Complete genome sequence and characterization of a novel Enterococcus faecium with probiotic potential isolated from the gut of Litopenaeus vannamei

Abstract

Litopenaeus vannamei, the Pacific whiteleg shrimp, is one of the most marketable species in aquaculture worldwide. However, it is susceptible to different infections causing considerable losses in production each year. Consequently, using prebiotics that promotes the proliferation of beneficial bacteria and strengthen the immune system is a current strategy for disease control. In this study, we isolated two strains of E. faecium from the gut of L. vannamei fed with agavin-supplemented diets. These isolates showed antibacterial activity against Vibrio parahaemolyticus, Vibrio harveyi and Vibrio alginolyticus , most likely due to peptidoglycan hydrolase (PGH) activity. Furthermore, we sequenced the genome of one isolate. As a result, we observed three proteins related to the production of bacteriocins, a relevant trait for selecting probiotic strains since they can inhibit the invasion of potential pathogens. Additionally, the genome annotation showed genes related to the production of essential nutrients for the host. It lacked two of the most common factors associated with virulence in Enterococcus pathogenic strains (esp and hyl). Thus, this host-probiotic-derived strain has potential application not only in shrimp health but also in alternative aquatic environments, as it is adapted to coexist within the gut shrimp microbiota, independently of the diet.

Data Summary

All supporting data, code and protocols have been provided within the article or through supplementary data files. Two supplementary figures and seven supplementary tables are available with the online version of this article.

Impact Statement.

We present here the functional and genomic characterization of a potential probiotic E. faecium strain isolated from the intestine of L. vannamei fed with prebiotics. This approach represents an ideal strategy to search for probiotics as part of a healthy, efficient and sustainable aquaculture. The combined application of prebiotics and probiotics is also the most appropriate approach to prevent and treat diseases in shrimp aquaculture avoiding the use of antibiotics.

Introduction

Aquaculture is crucial for ensuring global sustainable food production and regional food security participation [1]. One of the most successful species worldwide is Litopenaeus vannamei, the Pacific whiteleg shrimp. Its success lies in the broad adaptation to aquaculture environments. This shrimp specie has the best growth yields due to the higher availability of genetically selected viral-pathogenic free (SPF) and higher feed conversion ratios in domesticated broodstock. It has a low protein requirement, a lower susceptibility to pernicious viruses, and better tolerance to environmental fluctuations under captivity [2]. It currently accounts for over 50% of crustacean production [1] and over 80 % of shrimp aquaculture production [3] worldwide.

However, the prevalence of diseases is a critical challenge restraining the consolidation of aquaculture as an economically viable and ecologically sustainable activity, with viruses and bacteria as the primary pathogens in shrimp farming, causing an estimated annual loss in production of 1–4 billion dollars [4, 5]. One of the leading opportunistic pathogens in penaeid shrimp are species of the genus Vibrio , which produce vibriosis [6–8], and the pathogenic strains of Vibrio parahaemolyticus (Vp _AHPND) responsible for acute hepatopancreatic necrosis (AHPND) [9–11]. In 2013, global shrimp production dropped 10 % due to AHPND [12]. Furthermore, the use of antibiotics to treat infections in aquaculture has been discouraged due to the negative impact of its intensive application on the environment and human health and the growing emergence of resistant strains [13–16].

Consequently, current strategies to make aquaculture a sustainable and environmentally friendly activity focus on biological disease control. In this sense, prebiotics, defined as non-digestible food ingredients that promote the proliferation or activity of beneficial bacteria that strengthen the immune system and lead to animal well-being, is rising [17]. In particular, shrimp diets supplemented with prebiotics have enhanced the immune response and increased survival against pathogens, improving growth performance and food efficiency. The main prebiotics used include inulin [18], fructooligosaccharides or FOS [19–21], manooligosaccharides or MOS [22], galactooligosaccharides or GOS [23], isomaltooligosaccharides or IMOS [24], and xylooligosaccharides or XOS [25].

Fructans include agavins, a highly branched type of inulin synthesized by agave plants [26, 27]. Despite multiple studies showing the benefits of agavin use in human health, studies in L. vannamei are still scarce. However, there is already evidence of improved survival against pathogens due to the strengthened immune response derived from inulin consumption in the diet [28, 29]. In addition, the role of agavins as prebiotics on the gut and hepatopancreas microbiota of L. vannamei has been recently reported, as these fructans are associated with improved growth performance parameters and the presence of beneficial microbes in the shrimp microbiota [30]. Furthermore, these same researchers have studied the microbiome of healthy and diseased white shrimps, concluding that healthy shrimp have enriched bacterial genera with probiotic potential [31].

Indeed, similar to prebiotics, probiotics have emerged as an effective and environmentally friendly alternative to manage diseases in shrimp culture [32, 33]. Particularly in L. vannamei, several authors have reported that probiotics improve growth and survival by enhancing nutrient digestibility and absorption, fighting Vibrio pathogens, and stimulating immune components [34–40]. Several commercial probiotic products are available, including multi-species bacterial preparations in shrimp aquaculture, particularly lactic acid bacteria (LAB). Such is the case of AquaStar Growout [41], PrimaLac [42] and ECOFORCE [43, 44]. These products contain strains of Bacillus subtilis , Pediococcus acidilactici , Enterococcus faecium and Lactobacillus reuteri (AquaStar Growout), Lactobacillus acidophilus , Lactobacillus casei , Enterococcus faecium and Bifidobacterium bifidum (PrimaLac), and Streptococcus faecalis , Streptococcus faecium , Bacillus mesentericus, Bacillus subtilis , Bacillus natto and Clostridium butyricum (ECOFORCE). However, scarce efforts have been made to isolate endogenous probiotics or indigenous beneficial microorganisms directly from the gut microbiota of the white shrimp [45–47].

From the above, the isolation of bacteria with probiotic potential from the gut microbiota of healthy shrimp-fed agavin represents an ideal strategy for searching for and developing probiotics and indigenous microbes for shrimp aquaculture. Here we present the isolation and characterization of indigenous bacteria from the intestine of L. vannamei previously fed with prebiotics. This approach could represent an ideal strategy to develop probiotics to prevent and treat diseases in shrimp aquaculture, avoiding antibiotics.

Methods

Isolation of lactic acid bacteria from the gut of Litopenaeus vannamei

Guts of 4-month-old Litopenaeus vannamei were aseptically dissected in situ using shrimp living in semi-intensive culture ponds and fed with agavin-supplemented diets as the fibre source in an aquaculture farm located in northern Sinaloa state (28.4030° N–111.4513° W). Gut samples were stored at −80 °C until use.

Cultures were prepared with gut samples in De Man, Rogosa, and Sharpe liquid medium (MRS) supplemented with 0.5 g l^–1 l-Cysteine-HCl (Merck). The MRS medium was selected to facilitate LAB enrichment relative to other genera in the gut microbiota. Cultures were prepared in medium supplemented with 2 % glucose (culture 1) or 2 % agavin (w/v) (culture 2) as the carbon source: from these two cultures, bacterial stocks were prepared (Supplementary Material, available with the online version of this article). To reactivate the bacteria, O₂-free serum flasks containing 50 ml of medium were inoculated with 1 ml of cell stocks from cultures 1 and 2 (after two passes in the case of the agavin-supplemented medium), followed by overnight incubation at 37 °C and 180 r.p.m.; then, 2.5 ml of this inoculum was transferred to 50 ml of MRS/L-Cysteine-HCl medium. In the late exponential phase, serial dilutions were performed and 10 ml of MRS/L-Cysteine-HCl solid media supplemented either with glucose (culture 1) or agavin (culture 2) were inoculated with 500 µl of each dilution; both media were previously bubbled with CO₂ in O₂-free flasks to promote anaerobiosis. Flasks were incubated at 37 °C for 24 to 48 h. For subsequent analyses, colonies were selected based on the typical morphology of Gram-positive, non-sporulated and catalase-negative LAB, cocci or bacilli. To note, an isolated strain identified as Klebsiella pneumoniae was discarded as a potential probiotic at this stage for safety reasons, despite being capable of growing in agavin and testing catalase positive.

Agavin origin and structure

Agavins are inulins, i.e. fructose polymers (fructans); the ones obtained for the experiments in the present study were extracted from Agave tequilana Weber var. azul as described previously [30]. Agavins were structurally characterized by gel permeation chromatography (GPC) with a linear Ultrahydrogel column (Waters, Japan) using 0.1 mM NaNO₃ as eluent at 30 °C and 0.8 ml min⁻¹. Using sucrose, 1-ketose, nystose, fructosyl-nystose and dextrans as standards. We determined that these agavins had a weight average molecular weight (Mw) of 5890 Da and a number average molecular weight (Mn) of 3 000 Da, with a polydispersity index (PI) of 1.96. This is equivalent to an estimated degree of polymerization of 17. In terms of oligosaccharide content, agavins were characterized by HPAEC-PAD (high-performance anion exchange chromatography coupled to a pulsed amperometric detector) in a Dionex instrument with a CarboPac PA-200 (2 mm × 250 mm) column and an ED40 Electrochemical Detector. The column was equilibrated at 30 °C with 100 mM NaOH (J.T. Baker, Center Valley, USA) at a 0.5 ml min⁻¹ flow rate. Fructan oligosaccharides were eluted with a sodium acetate (J.T. Baker, Center Valley, USA) gradient: 5–100 mM for 20 min and 100–300 mM for 40 min, followed 300 mM for 15 min and then 15 min for initial conditions re-equilibration. A wide diversity of fructan oligosaccharides may be observed in the chromatogram obtained in this analysis, demonstrating the complex structure of this fructan source. This product profile is similar to the one reported for agavins composed of linear (β−2,1) and branched (β−2,6) graminans and neofructans.

V3-V4 sequencing of microbiota from enriched bacterial cultures from L. vannamei gut samples

From culture one stocks (culture enriched with gut bacteria grown in glucose), 500 µl were collected at the beginning and in the late exponential phase (6 h) and transferred to sterile 1.5 ml Eppendorf tubes. Samples were centrifuged (Sorvall Biofuge) at 15 000 r.p.m. and 4 °C for 15 min. Afterwards, pellets were resuspended in 1 ml RNAlater solution (ThermoFisher Scientific, Waltham, MA, USA), followed by freezing at −80 °C until treatment. DNA was extracted using the commercial kit Quick DNA Faecal/Soil Microbe Miniprep kit (Zymo Research, Irvine, CA, USA), following the manufacturer’s protocol. DNA integrity and concentration were determined by agarose gel electrophoresis and Qubit (Invitrogen, CA, USA), respectively. Next, the V3-V4 hypervariable region of the 16S rRNA gene was amplified using the universal primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 533R (5′-TTACCGCGGCTGCTGGCAC-3′) [48]. All the reactions were adjusted to a final volume of 25 µl and amplified under the following conditions: 95 °C for 3 min; 25 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, and a final extension step at 72 °C for 5 min. The resulting PCR products were purified with Ampure XP beads (Beckman Coulter, CA, USA) and barcoded according to the Illumina Sequencing Library Preparation users guide. Finally, each library concentration and size distribution were assessed with a Qubit fluorometer and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively. The libraries were sequenced in an Illumina MiSeq platform with a 2×250 Paired-End format at the Sequencing Unit of the Instituto Nacional de Medicina Genómica (National Institute of Genomic Medicine), Mexico.

Data preprocessing and taxonomy assignation

Pretreatment of the raw data included the following steps: removal of barcodes and primers, joining of paired ends, quality filtering of sequences with <Q20 in 4 bp sliding windows, and depletion of ambiguous bases. The sequences were clustered into operational taxonomic units (OTUs) based on 97 % sequence similarity against the Greengenes (GG) database (v13.8) using uclust. Singletons were excluded from downstream analyses. The OTU table generated was filtered to discard OTUs with total abundance <0.005 %, as previously suggested, to eliminate the least abundant OTUs. Finally, the alpha and beta diversity metrics from the OTU table were obtained using QIIME (v1.9).

Detection of beneficial microorganisms in the microbiota

We investigated the presence of E. faecium and K. pneumoniae in shrimp samples using the Silva132 database, which includes 16S rRNA reference sequences for 70 species previously reported as beneficial microorganisms [30]. To this end, we constructed a new BIOM table in which OTUs were generated with uclust and the Silva132 reference database with a 97 % identity level. From the newly generated BIOM table, the taxonomy was assigned at the genus level with Qiime (v1.9.1) using the command summary_taxa_through_plots.py. Finally, the relative abundance of the microorganisms: Enterococcus, Klebsiella, E. faecium and K. pneumoniae , was estimated from these taxonomy tables.

Identification of isolates by sequencing the complete 16S rRNA gene

Chromosomal DNA from isolates was extracted with the commercial UltraClean Microbial DNA Isolation Kit (MOBIO Laboratories, Carlsbad, CA, USA) as per the protocol. The complete sequence of the ribosomal 16S rDNA gene was amplified using the primers fD1 (forward) 5′-CCGAATTGTCGAACAGTTTGATCCTGGCTCAG-3′ and rD1 (reverse) 5′-CCCGCGATCCAGCTTAAGGGTCCAGCCAGCCAGCCAGT-3′ [49]. PCR reactions were performed with 50 ng of DNA from isolates in a final volume of 50 µl, with 2.5 U of Pfu DNA polymerase (Thermo Scientific, USA), 1 µl of primers, and 2.0 mM MgSO₄ under the following conditions: 1 cycle at 95 °C for 3 min; 30 cycles at 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 3 min; and a final step at 72 °C for 10 min. Amplicons were purified with the GeneJET PCR Purification Kit (Thermo Scientific, USA), following the supplier’s instructions. The purified products were sequenced at the Synthesis and Sequencing Unit, Instituto de Biotecnología (Institute of Biotechnology), UNAM. The resulting sequences were assembled with the DNA Baser Assembler program (v5.15.0) and analysed by blast (Basic Local Alignment Search Tool, National Center of Biotechnology Information) to define the identity of the sequences by local alignments of the sequence to be determined. The 16S ribosomal RNA Sequences database (Bacteria and Archaea) was used for this purpose.

Incubation conditions, growth kinetics and assessment of growth under different salinity and pH conditions

Each isolate was stored separately in an Eppendorf tube with MRS/glycerol medium (1 : 1 v/v) (refer to Supplemental Material A). Isolates were reactivated by transferring 100 µl to 10 ml of MRS medium, followed by incubation at 37 °C and 200 r.p.m. for 18 h in aerobic and anaerobic conditions. To perform the growth kinetics, 4 % (v/v) pre-inoculum was added to two flasks: one with fresh MRS medium for aerobic kinetics and another with degassed medium for anoxic conditions; both were incubated at 37 °C and 200 r.p.m. The evolution of the cultures was monitored over 24 h by measuring the pH (Metrohm) and the optical density at 600 nm (OD₆₀₀) (Perkin-Elmer spectrophotometer). For conducting growth tests at different pH values and salinity conditions, 10 ml of MRS medium was transferred to test tubes, and the pH was adjusted to 4.0, 5.0, 6.0, 7.0 and 8.0; also, tubes containing MRS media were supplemented with 6.5, 10 and 12 % (w/v) NaCl. One hundred μl of reactivated inoculum at the exponential growth stage was added to each tube and incubated at 37 °C for 24 h; afterwards, the growth of the strains was reported for each culture condition. In all cases, experiments were run in triplicate.

Detection of antibacterial activity of isolate strains

The qualitative antibacterial activity of the isolates was tested and compared versus microorganisms of the genus Vibrio reported as pathogens in shrimp aquaculture, as follows. A 100 µl aliquot with OD_{600 nm} adjusted to 1 of an overnight culture of isolates grown in MRS medium was spotted onto fresh MRS plates in quadruplicate and incubated at 37 °C for 18 h. The resulting growth was coated with 5 ml of soft Brain Heart Infusion agar (BHI agar, 0.75 %) containing 500 µl of a culture of pathogenic bacteria with a OD_{600 nm} adjusted to 0.18–0.2. Plates were incubated at 37 °C for 18–24 h and the inhibition halo produced was measured with a millimetre ruler. Control experiments were performed without the isolates. These tests were performed using the pathogens Vibrio parahaemolyticus NCTC 1093, Vibrio harveyi 89 375, and Vibrio alginolyticus 98 393, kindly supplied by Dr Armando Navarro Ocaña of the Department of Public Health of the Faculty of Medicine at UNAM.

Collection of supernatants and in vitro antibacterial activity assays by zymography

Culture supernatants of late logarithmic-phase isolates were collected by centrifugation at 8 000 r.p.m. and 4 °C for 15 min (Biofuge Primo R). The pH was adjusted to 7.0 using one eq/L NaOH and the supernatant was microfiltered through a sterile 0.22 µm membrane (Durapore Membrane Filters, Millipore, USA) and stored at 4 °C. The supernatants thus obtained were used for zymograms. Protein concentration in the supernatant was determined by the Bradford method using a commercial kit (Protein Assay Dye Reagent Concentrate, BioRad Laboratories) and a standard curve of bovine serum albumin in an Epoch plate reader (Biotex). All measurements were made in duplicate.

To evaluate extracellular lytic activity, electrophoresis was initially performed in 12 % sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). To this end, the supernatants were mixed with 4X load buffer (0.5 M Tris-HCl, 10 g/100 ml SDS, 20 g/100 ml glycerol, 0.005 g/100 ml bromophenol blue, pH 6.8). To avoid loss of enzyme activity, samples were neither heated nor treated with β-mercaptoethanol. A Mini Protean 3 System (BioRad Laboratories) was used at 80 V for 30 min, followed by 120 V for 2 h at 4 °C. Gels were stained with Coomassie blue and silver staining. For zymograms, renaturing SDS-PAGE electrophoresis was performed [50] using 12 % polyacrylamide gels and cells of target microorganisms as substrate. To this end, pathogen strains cultured for 12 h at 37 °C were centrifuged at 8 000 r.p.m. for 15 min at 4 °C; the pellets obtained were resuspended in acrylamide solution before the polymerization step. After running the gels under the same electrophoretic conditions, they were washed three times with distilled water and incubated in a renaturing solution (Tris-HCl 100 mM pH eight and Triton X-100 1 % v/v) for 18–24 h at 37 °C with gentle stirring. To enhance the contrast, isolates were stained with a 0.1 % (v/v) methylene blue solution in 0.01 % (v/v) KOH for 20 min, followed by de-staining with distilled water. Lytic activity was detected by the presence of translucent bands against an opaque blue background.

Images were captured using a gel imaging system (GelDoc, BioRad). The electrophoretic profile was compared with the Precision Plus Protein Unstained Standards (BioRad) molecular weight marker using the Gel Doc (BioRad) Image Lab software to estimate the approximate molecular weight of band(s) with lytic activity.

Sequencing of E. faecium and genome assembly

First, raw data were pretreated as follows: barcodes, sequences with an average quality <Q20 and sequences with length <75 nt were eliminated. Then, we used assemblers SPADES and IDBA independently for the de novo assembly using the recommended default parameters. Subsequently, we merged the assemblies with Metassambler. Afterward, we blasted the longest scaffold against the complete genomes reported for E. faecium and selected the best ones, consisting of 13 genomes. Then, we used the genomes selected and the Metassembler assembly as input to MEDUSA to fill the gaps in our assembly; we repeated this step until we obtained a single and final scaffold. Finally, we used bowtie2 to realign the quality-filtered sequences against the final assembly to validate it. We predicted 2 542 total proteins with Prodigal (https://github.com/hyattpd/Prodigal) using default parameters. All proteins were analysed against the non-redundant (nr) database with blastP, setting the E-value cutoff at 1.0E⁻³ in Blast2Go [51]. All proteins were functionally mapped to GO terms setting the following parameters: E-value-hi-filter: 1.0E⁻³; Annotation cutoff: 55; GO weight: 5, and Hsp-Hit Coverage cutoff: 0. Additionally, we used InterProScan [52] to annotate the protein families. The map of the assembled genome was built with DNAplotter.

GO and InterPro search associated with carbohydrate modification

The GO and InterPro annotation obtained with Blast2Go was used to search terms of interest such as ‘carbohydrate hydrolases’, ‘bacteriocin’ and ‘peptidoglycan hydrolases’. All proteins positive to the search were considered for the pie chart, and ‘carbohydrate hydrolases’ were further sorted according to their specificity.

Search of carbohydrate-active enzymes (CAZy)

The 2 542 predicted proteins from E. faecium were annotated with hmmscan against the carbohydrate-active enzymes (CAZy) database [53] from http://bcb.unl.edu/dbCAN2/download/Databases/dbCAN-old@UGA/ containing 921 174 sequences. This database was previously formatted with hmmpress using default parameters. The results were then filtered using hmmscan-parser.sh and in-house scripts. Finally, the CAZy family names (Glycoside Hydrolases, GlycosylTransferases, Carb-Esterases, Carb-binding module, Auxiliary Activities, Polysaccharide Lyases) were searched in the GO description obtained with Blast2Go. All proteins positive to the search were added to the list of carbohydrate-active proteins.

Identification of virulence factors, antibiotic resistance genes (ARGs) and KEGG metabolic pathways

The Virulence Factor Database (VFDB) [54] was used to identify virulence factors in the E. faecium assembly. Briefly, the assembly was analysed on the VFanalyzer website specifying Enterococcus as the genus and selecting the raw fasta sequence of a complete genome option. E. faecium NRRL B-2354 was selected as a representative genome and all E. faecium deposited strains were selected for comparison.

For the identification of antibiotic resistance genes (ARGs), the 2 542 proteins of E. faecium were analysed against the Comprehensive Antibiotic Resistance Database (v3.1.4) [55] with blastP, setting the E-value cutoff at 1.0E⁻³. The proteins were considered positive with an identity ≥80 % and a coverage ≥99 %.

Finally, the KEGG Automatic Annotation Server (KAAS) [56] was used to map all E. faecium proteins to KEGG metabolic pathways using the bi-directional best hit (BBH) method and default parameters.

Results and discussion

Structure of the bacterial community in enriched gut sample cultures

We dissected the gut from L. vannamei shrimps previously fed under farming conditions with a diet supplemented with agavin [30]. To isolate beneficial microbes, particularly lactic acid bacteria and Bifidobacterium , the gut samples were deposited in a MRS medium supplemented with either glucose (culture 1) or agavin (culture 2) as a carbon source. The genus at the beginning and after 6 h of culture 1 identified by 16S rRNA sequencing are shown in Figs 1 and 2.

Fig. 1.

Taxonomic assignment of bacterial genera present in a shrimp gut microbiota sample cultured in an enriched medium (culture 1, at 0 h). The taxonomic designation was carried out using the Green Genes database. Asterisk (*), LAB used as probiotics in aquaculture.

Fig. 2.

Taxonomic assignment of bacterial genera present in a shrimp gut microbiota sample cultured in an enriched medium (culture 1 after 6 h). The taxonomic designation was carried out using the Green Genes database. Asterisk (*) LAB used as probiotics in aquaculture. See Fig. S1 for a comparison of data at the genus level genera of Figs 1 and 2.

Interestingly the V3-V4 rRNA sequencing data of the bacteria present in a shrimps’ gut directly after dissection showed the presence of 147 genera, while in the MRS culture we observed 96 genera. This suggests that after cultivating the shrimps’ gut in a MRS medium, we keep 23.81 % of the original genera (Fig. S1, available with the online version of this article).

To note, the components of the MRS medium also promoted the growth of bacteria belonging to genera with previous reports of probiotic activity, such as Enterococcus , Lactococcus , Lactobacillus and Bacillus , which altogether accounted for about 61.0 % of the microbiota. In contrast, the presence of potentially pathogenic bacteria such as Vibrio , Anaerovibrio , and Pseudomonas decreased, even to undetectable levels, as was the case of Enterovibrio . As expected, the culture medium enriched a different abundance composition of the typical genus found in the gut shrimp microbiota [57].

A fact worth stressing is that the lower abundance of Vibrio after 6 h of culture may be the unfavourable result of competition with other bacteria for nutrients in the medium and the effect of antimicrobial factors, anaerobic conditions or the pH of the culture medium. This would also explain the findings observed for one member of the Clostridiaceae , a slow-growing bacterium that is also a poor competitor.

Another aspect to highlight is that 6 h of culture in an enriched medium yielded a higher relative abundance of Faecalibacterium sp., a strictly anaerobic bacterium that promotes human intestinal health. It has been shown that the Faecalibacterium genus is enriched in the microbiota of healthy L. vannamei populations compared to diseased shrimps [31]. It is essential to consider that LABs other than lactobacilli can also utilize the components of the MRS medium, which is evident in our study, given the remarkable growth of isolates of the genus Enterococcus . Coincidentally, it has been reported that enterococci strains grow adequately in MRS medium. Furthermore, this is the medium in which the highest production of antibacterial chemicals by Enterococcus has been reported [58], an additional element to explain the decrease of Clostridium and other pathogenic gut bacteria initially found.

Taxonomic identification of isolates by 16S rDNA sequencing and growth kinetics

Individual colonies were isolated in MRS agar from cultures 1 and 2. As a result, four strains were selected according to their morphological characteristics, isolated and designated as I3G, I3A, I4A1 and I4A2. These isolates were identified as E. faecium (strains I3G and I3A) and K. pneumoniae (strains I4A1 and I4A2) using the complete 16S rDNA sequence by Sanger, with identity values of 99 % for strains I3G and I3A and 98 % for strains 14A1 and I4A2.

A kinetic study of E. faecium I3G and I3A was carried out to have sufficient biomass of both strains to assess their production feasibility and evaluate their antibacterial capacity. The synthesis of Enterococcus antibacterial proteins is related to growth, with peak synthesis occurring during the late-exponential or early stationary phase [59]. Fig. 3 illustrates the growth kinetics of the two strains under aerobic and anaerobic conditions.

Fig. 3.

Growth kinetics of the two isolated strains of E. faecium I3A and I3G under aerobic and anaerobic conditions. In aerobiosis (a); in anaerobiosis (b), the left graphs belong to strain I3G; the right graphs correspond to strain I3A. Both strains were cultured in MRS medium at 37 °C and 200 r.p.m. Circles represent optical density (OD_{600 nm}); triangles correspond to pH; error bars represent the standard deviation of three independent experiments.

It is evident that in cultures of both strains in MRS medium, either with or without oxygen, the lag phase is virtually non-existent, and the exponential phase is rapidly attained. Furthermore, the shortest doubling time (1 h) equivalent to a specific growth rate (μ) of ≈ 0.69 h⁻¹ was observed for strain I3A in the presence of oxygen, while strain I3G showed adequate growth both in aerobic (μ ≈ 0.46 h⁻¹) and anaerobic (μ ≈ 0.54 h⁻¹) conditions.

In agreement with the literature, we found that E. faecium isolates grow in a pH range of 5 to 8, with improved growth in alkaline media as E. faecium grows within a pH range of 4.5 to 10, showing optimum growth at pH 7.5 [60]. When assessing the effect of salt concentration in the culture medium, we confirmed that the genus Enterococcus might grow in media with 6.5 % (w/v) NaCl, a phenotypic characteristic of the genus. In fact, we found that the strains may grow in the presence of up to 12 % (w/v) NaCl. The tolerance of E. faecium to alkaline pH values and high salt levels is due to P-ATPase activities [61, 62].

Although the E. faecium strains can grow in agavin, consumption of the high-molecular-weight fructans was not observed; the strains probably consumed only fructose, di-, and tri-fructooligosaccharides present in this complex sugar. It is a fact that several environmental factors affect the establishment of the gut microbiota in aquaculture systems. Therefore, it is reasonable to assume that the culture system, salinity, temperature, water quality, diet and dietary supplements, among others, influenced the establishment of the gut microbiota of L. vannamei samples from which E. faecium strains were isolated. This property is further discussed when describing the E. faecium genome.

The genus Enterococcus is a diverse group of ecological and clinical importance. Members of this genus are widespread in nature, including the gastrointestinal tract of humans and animals and in terrestrial and aquatic plants, sediments, and environments [63]. Enterococcus spp. is also an element of the native microbiota of fermented meat, vegetables and dairy products [64]. In addition, members of this genus can produce bacteriocins, which display activity against pathogens of importance in the food industry, such as species of Salmonella , Listeria and Clostridium , E. coli, S. aureus and others. On the other hand, it has been shown that some LAB species are common in fish, crustaceans, mollusks and shellfish, as well as in their aquatic environment and seafood. The genera Lactobacillus , Carnobacterium , Aerococcus , Enterococcus , Lactococcus , Leuconostoc , Pediococcus , Streptococcus , Vagococcus and Weissella are common components of the gut microbiota of aquatic animals [65, 66]. In particular, Enterococcus faecium has been isolated from various marine species such as white tuna, cod, squid, shrimp, crab, lobster, sea bass, ling, bivalve mollusks, octopus, Atlantic salmon, and rainbow trout, among others [67].

On the other hand, a recent study reported that LAB are the most abundant probiotic species in shrimp gut at the metagenomic level, with Enterococaceae as the most abundant family. In addition, strains of E. faecium displaying antimicrobial properties against pathogens of the whiteleg shrimp [68], antibacterial and adhesive activities [69], have been isolated from healthy shrimps.

As far as K. pneumoniae is concerned, to date it has been reported only as a potential probiotic of marine origin isolated from the shrimp Penaeus monodon and the lobster Panulirus ornatus [70]. This finding, together with some literature reports, points to the presence of K. pneumoniae in the gut microbiota of marine species and not only in livestock or humans.

However, as Klebsiella bacteria are a common source of hospital in-patient infections and animal disease, being resistant to antibiotics [71–73], we decided to proceed further only with the E. faecium isolates (strains I3G, I3A), given their probiotic potential. Recently, Du et al. [74], administered a commercial E. faecium powder produced by Yichun Strong Microbial Technology, Jiangxi, China, as a probiotic treatment in shrimp post-larvae gut. These authors found that the gut microbiota modifications correlate to both, shrimp survival and ingested E. faecium , concluding that these correlations uncover a promising strategy for developing novel probiotics through specific consortia of gut microbiota. However, this commercial strain was not isolated from the shrimp microbiota, suggesting a higher probiotic potential for a strain adapted to coexist in the shrimp gut and hepatopancreas, independently of the diet.

Tracking for the genera Klebsiella and Enterococcus in the shrimp microbiota under farming conditions, as well as alternative beneficial microbes

To know if the Klebsiella and Enterococcus genera are components of the shrimp microbiota from which the gut came, we used previously reported 16S rRNA sequencing data from the same set of shrimps fed diets containing 2 and 10 % (w/w) of agavin [30].

We found that both bacteria, i.e. E. faecium and K. pneumoniae, are part of the gut microbiota regardless of the shrimp diet, with an apparent abundance of K. pneumoniae when agavins are supplemented in the diet (Fig. 4a). The favourable in vitro K. pneumoniae growth, despite the presence of other bacteria from the gut sample, may be due to its ability to degrade agavins. In contrast, and as already mentioned, while the genus Enterococcus was enriched in agavin-fed shrimp, the presence of E. faecium was similar among diets and only appeared in a few samples (Fig. 4). However, when we analysed Enterococcus at the genus level, we observed enrichment in agavin diets (Fig. 4).

Fig. 4.

Relative abundance of K. pneumoniae , Enterococcus and E. faecium from 16S rRNA profiles of shrimp gut (Gut) samples fed with control, agavin 2 % and agavin 10 %.

We also analysed the abundance of already reported beneficial microorganisms for shrimp in culture 1. From 70 species previously suggested as producing a positive impact on shrimp health [30], we found 8 and 14 beneficial genera, respectively, in both culture times (Fig. 5). Interestingly, the probiotics of the genera Clostridium and Enterococcus accounted for about 80 % of all bacterial genera in both samples, and around 10 % of the species abundance, suggesting the potential of these cultures to provide novel indigenous and host-derived beneficial microorganisms for shrimp farming. We, therefore, proceeded with the characterization of the isolated E. faecium strains.

Fig. 5.

Relative abundance of probiotics in the 0 h and 6 h culture samples. Enterococcus lactis and E. faecium are the most abundant probiotics from the list of 70 probiotics species considering the Silva132 database and Uclust with 97 % of identity.

Antimicrobial effect of E. faecium on Vibrio

One of the basic criteria to define a probiotic strain is its antimicrobial activity against host pathogens. Thus, agar cell-to-cell tests were conducted to detect antimicrobial activity against Vibrio species known to cause disease in L. vannamei. The in vitro qualitative antimicrobial activity of E. faecium isolates against pathogenic Vibrio bacteria is shown in Fig. 6. Both strains exhibited significant antimicrobial activity against Vibrio ; however, when measuring the inhibition haloes’ average diameter, the I3G strain showed a significantly higher lytic activity.

Fig. 6.

Antimicrobial activities of E. faecium I3G and I3A against V. parahaemolyticus, V. harveyi and V. alginolyticus . Mean diameter values±sd inhibition zones (mm) observed for E. faecium I3G against V. parahaemolyticus and V. harveyi were 12.5±3.0 and 15.5±3.42, respectively. In the case of E. faecium I3A, values were 10.0±1.63 and 14.25±1.26, respectively. Values of inhibition zones against V. alginolyticus are omitted due to the heterogenous shape of the haloes.

E. faecium strains isolated from aquatic species have also demonstrated antimicrobial activity against fish pathogens such as Listonella anguillarum , Aeromonas hydrophila , Lactococcus garvieae , Streptococcus iniae , Streptococcus agalactiae , Photobacterium damselae , V. alginolyticus and V. campbellii . The antimicrobial activity of these strains has been attributed not only to the effect of organic acids, hydrogen peroxide and diacetyl, but also to protein compounds [67, 75].

Identification of lytic extracellular enzyme activity of E. faecium against Vibrio

The antibacterial activity of E. faecium may have resulted from the production of protein compounds such as bacteriocins or enzymes that degrade the cell wall of bacteria, like peptidoglycan hydrolases (PGHs); accordingly, zymography studies were carried out to identify these compounds. The results of these experiments detailed in the Methods and illustrated in Fig. 7, clearly show lytic activity via extracellular proteins for both strains of E. faecium against Vibrio parahaemolyticus , Vibrio harveyi and Vibrio alginolyticus , as evidenced by the presence of several protein bands with bacteriolytic activity on Vibrio strains.

Fig. 7.

Zymograms of E. faecium I3G and I3A cell-free extracts against V. parahaemolyticus , V. harveyi and V. alginolyticus . Lanes 1 and 4, zymogram against V. parahaemolyticus ; lanes 2 and 5, zymogram against V. harveyi ; lanes 3 and 6, zymogram against V. alginolyti cus; MW, high molecular weight marker (Bio-Rad). Lanes 1–3 correspond to E. faecium I3A, lanes 4–6 correspond to E. faecium I3G.

Enterococcus faecium I3G genome

To fully characterize the genomic potential of the strain and its properties, the genome of E. faecium strain I3G was sequenced and assembled as described in Methods. In a total of 2 542 proteins, we found that 99.8 % had a blastp annotation, 82.38 % had a GO term associated, and 90.28 % had an InterPro Scan match (see Fig. S2). Additionally, the term search showed 44 proteins annotated as carbohydrate hydrolases, 25 % related to glucosidase activities. All these proteins are listed in Table S1.

Production of antimicrobial compounds

Three genes encoding bacteriocins, antagonistic compounds against pathogenic bacteria, were identified. One of them was a bacteriocin identified as an integral membrane protein, along with two proteins related to bacteriocin immunity, an ABC bacteriocin-related transporter and a bacteriocin induction factor (Table S1). There are several reports of bacteriocins produced by E. faecium that are active against Gram-positive pathogens, mainly foodborne microbes such as L. monocytogenes , L. innocua , E. faecalis , S. aureus, S. epidermidis, Clostridium spp., Bacillus spp., and Propionibacterium spp. [76]. Studies addressing bacteriocins isolated from marine enterococci have also demonstrated to be active against common aquaculture pathogens such as V. penaeicida , Photobacterium damselae , V. harveyi, V. fischeri, V. anguillarum and V. parahaemolyticus [77, 78]. Additionally, bacteriocin production has been important for selecting probiotic strains, since they can inhibit the invasion of potential pathogens modulating the host microbiota in a positive manner [79]. Interestingly one study showed that mice colonized with a bacteriocinogenic E. faecium strain had a substantially greater abundance of Lactobacillus in their guts than mice colonized with a non-bacteriocinogenic E. faecium strain [80]. Thus, the production of class II bacteriocins is a positive antimicrobial mechanism exhibited by E. faecium strain I3G.

The functional annotation allowed the identification of PGH genes coding for N-acetylmuramoyl-l-alanine amidases and endo-beta-N-acetylglucosaminidases. In addition, several protein bands with lytic activity against pathogenic vibrios were identified in E. faecium I3G, with molecular masses of 42, 46, 51, 57, 65, 81 and 96 kDa. Previous reports on E. faecium have identified similar molecular mass bands of 95, 81 and 66 kDa through zymogram analysis against foodborne pathogens, and determined their identity as N-acetylglucosaminidases and N-acetylmuramoyl-l-alanine amidases by LC/ES/MS [81]. We therefore conclude that the compounds with bacteriolytic activity exhibited against vibrios by E. faecium I3G include PGHs. This constitutes a crucial potential probiotic asset of the isolated strain.

Analysis of CAZy enzymes

A set of 44 carbohydrate hydrolases were identified by genome annotation, mainly glycosidases and galactosidases. However, when a specific in-depth search for Carbohydrate Active Enzyme was performed among the 2 542 total proteins, the number of CAZy enzymes increased to 127, (Table S2). This plethora of hydrolases allows the strain to grow on diverse substrates, partially explaining its frequent presence in the shrimp microbiota and the media containing either glucose or agavin as carbon sources. Although no direct sequences corresponding to fructanase activity were identified, some glycosyl hydrolases may degrade oligosaccharides in complex carbon sources such as agavin, the fibre source. Among the glycoside hydrolases found, only one gene contained the GH32 domain, corresponding to a 55 KDa sucrose-6-phosphate hydrolase (E-value=0 and 100 % identity), a β-fructofuranosidase (E.C. 3.2.1.26), with FOS hydrolytic activity.

Analysis of virulence potential and antibiotic resistance

Strains from the Enterococcus genus are seldom considered as Generally Recognized As Safe (GRAS) microorganisms due to their association with antimicrobial resistance genes and virulence factors that can be transferred to other endogenous bacteria within the gastrointestinal tract. The genome analysis with the Virulence Factor of Bacterial Pathogens Database (VFDB) revealed eight proteins related to adherence mechanisms, three to capsule formation and two for biofilm formation (Table S3). These genes are also present in the reference strains deposited on the website for comparison. However, the presence of virulence genes does not convey that the carrying strain should be pathogenic or cause disease in a host. The simple presence of some players may be due to their contribution to the enterococci commensal lifestyle and their noninfectious interaction with the host [82]. On the other hand, some studies report that most E. faecium strains isolated from intra-hospital infection cases carry three virulence factors: enterococcal surface protein (esp), hyaluronidase (hyl) and collagen-binding adhesin (acm) [83]. From these, only the gene acm was identified in our isolate. Despite of the prevalence of the acm gen in E. faecium isolates, it is primarily functional in clinical multi-drug-resistant strains, and present as an inactive pseudogene within food-related isolates [84]. Similarly, determinants for collagen adhesion protein, aggregation substances, and capsule formation were identified in the probiotic E. faecalis Symbioflor1 strain [81] and virulence traits related to biofilm formation and adhesion in enterococci isolates from dairy products [85]. Hence, these genes may play a different role aiding in the colonization and proliferation of the gastrointestinal tract, a vital probiotic attribute, to combat pathogenic bacteria. This suggests that the strain I3G may have a lower potential of pathogenicity and may be innocuous to the host.

Genes related to macrolide, aminoglycoside and tetracycline resistance were detected in the E. faecium genome (Table S4). This resistance is intrinsic to the genus as it is the case for cephalosporins, beta-lactams, sulphonamides, clindamycin and aminoglycosides. Importantly, no vancomycin resistance genes were detected.

Metabolic activities associated with host-beneficial nutrient production

Given the relevance of vitamin and amino acid production as part of the nutritional elements of a probiotic to improve the host-health, genes encoding enzymes associated with the production of relevant nutrients for the host were identified using a KEGG metabolic pathway analysis. As a result, we found several genes that metabolize essential amino acids and vitamins for shrimp such as arginine, valine, methionine, valine and leucine (Tables S5 and S6).

Considering that a probiotic exerts a positive effect not only on its host but also on its environment, we searched for genes that participate in the degradation of common pollutants using the KEGG database. In this context, we found that E. faecium I3G is also capable of degrading xylene, styrene and dioxin, among other toxins (Table S7).

Conclusions

Previous studies have found favourable effects when agavin has been used as a fibre supplement in shrimp diets under farming conditions, particularly in a recent report from our group [30]. Here, we addressed the characterization and recovery of beneficial bacteria from the gut microbiota of shrimp samples fed agavin-supplemented diets. The sequencing of the V3-V4 16S rDNA allowed defining of the nature of LAB strains present in the bacteria culture of gut samples after incubation in a defined culture medium with glucose or agavin as a carbon source. In this sense, we confirmed that Enterococcus faecium and Klebsiella pneumoniae are components of the gut microbiota of L. vannamei and proceeded to isolate two strains of Enterococcus faecium , a species reported as having probiotic potential for application in aquaculture.

The Enterococcus faecium isolates exhibited antibacterial activity against V. parahaemolyticus , V. harveyi and V. alginolyticus – pathogens of L. vannamei in shrimp aquaculture – due to peptidoglycan hydrolase (PGH) activity, as demonstrated by the identification of PGH genes in the E. faecium I3G genome. The present finding is the first report of a PGH-type with bacteriolytic activity antagonistic to shrimp Vibrio pathogens. This is a potential antimicrobial mechanism in E. faecium against pathogen bacteria of significance in aquaculture. It may explain its presence in healthy shrimp microbiota. Moreover, the strain may be considered a valuable host-derived probiotic against Gram-positive and negative pathogenic bacteria, with the advantage of being effective even for antibiotic-resistant bacteria, given its mechanism of action [86]. We also showed that the strain has many hydrolytic enzymes that facilitate growth on diverse substrates, explaining its frequent presence in the shrimp microbiota. We acknowledge that even though the in vitro and genome characterization showed that E. faecium 13G isolate has the potential for beneficial functions in shrimps’ health, there is still the need for animal trials to confirm its probiotic character.