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Brazilian Journal of Microbiology

Print version ISSN 1517-8382On-line version ISSN 1678-4405

Braz. J. Microbiol. vol.41 no.1 São Paulo Jan./Mar. 2010

http://dx.doi.org/10.1590/S1517-83822010000100017 

FOOD MICROBIOLOGY

 

Use of Pseudomonas stutzeri and Candida utilis in the improvement of the conditions of Artemia culture and protection against pathogens

 

 

Mahdhi Abdelkarim*; Chaieb Kamel; Kammoun Fathi; Bakhrouf Amina

Laboratoire d'Analyse, Traitement et Valorisation des Polluants de l'Environnement et des Produits. Faculté de Pharmacie, 5000 Monastir. Tunisie

 

 


ABSTRACT

To evaluate the effect of two bacterial strains isolated from Artemia cysts and yeast (Candida utilis) on the survival, growth and total biomass production of its larvae, challenge tests were performed with Candida utilis, Pseudomonas stutzeri and Pasteurella haemolityca. In addition, a pathogenic strain of Vibrio alginolyticus was tested for comparative purposes.
Pseudomonas stutzeri and Candida utilis have no impact on survival, but enhance growth and total biomass production of the larvae. However, we noted that Pasteurella haemolityca affect negatively Artemia larvae. The adhesion and antagonism assay demonstrates that Candida utilis and Pseudomonas stutzeri are fairly adherent and play an important role in the enhancement of the protection of Artemia culture against pathogens. On the basis of these results, it's suggested that it's possible to use Candida utilis and Pseudomonas stutzeri, potential candidates, as probiotic for the culture of Artemia larvae.

Key words: Artemia, Probiotics, Adhesion, Yeast, Bacteria.


 

 

INTRODUCTION

One of the most significant advances in aquaculture farms and industrial hatcheries has been the use of Artemia as a food for marine finfish and crustacean around the world because of their nutritional profiles (15). It is presumed that Artemia nauplii have been used as a vector for the delivery of different nutrients (24), antibiotics (35) and probiotics (9, 24). However, these feeds can be a source of pathogenic bacteria in aquaculture farms (34) like the genus of Vibrio and Pasteurella, such as Pasteurella haemolityca which is one of the most common bacterial pathogen involved in veterinary diseases such as pneumonic pasteurellosis in sheep all over the world (30). It is responsible of much mortality in hatchery environments, in animal farming industry and it considered as an aetiological agent of fish pasteurellosis (13, 28, 25). For these reasons, it is necessary to study the bacterial interaction with crustaceans in order to distinguish between pathogens and potential probiotic candidate, because it is found that bacteria had a beneficial effect such as providing nutritional elements like vitamins, essential amino acid, fatty acids and enzymes (10, 36). Other microbiological studies have demonstrated that Bacillus sp. and Aeromonas hydrophila can protect Artemia against pathogens such us Vibrio proteolyticus and Vibrio campbelli (19).

Some yeast strains were used in the hatchery farms. Indeed, a high percentage of survival (100 % after 96 hour and more than 80 % after 144 hour) was obtained when autoclaved baker's yeast was used as food (23). Saccharomyces baulardii has been also utilized and it's found that this yeast can enhance protection of enrichment Artemia nauplii against Vibrio sp. (24). Recently, Marques et al. (19) used Saccharomyces cerevisea and demonstrated that this yeast can provide protection against different pathogens. This promising source of protein can also supply the feed with vitamins, mineral and other components, which could stimulate the resistance of aquatics animals (26). This study aimed to investigate the effects of Candida utilis and Pseudomonas stutzeri on the Artemia culture and to evaluate their role in the enhancement of Artemia nauplii resistance to Pasteurella haemolityca and Vibrio alginolyticus.

 

MATERIALS AND METHODS

Isolation and identification of bacterial strains

Bacterial strains associated with Artemia cysts were isolated according the following procedure: 0.01 g of Artemia cysts recovered from Sebkhat El Adhibet in the South East of Tunisia (33 º44 N - 10 º46 E) were incubated in 60 ml sterile falcons containing 30 ml of filtered and autoclaved sea water (FASW) (salinity 34 g l-1 and pH 7.99) at Shaker incubator (28 ºC, 120 rpm) and exposed to constant incandescent light of Shaker (Lab-Line Orbit Environ-Shaker). Under these conditions, hatched larvae appeared after 18 to 20 hours of incubation. Water samples (1 ml) were enriched 24 h at 30º C in nutrient broth sea water (NBSW) (salinity 34 g l-1 and pH 7.99) and speared on nutrient agar plate and incubated at 30 ºC. The most appeared colonies were reisolated on Petri plates with nutrient agar. Gram negative rods were identified using Rapid NF Plus strips (Remel. USA) according the manufacture's recommendation.

Isolation and identification of yeast strain (Candida utilis)

Pure culture of Candida utilis used in this study was isolated on Sabuoraud agar plate from the American biomass known as "Kambautcha" in our Laboratory. Biochemical characterization of Candida utilis was carried out by Api 20 C Aux (Bio Merieux, France) test.

Well diffusion agar assay (WDAA)

Potential probiotic strains were tested for their antagonistic activity using the well diffusion agar assay (WDAA) (34) against three target strains: Vibrio parahemolitycus ATCC17802, Vibrio alginolyticus ATCC17749, and Vibrio alginolyticus (S1) isolated from infected fish previously described by Ben Kahla-Nakbi et al.(2). The pathogenic bacteria were grown in 10 ml of nutrient broth and cultured for 24 hours on nutrient agar at 30ºC. The common colonies from pure culture were suspended in 10 ml of physiological medium and well mixed during 5 min. One ml was spread over the agar plates. Potential probiotic strains were cultured in 10 ml nutrient broth for 24 hours, 100 µl of the supernatant were introduced into the wells of the MH agar medium and incubated for a period of 24 h at 30 ºC. Antibacterial activity was defined as the diameter (mm) of the clear inhibitory zone formed around the well.

Adhesion assay

Adhesion ability of Pseudomonas stutzeri and Candida utilis grown in Trypticase Soy broth (TSB, Bio-Rad, France) was determined using a semi-quantitative adherence assay on 96-well tissue culture plates (Nunc, Roskilde, Denmark), as described previously (6, 18) with some modifications. Following overnight incubation at 37 ºC, the optical density at 620 nm (OD620) of these strains was measured. An overnight culture grown in TSB at 37 ºC was diluted to 1:100 in TSB with 2% (w/v) glucose. A total of 200 fl of these cell suspensions was transferredin a U-bottomed 96-well microtiter plate (Nunc, Roskilde, Denmark). Each strain was tested in triplicate. The plates were incubated aerobically at 37 ºC for 24 h. The cultures were removed and the microtiter wells were washed twice with phosphate-buffered saline (7 mM Na2HPO4, 3 mM NaH2PO4 and 130 mM NaCl at pH 7.4) to remove non-adherent cells and were dried in an inverted position. Adherent bacteria were fixed with 95% ethanol and stained with 100 fl of 1% crystal violet (Merck, France) for 5 min. The excess stain was rinsed and poured off and the wells were washed three times with 300 fl of sterile distilled water. The water was then cleared and the microplates were air-dried. The optical density of each well was measured at 620 nm (OD620) using an automated Multiskan reader (GIO. DE VITA E C, Rome, Italy). Adhesion ability was interpreted as highly (OD620 2 1), fairly (0.1 : OD620 < 1), or slightly (OD620< 0.1).

Inoculation of yeast and bacterial suspensions

Sterile 60 ml falcons were inoculated with bacterial strains and yeast (106 CFU ml-1) from pure culture in the exponential growth phase. The concentration of each bacterial strain and yeast was estimated through a regression analysis of the optical density of the pure culture. The number of CFU ml-1 was determined using Petri plates with marine agar.

Artemia gnotobiotic culture

Experiments were performed with Artemia cysts, collected from Sebkhat El Adhibet in the South East of Tunisia (33º44 N - 10º46 E). Bacteria-free cysts and nauplii were obtained via decapsulation as described by Sorgeloos et al. (32). Decapsulated cysts were washed with filtered and autoclaved sea water (FASW) over a 50fm sterile filter net. This procedure was repeated nine times, using new filtered and autoclaved sea water (FASW). After this step, washed decapsulated cysts were transferred to a sterile falcon containing 30 ml of FASW. The Falcon was capped and placed on Shaker incubator (28 ºC, 120 rpm) and exposed to constant incandescent light of Shaker. After 18 -20 h, ten axenic nauplii were picked and transferred to sterile falcons containing 30 ml of FASW, together with the amount of feed scheduled for day one. All manipulations were carried out under sterile conditions.

Experimental design

To evaluate the effect of the bacterial strains on Artemia culture, twelve challenge tests were performed. The first test was Artemia alone (Axe), the second was Artemia with Candida utilis (Art+Y). Tests 3, 4 and 5 were made with three bacterial strain: PS : Pseudomonas stutzeri (Art+y+PS ); PH : Pasteurella haemolityca (Art+Y+ PH) ; VA : Vibrio alginolyticus (Art+y+ VA). Test 6, 7 and 8 were carried out using Candida utilis and the same three bacterial strains: (Art+Y+PS ); (Art+Y+ PH) and (Art+Y+ VA). Tests 9 and 10 were made using a mixture of these bacterial strains: (Art+PS+PH); (Art+PS+VA), during tests 11 and 12 we added to the mixture of the bacteria used, the yeast Candida utilus: (Art+Y+PS+PH); (Art+Y+PS+VA). All challenge tests were made in two experiments: Experiment 1, Y and PS were added only at first day and PH and VA were provided at day three. Experiment 2, Y and PS were added during the first three day and pathogenic bacteria strains were provided only at day 3. Vibrio alginolyticus strain (S1) used for comparatives purposes was isolated from infected fish (2). Treatment of Artemia with Candida utilis (Art +Y) was chosen as a control treatment in the two experiments because larvae survival showed a higher percentage after 6 days of culture.

Methods used to verify axenity

At the beginning and end of each run of the procedure, absence of bacteria was monitored by transferring larvae and 100 µl of water to Petri plates in triplicate with marine agar.

Survival, growth and total biomass production of Artemia

During the challenge tests, the number of swimming larvae (survival percentage) was daily determined and the percentage of survival was calculated. At the end of each experiment, alive Artemia were fixed with lugol solution to measure their individual length (IL) under a binocular magnifying glass containing a graduated micrometer. As a criterion that combines both the effects of survival and IL, the total biomass production (TBP) was determined according to the following formula (19): TBP (millimeters per Falcon - mm / F) = number of survivor's × means IL.

 

STATISTICAL ANALYSIS

Values of survival larvae (percentage) were arcsine transformed, while values of IL and TBP were square root transformed to satisfy normal distribution and homoscedasticity requirements and differences on survival, individual length and total length of Artemia under different treatments were investigated with analysis of variances (ANOVA) and with Duncan's test using the Statistica 5.5 software.

 

RESULTS

Identification of bacterial strains

Bacterial strains isolated from Artemia cysts were identified as Pseudomonas stutzeri (PS) and Pasteurella haemolityca (PH).

Adherence and antagonisms assay

Pseudomonas stutzeri and Candida utilis are fairly adherent and showed inhibitory effect against Vibrio parahemolitycus ATCC 17802, Vibrio alginolyticus ATCC 17749, and Vibrio alginolyticus (S1). The diameters of the inhibitory zones around the growth of pathogenic strains tested were about 12-34 mm (Table 1 and 2).

 

 

Larval survival

During the first experiment, larval survival rates resulted in 0% on culture day 5 in treatment Artemia axenic (Art axe) (Fig. 1A). The addition of yeast (Y) on Artemia culture resulted in higher survival rate (76% at the end of culture day 6). Treatment "Artemia with PS" resulted in low survival rate (8% on culture day 5) (Fig. 1A) and showed difference with control treatment (Art + Y) (P < 0.05). When yeast (Y) and bacteria (PS) were used, Artemia culture presented a high survival rate and no significant difference was found in comparison to the control treatment (P > 0.05) (Fig. 1A). In presence or absence of yeast, Artemia nauplii challenged with PH and VA resulted in a low larval survival (0 % on culture day 5). Compared with control treatment, a significant difference was improved (P<0.05) (Fig. 1B). Treatments Artemia wih bacteria (Art+PS+PH and Art+PS+VA) and traetment with yeast and bacteria (Art+Y+PS+PH and Art+Y+PS+VA) resulted in low survival rate (0% on culture day 5) and no additional protection against PH and VA was noted in comparaison to the control treatment (P<0,05) (Fig.1C).

In the second expriment, results showed an improvement of survival rate in treatment Artemia with yeast (Y), with bactria (PS) and Artemia with yeast (Y) and bacteria (PS), but no significant difference was found (P > 0.05) (Fig. 4 A and B). It was demonstrated that when Artemia nauplii were fed during the first three days and challenged with PH and VA, the survival rate was not affected under treatments when yeast (Y) was used only or associated with bacteria (PS). No significant difference in comparaison with control treatment was noted (P > 0.05) (Fig. 4 A, B and C).

Larval development

In experiment 1, under axenic conditions, Artemia nauplii have an individual length ranged between 0.45 and 0.55 mm. In treatment "Artemia with yeast (Art + Y)", larvae reached a length varied between 0.9 - 1.2 mm. When Artemia was treated with Pasteurella haemolityca, in presence or in absence of yeast, the length of dead larvae was between 0.6 - 0.8 mm and a significant difference with control treatment was improved (P < 0.05). The same results were obtained when Artemia nauplii was challenged with VA (Fig. 2 A). The introduction of PS alone in Artemia culture increases the development (from 0.48 to 0.8 mm). When it is used in the presence of yeast, the larvae have a length of 1.4 mm and significant differences were found in comparison with the control treatment (Art + Y) (P>0.05) (Fig. 2 A). Results of interaction showed that the two strains, yeast (Y) and bacteria (PS), used separately or associated, did not confer protection for the larvae against pathogens. However, there was a significant difference in comparison with control treatment (P < 0.05) (Fig. 2 B).

During experiment 2, when bacteria (PS) was first used alone and then associated with yeast (Y), an improvement of the growth was demonstrated compared with treatment when PS was added just during the first day (P < 0.05). A significant difference was found compared to the control treatment (P < 0.05) when Artemia was supplied with PS in the presence of yeast. (Fig. 5). The addition of pathogens strains in culture medium enriched with Yeast (Y) didn't affect the growth of larvae which have a length ranging between 1.2 and 1.4 mm when pathogenic strains PH and VA were added. Also, treatment when bacteria PS was supplied with yeast, pathogens didn't affect the growth of Artemia larvae which have an individual length ranged between 1.4 and 1.5 mm. A significant difference was found compared with control treatment (P < 0.05) (Fig. 5). Compared with the first experiment, results demonstrate an improvement of growth in all challenged tests and a significant difference were found (P <0.05) (Fig. 2 A and B; Fig. 5).

 

 

 

 

Total biomass production

During the first experiment, in treatment Artemia with yeast (Art +Y) and bacteria PS (Art + Y+ PS), a light increase in the production of biomass, which is about 10.78 mm/F, was noted and a no significant difference was shown in comparison with the control treatment (Art + Y) (P > 0.05) (Fig. 3).

During the second experiment, in all challenged tests when PS was used only, Artemia nauplii demonstrate a low TBP, significant difference were found (P < 0.05), but the addition of yeast during these challenged tests resulted in an improvement of TPB were found. No significant difference was noted (P > 0.05) (Fig. 6).

 

 

DISCUSSION

The two identified strains have been isolated from Artemia culture and aquatic environments. Sahul Hameed and Balasubramanian, (29), have isolated Pseudomonas strains from Artemia nauplii. Pasteurella haemolityca was also isolated from egg, fry of Mekong giant catfish, water flea, brine shrimp Artemia, and from fish farms (Dicentrarchus labrax) with a percentage of 50% (25, 28).

The use of Candida utilis, for Artemia culture showed a positif effect. On the basis of these result, this yeast can be used as a source of nutrients for Artemia culture and in the enhancement of protection against pathogens Pasteurella haemolityca and Vibrio alginolyticus. The protection conferred by Candida utilis when it was used for three days can be related to its capacity of adhesion, its ability to inhibit the growth of pathogenic strains and to stimulate the innate immune system. In fact, some components presented in yeast cell wall are likely to boost the non specific immune response in Artemia against pathogenic bacteria such us β-glucans (5, 20) and chitin (1). Siwicki et al. (31) tested several immunostimulants such as lyophilised Candida utilis and S. cerevisiae, ß-glucans, deacylated chitin on rainbow trout and the most significant stimulations were generally observed with the two yeasts. The improvement of Artemia performance can be justified by the better quality of Candida utilis which is used for Tilapia culture as a source of protein and classified among the most interesting microorganisms for their protein content, which can account for up to 50% of the dry weight (22, 16). The apparent higher survival rate and resistance of Artemia in early developmental stages against pathogenic bacteria could be related to a variety of factors such as fewer attaching sites for bacteria which are available in the naupliar gastrointestinal tract and the presence of certain compounds sustaining naupliar resistance just after hatching. Stabili et al. (33) detected lysozyme and proteases in A. franciscana cysts and envelope before hatching. Lysozyme is a well-known antibacterial substance (e.g. direct bacteriolytic action, phagocytosis stimulation) in organisms unable to produce immunoglobulins (37) and proteases are involved in protein digestion and in various aspects of immunology (4).

The positive effect of PS and their potential to inhibit growth of pathogenic strains confirm that PS play an important role for Artemia culture. This role can be due to high doses which can induce its artificial and temporary dominance (8, 11), the ability of some microorganisms to adhere to intestinal tract and their effect in the digestive processes of aquatic animals (19, 27). However, in the interaction between Artemia and bacteria, a number of factors have been proposed such as the provision of additional digestive abilities and the use of bacterial biomass as food (7, 10, 14). Lone et al. (17) found that Pseudomonas fluorescens is able to inhibit Vibrio anguillarum and can be used as a possible probiotic treatment of fish.

Result obtained in the treatment ''Artemia with bacterial strains PH and VA", showed a lower survival and Artemia nauplii did not survive for more than 4 days (Fig. 1 A and C). These results can be explained by the negative effect of PH because of the components of extracellular matrix (ECM) such as fibronectin (Fn), collagen (Cn), laminin, and proteoglycans (3, 21). In addition, the attachment of some pathogenic bacteria like V. alginolyticus and V. parahaemolyticus to the body surface of Artemia, causing a deleterious effect, can explain the negative effect of V. alginolyticus (12).

In conclusion, this study shows that Artemia culture conditions can be controlled using Candida utilis and Pseudomonas stutzeri. Data suggest that protection against pathogenic bacteria depends on the nature of treatment and the quality and quantity of potentials probiotics used. It is hoped that more studies are required in order to clarify the exact contribution of potential candidates probiotics used in this study an improvement of Artemia performance and protection against pathogens using host gene - expression and overcome disease in aquaculture farms.

 

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Submitted: February 03, 2009; Approved: August 23, 2009.

 

 

* Corresponding Author. Mailing address: Laboratoire d'Analyse, de Traitement et de Valorisation des Polluants de l'Environnement et des Produits. Faculté de Pharmacie, 5000. Tunisie.; Tel: +216 97 59 89 16 Fax: +216 73 46 18 30. ; E-mail: abdelkarim_mh@yahoo.fr

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