Ann Microbiol (2015) 65:2343–2353 DOI 10.1007/s13213-015-1076-3

ORIGINAL ARTICLE

High variability of levels of Aliivibrio and lactic acid in the intestinal microbiota of farmed Atlantic salmon Salmo salar L.

Félix A. Godoy1 & Claudio D. Miranda2,3 & Geraldine D. Wittwer1 & Carlos P. Aranda1 & Raúl Calderón4

Received: 10 November 2014 /Accepted: 11 March 2015 /Published online: 16 April 2015 # Springer-Verlag Berlin Heidelberg and the University of Milan 2015

Abstract In the present study, the structure of the intestinal structure of the intestinal microbiota of farmed Atlantic salm- microbiota of Atlantic salmon (Salmo salar L.) was studied on enabling detection of a minority of taxa not previously using culture and culture-independent methods. Three adult reported as part of the intestinal microbiota of salmonids, in- specimens of S. salar were collected from a commercial salm- cluding the genera Hydrogenophilus, Propionibacterium, on farm in Chile, and their intestinal microbiota were studied Cronobacter, Enhydrobacter, Veillonella, Prevotella,and by partial sequencing of the 16S rRNA gene of pure cultures Atopostipes, as well as to evaluate the health status of farmed as well as of clone libraries. Out of the 74 bacterial isolates, fish when evaluating the dominance of potential pathogenic Pseudomonas was the most predominant genus among cul- and the incidence of . tured microbiota. In clone libraries, 325 clones were obtained from three adult fish, and a total of 36 operational taxonomic Keywords Aquaculture . Intestinal microbiota . Aliivibrio . units (OTUs) were identified. This indicated that lactic acid Salmon farming . Salmo salar bacteria (Weissella, Leuconostoc, and Lactococcus genera) comprised more than 50 % of identified clones in two fishes. This was in contrast with the high dominance of a single OTU Introduction (99 sequences) of Aliivibrio sp. related to the pathogenic Aliivibrio salmonicida species and the absence of lactic acid Chile is currently a worldwide leading salmon producer and bacteria in the third fish, suggesting a condition of an asymp- the Atlantic salmon Salmo salar L. is one of the main Chilean tomatic non-healthy carrier. It is clear that molecular identifi- salmonid farming products, constituting 182,712 tons from cation of 16S rRNA gene libraries obtained from intestinal January to June of 2014, and comprising 64 % of total salmo- content samples is effective in determining the overall nid exports (SalmonChile 2014). It is well established that the intestinal tract of reared fish harbors a microbiota that fulfill an important role in immunity, nutrition, and disease control of * Félix A. Godoy reared fishes (Trust and Sparrow 1974; Ringø and Birkbeck [email protected]; [email protected] 1999; Moffitt and Mobin 2006). To study the microbiota of the gastrointestinal tract of fish- 1 Centro i ~ mar, Universidad De Los Lagos, Camino Chinquihue km es, the general approach has been the use of conventional 6, Casilla 557, Puerto Montt, Chile culture methods (Ringø et al. 1995). However, it has been 2 Laboratorio de Patobiología Acuática, Departamento de Acuicultura, found that these methods present several disadvantages and Universidad Católica del Norte, Larrondo 1281, Coquimbo, Chile usually only detect aerobics and facultative anaerobic bacteria, 3 Centro de Estudios Avanzados en Zonas Áridas (CEAZA), Larrondo but do not detect slow-growing bacteria (Spanggaard et al. 1281, Coquimbo, Chile 2000;Nayak2010). Thus, molecular analysis of DNA extract- 4 Escuela de Ciencias Ambientales, Facultad de Recursos Naturales, ed directly from the sample has rapidly replaced cultivation in Universidad Católica de Temuco, Temuco, Chile the study of the structure of fish intestinal microbiota. 2344 Ann Microbiol (2015) 65:2343–2353

Nonetheless, when some phenotypic properties such as enzy- methods, and to characterize some of the metabolic and enzy- matic activities need to be studied in order to understand the matic capabilities of the isolated strains. potential role of the microbiota in improving fish nutrition, it is more appropriate to study the fish intestinal microbiota composition by culture techniques in combination with Materials and methods culture-independent methods (Bakke-McKellep et al. 2007; Kristiansen et al. 2011;Askarianetal.2012). Sampling The intestinal microbiota composition is known to depend on dietary factors (Gómez and Balcázar 2008;Nayak2010; Three apparently healthy adult specimens of Atlantic salmon Hartviksen et al. 2014). Navarrete et al. (2013), using micro- (Salmo salar L.) with an approximate weight of 2.5 kg were biological analysis, demonstrated that specific bacterial collected from three different rearing cages belonging to a groups were correlated with the administered diet, and commercial salmon farm located at Punta Quilque, X Region, Reveco et al. (2014) reported that intestinal microbiota of Chile (13 °C, water temperature; 32 g L-1, salinity). Samples Salmo salar is sensitive to dietary changes, observing that were packed in sterile bags, placed on ice, immediately the most dominant species were Lactococcus lactis, Weissella transported to the laboratory, and processed within 2 h of confusa,andPhotobacterium phosphoreum.Otherwise, collection. Navarrete et al. (2008) suggested that Atlantic salmon favors Pseudomonas establishment because this species was de- Sample processing and cultured bacterial count tected as the dominant component in most of the samples of juvenile farmed Atlantic salmon. There are other studies Adult salmon were externally washed with sterile deionized that describe the intestinal microbiota of farmed Atlantic water to reduce potential contamination with skin bacteria and salmon; however, the majority of these studies are associ- aseptically eviscerated. Salmon intestines were aseptically re- ated with fingerling or juvenile stages (Bakke-McKellep moved and placed in sterile Petri dishes and were divided into et al. 2007; Navarrete et al. 2008;Cantasetal.2011; proximal intestine (defined as the region between the distal Navarrete et al. 2013; Reveco et al. 2014). pyloric caeca and widening of the intestine and the appearance It has been reported that fish intestinal microbiota have an of transverse luminal folds) and distal intestine (the region important role in regulating nutrient digestion, immune re- from the widening of the intestine and the appearance of trans- sponses, and intestinal differentiation (Bates et al. 2006; verse luminal folds to anus). Then, digesta from proximal and Kanther and Rawls 2010;Merrifieldetal.2010; Nayak distal intestine were gently squeezed out and the two intestinal 2010; Ray et al. 2012), so physiological and biochemical char- segments were thoroughly rinsed three times using 3 mL of acterizations of the intestinal isolates are important in eluci- peptone water in order to collect both adherent and non- dating their functions in the gastrointestinal tract. Several stud- adherent bacteria (Ringø 1993). Culture counts of heterotro- ies reported that freshwater fish reared in warm waters harbor phic bacteria were determined by a spread plate method using proteolytic, amylolytic, and cellulolytic bacteria in their diges- Tryptic soy agar (TSA, Difco Labs). Salmon intestinal con- tive tracts (Bairaigi et al. 2002; Ghosh et al. 2002;Sahaetal. tents samples were aseptically weighed, ground, and added to 2006; Kar et al. 2008), whereas it was reported that an increase 5 mL of sterile physiological saline (0.85 %) (PS) to obtain a in proteolytic enzymes such as trypsin and chymotrypsin in homogenate as previously described by Miranda and salmon induces a better assimilation of proteins, as well as an Zemelman (2001). Appropriate tenfold dilution of the homog- increase in the growth and stimulation of immune and endo- enates in PS was prepared and 0.1 mL aliquots were inoculat- crine systems (Rungruangsak-Torrissen et al. 2009). Addition- ed in triplicate onto agar plates. All plates were incubated at ally, it has been observed that the different bacterial popula- 20 °C for 5–10 days and the bacterial numbers per g of tions composing the intestinal microbiota represent different sample were calculated as described in Miranda and metabolic groups, which can enhance the digestive capacity of Rojas (2007). A group of representative colonies from fish (Ringø and Olsen 1999). Hence, knowledge of the enzy- each sample was selected for purity. matic capacities of the gastrointestinal microbiota of farmed salmon could help regulate the intestinal microbiota enhanc- Bacterial isolation ing nutrition performance of farmed fishes under intensive rearing conditions. However, only a few studies on adult salm- Seventy-four isolates were recovered as a representative sam- on are available, and knowledge of the enzymatic properties ple of the intestinal cultured bacterial community of farmed of the intestinal microbiota of reared salmon is still scarce. salmon. From these, 26 strains were recovered from specimen The main aims of this study were to investigate the com- 1 (17 and nine strains from the proximal and distal intestine, position of intestinal microbiota of adult specimens of farmed respectively), 25 strains from specimen 2 (14 and 11 strains Atlantic salmon, Salmo salar L. by culture and cloning from the proximal and distal intestine, respectively) and 23 Ann Microbiol (2015) 65:2343–2353 2345 strains from specimen 3 (nine and 14 strains from the proximal the color allowed to develop for 5 min and test strips were and distal intestine, respectively). The strains were randomly read. All assays were performed twice. selected from plates with TSA medium and purified three times in TSA medium and stored at −85 °C in Tryptic soy 16S rRNA gene library construction and sequencing broth (Difco Labs) supplemented with 50 % glycerol (2:1) until use (Gherna 1994). From a sample of 200 μL of the homogenate of the complete intestinal content containing the proximal and distal intestine Bacterial identification by 16S rRNA sequence analysis (1:1), bacterial DNA was extracted using the QIAMP DNA Stool kit (QIAGEN), according to the manufacturer’sguide- Isolates were identified by bacterial 16S rRNA gene sequence lines. Bacterial DNA was verified by the amplification of a analysis. For amplification of the 16S rRNA genes, crude fragment of rRNA 16S gene using the 27 F and 907R univer- DNA extracts were obtained from pure bacterial isolates using sal primers, as was previously described and visualized with the Wizard genomic purification kit (Promega, Madison, WI, 1 % agarose gels. PCR products were purified from agarose USA). The 16S rRNA gene was amplified by the polymerase gels with the Wizard SV Gel kit and PCR Clean-up System chain reaction (PCR) using primers 27F 5’-AGAGTTTGAT (Promega) and cloned using the TOPO TAvector according to CMTGGCTCAG-3’,1492R5’-TACGGYTACCTTGTTA the procedures indicated by Invitrogen. Cultures of CGACTT-3’, and 907R 5’-CCGTCAATTCMTTTGAGTT Escherichia coli JM 107 strain were made competent using T-3’ (Lane 1991). PCR products were purified using the Wiz- the Transform Aid Bacterial Transformation Kit (Fermentas), ’ ard SV Gel kit and PCR Clean-up System (Promega) and following the manufacturer s guidelines. Each clone was pick- sequencing of amplicons was performed by Macrogen (Seoul, ed and cultured in LB broth with ampicillin for 16 h. To isolate μ Korea). Identification of partial sequences was performed plasmidic DNA with the insert, 100 L of liquid culture was using the NCBI BLAST (http://www.ncbi.nlm.nih.gov/), and centrifuged at 6000g for 30 min, the medium was discarded, μ strains were considered to belong to the same genus when and the pellet was resuspended in 100 L of sterile water, similarities in their sequences were ≥97 % (Rosselló-Mora incubated at 95 °C for 30 min to produce cellular lysis, and μ and Amman 2001). The partial 16S rRNA gene sequences then centrifuged at 6000g for 30 min. Finally, 5 Lofthe were submitted to the Genbank database and assigned lysate was amplified to detect the occurrence of the insert, accession numbers JF743668 to JF767415. using the M13F and M13 R primers. PCR products were verified in 1 % agarose gels, purified and sequenced by Macrogen (Seoul, Korea). Phenotypic characterization of isolated strains Sequence analysis The phenotypic tests of Gram's stain, cell morphology, and oxidase production were determined according to the proce- Partial sequences for chimeras using the Chimera Check pro- dures described in Barrow and Feltham (1993). Additional gram from RPD (Ribosomal Database Project) (http:// phenotypic characteristics were determined by using the API fungene.cme.msu.edu/FunGenePipeline/chimera_check/ ’ 20E system (bioMérieux, Marcy-l Etoile, France), and strains form.spr) were analyzed. Clone sequences in this study were ’ were inoculated according to the manufacturer s instructions. aligned using the INFERNAL aligner from RDP, secondary- API 20E strips were incubated at 20 °C for 48 h. structure aware aligner (Nawrocki and Eddy 2007). Se- quences with similarities over 97.0 % were defined as one Enzymatic activity of isolated strains phylotype, i.e., one operational taxonomic unit (OTU). The taxonomic affiliation of the aligned sequences was performed Enzyme production by the salmon intestinal strains were de- with Bayesian rRNA Classifier software from the RDP data- termined utilizing the API ZYM system (bioMérieux), accord- base, using a confidence threshold of 80 % (Wang et al. 2007). ing to the manufacturer’s guidelines. Briefly, isolated colonies For phylogenetic tree construction, sequences of clones from were cultured overnight in Tryptic soy broth, centrifuged at fish 2 classified as Aliivibrio sp. were clustered at 97 % se- 5000 g at 4 °C and resuspended in sterile 0.8 % (w/v) NaCl quence identity into OTUs, and aligned with 16S rRNA se- solution to obtain a turbidity of 6 McFarland (1.5–1.8× quences of the type strains of all species of the genus Aliivibrio 109 CFU mL−1). This suspension (65 μL) was added to each deposited in Genbank (NCBI) using Muscle in the MEGA 6 capsule, and the test strips were incubated for 8 h at 20 °C. software (Tamura et al. 2013). Phylogenetic tree was con- Following incubation, one drop of ZYM A (API; tris-hy- structed by the Maximum Likelihood method based on the droxymethyl-aminomethane, hydrochloric acid, sodium Tamura-Nei model with 1,000 resampling bootstrap analyses lauryl sulphate, H2O) and one drop of ZYM B (API; fast using MEGA 6 software (Tamura et al. 2013). The partial 16S blue BB, 2-methoxyethanol) were added to each capsule and rRNA gene sequences obtained have been deposited in 2346 Ann Microbiol (2015) 65:2343–2353

GenBank and assigned the accession numbers HQ897283- incidence of gram-positive organisms, belonging to the HQ897612. Staphylococcus and Bacillus genera (Table 2).

Diversity indices Enzymatic and metabolic properties of cultured intestinal microbiota Biodiversity indices were estimated from clone sequences and isolated strains. Simpson and Shannon indices were calculated No notable differences in the enzymatic profiles obtained by using software EstimateS 9.1 (http://viceroy.eeb.uconn.edu/ the API ZYM tests were observed among the intestinal micro- estimates/). biota strains from the sampled fishes. A high incidence of strains exhibiting the ability to produce the alkaline phospha-

tase, esterase (C4), esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, and naphthol-AS-BI- Results phosphohydrolase enzymes (Table 3) was observed. On the other hand, the production of the β-glucoronidase, N- Total cultured bacteria acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase enzymes was rare (Table 3). When salmon intestinal strains In general, intestinal samples of the studied salmon exhibited were analyzed for their capacity to assimilate various sub- similar cultured bacterial levels, with bacterial counts on TSA strates, no notable differences among fish samples as well as decreasing from the foregut to the hindgut. Bacterial counts between strains isolated from different intestinal portions were from proximal portions were always 1 log higher than those of detected. A high incidence of assimilation of citrate, malate, the distal portions of fish intestine. Proximal portions of fish − glucose, and mannose, contrasting with a lower assimilation intestines were >105 CFU g 1, whereas samples from distal of phenyl-acetate, adipate, and maltose was observed portions of the intestines from all fishes ranged from 103 to − (Table 4). 104 CFU g 1, as indicated in Table 1.

Diversity of intestinal microbiota by molecular cloning Diversity of cultured intestinal microbiota When intestinal microbiota were studied by using 16S rRNA Using culture methods, 74 strains were isolated from intestinal cloning, 325 clones were selected and analyzed, mainly from samples of farmed Atlantic salmon, S. salar. Then the repre- fishes 1 and 2 (118 and 143 sequences, respectively). An sentative isolates were identified based on 16S rRNA gene important degree of variability in the taxonomic diversity of sequencing including , Actinobacteria, and clones obtained from intestinal samples of sampled fishes was phyla. Most of the isolated cultured bacteria detected (Fig. 1). A significant dominance of Weissella belonged to the Proteobacteria Phylum, with a high number (48.3 %) and Leuconostoc (22.0 %) genera was observed of representatives of the γ-proteobacteria group. Among the- among the intestinal clones from fish 1, whereas Aliivibrio se, Pseudomonas (31 strains), Acinetobacter (11 strains), and (81.1 %) was the most dominant genus among the intestinal Psychrobacter (seven strains) were identified as the most im- clones from fish 2. However, no dominance was detected portant genera among the cultured intestinal microbiota; they among the intestinal clones from fish 3, though Weissella were being detected in all the sampled fishes (Table 2). No (25.0 %), Aliivibrio (15.6 %), Leuconostoc (12.5 %), significant differences among the three sampled fishes as well Acinetobacter (10.9 %), and Lactococcus (10.9 %) were the as between bacterial strains isolated from proximal and distal more frequent genera (Fig. 1). Using cloning methodologies portions of the intestine were detected, with a noticeable low to identify the salmon intestinal microbiota, an important in- Table 1 Heterotrophic plate counts (Mean±SD of 3 replicates) from cidence of lactic acid bacteria in fish 1 (78.0 %) and 3 (50.0 %) intestinal content samples of farmed salmon Salmo salar L was observed. It was comprised of the Weissella, Leuconostoc, Lactococcus, and Enterococcus genera, whereas −1 Sample Intestine section Cultured count±SD (CFU g ) only 0.7 % of the intestinal microbiota of fish 2 corresponded Fish 1 Proximal 5.23×105±5.77×103 to lactic acid bacteria (Fig. 1). Distal 3.07×104±3.46×103 When the number of genera per sample was considered a Fish 2 Proximal 2.27×105±2.52×104 measure of richness, it was observed that the not cultured Distal 7.67×103±1.53×103 microbiota from fishes 1 and 3 exhibited remarkable richness 5 4 values greater than those of the cultured intestinal microbiota, Fish 3 Proximal 3.07×10 ±7.50×10 whereas fish 2 showed the opposite due to the high incidence Distal 1.77×104±2.08×103 of representatives of Aliivibrio genus. Ann Microbiol (2015) 65:2343–2353 2347

Table 2 Identification of cultured intestinal microbiota of farmed salmon Salmo salar

Phylum and/or Class Genus similarity (%) Similarity (%) Number of strains

Fish 1 Fish 2 Fish 3

P(n=17) D (n=9) P (n=14) D (n=11) P (n=9) D (n=14)

α-Proteobacteria Agrobacterium 91.3–99.4 1 1 2 1 α-Proteobacteria Brevundimonas 100.0 1 γ-Proteobacteria Acinetobacter 98.8–100.0 3 2 2 1 3 γ-Proteobacteria Lelliottia 100.0 1 γ-Proteobacteria Luteimonas 98.2–98.3 1 1 γ-Proteobacteria Pseudomonas 97.5–100.0 9 5 5 6 1 5 γ-Proteobacteria Psychrobacter 98.7–100.0 1 1 1 4 γ-Proteobacteria Stenotrophomonas 99.0–100.0 2 1 2 Actinobacteria Brachybacterium 99.6 1 Actinobacteria Kocuria 99.8–99.9 1 1 Actinobacteria Microbacterium 97.4 1 Actinobacteria Rhodococcus 100.0 1 Firmicutes/ Bacillus 94.6–100.0 1 1 Firmicutes/Bacilli Staphylococcus 98.1–100.0 2 1

P Proximal section of intestine, D Distal section of intestine

Table 3 Enzymatic activities of intestinal microbiota of farmed Activity Percentage of enzymatic activity salmon determined using the API ZYM system (bioMérieux) Fish 1 Fish 2 Fish 3

P D P D P D (n=17) n=11) (n=10) (n=13) (n=12) (n=12)

Alkaline phosphatase 94.1 100.0 100.0 92.3 100.0 91.7

Esterase (C4) 64.7 54.5 60.0 76.9 91.7 83.3

Esterase lipase (C8) 88.2 81.8 90.0 92.3 83.3 66.7

Lipase (C14) 47.1 18.2 20.0 23.1 16.7 0.0 Leucine arylamidase 94.1 100.0 100.0 100.0 100.0 100.0 Valine arylamidase 82.4 45.4 40.0 76.9 66.7 58.3 Cystine arylamidase 17.6 18.2 10.0 30.8 16.7 8.3 Trypsin 64.7 81.8 60.0 76.9 50.0 16.7 α-Chymotrypsin 29.4 18.2 20.0 7.7 33.3 0.0 Acid Phosphatase 100.0 100.0 100.0 100.0 100.0 91.7 Naphthol-AS-BI- 94.1 100.0 80.0 100.0 91.7 91.7 phosphohydrolase α-Galactosidase 11.8 9.1 10.0 7.7 8.3 8.3 β-Galactosidase 23.5 18.2 20.0 30.8 16.7 8.3 β-Glucoronidase 5.9 0.0 0.0 0.0 0.0 8.3 α-Glucosidase 29.4 27.3 30.0 38.5 33.3 16.7 β-Glucosidase 41.2 54.5 30.0 46.2 25.0 8.3 N-Acetyl-β-glucosaminidase 17.6 0.0 0.0 7.7 0.0 0.0 α-Mannosidase 0.0 0.0 0.0 15.4 0.0 0.0 α-Fucosidase 0.0 0.0 0.0 7.7 0.0 0.0 Gelatinase* 35.3 45.4 100.0 69.2 58.3 41.7

P Proximal section of intestine, D Distal section of intestine, * Determined using the API 20NE system 2348 Ann Microbiol (2015) 65:2343–2353

Table 4 Metabolic activities of intestinal microbiota of farmed Substrate assimilated Percentage of metabolic activity salmon determined using the API 20NE system (bioMérieux) Fish 1 Fish 2 Fish 3

P(n=17) D (n=9) P (n=12) D (n=14) P (n=10) D (n=11)

Glucose 76.5 100.0 91.7 92.9 90.0 63.6 Arabinose 64.7 88.9 83.3 85.7 70.0 63.6 Mannose 70.6 88.9 83.3 92.9 90.0 81.8 Mannitol 52.9 88.9 83.3 92.9 80.0 45.4 N-acetyl glucosamine 70.6 77.8 83.3 85.7 60.0 45.4 Maltose 29.4 11.1 41.7 35.7 30.0 45.4 Gluconate 64.7 88.9 91.7 85.7 80.0 63.6 Caprate 94.1 77.8 75.0 85.7 60.0 72.7 Adipate 52.9 55.6 25.0 50.0 10.0 9.1 Malate 94.1 100.0 100.0 92.8 90.0 72.7 Citrate 82.4 100.0 100.0 85.7 90.0 81.8 Phenyl-acetate 23.5 22.2 0.0 0.0 10.0 18.2

P Proximal section of intestine, D Distal section of intestine

A total of 113 clones classified as Aliivibrio sp. and clustered whereas only six genera were detected among the intestinal into 14 OTUs were analyzed to provide phylogenetic informa- microbiota of fish 3 (Table 5). When diversity indices of tion. Phylogenetic analysis shows a dominant OTU (represent- clones from intestinal samples were compared to those of ed by clone NG1) containing 99 sequences (87.6 % of all the cultured bacteria, the not cultured bacterial diversity indi- sequences), which is closely related to pathogenic species ces were slightly higher than the cultured ones of fishes 1 and Aliivibrio salmonicida and Aliivibrio logei (Fig. 2). 3, but in fish 2 diversity indices of cultured bacteria were When diversity indices of the salmon intestinal clones were double the diversity indices of intestinal clones (Table 5). estimated, fish 3 presented the highest diversity, with Simpson and Shannon-Wiener diversity indices of 0.86 and 2.22, re- spectively, contrasting with the lowest diversity of intestinal Discussion microbiota of fish 2 with Simpson and Shannon-Wiener di- versity indices of 0.32 and 0.68, respectively (Table 5). Oth- Most of the currently available information on the intestinal erwise, no important differences in the number of genera were microbiota of Atlantic salmon Salmo salar refers to the early observed between the fishes 1 and 3 (16 and 14, respectively), stages of growth, mainly juveniles (Navarrete et al. 2008,

Fig. 1 Relative abundance of 100% bacterial genera in 16S rRNA gene clone libraries constructed 90% Acinetobacter Aeromonas from DNA obtained from intestinal microbiota of farmed 80% Aliivibrio Atopostipes Atlantic salmon (Salmo salar L.). Bacillus Citrobacter Genus-level classification was 70% based on the Classifier tool of the Cronobacter Enhydrobacter Ribosomal Database Project 60% Enterobacter Enterococcus (http://rdp.cme.msu.edu/ classifier/classifier.jsp) 50% Enterovibrio Hydrogenophilus Lactococcus Leuconostoc 40% Photobacterium Prevotella Relative abundance 30% Propionibacterium Pseudomonas

20% Stenotrophomonas Streptococcus

10% Unclassified Veillonella Vibrio Weissella 0% Fish 1 Fish 2 Fish 3 Ann Microbiol (2015) 65:2343–2353 2349

Fig. 2 Phylogenetic tree showing the relationships between 16S rRNA sequences of classified OTUs as Aliivibrio according to RDP from fish 2 and 16S rRNA sequences of the type strains of all species of the genus Aliivibrio deposited in Genbank (NCBI). A bootstrap analysis was performed with 1,000 repetitions. Sequences of clones are represented by open circles and sequences representatives of the genus Aliivibrio are represented by filled circles. Numbers in parentheses indicate the number of sequences per OTUs

2009; Cantas et al. 2011), but knowledge of intestinal micro- experimental diets (Bakke-McKellep et al. 2007), whereas biota of adults of Atlantic salmon is still scarce. To our knowl- Hovda et al. (2007) determined bacterial levels in different sec- edge, this is the first study of the intestinal microbiota of adult tions of the Atlantic salmon gut finding 7.94×103 CFU g−1 in S. salar from intensive farming in Chile. the foregut, and 5.01×103 CFU g−1 in the midgut. Most of the previous studies analyzing the cultured fraction In this study, the dominant genera identified among the of intestinal microbiota of Atlantic salmon using TSA, report- cultured intestinal microbiota isolated using TSA medium ed similar levels of cultured bacteria as found in this study were Pseudomonas, Acinetobacter, and Psychrobacter (Yoshimizu et al. 1976; Huber et al. 2004). Other authors (41.89, 14.86, and 9.46 %, respectively). This is in agreement reported lower levels of cultured counts from salmon intestine with Navarrete et al. (2008) who reported that microbiota from samples such as Navarrete et al. (2008) who found mean untreated Atlantic salmon in Chile were mainly composed of values ranging from 5.01×102 to 6.31×103 CFU g−1 of intes- Pseudomonas, Acinetobacter, Bacillus, Flavobacterium, tinal content in juveniles of reared Atlantic salmon, and Ringø Psychrobacter,andBrevundimonas.Thisalsoagreeswitha et al. (2014) who found mean values of 4.17×102,1×103 and more recent study by Cantas et al. (2011), which reported a 2.82×102 CFU g−1 of proximal, midintestine and distal intes- dominance of representatives of the genera Pseudomonas, tine of S. salar, respectively. In other studies, total viable Acinetobacter, and Psychrobacter among the gut bacteria of counts were determined from adherent and digesta from juvenile Salmo salar, identified by bacterial culturing and 16S midintestine (4.26×103–9.77×103 CFU g−1 and 4.07×103– rRNA PCR techniques. In addition, Pseudomonas sp. and 1.62×105 CFU g−1, respectively) and distal intestine (8.32× Acinetobacter sp.havepreviouslybeenreportedasconstitut- 103–2.57×104 CFU g−1 and 9.77×103–2.63×105 CFU g−1, ing an important part of the intestinal microbiota of salmonids respectively) of juveniles of S. salar fedwithvarious (Cahill 1990; Ringø et al. 2005; Romero and Navarrete 2006;

Table 5 Diversity of cultured and not cultured intestinal Fish 1 Fish 2 Fish 3 microbiota of farmed salmon CM UM CM UM CM UM

Number of strains or clones 26 118 25 143 23 64 Number of genera or OTUs 7 16 10 6 9 14 Simpsondiversityindex0.660.720.760.320.830.86 Shannon-Wienerindex1.421.771.870.681.962.22

CM Cultured microbiota, UM Uncultured microbiota using 16S rRNA clone libraries 2350 Ann Microbiol (2015) 65:2343–2353

Hovda et al. 2007). Furthermore, recent reports have demon- salmonids (Liu et al. 2009; Figueiredo et al. 2012), as well as strated the presence of different Psychrobacter species in the the experimental development of disease after its inoculation, alimentary tract of Atlantic salmon (Ringø et al. 2006a; 2008), demonstrated the role of the primary pathogen of some strains as well as the distal portion of the intestine of Arctic charr identified as Weissella sp., confirming Weissellosis as an (Ringø et al. 2006b). emerging disease in rainbow trout aquaculture (Marancik In this study a high proportion of the isolated bacteria from et al. 2013; Welch and Good 2013). the intestinal content of Atlantic salmon exhibited enzymatic When comparing diversity of the intestinal microbiota of activities, suggesting a potential role in the degradation and farmed Atlantic salmon obtained by analysis of the 16S rRNA assimilation of nutrients, contributing to the nutrition of reared gene of cultured strains and clone libraries, the diversity of salmon, but the low bacterial counts suggest a poor contribu- clone libraries was higher than those from the cultured micro- tion of bacterial enzymes to the degradation of macronutrients. biota, with the exception of fish 2, which exhibited an unex- Furthermore, it is important to note that intestinal transit is pectedly high dominance of Aliivibrio representatives when short, and the rearing temperature is much lower than those its clone library was identified. Despite the absence of external used for in vitro enzymatic assays and the API ZYM profiles and internal symptoms of vibriosis when fish 2 was sampled, are insufficient and other enzymatic assays are required to there was a high incidence of Aliivibrio genus in the intestinal evaluate the possible contribution to digestion by gut micro- microbiota, suggesting that the sampled fish was under an biota. More research is required to understand the potential initial stage of infection by this strain. On the other hand, the function of intestinal microbiota as a source of digestive en- absence of Aliivibrio in the cultured microbiota is probably zymes in farmed salmon, and the feasibility of its use in en- due to the use of TSA medium without addition of NaCl. hancing nutrient utilization and growth rate when they are fed Currently, Aliivibrio genus comprises five species: A. fischeri, with formulated diets. A. logei, A. salmonicida, A. wodanis, and A. finisterrensis As has been noted, when intestinal microbiota were studied (Beaz-Hidalgo et al. 2010). From these species, A. salmonicida by using culture-dependent methods, most of the cultured or- (Egidius et al. 1986), A. wodanis (Lunder et al. 2000), and ganisms belonged to the γ-subclasses of the proteobacteria A. logei (Benediktsdottir et al. 1998) have been associated (Pseudomonas sp. and Acinetobacter sp.), in contrast to the with disease in Atlantic salmon. Furthermore, it has been intestinal microbiota revealed by the results of direct cloning demonstrated that A. salmonicida is able to colonize the fish methods, that exhibited a predominance of lactic acid bacteria, intestinal tract (Hansen and Olafsen 1999). Further studies which comprised more than 36 % of the identified clones. are needed to determine whether high levels of Aliivibrio This proportion could have been higher, but for the unexpect- spp. as observed in fish 2, are detrimental to the sanitary ed high dominance of representatives belonging to the status of reared salmon under intensive conditions, because Aliivibrio genus in fish 2. The fact that lactic acid bacteria the increase in the concentration of this genus could be a from fish are commonly slow growing, requiring growth con- response to an infection with a pathogenic Aliivibrio strain ditions on agar-media at low temperatures for up to four weeks causinganimbalanceinthestructure of the intestinal (Ringø and Gatesoupe 1998), would explain why lactic acid microbiota. bacteria were only detected from 16S rDNA clones libraries, It is important to note that fish 2 not only exhibited a and not from the intestinal microbiota obtained after cultured remarkably higher level of Aliivibrio sp. than that ob- on TSA, given that this medium is not suitable for growth of served in the other two sampled individuals, but also this bacterial group. showed almost an absence of lactic acid bacteria, contrast- It is well established that lactic acid bacteria constitute a ing with the observations in the other sampled fishes. It part of the native microbiota of fish (Ringø 2004; Hovda et al. must be noted that the importance of the interaction be- 2012; Ringø et al. 2014). Our results are in agreement with a tween lactic acid bacteria and pathogens in the intestines recent study, showing the taxonomic affiliation of DGGE of salmon species prevents intestinal cellular damage band sequences from the midintestine and distal intestine con- (Ringø et al. 2010). tent of Atlantic salmon.This study is based on known se- In a previous study, it was demonstrated that Vibrio quences of 16S rRNA genes that indicated a high dominance (Aliivibrio) salmonicida can colonize the salmon intestine, of lactic acid bacteria mainly composed of the Weissella and which creates healthy carriers (Bjelland et al. 2012), but this Lactococcus genera, whereas Photobacterium was the most state only occurs without the bacteria dominating the ubiqui- representative of γ-proteobacteria (Reveco et al. 2014). Along tous gut microbiota. In this study, the high predominance of with this, within the lactic acid bacteria there is a high domi- Aliivibrio in the intestinal microbiota of fish 2 suggests that nance of the genus Weissella from specimens 1 and 3. Various fish 2 was an asymptomatic non-healthy carrier, but the other strains belonging to this genus have previously been proposed two sampled individuals were healthy carriers. It is important as potential probiotics for various fish species (Cai et al. 1998; to note that all sampled fishes had the same origin and rearing Balcázar et al. 2008) However, recent cases of Weissellosis in history. The only difference was that they came from different Ann Microbiol (2015) 65:2343–2353 2351 cages from adjacent cage blocks belonging to the same Bakke-McKellep AM, Penn MH, Salas PM, Refstie S, Sperstad S, company. 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