Effect of Thymus Vulgaris Essential Oil on Intestinal Bacterial Microbiota of Rainbow Trout, Oncorhynchus Mykiss (Walbaum) and Bacterial Isolates

Aquaculture Research, 2010, 41, e 667 ^ e 678 d o i: 10.1111/ j.1365 -2109.2010.02590. x

Effect of Thymus vulgaris essential oil on intestinal bacterial microbiota of rainbow trout, Oncorhynchus mykiss (Walbaum) and bacterial isolates

Paola Navarrete1, Isabel Toledo2, Pamela Mardones1, Rafael Opazo1, Romilio Espejo1 & Jaime Romero1 1Laboratorio de Biotecnolog|¤a, Instituto de Nutricio¤n y Tecnolog|¤a de los Alimentos (INTA), Universidad de Chile, Macul, Santiago, Chile 2Laboratorio de Cultivo de Peces, Escuela de Ciencias del Mar, Ponti¢cia Universidad Cato¤lica deValpara|¤so,Valpara|¤so, Chile

Correspondence: Prof. J Romero, Laboratorio de Biotecnolog|¤a, Instituto de Nutricio¤n y Tecnolog|¤a de los Alimentos (INTA), Universidad de Chile, Macul 5540, Macul, Santiago, Chile. E-mail: [email protected]

Abstract However, intensive ¢sh farming in Chile has pro- moted the growth of several bacterial diseases, lead- The application of natural and innocuous com- ing to an elevated use of antimicrobials (Miranda & pounds has potential in aquaculture as an alterna- Zemelman 2002; Miranda & Rojas 2007). Concerns tive to antibiotics. We evaluated the e¡ect of diet regarding antibiotic resistance in bacteria associated supplementation with Thymus vulgaris essential oil with salmon as well as the dissemination of bacterial (TVEO) on the allochthonous microbial composition resistance among environmental microbiota have in- of rainbow trout. DNA was extracted directly from creased. Recently, commensal bacterial populations the intestinal contents, and the V3-V4 regions of the of the gastrointestinal tract have been proposed as re- 16S rRNA genes were ampli¢ed by PCR.The bacterial servoirs for antibiotic resistance determinants that composition was analysed using temporal tempera- could disperse resistance via horizontal gene transfer ture gradient electrophoresis (TTGE). No signi¢cant (Salyers, Gupta & Wang 2004; Salyers & Shoemaker changes (P40.05) were detected in theTTGE pro¢les 2006). In the Atlantic salmon (Salmo salar)gut,anti- of TVEO-treated trout compared with the controls. biotic treatment has been reported to reduce bacter- The Dice similarity index revealed a high stability ial diversity and lead to the development of (Cs 470%) of the intestinal microbiota in both opportunistic Aeromonas harbouring antibiotic resis- groups during the 5-week period. Sequence analyses tance (Navarrete, Mardones, Opazo, Espejo & Romero of theTTGE bands revealed the same bacterial com- 2008). These opportunistic bacteria are normally position in both groups, with most bacteria belong- present in low numbers, and therefore, they do not ing to the Proteobacteria and Firmicutes phyla. The present a signi¢cant risk; however, they can grow in vitro antibacterial activity of TVEO was assessed and reach signi¢cant numbers after antibiotic treat- using a range of normal intestinal isolates and ¢sh ment due to the reduction in bacterial diversity, at pathogens. The inhibitory concentrations for all the which time, they may occupy previously unavailable tested bacteria were higher than theTVEO levels used ecological niches. Concerns about bacterial resis- in trout, which may explain the in vivo results. tance and antibiotic residues have contributed to in- creased caution in the use of antibiotics in animal Keywords: microbiota, PCR^TTGE,16S rRNA gene, production and especially in aquaculture (Cabello Thymus vulgaris, essential oil, Oncorhynchus mykiss 2006; Mo⁄tt & Mobin 2006; Benchaar, Calsamiglia, (Walbaum) Chaves, Fraser, Colombatto, McAllister & Beauche- min 2008; Navarrete et al. 2008) and have encour- aged research for alternatives to antibiotics. Introduction Essential oils (EOs) are natural components of Chile is currently one of the leading salmon produ- plants that are generally recognized as safe sub- cers worldwide (Vielka, Morales & Moreno 2006). stances (GRAS) (http://www.cfsan.fda.gov/ dms/

r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd e667 Antibacterial e¡ect of T. vulgaris essential oil P Navarrete et al. Aquaculture Research, 2010, 41,e667^e678 eafus.html). Because of their antimicrobial proper- tion (Rawls, Samuel & Gordon 2004; Bates, Mittg, ties, these oils may constitute viable alternatives to Kuhlman, Baden, Cheesman & Guidulain 2006; Go- antibiotics as prophylactic and therapeutic agents in mez & Balcazar 2008). animal production and aquaculture systems. These The objective of this study was to evaluate the e¡ect antibacterial properties have been examined in sev- of a diet supplemented withTVEO on the composition eral studies (Kim, Marshall, Cornell, Preston & Wei of rainbow trout intestinal microbiota using molecu- 1995; Lambert, Skandamis, Coote & Nychas 2001; De- lar pro¢ling methods based on16S rRNA gene analy- laquis, Stanich, Girard & Massa 2002; Mejlholm & sis [restriction fragment length polymorphism Dalgaard 2002; Mourey & Canillac 2002; Burt (RFLP)] and PCR^temporal temperature gradient 2004). However, most of the research has been con- electrophoresis (PCR^TTGE). In addition, in vitro de- ducted to extend the shelf-life of di¡erent foods, and termination of the antibacterial activity of TVEO was so these reports have focused on the in vitro evalua- performed using several bacteria isolated from the tion of EO e¡ectiveness against food-borne pathogens gut of healthy trout as well as some pathogens. (Nascimento, Locatelli, Freitas & Silva 2000) and spoilage microorganisms (Mejlholm & Dalgaard 2002; Mahmoud, Yamazaki, Miyashita, Kawai, Shin Materials and methods & Suzuki 2006). Thymol is the principal component of the Thymus vulgaris essential oil (TVEO), and it Diets, ¢sh and sample collection has the widest spectrum of antimicrobial activity To test the e¡ect of TVEO on trout microbiota, four (Dorman & Deans 2000; Burt 2004). Nevertheless, diets containing 0, 5, 10 and 20 mg TVEO kg 1feed there is limited information about the in vivo e¡ects were prepared using a base of commercial pellets of theTVEO. from EWOS Aquaculture International. The composi- The basis for the antibacterial action of EOs is tion of this basic feed is presented inTable1; proximal poorly understood. It has been suggested that they analysis was performed in the food laboratory of the can disturb the permeability of the bacterial cell Universidad Cato¤lica de Valpara|¤so. The composition membrane, leading to a disruption of the proton mo- of the TVEO used in this study was determined by tive force, electron £ow and active transport (Conner gas chromatography in the analytical laboratory of & Beuchat 1984; Cox, Gustafson, Mann, Markham, Universidad Federico Santa Mar|¤a (Table 2). The Liew, Hartland, Bell,Warmington & Wyllie 1998; He- amount of TVEO was selected based on a previous lander, Alakomi, Latva-Kala, Mattila-Sandholm, Pol, toxicological experiment performed in trout (Stroh, Smid & von Wright 1998; Lambert et al. 2001; Ultee, Wan, Isman & Moul 1998). Bennik & Moezelaar 2002). Other potential mechan- Commercial pellets were ground in a lab mixer isms are related to the coagulation of cell contents (450 W) and then milled using an IKA-Werke A11ba- (Sikkema, De Bont & Poolman 1995; Lambert et al. 2001) and/or the inactivation of the genetic material Table 1 Diet composition: components and proximal ana- (Kim, Marshall & Wei1995). lysis of pelleted feed used as a base for TVEO supplementa- A new report byYeh, Shiu, Shei, Cheng, Huang, Lin tion diet and Liu (2009) led to a new perspective on the use of natural extracts in aquaculture. This study reported Ingredients g kg 1 feed that extracts from Cinnamomum kanehirae (stout camphor tree) showed antibacterial e¡ects against Fish meal 480 Poultry by-products 115 di¡erent pathogens of aquatic animals. Shrimp trea- Starch sources 107 ted with this extract exhibited an enhanced disease Wheat gluten 16 resistance toVibrio alginolyticus (Yeh et al.2009) Corn gluten meal 78 Oral administration of antibacterial chemicals Oil 172 may produce an alteration in the gut microbiota or Others (vitamins, minerals, amino acids) 32 Proximal analysis may even facilitate the establishment of opportunis- Crude protein 500 tic pathogenic bacteria (Navarrete et al.2008). Recent Fat 220 studies have revealed that, depending on the kind of Humidity 90 bacteria present, gut microbiota induce several Ash 85 important host responses related to nutrient metabo- Crude ﬁbre 10 Nitrogen-free extract 95 lism, the innate immune system and gut di¡erentia-

r 2010 The Authors e668 Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41,e667^e678 Aquaculture Research, 2010, 41, e667^e678 Antibacterial e¡ect of T. vulgaris essential oil P Navarrete et al.

Table 2 Major component of Thymus vulgaris essential oil 27F and 1492R (De Long 1992). Analysis of the RFLP (TVEO) used in this study using AluI, Rsa IandHaeIII(Invitrogen)wasper- formed to examine the 16S rRNA gene as described 1 Chemical component g kg previously (Romero & Navarrete 2006). Ampli¢ca-

Thymol 400 tion of the V3-V4 region of the 16S rRNA gene was p-Cymene 130 carried out as described previously (Magne, Abely, Linalool 39 Boyer, Morville, Pochart & Suau 2006) using the con- Carvacrol 22 served bacterial domain-speci¢c primers 341F (50- Borneol 8 CCTACGGGAGGCAGCAG-30 with a GC clamp at the a-Pinene 7 0 Eucaliptol 5 5 end; McCracken, Simpson, Mackle & Gaskins 0 0 b-Pinene 2 2001) and 788R (5 - GGACTACCAGGGTATCTAA-3 ; Magne et al. 2006). TTGE was performed according to Magne et al. (2006). Each gel included standards sic analytical mill (3 mm size) (IKA Werke GmbH, containing PCR amplicons of known bacterial se- Staufen, KG, Germany).The resultant powder was sequences (%GC) to validate comparisons between gels. parated into portions of equal weight before mixing The TTGE pro¢les were analysed using GELCOMPAR II with the TVEO suspensions. The TVEO suspensions software (Applied Maths, Sint-Martens-Latem, Bel- were prepared in phosphate-bu¡ered saline (PBS) to gium) and by applying the Dice similarity index (C ). obtain the di¡erent ¢nal concentrations. These sus- s Statistical analyses were performed using the pensions were mixed with the powdered basic feed non-parametric Kruskal^Wallis test. The pairwise and then homogenized. The mixtures were pelleted similarity coe⁄cient (C ) is a similarity index used to and dried at room temperature. s compare the bacterial compositions of di¡erent sam- One hundred and twenty Oncorhynchus mykiss ples (McCracken et al. 2001). Two identical pro¢les (Walbaum) rainbow trouts with an average weight generate a Cs 5100%, whereas completely di¡erent of 25 5 g were used in this study. They were ran- pro¢les generate a C 5 0%. Each sample can be com- domly and equally distributed into eight tanks. The s pared with every other sample; therefore, the mean ¢sh were fed to visual satiation twice a day for 5 percentage similarities (C values) can be used to weeks using the experimental diets (0, 5, 10 and s compare each diet/treatment group with itself and 20 mg TVEO kg^1 feed). Each diet was assessed in du- with all the other groups (McCracken et al.2001).For plicate using two tanks containing freshwater (50 L); intragroup comparisons (i.e., ¢sh under the same the temperature and pH were set at 14 2 1Cand treatment), the Dice index was calculated using pro- 8.0 0.5 respectively. The ¢sh were monitored for ¢les derived from samples collected at the same time their health status, speci¢c growth rates and condi- and from duplicate tanks. For intergroup compari- tion factors (Sveier,Whathne & Lied1999).The micro- sons, C values were calculated using pro¢les derived biota was analysed every week; 15 trout per tank s from samples collected at the same time but corre- were anaesthetized and the intestinal contents were sponding to di¡erent treatments (with TVEO versus collected by squeezing the abdomens. The intestinal without TVEO). To estimate the stability of the micro- contents of three to four individuals per tank were biota composition during the experiment, the C va- pooled before DNA extraction (Romero & Navarrete s lues were calculated for each treatment group using 2006; Navarrete, Espejo & Romero 2009). Three sam- the microbial pro¢les obtained at the beginning of ples per tank were analysed, and the intestinal con- the trial (time 5 0 weeks) and those obtained in sub- tents were maintained at 20 1Cuntiluse. sequent weeks.

DNA extraction and PCR ampli¢cation Identi¢cation of bacterial components in the TTGE pro¢les DNA from the trout intestinal contents was obtained from the pooled samples using the Power Soil DNA All bands visually recognized in each TTGE pattern kit according to the manufacturer’s instructions were excised from the gel and eluted overnight in (MOBIO Laboratories, Carlsbad, CA, USA). To obtain a 50 mL of MilliQ water (Millipore, Bedford, MA, USA). rapid overview of the e¡ect of TVEO on the microbio- All samples (1 mL) were reampli¢ed, as described ta pro¢les of di¡erent samples, the 16S rRNA gene above, except by using the forward primer without was PCR ampli¢ed with the bacteria-speci¢c primers the GC clamp.

r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41,e667^e678 e669 Antibacterial e¡ect of T. vulgaris essential oil P Navarrete et al. Aquaculture Research, 2010, 41,e667^e678

The 16S rRNA sequences from the reampli¢ed bacterial inoculum was 5.0 105 CFUmL 1 in each bands were sent to the Macrogen Sequencing Service well. Positive and negative growth controls were in- Center (Seoul, Korea) for puri¢cation and sequencing cluded with every bacterial strain tested; all analyses with the 788R primer. The retrieved sequences were performed in triplicate.Vibrio ordalii (Vo),Vibrio were deposited in GenBank (EU861368^EU861387, anguillarum (Va),Vibrio parahaemolyticus (Vp) and Fla- EU888865^EU888879) and aligned with reference se- vobacterium psychrophilum (Fp) were tested in Luria^ quences using Sequence Match from the Ribosomal Bertani (LB) broth with 1% NaCl, tryptic soy broth Database Project II (RDP II) website (Cole, Chai, Far- (TSB) with1% NaCl, LB with 3% NaCl and nutrient ris,Wang, Kulam-Syed-Mohideen, McGarrell, Bande- broth with 0.5% tryptone, 0.05% yeast extract, la, Cardenas, Garrity & Tiedje 2007). 0.02% sodium acetate and 0.02% meat extract respectively. All indigenous bacteria were grown in TSB at 30 1C for 24 h. Pathogenic bacteria (Vp,Vo,Va Bacterial strains and isolates and Fp) were grown in TSB at 17 1C for 48 h. These cultures were then used to inoculate plates supple- Pathogenic collection strains Vibrio anguillarum mented with 0.5% Tween 80. All plates were incu- ATCC19264, Flavobacterium psychrophilum ATCC49418, bated aerobically at17 1Cfor48h. Vibrio ordalii ATCC33509 and Vibrio parahaemolyticus RIMD 2210633were obtained from theATCC and RIMD collections. Pathogenic isolates (ThV-1, ThV-2, ThV-5, ThV-6) were kindly provided by a diagnostic labora- Results tory in Puerto Montt, Chile. These pathogenic iso- E¡ect of TVEO dietary inclusion on ¢sh lates were obtained from sick ¢sh using di¡erent growth tissues and media depending on the bacterial type; Vibrio were isolated in TCBS and Streptococcus and During the 5-week trial, the ¢sh were evaluated Lactococcus in blood agar (Valde¤s, Jaureguiberry, based on growth parameters. No signi¢cant di¡er- Romalde, Toranzo, Magarinos & Avendano-Herrera ences were observed in these parameters between 2009). Indigenous bacterial isolates were obtained untreated ¢sh and those treated with TVEO. In the previously from the intestinal contents of healthy untreated ¢sh, the average speci¢c growth rate rainbow trout after culture in tryptic soy agar and in- (SGR) was 2.70 0.22 and the average condition fac- cubation at 17 1C. The indigenous bacteria A8P1-8, tor (K) was 1.27 0.05. In theTVEO-treated ¢sh, SGR A8P1-9, B8P3-1, ThV-A, ThV-E, ThV-F, ThV-G, ThV-H was 2.70 0.25 and K was 1.25 0.07. During the and ThV-I were isolated in a previous study (Navar- treatment, the ¢sh displayed normal behaviour and rete, Magne, Mardones, Riveros, Opazo, Suau, Po- no signs of illness; thus, no particular therapy was chart & Romero 2010); I8 and P1were obtained from provided. No mortality was observed during the ex- healthy Salmo salar gut (Navarrete et al. 2009). Mole- periment. cular identi¢cation of pathogenic and indigenous isolates was carried out by16S rDNA sequencing. E¡ect of TVEO dietary inclusion on the composition of the microbiota Assessment of TVEO in vitro antibacterial To obtain a rapid overview of the e¡ect of TVEO on activity bacterial populations, the microbiota pro¢les from The antibacterial activity of TVEO was assessed all groups (0,5,10 and 20 mg TVEO kg 1 feed) were using several isolates obtained from faecal samples obtained by digesting the PCR-ampli¢ed 16S rRNA of rainbow trout and pathogenic bacteria from a ve- gene with AluI, RsaIandHaeIII.The RFLP pro¢les re- terinary laboratory. Minimum inhibitory concentra- vealed a mixture of bands, indicating the coexistence tions (MICs) of TVEO were determined using a of di¡erent kinds of microorganisms. The bacterial previously described broth microdilution method pro¢les of untreated and TVEO-treated groups re- (Cosentino, Tuberoso, Pisano, Satta, Mascia, Arzedi mained highly similar (Fig.1). & Palmas 1999). Brie£y, serial doubling dilutions of To identify the bacterial components of the micro- TVEO were performed in a 96-well microtitre plate biota and to evaluate the stability as well as the intra- (Nunc, Copenhagen, Denmark) over a range of 2.5^ and intergroup variabilities, PCR^TTGE from the 0 1280 mgmL 1 (ppm).The ¢nal concentration of each and 20 mg TVEO kg 1feed groups were analysed.

r 2010 The Authors e670 Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41,e667^e678 Aquaculture Research, 2010, 41, e667^e678 Antibacterial e¡ect of T. vulgaris essential oil P Navarrete et al.

Figure 1 Dice’ssimilarity indices (Cs) were calculated to compare the16SrRNA RFLP patterns obtained using AluI, RsaI and HaeIII. Each pro¢le was derived from the pooled intestinal contents of three to four ¢sh, which were obtained after 5 weeks of treatment (0,5,10 and 20 mg TVEO kg 1 feed). Bands marked with arti¢cial lines were included in the comparison. Silver stain reveals single DNA strands like red bands that have limited migration and remain on the top of the gel. These bands were not included in the analysis.

The variabilities of the microbiota pro¢les in the repli- Identi¢cation of TTGE bands by sequencing cates (intragroup) for the control and theTVEO-trea- Sequencing was used to identify the bands that were ted groups showed high coincidences, with values visually detected in TTGE from the untreated and the ranging from 70% to 90%. These results indicated TVEO-treated groups. Partial sequences of the 16S that the microbiota was homogenous enough to rRNA gene (400 bp) were compared with sequences allow diet and temporal comparisons (Fig. 2a, in- available in RDP II, and their identities are shown in tragroup comparison). Comparison of the microbiota Table 3. Our results indicated that microorganisms pro¢les using the Dice index (C ) showed high simila- s represented by the main bands belong to the phyla rities (471%) between the 20 mg TVEO kg 1 feed- Proteobacteria and Firmicutes. Speci¢cally, they were treated and -untreated trout. No statistical di¡er- related to Gram-negative bacteria from the genera ences were observed between the TVEO-treated and Moraxella, Vibrio, Butiauxella and Legionella and to the untreated ¢sh (P40.05, Fig. 2b, intergroup com- Gram-positive organisms such as Streptococcus and parison). Thus, for these concentrations, TVEO in- Alicyclobacillus. An additional weak band corre- duced no changes in the pro¢les of the intestinal sponding to Streptomyces of the Actinobacteria phy- microbiota of the ¢sh.These results are in accordance lum was also observed. The most frequent bands with the similarity of the microbiota pro¢les ob- observed in both the untreated and theTVEO-treated served in the RFLP analysis described above. groups correspond to Moraxella,Vibrio and Legionella When the TTGE pro¢les within the same groups (Table 4). (treated or untreated) were compared throughout the collection period, several common bands were observed. These bands were persistent throughout Antibacterial activity of TVEO on bacterial the trial, indicative of the stability of the microbiota ¢sh pathogens and bacteria isolated from the composition in both TVEO-treated and -untreated salmonid gut ¢sh. The stability of the TTGE pattern over time was revealed by the Dice index (Cs), which showed aver- Table 5 summarizes the identi¢cation of bacterial age values 465% for both TVEO-treated and -un- isolates by partial sequencing of the 16S rRNA gene treated trout (Fig. 2c). and the antibacterial activity of TVEO.The MIC range

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Discussion

In the present study,we evaluated the in vivo e¡ect of 20 mg TVEO kg 1feed (8 mg thymol kg 1 feed) in- troduced into the diet on the intestinal microbiota of rainbow trout. The level of TVEO was selected based on a previous study that reported an in vivo toxic effect on rainbow trout and salmon at concentrations above 20 mg kg 1 (Stroh et al.1998). The e¡ect of EO inclusion on the microbiota composition was evaluated using PCR^TTGE. This method has been useful for detecting changes in the composition of microbiota after treatment with antibacterial agents in several hosts such as mice, humans and rabbits (McCracken et al. 2001; De la Cochetie' re, Durand, Lepage, Bour- reille, Galmiche & Dore¤2005; Abecia, Fondevila, Bal- cells, Lobley & McEwan 2007). For this study, we focused on bacteria populations that could be ex- posed to feed ingredients (i.e.,TVEO). Allochthonous microbiota is likely to be more a¡ected by these ingredients. Because bacterial communities were extracted from the intestinal contents of trout, the microbiota analysed comprised mainly allochthonous bacteria (transient or associated with digesta; Salinas, Myklebust, Esteban, Olsen, Meseguer & Ring 2008). Our results showed that the TVEO concentration did not signi¢cantly alter the bacterial populations of trout intestines, as assessed by PCR^TTGE. Se- quencing of the TTGE bands showed that the intestinal microbiota of trout was composed of three phyla: Proteobacteria, Firmicutes and Actinobacteria (Table 3). These taxa have been reported previously in salmonids, and they represent the abundant bac-

Figure 2 Dice’s similarity indices (Cs) were calculated to terial populations present in the gut of these ¢sh compareTTGE patterns.Values represent the average simi- (Holben, Williams, Gilbert, Saarinen, Sarkilahti. & larities and SEMs for three groups of pooled intestinal Apajalahti 2002; Huber, Spanggaard, Appel, Rossen, contents from three to four ¢sh in each experimental Nielsen & Gram 2004; Romero & Navarrete 2006; treatment from weeks 0, 1, 2, 3, 4 and 5. (a) Intragroup Skrodenyte-Arbaciauskiene, Sruoga & Butkauskas comparisons (replicate): 20 mg TVEO kg 1 feed-treated 2006). In our previous investigations, we consistently and untreated groups. (b) Intergroup comparison: un- found the predominance of a few bacterial groups in treated versus TVEO 20 mg TVEO kg 1 feed-treated groups. (c) Stability considering the TTGE pro¢le of week the salmonid guts within Chilean farms (Romero & Navarrete 2006; Navarrete et al.2009,2010), in accor- 0 as a reference. The Cs values did not di¡er signi¢cantly (ANOVA, P40.05). dance with the observation by Holben et al.(2002). Previous investigations have reported that the genera of TVEO against all bacteria was 80^1280 mg L 1.In Lactococcus (Navarrete et al.2010),Streptococcus general, bacterial isolates from the intestine of (Ring, Bendiksen,Wesmajervi, Olsen, Jansen & Mik- healthy ¢sh were more resistant than pathogenic iso- kelsen 2000), Streptomyces (Merri¢eld, Dimitroglou, lates to TVEO with higher MIC values (640^ Bradley, Baker & Davies 2009), Buttiauxella (Kim, 1280 mg L 1). The most potent activity of TVEO was Brunt & Austin 2007; Navarrete et al.2010),Kluyvera demonstrated against Vibrio anguillarum, with an (Kim et al. 2007; Navarrete et al.2010),Hafnia (Kim MIC of 80 mg L 1. et al. 2007; Navarrete et al.2010)andCitrobacter

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Table 3 Nearest-match identi¢cation of16S rDNA sequences obtained with PCR-TTGE approach from rainbow trout, Oncor- hynchus mykiss (Walbaum), to known sequences in the RDP II database

Frequency of band detection in replicates

Accession 0mgTVEO 20 mgTVEO Band name number % identity Affiliation phylum/class Closest sequence kg 1 feed kg 1 feed

P 0–6 EU861369 90.7 Firmicutes/clostridia Acetanaerobacterium elongatum 1/6 1/6 (AY487928) P 0–7 EU861370 99.5 Firmicutes/bacilli Streptococcus bovis (AY442813) 1/6 0/6 P 1–5 EU861368 100 Proteobacteria/g- Vibrio sp. (EU854882) 1/6 0/6 proteobacteria P 2–1 EU861371 88 Proteobacteria/g- Legionella brunensis (Z32636) 3/6 2/6 proteobacteria P 2–4 EU861372 99.7 Proteobacteria/g- Vibrio sp. (AJ316187) 1/6 2/6 proteobacteria P 3–2 EU861373 89.7 Proteobacteria/g- Legionella sp. (X97365) 6/6 6/6 proteobacteria P 5–2 EU872321 87.6 Proteobacteria/g- Uncultured bacterium (AY661981) 6/6 5/6 proteobacteria 75.1 xMoraxella caprae (DQ156148) P 5–3 EU861374 96.2 Proteobacteria/g- Legionella sp. (X97359) 1/6 1/6 proteobacteria P 5–5 EU861375 95.5 Proteobacteria/g- Legionella quateirensis (Z49732) 2/6 3/6 proteobacteria P 5–7 EU861376 97.7 Proteobacteria/g- Legionella rubrilucens (Z32643) 5/6 5/6 proteobacteria P 5–8 EU861377 96.4 Proteobacteria/g- Legionella worsleiensis (Z49739) 1/6 0/6 proteobacteria Q 1–4 EU861378 100 Proteobacteria/g- Vibrio sp. (AY542526) 5/6 4/6 proteobacteria Q 1–8 EU861379 99.5 Actinobacteria/ Streptomyces sp. (AB052845) 1/6 2/6 actinobacteria Q 2–2 EU861380 99.5 Proteobacteria/g- Moraxella sp.(X95304) 2/6 3/6 proteobacteria Q 2–7 EU861381 99.7 Proteobacteria/a- Paracoccus sp. (AY515424) 0/6 1/6 proteobacteria Q 2–8 EU861382 100 Proteobacteria/g- Buttiauxella sp. (AJ293683) 1/6 1/6 proteobacteria 100 Pantoea sp. (AF227860) 100 Kluyvera intermedia (AF310217) Q 4–6 EU861383 98.9 Proteobacteria/g- Vibrio sp. (AJ316187) 1/6 2/6 proteobacteria Q 4–7 EU861384 99.7 Proteobacteria/g- Vibrio sp. (AJ316187) 5/6 4/6 proteobacteria Q 4–8 EU861385 92 Proteobacteria/g- Legionella erythra (Z32638) 0/6 1/6 proteobacteria Q 4–15 EU861386 92 Proteobacteria/g- Legionella birminghamensis 1/6 1/6 proteobacteria (Z49717) Q 4–16 EU861387 98.7 Firmicutes/bacilli Alicyclobacillus pohliae (AJ564766) 0/6 1/6

PCR-TTGE pro¢les were obtained from DNA extracted from the intestinal content of trout fed after 5 week with a diet supplemented with 0 and 20 mg TVEO kg 1 feed.

(Kim et al. 2007; Navarrete et al. 2010) are indigenous elongatum and Alicyclobacillus pohliae have never microbiota of healthy salmonids. Moreover, Legionel- been described as ¢sh gut microbiota. However, the la, Moraxella and Paracoccus have been retrieved from latter has been identi¢ed in the guts of termites the guts of healthy zebra¢sh (Rawls et al. 2004). In (Yang, Schmitt-Wagner, Stingl & Brune 2005), chick- contrast, to our knowledge, Acetanaerobacterium ens (Gong, Si, Forster, Huang, Yu, Yin, Yang & Han

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Table 4 Bacteria present in the intestinal microbiota of trout fed after 5 weeks with a diet supplemented with 0 and 20 mg TVEO kg 1 feed

mgTVEO kg 1 feed

020

Ta n k 1 Ta n k 2 Ta n k 1 Ta n k 2

Bacterial group P1 P2 P3 P1 P2 P3 P1 P2 P3 P1 P2 P3

Legionella spp. x x x x x x x x x x x x Vibrio sp. x x x x x x x x x Moraxella sp. xxxxxxxxx xx Paracoccus sp. x Streptococcus sp. x Streptomyces sp. x x x Butiauxella/Pantoea/Kluyvera intermedia xx Acetanaerobacterium elongatum xx Alicyclobacillus pohliae x

Each diet was assessed in duplicate using two tanks (15 trout per tank) and the intestinal contents of three to four individuals per tank were pooled (P1, P2 and P3) before DNA extraction.

2006) and humans (Kassinen, Krogius-Kurikka, M- chickens fed with a herbal mix (100g oil kg 1 herb, kivuokko, Rinttil, Paulin, Corander, Malinen, Apaja- including thyme) showed no changes in the viable lahti & Palva 2007). counts of several bacterial groups, including lactic Despite an interest in developing new alternatives acid bacteria, coliforms and Clostridium perfringens to antibiotics in aquaculture, our work is the ¢rst (Cross, McDevitt, Hillman & Acamovic 2007). In con- study to address the invivo e¡ect of EOs on the bacter- trast, Janczyk, Trevisi, Sou¡rant and Bosi (2008) ial composition of ¢sh. Although some studies have showed that the inclusion of thymol [1% (w/w)] in reported the e¡ect of di¡erent dietary oils on the gut the pig diet caused clear changes in the small intes- microbiota (Ring, Bendiksen, Gausen, Sundsfjord & tine microbial community,notably decreasing theAc- Olsen 1998; Ring, Ldemel, Myklebust, Jensen, tinobacillus to undetectable levels. These results Lund, Mayhew & Olsen 2002), EOs have only been suggest that the e¡ect of dietary inclusion of EO on used as preserving agents in seafood. For example, the microbiota may depend on the susceptibility of dipping carp ¢llets into a solution containing both the bacterial group. carvacrol and thymol led to reduced growth and The antibacterial activity of TVEO was assessed in numbers of bacteria, consequently extending the vitro using ¢sh pathogens and common bacteria iso- shelf-life of the ¢llets (Kim, Marshall, Cornell et al. lated from healthy salmonids. The MICs were similar 1995; Mejlholm & Dalgaard 2002; Mahmoud et al. to those reported for food-borne pathogens (Cosenti- 2006). In aquaculture systems, examples for in vivo no et al. 1999; Burt 2004). Noticeably, the pathogens use of EOs are rare but promising. Yeh et al.(2009) seemed to be more susceptible than the indigenous showed that shrimp treated with an extract of C. ka- bacterial microbiota. This di¡erence may be related nekirae exhibited enhanced disease resistance toV. a l - to membrane permeability; however, there is no clear ginolyticus. mechanism of TVEO action. Notably, the pathogenic The results of the in vivo studies of EOs have been Lactococcus piscium was clearly more susceptible to contradictory, and these studies were mostly per- TVEO than those isolates belonging to indigenous formed in cows, chickens and pigs. In cows, viable microbiota. The cell-surface components of this bac- bacteria, cellulolytic bacteria and protozoa were not terial group showed considerable diversity within in£uenced by a mixture of EO supplementation, in- several lactococcal strains, with multiple di¡erences cluding thymol (Benchaar et al. 2008). In broiler di- observed between manyof the strain pairs (Crow,Go- gesta, a diet supplemented with EOs (thymol at pal & Wicken 1995; Giaouris, Chapot-Chartier & 50 mg kg 1 feed) induced a decrease in the E. coli Briandet 2009), such as hydrophobicity,extracellular CFU counts, whereas the Lactobacillus counts were lipoteichoic acid concentration, molecular weight not a¡ected (Jang, Ko, Kang & Lee 2007). However, pro¢le of proteins and amount of protein. These dif-

r 2010 The Authors e674 Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41,e667^e678 Aquaculture Research, 2010, 41, e667^e678 Antibacterial e¡ect of T. vulgaris essential oil P Navarrete et al.

Table 5 Minimum inhibitory concentration of TVEO on bacteria isolated from salmon

Accession Affiliation phylum/ MIC (mg L 1) Isolate name number % identity class Closest sequence Gram pH 7.0

Pathogenic isolates ThV-1 EU888876 100 Firmicutes/Bacilli Lactococcus piscium (DQ343754) 1 320 ThV-2 EU888877 100 Firmicutes/Bacilli Lactococcus piscium (DQ343754) 1 320 ThV-5 EU888878 97 Firmicutes/Bacilli Streptococcus phocae (EF599165) 1 640 ThV-6 EU888879 100 Firmicutes/Bacilli Streptococcus phocae (EF599165) 1 640 ATCC 49418 Flavobacterium psychrophilum 320 ATCC 33509 Proteobacteria/g- Vibrio ordalii 320 proteobacteria ATCC 19264 Proteobacteria/g- Vibrio anguillarum 80 proteobacteria RIMD 2210633 Proteobacteria/g- Vibrio parahaemolyticus 320 proteobacteria Microbiota isolates I8 EU888874 99.3 Proteobacteria/g- Shewanella baltica (AB205578) 640 proteobacteria P1 EU888875 99.9 Proteobacteria/g- Pseudomonas sp. (AY456701) 640 proteobacteria A8P1-8 EU888871 99.7 Proteobacteria/g- Kluyvera intermedia (AF047187) 1280 proteobacteria A8P1-9 EU888872 100 Proteobacteria/g- Citrobacter gillenii (AF025367) 1280 proteobacteria B8P3-1 EU888873 99.9 Proteobacteria/g- Hafnia alvei (DQ412565) 1280 proteobacteria ThV-A EU888865 100 Proteobacteria/g- Psychrobacter sp.(DQ337539) 1280 proteobacteria ThV-E EU888866 100 Firmicutes/Bacilli Lactococcus lactis (AJ488176) 1 1280 ThV-F EU888867 100 Firmicutes/Bacilli Lactococcus lactis subsp. lactis 1 1280 (DQ173744) ThV-G EU888868 100 Firmicutes/Bacilli Lactococcus lactis subsp. lactis bv. 1 1280 diacetylactis (AB100805) ThV-H EU888869 99.7 Actinobacteria/ Arthrobacter sp. (EU034524) 1 1280 Actinobacteria ThV-I EU888870 100 Actinobacteria/ Arthrobacter sp. (AJ786821) 1 1280 Actinobacteria

Pathogenic isolates were obtained from di¡erent tissues of sick ¢sh. Microbiota isolates were obtained from faeces of healthy ¢sh. Nearest-match identi¢cation of 16S rDNA sequences obtained to known sequences in the RDP II database.

ferences have important biological e¡ects and could Some authors have suggested that the preservation explain the susceptibility to TVEO. It should be no- of a diverse microbial community that includes in- ticed that the level of TVEO required to inhibit bacter- nocuous and bene¢cial bacteria is key to managing ial pathogen (480 mg L 1) is higher than the level a successful hatchery (Schulze, Alabi,Tattersall-Shel- used in the in vivo study (20 mg TVEO kg^1 feed). drake & Miller 2006), which is important to consider Therefore, more in vivo studies are needed to evaluate in terms of antibiotic use and abuse. Antibiotic treat- the e¡ect of a higher TVEO concentration in gut bac- ments can eradicate susceptible microorganisms, teria. Also, it would be necessary to evaluate whether which may promote colonization by resistant oppor- these higher concentrations can alter feed £avour or tunist bacteria (Mo⁄tt & Mobin 2006; Navarrete et al. induce toxic responses in the ¢sh (Stroh et al.1998). 2008). BecauseTVEO is e¡ective with pathogens and The encapsulation of EOs could be a plausible alter- permissive with indigenous microbiota, we suggest native to deliver active EOs into the ¢sh gut, as this that natural EOs could be used as alternatives for would reduce interactions with the food matrix and managing bacterial populations and avoiding bacter- possibly reduce toxic e¡ects (Wang, Gong, Huang,Yu ial resistance. However, more studies are needed to &Xue2009). evaluate the in vivo e¡ects of TVEO in ¢sh.

r 2010 The Authors Aquaculture Research r 2010 Blackwell Publishing Ltd, Aquaculture Research, 41,e667^e678 e675 Antibacterial e¡ect of T. vulgaris essential oil P Navarrete et al. Aquaculture Research, 2010, 41,e667^e678

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