Effect of the addition of Lactobacillus delbrueckii subsp. delbrueckii on the gut microbiota composition and contribution to the well-being of European sea bass (Dicentrarchus labrax, L.)

Abstract

The present study aimed to test the effects of probiotic treatment on gut microbiota and the contribution to the well-being of European sea bass (Dicentrarchus labrax, L.). A bacterial strain of Lactobacillus delbrueckii subsp. delbrueckii (AS13B), isolated from adult European sea bass gut, was administered during sea bass development using Brachionus plicatilis and/or Artemia salina as carriers. The effective strain colonization and modulation of the gut microbiota, the mortality and the cortisol levels of sea bass were monitored during early developmental stages. The L. delbrueckii subsp. delbrueckii strain showed good capability to colonize the gut of sea bass larvae, therefore modifying the gut microbiota, and exerting positive effects on the survival of the treated sea bass. L. delbrueckii subsp. delbrueckii as a probiotic dietary supplement represents an advantageous and ecological strategy for improving the health of fish in aquaculture and increasing fish production.

• Lactobacillus delbrueckii subsp. delbrueckii
• probiotic strain
• Dicentrarchus labrax
• aquaculture
• gut microbiota
• mortality
• stress

Introduction

Aquaculture plays an increasingly significant role in European fish supplies and has particular social and economic significance in rural coastal communities. European sea bass (Dicentrarchus labrax L.) and sea bream (Sparus aurata L.) are currently produced in most countries bordering the Mediterranean. Italy has the largest market for sea bass, with around 75% of demand being met by imports, mainly from Greece and Turkey [1]. However, this rapidly growing industry for intensive seafood production under ‘controllable’ conditions, has experienced relatively severe disease problems owing to lack of control of the microbiota in rearing systems [2].

In intensive cultivation of marine fish, mass mortality of larvae is a major complication for regular production of high quality juveniles. Often the mortality cannot be attributed to specific obligate pathogenic bacteria, but rather to the proliferation of opportunistic pathogenic bacteria [3–5]. Disease outbreaks are being increasingly recognized as a significant constraint on aquaculture production and trade, affecting the economic development of the sector in many countries. Furthermore, there is a growing concern about the use and, particularly, the abuse of antimicrobial drugs, not only in human medicine and agriculture, but also in aquaculture.

The massive use of antimicrobials for disease control and growth promotion in animals increases the selective pressure exerted on the microbial world and encourages the natural emergence of bacterial resistance [6].

In agreement with the warning issued by the international scientific community, a common position [7] has been adopted by the Council with a view to adopting a Regulation of the European Parliament on additives for use in animal nutrition. The proposal is to withdraw from use of antimicrobial agents in animal foods as soon as possible [7] and then to abolish them.

In the light of this, several alternative strategies for the use of antimicrobials in disease control have been proposed and have already been applied very successfully in aquaculture [8–10].

One of the most significant methodologies that has evolved in response to disease control problems is the use of probiotics. Probiotics are live microorganisms that when administered in adequate amounts confer a health benefit on the host [11]. The successful use of probiotics has been reported in a wide range of invertebrates and vertebrates [12], [13]. Most studies of the effects of probiotics on cultured aquatic animals have emphasized laboratory rather than field results.

The purpose of this study was to administer a bacterial strain, isolated from adult European sea bass gut, as a probiotic, to improve sea bass well-being during development. The bacteria were administered using rotifers and Artemia nauplii as carriers, and added to pelleted dry feed. The effective strain colonization of sea bass gut was monitored during early development stages. To evaluate the effects of probiotic administration in treated fish, we monitored changes in gut microbiota, the larvae mortality and the cortisol levels.

Materials and methods

Probiotic strain

The probiotic autochthonous strain used was L. delbrueckii subsp. delbrueckii (AS13B) previously isolated from adult European sea bass (Dicentrarchus labrax, L.) gut. The isolation was performed by plating on MRS (de Man, Rogosa, Sharpe) agar plates (Oxoid, Basingstoke, UK) 100 µl of a pool of homogenate caecal content in sterile dilution blank [14]. All non-spore-forming straight rods from colonies grown on MRS agar were tested by API 50CHL (bioMerieux, Marcy l'Etoile, France) [10]. The strain identification based on morphological and cultural parameters, biochemical profile has been confirmed using a molecular method [15]. The sequence of 16S rDNA of the strain has been determined. DNA extraction was carried out by using the DNeasy Tissue kit (Qiagen, Milan, Italy). PCR reaction was prepared using primers P0 and P6 corresponding to position 27f (forward) and 1495r (reverse) of Escherichia coli 16S rDNA. After determining the amplification by gel electrophoresis the PCR products were purified by QIAquick PCR purification kit (Qiagen) and then sequenced. The sequences obtained have been compared to sequence information available in the GenBank DNA database (accession no. AY050172).

Among Lactobacillus spp. isolated from European adult sea bass gut, L. delbrueckii subsp. delbrueckii showed the greatest ability to survive aerobiosis at 17–20°C (data not shown) in typical tank conditions, and it was then chosen as the strain to administer during sea bass larvae rearing.

This bacterial strain had good growth capacity in biomass production and good viability during storage conditions (data not shown). The cultivation of strain and the bacterial preparation for administration were performed as reported by Carnevali et al. [10].

Experimental design

Three experimental groups (each in duplicate) composed of 40 000 larvae of European sea bass (D. labrax, L.) were set up at Panittica Pugliese Fish Farm (Torre Canne di Fasano, BR, Italy): i.e. the control group, group A, and group B. They were reared for a period of 70 days. Group A (early treatment) received the bacterial strain at a final concentration of 10⁵ bacteria ml⁻¹ via rotifers from 11 to 29 days post hatching (p.h.); from 30 to 70 days p.h., the bacterial administration was performed with Artemia nauplii as carrier. Group B (later treatment) received the bacterial strain at the same concentration as group A, but the administration started from day 30 to 70 p.h. with Artemia solely as carrier. In the control group (group C) no bacteria were added.

The bacterial strain (at a concentration of 10¹⁰ CFU g⁻¹) was added to live food (either rotifers or Artemia) 15 min before administration. Viable counts of bacteria from sampling of rotifers/Artemia were performed to check the bacterial viability.

Live food

The farming of the most relevant sea water species needs the use of live food as first feeding. In aquaculture the most standardized live-chain diet includes rotifers (Brachionus plicatilis) and Artemia that guarantee similar properties to the zooplankton utilized in the natural environment by the growing larvae.

For this purpose, B. plicatilis (100–400 µm maximum length) was cultured in F/2 medium [16] in 1600 l square tanks, and fed with Chlorella spp., Saccharomyces cerevisiae (1 g/10⁻⁶) and DHA-Selco at a 10:1 weight ratio [17]. The rotifers were short-term enriched for 14–16 h in 2 mg 1/10⁻¹ protein Selco (INVE Aquaculture, Belgium) to increase their nutritional value [18].

Cysts of Artemia salina (EG grade INVE Aquaculture, Belgium) were decapsulated with NaOH and hypochlorite at a concentration of 100 g of cyst l⁻¹ incubated for 24 h at 28°C and 35% salinity under strong illumination and aeration and fed for 3 days p.h. with commercial products (oils, vitamins and essential fatty acids) at 28°C. Three-day-old Artemia nauplii were administered to fry.

First feeding

The first feeding live food – both rotifers and Artemia – were utilized as carrier for the L. delbrueckii subsp. delbrueckii administration.

Sea water was UV treated and filtered with sand filter, and approximately 40 000 sea bass (D. labrax L.) larvae were distributed in 400 l tanks and fed as described below. The flow was initiated on day 1 and was set to 30 l h⁻¹. The tanks were aerated through air-stones at a low level. B. plicatilis was added to the tanks at a final density of 3 rotifers ml⁻¹, the quantity being gradually increased until a density of 15 ml⁻¹ was reached at day 26.

Feeding with Artemia (AF 430, INVE Technologies, Belgium) started at day 30 p.h. at a density of 1 ind. ml⁻¹ and was gradually increased, reaching a density of 15 ind. ml⁻¹ at the end of administration (day 70 p.h.). At day 42, concomitantly with Artemia, the dry food (Trouwit, Hendrix, 100–1200 µm) was administered at a final quantity of 25 g tank⁻¹ given at five different times.

Sampling and microbial analysis of larvae and juveniles

Five samplings were conducted on different days, i.e. at 11, 22, 30, 50 and 70 days p.h. For each sampling all tanks were sampled according to the method of Carnevali et al. [10]. The larvae were surface-disinfected with benzalkonium chloride (0.1% w/v) for 30 s, rinsed three times in autoclaved water and homogenized in 3 ml of blank solution [10]. The homogenate was serially diluted in blank solution and plated in duplicate. The total counts of aerobic and anaerobic bacteria were performed on Columbia blood agar base (Oxoid). MacConkey agar plates (Oxoid) were used for viable counts of Enterobacteriaceae as reported by Rollo et al. [19]. At the same time Lactobacillus counts were conducted on MRS agar. The plates were incubated in aerobic conditions for 48–72 h at 17–20°C and in anaerobic conditions at the same temperatures inside an anaerobic cabinet (Don Whitley Scientific, Shipley, UK). Bacteria from colonies grown on MRS agar were examined microscopically. All Gram-positive straight rods, non-spore-forming, catalase-negatives and non-motiles were tested by API 50 CHL (bioMérieux) to identify LAB species and detect the presence of L. delbrueckii subsp. delbrueckii.

Survival rate

The total numbers of live juveniles were counted at day 70 p.h. for each experimental group.

Cortisol analysis

Cortisol extraction was performed in whole-body larvae and juveniles. Samples from the different groups and at different times of growth were processed following the procedures described by Carnevali et al. [20].

Statistical analysis

Data are presented as mean±SD of means. Results were examined by one-way ANOVA followed by Student-Newman-Keuls test or the Student's t test as appropriate, using a statistical software package, Stat View 512 + TM (Brain Power Inc., USA). A p value of 0.05 was used as the limit of statistical significance.

Results

Analysis of gut microbiota in sea bass larvae

The microbial analyses were conducted at different growth phases: 11, 22, 30, 50 and 70 days p.h. Some of the sampling days correspond to critical developmental phases of digestive functions of sea bass larvae. At 11 days p.h. no differences were detected among the three experimental groups as regards all the bacterial counts (Table I). At this stage sea bass larvae had low levels of anaerobic and aerobic bacteria in all three groups, averaging 10⁶ CFU g⁻¹ and also low levels of enterobacteria (<10⁵ CFU g⁻¹) and Lactobacillus spp. (<10⁴ CFU g⁻¹).

Table I.  Total bacterial counts of anaerobes, aerobes, Enterobacteriaceae and Lactobacillus spp. at 11, 22, 30, 50 and 70 days post hatching (p.h.) in larvae of European sea bass gut.

Days p.h.	11			22			30
Group	A	B	C	A	B	C	A	B	C
Total anaerobes	5.75±0.30*	5.74±0.82	5.73±0.87	9.01±0.87	8.82±0.22	8.80±0.21	5.85±0.39^d	7.89±0.55^c	7.86±0.51^c
Total aerobes	5.25±0.09	5.27±0.42	5.29±0.41	8.88±0.14	8.38±0.38	8.34±0.36	5.54±0.49	6.59±0.56	6.54±0.50
Enterobacteriaceae	4.66±0.11	4.68±0.48	4.66±0.40	7.97±0.19^b	7.38±0.24^ab	7.35±0.20^a	4.61±0.49	5.32±0.83	5.30±0.80
Lactobacillus spp.	3.27±0.08	3.27±0.16	3.27±0.14	3.33±0.01	3.30±0.22	3.32±0.24	3.73±0.42	3.67±0.13	3.62±0.10
Days p.h.	50			70
Group	A	B	C	A	B	C
Total anaerobes	5.13±0.87^f	9.56±0.87^e	8.93±0.34^e	7.04±0.09^h	7.71±0.22^g	7.74±0.14^g
Total aerobes	6.04±0.39^f	8.32±0.39^e	7.87±0.24^e	6.62±0.63^h	7.89±0.33^g	7.85±0.49
Enterobacteriaceae	4.04±0.24^f	6.06±1.30^ef	5.67±0.38^e	5.18±0.56^h	5.35±0.30^h	6.45±0.49^g
Lactobacillus spp.	4.99±0.22^f	6.38±0.49^e	6.26±0.48^e	4.53±0.08^h	5.68±0.19^i	4.48±0.56^g

Group A, early treatment; group B, later treatment; group C, control. Different superscripts indicate that values are significantly different (p < 0.05, ANOVA) when compared at 22, 30, 50, 70 days p.h.

*Values are expressed as log CFU g⁻¹.

There was an increasing effect of the total microbiota at 22 days p.h. when the larvae of group A (early treatment), group B (later treatment) and control group were on live feeding. The anaerobic, aerobic and enterobacteria counts had the maximum observed level of >10⁹ CFU g⁻¹, 10⁹ CFU g⁻¹ and 10⁸ CFU g⁻¹, respectively. In contrast, the Lactobacillus spp. count had a minimum level of about 10³ CFU g⁻¹.

At 30 days p.h., this effect slightly decreased in larvae of group B and control groups, while group A, already under probiotic treatment, presented values similar to the beginning of the experiment; all the counts had values <10⁶ CFU g⁻¹.

At 50 days p.h. the total number of anaerobic bacteria in group A was about 10 000-fold less than in the other experimental groups, averaging 10⁵ CFU g⁻¹. Also the total aerobe, enterobacteria and Lactobacillus spp. counts in group A presented values significantly lower (p < 0.05) than those for group B and the control group. The observed values were 10⁶ CFU g⁻¹, 10⁴ CFU g⁻¹ and 10⁵ CFU g⁻¹, respectively.

At 70 days p.h., the anaerobes and aerobes decreased in group B and the control group, while they increased in group A, maintaining their number significantly lower (p < 0.05) than in both group B and the control group. The sea bass fry belonging to both the treated groups presented levels of enterobacteria of about 10⁵ CFU g⁻¹. These values were significantly lower (p < 0.05) than that in the control group.

The total Lactobacillus spp. decreased in all the experimental groups, although group B had Lactobacillus spp. level averaging 10⁶ CFU g⁻¹, a significantly higher number (p < 0.05) than the control group (Table I).

The species of lactic acid bacteria identified in the three experimental groups were: Aerococcus viridans, Lactobacillus acidophilus, L. brevis, L. curvatus, L. delbrueckii subsp. delbrueckii, L. fructivorans, L. lindneri, Lactobacillus spp., L. viridiscens, Lactococcus lactis subsp. lactis and Leuconostoc mesenteroides subsp. mesenteroides.

At 30 days p.h. all three experimental groups reached the highest number of LAB genera and species with respect to the other growth phases (11 and 22 days p.h.). Group A presented 5 of 11 different species: A. viridans (11.7%), L. brevis (17.2%), L. delbrueckii subsp. delbrueckii (41.8%), Lactobacillus spp. (15.0%) and Lact. lactis subsp. lactis (14.3%). At the same time, group B and the control group presented the same 6 of 11 species: L. brevis (29.3% and 31.3%, respectively), L. curvatus (17.4% and 16.7%), L. delbrueckii subsp. delbrueckii (8.5% and 7.4%), L. fructivorans (7.1% and 8.3%), Lactobacillus spp. (19% and 18.5%) and Lact. lactis subsp. lactis (18.7% and 17.8%).

At 50 days p.h. the biodiversity of LAB species slightly decreased in all groups. At 70 days p.h. the treated groups (A and B) showed almost the complete presence of the probiotic strain L. delbrueckii subsp. delbrueckii (96.0% and 95.0% of the total LAB, respectively). In both groups Lactobacillus spp. represented the remaining percentage. On the contrary the control group retained the five different species of LAB until the end of the experiment: L. acidophilus (27.0%), L. delbrueckii subsp. delbrueckii (10%), L. fructivorans (14.0%), L. lindneri (46%), Lactobacillus spp. (4.0%).

The probiotic strain L. delbrueckii subsp. delbrueckii was present in group A from day 11 p.h., then after the probiotic administration the percentage changed from about 17% at the beginning of the study to 96% at the end. Group B presented low percentages of L. delbrueckii subsp. delbrueckii before the probiotic treatment. From day 50 p.h. the percentage of the probiotic strain increased to 35% and then to 95% of the total LAB at 70 days p.h. During the whole period the control group showed percentages of L. delbrueckii subsp. delbrueckii lower than 32%, with the contemporaneous presence of other LAB species.

Survival rates

At 70 days p.h. the sea bass fry of group A (early treatment) showed a threefold higher vitality rate than those of control group C. An increase of vitality, even if less marked, was also observed in sea bass fry of group B (later treatment) (Figure 1).

Figure 1.  Survival of European sea bass fry at 70 days post hatching (p.h.): group A (early treatment), group B (later treatment), group C (control). Different superscripts indicate significant difference between groups (p < 0.05, ANOVA).

Cortisol levels

Cortisol levels obtained from larvae and juveniles 30, 50 and 70 days p.h. were significantly lower in group A than in the control group (p < 0.05) (Figure 2). Also group B (later treatment) had significantly lower cortisol levels than the control group (p < 0.05), even if slightly higher than group A (Figure 2).

Figure 2.  Whole-body cortisol levels in larvae and juveniles, measured at different growth phases, 30, 50 and 70 days post hatching (p.h.); group C (control, black bars), group A (early treatment, grey bars), group B (later treatment, white bars). Bars with different superscripts are significantly different (p < 0.05, ANOVA) when compared at 30, 50, 70 days p.h. ND, not determined.

Discussion

In the light of the importance of reducing mortality due to infection diseases during larval rearing and abolishing the use of antimicrobial compounds (particularly antibiotics), the administration of probiotic bacteria assumes a major significance. A probiont should be able to both colonize the gut and improve the well-being of animals. Probionts should be isolated from healthy animals, and administered to the larvae at a very early stage [21]. L. delbrueckii subsp. delbrueckii (AS13B) isolated from healthy adult European sea bass, administered as probiotic strain, has a modulatory effect on gut microbiota of larvae and fry. The dramatic increase of total gut microbiota in all three experimental groups at 22 days p.h. could be explained by the feeding on live feed started 10 days before and also by the beginning of sea bass mortality [22]. These facts produce a remarkable organic contribution in the experimental tanks and consequently also in the larvae gut, not yet colonized or colonized by temporarily present bacterial species [23], [24].

Furthermore, the larval forms of most fish and shellfish are released in the external environment at an early stage of development. These larvae are highly exposed to gastrointestinal microbiota-associated disorders, because they start feeding even though the digestive tract is not yet fully developed [25] and the immune system is still incomplete [26].

With the exception of this episode at 22 days p.h., the total gut microbiota (anaerobes and aerobes) decreased with the administration of the probiotic strain. In fact group A, treated earlier, had a lower microbial level than both of the other groups (group B not treated yet and the control). Also, when treated, group B had a decrease of the gut microbiota with respect to the control.

It is interesting to note that the enterobacteriaceae seem to be particularly affected by the probiotic treatment. This potentially pathogenic bacterial group [27] decreased by the end of the probiotic treatment in both of the treated groups, in agreement with other studies [19]. Moreover, group A showed a lower presence of this bacteria as it had been probiotic-treated for longer. Thus, L. delbrueckii subsp. delbrueckii showed the ability to inhibit colonization and proliferation of a bacterial family that includes some opportunistic pathogenic strains that are often involved in larval and adult mortality [28]. A number of mechanisms of microbial action might have been responsible for suppression of potential pathogens, for example, the production by probiotic bacteria of inhibitory substances to pathogenic bacteria, the competition between probiotic and pathogenic bacteria for adhesion sites and sources of nutrients, and also immune response enhancement [29–32].

The L. delbrueckii subsp. delbrueckii colonization was evident in group A, which showed an increasing percentage of this species during the experimental period and a presence higher than the control group. The later treatment of group B likewise allowed a good colonization with higher percentage values of L. delbrueckii subsp. delbrueckii than the control group at the end of the treatment. The L. delbrueckii subsp. delbrueckii strain showed a good capability to colonize the gut of European sea bass larvae and to exert beneficial effects on well-being and growth. This is evident looking at the results of the survival rate. Group A (on early probiotic treatment) showed a higher number of live fry at 70 days p.h. Also, group B (treated later) had a higher number of survivors than the control. These results on larval mortality are in agreement with the data obtained in sea bream by Rollo et al. [19] and may confirm that the use of L. delbrueckii subsp. delbrueckii is an important defence mechanism.

The positive effect of probiotics on the health status of sea bass is also marked by a contemporaneous higher body weight and the standard length of the treated sea bass, as reported previously by Carnevali et al. [20]. Furthermore, in agreement with Rollo et et al. [19] and Carnevali al. [20], we detected lower levels of cortisol, a hormone directly involved in stress responses, in the probiotic-treated groups with respect to the control group. In teleosts, cortisol functions as glucorticoid, affecting both metabolic and osmoregulatory pathways. Cortisol also plays an important role in larval survival [33], salinity adaptation [34], drinking activity [35] and growth [36].

Moreover, probiotics positively affected the expression of genes involved in muscular growth (IGF-I and MSTN), confirming the beneficial role of probiotic integrated with the diet [20].

This research represents one of the few field studies where an autochthonous strain was used successfully as a probiotic integrator in the diet of European sea bass in rearing conditions. The use of a probiotic-supplemented diet allows the treatment of fish larvae and fry from an early stage of development, offering a good means of stimulating the gut immune system [32] and for replacing conventional methods of disease prevention and control in aquaculture and in turn for the health of consumer.

Acknowledgements

The authors wish to thank all of the technical staff of Panittica Pugliese Fish Farm. This study was supported by funds from the Italian Ministry for Agriculture (MIPAF) to O.C. and A.C.