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Latin american journal of aquatic research

On-line version ISSN 0718-560X

Lat. Am. J. Aquat. Res. vol.43 no.4 Valparaíso Sept. 2015

http://dx.doi.org/10.3856/vol43-issue4-fulltext-15 

Research Article

 

Probiotic modulation of the gut bacterial community of juvenile Litopenaeus vannamei challenged with Vibrio parahaemolyticus CAIM 170

Modulación por probióticos de la comunidad bacteriana intestinal de juveniles de Litopenaeus vannamei infectados con Vibrio parahaemolyticus CAIM 170

 

Irasema E. Luis-Villaseñor1,2, Domenico Voltolina3, Bruno Gomez-Gil4, Felipe Ascencio2 Ángel I. Campa-Córdova2, Juan M. Audelo-Naranjo1 & Olga O. Zamudio-Armenta1

1Facultad de Ciencias del Mar, Universidad Autónoma de Sinaloa (UAS) Mazatlán, Sinaloa, CP 82000, México
2Centro de Investigaciones Biológicas del Noroeste (CIBNOR), La Paz, B.C.S., CP 23096, México
3
Centro de Investigaciones Biológicas del Noroeste, Laboratorio UAS-CIBNOR Mazatlán, Sinaloa, CP 82000, México
4
Centro de Investigación en Alimentación y Desarrollo (CIAD), Mazatlán Unit for Aquaculture
Mazatlán, Sinaloa, CP 82000, México

Corresponding author: Ángel I. Campa-Córdova (angcamp04@cibnor.mx)
Corresponding editor: Sandra Bravo


ABSTRACT. The protective effects of two probiotic mixtures was studied using the fingerprints of the bacterial community of Litopenaeus vannamei juveniles exposed to probiotics and challenged with Vibrio parahaemolyticus CAIM 170. Fingerprints were constructed using 16S rRNA gene and the PCR-SSCP (Single strand conformation polymorphism) technique, and the probiotics used were an experimental Bacillus mixture (Bacillus tequilensis YC5-2 + B. endophyticus C2-2 and YC3-B) and the commercial probiotic Alibio. The DNA for PCR-SSCP analyses was extracted directly from the guts of shrimps treated for 20 days with the probiotics and injected with 2.5*105 CFU g-1 of V. parahaemolyticus one week after suspension of the probiotic treatment. Untreated shrimps served as positive (injected with V. parahaemolyticus) and negative (not injected) controls Analysis of the bacterial community carried out after inoculation and 12 and 48 h later confirmed that V. parahaemolyticus was present in shrimps of the positive control , but not in the negative control or treated with the probiotic mixtures. A significant difference in the diversity of the bacterial community was observed between times after infection. The band patterns in 0-12 h were clustered into a different group from that determined after 48 h, and suggested that during bacterial infection the guts of whiteleg shrimp were dominated by gamma proteobacteria represented by Vibrio sp. and Photobacterium sp. Our results indicate that the experimental and the commercial mixtures are suitable to modulate the bacterial community of L. vannamei and could be used as a probiotic to control vibriosis in juvenile shrimp.

Keywords: Litopenaeus vannamei, Bacillus mix, Vibrio parahaemolyticus, bacterial community, aquaculture.


RESUMEN. Se estudiaron los perfiles de bandeo de la comunidad bacteriana de juveniles de Litopenaeus vannamei tratados con dos probióticos y expuesto a la bacteria patógena Vibrio parahaemolyticus CAIM 170. Los perfiles de bandeo se construyeron usando el gen 16S rRNA y la técnica PCR-SSCP (Polimorfismo conformacional de cadena sencilla) y los probióticos fueron una mezcla experimental de Bacillus (Bacillus tequilensis YC5-2 y B. endophyticus C2-2 y C3-B) y el probiótico comercial Alibio. El ADN para el análisis PCR-SSCP se obtuvo de los intestinos de camarones tratados durante 20 días con los probióticos, inyectados con 2,5*105 UFC g-1 de V. parahaemolyticus una semana después de la suspensión del tratamiento con probióticos. Camarones no tratados con probióticos sirvieron como control positivo (inyectados con V. parahaemolyticus) y negativo (no inyectados). El análisis de la comunidad bacteriana durante el reto confirmó la presencia del patógeno inyectado en el control positivo y su ausencia en el negativo y en los organismos tratados con probióticos. Durante las 48 h del período experimental se observó una diferencia significativa en la diversidad de la comunidad bacteriana.

Los patrones de bandas se agruparon en un grupo a las 0-12 h y en uno diferente después de 48 h y sugirieron que los intestinos de camarón blanco fueron dominados por gamma proteobacteria representados por Vibrio sp. y Photobacterium sp. durante la infección bacteriana. Estos resultados indican que las dos mezclas pueden modular la comunidad bacteriana y pueden ser usadas como probióticos para controlar la vibriosis en camarones juveniles.

Palabras clave: Litopenaeus vannamei, mezcla de Bacillus, Vibrio parahaemolyticus, comunidad bacteriana, acuicultura.


 

INTRODUCTION

The bacterial genus Vibrio is common and widely distributed in the natural marine environment and in the microbiota of farmed shrimp ponds (Gopal et al., 2005), where some of its species may become opportunistic pathogens and sources of major diseases when the natural defense mechanisms of cultured shrimp are suppressed (Lightner, 2005).

Under the common name of vibriosis, these diseases may cause considerable economic losses, and are considered among the most serious limiting factors for the success of marine aquaculture (Lightner, 2005; Chatterjee & Haldar, 2012). Among the etiological agents, Vibrio harveyi, V. vulnificus, V. parahaemolyticus, V. campbelli, V. alginolyticus and V. penaeicida have been associated with cultured shrimp diseases (Ishimaru et al., 1995; Sahul-Hameed et al., 1996; Jayasree et al., 2006), and among these V. harveyi and V. alginolyticus are thought to be the most common causes of disease during larval and postlarval development (Manefield et al., 2000; Abraham & Palaniappan, 2004).

The addition of probiotic bacteria to culture systems has gained attention as a precautionary measure against pathogens. This addition aims to reduce or eliminate selected pathogenic species and to improve growth and survival of the cultured species through the modulation of the microbial communities of the culture environment (Balcázar et al., 2006; Martínez-Cruz et al., 2012), because bacteria may affect growth and survival of aquatic organisms and are a major element in their well being, since they play distinct roles in the host organism, which are associated with nutrition, immune responses and disease resistance (Austin, 2006; Chaiyapechara et al., 2011; Tuyub-Tzuc et al., 2014).

Culture-independent techniques for population fingerprinting, such as denaturing gradient gel electrophoresis (DGGE) and single strand conformation polymorphism (SSCP), are effective tools for a more complete and rapid assessment of microbial diversity, especially of complex ecosystems such as intestinal microbiota (Muyzer & Smalla, 1998; Dohrmann & Tebbe, 2004; Hassan, 2012).

Previous experiments showed that a Bacillus mixture which improved survival and development of Litopenaeus vannamei larvae caused also an increase in diversity and evenness of the bacterial community of the larval gut, thus increasing resistance to V. parahaemolyticus infection (Luis-Villaseñor et al., 2011, 2013). However, there is no information on the effect of this or other probiotics on the structure of the bacterial community of the intestinal tract of juvenile or adult shrimp challenged with pathogenic bacteria. This study aimed to evaluate the changes induced by the same Bacillus mixture on the gut bacterial community of juvenile Litopenaeus vannamei (Pacific whiteleg shrimp) infected with V. parahaemolyticus.

MATERIALS AND METHODS

Probiotic strains

Cultures of the bacteria Bacillus tequilensis YC5-2, B. endophyticus C2-2 and B. endophyticus YC3-B were grown at 37°C for 24 h in 200 mL Erlenmeyer flasks with 100-mL of TSB medium, and concentrated by centrifugation at 5000×g for 10 min. Each pellet was suspended in a sterile saline solution containing 3% (w/v) NaCl (S-7653, Sigma, St. Louis, MO). The absorbance was adjusted to an optical density of 1 at 600 nm (approximately 1×109 CFU mL-1), and the resulting suspensions were added to the shrimp rearing system at a final concentration of 1×105 CFU mL-1.

Pathogenic bacterium

Strain Vibrio parahaemolyticus CAIM 170, obtained from the Colección de Microorganismos de Importancia Acuicola (CIAD, Mazatlan, Mexico, www.ciad.mx/caim), grown in trypticase soy broth (TS#236950, Difco, Franklin Lakes, NJ) with 3% (w/v) NaCl, was centrifuged at 5000×g for 10 min; the pellet was suspended in 3% (w/v) sterile saline solution. The bacterial suspension was diluted with filtered sterile seawater to an optical density of 1.0 (approximate concentration: 1×109 CFU mL-1), and a 1:10 dilution of this suspension was used for the challenge experiment.

Probiotic treatment and infection

Juvenile shrimps (mean live weight 8 ±1 g) were obtained from a commercial hatchery and acclimated for five days to laboratory conditions, which did not change throughout the experiment (5-μm filtered seawater, 29°C and salinity 36) in a common tank. After acclimation, 16 groups of 21 shrimps were placed in 80-L aquaria. Five aquaria (treatment A) were added daily 1×105 CFU mL-1 of the Bacillus mixture. A second group of five aquaria (treatment B) received the dose used by local shrimp farmers (1 mL L-1, with 1×106 CFU mL-1) of a commercial probiotic mixture (Alibio2135 + AlibioAC + Alibio Bionutre) activated as recommended by the manufacturer (AliBio S.A. de C.V., Mexico City). The remaining six aquaria served as triplicate positive and negative (unchallenged) controls (treatments C and D, respectively).

Addition of probiotics was suspended after 20 days, and seven days later all shrimps of treatments A, B and C were injected into the fifth abdominal section with 20 μL of Vibrio suspension (= 1×108 CFU mL-1), giving 2×106 CFU/shrimp. Shrimps of treatment D were injected with a sterile saline solution.

Throughout the experiment all treatments were fed ad libitum a 35% protein commercial diet. Continuous aeration was maintained in all aquaria, which were maintained with 50% daily water exchanges.

Sample collection and DNA extraction

One shrimp was randomly selected from each container immediately after Vibrio injection (time 0) (5 shrimps for each probiotic treatment and 3 shrimps for positive and negative controls), and sampling was repeated after 12 and 48 h, in the first case because this time coincided with the first case of mortality, while the last was observed 12 h later. Consequently, samples of live shrimp were taken at times 12 h (onset of mortality) and 48 h, giving the surviving shrimp 24 to recover after the last observed death. Immediately after sampling, the body surface of each shrimp was washed with sterile seawater, disinfected with 70% ethanol, dissected with sterile instruments and the entire intestinal tract was removed, excised with sterile forceps and scissors, and preserved at -80°C in individual screw-capped tubes with 1 mL absolute ethanol.

At the end of the experiment, the chromosomal DNA was extracted to assay for the diversity of the intestinal communities, using Wizard genomic DNA purification kits (Promega, Madison, WI) according to the manufacturer's instructions.

Amplification of 16S rRNA

The universal bacterial primers Com1 and Com2ph were used to amplify a 407 bp fragment corresponding to positions 519 to 926 (E. coli positions; including variable regions 4 and 5 of the 16S gene). The Com1 sequence was 5'-CAGCAGCCGCGGTAATAC and Com2ph was 3'-CCGTCAATTCCTTTGAGTTT (Schwieger & Tebbe, 1998). Each PCR was performed in a total volume of 50 μL in 0.2 mL micro tubes. The reaction mixtures were contained in 1×PCR buffer with 1.5 mM MgCl2, 0.5 μΜ of each primer, 200 μΜ of each dNTP, and 2.5 U Taq polymerase (GoTaq, Promega). The total amount of genomic DNA added to the PCR mixtures was 250 ng. Thermocycling (Peltier Thermal Cycler, Bio-Rad Laboratories, Hercules, CA) started with an initial denaturation for 3 min at 94°C, followed by 30 cycles of 60 s at 94°C, one cycle for of 60 s at 53°C and one of 90 s at 72°C, ending with a final extension for 5 min at 72°C. The presence of specific PCR products was confirmed on 1% (w/v) agarose gel.

Single-strand conformation polymorphism

The single-strand removal method (Schwieger & Tebbe, 1998) was used for profiling bacterial communities. All PCR products of each replicate were purified (PCR purification kit, Qiagen, Hilden, Germany) and diluted in Tris-HCl buffer to a final volume of 20 μL. Samples were digested for 45 min at 37°C with 1 μL (5 U) of lambda-exonuclease solution (New England Biolabs, Ipswich, MA), with 3 μL exonuclease buffer and 6 μL milli-Q H2O, for a total volume of 30 μL. Digestion was stopped with the first step of purification with spin columns (MiniElute Kit, Qiagen), and diluted in 10 μL Tris-HCl buffer.

A 9 μL denaturing loading buffer containing 95% formamide (v/v), 10 mM NaOH (w/v), 0.25% bromophenol blue (w/v), and 0.25% xylene cyanole (w/v) was added before electrophoretic analysis. Samples were incubated at 95°C for 2 min and immediately cooled on ice. After 3 min, samples were loaded onto polyacrylamide gels of 0.625% MDE (Cambrex, East Rutherford, NJ), and electrophoresis at 260 V at 20°C was carried out for 18 h (DCode Universal Mutation System, Bio-Rad Laboratories, Hercules, CA). After electrophoresis was completed, the gel was stained with AgNO3 (Benbouza et al., 2006) and scanned using Power Look III (Umax Systems, Willich, Germany).

Analysis of SSCP profiles

Gel analysis software (Gel Compar II, Applied Maths, Sint-Martens-Latem, Belgium) was used to calculate similarities between profiles of bacteria obtained from the different treatments and times of inoculation, after image normalization with bacteria markers (B. licheniformis, Rhizobium trifolii, Flavobacterium johnsoniae, and R. radiobacter). The calculation of the similarity matrix was based on Pearson's correlation coefficients. The clustering method was the unweighted pair group method with arithmetic averages (UPGMA).

Elution of bands and DNA sequencing

Bands of interest were cut from the silver-stained polyacrylamide SSCP gel with a sterile scalpel. The single-stranded DNA was eluted from the gel by the crush and soak procedure (Sambrook & Russell, 2001), resuspended in 12 μL Tris buffer (10 mMTris-HCl, pH 8.0), and amplified via PCR using primers Com1 and Com2ph under the conditions previously described. The PCR-amplified products were sequenced by a commercial firm (Genewiz, South Plainfield, NJ).

The sequences were compared with sequences in the GenBank database. The BLAST search of the National Center for Biotechnology Information and the EzTaxon server database (www.eztaxon.org; Chun et al., 2007) were used to determine the closest relationships of the 16s rRNA sequences.

Statistical analysis

To determine the similarity between treatments, the data of the metrics obtained from each sample were exported as a binary matrix (PAST software, palaeo-electronica.org). A PCA was performed from the correlation matrices generated from a binary matrix, which was expressed as a value of Pearson's similarity coefficient (Fromin et al., 2002). A PCA analysis was conducted with software Statistica 6.0 (StatSoft, Tulsa, OK).

RESULTS

Modulation of intestinal microbiota

The dendograms showed a clear modulation of the intestinal microbiota from 12 h post-infection (onset of death) to 48 h (organisms recovered), divided into two clusters. One cluster (48 h) had a similarity value of 40.81% with respect to the second cluster, including times 0 and 12 h, with a percentage of similarity between 49.64%. Samples Start 1 and Start 2 (samples taken before probiotic treatment) were within the same cluster as time 48 h with 54% similarity, indicating a recovery of the initial microbiota similar to the bioassay (Fig. 1).

 

Figure 1. Acrylamide gel-generated via SSCP dendrogram illustrating the relationship
(percent similarity) between bacterial communities in gut of shrimp at time 0 h, 12 h and
48 h: M1-M5 (Bacillus mix), A1-A5 (commercial probiotic), C1(-)-C3(-): (without probiotics
and injected with saline solution) C1(+)-C3(+): (without probiotics and injected with
pathogenic bacteria). Start 1 and 2: initial profiles, before probiotic treatment. The 40-100
scale of the dendrogram shows percent of similarity of the clusters. The dendrogram was
calculated with UPGMA and the Dice coefficient.

 

Bacterial community of juvenile shrimp infected by Vibrio parahaemolyticus

The results from the SSCP fingerprint showed that the taxonomic group Flavobacteria was dominant: α proteobacteria (mainly Rugeria lacuscaerulensis), γ-proteobacteria, fusobacteria and Cytophagaceae, represented by Wandonia haliotis Haldis.

V. parahaemolyticus and Vibrio sp. were present only in the positive control, Cytophaga fermentans was present only in organisms treated with Alibio while Photobacterium damselae subs. piscicida was present in all treatments but not in the positive control. The individual bands present in the Bacillus mix were identified as Candidatus bacilloplasma mollicute and Nautella italica (Table 1). Unidentified bands (Uncultured bacteria) were also present in the treatment of the Bacillus mix.

 

Table 1. Closest relative, as determined by Blast search, with similarity
(SIM, in %) to the major OTUs from the 16S rRNA V4 and V5 SSCP gels.

 

Maribius salinus and Donghicola eburneus (α-proteobacteria) were detected only in the Bacillus mix and the positive control groups (Table 1). Bacteria species unique to the negative control were Thioprofundum lithotrophica, Sebaldella termitidis, Elizabethkingia anophelis, Oceanicola sp., and Thioclava pacifica. Thalassobium sp. was detected in both control groups.

PCA Analysis

Principal Component Analysis (PCA) showed that two of the components explained 91% of the total variance in the data (CP1 and CP2: 64.8 and 26.2%, respectively) (Fig. 2), and that their factor loadings were considered significant at values greater than 0.70. No significant differences were observed between the banding profiles at time 0 h and 12 h, but their trends are separated clearly from that determined after 48 h. These results coincide with the indications of the similarity dendrogram.

 

Figure 2. Principal components analysis using the Dice coefficient of single strand
conformation SSCP profiles associated with the intestines of individual L. vannamei
inoculated with the treatments and challenged with V. parahaemolyticus for each
time: Time start (), 0 h (Δ), 12 h (), 48 h (×). Each point represents a SSCP profile
from one shrimp.

 

DISCUSSION

Manipulation of microbiota with probiotics may be a convenient practice to control or inhibit pathogenic bacteria in aquaculture, as well as to improve growth performance and digestive enzymes activities, and enhance immune responses against pathogens or physical stress (Balcázar et al., 2006; Pérez et al., 2010; Zokaeifar et al., 2012).

The Bacillus mix used in this work showed several effects which may be useful for L. vannamei culture: after a V. parahaemolyticus challenge which caused >90% mortality in the control group, juvenile shrimps treated with this mix had significant higher survival, different total hemocyte concentrations and a higher diversity and evenness of their bacterial gut community than those treated with the commercial product Alibio, and demonstrated efficient probiotic protection (Luis-Villaseñor et al., 2013). However, the underlying mechanisms for this protection remained unclear.

In this work we showed how the effect of V. parahaemolyticus CAIM 170 on the bacterial community of juvenile shrimp may be at least partially avoided in shrimps treated with the Bacillus mix even after one week after suspension of the treatment. Although the SSCP distribution profiles displayed little variation of the number of bands (OTU) at each sampling period, the greatest variation observed was their increase 12 h after infection in all treatments with Vibrio.

The PCA of 0 h and 12 h indicated no evidence of clustering of individual probiotics groups and no statistically significant deviation from the baseline SSCP profile. However, the PCA conducted on the 48 h gut samples showed the probiotic groups clustered separately from those at the beginning and those at 12 h post-infection. This indicated that the gut microbial population ecology of the animals at 48 h was significantly different from that at 0 h and 12 h after infection, and the separation more evident was that between probiotic-treated and V. parahaemolyticus-infected shrimps.

Recent studies on the modulastion and stabilization of gut microbiota by probiotic treatment suggest that probiotics can exert a positive effect on uncultivable gut microbiota (Sáenz de Rodrigáñez et al., 2009; Sun et al., 2012a, 2012b; Yang et al., 2012), which coincides with the modification of the gut microflora and the increase in bacterial diversity after probiotic administration reported in Solea senegalensis by Tapia-Paniagua et al. (2010). Several studies indicated that the culturable intestinal microbial community of shrimp was mainly composed of Aeromonas, Plesiomonas, Photobacterium, Pseudoalteromonas, Pseudomonas and Vibrio species (Moss et al., 2000; Oxley et al., 2002). Of these, only Photobacterium and Pseudoalteromonas were detected in this work whereas Vibrio species were detected only in the positive control (V. parahaemolyticus, confirming the induced infection, and Vibrio sp.), and in shrimps treated with Alibio (Vibrio sp.), possibly because several Vibrio or Vibrio-related species are common in commercial probiotic mixtures (Verschuere et al., 2000; Qi et al., 2009).

In our case, the indigenous intestinal microbiota tended to be dominated by Wandonia haliotis Haldis, which may be considered a commensal, because it was present at all times and in all treatments. The presence of V. mediterranei in the negative control may be due to its occurrence in natural microbiota, because this species is commonly associated to a wide-range of hosts, with mutual interactions which may range from mutualism or symbiosis to a pathogenic relation (Turner et al., 2009; Senderovich et al., 2010).

The fact that shrimps treated with the Bacillus mix did not show the presence of the pathogen injected may be taken as an indication of a protective effect of this probiotic, similar to the effect against V. parahaemolyticus of the indigenous intestinal microbiota modified with a Bacillus-based probiotic observed by Wu et al. (2014) in the mud crab S. paramamosain. Modifications of the intestinal microflora by a probiotic Bacillus resulting in inhibition of growth of intestinal Vibrio spp. have been reported also in Penaeus monodon by Rengpipat et al. (2000) and by Vaseeharan & Ramasamy (2003), who also noted a positive effect in the external water environment.

Photobacterium damselae subsp. piscicida was observed in all our treatments. This microorganism (formerly Pasteurella piscicida) is a highly pathogenic bacterium that causes photobacteriosis and does not show host specificity (Toranzo et al., 1991; Noya et al., 1995) but, in spite of its generalized presence it did not show any pathogenic effect, possibly because the presence of the probiotic Bacillus strains, since these are known to modulate shrimp gut bacterial communities (Luis-Villaseñor et al., 2013), thereby improving their immune response against pathogenic bacteria (Zokaeifar et al., 2012, 2014).

The effect of probiotic protection on the structure of the intestinal bacterial community of shrimp infected with pathogenic bacteria was unknown, and this work shows that both probiotic mixtures, Alibio and Bacillus mix, helped to maintain a natural balance in the bacterial community of the shrimps intestine, modulating and increasing diversity and evenness of bacterial species in shrimps challenged by bacterial infection.

ACKNOWLEDGMENTS

Supported by SEP-CONACyT Grant 25981. Hector Acosta of CIBNOR provided technical assistance during the preparation of the manuscript.

 

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Received: 5 February 2015;
Accepted: 4 August 2015

 

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