Author: Sarah Godwin
Institution: Amherst College
Date: April 2005
Abstract
The 16S rDNA genes of fourteen bacterial isolates derived from shrimp larvae were successfully amplified, cloned and sequenced. Comparison of the sequences with those available at the NCBI web site using BLAST (nr database) allowed for bacterial classification based on similarity indices of greater than or equal to 96%. These identifications were consistent with previous phenotypic analyses of the same isolates by their carbon source utilization patterns and fatty acid methyl esters (FAME). Use of 16S sequencing to identify bacteria to species level is not without problems. Specifically, databases may not contain the sequence submitted for identification or species may be too genetically similar for 16S sequencing to be used as a differentiation method. Even with fatty acid, Microlog, 16S sequencing, and classic bacterial assays, it can be problematic to make definitive species designations. These results highlight the difficulty in identifying a specific species and suggest that much of the literature dedicated to bacterial pathogens in shrimp culture may have inappropriately relied on single identification protocols when a full polyphasic approach should have been taken.
Introduction
Shrimp aquaculture is threatened by diseases attributed to a variety of bacterial microorganisms introduced to the hatchery environment by means of water supply and food sources. A number of these microbes are opportunistic pathogens that become virulent in response to environmental cues that favor their survival over that of the host (Aguirre-Guzman et al., 2001). Microbial infection has been associated with an increase in larval deformities and mortality rates (Vandenberghe et al., 2002). Bacteria of the genus Vibrio have been specifically implicated as shrimp pathogens because they are regularly found in high numbers during periods of high larval mortality and disease outbreak in grow out ponds (Saulnier et al., 2000). Vibrio spp. are Gram-negative, motile, oxidase-positive (in most species), straight or curved rod-shaped, facultative anaerobes (Sung et al., 2001). Numerous attempts have been made to identify and characterize Vibrio spp. in order to understand what distinguishes the pathogenic from the non-pathogenic members of this genus (Vandenberghe et al., 2002). One popular technique is the Microlog Identification System (Biolog Inc., Hayward, CA), which has been used as a means to test an array of phenotypic characters. The great biochemical diversity of the genus, however, has posed a challenge for distinguishing between pathogenic and non-pathogenic strains that are genotypically similar (Vandenberghe et al., 2002).
Table 1. Previous identifications of bacterial isolates from shrimp aquaculture. Fatty Acid Analysis refers to a commercial assay of fatty acids present in cell walls. Microlog refers to metabolic fingerprints obtained by substrate-growth analysis. Sim refers to a similarity score reported for the assay.
Characterization of strains associated with shrimp larval cultivation allows determination of typical organism profiles and provides a basis for future work characterizing shrimp pathogens through pathogenicity experiments. This work focused on the relative compatibility and accuracy of several techniques used to identify bacterial isolates. As summarized in Table 1, previous work on these isolates included Microlog and fatty acid analysis. Since preliminary data showed discrepancies across different forms of identification, the current study aimed to generate additional molecular identification data for the strains. The method used for this characterization was 16S rDNA sequencing, which is a genomic approach to the classification of bacteria based upon the 16S ribosomal RNA gene (Lane et al., 1985). This gene is both ubiquitous and highly conserved among prokaryotic organisms, making it a useful primer site for gene amplification by PCR (Lin et al., 2003). Results emphasized the need to take a polyphasic approach when isolating and identifying bacterial isolates, particularly when they are to be used in future pathogenicity studies.
Methods and Materials
Phenotypic and Bacteriological Tests
To ensure strain purity, and to characterize basic bacteriological characteristics, the bacterial isolates were submitted to a series of bacteriological tests, including growth on differential media, Gram staining, KOH testing, O/F testing, and motility testing. Motility was indicated by an even halo of growth on TSA agar plates. The results of these bacteriological tests are listed in Table 2. More metabolic profiles of the fourteen bacterial isolates were obtained using the Microlog system (Biolog Inc., Hayward, CA). For this characterization, the GN-2 microplate protocol (data not shown) was followed and results were compared with available metabolic fingerprints. The presence of specific fatty acid methyl esters (FAME) was determined by gas chromatography and the data fingerprint used to identify each strain. (Microbial ID, Newark, Delaware).
As a quality control, prior to 16S sequencing in the current work, bacterial isolates were submitted to a series of bacteriological tests, including growth on differential media, Gram staining and KOH testing, O/F testing, and motility testing. A test of motility was carried out on TSA agar plates; motility was indicated by an even halo of growth. The results of these bacteriological tests are listed in Table 2.
16S Sequencing
Table 2. Bacteriological tests on bacterial isolates from shrimp aquaculture as quality control. TCBS, YDC and TZC are differential growth media. Gram stain and KOH tests were used to differentiate cell morphology. In the KOH test, bacterial isolates either lysed and released a string of DNA (S) or did not lyse (NS). O/F test was used to differentiate between organisms that have oxidative or fermentative metabolism. Organisms were determined to be motile or not-motile. Organism shape was assessed under a light-microscope.
DNA Extraction
Bacterial DNA was extracted from isolates following the protocol of the Promega Wizard Genomic DNA purification kit (Promega, USA) for Gram-positive and Gram-negative bacteria. Aliquots (1ml) of overnight cultures were centrifuged at 16,000 x g for 2 minutes to pellet the cells. The supernatant was discarded. Gram positive bacteria were resuspended thoroughly in 480ul of 50 mM EDTA, while gram negative bacteria remained on ice until the addition of Nuclei Lysis Solution. 10 mg/ml of lysozyme and 10 mg/ml of proteinase K were added to the resuspended cell pellet in a total volume of 120L, which was mixed by pipet. The samples were incubated at 37° C for 60 minutes, and then centrifuged for 2 minutes at 16,000 x g and the supernatant removed. 600L of Nuclei Lysis Solution was added to both Gram positive and Gram negative bacteria and the cells were resuspended by gentle pipeting. Samples were incubated at 80° C for 5 minutes to lyse the cells; then cooled to room temperature. Three L of RNase Solution was added to the cell lysate and tubes were inverted 2-5 times to mix. Samples were incubated at 37° C for 30 minutes; then cooled to room temperature. 200L of Protein Precipitation Solution was added to the RNase-treated cell lysate and then tubes were vortexed vigorously at high speed for 20 seconds. Samples were incubated on ice for 5 minutes and then centrifuged at 16,000 x g for 3 minutes. The supernatant containing the DNA was transferred to a clean 1.5 ml microcentrifuge tube containing 600L of room temperature isopropanol. Samples were gently mixed by inversion until the thread-like strands of DNA formed a visible mass. Samples were centrifuged at 16,000 x g for 2 minutes. The supernatant was carefully pipetted off and 600L of room temperature 70% ethanol was added; the tube was gently inverted to wash the DNA pellet. Samples were centrifuged at 16,000 x g for 2 minutes and then the ethanol was pipetted off. Samples were vacuum dried for 45 minutes. 100L of DNA Rehydration Solution was added and tubes were incubated at 65° C for 1 hour.
PCR Amplification
PCR of the 16S gene for the bacterial isolates was conducted according to Expand High Fidelity PCR System protocol (Roche). The primers used for amplification were 16S_1492R: 5'-ACGGCTACCTTGTTACGACTT, and 16S_27F:5'-AGAGTTTGATCCTGGCTCAG. Aliquots of 49 of PCR master mix were made with 1L of each sample added to its own aliquot of PCR master mix. PCR reactions were run with a 5 minute denature step at 96° C; 35 cycles of 40 seconds at 96° C, 45 seconds at 50° C, 2 minutes at 72° C; final annealing step at 72° C for 10 minutes. Reactions were kept at 4° C until loaded onto the gel. 8L of PCR product was mixed with 2L of 10x loading dye. A 1% agarose gel containing ethidium bromide was loaded, with the product/dye mix then electrophoresed at 80V for 1.5 hours.
Cloning of PCR Product
The PCR products were cloned following the TOPO Cloning Kit (Invitrogen, USA) protocol. Four L of fresh PCR product was added to its own aliquot of 1L Salt Solution plus 1L TOPO vector. The reaction was mixed gently and incubated for 5 minutes at room temperature. The TOPO Cloning reaction was placed on ice preceding the transformation reaction.
Transformation of Clones
2L of each TOPO Cloning reaction was added to its own vial of One Shot chemically competent E. coli cells and gently mixed; vials were incubated on ice for 10 minutes. Cells were heat-shocked for 30 seconds at 42° C without shaking and then tubes were immediately transferred to ice. 250L of room temperature S.O.C medium was added to each tube; tubes were capped and shaken horizontally (200 rpm) at 37° C for 1 hour. A pre-warmed selective plate was spread with 20 or 40 from each transformation and incubated overnight at 37° C. Selective plates contained Luria-Bertani Medium (1.0% Tryptone, 0.5% Yeast Extract, 1.0% NaCl, ph 7.0) and 50 g/ml of kanamycin. Each plate was spread with 40 mg /ml of X-gal (dimethyl formimide); plates were incubated at 37° C for 30 minutes before use. Cloning reactions produced plates containing blue and white colonies. Two white colonies were chosen for each strain for analysis and cultured overnight in 1.5 ml of LB medium containing 50g/ml kanamycin.
Plasmid Extraction
Plasmids were extracted from the 1.5 ml overnight cultures of E.coli following the QIAprep Miniprep kit protocol (Qiagen, Valencia, CA). Bacterial cultures were centrifuged for 7 minutes at 14,000 x g. The supernatant was discarded and the pelleted cells were resuspended in 250 L of Buffer P1 and transferred to a microcentrifuge tube. 250 L of Buffer P2 was added; tubes were inverted 4-6 times to mix. 350 L of Buffer N3 was added and the tubes were immediately inverted 4-6 times to mix. Tubes were centrifuged for 10 minutes at 15,000 x g; the supernatants were applied to the QIAprep column by decanting. Tubes were centrifuged for 60 seconds and the flow-through was discarded. The QIAprep spin column was washed by adding 0.5 ml Buffer PB and centrifuged for 60 seconds and the flow-through was discarded. The QIAprep spin column was washed by adding 0.75 ml Buffer PE and centrifuged for 60 seconds and the flow-through was discarded and the tubes centrifuged for an additional 1 minute to remove residual wash buffer. The QIAprep columns were placed in clean 1.5 ml microcentrifuge tubes and the DNA was eluted by the addition of 50 L of Buffer EB (10 mM Tris-Cl, pH 8.5) to the center of each QIAprep column. Columns were allowed to stand for 1 minute and then centrifuged for 1 minute. The verification of plasmid inserts was achieved by a restriction enzyme digest. 2 L of each sample of plasmid DNA was added to its own aliquot of Digest master-mix containing 0.5 L of EcoR1, 1L of Buffer, and 6.5L of water. Reactions were kept at 4 degrees C until loaded onto the gel. 8 L of PCR product or plasmid DNA was mixed with 2 L of 10x loading dye. A 1.5% agarose gel containing ethidium bromide was loaded with the product/dye mix then electrophoresed at 125V for 1.5 hours.
Figure 1. Construction of total 16S sequence from forward and reverse partial sequences.
Sequencing
12 L of each sample (1L plasmid DNA, 2uL primer, 9 uL nanopure water) were supplied to the Core Facility (University of Hawaii, Manoa) for sequencing using an Applied Biosystems 377XL DNA Sequencer. The primers used were the M13 forward and reverse primers. Vector NTI software was used to analyze and construct the final sequences. A diagram of the method for sequence construction is provided in Figure 1.
Results
To ensure 16S sequencing was performed on pure cultures and to obtain classic bacteriological profiles, a number of accepted bacteriological tests (i.e. growth on differential media, O/F tests, and motility) were executed (see Table 2). The fourteen bacterial isolates were confirmed to be pure strains and were classified accordingly. Only two of the fourteen cultures (20 and 53) were Gram-negative, fermentative and motile. In the KOH test (for lysis of Gram-negative bacteria), DNA was released from the lysed cells of isolates 20 and 53, which matched the results of the Gram stain. Of the fourteen strains, the predominant physical shapes were cocci or short, thin rods. The two Gram-negative strains are the only two isolates that have characteristics of the Vibrio species, which is noteworthy because previous attempts to identify the fourteen strains had not applied these initial bacteriological tests and this oversight led to several incorrect assumptions and incorrect identifications.
Table 3. 16S sequence matching to NCBI nr database for bacterial isolates from shrimp aquaculture. The first column contains the culture identification number. The next pair of columns shows the species identification that had the highest percent match with the sequence submitted for the culture. The Expectation value, E is the amount of times a database match is expected. The next pair of columns shows the species identification that had the highest percent match with a species different from that of the top match. The final pair of columns shows the top match for a different genus.
After the cloning reaction, an EcoR1 restriction enzyme digest was performed on the transformed clones and gel electrophoresis was used to verify cloning success (see Figure 2). The final culture identifications for the 14 bacterial isolates are shown in Table 3. Two of the isolates were closest in identity to Vibrio species (V. harveyi and V. natriegens). Of the remaining isolates, one was most similar to Gordonia sputi, and seven matched the genus Staphylococcus (S. gallinarum, S. epidermis, and S. hominis-hominis). Three bacteria matched the genus Brevibacterium (B. casei, B. epidermis/iodinum, and B .casei), and one isolate matched the species Micrococcus luteus. Additionally, using metabolic fingerprint analysis, two of the isolates were closest in identity to Vibrio species (V. metchnikovii and V. alginolyticus). Of the remaining isolates, one was most similar to Corynebacterium mycetoides, and six were of the genus Staphylococcus (including S. equorum, S. sciuri, S. epidermis, and S. hominis). Four bacteria matched the genus Brevibacterium (B. otidis, and B. casei), and one isolate matched the species Micrococcus luteus. Both phenotypic and genotypic designations offered a consensus for each strain of bacteria at the genus level, with the exception of isolate 12WA, which is matched to both Staphylococcus and Brevibacterium. In the case of isolate 30, there is a full consensus at Micrococcus luteus for all three methods. Comparison of the sequences with the NCBI database showed that isolates matched bacteria of the genus Vibrio (20, 53), Staphylococcus (12WA, 13WA, 16), Brevibacterium (12Y, 18) and Micrococcus (30) (Table 3).
The identifications of bacterial isolates based on 16S rDNA gene sequencing were determined by choosing the species of bacteria with the highest identity match. Identifications were based on identity values of greater than or equal to 96% similarity. It should be noted, however, that a reduction by as little as one percent in the identity match value can result in a different species designation (see Table 3).
Figure 2. Electrophoresis gel used to verify cloning success. Three lanes for each isolate represent PCR amplified 16S sequence and two (A and B) digested plasmid preparations of transformed clones. EcoR1 used as restriction enzyme for digestion.
Discussion
Shrimp aquaculture is threatened by diseases attributed to a variety of bacterial microorganisms introduced to the hatchery environment by means of water supply and food sources. A number of these microbes can be opportunistic pathogens that become virulent in response to environmental cues that favor their survival over that of the host (Aguirre-Guzman et al., 2001). Microbial infection has been associated with an increase in larval deformities and mortality rates (Vandenberghe et al., 2002). Bacteria of the genus Vibrio have been specifically implicated as shrimp pathogens because they are regularly found in high numbers during periods of high larval mortality and disease outbreak in grow out ponds (Saulnier et al., 2000).
The present study genetically characterized fourteen bacterial isolates derived from shrimp aquaculture and compared these results against bacteriological and metabolic identification techniques to assess the need for a polyphasic approach when identifying bacteria from shrimp larvae. As bacteriological and metabolic data showed discrepancies across different phenotypic analyses, our results suggest the need to incorporate 16S rDNA analysis to identify the strains.
The analysis of bacteriological features in this study provided key initial characteristics for grouping strains, as well as a quality control to ensure that cultures were pure. High precision and attention to detail is needed at this stage because as these bacteria were mostly sampled from surfaces and biofilms, cell-cell adhesion between strains is a reality and can lead to difficulty when attempting to isolate pure strains via spreading and colony growth on agar plates. These tests include motility, shape, Gram stain, oxidative or fermentative metabolism, and colony morphology on differential media (e.g. TCBS, YDC, and TZC plates). Coupling these tests with 16S rDNA analysis led to the strain identifications in Table 3. Only two of the fourteen cultures (20 and 53) were Gram-negative, fermentative and motile. These are the only two isolates that have characteristics of the Vibrio species.
The method used for the genetic characterization of the fourteen isolates was 16S rDNA sequencing, which is a genomic approach to the classification of bacteria based upon the 16S ribosomal RNA gene. The 16S rDNA database matches were consistent with the bacteriological findings, in that Gram-negative and Gram-positive identifications matched (Tables 2 and 3). For example, isolates 20 and 53 (identified by bacteriological tests to be Gram- negative) matched V. parahaemolyticus and V. alginolyticus with similarities of 98% and 96% respectively (Table 3). The remaining 12 isolates (identified by bacteriological tests to be Gram-positive) were likewise identified as gram positive by the 16S sequence and phylogenetic analysis. It is not possible, however, to distinguish between different isolates to the species level with this database if identity match values are equivalent. For example, isolate 20 was 98% identical to both V.parahaemolyticus, and V. natriegens, while it was 97% identical to V. harveyi. Isolate 53 was 96% identical to V. alginolyticus and V.campbelli, while it was 95% identical to V.natriegens. It was also 95% identical to a bacterium of a different genus, Photobacterium phosphoreum.
Because the 16S rDNA gene is highly conserved, determination of species and strain distinctions relies upon the resolution of small differences between sequences. Differences as small as 17 base pairs in the 16S gene have been associated with pathogenicity changes (Nilsson et al., 2003). Over the entire 16S gene, such changes require an accuracy of approximately 1% in matching to resolve. Ideally, a resolution of less than 0.1% (single base pair) would be required. In the current study, the entire 16S gene sequence (~1500bp) was assembled from a single primer pair (16S-27F, 16S-1492R), requiring use of sequence data ~750bp in each direction. Because sequencing error rate increased after ~500bp in the current study, overall resolution in the current study was decreased by errors concentrated in the constructed middle region of the sequence. To increase resolution, additional 16S primers covering the middle region would allow accurate sequencing of the entire 16S gene and increased resolution of species differences.
Knowledge of the 16S rDNA sequences of the fourteen bacterial isolates could be used to facilitate the design and implementation of an assay that could characterize the microbial population in a shrimp aquaculture system as well as identify the presence of potential pathogens. For example, recent work by Ji et al.. (2004), has shown that universal primer PCR (UPPCR) and denaturing gradient gel electrophoresis (DGGE) can provide a rapid method for detecting pathogenic bacteria in fish. DGGE has emerged as a powerful tool for its sensitivity in resolving DNA fragments that differ by as little as one nucleotide. Thus, once PCR-amplified fragments coding for 16S rDNA have been generated, they can undergo DGGE such that different sequences within the 16S rDNA gene can be distinguished (Ji et al., 2004). UPPCR-DGGE assays could potentially provide a means of rapid detection of shrimp pathogens in shrimp aquaculture systems.
Conclusions
The 16S rDNA genes of fourteen bacterial isolates derived from shrimp larval aquaculture were successfully amplified, cloned and sequenced. Comparison of the sequences with those available at the NCBI web site using BLAST (nr database) allowed for bacterial classification based on similarity indices of greater than or equal to 96%. Although these identifications were consistent with previous phenotypic analyses of the same isolates (Microlog, Fatty Acid Analysis), there were problems using 16S sequencing to identify the bacterial isolates to species level. Specifically, databases may not contain the sequence submitted for identification, or the species may be too genetically similar for 16S sequencing to be used as a differentiation method. Some increase in resolution over the current study, however, could be expected using additional primers covering overlapping regions of the 16S gene.
More specific designations will require a merging of the 16S method with identification techniques that more accurately reflect the metabolic profile of the isolate. This so called "polyphasic" approach has recently gained attention in the literature (Vandamme, 1996). Such an integrated genetic and phenotypic means of taxonomic classification may offer the most accurate way of identifying bacteria. Such a polyphasic taxonomic system would be possible with software designed to use mathematical and informational strategies to classify bacteria (Vandamme, 1996).
Acknowledgements
The author would like to acknowledge funding support from NSF grant #023600 and the University of Hawaii Sea Grant College Program. In addition, thanks are to the Alvarez Laboratory, Anne Alvarez, Wayne Johnston, Michael Cooney, Grieg Steward, and Olivia Nigro.
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