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Predominant Bacteria Associated with Red Snapper from the Northern Gulf of Mexico a
a
Covadonga R. Arias , Kevin Koenders & Andrea M. Larsen
a
a
Aquatic Microbiology Laboratory, Department of Fisheries and Allied Aquacultures, Auburn University , 253 Upchurch Hall, Auburn , Alabama , 36849 , USA Published online: 09 Dec 2013.
To cite this article: Covadonga R. Arias , Kevin Koenders & Andrea M. Larsen (2013) Predominant Bacteria Associated with Red Snapper from the Northern Gulf of Mexico, Journal of Aquatic Animal Health, 25:4, 281-289, DOI: 10.1080/08997659.2013.847872 To link to this article: http://dx.doi.org/10.1080/08997659.2013.847872
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Aquatic Animal Health 25:281–289, 2013 C American Fisheries Society 2013 ISSN: 0899-7659 print / 1548-8667 online DOI: 10.1080/08997659.2013.847872
ARTICLE
Predominant Bacteria Associated with Red Snapper from the Northern Gulf of Mexico Covadonga R. Arias,* Kevin Koenders, and Andrea M. Larsen
Downloaded by [University of Sherbrooke] at 14:53 19 June 2014
Aquatic Microbiology Laboratory, Department of Fisheries and Allied Aquacultures, Auburn University, 253 Upchurch Hall, Auburn, Alabama 36849, USA
Abstract Since the Deepwater Horizon oil spill in 2010, anecdotal observations of Red Snapper Lutjanus campechanus from the northern Gulf of Mexico exhibiting unusual external lesions have been reported. Two opportunistic bacterial fish pathogens, Vibrio vulnificus and Photobacterium damselae, were recovered from the fish and were deemed responsible for the abnormalities. However, the culturable microbiota of healthy Red Snapper has not yet been characterized. We analyzed the heterotrophic bacteria associated with healthy Red Snapper caught off the Louisiana coast. In total, 179 isolates from 60 fish were recovered from skin and mucus, and 43 isolates were obtained from anterior kidney. All isolates were identified by 16S ribosomal RNA gene sequencing. The Proteobacteria was the predominant phylum in both external and internal samples, followed by the Firmicutes and the Actinobacteria. Within the Proteobacteria, most isolates were members of the genera Vibrio and Photobacterium, and V. natriegens and P. damselae were the predominant species. The results of this study suggest that both Vibrio spp. and Photobacterium spp. are associated with the normal microbiota of healthy Red Snapper. Thus, the opportunistic fish pathogens recovered in previous studies cannot be deemed lesion-forming until Koch’s postulates are fulfilled.
The Red Snapper Lutjanus campechanus is an emblematic fish species of the Gulf of Mexico, with an estimated commercial and recreational fisheries value of $60 million per year in the USA (Gallaway et al. 2009). The Red Snapper, like many other fishery species throughout the world, had been consistently overfished to the point of making commercial fishing nonviable. However, a management plan was implemented by the Gulf of Mexico Fishery Management Council in 1989, and since then the Red Snapper stock has been on the path to recovery (Cowan et al. 2011). Currently, the Red Snapper is considered one of the most valuable sport fish species in the USA. Shortly after the Deepwater Horizon oil spill in 2010, Red Snapper became an object of controversy over the spill’s effects on marine ecosystems. Different media outlets, including local and national newspapers, television channels, and Internet blogs, reported cases in which local fishermen had encountered Red Snapper displaying “unusual” lesions and ulcers on their bodies. Researchers from Louisiana State University analyzed
some of those fish and reported the isolation of Photobacterium damselae and Vibrio vulnificus from the ulcers (Rogers 2011). Since both of these bacteria can behave as opportunistic fish pathogens, a causal association between “sick” Red Snapper and the oil spill was soon made on the basis of the fish being immunocompromised due to exposure to toxic chemicals (Pittman 2011), although no peer-reviewed work on this subject exists to date. Moreover, because both bacterial species were reported to be opportunistic pathogens of humans (Penman et al. 1995; Goodell et al. 2004), the “sick” fish were described as vectors for zoonotic diseases, leading the National Oceanic and Atmospheric Administration (NOAA) to issue a warning on handling finfish with lesions in areas affected by the spill. These reports contrasted with the information disseminated by federal public health authorities to reassure the public that Gulf of Mexico seafood was safe (Ylitalo et al. 2012). Because of the media turmoil during the spill and the economic importance of Red Snapper to the Gulf states, local and federal agencies
*Corresponding author: [email protected] Received March 1, 2013; accepted September 4, 2013
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launched several campaigns to assess the health of this species in the area. In Alabama, nearly 3,000 Red Snapper were examined in 2011, with no significant lesions found (900 bp) 16S rRNA gene sequences were obtained from isolates belonging to the genera
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Vibrio and Photobacterium (using the above-mentioned protocol but with purified DNA as the template for PCR; see Tao et al. 2012 for DNA extraction details) and were imported into BioNumerics version 7.0 (Applied Maths, Sint-Martens-Latem, Belgium) for phylogenetic analysis. Multiple alignment and cluster analysis of sequences was conducted by using the Alignment and Mutation Analysis tool according to BioNumerics’ own proprietary algorithm. BioNumerics was used to calculate similarity matrices, distances (using the Jukes–Cantor correction; Jukes and Cantor 1969), and phylogenetic trees constructed with neighbor-joining (Saitou and Nei 1987) and maximum parsimony algorithms. The ERIC-PCR profiles were also processed with BioNumerics. After conversion, normalization, and background subtraction with mathematical algorithms, levels of similarity between fingerprints were calculated with Pearson’s product-moment correlation coefficient. Cluster analysis was performed according to Tao et al. (2012). TABLE 1.
RESULTS In total, 60 Red Snapper (of the 88 fish harvested) were sampled for bacterial analysis during the study; fish that remained on deck for more than 7 min were not used for bacterial analysis. Fish were examined for external lesions and signs of internal disease during necropsy, and all harvested individuals were deemed healthy. Culture plates yielded approximately 400 bacterial isolates, of which 222 isolates were randomly selected for analysis. External samples (skin and mucus) yielded 179 bacterial isolates, while 43 cultures were recovered from samples of anterior kidney. Fifteen of the 60 fish harbored bacteria in their blood. All sequences found a match in GenBank at 94% maximum identity or higher. Table 1 summarizes the distribution of phyla and genera isolated from Red Snapper. Overall, the majority of the isolates belonged to the phylum Proteobacteria (66.5% of the total), followed by the phylum Firmicutes (23.1%) and the phylum
Bacterial genera isolated from 60 Red Snapper collected off the coast of Louisiana.
Skin and mucus Genus
No. of isolates
% of isolates
Anterior kidney No. of isolates
Vibrio Photobacterium Enterobacter Psychrobacter Shewanella Alteromonas Halomonas Kushneria Pseudoalteromonas Pseudomonas Stenotrophomonas
59 37 5 2 3 1 1 1 1 0 0
Phylum Proteobacteria 33.1 14 20.6 15 2.8 2 1.1 0 1.7 1 0.6 0 0.6 0 0.6 0 0.6 1 0 1 0 3
Macrococcus Bacillus Staphylococcus Exiguobacterium Salinicoccus
20 14 9 3 1
Phylum Firmicutes 11.2 0 7.9 2 5.0 1 1.7 2 0.6 0
Kocuria Microbacterium Micrococcus Rhodococcus Arthrobacter Brachybacterium Corynebacterium Rothia Total
6 6 3 3 1 1 1 1 179
Phylum Actinobacteria 3.4 0 3.4 1 1.7 0 0.6 0 0.6 0 0.6 0 0.6 0 0.6 0 100 43
Total
% of isolates
No. of isolates
% of isolates
31.8 36.4 4.5 0 2.7 0 0 0 2.7 2.7 6.8
73 52 7 2 4 1 1 1 2 1 3
32.9 23.4 3.2 0.9 1.8 0.5 0.5 0.5 0.9 0.5 1.4
0 4.5 2.7 4.5 0
20 16 10 5 1
9.0 7.2 4.5 2.3 0.5
0 2.3 0 0 0 0 0 0 100
6 7 3 3 1 1 1 1 222
2.7 3.2 1.4 1.4 0.5 0.5 0.5 0.5 100
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Actinobacteria (10.4%). Isolates recovered from skin and mucus belonged mainly to the genera Vibrio (33.1% of external isolates) and Photobacterium (20.2%). Proteobacteria genera that were represented by more than one isolate included Enterobacter, Psychrobacter, and Shewanella. Predominant genera within the phylum Firmicutes were Macrococcus (11.2%), Bacillus (7.9%), and Staphylococcus (5.0%). Within the Actinobacteria, the genera Kocuria and Microbacterium accounted for 55% of all external isolates. Among isolates recovered from anterior kidney, the overall distribution of phyla was similar to that observed in external samples, with the Proteobacteria being the predominant phylum (83.4% of all internal isolates), followed by the Firmicutes (11.7%) and the Actinobacteria (2.3%). Of the 43 internal isolates, 14 belonged to the genus Vibrio and 16 belonged to Photobacterium. Pseudomonas and Stenotrophomonas were the only two genera that were recovered solely from internal organs (i.e., were not isolated from external samples). Figure 1 shows the phylogenetic tree that was obtained when partial 16S rRNA gene sequences of the Vibrio spp. isolated from Red Snapper were compared with those from closely related species according to BLAST analysis. Partial 16S sequences from the type strains of Vibrio species were used as references. Only high-quality sequences longer than 930 bp were included in the phylogenetic analysis (58 total sequences). The topologies of the maximum parsimony and neighbor-joining trees were in agreement. Based on the phylogenetic tree, sequences of Vibrio isolated from Red Snapper shared high levels of similarity with V. natriegens (13 isolates), V. rotiferianus (5 isolates), V. harveyi (3 isolates), V. parahaemolyticus (1 isolate), and V. xuii (1 isolate). Interestingly, three groups of sequences did not cluster with any Vibrio reference strain (although they were putatively identified as V. natriegens or V. rotiferianus based on BLAST analysis). These three clusters could represent new species of Vibrio; however, further analyses will be needed to fully characterize these isolates, as only partial 16S rRNA sequences were used in the present study. Figure 2 presents the phylogenetic analysis of the Photobacterium 16S rRNA sequences. Only three species of Photobacterium were recovered from Red Snapper: P. damselae (37 isolates), P. leiognathi (12 isolates), and P. angustum (1 isolate). Ascription to the species level was clearer for these isolates than for representatives of the genus Vibrio. Phylogenetic analysis of Photobacterium isolates recovered from Red Snapper showed that all of the isolates grouped with reference strains. Therefore, no putative new species were identified within this genus. Intraspecies characterization of P. damselae isolates (i.e., using API 20E strips) revealed that eight isolates displayed the typical API 20E profile (2005004) of P. damselae subsp. piscicida, while 29 isolates were urease positive (API 20E profile 2015004) and were identified as P. damselae subsp. damselae. This identification was further corroborated by the P. damselae subsp. piscicida isolates’ inability to grow on TCBS. However, it should be noted that some of the P. damselae subsp. piscicida
isolates were originally recovered on TCBS medium but were unable to grow on this medium after being stored at −80◦ C for more than a year. Figure 3 shows the clustering analysis obtained using ERIC-PCR, wherein all of the P. damselae subsp. piscicida isolates formed a separate cluster from the P. damselae subsp. damselae isolates.
DISCUSSION Predominant bacteria (>60%) associated with Red Snapper from the northern Gulf of Mexico belonged to the phylum Proteobacteria. Previous studies on naturally occurring bacteria of wild-caught marine fish also reported Proteobacteria as the main bacterial phylum associated with the skin and mucus of fishes (Georgala 1958; Liston 1957; Horsley 1977). However, in those studies, Pseudomonas was identified as the most abundant genus in teleosts and elasmobranchs (Liston 1957; Colwell and Liston 1962; Horsley 1977), whereas in our study Vibrio and Photobacterium were the most frequently recovered bacteria. It is well established that culture media and growing conditions influence the growth of bacteria and may favor the isolation of particular types (Barer and Harwood 1999). For skin and mucus samples, we used a general medium supplemented with salt (TSA-2) and a selective medium for Vibrio (TCBS); for internal samples, we used a rich medium (blood agar). These culture media are typically recommended for the examination of marine fishes (AFS-FHS 2004). Vibrio and Photobacterium were recovered in abundance on both TSA-2 and TCBS, and we did not observe a bias based on culture medium. Isolation of bacteria from anterior kidney was somewhat unexpected since the internal organs of fish are presumed to be sterile, although diverse bacterial genera have been found in the internal organs of healthy fish (Horsley 1977; Toranzo et al. 1993; Gomez-Gil et al. 2007). The reasons for the presence of these bacteria are unclear but warrant further investigation. Despite the value of snappers as commercial, recreational, and potential aquaculture species, there is almost no information on the normal microbiota of snappers. Gomez-Gil et al. (2007) characterized the Vibrionaceae of Spotted Rose Snapper Lutjanus guttatus (collected along the Pacific coast of Mexico); those authors identified P. damselae subsp. damselae and V. harveyi as the predominant species. Our results were similar to theirs, as we found a high percentage (20.6%) of P. damselae isolates (16S rRNA sequencing does not allow ascription to subspecies within P. damselae). However, the Vibrio species most frequently recovered from Red Snapper was V. natriegens followed by V. rotiferianus, while only three isolates were identified as V. harveyi. As with the study by Gomez-Gil et al. (2007), we found significant clusters of Vibrio sequences that could not be clearly ascribed to any species after running the phylogenetic analysis, and these could represent new Vibrio species. Our data show that P. damselae is a commensal bacteria species on Red Snapper skin and mucus that is frequently (35% of fish yielded this bacterium) associated with apparently
285
BACTERIA ASSOCIATED WITH RED SNAPPER V. cholerae ATCC 14731 V. fortis LMG 21557 V. proteolyticus ATCC 15338
95 87
V. xuii DSM 17185 2S1 DSM 21326 90
100
14T1 98 83
V. mytilii CECT 632 V. diabolicus LMG 19805 V. parahaemolyticus ATCC 17802
19S3 71T2
100
50S2 62T4
75
38S1 .65S2 65S3
85 81
65S1 59T1 51S3 76 54S5 76 62T3 65S4 50S1 64S1 100 19S1 89
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75
99 99
47B4 85T1 29S1 19S4
47B2 58B11
77
80
47B3 18S3 18S1 88S2 88S1 14S2 13S1 V. azureus NBRC 104587 24B1 82 24B1 82 100 24S4 V. harveyi NCIMB 1280
12T8 18T1 47B1 60B1 12T1 V. campbellii ATCC 25920 V. rotiferianus LMG 21460 12T2 60B2 2B2 10B4 24T1 V. alginolyticus ATCC 17749 68S1 19S2 66S3 62S2 66S4 V. natriegens ATCC 14048 12T2 74S3
87
62S2 77T1 12T3 67S2 77S2 14T4
FIGURE 1. Phylogenetic tree (derived from partial 16S ribosomal RNA gene sequences) of the Vibrio isolates recovered from Red Snapper that were captured off the coast of Louisiana. Isolate codes reflect the fish number (first digits), followed by the isolation medium (T = thiosulfate–citrate–bile salts–sucrose agar; S = tryptone soy agar supplemented with 2% NaCl; B = blood agar) and colony number (1–5). Sequences from type strains are included for comparison (ATCC = American Type Culture Collection; DSM = German Collection of Microorganisms and Cell Cultures; CECT = Spanish Type Culture Collection; LMG = Laboratory of Microbiology, Ghent University [Belgium]; NBRC = Biological Resource Center, NITE [Japan]; NCIMB = National Collection of Industrial, Food, and Marine Bacteria [UK]). The tree topology was obtained by the neighbor-joining method (Jukes–Cantor correction). Numbers at nodes indicate bootstrap values (1,000 replicates) higher than 50%; the scale bar represents 1% sequence divergence.
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ARIAS ET AL. 2B1 97 87
57S4 9B1
100
1B3 48B2 58S3 91
14B2 1B4 P. angustum M9A2
100
57S3
100
P. leiognathi L1 2S3 2S4 15S4 2B3 11S2 51T1
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24S1 39S1 70S4 77T2 75
100
98 98 99
. TOS1 P. damselae subs. piscicida P. damselae subs. damselae ATCC 33539
57T2
39S4
16T2 51S6 19T1 16T1 48S5 37S2 23B1 70S3 57B1 40S1 70S6 58S4 19T3 24S2 34S2 54S1 57S1 78S1 3T2 48S2 57T1 10B3 58B9 16B3 14B1 16B2 19T2 24T3 11S5
FIGURE 2. Phylogenetic tree (derived from partial 16S ribosomal RNA gene sequences) of the Photobacterium isolates recovered from Red Snapper that were captured off the coast of Louisiana. Red Snapper isolates reflect the fish number (first digits), followed by the isolation medium (T = thiosulfate–citrate–bile salts–sucrose agar; S = tryptone soy agar supplemented with 2% NaCl; B = blood agar) and colony number (1–5). Sequences from type strains are included for comparison (ATCC = American Type Culture Collection). The tree topology was obtained by the neighbor-joining method (Jukes–Cantor correction). Numbers at nodes indicate bootstrap values (1,000 replicates) higher than 50%; the scale bar represents 1% sequence divergence.
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BACTERIA ASSOCIATED WITH RED SNAPPER
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FIGURE 3. Enterobacterial repetitive intergenic consensus PCR patterns of Photobacterium damselae isolates. The dendrogram was derived from a cluster analysis (based on the unweighted pair group method with arithmetic mean) following the calculation of similarities via Pearson’s product-moment correlation coefficient. The tracks show the processed band patterns after conversion, normalization, and background subtraction (BioNumerics version 7.0). Clusters corresponding to P. damselae subsp. damselae and P. damselae subsp. piscicida are indicated. Cophenetic correlation coefficients, reflecting the robustness of each node, are indicated (only values ≥ 75% are shown).
healthy fish. Although both subspecies of P. damselae can be pathogenic for fish (Labella et al. 2011), P. damselae subsp. damselae is considered an opportunistic pathogen, whereas P. damselae subsp. piscicida is a serious fish pathogen that causes great economic losses in marine aquaculture (Romalde 2002). Although the majority (78%) of our P. damselae isolates were identified as P. damselae subsp. damselae, some (22%) of the isolates were ascribed to P. damselae subsp. piscicida based on API 20E profiles. Similarly, we determined that Vibrio spp., particularly V. natriegens and V. rotiferianus, are part of the autochthonous microbiota of Red Snapper. Interestingly, V. natriegens was first classified as Pseudomonas natriegens (Eagon 1962) and later reclassified as Beneckea (Payne et al. 1961) and then Vibrio
(Baumann et al. 1980). This bacterium has one the shortest generation times of any bacteria (
Journal of Aquatic Animal Health Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uahh20
Predominant Bacteria Associated with Red Snapper from the Northern Gulf of Mexico a
a
Covadonga R. Arias , Kevin Koenders & Andrea M. Larsen
a
a
Aquatic Microbiology Laboratory, Department of Fisheries and Allied Aquacultures, Auburn University , 253 Upchurch Hall, Auburn , Alabama , 36849 , USA Published online: 09 Dec 2013.
To cite this article: Covadonga R. Arias , Kevin Koenders & Andrea M. Larsen (2013) Predominant Bacteria Associated with Red Snapper from the Northern Gulf of Mexico, Journal of Aquatic Animal Health, 25:4, 281-289, DOI: 10.1080/08997659.2013.847872 To link to this article: http://dx.doi.org/10.1080/08997659.2013.847872
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Aquatic Animal Health 25:281–289, 2013 C American Fisheries Society 2013 ISSN: 0899-7659 print / 1548-8667 online DOI: 10.1080/08997659.2013.847872
ARTICLE
Predominant Bacteria Associated with Red Snapper from the Northern Gulf of Mexico Covadonga R. Arias,* Kevin Koenders, and Andrea M. Larsen
Downloaded by [University of Sherbrooke] at 14:53 19 June 2014
Aquatic Microbiology Laboratory, Department of Fisheries and Allied Aquacultures, Auburn University, 253 Upchurch Hall, Auburn, Alabama 36849, USA
Abstract Since the Deepwater Horizon oil spill in 2010, anecdotal observations of Red Snapper Lutjanus campechanus from the northern Gulf of Mexico exhibiting unusual external lesions have been reported. Two opportunistic bacterial fish pathogens, Vibrio vulnificus and Photobacterium damselae, were recovered from the fish and were deemed responsible for the abnormalities. However, the culturable microbiota of healthy Red Snapper has not yet been characterized. We analyzed the heterotrophic bacteria associated with healthy Red Snapper caught off the Louisiana coast. In total, 179 isolates from 60 fish were recovered from skin and mucus, and 43 isolates were obtained from anterior kidney. All isolates were identified by 16S ribosomal RNA gene sequencing. The Proteobacteria was the predominant phylum in both external and internal samples, followed by the Firmicutes and the Actinobacteria. Within the Proteobacteria, most isolates were members of the genera Vibrio and Photobacterium, and V. natriegens and P. damselae were the predominant species. The results of this study suggest that both Vibrio spp. and Photobacterium spp. are associated with the normal microbiota of healthy Red Snapper. Thus, the opportunistic fish pathogens recovered in previous studies cannot be deemed lesion-forming until Koch’s postulates are fulfilled.
The Red Snapper Lutjanus campechanus is an emblematic fish species of the Gulf of Mexico, with an estimated commercial and recreational fisheries value of $60 million per year in the USA (Gallaway et al. 2009). The Red Snapper, like many other fishery species throughout the world, had been consistently overfished to the point of making commercial fishing nonviable. However, a management plan was implemented by the Gulf of Mexico Fishery Management Council in 1989, and since then the Red Snapper stock has been on the path to recovery (Cowan et al. 2011). Currently, the Red Snapper is considered one of the most valuable sport fish species in the USA. Shortly after the Deepwater Horizon oil spill in 2010, Red Snapper became an object of controversy over the spill’s effects on marine ecosystems. Different media outlets, including local and national newspapers, television channels, and Internet blogs, reported cases in which local fishermen had encountered Red Snapper displaying “unusual” lesions and ulcers on their bodies. Researchers from Louisiana State University analyzed
some of those fish and reported the isolation of Photobacterium damselae and Vibrio vulnificus from the ulcers (Rogers 2011). Since both of these bacteria can behave as opportunistic fish pathogens, a causal association between “sick” Red Snapper and the oil spill was soon made on the basis of the fish being immunocompromised due to exposure to toxic chemicals (Pittman 2011), although no peer-reviewed work on this subject exists to date. Moreover, because both bacterial species were reported to be opportunistic pathogens of humans (Penman et al. 1995; Goodell et al. 2004), the “sick” fish were described as vectors for zoonotic diseases, leading the National Oceanic and Atmospheric Administration (NOAA) to issue a warning on handling finfish with lesions in areas affected by the spill. These reports contrasted with the information disseminated by federal public health authorities to reassure the public that Gulf of Mexico seafood was safe (Ylitalo et al. 2012). Because of the media turmoil during the spill and the economic importance of Red Snapper to the Gulf states, local and federal agencies
*Corresponding author: [email protected] Received March 1, 2013; accepted September 4, 2013
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launched several campaigns to assess the health of this species in the area. In Alabama, nearly 3,000 Red Snapper were examined in 2011, with no significant lesions found (900 bp) 16S rRNA gene sequences were obtained from isolates belonging to the genera
283
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Vibrio and Photobacterium (using the above-mentioned protocol but with purified DNA as the template for PCR; see Tao et al. 2012 for DNA extraction details) and were imported into BioNumerics version 7.0 (Applied Maths, Sint-Martens-Latem, Belgium) for phylogenetic analysis. Multiple alignment and cluster analysis of sequences was conducted by using the Alignment and Mutation Analysis tool according to BioNumerics’ own proprietary algorithm. BioNumerics was used to calculate similarity matrices, distances (using the Jukes–Cantor correction; Jukes and Cantor 1969), and phylogenetic trees constructed with neighbor-joining (Saitou and Nei 1987) and maximum parsimony algorithms. The ERIC-PCR profiles were also processed with BioNumerics. After conversion, normalization, and background subtraction with mathematical algorithms, levels of similarity between fingerprints were calculated with Pearson’s product-moment correlation coefficient. Cluster analysis was performed according to Tao et al. (2012). TABLE 1.
RESULTS In total, 60 Red Snapper (of the 88 fish harvested) were sampled for bacterial analysis during the study; fish that remained on deck for more than 7 min were not used for bacterial analysis. Fish were examined for external lesions and signs of internal disease during necropsy, and all harvested individuals were deemed healthy. Culture plates yielded approximately 400 bacterial isolates, of which 222 isolates were randomly selected for analysis. External samples (skin and mucus) yielded 179 bacterial isolates, while 43 cultures were recovered from samples of anterior kidney. Fifteen of the 60 fish harbored bacteria in their blood. All sequences found a match in GenBank at 94% maximum identity or higher. Table 1 summarizes the distribution of phyla and genera isolated from Red Snapper. Overall, the majority of the isolates belonged to the phylum Proteobacteria (66.5% of the total), followed by the phylum Firmicutes (23.1%) and the phylum
Bacterial genera isolated from 60 Red Snapper collected off the coast of Louisiana.
Skin and mucus Genus
No. of isolates
% of isolates
Anterior kidney No. of isolates
Vibrio Photobacterium Enterobacter Psychrobacter Shewanella Alteromonas Halomonas Kushneria Pseudoalteromonas Pseudomonas Stenotrophomonas
59 37 5 2 3 1 1 1 1 0 0
Phylum Proteobacteria 33.1 14 20.6 15 2.8 2 1.1 0 1.7 1 0.6 0 0.6 0 0.6 0 0.6 1 0 1 0 3
Macrococcus Bacillus Staphylococcus Exiguobacterium Salinicoccus
20 14 9 3 1
Phylum Firmicutes 11.2 0 7.9 2 5.0 1 1.7 2 0.6 0
Kocuria Microbacterium Micrococcus Rhodococcus Arthrobacter Brachybacterium Corynebacterium Rothia Total
6 6 3 3 1 1 1 1 179
Phylum Actinobacteria 3.4 0 3.4 1 1.7 0 0.6 0 0.6 0 0.6 0 0.6 0 0.6 0 100 43
Total
% of isolates
No. of isolates
% of isolates
31.8 36.4 4.5 0 2.7 0 0 0 2.7 2.7 6.8
73 52 7 2 4 1 1 1 2 1 3
32.9 23.4 3.2 0.9 1.8 0.5 0.5 0.5 0.9 0.5 1.4
0 4.5 2.7 4.5 0
20 16 10 5 1
9.0 7.2 4.5 2.3 0.5
0 2.3 0 0 0 0 0 0 100
6 7 3 3 1 1 1 1 222
2.7 3.2 1.4 1.4 0.5 0.5 0.5 0.5 100
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Actinobacteria (10.4%). Isolates recovered from skin and mucus belonged mainly to the genera Vibrio (33.1% of external isolates) and Photobacterium (20.2%). Proteobacteria genera that were represented by more than one isolate included Enterobacter, Psychrobacter, and Shewanella. Predominant genera within the phylum Firmicutes were Macrococcus (11.2%), Bacillus (7.9%), and Staphylococcus (5.0%). Within the Actinobacteria, the genera Kocuria and Microbacterium accounted for 55% of all external isolates. Among isolates recovered from anterior kidney, the overall distribution of phyla was similar to that observed in external samples, with the Proteobacteria being the predominant phylum (83.4% of all internal isolates), followed by the Firmicutes (11.7%) and the Actinobacteria (2.3%). Of the 43 internal isolates, 14 belonged to the genus Vibrio and 16 belonged to Photobacterium. Pseudomonas and Stenotrophomonas were the only two genera that were recovered solely from internal organs (i.e., were not isolated from external samples). Figure 1 shows the phylogenetic tree that was obtained when partial 16S rRNA gene sequences of the Vibrio spp. isolated from Red Snapper were compared with those from closely related species according to BLAST analysis. Partial 16S sequences from the type strains of Vibrio species were used as references. Only high-quality sequences longer than 930 bp were included in the phylogenetic analysis (58 total sequences). The topologies of the maximum parsimony and neighbor-joining trees were in agreement. Based on the phylogenetic tree, sequences of Vibrio isolated from Red Snapper shared high levels of similarity with V. natriegens (13 isolates), V. rotiferianus (5 isolates), V. harveyi (3 isolates), V. parahaemolyticus (1 isolate), and V. xuii (1 isolate). Interestingly, three groups of sequences did not cluster with any Vibrio reference strain (although they were putatively identified as V. natriegens or V. rotiferianus based on BLAST analysis). These three clusters could represent new species of Vibrio; however, further analyses will be needed to fully characterize these isolates, as only partial 16S rRNA sequences were used in the present study. Figure 2 presents the phylogenetic analysis of the Photobacterium 16S rRNA sequences. Only three species of Photobacterium were recovered from Red Snapper: P. damselae (37 isolates), P. leiognathi (12 isolates), and P. angustum (1 isolate). Ascription to the species level was clearer for these isolates than for representatives of the genus Vibrio. Phylogenetic analysis of Photobacterium isolates recovered from Red Snapper showed that all of the isolates grouped with reference strains. Therefore, no putative new species were identified within this genus. Intraspecies characterization of P. damselae isolates (i.e., using API 20E strips) revealed that eight isolates displayed the typical API 20E profile (2005004) of P. damselae subsp. piscicida, while 29 isolates were urease positive (API 20E profile 2015004) and were identified as P. damselae subsp. damselae. This identification was further corroborated by the P. damselae subsp. piscicida isolates’ inability to grow on TCBS. However, it should be noted that some of the P. damselae subsp. piscicida
isolates were originally recovered on TCBS medium but were unable to grow on this medium after being stored at −80◦ C for more than a year. Figure 3 shows the clustering analysis obtained using ERIC-PCR, wherein all of the P. damselae subsp. piscicida isolates formed a separate cluster from the P. damselae subsp. damselae isolates.
DISCUSSION Predominant bacteria (>60%) associated with Red Snapper from the northern Gulf of Mexico belonged to the phylum Proteobacteria. Previous studies on naturally occurring bacteria of wild-caught marine fish also reported Proteobacteria as the main bacterial phylum associated with the skin and mucus of fishes (Georgala 1958; Liston 1957; Horsley 1977). However, in those studies, Pseudomonas was identified as the most abundant genus in teleosts and elasmobranchs (Liston 1957; Colwell and Liston 1962; Horsley 1977), whereas in our study Vibrio and Photobacterium were the most frequently recovered bacteria. It is well established that culture media and growing conditions influence the growth of bacteria and may favor the isolation of particular types (Barer and Harwood 1999). For skin and mucus samples, we used a general medium supplemented with salt (TSA-2) and a selective medium for Vibrio (TCBS); for internal samples, we used a rich medium (blood agar). These culture media are typically recommended for the examination of marine fishes (AFS-FHS 2004). Vibrio and Photobacterium were recovered in abundance on both TSA-2 and TCBS, and we did not observe a bias based on culture medium. Isolation of bacteria from anterior kidney was somewhat unexpected since the internal organs of fish are presumed to be sterile, although diverse bacterial genera have been found in the internal organs of healthy fish (Horsley 1977; Toranzo et al. 1993; Gomez-Gil et al. 2007). The reasons for the presence of these bacteria are unclear but warrant further investigation. Despite the value of snappers as commercial, recreational, and potential aquaculture species, there is almost no information on the normal microbiota of snappers. Gomez-Gil et al. (2007) characterized the Vibrionaceae of Spotted Rose Snapper Lutjanus guttatus (collected along the Pacific coast of Mexico); those authors identified P. damselae subsp. damselae and V. harveyi as the predominant species. Our results were similar to theirs, as we found a high percentage (20.6%) of P. damselae isolates (16S rRNA sequencing does not allow ascription to subspecies within P. damselae). However, the Vibrio species most frequently recovered from Red Snapper was V. natriegens followed by V. rotiferianus, while only three isolates were identified as V. harveyi. As with the study by Gomez-Gil et al. (2007), we found significant clusters of Vibrio sequences that could not be clearly ascribed to any species after running the phylogenetic analysis, and these could represent new Vibrio species. Our data show that P. damselae is a commensal bacteria species on Red Snapper skin and mucus that is frequently (35% of fish yielded this bacterium) associated with apparently
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BACTERIA ASSOCIATED WITH RED SNAPPER V. cholerae ATCC 14731 V. fortis LMG 21557 V. proteolyticus ATCC 15338
95 87
V. xuii DSM 17185 2S1 DSM 21326 90
100
14T1 98 83
V. mytilii CECT 632 V. diabolicus LMG 19805 V. parahaemolyticus ATCC 17802
19S3 71T2
100
50S2 62T4
75
38S1 .65S2 65S3
85 81
65S1 59T1 51S3 76 54S5 76 62T3 65S4 50S1 64S1 100 19S1 89
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75
99 99
47B4 85T1 29S1 19S4
47B2 58B11
77
80
47B3 18S3 18S1 88S2 88S1 14S2 13S1 V. azureus NBRC 104587 24B1 82 24B1 82 100 24S4 V. harveyi NCIMB 1280
12T8 18T1 47B1 60B1 12T1 V. campbellii ATCC 25920 V. rotiferianus LMG 21460 12T2 60B2 2B2 10B4 24T1 V. alginolyticus ATCC 17749 68S1 19S2 66S3 62S2 66S4 V. natriegens ATCC 14048 12T2 74S3
87
62S2 77T1 12T3 67S2 77S2 14T4
FIGURE 1. Phylogenetic tree (derived from partial 16S ribosomal RNA gene sequences) of the Vibrio isolates recovered from Red Snapper that were captured off the coast of Louisiana. Isolate codes reflect the fish number (first digits), followed by the isolation medium (T = thiosulfate–citrate–bile salts–sucrose agar; S = tryptone soy agar supplemented with 2% NaCl; B = blood agar) and colony number (1–5). Sequences from type strains are included for comparison (ATCC = American Type Culture Collection; DSM = German Collection of Microorganisms and Cell Cultures; CECT = Spanish Type Culture Collection; LMG = Laboratory of Microbiology, Ghent University [Belgium]; NBRC = Biological Resource Center, NITE [Japan]; NCIMB = National Collection of Industrial, Food, and Marine Bacteria [UK]). The tree topology was obtained by the neighbor-joining method (Jukes–Cantor correction). Numbers at nodes indicate bootstrap values (1,000 replicates) higher than 50%; the scale bar represents 1% sequence divergence.
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57S4 9B1
100
1B3 48B2 58S3 91
14B2 1B4 P. angustum M9A2
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57S3
100
P. leiognathi L1 2S3 2S4 15S4 2B3 11S2 51T1
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24S1 39S1 70S4 77T2 75
100
98 98 99
. TOS1 P. damselae subs. piscicida P. damselae subs. damselae ATCC 33539
57T2
39S4
16T2 51S6 19T1 16T1 48S5 37S2 23B1 70S3 57B1 40S1 70S6 58S4 19T3 24S2 34S2 54S1 57S1 78S1 3T2 48S2 57T1 10B3 58B9 16B3 14B1 16B2 19T2 24T3 11S5
FIGURE 2. Phylogenetic tree (derived from partial 16S ribosomal RNA gene sequences) of the Photobacterium isolates recovered from Red Snapper that were captured off the coast of Louisiana. Red Snapper isolates reflect the fish number (first digits), followed by the isolation medium (T = thiosulfate–citrate–bile salts–sucrose agar; S = tryptone soy agar supplemented with 2% NaCl; B = blood agar) and colony number (1–5). Sequences from type strains are included for comparison (ATCC = American Type Culture Collection). The tree topology was obtained by the neighbor-joining method (Jukes–Cantor correction). Numbers at nodes indicate bootstrap values (1,000 replicates) higher than 50%; the scale bar represents 1% sequence divergence.
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FIGURE 3. Enterobacterial repetitive intergenic consensus PCR patterns of Photobacterium damselae isolates. The dendrogram was derived from a cluster analysis (based on the unweighted pair group method with arithmetic mean) following the calculation of similarities via Pearson’s product-moment correlation coefficient. The tracks show the processed band patterns after conversion, normalization, and background subtraction (BioNumerics version 7.0). Clusters corresponding to P. damselae subsp. damselae and P. damselae subsp. piscicida are indicated. Cophenetic correlation coefficients, reflecting the robustness of each node, are indicated (only values ≥ 75% are shown).
healthy fish. Although both subspecies of P. damselae can be pathogenic for fish (Labella et al. 2011), P. damselae subsp. damselae is considered an opportunistic pathogen, whereas P. damselae subsp. piscicida is a serious fish pathogen that causes great economic losses in marine aquaculture (Romalde 2002). Although the majority (78%) of our P. damselae isolates were identified as P. damselae subsp. damselae, some (22%) of the isolates were ascribed to P. damselae subsp. piscicida based on API 20E profiles. Similarly, we determined that Vibrio spp., particularly V. natriegens and V. rotiferianus, are part of the autochthonous microbiota of Red Snapper. Interestingly, V. natriegens was first classified as Pseudomonas natriegens (Eagon 1962) and later reclassified as Beneckea (Payne et al. 1961) and then Vibrio
(Baumann et al. 1980). This bacterium has one the shortest generation times of any bacteria (
