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Article

Allium-Derived Compound Propyl Propane Thiosulfonate (PTSO) Reduces Vibrio Populations and Increases Body Weight of European Seabass (Dicentrarchus labrax) Juveniles

by
Miguel Rabelo-Ruiz
1,
Juan Manuel Peralta-Sánchez
1,
Antonio Manuel Martín-Platero
1,*,
Ana J. Ruiz
1,
María del Mar Agraso
2,
Laura Bermúdez
2,
Juan José Ariza
2,
Alberto Baños
3,
Eva Valdivia
1,4 and
Manuel Martínez-Bueno
1,4
1
Departament of Microbiology, University of Granada, Avda. Fuentenueva, s/n, 18071 Granada, Spain
2
Aquaculture Technology Centre of Andalusia, CTAQUA. Muelle Comercial s/n, El puerto de Santa María, 11500 Cádiz, Spain
3
Departament of Microbiology and Biotecnology, DMC Research Center, Camino de Jayena s/n, 18620 Granada, Spain
4
Institute of Biotecnology, University of Granada, 18071 Granada, Spain
*
Author to whom correspondence should be addressed.
Antibiotics 2023, 12(1), 134; https://doi.org/10.3390/antibiotics12010134
Submission received: 13 December 2022 / Revised: 2 January 2023 / Accepted: 6 January 2023 / Published: 10 January 2023
(This article belongs to the Section Novel Antimicrobial Agents)
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Abstract

:
The global demand for fish products is continuously increasing as the population grows, and aquaculture plays an important role in supplying this demand. However, industrial antibiotic misuse has contributed to the spread of antimicrobial resistance among pathogenic bacteria, therefore, several antibiotic alternatives have been proposed. In this study, we have analyzed the effects of Allium-derived propyl propane thiosulfonate (PTSO) in European seabass juveniles’ growth and performance. These effects were tested by measuring the body weight and analyzing the gut microbiome of fish after 89 days of feeding trial. The relative abundance of potentially pathogenic Vibrio in the foregut and hindgut of supplemented fish decreased, while Pseudomonas and Kocuria increased significantly. Alpha diversity indices significantly decreased in both gut regions of fish fed with Allium-derived PTSO supplemented diet, as well as between bacterial community composition. These results may indicate a positive effect of the supplementation in the diet with Allium-derived PTSO, reducing potentially pathogenic Vibrio and increasing body weight at the end of the experiment (89 days). However, this supplementation with Allium-derived PTSO produces changes in the diversity and composition of microbial communities, so further experiments would be necessary to explore bacterial community composition and health relationship.

1. Introduction

World population has increased exponentially in the last years, and it is expected to continue growing in the coming years, reaching 9.7 billion in the year 2050 and almost 11 billion people worldwide in the year 2100 [1]. This increase in world population implies an increase in food demand, which can be partially covered by aquaculture products, given the high impact of land-based animal production and the stagnation of wild fishery catches [2]. Currently, this industry plays an important role in supplying the world food demand and protein source, with a global aquaculture production of 82 million tons in 2018 and an economic value of USD $250 billion [3]. However, economic profits in the industry are affected by fish diseases caused by several pathogenic bacteria such as Aeromonas, Vibrio, or Photobacterium [4]. Infections of these pathogenic bacteria are treated by high doses of antibiotics, given the high fish stocking densities and the impossibility of individual treatment [5]. Furthermore, in the aquaculture industry, antibiotics have been used as growth promoters (antibiotic growth promoters, AGP) for several years, showing an improvement in feed efficiency and growth performance in different fish species [6,7]. However, the extensive use of antibiotics for growth-promoting and therapeutic purposes in aquaculture systems has increased the antibiotic resistance of pathogenic bacteria [8]. Therefore, a worldwide effort is necessary to reduce and rationalize the use of antibiotics in livestock and aquaculture. For this reason, the use of antibiotic growth promoters (AGPs) in animal feed was banned by the European Union in 2006 [9] and by other countries in the following years [10,11].
Several feed additives have been proposed as alternatives to AGPs in the aquaculture industry. The most promising alternatives include enzymes, bacteriophages, probiotics, prebiotics, and phytobiotics [12,13,14]. Phytobiotics are defined as plant-derived bioactive compounds supplemented in the diet to improve animal productivity [15]. Phytobiotics are known to have antimicrobial activity against pathogenic bacteria and can act as prebiotics, facilitating a continuous supply of specific substrates for intestinal microbiota or minimizing the risk of pathogenic bacteria development [16]. These products also act as stimulant of saliva and bile secretion, which helps to increase productive parameters [17]. Many potential herbal plants have been identified and used in aquaculture for improvement of fish health, including more than 60 different medicinal plant species [18,19].
Allium species, mainly garlic (Allium sativum) and onion (Allium cepa), produce a wide variety of bioactive compounds with antifungal, antimicrobial, and antioxidant activity [20]. Dietary supplementation of these compounds has shown promising results, improving the health and productive parameters of goats, cattle, pigs and poultry [21]. The use of A. cepa extract in cattle produced no changes in milk attributes [22], while in goats, A. sativum oil showed a beneficial effect in the milks’ fatty acid profile [23]. The inclusion of Allium in growing finishing pigs showed a reduction of Salmonella, an increase in Lactobacillus and acid levels in feces [24], and an increased growth performance [25]. In the poultry industry, Allium supplementation in laying hens improved health status, intestinal microbiota, and increased egg size and weight [26,27]; it also increased growth performance, immunity, and antioxidant status of broiler chickens [28,29]. Application of Allium species in fish farming has become popular for promoting growth, improving the activity of defense systems, and protecting against diseases caused by pathogenic bacteria [21,30]. The inclusion of onion (A. cepa) powder in the diet of beluga juveniles (Huso huso) improved growth performance, immune function, and blood parameters [31]. Regarding the dietary supplementation of garlic (A. sativum) extract, it has been proven that it promotes growth, enhances the immune system, and improves the control of pathogens [30,32]. Inclusion of garlic in diet showed an increase in weight gain and growth rate of rainbow trout (Oncorhynchus mykiss) [33], an improvement of food digestibility and biochemical and immunohematological effects of Eurasian perch (Perca fluviatilis) juveniles [34], as well as an increase in the immune parameters of skin mucus of guppy fish (Poecilia reticulata) [35]. Furthermore, dietary inclusion of garlic has demonstrated its ability to control pathogens of host, showing antimicrobial activity against fungi and bacteria, including Pseudomonas fluorescens or Vibrio anguillarum [30,32].
The activity of these plant compounds has been related to secondary metabolites, volatile organosulfur compounds such as ajoene, allicin, isoalliin, methiin, propiin, propyl propane thiosulfonate (PTSO), and propyl propane thiosulfinate (PTS) [30,36]. PTS and PTSO (Supplementary Figure S1) have shown antibacterial, antifungal [37,38], and anticoccidial activity [39]. Furthermore, PTSO showed beneficial effects on intestinal health in several animal species [36] and changes in gut microbiota and growth performance of different livestock animals, such as mice, broiler chickens, laying hens, and pigs [24,27,40,41,42,43,44]. In addition, recent studies using experimental animals have shown that PTSO is a toxicologically safe compound [45]. However, the potential effects of Allium-derived PTSO on intestinal microbiota and body weight of European seabass juveniles has not yet been explored.
Hence, our aim in this study was to evaluate the effects of Allium-derived PTSO on the European seabass (D. labrax) juveniles’ growth performance, as well as its foregut and hindgut microbiota via high-throughput sequencing of the V6-V8 region of 16S rRNA gene. As described below, this approach shows that the inclusion of this Allium-based product increases fish growth performance and induces changes in the gut microbiota after 89 days of feeding trial, including the reduction of potential pathogens such as Vibrio populations.

2. Results

2.1. Effect of Feeding Diet on European Seabass Juvenile Growth Performance

No differences appeared in the initial body weight between the fish fed with the control or Allium-derived PSTO supplemented diet (Table 1, Figure 1). European seabass juveniles supplemented with Allium-derived PTSO showed an increase in body weight at the end of the feeding trial (Day 89) (Table 1, Figure 1). However, no differences in body weight appeared between fish fed with a control diet or Allium-derived PTSO along the experiment, showing similar body weight at days 12, 26, 42, and 63 (Table 1, Figure 1).

2.2. Bacterial Community Composition

The foregut microbiota of juvenile European seabass was dominated at class level by Gammaproteobacteria (47%), Alphaproteobacteria (25%), Betaproteobacteria (9%), Actinobacteria (8%), and Bacilli (8%) (Figure 2). Fish supplemented with Allium-derived PTSO showed a significant decrease in Alphaproteobacteria (13%), Betaproteobacteria (7%), and Bacilli (7%), as well as an increase in Actinobacteria (21%) (Figure 2). At the genus level, the foregut of control fish was dominated by Ochrobactrum (22%), Pseudomonas (19%), and Vibrio (19%). In the foregut of Allium-derived PTSO fish an increase in Pseudomonas (40%) and Kocuria (15%), and a decrease in Vibrio (<1%) and Ochrobactrum (11%) were observed (Figure 3 and Figure 4).
The bacterial composition of the hindgut of control fish was similar to their foregut, dominated at class level by Alphaproteobacteria (41%), Gammaproteobacteria (40%), Betaproteobacteria (7%), Bacilli (5%), and Actinobacteria (5%) (Figure 2). The hindgut microbiota of Allium-derived PTSO supplemented fish showed a significant increase in Actinobacteria (13%) and decrease in Betaproteobacteria (4%) and in the minority class Mollicutes (Figure 4). At the genus level, the foregut of control fish was dominated by Ochrobactrum (39%), Pseudomonas (18%), and Vibrio (15%). In the hindgut of Allium-derived PTSO supplemented fish, an increase in Kocuria (9% respect to 1% in control fish) and Pseudomonas (41%) was observed, as well as a decrease in Vibrio (<1%) (Figure 3 and Figure 4).

2.3. Effect of Feeding Diet on Alpha and Beta Diversity

Supplementing the diet of European seabass juveniles with Allium-derived PTSO affected alpha diversity indices (Table 2). Allium-derived PTSO supplemented fish showed a reduction in alpha diversity respect to control fish. However, no differences appeared between gut region, showing both foregut and hindgut similar levels of alpha diversity. Furthermore, no differences appeared in the interaction of diet and gut region, indicating that the changes in diversity between both gut regions occurs in the same way in both feeding diets (see Diet*Gut Region interaction term in Table 2).
The bacterial community of European seabass juveniles varied significantly between the two diets, considering both the most abundant bacterial ASVs (weighted UniFrac) and minority ASVs (unweighted UniFrac) (Table 3, Figure 5). Regarding both gut regions separately, significant differences appeared in both regions. In the foregut, differences between experimental diets were observed among both majority (GLMM, weighted UniFrac, diet as factor, Pseudo-F1,84 = 18.79, p = 0.001) and minority ASVs (GLMM, unweighted UniFrac, diet as factor, Pseudo-F1,84 = 4.99, p = 0.001). In the hindgut, results were similar, with differences in diet with both majority (GLMM, weighted UniFrac, diet as factor, Pseudo-F1,92 = 13.03, p = 0.001) and minority ASVs (GLMM, unweighted UniFrac, diet as factor, Pseudo-F1,92 = 4.89, p = 0.001).

3. Discussion

In this study, juvenile European seabass supplemented with an Allium-derived organosulfur compound, such as propyl propane thiosulfonate (PTSO), produced an increased in body weight at the end of the feeding trial (89 days). This increase in growth performance was accompanied by significant changes in bacterial communities and in some bacterial groups in both foregut and hindgut, as well as a decrease in alpha diversity in PTSO supplemented fish.
The spread of antimicrobial resistance requires an urgent quest in searching for new alternatives to AGP in aquaculture. However, these new products must ensure animal welfare. Some compounds have been proposed as good AGP alternatives, such as probiotics, prebiotics, organic acids, and plant extracts [46]. Plant extracts, also known as phytobiotics, include a wide range of plant-derived products, such as essential oils, herbs, and oleoresins [17]. Phytobiotics have been proposed as good and safe AGP alternatives, capable of modulating intestinal microbiota and increasing productive parameters, while also containing anti-pathogenic and appetite stimulation properties of both terrestrial and aquatic animals [15,47]. The phytobiotics used in the animal feed come from different plant species, being the products derived from Allium plants the most widely used, mainly garlic (Allium sativum) and onion (Allium cepa) [36,48]. Organosulfur compounds are the most important bioactive compounds derived from Allium, showing antibacterial, antifungal, antiviral, anti-inflammatory, and antioxidant activities [37,38,39]. Some of the most Allium-derived organosulfur compounds used for animal feed include ajoene, allicin, isoalliin, methiin, propiin, propyl propane thiosulfinate (PTS), and propyl propane thiosulfonate (PTSO) [30,36]. PTSO addition has shown beneficial effects in different farm animals. In poultry, different doses of PTSO in broiler chickens improved food digestibility and growth performance and produced changes in gut microbiota [43,49,50]. Additionally, in laying hens, PTSO increased the number and the size of eggs laid and produced an increase in potentially beneficial bacteria in the intestine [27,41]. In pig industry, PTSO has shown beneficial effects in intestinal microbiota and increased growth performance in piglets and growing-finishing pigs [24,42]. The use of Allium-derived PTSO in aquaculture has only been studied in gilthead seabream (Sparus aurata) juveniles, showing potentially beneficial changes in gut microbiota and producing no changes in growth performance [51]. However, in the present study, fish supplemented with PTSO additive showed a higher body weight gain at the end of the experimental trail than control ones, supporting previous positive results of such kind of supplements in other farm animals.
Despite the few research articles on the use of PTSO in aquaculture, other Allium-based compounds have been used in aquafeeds in different studies and with different fish species [30]. Dietary inclusion of onion (Allium cepa) powder produced an increase in body weight, SGR, and immune parameters of beluga juveniles [31]. Supplementing the diet with garlic (Allium sativum) showed an increase in growth performance in Asian seabass (Lates calcarifer) [52,53]. The use of crude polysaccharides from garlic produced an increase in body weight and SGR in rainbow trout (Onchorhynchus mykiss) [33]. Other studies using allicin, a garlic-derived organosulfur compound, showed its benefits as growth promoter, antimicrobial agent, and feed stimulator [32]. However, the results of different studies are controversial because other studies noted the lack of effect of Allium extract and Allium-derived compounds on different fish species in aquaculture [54,55]. In fact, in a previous study using PTSO in gilthead seabream, the inclusion of this Allium-derived compound produced no changes in growth performance [51]. Our results with the European seabass juveniles showed no differences in body weight between control and Allium-derived PTSO supplemented fish along the experiment, although we found a significant increase in body weight at the end of the experimental period (after 89 days of treatment). Further studies are needed to clarify differences between phytobiotic presentation and fish species.
Our study showed a significant decrease in all the alpha diversity indices studied in the foregut and hindgut of European seabass juveniles supplemented with PTSO, except in the hindgut with Shannon diversity index. Some studies have shown that reduction in alpha diversity increased body weight in birds, and obesity in humans [56,57]. In aquaculture, results relating alpha diversity and body weight are disparate. In a previous study, [58] they found that differences in bacterial diversity did not translate into differences in body weight of largemouth bronze gudgeon (Coreius guichenoti). However, a study with rainbow trout (Onchorhynchus mykiss) suggested a correlation between an increase in body weight and an increase in bacterial diversity [59]. Previous results from our research group [51] showed no differences in body weight accompanied by an increase in alpha diversity indices in gilthead seabream juveniles supplemented with Allium-derived PTSO. Our results with European seabass showed an opposite trend; an increase in body weight is related with a reduction in alpha diversity. This negative association between body weight gain and bacterial diversity has been found in humans [57]. In pigs, the use of an Allium extract similar to the supplement we provided to our gilt-head breams reduced bacterial alpha diversity and increased body weight [42]. We cannot discard that the relation between alpha diversity and body weight could be species-dependent, so standardization in experimental setups, diets, and products might disentangle this association between body weight and alpha diversity. Moreover, we have explored the effects of PTSO in juvenile growth. Longitudinal studies along the productive life of fish would show long-term effects of PTSO supplementation in growth and microbiota of fish.
Intestinal community differed between the control and Allium-derived PTSO diets, either when considering majority ASVs (Weighted UniFrac) or minority ASVs (Unweighted UniFrac). These community differences are in accordance with changes in some of the majority genera of the intestinal microbiota in supplemented fish with respect to those of the control fish. The relative abundance of Pseudomonas increased in Allium-derived PTSO supplemented fish in both foregut and hindgut regions. These results could be a negative trade-off since, despite the fact that Pseudomonas have been described as an ubiquitous bacterial genus, some species are emergent opportunistic fish pathogens [60]. P. anguilliseptica is considered a fish pathogen, and it is the main causative agent of winter disease, an illness associated with several farmed fish, such as seabass, cod, and gilthead seabream [61]. Other Pseudomonas species such P. aeruginosa, P. putida or P. fluorescens are considered opportunistic pathogens in aquaculture [62]. However, different strains of P. fluorescens have shown probiotic properties in fish, improving immune system [63] or inhibiting the fish pathogenic bacteria Vibrio anguillarum [64]. As with many other bacteria, the pathogenic or symbiotic trait in some bacteria depends on the species and the strains. We have also found that the relative abundance of Vibrio in both foregut and hindgut significantly decreased in the European seabass juveniles supplemented with Allium-derived PTSO. Vibrio species are ubiquitous in marine environments, and some species are considered potentially pathogenic, causing clinical diseases as vibriosis [13,65]. V. anguillarum, V. salmonicida, V. alginolyticus, V. harveyi, or V. parahaemolyticus are some of the Vibrio species which cause the most devastating effects on marine fish [66]. Some plant extracts have demonstrated antimicrobial activity against different Vibrio species in aquaculture. Ginger powder and garlic powder showed antimicrobial effects against V. harveyi in Asian seabass [67]. The use of garlic has shown antimicrobial effects against Vibrio species in aquaculture [30]. A previous study [68] showed in vitro inhibitory activity of garlic (A. sativum) against V. anguillarum, V. alginolyticus, and V. harveyi. This is also true for PTSO, which has shown direct inhibition in vitro against Vibrio, Pseudomonas, Enterobacteria, and several Gram-positive bacteria [37,69]. Among these, Vibrio parahaemolyticus was the most sensitive strain against PTSO, which may explain the Vibrio reduction observed in this study. Further research is necessary to explore in detail different Vibrio and Pseudomonas strains in order to untangle the antagonistic relationships between bacterial species. Future studies should address the limitations of the current study, including increasing the experimental timeline to adult stage of the seabass and observing how the treatment affects the morphology of the intestinal mucosa. Peinado and colleagues [49] showed a significant increase in histometrical parameters of the small intestinal, such as villus height, width, and surface area in birds fed with 90 mg/kg of PTSO, which could explain the body weight gain due to an increased nutrient absorption via an increase in surface area.

4. Materials and Methods

4.1. Animals, Experimental Design and Fish Sampling

European seabass (Dicentrarchus labrax) juveniles (n = 780) were randomly assigned to two experimental groups (390 fish per group), consisting of triplicate tanks (400 L; 130 fish per tank). Fish were kept in a recirculating RAS D-400 water system equipped with physical and biological filters. An amount of 5–10% of the water was renewed daily, depending on the quality of water. The temperature was adjusted at 21 ± 1 °C, and a photoperiod regime of 12L/12D hours was applied. All studied fish were handled in accordance with the European Union Guidelines (Directive 2010/63/UE) for the use of laboratory animals. The Ethical Committee at the University of Granada approved the experiments, and they were endorsed by the regional government (Junta de Andalucía, Spain, ref. no. 13/04/2018/048).
The experimental diet consisted of commercial fishmeal (NUTRAPLUS, Dibaq, Spain) and the addition of the Allium-based product (150 mg of PTSO/kg of fishmeal) (Supplementary Table S1). After the meal homogenization, the granulated fish feed was manufactured by SPAROS I&D Nutrition in Aquaculture (Olhão, Portugal). The same diet without Allium-based additive was prepared as a control. SPAROS I&D Nutrition in Aquaculture checked PTSO concentration by UHPLC-ESI-MS/MS analyses, according to the method described in [70]. The Allium-based product used is commercialized under the trademark AquaGarlic® and was supplied by DOMCA S.A. (Granada, Spain). This product is standardized in propyl propane thiosulfonate (PTSO) at a concentration of 10% and presented as a powder on inert sepiolite.
At the beginning of the experiment, fish were randomly housed in different tanks, obtaining the same initial biomass in each tank. After 2 weeks of acclimatization, fish were anesthetized with 80 mg/L of tricaine methanesulfonate (MS-222) and weighed, with average initial body weight (BW) of 3.78 ± 0.09 g. During the feeding trial (89 days), fish were fed 3–4 times per day, 6 days per week. All fish from each tank were collected, anesthetized using MS-222, and weighed on days 0, 12, 26, 42, 63 and 89. At the end of the feeding trial (89 days, according to the facilities availability and ensuring enough time for testing the experimental effect of PTSO), 20 fish per experimental tank were euthanized by an overdose of anesthesia MS-222 (400 mg/L), followed by spine severing. Fish were immediately dissected and the whole intestine was collected with sterile material. Intestines were stored in sterile 90 mm Petri dishes and transported to the laboratory, where they were kept at −80 °C until DNA extraction.

4.2. DNA Extraction

Intestinal pieces of approximately 100 mg were dissected from the foregut and hindgut of European seabass (D. labrax) juveniles using a sterile scalpel. DNA extraction was carried out following the modified salting out procedure (MSOP) proposed by [71]. An initial mechanical lysis step using a cell disrupter FastPrep FP120 (BIO 101, Thermo Savant, Irvine, CA, USA) was introduced to increase cell lysis. In summary, intestine pieces of about 100 mg were introduced in a 2 mL microcentrifuge screw cap tube filled with 100 mg of 2 mm zirconia beads and homogenized by two consecutive pulses of 30 s at speed 5 in FastPrep FP120. After this previous step, the MSOP protocol was followed. The yield of the DNA extraction was checked by 0.7% agarose gel electrophoresis. DNA concentration was measured using NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and then DNA was stored at −20 °C until PCR amplification.

4.3. V6-V8 16S rRNA Gene Amplification and High-Throughput Sequencing

V6-V8 region of 16S rRNA gene libraries were constructed using the primer pair B969F (5′-ACGCGHNRAACCTTACC-3′) and BA1406R (5′-ACGGGCRGTGWGTRCAA-3′) [72] with Illumina adapter overhang sequences. PCR amplification was carried out using the iProof™ High-Fidelity DNA Polymerase (Bio-Rad®, Hercules, CA, USA) following Rabelo-Ruiz et al. [42]. The PCR products were purified and then used as template for a second PCR. In this second PCR amplification, a unique combination of two Illumina compatible barcodes were index to each sample. This unique barcoding allow that the derived sequences can be demultiplexed into their respective samples in downstream analysis. The barcodes overlapped with the sequence of the primers used in the first PCR. All PCR amplicon purifications were made using DNA Purification SPRI Magnetic Beads (Canvax®, Córdoba, Argentina) following the manufacturer’s instructions. PCR amplicons were checked by 1% agarose gel electrophoresis, and DNA concentrations were measured using Qubit® 3.0 Fluorometer (Invitrogen™, Carlsbad, CA, USA). Afterwards, PCR amplicons were pooled in equimolar concentrations, and high-throughput sequencing was carried out with Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA) in paired-ends reads of 2 × 300 bp length. Sequencing was carried out in the Illumina MiSeq platform in the Institute of Parasitology and Biomedicine “López-Neyra” (Granada, Spain).

4.4. Sequences Processing and Data Analysis

16S rRNA reads generated from Illumina MiSeq sequencer were analyzed using the Quantitative Insights Into Microbial Ecology (QIIME2 v2020.11; [73]) software. At the beginning, primer trimming was performed using cutadapt plugin [74], and pair joining was carried out using default parameters. Quality filtering was performed with a threshold of 20 Phred score. Afterward, Deblur algorithm was used for sequence clustering into ASVs (Amplicon Sequence Variants) in order to remove sequencing errors [75]. Sequences that passed quality filters were trimmed to 400 bp, giving a dataset of 10,832,912 total reads with a mean of 51,098.64 reads per sample. The fragment insertion script implemented in QIIME2 was used to align the sequences and build a bacterial phylogenetic tree based on a reference phylogenetic tree (SEPP reference Greengenes 13.8; [76]). The taxonomy was assigned based on a pretrained classifier on Greengenes 13.08 with a similarity of 99% [77]. Finally, sequences belonging to chloroplast, mitochondria or non-bacterial DNA were filtered of the ASVs table.

4.5. Statistics

To test the effect of different diets on body weight, we performed generalized linear mixed models (GLMM). We used mean body weight per tank as experimental unit with diet as fixed factor.
For alpha and beta diversity analyses, the ASV table was rarified at 10,000 sequencing depth per sample. Samples that did not reach this sequencing depth were excluded from subsequent analyses (this was an 11% of the samples, i.e., 22 out of a total of 200 samples). Four alpha diversity indices were calculated, i.e., Shannon diversity index [78], chao1 index [79], Faith phylogenetic diversity index [80], and OTU Richness. We used GLMM to explore the effect of diet and gut region as fixed factors in both alpha diversity indices. In alpha and beta diversity analysis, fish was used as the experimental unit.
Body weight and alpha diversity analyses were performed using STATISTICA 10.0 (StatSoft).
Differences in genera and classes abundances between control and Allium-derived PTSO supplemented fish were explored by means of linear discriminant analysis effect size (LEfSe) [81]. LEfSe analyses were performed on the Galaxy web platform, implemented on the public server https://huttenhower.sph.harvard.edu/galaxy/ (accessed on 4 July 2022).
Beta diversity distance matrixes were calculated using UniFrac index. Both weighted and unweighted UniFrac indices [82,83] were used for subsequent analysis. Weighted UniFrac considers the relative abundance of bacteria shared between samples, giving more importance to the most abundant bacteria. Unweighted UniFrac gives more importance to rare bacteria in the ASVs as it only considers their presence or absence irrespective of their abundance. Permutational ANOVA (PERMANOVA) was performed to test these effects on both UniFrac distance matrixes using PRIMER-7 software (PRIMER-e), implemented with PERMANOVA plugin. Principal coordinate analyses (PCoA) were performed in order to visualize the 2 first axes using EMPeror 2018.2.0 [84,85].

5. Conclusions

The worlds’ food demand and the ban of antibiotics as growth promoters are enhancing the appearance of new alternatives for animal production and welfare. Phytobiotics may play an important role as food additives due to their positive effect on growth performance and antimicrobial activity against certain pathogens. Although the ultimate cause has not been elucidated yet, their positive effects in animal production might be associated with change in the bacterial community composition. Our experimental supplementation of the diet of European seabass juveniles with Allium-derived PTSO produces a final increase in fish body weight accompanied with changes in bacterial community composition. Moreover, Allium-derived PTSO induced changes in some bacterial groups, especially a reduction in Vibrio, a potential pathogen. Our results support the positive association between diet and performance in fish. However, further research is necessary to study how this Allium-derived PTSO affects specific pathogenic strains and how this phytobiotic product affects the immune system and health status of fish.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/antibiotics12010134/s1, Figure S1: Structure of PTS and PTSO; Table S1: Diet composition of the fish experimental diet.

Author Contributions

Conceptualization, J.J.A., A.B. and M.M.-B.; methodology, M.R.-R., J.J.A., A.J.R., M.d.M.A., L.B., A.M.M.-P., A.B., E.V., M.M.-B. and J.M.P.-S.; validation, A.B., M.M.-B. and J.M.P.-S.; formal analysis, M.R.-R., J.J.A. and J.M.P.-S.; resources, A.B. and M.M.-B.; data curation, M.R.-R. and J.M.P.-S.; writing—original draft preparation, M.R.-R.; writing—review and editing, M.R.-R., J.J.A., A.J.R., M.d.M.A., L.B., A.M.M.-P., A.B., E.V., M.M.-B. and J.M.P.-S.; supervision, A.B., M.M.-B. and J.M.P.-S.; project administration, A.B. and M.M.-B.; funding acquisition, A.B. and M.M.-B. All authors have read and agreed to the published version of the manuscript.

Funding

This study was financially supported by the AQUAHEALTH project (IDI-20170032, Operational Programme Smart Growth 2014–2020) and FEDER-INNTERCONECTA-CDTI 2018 (CDTI, Spanish Ministry of Economy and Competitiveness; ALQUABIOTIC project, ITC-20181099). M.R-R. was funded by Programa Operativo de Empleo Juvenil (Fondo Social Europeo, Junta de Andalucia, Ref. 6127).

Institutional Review Board Statement

All studied fish were handled in accordance with the European Union Guidelines (Directive 2010/63/UE) [86] for the use of laboratory animals, and experiments were endorsed by the regional government (Junta de Andalucía, Spain, ref. no. 13/04/2018/048).

Informed Consent Statement

Not applicable.

Data Availability Statement

Raw sequences are available at the Sequence Read Archive (SRA) in Genbank (https://www.ncbi.nlm.nih.gov/sra/, accessed on 22 February 2022) under accession numbers SAMN26177506 to SAMN26177705.

Acknowledgments

We thank the anonymous reviewers for their useful suggestions that undoubtedly have improved the quality of the present paper.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Department of Economic and Social Affairs, Population Division. World Population Prospects 2019: Highlights (ST/ESA/SER.A/423). 2019. Available online: https://population.un.org/wpp/publications/files/wpp2019_highlights.pdf (accessed on 13 December 2022).
  2. Merino, G.; Barange, M.; Blanchard, J.L.; Harle, J.; Holmes, R.; Allen, I.; Allison, E.H.; Badjeck, M.C.; Dulvy, N.K.; Holt, J.; et al. Can Marine Fisheries and Aquaculture Meet Fish Demand from a Growing Human Population in a Changing Climate? Glob. Environ. Change 2012, 22, 795–806. [Google Scholar] [CrossRef]
  3. Food and Agriculture Organization (FAO). The State of World Fisheries and Aquaculture 2020. Sustainability in Action; FAO: Rome, Italy, 2020; ISBN 978-92-5-132692-3. [Google Scholar]
  4. Yukgehnaish, K.; Kumar, P.; Sivachandran, P.; Marimuthu, K.; Arshad, A.; Paray, B.A.; Arockiaraj, J. Gut Microbiota Metagenomics in Aquaculture: Factors Influencing Gut Microbiome and Its Physiological Role in Fish. Rev. Aquac. 2020, 12, 1903–1927. [Google Scholar] [CrossRef]
  5. Resende, J.A.; Silva, V.L.; Fontes, C.O.; Souza-Filho, J.A.; Lopes, T.; De Oliveira, R.; Coelho, C.M.; César, D.E.; Diniz, C.G. Multidrug-Resistance and Toxic Metal Tolerance of Medically Important Bacteria Isolated from an Aquaculture System. Microbes Environ. 2012, 27, 449–455. [Google Scholar] [CrossRef] [Green Version]
  6. Lulijwa, R.; Rupia, E.J.; Alfaro, A.C. Antibiotic Use in Aquaculture, Policies and Regulation, Health and Environmental Risks: A Review of the Top 15 Major Producers. Rev. Aquac. 2020, 12, 640–663. [Google Scholar] [CrossRef]
  7. He, S.; Wang, Q.; Li, S.; Ran, C.; Guo, X.; Zhang, Z.; Zhou, Z. Antibiotic Growth Promoter Olaquindox Increases Pathogen Susceptibility in Fish by Inducing Gut Microbiota Dysbiosis. Sci. China Life Sci. 2017, 60, 1260–1270. [Google Scholar] [CrossRef] [PubMed]
  8. Cañada-Cañada, F.; La Peña, A.M.D.; Espinosa-Mansilla, A. Analysis of Antibiotics in Fish Samples. Anal. Bioanal. Chem. 2009, 395, 987–1008. [Google Scholar] [CrossRef] [PubMed]
  9. Commision, E. Regulation (EU) 2019/4 of the European Parliament and of the Council of 11 December 2018 on the Manufacture, Placing on the Market and Use of Medicated Feed, Amending Regulation (EC) No 183/2005 of the European Parliament and of the Council and Repealing. Off. J. Eur. Union 2018, 62, L:2019:004:TOC. [Google Scholar]
  10. Maron, D.F.; Smith, T.J.S.; Nachman, K.E. Restrictions on Antimicrobial Use in Food Animal Production: An International Regulatory and Economic Survey. Glob. Health 2013, 9, 48. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  11. U.S. Food and Drugs Administration. FDA Reminds Retail Establishments of Upcoming Changes to the Use of Antibiotics in Food Animals; U.S. Food and Drugs Administration: Silver Spring, MD, USA, 2016.
  12. Dawood, M.A.O.; Koshio, S.; Esteban, M.Á. Beneficial Roles of Feed Additives as Immunostimulants in Aquaculture: A Review. Rev. Aquac. 2018, 10, 950–974. [Google Scholar] [CrossRef]
  13. Pérez-Sánchez, T.; Mora-Sánchez, B.; Balcázar, J.L. Biological Approaches for Disease Control in Aquaculture: Advantages, Limitations and Challenges. Trends Microbiol. 2018, 26, 896–903. [Google Scholar] [CrossRef]
  14. Sapsuha, Y.; Suprijatna, E.; Kismiati, S.; Sugiharto, S. Combination of probiotic and phythobiotic as an alternative for antibiotic growth promoter for broilers chickens-a review. Livest. Res. Rural. Dev. 2021, 33, 49. [Google Scholar]
  15. Windisch, W.; Schedle, K.; Plitzner, C.; Kroismayr, A. Use of Phytogenic Products as Feed Additives for Swine and Poultry. J. Anim. Sci. 2008, 86, E140–E148. [Google Scholar] [CrossRef] [Green Version]
  16. Vidanarachchi, J.; Sims, I.M.; Iji, P. Phytobiotics: Alternatives to Antibiotic Growth Promoters in Monogastric Animal Feed. Recent Adv. Anim. Nutr. Aust. 2005, 15, 131–144. [Google Scholar]
  17. Gheisar, M.M.; Kim, I.H. Phytobiotics in Poultry and Swine Nutrition—A Review. Ital. J. Anim. Sci. 2018, 17, 92–99. [Google Scholar] [CrossRef] [Green Version]
  18. Hai, N. Van The Use of Medicinal Plants as Immunostimulants in Aquaculture: A Review. Aquaculture 2015, 446, 88–96. [Google Scholar] [CrossRef]
  19. Bulfon, C.; Volpatti, D.; Galeotti, M. Current Research on the Use of Plant-Derived Products in Farmed Fish. Aquac. Res. 2015, 46, 513–551. [Google Scholar] [CrossRef]
  20. Kyung, K.H. Antimicrobial Properties of Allium Species. Curr. Opin. Biotechnol. 2012, 23, 142–147. [Google Scholar] [CrossRef] [PubMed]
  21. Chen, J.; Wang, F.; Yin, Y.; Ma, X. The Nutritional Applications of Garlic (Allium sativum) as Natural Feed Additives in Animals. PeerJ 2021, 9, e11934. [Google Scholar] [CrossRef] [PubMed]
  22. Abad, P.; Arroyo-Manzanares, N.; Gil, L.; García-Campaña, A.M. Use of Onion Extract as a Dairy Cattle Feed Supplement: Monitoring Propyl Propane Thiosulfonate as a Marker of Its Effect on Milk Attributes. J. Agric. Food Chem. 2017, 65, 793–799. [Google Scholar] [CrossRef]
  23. Zhu, Z.; Hang, S.; Zhu, H.; Zhong, S.; Mao, S.; Zhu, W. Effects of Garlic Oil on Milk Fatty Acid Profile and Lipogenesis-Related Gene Expression in Mammary Gland of Dairy Goats. J. Sci. Food Agric. 2013, 93, 560–567. [Google Scholar] [CrossRef]
  24. Sánchez, C.J.; Martínez-Miró, S.; Ariza, J.J.; Madrid, J.; Orengo, J.; Aguinaga, M.A.; Baños, A.; Hernández, F. Effect of Alliaceae Extract Supplementation on Performance and Intestinal Microbiota of Growing-Finishing Pig. Animals 2020, 10, 1557. [Google Scholar] [CrossRef]
  25. Zivkovic, V.; Stankovic, B.; Radovic, C.; Gogic, M.; Stanojkovic, A.; Obradovic, S.; Stojiljkovic, N. Garlic as Alternative for Antibiotics in Diet for Growing Pigs. Biotechnol. Anim. Husb. 2019, 35, 281–287. [Google Scholar] [CrossRef]
  26. Omer, H.A.A.; Ahmed, S.M.; Abdel-Magid, S.S.; El-Mallah, G.M.H.; Bakr, A.A.; Fattah, M.M.A. Nutritional Impact of Inclusion of Garlic (Allium sativum) and/or Onion (Allium cepa L.) Powder in Laying Hens’ Diets on Their Performance, Egg Quality, and Some Blood Constituents. Bull. Natl. Res. Cent. 2019, 43, 23. [Google Scholar] [CrossRef]
  27. Abad, P.; Arroyo-Manzanares, N.; Ariza, J.J.; Baños, A.; García-Campaña, A.M. Effect of Allium Extract Supplementation on Egg Quality, Productivity, and Intestinal Microbiota of Laying Hens. Animals 2021, 11, 41. [Google Scholar] [CrossRef]
  28. Ismail, I.E.; Alagawany, M.; Taha, A.E.; Puvača, N.; Laudadio, V.; Tufarelli, V. Effect of Dietary Supplementation of Garlic Powder and Phenyl Acetic Acid on Productive Performance, Blood Haematology, Immunity and Antioxidant Status of Broiler Chickens. Asian-Australas. J. Anim. Sci. 2020, 34, 363–370. [Google Scholar] [CrossRef] [PubMed]
  29. Omar, A.E.; Al-Khalaifah, H.S.; Mohamed, W.A.M.; Gharib, H.S.A.; Osman, A.; Al-Gabri, N.A.; Amer, S.A. Effects of Phenolic-Rich Onion (Allium cepa L.) Extract on the Growth Performance, Behavior, Intestinal Histology, Amino Acid Digestibility, Antioxidant Activity, and the Immune Status of Broiler Chickens. Front. Vet. Sci. 2020, 7, 728. [Google Scholar] [CrossRef] [PubMed]
  30. Valenzuela-Gutiérrez, R.; Lago-Lestón, A.; Vargas-Albores, F.; Cicala, F.; Martínez-Porchas, M. Exploring the Garlic (Allium sativum) Properties for Fish Aquaculture. Fish Physiol. Biochem. 2021, 47, 1179–1198. [Google Scholar] [CrossRef] [PubMed]
  31. Akrami, R.; Gharaei, A.; Mansour, M.R.; Galeshi, A. Effects of Dietary Onion (Allium cepa) Powder on Growth, Innate Immune Response and Hemato–Biochemical Parameters of Beluga (Huso Huso Linnaeus, 1754) Juvenile. Fish Shellfish. Immunol. 2015, 45, 828–834. [Google Scholar] [CrossRef] [PubMed]
  32. Lee, J.-Y.; Gao, Y. Review of the Application of Garlic, Allium sativum, in Aquaculture. J. World Aquac. Soc. 2012, 43, 447–458. [Google Scholar] [CrossRef]
  33. Büyükdeveci, M.E.; Balcázar, J.L.; Demirkale, İ.; Dikel, S. Effects of Garlic-Supplemented Diet on Growth Performance and Intestinal Microbiota of Rainbow Trout (Oncorhynchus mykiss). Aquaculture 2018, 486, 170–174. [Google Scholar] [CrossRef]
  34. Zare, M.; Tran, H.Q.; Prokešová, M.; Stejskal, V. Effects of Garlic Allium sativum Powder on Nutrient Digestibility, Haematology, and Immune and Stress Responses in Eurasian Perch Perca fluviatilis Juveniles. Animals 2021, 11, 2735. [Google Scholar] [CrossRef]
  35. Motlagh, H.A.; Safari, O.; Selahvarzi, Y.; Baghalian, A.; Kia, E. Non-Specific Immunity Promotion in Response to Garlic Extract Supplemented Diets in Female Guppy (Poecilia reticulata). Fish Shellfish. Immunol. 2020, 97, 96–99. [Google Scholar] [CrossRef]
  36. Guillamón, E.; Andreo-Martínez, P.; Mut-Salud, N.; Fonollá, J.; Baños, A. Beneficial Effects of Organosulfur Compounds from Allium cepa on Gut Health: A Systematic Review. Foods 2021, 10, 1680. [Google Scholar] [CrossRef]
  37. Sorlozano-Puerto, A.; Albertuz-Crespo, M.; Lopez-Machado, I.; Ariza-Romero, J.J.; Baños-Arjona, A.; Exposito-Ruiz, M.; Gutierrez-Fernandez, J. In Vitro Antibacterial Activity of Propyl-Propane-Thiosulfinate and Propyl-Propane-Thiosulfonate Derived from Allium spp. Against Gram-Negative and Gram-Positive Multidrug-Resistant Bacteria Isolated from Human Samples. BioMed Res. Int. 2018, 2018, 7861207. [Google Scholar] [CrossRef] [Green Version]
  38. Sorlozano-Puerto, A.; Albertuz-Crespo, M.; Lopez-Machado, I.; Gil-Martinez, L.; Ariza-Romero, J.J.; Maroto-Tello, A.; Baños-Arjona, A.; Gutierrez-Fernandez, J. Antibacterial and Antifungal Activity of Propyl-Propane-Thiosulfinate and Propyl-Propane-Thiosulfonate, Two Organosulfur Compounds from Allium cepa: In Vitro Antimicrobial Effect via the Gas Phase. Pharmaceuticals 2021, 14, 21. [Google Scholar] [CrossRef] [PubMed]
  39. Kim, D.K.; Lillehoj, H.S.; Lee, S.H.; Lillehoj, E.P.; Bravo, D. Improved Resistance to Eimeria acervulina Infection in Chickens Due to Dietary Supplementation with Garlic Metabolites. Br. J. Nutr. 2013, 109, 76–88. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  40. Peinado, M.J.; Ruiz, R.; Echávarri, A.; Aranda-Olmedo, I.; Rubio, L.A. Garlic Derivative PTS-O Modulates Intestinal Microbiota Composition and Improves Digestibility in Growing Broiler Chickens. Anim. Feed. Sci. Technol. 2013, 181, 87–92. [Google Scholar] [CrossRef]
  41. Rabelo-Ruiz, M.; Ariza-Romero, J.J.; Zurita-González, M.J.; Martín-Platero, A.M.; Baños, A.; Maqueda, M.; Valdivia, E.; Martínez-Bueno, M.; Peralta-Sánchez, J.M. Allium-Based Phytobiotic Enhances Egg Production in Laying Hens through Microbial Composition Changes in Ileum and Cecum. Animals 2021, 11, 448. [Google Scholar] [CrossRef]
  42. Rabelo-Ruiz, M.; Teso-Pérez, C.; Peralta-Sánchez, J.M.; Ariza, J.J.; Martín-Platero, A.M.; Casabuena-Rincón, Ó.; Vázquez-Chas, P.; Guillamón, E.; Aguinaga-Casañas, M.A.; Maqueda, M.; et al. Allium Extract Implements Weaned Piglet’s Productive Parameters by Modulating Distal Gut Microbiota. Antibiotics 2021, 10, 269. [Google Scholar] [CrossRef] [PubMed]
  43. Ruiz, R.; Peinado, M.J.; Aranda-Olmedo, I.; Abecia, L.; Suárez-Pereira, E.; Melle, C.O.; Fernández, J.M.G.; Rubio, L.A. Effects of Feed Additives on Ileal Mucosa-Associated Microbiota Composition of Broiler Chickens. J. Anim. Sci. 2015, 93, 3410–3420. [Google Scholar] [CrossRef] [Green Version]
  44. Vezza, T.; Garrido-Mesa, J.; Diez-Echave, P.; Hidalgo-García, L.; Ruiz-Malagón, A.J.; García, F.; Sánchez, M.; Toral, M.; Romero, M.; Duarte, J.; et al. Allium-Derived Compound Propyl Propane Thiosulfonate (PTSO) Attenuates Metabolic Alterations in Mice Fed a High-Fat Diet through Its Anti-Inflammatory and Prebiotic Properties. Nutrients 2021, 13, 2595. [Google Scholar] [CrossRef]
  45. Lira, A.C.; Prieto, A.I.; Baños, A.; Guillamón, E.; Moyano, R.; Jos, A.; Cameán, A.M. Safety Assessment of Propyl-Propane-Thiosulfonate (PTSO): 90-Days Oral Subchronic Toxicity Study in Rats. Food Chem. Toxicol. 2020, 144, 111612. [Google Scholar] [CrossRef] [PubMed]
  46. Romero, J.; Feijoo, C.G.; Navarrete, P. Antibiotics in Aquaculture—Use, Abuse and Alternatives. In Health and Environment in Aquaculture; IntechOpen Limited: London, UK, 2012. [Google Scholar]
  47. Reverter, M.; Bontemps, N.; Lecchini, D.; Banaigs, B.; Sasal, P. Use of Plant Extracts in Fish Aquaculture as an Alternative to Chemotherapy: Current Status and Future Perspectives. Aquaculture 2014, 433, 50–61. [Google Scholar] [CrossRef]
  48. Kothari, D.; Lee, W.-D.; Niu, K.-M.; Kim, S.-K. The Genus Allium as Poultry Feed Additive: A Review. Animals 2019, 9, 1032. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  49. Peinado, M.J.; Ruiz, R.; Echavarri, A.; Rubio, L.A. Garlic Derivative Propyl Propane Thiosulfonate Is Effective against Broiler Enteropathogens in Vivo. Poult. Sci. 2012, 91, 2148–2157. [Google Scholar] [CrossRef]
  50. Rubio, L.A.; Peinado, M.J.; Ruiz, R.; Suárez-Pereira, E.; Mellet, C.O.; Fernández, J.M.G. Correlations between Changes in Intestinal Microbiota Composition and Performance Parameters in Broiler Chickens. J. Anim. Physiol. Anim. Nutr. 2015, 99, 418–423. [Google Scholar] [CrossRef]
  51. Rabelo-Ruiz, M.; Newman-Portela, A.M.; Peralta-Sánchez, J.M.; Martín-Platero, A.M.; Agraso, M.d.M.; Bermúdez, L.; Aguinaga, M.A.; Baños, A.; Maqueda, M.; Valdivia, E.; et al. Beneficial Shifts in the Gut Bacterial Community of Gilthead Seabream (Sparus aurata) Juveniles Supplemented with Allium-Derived Compound Propyl Propane Thiosulfonate (PTSO). Animals 2022, 12, 1821. [Google Scholar] [CrossRef] [PubMed]
  52. Abdelwahab, A.M.; El-Bahr, S.M.; Al-Khamees, S. Influence of Dietary Garlic (Allium sativum) and/or Ascorbic Acid on Performance, Feed Utilization, Body Composition and Hemato-Biochemical Parameters of Juvenile Asian Sea Bass (Lates calcarifer). Animals 2020, 10, 2396. [Google Scholar] [CrossRef] [PubMed]
  53. Talpur, A.D.; Ikhwanuddin, M. Dietary Effects of Garlic (Allium sativum) on Haemato-Immunological Parameters, Survival, Growth, and Disease Resistance against Vibrio Harveyi Infection in Asian Sea Bass, Lates Calcarifer (Bloch). Aquaculture 2012, 364–365, 6–12. [Google Scholar] [CrossRef]
  54. Motlagh, H.A.; Paolucci, M.; Bami, M.L.; Safari, O. Sexual Parameters, Digestive Enzyme Activities, and Growth Performance of Guppy (Poecilia reticulata) Fed Garlic (Allium sativum) Extract Supplemented Diets. J. World Aquac. Soc. 2020, 51, 1087–1097. [Google Scholar] [CrossRef]
  55. Nya, E.J.; Dawood, Z.; Austin, B. The Garlic Component, Allicin, Prevents Disease Caused by Aeromonas Hydrophila in Rainbow Trout, Oncorhynchus mykiss (Walbaum). J. Fish Dis. 2010, 33, 293–300. [Google Scholar] [CrossRef]
  56. Bae, Y.; Koo, B.; Lee, S.; Mo, J.; Oh, K.; Mo, I.P. Bacterial Diversity and Its Relationship to Growth Performance of Broilers. Korean J. Vet. Res. 2017, 57, 159–167. [Google Scholar] [CrossRef]
  57. Menni, C.; Jackson, M.A.; Pallister, T.; Steves, C.J.; Spector, T.D.; Valdes, A.M. Gut Microbiome Diversity and High-Fibre Intake Are Related to Lower Long-Term Weight Gain. Int. J. Obes. 2017, 41, 1099–1105. [Google Scholar] [CrossRef] [PubMed]
  58. Li, X.; Yan, Q.; Ringø, E.; Wu, X.; He, Y.; Yang, D. The Influence of Weight and Gender on Intestinal Bacterial Community of Wild Largemouth Bronze Gudgeon (Coreius guichenoti, 1874). BMC Microbiol. 2016, 16, 191. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  59. Betiku, O.C.; Yeoman, C.J.; Gaylord, T.G.; Americus, B.; Olivo, S.; Duff, G.C.; Sealey, W.M. Water System Is a Controlling Variable Modulating Bacterial Diversity of Gastrointestinal Tract and Performance in Rainbow Trout. PLoS ONE 2018, 13, e0195967. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  60. Fadel, A.; Mabrok, M.; Aly, S. Epizootics of Pseudomonas Anguilliseptica among Cultured Seabream (Sparus aurata) Populations: Control and Treatment Strategies. Microb. Pathog. 2018, 121, 1–8. [Google Scholar] [CrossRef]
  61. Wiklund, T. Pseudomonas Anguilliseptica Infection as a Threat to Wild and Farmed Fish in the Baltic Sea. Microbiol. Aust. 2016, 37, 135. [Google Scholar] [CrossRef] [Green Version]
  62. Altinok, I.; Kayis, S.; Capkin, E. Pseudomonas Putida Infection in Rainbow Trout. Aquaculture 2006, 261, 850–855. [Google Scholar] [CrossRef]
  63. Giri, S.S.; Sen, S.S.; Sukumaran, V. Effects of Dietary Supplementation of Potential Probiotic Pseudomonas Aeruginosa VSG-2 on the Innate Immunity and Disease Resistance of Tropical Freshwater Fish, Labeo Rohita. Fish Shellfish. Immunol. 2012, 32, 1135–1140. [Google Scholar] [CrossRef] [PubMed]
  64. Gram, L.; Melchiorsen, J.; Spanggaard, B.; Huber, I.; Nielsen, T.F. Inhibition of Vibrio Anguillarum by Pseudomonas Fluorescens AH2, a Possible Probiotic Treatment of Fish. Appl. Environ. Microbiol. 1999, 65, 969–973. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  65. Abdel-Aziz, M.; Eissa, A.E.; Hanna, M.; Okada, M.A. Identifying Some Pathogenic Vibrio/Photobacterium Species during Mass Mortalities of Cultured Gilthead Seabream (Sparus aurata) and European Seabass (Dicentrarchus labrax) from Some Egyptian Coastal Provinces. Int. J. Vet. Sci. Med. 2013, 1, 87–95. [Google Scholar] [CrossRef] [Green Version]
  66. Mohamad, N.; Amal, M.N.A.; Yasin, I.S.M.; Saad, M.Z.; Nasruddin, N.S.; Al-saari, N.; Mino, S.; Sawabe, T. Vibriosis in Cultured Marine Fishes: A Review. Aquaculture 2019, 512, 734289. [Google Scholar] [CrossRef]
  67. Stratev, D.; Zhelyazkov, G.; Noundou, X.S.; Krause, R.W.M. Beneficial Effects of Medicinal Plants in Fish Diseases. Aquac. Int. 2018, 26, 289–308. [Google Scholar] [CrossRef]
  68. Natasya-Ain, R.; Eirna-Liza, N.; Jasmin, M.Y.; Karim, M. Antibacterial Activity of Garlic Extracts on Fish Pathogenic Bacteria. J. Environ. Biol. 2018, 39, 808–812. [Google Scholar] [CrossRef]
  69. Llana-Ruiz-Cabello, M.; Gutiérrez-Praena, D.; Puerto, M.; Pichardo, S.; Moreno, F.J.; Baños, A.; Nuñez, C.; Guillamón, E.; Cameán, A.M. Acute Toxicological Studies of the Main Organosulfur Compound Derived from Allium Sp. Intended to Be Used in Active Food Packaging. Food Chem. Toxicol. 2015, 82, 1–11. [Google Scholar] [CrossRef]
  70. Abad, P.; Arroyo-Manzanares, N.; García-Campaña, A.M. A Rapid and Simple UHPLC-ESI-MS/MS Method for the Screening of Propyl Propane Thiosulfonate, a New Additive for Animal Feed. Anal. Methods 2016, 8, 3730–3739. [Google Scholar] [CrossRef]
  71. Martín-Platero, A.M.; Valdivia, E.; Maqueda, M.; Martínez-Bueno, M. Fast, Convenient, and Economical Method for Isolating Genomic DNA from Lactic Acid Bacteria Using a Modification of the Protein “Salting-out” Procedure. Anal. Biochem. 2007, 366, 102–104. [Google Scholar] [CrossRef]
  72. Comeau, A.M.; Li, W.K.W.; Tremblay, J.-É.; Carmack, E.C.; Lovejoy, C. Arctic Ocean Microbial Community Structure before and after the 2007 Record Sea Ice Minimum. PLoS ONE 2011, 6, e27492. [Google Scholar] [CrossRef]
  73. Bolyen, E.; Rideout, J.R.; Dillon, M.R.; Bokulich, N.A.; Abnet, C.C.; Al-Ghalith, G.A.; Alexander, H.; Alm, E.J.; Arumugam, M.; Asnicar, F.; et al. Reproducible, Interactive, Scalable and Extensible Microbiome Data Science Using QIIME 2. Nat. Biotechnol. 2019, 37, 852–857. [Google Scholar] [CrossRef]
  74. Martin, M. Cutadapt Removes Adapter Sequences from High-Throughput Sequencing Reads. EMBnet J. 2011, 17, 10. [Google Scholar] [CrossRef]
  75. Amir, A.; McDonald, D.; Navas-Molina, J.A.; Kopylova, E.; Morton, J.T.; Xu, Z.Z.; Kightley, E.P.; Thompson, L.R.; Hyde, E.R.; Gonzalez, A.; et al. Deblur Rapidly Resolves Single-Nucleotide Community Sequence Patterns. mSystems 2017, 2, e00191-16. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  76. Janssen, S.; McDonald, D.; Gonzalez, A.; Navas-Molina, J.A.; Jiang, L.; Xu, Z.Z.; Winker, K.; Kado, D.M.; Orwoll, E.; Manary, M.; et al. Phylogenetic Placement of Exact Amplicon Sequences Improves Associations with Clinical Information. mSystems 2018, 3, e00021-18. [Google Scholar] [CrossRef] [Green Version]
  77. DeSantis, T.Z.; Hugenholtz, P.; Larsen, N.; Rojas, M.; Brodie, E.L.; Keller, K.; Huber, T.; Dalevi, D.; Hu, P.; Andersen, G.L. Greengenes, a Chimera-Checked 16S RRNA Gene Database and Workbench Compatible with ARB. Appl. Environ. Microbiol. 2006, 72, 5069–5072. [Google Scholar] [CrossRef]
  78. Shannon, C.E. A Mathematical Theory of Communication. Bell Syst. Tech. J. 1948, 27, 379–423. [Google Scholar] [CrossRef] [Green Version]
  79. Chao, A. Nonparametric Estimation of the Number of Classes in a Population. Scand. J. Stat. 1984, 11, 265–270. [Google Scholar]
  80. Faith, D.P. Conservation Evaluation and Phylogenetic Diversity. Biol. Conserv. 1992, 61, 1–10. [Google Scholar] [CrossRef]
  81. Segata, N.; Izard, J.; Waldron, L.; Gevers, D.; Miropolsky, L.; Garrett, W.S.; Huttenhower, C. Metagenomic Biomarker Discovery and Explanation. Genome Biol. 2011, 12, R60. [Google Scholar] [CrossRef] [Green Version]
  82. Lozupone, C.A.; Hamady, M.; Kelley, S.T.; Knight, R. Quantitative and Qualitative β Diversity Measures Lead to Different Insights into Factors That Structure Microbial Communities. Appl. Environ. Microbiol. 2007, 73, 1576–1585. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  83. Lozupone, C.; Knight, R. UniFrac: A New Phylogenetic Method for Comparing Microbial Communities. Appl. Environ. Microbiol. 2005, 71, 8228–8235. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  84. Vázquez-Baeza, Y.; Gonzalez, A.; Smarr, L.; McDonald, D.; Morton, J.T.; Navas-Molina, J.A.; Knight, R. Bringing the Dynamic Microbiome to Life with Animations. Cell Host Microbe 2017, 21, 7–10. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  85. Vázquez-Baeza, Y.; Pirrung, M.; Gonzalez, A.; Knight, R. EMPeror: A Tool for Visualizing High-Throughput Microbial Community Data. GigaScience 2013, 2, 16. [Google Scholar] [CrossRef] [PubMed]
  86. European Commission. Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the Protection of Animals Used for Scientific Purposes; No 2010/63/EU; European Commission: Brussels, Belgium, 2010; pp. 33–79.
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Figure 1. Evolution of growth performance of European seabass (D. labrax) juveniles fed with control diet or supplemented with Allium-derived PTSO along the feeding trial. Error bars show standard error.
Figure 1. Evolution of growth performance of European seabass (D. labrax) juveniles fed with control diet or supplemented with Allium-derived PTSO along the feeding trial. Error bars show standard error.
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Figure 2. Microbial composition at the class level of juvenile European seabass gut microbiota group by experimental diet (control and Allium-derived PTSO). Classes in the legend are sorted from most abundant to lowest abundant.
Figure 2. Microbial composition at the class level of juvenile European seabass gut microbiota group by experimental diet (control and Allium-derived PTSO). Classes in the legend are sorted from most abundant to lowest abundant.
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Figure 3. Microbial composition at the genus level of juvenile European seabass gut microbiota group by diet (control and Allium-derived PTSO). Genera in the legend are sorted from most abundant to lowest abundant.
Figure 3. Microbial composition at the genus level of juvenile European seabass gut microbiota group by diet (control and Allium-derived PTSO). Genera in the legend are sorted from most abundant to lowest abundant.
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Figure 4. LDA Effect Size (LEfSe) analyses showing bacterial classes and genera that differ significantly between control fish and those supplemented with Allium-derived PTSO, in the foregut and in the hindgut of European seabass juveniles. Significant LDA Score > 4.0.
Figure 4. LDA Effect Size (LEfSe) analyses showing bacterial classes and genera that differ significantly between control fish and those supplemented with Allium-derived PTSO, in the foregut and in the hindgut of European seabass juveniles. Significant LDA Score > 4.0.
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Antibiotics 12 00134 g005 550
Figure 5. Dimensional figures showing the first two axes of principal coordinate analysis and representing bacterial communities of foregut and hindgut of juvenile European seabass fed with control diet or supplemented with Allium-derived PTSO using unweighted and weighted UniFrac distance matrixes. Samples are colored by gut region and experimental diet (foregut control—red; hindgut control—blue; foregut Allium-derived PTSO—yellow; hindgut Allium-derived PTSO—green). Proportion of explained variance by each PCo axis is shown.
Figure 5. Dimensional figures showing the first two axes of principal coordinate analysis and representing bacterial communities of foregut and hindgut of juvenile European seabass fed with control diet or supplemented with Allium-derived PTSO using unweighted and weighted UniFrac distance matrixes. Samples are colored by gut region and experimental diet (foregut control—red; hindgut control—blue; foregut Allium-derived PTSO—yellow; hindgut Allium-derived PTSO—green). Proportion of explained variance by each PCo axis is shown.
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Table
Table 1. General linear mixed models exploring the effects of diet in body weight in European seabass juveniles fed with a control diet or supplemented with Allium-derived PTSO along 89 days of experiment. Significant p-values are shown in bold.
Table 1. General linear mixed models exploring the effects of diet in body weight in European seabass juveniles fed with a control diet or supplemented with Allium-derived PTSO along 89 days of experiment. Significant p-values are shown in bold.
ControlAllium-Derived PTSOp
Body Weight Day 0 (g/fish)3.72 ± 0.053.84 ± 0.020.110
Body Weight Day 12 (g/fish)4.64 ± 0.064.70 ± 0.040.438
Body Weight Day 26 (g/fish)5.90 ± 0.125.87 ± 0.090.818
Body Weight Day 42 (g/fish)8.21 ± 0.128.14 ± 0.080.640
Body Weight Day 63 (g/fish)11.96 ± 0.0912.25 ± 0.100.089
Body Weight Day 89 (g/fish)21.14 ± 0.2122.08 ± 0.080.013
Table
Table 2. General linear mixed models exploring the effects of fish experimental diet (control and supplemented with Allium-derived PTSO) and gut region in different alpha diversity indices of bacterial community of juvenile European seabass. D.f. refers to degree of freedom. Significant p-values are shown in bold.
Table 2. General linear mixed models exploring the effects of fish experimental diet (control and supplemented with Allium-derived PTSO) and gut region in different alpha diversity indices of bacterial community of juvenile European seabass. D.f. refers to degree of freedom. Significant p-values are shown in bold.
Alpha Diversity IndexExplanatory VariablesD.fFp
Diet117783.97<0.001
Chao1 IndexGut Region11770.020.899
Diet*Gut Region11771.520.220
Diet117779.90<0.001
Faith PDGut Region11770.020.903
Diet*Gut Region11770.360.547
Diet117795.10<0.001
OTUs RichnessGut Region11770.210.652
Diet*Gut Region11770.550.459
Diet11776.510.012
Shannon Diversity IndexGut Region117715.23<0.001
Diet*Gut Region11770.960.330
Table
Table 3. Permutational ANOVA (PERMANOVA) exploring the effects of diet, gut region, and their interaction in beta diversity indices of bacterial community of European seabass juveniles fed with control diet or supplemented with Allium-derived PTSO. D.f. refers to degree of freedom. Significant p-values are shown in bold.
Table 3. Permutational ANOVA (PERMANOVA) exploring the effects of diet, gut region, and their interaction in beta diversity indices of bacterial community of European seabass juveniles fed with control diet or supplemented with Allium-derived PTSO. D.f. refers to degree of freedom. Significant p-values are shown in bold.
β-Diversity Distance MatrixExplanatory VariablesD.fPseudo-Fp
Diet117731.510.001
Weighted UniFracGut Region117714.000.001
Diet*Gut Region11770.980.409
Diet11778.890.001
Unweighted UniFracGut Region11771.050.325
Diet*Gut Region11770.940.595
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MDPI and ACS Style

Rabelo-Ruiz, M.; Peralta-Sánchez, J.M.; Martín-Platero, A.M.; Ruiz, A.J.; Agraso, M.d.M.; Bermúdez, L.; Ariza, J.J.; Baños, A.; Valdivia, E.; Martínez-Bueno, M. Allium-Derived Compound Propyl Propane Thiosulfonate (PTSO) Reduces Vibrio Populations and Increases Body Weight of European Seabass (Dicentrarchus labrax) Juveniles. Antibiotics 2023, 12, 134. https://doi.org/10.3390/antibiotics12010134

AMA Style

Rabelo-Ruiz M, Peralta-Sánchez JM, Martín-Platero AM, Ruiz AJ, Agraso MdM, Bermúdez L, Ariza JJ, Baños A, Valdivia E, Martínez-Bueno M. Allium-Derived Compound Propyl Propane Thiosulfonate (PTSO) Reduces Vibrio Populations and Increases Body Weight of European Seabass (Dicentrarchus labrax) Juveniles. Antibiotics. 2023; 12(1):134. https://doi.org/10.3390/antibiotics12010134

Chicago/Turabian Style

Rabelo-Ruiz, Miguel, Juan Manuel Peralta-Sánchez, Antonio Manuel Martín-Platero, Ana J. Ruiz, María del Mar Agraso, Laura Bermúdez, Juan José Ariza, Alberto Baños, Eva Valdivia, and Manuel Martínez-Bueno. 2023. "Allium-Derived Compound Propyl Propane Thiosulfonate (PTSO) Reduces Vibrio Populations and Increases Body Weight of European Seabass (Dicentrarchus labrax) Juveniles" Antibiotics 12, no. 1: 134. https://doi.org/10.3390/antibiotics12010134

APA Style

Rabelo-Ruiz, M., Peralta-Sánchez, J. M., Martín-Platero, A. M., Ruiz, A. J., Agraso, M. d. M., Bermúdez, L., Ariza, J. J., Baños, A., Valdivia, E., & Martínez-Bueno, M. (2023). Allium-Derived Compound Propyl Propane Thiosulfonate (PTSO) Reduces Vibrio Populations and Increases Body Weight of European Seabass (Dicentrarchus labrax) Juveniles. Antibiotics, 12(1), 134. https://doi.org/10.3390/antibiotics12010134

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