Control

 

Inhibition of Aeromonas hydrophila by probiotic strains isolated from the digestive tract of Pterophyllum scalare

 

Inhibición de Aeromonas hydrophila por cepas probióticas aisladas del tracto digestivo de Pterophyllum scalare

 

M.C. Monroy-Dosta¹*, T. Castro-Barrera¹, F.J. Fernández-Perrino² and L. Mayorga-Reyes³

 

¹ Laboratorio de Alimento Vivo, Departamento El Hombre y Su Ambiente, Universidad Autónoma Metropolitana-Xochimilco, Calzada del Hueso 1100, Colonia Villa Quietud, C. P. 04960, México, D. F. *Corresponding author. E-mail: monroydosta@hotmail.com Tel. (52) 55-5483-7151 Fax (52) 55-5483-7469.

² Departamento de Biotecnología, Universidad Autónoma Metropolitana-Iztapalapa Av. San Rafael Atlixco 186 Colonia Vicentina. C. P. 09340 México D. F.

³ Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana-Xochimilco, Calzada, del Hueso 1100, Colonia Villa Quietud, C. P. 04960, México, D. F.

 

Received 1 of October 2009;
Accepted 22 of December 2010

 

Abstract

The effect of three different probiotic strains of Bacillus (B1, B2 and B3) isolated from the digestive tract of Pterophyllum scalare against Aeromonas hydrophila was evaluated. All the fish that where fed with the different probiotic strains obtained higher resistance to the pathogen inoculation since they did not developed any signs of illness nor lesions and they stayed healthy until the end of the experiment, with an observed survival of 100 %. On the other hand, the fish where the probiotic bacteria were not administrated developed the infectious process caused by Aeromonas hydrophila with signs of illness and lesions in the gills and hemorrhagic eyes, irregular swim, and injuries on the skin. The survival in these treatments was barely up to 8%.

Keywords: Aeromonas hydrophila, Bacillus sp, Probiotics, Pterophyllum scalare.

 

Resumen

El efecto antagonista de tres diferentes cepas prebióticas de Bacillus (B1,B2 y B3) aisladas del tracto digestivo de Pterophyllum scalare frente a Aeomonas hydrophila fue evaluado. Todos los peces que fueron alimentados con las diferentes cepas prebióticas obtuvieron alta resistencia a la inoculación del patógeno y no mostraron signos de lesiones o enfermedad y permanecieron saludables hasta el final del experimento con una sobrevivencia del 100%. Los peces que no fueron alimentados con prebióticos desarrollaron el proceso infeccioso observándose signos y lesiones de enfermedad como branquias y ojos hemorrágicos, nado irregular y lesiones en la piel. La sobrevivencia en estos tratamientos fue escasamente del 8 %.

Palabras clave: Aeromonas hydrophila, Bacillus sp, probióticos, Pterophyllum scalare.

 

1. Introduction

During the last few years, several papers have been directed to the use of probiotic microorganisms with the objective of reducing the amount of diseases during the culture of ornamental fish and restrict or reduce the use of antibiotics, as these compounds have provoked bacterial resilience, destruction of ecosystems, and high cost of production (Westerdahl et al., 1991; Maeda 1994; Abraham et al. 2001; Nikoskelainen et al., 2003). The probiotics are microorganisms that adhere to the gastrointestinal tract forming a thin biofilm and have benefic effects on the host, including improvements on digestion, immunity, and resistance against diseases as they produce substances like bacteriocins, acetic acid and lactic acid that inhibit the proliferation of pathogenic bacteria (Rengpipat et al., 2000; Irianto and Austin, 2002; Vine et al., 2004; Gullian et al., 2004; Balcázar et al., 2006).

Amongst the most studied probitic strains, we find: the lactic bacteria, bifidobacteria, and yeasts (Abraham et al., 2001; Singh et al., 2001; Jameson, 2003). However, one of the problems on the use of probitics is the method to select them, according to Gaomez and Roque (1998) in most of the cases this process is based only on empiric observations and with limited scientific evidence as the bacteria used on fish culture are isolated from the digestive tract of humans or other mammals. Thus, the use of strains isolated from fishes might be an interesting possibility to obtain better results. The objective of the present work was to evaluate the response of Pterophylum scalare fed with three strains of Bacillus (isolated from fish) against Aeromonas hydrophila, a common bacteria found on infectious processes in aquatic organisms.

 

2. Materials and methods

2.1. Microorganisms

The three strains that were used in this study were previously isolated from the digestive tract of health fish P. scalare in the laboratory. It is noteworthy that the molecular evidence to date indicates that these are three different strains of Bacillus, so to ease the handling during the experiment they were assigned with the nomenclature B1, B2 and B3.

2.2. Isolation and identification of A. hydrophila obtained from the fish's kidney.

The strain of A. hydrophila was isolated from an ornamental fish farm during an infectious process. Samples of kidney and injuries on the skin, gills, fins, and eyes were collected and were placed in Petri dishes with TBCS agar. Colonies were purified by successive re-seeding in BHI agar until a homogeneous cellular morphology was obtained. Gram staining and some confirmative biochemical test were performed (motility, citocrome C, glucose oxide fermentation, NaCl tolerance, catalase and resistance against the vibrostatic agent 0/129).

The molecular identification of A. hydrophila was done by DNA isolation (16s rDNA) with the DNA-EASY kit (Qiagen) by following the manufacturer's instruction. To establish the presence of A. hydrophila in the samples, PCR technique was performed by using the sequence oligo Aer8-5'- TGCTGGCTGTGACGTTACTCGCAG-3' and Aer9-5'-TTCGCCACCGGTATTCCTCCAGATC-3' (Martínez-Murcia et al., 1992). Amplification reactions were done on a thermocycler (Amplitron II Thermolyne Barnstead International) under the following conditions: pre-incubation: 95°C during 10 min; denaturalization: 30 cycles at 95°C for 1 min; aligning: 68^°C for 1 min, extension: 72^°C for 30 seconds and pause of 4^° C. The PCR products were analyzed on 1 % agarose gel with a photodocumentator GelDoc 2000 (Bio-Rad, Hercules, California). A DNA sample of the strain A. hydrophila ATCC356554A was used as positive control.

2.3. Probiotic preparation

A sample of each Bacillus was added to 500 mL of TSA broth, incubating them at 30^°C for 48 hrs or until achieving a 10⁷ CFU/mL concentration. To measure the required bacterial concentration, a JENWAY 6400(g) spectrometer was used using a 620nm wave length. Also an CFU/mL count was done. The relationship between the obtained value by spectrometry and the CFU/mL was done according to Gullian et al. (2004).

2.4. Enrichment Artemia franciscana adults

In a 200 mL beaker previously sterilized, 50 adults of A. franciscana were placed and 10mL of the different probiotic strains were added at a concentration of 10⁷ CFU/mL. Enrichment was performed during 30 min with continuous aeration and after this period, the adults of Artemia were observed on a stereoscopic microscope to ensure that digestive tract was full with the bacteria, and them Artemia was washed with tap water and fed to the fish. Same procedure was followed to enrich the Artemia adults with the rest of the probiotic bacteria and the pathogen Aeromonas hydrophila.

2.5. Treatments

150 healthy juvenile fish (that not presented signs of infection or lesions) of the species Pterophyllum scalare were used. After a period of acclimatization of 30 days the experimental phase began. In 12 aquariums, each with a 40L capacity, 10 juvenile fish were distributed, on each of the aquariums at temperature of 28ºC, pH 7, and 5 mg/L of dissolved oxygen and afterwards the following treatments were carried out:

Treatment 1 (control): Fish fed with Artemia adults (without any probiotics and A. hydrophila) during 30 days.

Treatment 2: Fish fed with Artemia adults inoculated with A. hydrophila.

Treatment 3: Fish fed with the combination of the probiotic bacteria (B1, B2y B3) during 7 days, on the eighth day A. hydrophila was inoculated in the Artemia and then fed to the fish.

For treatments 4, 5, and 6, the fish were fed with Artemia adults enriched with the different probiotic bacteria and A. hydrophila was administrated in the same way that in the previous treatment.

2.6. Characterization of the signs of injuries observed on the fish

24 hrs after the administration of the pathogen, mortality, changes in behavior, and signs of injuries on the skin were observed carefully, with the objective of developing a clinic history.

2.7. Pathogen recovery

To ensure that A. hydrophila was the actual agent that produced the infection and/or the fish's death, samples were taken from the injuries and kidneys of the diseased animals. Samples were placed on a TCBS medium and were isolated afterwards on BHI medium. The presence of A. hydrophila was confirmed on samples using the PCR technique described previously.

2.8. Statistical analysis

To determine which organ of the fish were the most affected by the inoculation of A. hydrophila a discriminate analysis was performed. The following variables were considered: coloration, skin, scales, fins and tail, mouth, gills, eyes, body, swimming, behavior, digestive tube, kidney, liver, swimming bladder, bile vesicle, heart, gonads and the grade of injuries: 0 when no damage or injuries were observed, 1 was associated with minor damage, 2 with major damage and 3 with severe damage.

 

3. Results

3.1 . Signs and lesions presented by the fish

24h after the pathogen administration, the infectious process began in the treatments where no probiotic was administrated. Characteristic signs of lesions and illness that were observed are shown on Table 1. When making the statistical analysis using the Canonical discriminant functions (standardized by within variances), it was considered that the infection signs that developed the most after the pathogen administration were: hemorrhagic gills and eyes, irregular swim and skin lesions.

3.2. Survival

The inoculation of A. hydrophila in the fish that were not fed with the probiotics caused a mortality of more than 90 % of the organisms, while the fish tried with the different Bacillus strains resisted the pathogen administration better with a survival between 88 and 100% (Fig. 1).

3.3. Pathogen recovery

A. hydrophila was found in every sample gathered from the infected lesions. This microorganism was identified by PCR by amplifying a 400-bp fragment that corresponded to an A. hydrophila positive control, thereby confirming the presence of the pathogen and its relationship with the infectious process in the fish not fed the probiotic strains (Figs. 2 and 3).

 

4. Discussion

Different studies have demonstrated the capacity of different microorganisms to improve the fish survival during their culture (Gatesoupe, 1994; Gullian et al., 2004; Venkant et al., 2004; Bagheri et al., 2008). In agreement with the present work the use of the probiotic strains B1, B2, and B3, isolated from the digestive tract of P. scalare, used in an individual way or in a combined way they remarkably improved the survival of this ci-chlid, showing higher resilience to the inoculation of A. hydrophila. The survival rate was of 100% in the treatments B1, B3, and the combination of the three bacteria. These results improved those obtained by Martinez et al (2008) who infected tilapias with different pathogens, after the challenge with the pathogenic bacteria, the highest survival was obtained in those treatments that the supplement Bacillussp. and Lacto Bacillus casei in comparison the treatment that was not given any bacteria (control). Gatesoupe (1994), improved the survival of Scophthalmus maximus larvae when gave them acid lactic bacteria; Lara (1999) carried out an investigation on the effect of three different probiotics fed to Tilapia nilotica (Oreochromis niloticus) subjected to different stress conditions, obtaining the best results in growth and survival with the addition of Saccharomyces cerevisae.

On investigations on the use of probiotics for the pathogens exclusion in aquaculture Aly et al., (2008), mention the exclusion of Aeromonas hydrophila with Bacillus subtilis and Lacto Bacillus acidophillus in Tilapia nilotica. However concerning to the use of specific probiotic strains for ornamental fish few studies have been made. This investigation reports the first advances on the use of bacteria isolated from the digestive tract of P. scalare with probiotic capacities; ornamental fish of great commercial importance that has been affected by infectious processes in those that frequently has been isolated bacterias like A. hydrophila (Cipriano et al., 1984; Dixon and Issvoran, 1993, Baez et al., 2008). The results obtained in this work are very encouraging because they demonstrate the live antagonistic effect of the three strains of Bacillus sp. setting them as specific probiotics for P. scalare due to the fact that they do not allow the development of the infection process in the treatments where this bacteria was administrated, also, after concluding the experiment their presence was verified inside the digestive tract of the fish which confirms their capacity to adhere to the digestive tract and their antagonistic potential. Therefore it is recommended the use of this strains that where isolated to battle against illnesses like hemorrhagic septicemia caused by A. hydrophila which occupies a highlighted place due to the economical losses of those that produce species that are specially important such as salmons, flounders, basses or sea basses, amongst some other and a great number of ornamental species (Sumawidjaja et al., 2001; Rodríguez, 2002; Harikrishnan and Balasundaram, 2005).

 

Acknowledgements

This work is part of the doctoral thesis of first author, inscribed in the Ph.D. program in Biological Sciences from the Universidad Autónoma Metropolitana and funded by the National Science and Technology of México (CONACyT) Record: 200491.

 

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