isolated from different outbreaks

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affecting cultured Sparus aurata and Dicentrarchus labrax. The aim of the present study

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was to characterize the phenotype and genotype of 12 Photobacterium damselae subsp.

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damselae strains isolated from these outbreaks. The roles of skin mucus in resistance to

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the initial infection steps have been studied. All tested strains resisted the bactericidal

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activity of the mucus and showed an ability to adhere to it, but only those showing

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hemolytic activity were found to be virulent by intraperitoneal injection. Phenotypic and

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genetic characterization revealed a considerable degree of variability within the

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subspecies. We found that RAPD-PCR represents a quick tool to generate information on

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intraspecific differences in environmental strains. We found, that some biotypes are more

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pathogenic than others, which by doing correlation between adhesion profile, enzymatic

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and hemolytic activity.

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Key words: Photobacterium damselae subsp. damselae, RAPD, biofilm, virulence

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properties.

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*Corresponding author: Phone: +216 73461000; Fax: +216 73461830; E-mail: [email protected]

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1. Introduction Photobacterium damselae includes Gram-negative marine bacteria that comprises, the fish

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virulent isolates belonging to two different subspecies, namely, Photobacterium damselae

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subsp. piscicida (formerly Pasteurella piscicida) [1] and Photobacterium damselae subsp.

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damselae (formerly Vibrio damsela) [1]. The two subspecies differ in important biochemical

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and physiological traits, such as motility, gas production from glucose, nitrate reduction,

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urease, lipase, amylase, hemolysin production, and the ranges of temperature and salinity for

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growth, as well as host specificity.

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Photobacterium damselae subsp. damselae, upon first isolation from skin ulcers of

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damselfish [2], was named Vibrio damsela [3]. Following 5S rRNA sequence analysis, it was

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assigned to the new genus Listonella and following analysis of phenotypic traits, it was

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reassigned to the genus Photobacterium as Photobacterium damselae [4]. The species also

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comprises the fish-virulent isolates formerly assigned to the species Pasteurella piscicida,

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now P. damselae subsp. piscicida. The subspecies are phenotypically dissimilar, develop

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different diseases and only one of them (the subspecies damselae) is pathogenic for humans

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[5].

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P. damselae subsp. damselae has been recognized as an opportunistic pathogen for a wide

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variety of fish [6] and mammalians, including humans hosts [5] . These strains cause

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septicaemia in warm and cold water fish such as damselfish (Chromis punctipinnis), eels

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(Anguilla

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quinqueradiata), seabream (Sparus aurata) and turbot (Scophthalmus maximus) [6-9].

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Clinical symptoms of infection with this bacterium include skin ulcerative lesions and

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extensive hemorrhages, especially in mouth, eyes and musculature [10].

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anguilla),

brown

shark

(Carcharhinus

plumbeus),

yellowtail

(Seriola

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Marine fish farming is a very important activity of the Tunisian aquaculture industry. The

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main marine fish species intensively cultured are gilt-head sea bream (Sparus aurata) and

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ACCEPTED MANUSCRIPT European sea bass (Dicentrarchus labrax) [11, 12]. However, the intensive culture of these

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fish species has favored the appearance of several outbreaks with varied mortality rates.

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Therefore, there is a necessity to evaluate the impact of Photobacterium damselae to

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aquaculture in Tunisia and improve our understanding of the interaction between this bacteria

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and fish. The objective of the present study was to study the phenotypic, genomic diversity

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and virulence properties within Photobacterium damselae strains isolated during a mass

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mortality of S. aurata and D. labrax in 2012.

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2. MATERIALS AND METHODS

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2.1. Sampling and bacterial isolation

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Photobacterium damselae subsp. damselae strains were isolated from diseased Sea bass

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(Dicentrarchus labrax) and Gilthead sea bream (Sparus aurata L.) obtained from a Tunisian

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farm located along the Mediterranean Sea coast (Tbolba, Centre of Tunisia).

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The first mortality occurred when water temperature increased from 20 to 25 °C and salinity

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was 37‰. Cumulative fish losses were approximately 5 % of the stocks in the two outbreaks.

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Samples from liver, spleen, kidney and external lesions of older D. labrax specimens (weight:

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220 g and length: 20 cm) and S. aurata (weight: 130 g and length: 12 cm) obtained from two

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outbreak were cultured on tryptic soy agar and in tryptic soy broth (Difco) supplemented with

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1% NaCl (TSA 1% and TSB 1%, respectively), as well as on thiosulphate–citrate–bile salt–

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sucrose agar (TCBS, Oxoid)

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2.2. Bacterial strains characterization

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All strains were subjected to standard morphological, physiological and biochemical plate

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and tube tests using the procedures described in Bergey’s Manual of Systematic Bacteriology.

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A commercial miniaturized Api 20NE Kit (Bio-Mérieux, Inc) was also used for further

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physiological identification of the bacterial strains.

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ACCEPTED MANUSCRIPT The API 20NE strips were inoculated, read and interpreted according to the

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manufacturer’s instructions, with the following modifications. All test strips were inoculated

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with a 2.5% NaCl solution, while the AUX Medium for the assimilation tests was

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supplemented with 0.5 ml of a concentrated sterile saline solution to reach the same NaCl

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final concentration. The results were interpreted using APILAB PLUS 3.3.3 software

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(bioMerieux, Inc).

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The identities of the isolated bacteria were confirmed using PCR assay. For the extraction

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of bacterial DNA, individual colonies grown in TSB 1% were picked and re-suspended in 500

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µl of sterilized double distilled water. Bacterial DNA was then extracted by boiling the

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bacterial cells for 5 min followed by centrifugation at 12,000 × g for 5 min. The upper

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aqueous phase containing the bacterial DNA was transferred to a sterile 1.5 mL

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microcentrifuge tube and stored at −20°C until needed.

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The DNA concentration of each sample was adjusted to (40 ng/µl) for PCR by diluting DNA

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in sterilized double distilled water. The ureC gene species-specific forward primer Ure-F (5'-

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TCCGGAATAGGTAAAGCGGG-3')

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ATCCATCTCATCTGC-3') for the detection of Photobacterium damselae subsp. damselae

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[13]. Amplification consisted of: an initial denaturation of 95°C for 4 min, followed by 30

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cycles of a denaturation of 1 min at 95°C ,1 min at 60°C and 40 sec at 72°C with a final

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extension step of 5 min at 72°C [13]. The PCR fragments were subjected to electrophoresis on

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a 1.5% agarose gel in Trisborate- EDTA buffer (0.89 M tris, 0.89 M boric acid, 0.02 M

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EDTA, pH 8.0) and visualized by ethidium bromide staining and photographed using Gel Doc

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XR apparatus (Biorad, USA) to confirm reaction specificity and the size of the PCR product.

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reverse

primer,

Ure-R

(5'CTTGAAT

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2.3. Extracellular products preparation

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Bacterial extracellular products (ECPs) were obtained by the cellophane overlay method

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described by Liu (1957). Briefly, separate tubes containing 5 ml of TSB 1% were inoculated

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ACCEPTED MANUSCRIPT with one bacterial colony obtained from a 24h TSA 1% NaCl culture of each of the twelve P.

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damselae subsp. damselae strains and incubated at 30°C for 24 h. An aliquot (0.2 ml) of each

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culture was subsequently transferred onto a sterile cellophane sheet that had been placed on

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the surface of separate TSA 1% NaCl plates. After incubation at 30°C for 48 h, each

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cellophane sheet was removed and washed using phosphate-buffered saline (7mM Na2HPO4,

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3mM NaH2PO4 and 130mM NaCl at pH 7) in 15 ml tube and the cells were removed by

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centrifugation at 10,000 × g and 4°C for 30 min [12]. The supernatant containing the ECPs

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was recovered and sterilized by filtration (0.22 µm) and stored at –80°C until use.

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Enzymatic activities of the extracellular products were evaluated with the API Zym

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System (BioMérieux, Inc) composed of 19 enzymatic substrates. The activities of various

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other enzymes were determined following inoculation of 20 µl of ECPs onto agarose plates

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(0.8% agarose in 0.1 M PBS, pH 7) to which the following substrates had been added: 0.2%

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(wt/vol) starch for amylase and 1% skim milk for caseinase. 0.4% Gelatin for gelatinase had

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been added to TSA 1% NaCl plates. Zone of gelatinase activity around the colonies may be

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demonstrated if the plate is flooded with aqueous tannic acid (1%), the medium becomes

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opaque but zones of gelatinase activity are more opaque [14].

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Sterile PBS was inoculated onto the plates and served as a negative control, whereas

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Staphylococcus aureus ATTC 25923 ECPs were used as a positive control. The expression of

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DNase and haemolysin activities was determined with whole viable cells. Bacteria grown

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overnight in TSA 1% NaCl at 30°C were spot inoculated onto the assay media described

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below. DNase activity was studied using DNase test agar (DNase Agar, Scharlau

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Microbiogy). The production of haemolysin was detected on blood agar plates with 5% of sea

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bass erythrocytes. Haemolysis was evident after incubation at 30°C for 24 h.

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2.4. Antibacterial bioassay with fish mucus

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ACCEPTED MANUSCRIPT The antibacterial activity of skin mucus isolated from Gilthead sea bream and sea bass were

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evaluated using a disc diffusion method on agar plates (Fouz et al. 1990). Surface mucus was

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collected by scraping the skin, using sterile glass slides, of five healthy specimens of S. aurata

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and D. labrax older fish (Weight 200-300 g) collected from the fish farm installed near the

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site of study. The mucus was subsequently filtered using 0,45 and 0,2 µm pore-size

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membranes (Millipore, Sartorius Minisart CE 0297, Germany).

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Photobacterium damselae subsp. damselae ATCC 33539 and Staphylococcus aureus ATTC

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25923 were used as positive (resistant) and negative (sensitive) controls, respectively (Fouz et

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al. 1990). Briefly, sterilized 6 mm diameter discs (Oxoid) impregnated with 20 µl of the

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mucus solution was aseptically transferred onto the surface of plates recently inoculated with

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a bacterial lawn containing about105 cfu ml-1 of either positive or negative control bacterium

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or one of the twelve isolated Photobacterium strains. All tests were performed in triplicate.

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After 24 h of incubation at 30 °C, the appearance of a growth inhibition halo around the discs

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indicated that antibacterial substances were present in the mucus preparation.

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The diameters of the inhibition zones were measured and the ratio (equal to or larger than

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1 cm) was used as index to represent the intensity of antibacterial activity against the tested

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strains.

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2.5. Assays of adherence to fish mucus

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In order to examine the degree of biofilm formation in the presence of fish mucus,

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biofilm production by the isolated Photobacterium strains was determined using a semi-

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quantitative adherence assay on 96-well tissue culture plates [15]. The assay was performed

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as follows: 2 ml mucus solutions were transferred to 96-well tissue culture plates (20 µl in

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each well), left overnight for air-drying, and fixed for 20 min with absolute methyl alcohol.

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One hundred microlitres of a cell suspension from each of the 12 isolated Photobacterium

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damselae subsp. damselae and the ATCC 33539 strains was added to separate wells of the 96-

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well microtitre plates coated with fish mucus before adding equal volumes of PBS supplement

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with 2% glucose, to each well. As a positive control of the adhesion ability, we tested

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bacterial adherence in the presence of PBS in 96-well polyesters plate. Sterile PBS was added as an additional control to ensure that the media remained sterile

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during the course of the experiment. The cultures were added into the wells in triplicate. The

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plates were incubated at 30°C for 24 h without shaking to allow cell attachment and biofilm

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development. The cultures were removed and the microtiter wells were washed twice with

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PBS to remove non-adherent cells and dried in an inverted position. Thereafter, plates were

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rinsed three times with PBS to remove loosely attached cells, dried in an inverted position,

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and then 180 µl of PBS and 20 µl of the XTT-menadione solution (12.5 volume of XTT

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solution was mixed with 1 volume of menadione solution) were added to each well and the

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plates were incubated at 30 °C in the dark for 3 h [16].

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The XTT assay was used to quantify the number of viable cells in each of the wells

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following mucus supplementation in comparison with mucus-free controls. This method has

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been used extensively for the quantification of bacterial biofilm based in oxidative metabolic

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activity. It measures the reduction of a tetrazolium salt (2, 3-bis [2-methyloxy-4-nitro-5-

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sulfophenyl]-2H-tetrazolium-5 carboxanilide (XTT)) by metabolically active cells to a

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colored water soluble Formosan derivative that can be easily quantified colorimetrically.

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Briefly, XTT solution (1 mg/ml) was prepared in PBS, sterilized through a 0.22 µm pore size

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filter (Millipore, Sartorius Minisart CE 0297, Germany) and stored at -70°C. Menadione

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(SigmaeAldrich, Switzerland) solution (0.4mM) was prepared in acetone and sterilized

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instantaneously before each assay [16]. Reduction of XTT (oxidative activity) was then

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measured at 492 nm using automated Multiskan reader (GIO, Rome, Italy).

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formation was interpreted as highly positive (OD492 ≥1), low grade positive (0.1 ≤OD492<1),

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or negative (OD492< 0.1).

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Biofilm

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Biofilm biomass was assessed, and the results were expressed as percentage of biofilm

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reduction percentage (BRP): BRP= [(OD Without fish mucus - OD With fish mucus) / OD Without fish

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x 100.

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mucus]

2.6. Infectivity tests The median lethal dose (LD50) test was conducted by intraperitoneal (i.p.) injection as

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previously described [17]. The P. damselae subsp. damselae strains to be tested (n=12

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isolated P. damselae subsp. damselae and ATCC 33539 strain) were grown overnight in TSA

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1% NaCl at 30°C, from which one colony was sub-cultured in 40 ml fresh medium (TSB 1%

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NaCl) and grown for a further 24 h at 30°C . The cells were harvested by centrifugation (5000

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× g, 10 min), washed two times and resuspended in PBS to OD600 nm of 0.2 to 0.9, so that the

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bacterial concentration ranged from 102 to 108 cfu ml-1 as determined by the dilution-plate

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method. Sterile PBS was injected i.p. into fish as control. Dicentrarchus labrax (weight 8 to

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10 g, length 8 cm, 20 individuals per testing dose) were acclimated in sea water maintained at

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of 25°C for 4–5 days before testing. Sea water was kept in each tank at salinity of 37‰ and

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under continuous aeration. Before infection the fish were fed with commercial food (INVE

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Aquaculture Nutrition), while feeding was suspended during the virulence test. Each bacterial

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dilution containing from 102 cfu ml-1to 108 cfu ml-1 was tested by i.p. injection of 50 µl of

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suspension (20 individuals per testing dose). Bacterial strains were considered virulent

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whenever the LD50 was less than 108 cfu ml-1 [18]. Mortalities were recorded daily for 7 days

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and fish were only considered infected if P. damselae subsp. damselae was recovered from

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experimentally infected fish. The LD50, was calculated by simple method for estimating fifty

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per cent endpoints [19].

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2.7. RAPD-PCR fingerprinting of the isolates

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PCR reaction conditions were optimized for important parameters such as primer

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annealing temperature, MgCl2 concentrations, template DNA, Taq DNA polymerase, and

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ACCEPTED MANUSCRIPT dNTPs. Two primers were selected for further data analysis, P5 (5’-AACGCGCAAC-3’) and

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P6 (5’-CCCGTCAGCA-3’). The PCR reaction mixture consisted of 2.0 µL of Taq reaction

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buffer (100 mM Tris–HCl pH 8.3, 500 mM KCl, 20 mM MgCl2 and 0.001% gelatin), 0.125

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mM dNTPs (Promega, USA), 1 unit of Taq DNA polymerase (Promega, USA), 30 pM of

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each primer and 40 ng of the DNA template in a total reaction volume of 20 µL. RAPD was

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performed with the following parameters: an initial denaturation cycle at 94°C for 2 min,

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followed by 45 cycles of denaturation at 94 °C for 1 min, primer annealing at 36°C for 1 min

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and primers extension at 72 °C for 2 min. The program also included a final primer extension

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step at 72°C for 5 min. The ramp from primer annealing to primer extension temperature was

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set at 30s. Bands were visually read from fingerprints generated by two primers and a data

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matrix was generated for each strain by giving scores of 0 and 1 for the absence or presence

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of bands, respectively, at each band position for all strains. The data matrix of individual

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primers was finally joined to form a single matrix. A dendrogram was constructed using the

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data matrix of all 13 strains of bacteria based on unweighted pair group method with

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arithmetic means (UPGMA) [20].

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2.8. Statistical analysis

Each analysis was performed using the SPSS 17.0 statistics package for Windows. The

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differences in the degree of biofilm formation as a function of mucus supplementation were

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examined by the Friedman test, followed by the Wilcoxon signed ranks test. P < 0.05 was

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considered significant.

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3. RESULTS

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3.1. Bacterial strains characterization

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Isolated bacteria were Gram-negative rod-cocci, cytochrome-oxidase positive organisms and

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were identified as Photobacterium damselae using the API-20E system (Table 1). The PCR

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technique confirmed the 12 strains studied as P. damselae ssp. damselae, because all of them

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yielded amplification fragments of 448 bp, corresponding to ureC gene (Fig 1).

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3.2. Bacterial Extracellular products P. damselae subsp. damselae extracellular products tested positive for four exoenzymatic

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activity patterns out of the 19 included in API-ZYM test strip kit. The majority of strains

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tested positive for leucine arylamidase, acid phosphatase, the phosphohydrolase, the esterase,

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the esterase–lipase and alkaline phosphatase (Table 2).

3.3. Hemolytic phenotypes in P. damselae subsp. damselae isolates

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Among the 12 strains, 4 showed a large hemolysis halo (15 and 18 mm), three were

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nonhemolytic, three showed moderate hemolysis (10 and 11 mm), and the remaining 2 strains

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yielded a small hemolytic halo on sea bass erythrocytes agar plates (Table 1).

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3.4. Biofilm and resistance to fish mucus

All assayed strains of P. damselae subsp. damselae, irrespective of their origin and degree of

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virulence, were resistant to the antimicrobial action of skin mucus from sea bream and sea

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bass. Fifty percent of P. damselae subsp. damselae strains (6/12) were strongly adhesive to

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polystyrene with a values ranging from (1.153) to (1.638) at 492 nm (P >0.05). For these

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same strains, even in the presence of mucus, they remain strongly adhesive to polystyrene

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with biofilm reduction percentage between 0.2 and13.98 % with Sparus aurata mucus and 2.6

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% and 12.58 with Dicentrarchus labrax mucus.

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Only two strains (S8, S9) were non-biofilm forming. ATCC 33539 (R1) strain displays the

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highest oxidative activity (2.17 nm). Moreover, it was found that majority strains adhered to

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the fish skin mucus fixed on microtitre plate. All these data were summarized in table 3.

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3.5. Infectivity tests

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Experimentally infected fish showed clinical signs of infection similar to those observed

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during the two outbreaks.

During the determination of the LD50, all strains showing

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ACCEPTED MANUSCRIPT hemolytic activity were virulent and caused mortality during the infection tests. Pure cultures

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of P. damselae subsp. damselae were isolated from liver and skin tissues of moribund fish’s

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and no mortality were detected in the control group. Sign of infection began to appear on the

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2nd day following infection with strains R1 and S4, whereas for all other strains clinical signs

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of infection were evident only from the 4th day. The LD50 of the pathogenic strains ranged

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between 1.78102 and 3107 cfu fish-1 (Table 3). Healthy fish inoculated with cells of virulent

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strains of P. damselae subsp. damselae developed skin ulcers, similar to those observed on the

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surface of naturally diseased sea bass, whereas, fish injected with isolates S5, S6, S7, S8 and

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S9 strains showed no signs of infection throughout the experiments.

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3.6. RAPD-PCR fingerprinting of the isolates

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According to their RAPD genetic profiles, the strains of Photobacterium damselae subsp.

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damselae isolated during this study, were grouped for a degree of similarity and clustered in

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seven groups using primers P5 and P6. Each strain rendered a unique profile, which differed

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in the number of bands (from 1 to 3) and in their molecular size (between 90 and 2200 bp),

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with a band of 200 bp who is present in several strains (fig 2). We also observed that some

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strains shared the same profile (S10-S1), (S8-S9) and (S2-S4-S11-S12 and R1) (Fig.3). The

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repeatability of fingerprints generated by the two primers was evaluated by amplifying the

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genomic DNA of each of the bacterial strains three times and comparing the results. Since

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these two primers were found to give consistent and repeatable band patterns.

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4. Discussion

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The development of a fish disease is the result of the interaction among pathogen, host

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and environment. In the present study, the description and the characterization of the

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Photobacterium damselae subsp. damselae etiological agents involved in two separate

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outbreaks are described in detail. All strains, (including the reference strain ATCC 33539),

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isolated were Gram-negative, rod shaped cells that were oxidase and catalase positive and

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ACCEPTED MANUSCRIPT capable of fermenting melibiose and maltose. The biochemical activities of 13 strains tested

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on Api 20NE strips demonstrated the heterogeneity of the strains isolated in this study. In fact,

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out of the 13 Photobacterium damselae subsp. damselae strains tested, 9 biotypes were

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identified (Table 1). Several studies describe the variation in the phenotypic typing of this

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specie and demonstrates a variation among strains isolated from clinical isolates and

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environmental isolates [5].

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The extracellular products (ECPs) produced by bacterial pathogens, facilitate the uptake

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of nutrients from the surrounding environment and/or the successful penetration and survival

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of pathogens inside the host [21, 22]. Adhesion and hydrolytic activities are essential factors

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for the infection and disease symptoms of P. damselae subsp. damselae [23]. Most of the

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isolates of P. damselae subsp. damselae in the present study produced gelatinase, lipase,

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DNase and caseinase, all probable virulence factors that might help the organisms to invade

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and proliferate. Labella et al. (2010), studying the pathogenicity of P. damselae subsp.

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damselae strains isolated from cultured fish, demonstrated that the intraperitoneal injection of

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fish with heated ECPs (100ºC, 10 min), did not result in mortality, which suggests that the

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active toxic fraction present in the ECPs is secreted and thermosesitive.

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Hemolysins are one of the major virulence factors that enable P. damselae subsp.

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damselae to cause septicemia in aquatic animals. The existence of correlation was initially

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observed between the ability of P. damselae subsp. damselae to cause disease in mice and the

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production of large amounts of a heat-labile cytolytic toxin in vitro [24]. In subsequent

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studies, this toxin was named damselysin (Dly) [25].

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The degree of hemolysis varies among P. damselae subsp. damselae isolates. Two main

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distinct hemolytic phenotypes could be observed on blood agar plates, with strains showing a

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large hemolysis halo (LH) and strains producing a small hemolysis halo (SH) [26]. We

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observed a variation in the hemolytic and virulence activity despite that all the strains were

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ACCEPTED MANUSCRIPT isolated from fish outbreaks. This may be due to the effect of changes in environmental

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conditions between the isolation of the strains and the in vitro tests. The environmental factors

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such as iron starvation and salt concentration were found to regulate the expression of the

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hemolysins in P. damselae subsp. Damselae [27] .

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Rivas et al. (2011), provide evidence that, in addition to Dly, a pore-forming toxin hemolysin

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of the HlyA family contribute to the hemolysis and the virulence of P. damselae subsp.

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damselae. It was also demonstrated by several authors that presence of dly is not a pre

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requisite for the hemolytic activity and for the pathogenicity for mice or fish, since dly

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negative strains bear virulence potential for animals and also toxicity for homeotherm and

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poikilotherm celllines [23, 28]. Furthermore the contributions, in terms of the individual and

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combined effects, of the Dly and hlyA hemolysins to hemolysis and virulence varied

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depending on the animal species tested [27].

313

In the present study, XTT assay was performed for evaluating biofilm formation by P.

314

damselae subsp. damselae strains. The XTT indirectly measures the microbial activity; it is

315

reduced by the dehydrogenase enzymes present in the electron transport system (ETS) to a

316

water-soluble formazan dye [29, 30]. The XTT colorimetric assay was used to determine the

317

percentage of viable cells in biofilm with the presence of mucus or not. To determine whether

318

fish mucus would influence biofilm formation, we investigated the ability of the tested strains

319

to form biofilm in response to Sparus aurata and Dicentrarchus labrax mucus presence.

320

Analysis of the data obtained with two fish mucus showed that biofilm formation by the P.

321

damselae subsp. damselae strains is slightly affected by the mucus presence, with very low

322

biofilm percentage reduction.

323

Resistance to the bactericidal mechanisms of fish mucus appears to be an important

324

contributor to the virulence of fish pathogen. Complement activity in fish mucus is known to

325

play an important role in the defence against bacterial pathogens [18]. Photobacterium

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13

ACCEPTED MANUSCRIPT damselae subsp. damselae isolates showed a resistance to complement activity of the sea bass

327

and gilthead sea bream mucus. The skin, which forms a physical barrier between the external

328

and internal environments of the fish, is covered by a mucous layer containing proteases,

329

lysozyme, antibodies, complement [31]. As a consequence, the skin acts as a primary line of

330

defense against pathogenic micro-organisms. P. damselae subsp. damselae tested during this

331

study show potential capability to avoid skin mucus, how can facilitates fish colonization and

332

increase his pathogenicity. The resistance to fish skin mucus bactericidal activity can

333

represent a determinant factor in pathogenic action of Photobacterium damselae subsp.

334

damselae strains.

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326

The results of lethal concentration (LD50) tests obtained showed that the LD50 values

336

ranged from 1.78102 and 3107 cfu fish-1. According to these results there seemed to be a

337

correlation between virulence, adhesive capability to skin mucus and extracellular products,

338

including hemolytic activity. In agreement with findings of Pedersen et al. (2009), strains that

339

had haemolytic activity and those with ability to adhere to mucus seemed to be the most

340

pathogenic [6].

341

The intraspecific typing of Photobacterium damselae subsp. damselae can be of great value

342

for the recognition of clone relationships among isolates causing disease outbreaks. Our

343

results, in agreement with previous studies, confirmed the high intraspecific variability among

344

these strains at both phenotypic and genetic levels [5, 9]. This suggests existence of different

345

clonal lineages that coexist in the same geographic area, within a short period of time, which

346

in our case is during a period less to a year. Using ribotyping, amplified fragment length

347

polymorphism and pulsed-field gel electrophoresis, clinical P. damselae subsp. damselae

348

isolates causing fatal cases in humans had similar genotypes, but they were not clearly

349

distinguishable from environmental isolates [32]. We found that RAPD-PCR represents quick

350

tools to generate information on intraspecific differences in environmental strains. We

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ACCEPTED MANUSCRIPT observe a high similarity between strains belonging to the same cluster. Correlation between

352

adhesion profiles, enzymatic activity and hemolytic activity confirm the idea that some

353

biotypes are more pathogenic than other. P. damselae subsp. damselae strains can be divided

354

into two groups according to the presence of hemolytic activity or not. Strains (S8 and S9)

355

and (S5, S6 and S7), may be clustered in very close two biotypes. They are biofilms low-

356

producer and these same strains were found negative for hemolytic activity and don’t cause

357

morality during infection tests. While, all highly biofilm positive strains shows hemolytic

358

activity and they are pathogenic to fish.

359

In conclusion, all strains showing haemolytic activity (S1, S2, S3, S4, S10, S11 and S12)

360

against sea bass erythrocytes and resistance to the mucus bactericidal activity of were found

361

the most pathogenic. Therefore, the wide variability in enzymatic functions makes us think

362

that other virulence factors may be involved in the pathological damages caused by this

363

microorganism in infected fish. The RAPD-PCR techniques are confirmed as good tools for

364

molecular typing, because they allow discrimination between P. damselae subsp. damselae

365

strains isolated within the same outbreak.

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[1] Gauthier G, Lafay B, Ruimy R, Breittmayer V, Nicolas JL, Gauthier M, et al. Small-subunit rRNA sequences and whole DNA relatedness concur for the reassignment of Pasteurella piscicida (Snieszko et al.) Janssen and Surgalla to the genus Photobacterium as Photobacterium damsela subsp. piscicida comb. nov. Int J Syst Bacteriol. 1995;45:139-44. [2] Love M, Teebken-Fisher D, Hose JE, Farmer JJ, 3rd, Hickman FW, Fanning GR. Vibrio damsela, a Marine Bacterium, Causes Skin Ulcers on the Damselfish Chromis punctipinnis. Science. 1981;214:1139-40. [3] Pillidge CJ, MacDonell MT, Colwell RR. Nucleotide sequence of the 5S rRNA from Listonella (Vibrio) aestuarianus ATCC 35048. Nucleic Acids Res. 1987;15:1879. [4] Smith SK, Sutton DC, Fuerst JA, Reichelt JL. Evaluation of the genus Listonella and reassignment of Listonella damsela (Love et al.) MacDonell and Colwell to the genus Photobacterium as Photobacterium damsela comb. nov. with an emended description. Int J Syst Bacteriol. 1991;41:52934. [5] Takahashi H, Miya S, Kimura B, Yamane K, Arakawa Y, Fujii T. Difference of genotypic and phenotypic characteristics and pathogenicity potential of Photobacterium damselae subsp. damselae between clinical and environmental isolates from Japan. Microb Pathog. 2008;45:150-8. [6] Pedersen K, Skall HF, Lassen-Nielsen AM, Bjerrum L, Olesen NJ. Photobacterium damselae subsp. damselae, an emerging pathogen in Danish rainbow trout, Oncorhynchus mykiss (Walbaum), mariculture. J Fish Dis. 2009;32:465-72.

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References

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[7] Fouz B, Toranzo AE, Marco-Noales E, Amaro C. Survival of fish-virulent strains of Photobacterium damselae subsp. damselae in seawater under starvation conditions. FEMS Microbiol Lett. 1998;168:181-6. [8] Fouz B, Toranzo AE, Milan M, Amaro C. Evidence that water transmits the disease caused by the fish pathogen Photobacterium damselae subsp. damselae. J Appl Microbiol. 2000;88:531-5. [9] Labella A, Manchado M, Alonso MC, Castro D, Romalde JL, Borrego JJ. Molecular intraspecific characterization of Photobacterium damselae ssp. damselae strains affecting cultured marine fish. J Appl Microbiol. 2010;108:2122-32. [10] Fouz B, Larsen JL, Nielsen BB, Barja JL, Toranzo AE. Characterization of Vibrio damsela strains isolated from turbot Scophthalmus maximus in Spain. Diseases of Aquatic Organisms. 1992:155–66. [11] Bakhrouf A, Jeddi M, ouda HB. Essai d’identification de deux vibrions isolés dans une zone de pisciculture. Microb Hyg Alim. 1992;4 (9) 3-6. [12] Ben Kahla-Nakbi A, Chaieb K, Bakhrouf A. Investigation of several virulence properties among Vibrio alginolyticus strains isolated from diseased cultured fish in Tunisia. Dis Aquat Organ. 2009;86:21-8. [13] Osorio CR, Toranzo AE, Romalde JL, Barja JL. Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae. Dis Aquat Organ. 2000;40:177-83. [14] Barer G. The rapid detection of gelatin-liquefying organisms. Mon Bull Minist Health Emerg Public Health Lab Serv. 1946;5:28. [15] Brown NF, Boddey JA, Flegg CP, Beacham IR. Adherence of Burkholderia pseudomallei cells to cultured human epithelial cell lines is regulated by growth temperature. Infect Immun. 2002;70:97480. [16] Sandasi M, Leonard CM, Viljoen AM. The in vitro antibiofilm activity of selected culinary herbs and medicinal plants against Listeria monocytogenes. Lett Appl Microbiol. 2010;50:30-5. [17] Alcaide E, Amaro C, Todoli R, Oltra R. Isolation and characterization of Vibrio parahaemolyticus causing infection in Iberian toothcarp Aphanius iberus. Diseases Of Aquatic Organisms. 1999;35:7780. [18] Amaro C, Biosca EG, Fouz B, Alcaide E, Esteve C. Evidence that water transmits Vibrio vulnificus biotype 2 infections to eels. Appl Environ Microbiol. 1995;61:1133-7. [19] Reed M, Muench H. A simple method for estimating fifty per cent endpoints. American journal of hygiene. 1938;27:493-7:. [20] Sneath PH. Principles of bacterial taxonomy. Proc R Soc Med. 1972;65:851-2. [21] Hanif A, Bakopoulos V, Leonardos I, Dimitriadis GJ. The effect of sea bream (Sparus aurata) broodstock and larval vaccination on the susceptibility by Photobacterium damsela subsp. piscicida and on the humoral immune parameters. Fish Shellfish Immunol. 2005;19:345-61. [22] Bakopoulos V, Hanif A, Poulos K, Galeotti M, Adams A, Dimitriadis GJ. The effect of in vivo growth on the cellular and extracellular components of the marine bacterial pathogen Photobacterium damsela subsp. piscicida. J Fish Dis. 2004;27:1-13. [23] Labella A, Sanchez-Montes N, Berbel C, Aparicio M, Castro D, Manchado M, et al. Toxicity of Photobacterium damselae subsp. damselae strains isolated from new cultured marine fish. Dis Aquat Organ. 2010;92:31-40. [24] Kreger AS. Cytolytic activity and virulence of Vibrio damsela. Infect Immun. 1984;44:326-31. [25] Kothary MH, Kreger AS. Purification and characterization of an extracellular cytolysin produced by Vibrio damsela. Infect Immun. 1985;49:25-31. [26] Rivas AJ, Balado M, Lemos ML, Osorio CR. The Photobacterium damselae subsp. damselae hemolysins damselysin and HlyA are encoded within a new virulence plasmid. Infect Immun. 2011;79:4617-27. [27] Rivas AJ, Balado M, Lemos ML, Osorio CR. Synergistic and additive effects of chromosomal and plasmid-encoded hemolysins contribute to hemolysis and virulence in Photobacterium damselae subsp. damselae. Infect Immun. 2013;81:3287-99.

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[28] Osorio CR, Romalde JL, Barja JL, Toranzo AE. Presence of phospholipase-D (dly) gene coding for damselysin production is not a pre-requisite for pathogenicity in Photobacterium damselae subsp. damselae. Microb Pathog. 2000;28:119-26. [29] Bensaid A, Thierie J, Penninckx M. The use of the tetrazolium salt XTT for the estimation of biological activity of activated sludge cultivated under steady-state and transient regimes. J Microbiol Methods. 2000;40:255-63. [30] McCluskey C, Quinn JP, McGrath JW. An evaluation of three new-generation tetrazolium salts for the measurement of respiratory activity in activated sludge microorganisms. Microb Ecol. 2005;49:379-87. [31] Austin B, Austin D, Sutherland R, Thompson F, Swings J. Pathogenicity of vibrios to rainbow trout (Oncorhynchus mykiss, Walbaum) and Artemia nauplii. Environ Microbiol. 2005;7:1488-95. [32] Botella S, Pujalte MJ, Macian MC, Ferrus MA, Hernandez J, Garay E. Amplified fragment length polymorphism (AFLP) and biochemical typing of Photobacterium damselae subsp. damselae. J Appl Microbiol. 2002;93:681-8.

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437 438 439 440 441 442 443 444 445 446 447 448 449 450 451

Figure 1. Agarose gel electrophoresis of DNA amplification of selected isolate (S1, S5,

454

S12) s obtained with the ureC gene primers Ure-F and Ure-R: isolated strain (M): DNA

455

molecular weight marker, (R1) Photobacterium damselae subsp. damselae ATCC 33539 and

456

(T) negative control

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452 453

458 459

Figure 2. RAPD-PCR profiles of 13 bacterial strains generated after amplification with

460

primers P5 and P6. The order of samples in lanes 1-12 is as shown in Table 1. (M): DNA

461

molecular weight marker, (R1) Photobacterium damselae subsp. damselae ATCC 33539 and

462

(T) negative control.

463

17

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Β- Hemolytic

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Β- Hemolytic

464 465

Figure 3. Dendrogram based on the unweighted pair group method of arithmetic averages

466

and Jaccard’s correlation coefficient derived from the RAPD profiles of 13 Photobacterium

467

damselae subsp. Damselae strains.

471 472 473 474 475 476 477 478

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469

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468

479 480 481 482

18

ACCEPTED MANUSCRIPT 483 484

Table 1. Phenotypic characterization of the different isolates of Photobacterium damselae subsp. damselae.

485 Hydrolytic enzymes Biotype on Api 20 NE

Origin

Season

S1

3300004

Gills (D.L)

Spring 2012

+

+

+

+

-

+

S2

3340204

Gills (D.L)

Spring 2012

+

+

+

+

-

+

S3

3340244

Gills (D.L)

Spring 2012

+

-

+

+

+

+

S4

3340246

Liver (S.A)

Spring 2012

+

-

+

+

S5

3340204

Liver (S.A)

Spring 2012

+

+

+

+

S6

3340244

Liver (S.A)

Spring 2012

+

S7

3340046

Liver (S.A)

Spring 2012

+

S8

5240046

Heart (D.L)

Summer 2012

+

S9

6012004

Heart (D.L)

Summer 2012

+

S10

2012004

Spleen (D.L)

Summer 2012

+

S11

6010004

Spleen (D.L)

Summer 2012

S12

6010004

Liver (D.L)

R1a

3340204

ATCC 33539

A

G

C

L

ΒHaemolysis

10± 1

+

11± 1

+

15± 1

+

+

+

18± 1

-

-

-

3± 1

+

+

+

-

-

0

+

+

+

+

-

-

0

+

+

+

+

+

-

5± 1

+

+

+

+

+

-

0

+

+

+

-

+

+

18± 1

+

+

+

+

+

-

+

10± 1

Summer 2012

+

+

+

+

+

+

+

15± 1

Type strain

+

+

+

+

+

+

+

11± 1

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-

487 488 489

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S.A= Sparus aurata ; D.L= Dicentrarchus labrax, G: Gélatinase, C: Caseinase, L Lecithinase a: Photobacterium damselae subsp. damselae ATCC 33539

486

Haemolytic Diameter (mm)

+

SC

Dnase

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Strain

490 491 492

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Table 2. Extracellular products of the different Photobacterium damselae subsp. damselae isolates.

495

Enzymes

α-fucisidase

S3 +

S4 +

S5 +

S6 +

S7 +

S8 +

S9 +

S10 +

S11 +

S12 +

R1a +

+ + -

+ + -

+ + -

+ + (+) -

(-) + -

+ + (+) -

+ + -

(-) + -

+ + -

+ + -

+ + -

+ + -

+ + -

+ -

+ -

+ -

+ -

(+) + (+)

+ -

+ -

(+) + (+)

+ -

+ -

+ -

+ -

+ -

(+)

(+)

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

EP

SC

. +: positif character, - : negative character, ( ): different character.

497 498 499 500

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a: Photobacterium damselae subsp. damselae ATCC 33539.

496

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S2 +

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α -chymotrypsin Acid phosphatase Naphtol-AS-BIphosphohydrolase α-galactosidase β-galactosidase β-glucuronidase α-glucosidase β-glucosidase N-acetyl-β glucosaminidase α-mannosidase

S1 +

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control Alcaline phosphatase Esterase (C4) Esterase Lipase (C8) Lipase (C14) Leucine arylamidase Valine arylamidase Cystine arylamidase Trypsin

Strains

501 502 503 504 505 506 20

ACCEPTED MANUSCRIPT 507 508 509

Table 3. Results tests for biofilm oxidative activity and virulence of different strains isolated.

Strains

S9

S10

S11

S12

R 1a

N

L

H

H

H

0.048 ±0.08

0.35 ±0.04

1.43 ±0.06

1.62 ±0.051

2.17 ±0.13

0.31 ±0.07

1.23 ±0.04

1.548 ±0.05

2.03 ±0.3

0

11.42 ±0.04

13.98 ±0.04

4.93 ±0.03

6.45 ±0.4

0.05 ±0.04

0.048 ±0.04

0.33 ±0.04

1.25 ±0.04

1.419 ±0.05

2.09 ±0.087

1.33 ±0.4

0

0

5.71 ±0.04

12.58 ±0.06

12.4 ±0.05

3.68 ±0.04

>108

>108

>108

5.2 105

4 106

2.9 103

1.78 102

S2

S3

S4

S5

S6

S7

S8

Biofilm formation

L

H

H

H

L

L

L

N

OD492± SD Without mucus

0.375 ±0.3

1.352 ±0.051

1.153 ±0.03

1.638 ±0.12

0.776 ±0.051

0.48 ±0.04

0.375 ±0.03

0.05 ±0.09

0.36 ±0.06

1.32 ±0.03

1.15 ±0.09

0.548 ±0.02

0.22 ±0.04

0.39 ±0.12

0.35 ±0.04

4 ±0.06

2.36 ±0.043

0.2 ±0.07

66,54 ±0.04

71.64 ±0.04

18.75 ±0.04

6.66 ±0.04

0

OD492± SD With D.L mucus

0.355 ±0.06

1.24 ±0.04

1.12 ±0.04

0.419 ±0.04

0.274 ±0.05

0.34 ±0.04

0.37 ±0.2

Biofilm reduction percentage (%)

5.33 ±0.06

8 ±0.04

2.86 ±0.05

74.42 ±0.04

64.69 ±0.12

29.16 ±0.04

D.L DL50 cfu fish-1

3 107

5 104

2.3 104

3.3 103

>108

>108

SC

S1

0.05 ±0.04

0.048 ±0.14

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OD492± SD With S.A mucus Biofilm reduction percentage (%)

RI PT

Enzymes

EP

S.A= Sparus aurata; D.L= Dicentrarchus labrax; H: highly positive (OD492 ≥1), L: low grade positive (0.1 ≤OD492<1), N: negative (OD492< 0.1), SD: standard deviation.

510 511 512 513

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a: Photobacterium damselae subsp. damselae ATCC 33539

21

ACCEPTED MANUSCRIPT •

During 2012 we had several deaths in the sea bass fish farm,

•

We isolated a few strains that have proven are Photobacterium damselae subsp. damselae.

•

We tried to do a complete characterization of these strains.

•

This is the first case sea bass mortality repotted to Photobacterium damselae subsp.

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damselae.