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Food Control 22 (2011) 1742e1747
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A model to develop biological probes from microflora to assure traceability of tilapia Darawan Ruamkuson a, Saowanit Tongpim c, Mariena Ketudat-Cairns a, b, * a
School of Biotechnology, Institute of Agricultural Technology, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima 30000, Thailand Embryo Technology and Stem Cell Research Center, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima 30000, Thailand c Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 February 2011 Received in revised form 5 April 2011 Accepted 9 April 2011
The bacterial community of Suranaree University of Technology (SUT) tilapia was studied with the aim to develop a model for traceable biological markers. Five fish per time were collected from SUT farm and other sources. Total viable counts (TVC) of bacteria from SUT farm were quite similar in all seasons. Seventy three percent of the bacteria were Gram-negative. Total DNA extracted from the fish gills and intestines were used as template to amplify bacterial 16S rDNA V3 region using GC clamp primer to identify specific bacteria of fish origin by PCR-DGGE technique. The results showed 3 DNA bands on DGGE gel that were specific to only bacterial DNA of SUT tilapia when compared to the other sources. The 3 DNA bands were sequenced and identified as uncultured bacteria of different species. Primers were designed from the 3 specific sequences and used to amplify DNA samples from four sources and some pure cultured bacteria. The results indicated that, only one primer pair can amplify DNA samples from SUT farm but not other samples. This primer pair can be used to identify and trace samples from SUT farm. This method can be used as a model to develop bacterial primer specific location for other food products. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Tilapia microflora 16S rDNA-DGGE GC clamp
1. Introduction The aquaculture industry is probably the fastest growing foodproducing industry in the world. Aquaculture has become an important part of the Thai fishing industry. Furthermore, the Thai fishing segment represents one of the largest national industries in terms of economic value. The export of traditional fish products especially, tilapia from Thailand is increasing and has very good further potential. The demand for tilapia products picked up in Thailand after the economic recession of 1997 and 1998. Currently, tilapia in Thailand is being exported to nearby markets like Singapore and Europe in live, chilled and frozen forms (FAO, 2008). However, fish is perishable product and is suitable substrate for bacterial growth especially pathogenic bacteria, which can cause consumers concerned. So, the food safety and security needed to be monitored throughout the supply chain (EU regulation 178/2002).
* Corresponding author. Embryo Technology and Stem Cell Research Center, Institute of Agricultural Technology, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima 30000, Thailand. Tel.: þ66 4422 4355; fax: þ66 4422 4154. E-mail address:
[email protected] (M. Ketudat-Cairns). 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.04.008
The ability to identify and validate some pertinent biological markers (bacteria) from the environment of the fish to assure traceability would be of great benefit. Currently, there are no existing scientific methods that can follow or determined precisely to the food origin but Le Nguyen, Ngoc, Dijoux, Loiseau, & Montet (2008) propose a molecular solution linking ecology of bacteria to geographical origin of fish. For traditional analysis, microflora will be cultured on specific or non-specific growth media. This method consists of colony isolation, phenotypic characterization and biochemical test (Colwell, 1962; Huber et al., 2004). However, the weaknesses of phenotypic methods are poor reproducibility and laborious investigations are needed. Genotypic or molecular methods have been developed for the detection, identification and characterization of microorganisms found in environmental samples, foods and other complex ecosystems (Murray, Hollibaugh, & Orrego, 1996). Investigation of the bacterial diversity in fish can be performed by polymerase chain reaction (PCR) amplification and the most common target region is the 16S rDNA of the bacterial genome (Ampe, Omar, Moizan, Wacher, & Guyot, 1999; Moeseneder, Arrieta, Muyzer, Winter, & Herndl, 1999; Murray et al., 1996; Øvreås, Forney, Daae, & Torsvik, 1997; Riemann, Fandino, Campbell, Landry, & Azam, 1999). The denaturing gradient gel electrophoresis (DGGE) has been used and
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applied for the identification of bacterial microflora in fish in Cameroon (Tatsadjieu et al., 2010). In this research, bacterial microflora from tilapia from different seasons were isolated and compared with tilapia from three other sources by cultivable technique and PCR-DGGE technique. Genetic markers of bacteria specific to SUT farm were designed and used as a model of biological probe to assure traceability. 2. Materials and methods 2.1. Fish sampling The tilapia samples were collected (five fish/time) five times during 12 months (June 2007eApril 2008 for all seasons) from SUT farm and 3 other sources (2 farms and Nakhon Ratchasima moat) in only rainy season. The samples were transferred to storage bags and maintained on ice until transported to laboratory. Then, the samples were measured and weighed. Gills and intestines were aseptically removed from each sample then, some of the gills and intestines were suspended in 0.85% NaCl to isolate bacterial microflora. The remnant of gills and intestines were used for genomic DNA (gDNA) extraction. 2.2. Screening of bacterial community Each gills and intestines sample was serially diluted in sterile 0.85% NaCl to 106, then spreaded onto Plate Count Agar (PCA, Himedia, India), Potato Dextrose Agar (PDA, Himedia, India) supplemented with 5 mg/ml chlortetracycline,HCl and 5 mg/ml chloramphenicol (Oxoid, UK), Thiosulfate Citrate Bile Salts Sucrose Agar (TCBS, Oxoid, UK), Aeromonas agar base supplemented with 5 mg/ml ampicillin (Oxoid, UK), Pseudomonas agar base contained 5 mg/ml Cetrimide-Fucidin-Cephalosporin (CeFeC) supplement (Oxoid, UK) and de Man, Rogosa and Sharpe (MRS) contained CaCO3 (Oxoid, UK), in duplicate. Aeromonas agar base plates were incubated for 18e24hr, MRS plates for 48hr and PDA plates for 3e5 days at 30 C. PCA plates were incubated for 24hr and TCBS agar plates for 18e24 hr at 37 C and Pseudomonas agar plates for 18e48 hr at 25 C.
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2.5. PCR-DGGE analysis Partial sequence of bacterial 16S rDNA V3 region were amplified using primers GC338f (50 -CGCCCGCCGCGCGCGGCGGGCGGGGCG GGGGCACGGGGGGACTCC TACGGGAGGCAGCAG-30 ) and 518r (50 ATTACCGCGGCTGCTGG-30 ) (Øvreås et al., 1997; Ampe et al., 1999). Each mixture (final volume 50 ml) contained 2 ng template DNA, 0.4 mM primers, 0.2 mM dNTP, 2 mM MgCl2, 1 of MgCl2 free buffer (Promega, USA) and Taq polymerase (homemade at SUT). The PCR cycle were performed with the first stage; 1 cycle at 94 C for 5 min, second stage; 35 cycles at 94 C for 30 s, 55 C for 30 s and 72 C for 30 s and the last stage; at 72 C for 7 min. PCR products (3 ml) were analyzed first by conventional electrophoresis on 2% (w/v) agarose gel with 1 TAE buffer and quantified by using standard DNA mass ladder 100 base pair (bp) (Promega, USA). The remnant PCR products were then analyzed by DGGE using DCodeÔ universal mutation detection system (Bio-Rad Laboratories, USA). PCR products were separated on 8% (w/v) polyacrylamide gel, 30e60% denaturing gradient, 100 V with 18hr run time (Ruamkuson & Ketudat-Cairns, 2009). Electrophoresis experiments were performed at 60 C in 1 TAE buffer. The gels were stained for 1 min with 1.0 mg/ml ethidium bromide and rinsed for 20 min in water and then photographed on an UV transilluminator. 2.6. Image and statistical analysis The DGGE fringerprints were manually scored by the presence and absence of co-migrating bands, independent of intensity. Data analysis was done using the Dice similality coefficient (SD)
2.3. Purification and Gram’s stain The colonies were re-streaked to confirm the purification and kept as glycerol stock at 80 C. The purified colonies were Gram’s stained followed the protocol of Rollins, Temenak, Shields, & Joseph, 2003 and the morphologies were visualized under microscope. 2.4. Total DNA extraction The protocol of Le Nguyen et al. (2008) was followed with minor modifications, briefly, gills and intestines were crushed in liquid nitrogen then, 0.2 g were transferred into 1.5 ml microcentrifuge tube and 720 ml extraction buffer (1M TriseHCl pH8, 0.5 M EDTA, 5 M NaCl, 10% SDS) were added. The tubes were incubated at 65 C in heat box and vortex every 5 min 3 times then, 225 ml of 5 M potassium acetate were added and mixed, after that, the tubes were incubated on ice with shaking for 20 min. The suspensions were then centrifuged at 16,000g for 15 min and 750 ml of supernatant was transferred to new microcentrifuge tubes. The DNA was precipitated with 500 ml of cold isopropanol then, centrifuged at 16,000g for 10 min. The supernatant was removed and the pellet was washed with 300 ml of 70% of cold ethanol and centrifuged at 8160g for 10 min then, the supernatant were removed and air dried at room temperature. Finally, the DNA were re-suspended with 30 ml of TE buffer and stored at 20 C until analysis.
Fig. 1. PCR-DGGE 16S rDNA banding profiles of fish bacteria from SUT farm: influence season and fish organ.(H) hot season, (C) cool season and (R) rainy season, I: intestine; G: gill.
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Fig. 2. PCR-DGGE 16S rDNA banding profiles of fish bacteria from 3 sources in rainy season (R). (a) F1: farm #1; (b) F2: farm #2; (c) NM: Nakhon Ratchasima moat; I: intestine; G: gill; 1e5: replicate of fish.
according to Heyndrickx, Vauterin, Vandamme, Kersters, & De Vos, 1996. Dendogram was constructed using the STATGRAPHICS plus v5.1 software (Statpoint Technologies, Inc., USA). Similarity was determined using the cluster analysis with Euclidian distance measure. 2.7. Sequencing and alignment The DNA bands specific for only SUT samples from all seasons were eluted from polyacrylamide gel (DGGE) and purified and then sent to Macrogen (Korea) for sequencing. The sequencing results were aligned using NCBI blast with databases.
DNASTAR Lasergene v7 program (DNASTAR, Inc., USA). The extracted DNA samples were used as template to amplify using these newly designed specific primers. Firstly, PCR products were analyzed by conventional electrophoresis on 2% (w/v) agarose gel with 1 TAE buffer and quantified by using DNA mass ladder (100 bp, promega) as standard. DNA samples from other sources were amplified also to make sure that these primers were specific only for samples from SUT. 3. Results and discussion 3.1. Screening of bacterial community using media and Gram’s staining
2.8. Specific primers development The specific oligonucleotide primers for these 16S rDNA sequences of specific bacteria from SUT farm were designed using
When bacterial load of samples from SUT in all seasons (rainy, cool and hot) were compared, the results indicated that, total viable count of bacterial load were not significant different even when the
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temperature were quite different (varied from 25e35 C). The bacterial load varied between 1.6 106 and 5.1 107 colony forming units per gram (cfu.g1) in rainy season, 8.9 105 and 1.3 107 cfu g1 in cool season and 6.8 106 and 7.5 107 cfu g1 in hot season. When bacterial load from SUT and three other sources in rainy season were compared, the results indicated that, fish from SUT farm have more bacterial load than the three other sources (data not shown). This is due to the tilapia from SUT farm is raised in close system, which leads to more colonization of bacteria in the system. For the two other farms, tilapia are raised and fed in open systems in float baskets that water flow all the time therefore, lower bacterial number were found. For Nakhon Ratchasima moat, less bacterial load than SUT farm was found. In the rainy season, the moat water was high, so less bacterial number was found. When compared with the two other farms, fish from Nakhon Ratchasima moat have more bacterial load than the two other farms because, it is more of a close system and the water was not as clean as the farms, with the water flow all the time so, more colonization of bacteria were observed. Total of 249 bacterial isolates from all samples were Gram’s stained. Most of the bacteria (73%) were Gram-negative bacteria because they are normal flora of healthy fish (Hatha, Kuruvilla, & Cheriyan, 2000). Actually, they are opportunistic pathogen that can not harm healthy fish but they can cause diseases when fish are under stress conditions (Al-Harbi & Uddin, 2004; Harish, Nisha, & Mohamed Hatha, 2003) e.g. stocked at high density or subjected to poor environmental conditions.
3.2. PCR-DGGE analysis PCR amplification was done using GC clamp primer and 2 ng total DNA templates to amplify bacterial 16S rDNA. The results showed that, the PCR products were 220 bp on agarose gel as expected (data not shown). The DGGE results of samples from SUT farm in three seasons and farm #1, farm #2 and Nakhon Ratchasima moat in only rainy season are shown in Figs. 1 and 2 respectively. The same pattern of DNA bands on the polyacrylamide gel can be
Fig. 3. PCR-DGGE 16S rDNA banding profiles of fish bacteria from four sources. SUT farm (S) in the hot season (H), cool season (C) and rainy season (R); farm #1 (F1), Nakhon Ratchasima moat (NM) and farm #2 (F2) in rainy reason (R); I: intestine; G: gill. Arrow show specific bands (a, b and c) found in only sample from SUT fish.
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seen from the samples of the same source even though the DNA samples were from different tilapia (Fig. 2). The patterns of DNA bands on the polyacrylamide gel were quite similar in all seasons of samples from SUT farm (Fig. 1). Fig. 3 shows the comparison of DNA bands from samples of SUT farm, farm #1, farm #2 and Nakhon Ratchasima moat. When compared the pattern of DNA bands on polyacrylamide gel from all four sources, the results indicate that, SUT farm showed more bands than the other two farms (Fig. 3). This is due to the tilapia from SUT farm is raised in close system which leads to more bacteria in the system. For the other two farms, tilapia are raised and fed in the open running water system that water flow all the time. Therefore, the samples from SUT farm showed more DNA bands indicating more bacterial diversity than the other two farms. For samples from Nakhon Ratchasima moat, even though it not really running water system, but there was no regular feeding of the fish so, no contamination of bacteria from leftover feed were found in the system. Therefore, the samples from Nakhon Ratchasima moat showed less DNA bands indicating less bacterial diversity than SUT farm. Moreover, three specific bands of the samples from SUT farm were found (Fig. 3 bands a, b and c). The three bands showed up at different positions indicating that they are different bacterial species. The cluster analysis obtained by STATGRAPHICS plus v5.1 software (Statpoint Technologies, Inc., USA) of the DGGE pattern from four different sources showed a community similarlity among the geographical locations where the fish samples were collected (Fig. 4). At 70.6% of similarity level, two main clusters were obtained: the first cluster comprised the samples from SUT farm from all seasons, the second cluster included the samples from farm #1, farm #2 and Nakhon Ratchasima moat. 3.3. Specific primers development The specific DNA bands; a, b and c from samples of SUT farm in Fig. 3 were named SUT1, SUT2 and SUT3, respectively. They were also sequenced. The sequences were blasted and the results indicated that they were uncultured bacteria of different species and
Fig. 4. Cluster analysis of 16S rDNA banding profiles of fish from four sources. SUT farm (S) in the hot season (H), cool season (C) and rainy season (R); farm #1 (F1), Nakhon Ratchasima moat (NM) and farm #2 (F2) in rainy reason (R); I: intestine; G: gill.
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Table 1 Sequences of three specific primers, Tm and the size of PCR products. Name of primers
Sequences of primers
Tm ( C)
SUT1_F SUT1_R
50 -GATGAAGGATCGTGGTC-30 50 -CGGCTGGCACAGAGTTAG-30
52.8 58.2
90
SUT2_F SUT2_R
50 -CCACAAGCCTGATCCAGC-30 50 -CTGGCACGTACTTAGC-30
58.2 51.7
122
SUT3_F SUT3_R
50 -CTTCGGGTTGTAGAGTAC-30 50 -CTGCAGGTAACGTCAACTC-30
53.7 56.7
77
Expected product size (bp)
the sequences are submitting to GenBank. These sequences were used to design specific primers using DNASTAR Lasergene v7 program (DNASTAR, Inc., USA). These specific primers (Table 1) were used to amplify DNA samples from SUT farm and other sources to confirm the specificity of these primers to samples of only SUT farm. Firstly, the results showed lots of non-specific bands (data not showed). The results of amplification with primers SUT1_F, SUT1_R and SUT3_F, SUT3_R did not show expected size band from any samples or if there was any, it was not clear. So, these primer pairs were discarded. For the primers SUT2_F and SUT2_R, the target band from SUT samples show the expected size of about 120 bp. More detail study was done using this primer pair. Optimizations of the PCR conditions were done. The annealing temperature was optimized using gradient PCR of annealing temperature of 50e60 C with 1 C integral. The results indicated that, lower non-specific bands were seen at higher annealing temperature. However, the intensity of the specific band of 120 bp was also lower. Finally, the annealing temperature of 57 C was used (Fig. 5). Fig. 5, upper gel showed specific band of about 120 bp,
which is not seen from samples of other sources (lower gel) or in the pure cultured and negative control. However, when DNA from other sources were used as template (lower gel) high intensity of primer dimer were seen. In any case, these primers SUT2_F and SUT2_R can be used to distinguish bacteria from SUT farm when compared to other sources. 4. Conclusion Bacterial community in fish is related to habitat and management of the farm. Although, most of the bacteria are opportunistic pathogens but they do not harm healthy fish (Harish et al., 2003). Moreover, they synthesized vitamins that are lack in feed and have been demonstrated to enhance the growth of fish (Limsuwan & Lovel, 1981). Specific bacteria from fish can be used to identify and trace the location of fish (Tatsadjieu et al., 2010). Statistical analysis of DGGE pattern in this study indicated that there were enough environmental differences between four sources to obtain a major effect on bacterial ecology. However, the aim of this study was to find specific biological probes to be a model for traceability of fish. Therefore, the PCR-DGGE technique was used to specify bacterial DNA. In this study, three specific DGGE bands of the V3 region of the 16S rDNA of bacteria from SUT fish were sequenced. They were identified as uncultured bacteria of different species. Three specific primer pairs were designed from these sequences. Only primer SUT2_F and SUT2_R can be used to specify and trace samples from SUT farm. This research can be used as a model to develop traceability marker for products of interest. Acknowledgment DR was supported by OROG-SUT scholarship. This work was supported by SUT 3-304-48-36-31 National Research Council, Thailand (NRCT) grant to MKC. D. Montet (CIRAD, France) is acknowledged for initiating the idea for the project. References
Fig. 5. Specific PCR banding profiles of fish bacteria from four sources and pure cultured (p) of bacteria isolated from SUT fish using primers SUT2_F and SUT2_R. SUT farm (S) in the hot season (H), cool season (C) and rainy season (R); farm #1 (F1), Nakhon Ratchasima moat (NM) and farm #2 (F2) in rainy reason (R); I: intestine; G: gill; PR: Gram-positive rod; PC: Gram-positive cocci; NR: Gram-negative rod; NC: Gram-negative cocci; 1e2: replicate of sampling.
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