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Keywords:

  • coral;
  • deep sea;
  • cold water;
  • bacteria;
  • Lophelia

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Microorganisms associated with corals are hypothesized to contribute to the function of the host animal by cycling nutrients, breaking down carbon sources, fixing nitrogen, and producing antibiotics. This is the first study to culture and characterize bacteria from Lophelia pertusa, a cold-water coral found in the deep sea, in an effort to understand the roles that the microorganisms play in the coral microbial community. Two sites in the northern Gulf of Mexico were sampled over 2 years. Bacteria were cultured from coral tissue, skeleton, and mucus, identified by 16S rRNA genes, and subjected to biochemical testing. Most isolates were members of the Gammaproteobacteria, although there was one isolate each from the Betaproteobacteria and Actinobacteria. Phylogenetic results showed that both sampling sites shared closely related isolates (e.g. Pseudoalteromonas spp.), indicating possible temporally and geographically stable bacterial–coral associations. The Kirby–Bauer antibiotic susceptibility test was used to separate bacteria to the strain level, with the results showing that isolates that were phylogenetically tightly grouped had varying responses to antibiotics. These results support the conclusion that phylogenetic placement cannot predict strain-level differences and further highlight the need for culture-based experiments to supplement culture-independent studies.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Coral-associated bacteria are hypothesized to perform many functions. They can potentially be probiotic, secrete bioactive compounds that prevent biofouling of the coral tissue and skeleton, or aid in cycling nutrients and carbon (Dobretsov & Qian, 2004; Reshef et al., 2006; Ritchie, 2006; Neulinger et al., 2008). While it is widely understood that shallow-water tropical corals depend on carbon translocation from symbiotic zooxanthellae, the degree to which corals rely on bacterial symbiosis is not fully known. In the case of deep-water corals, which lack zooxanthellae, the symbiosis between the coral and its associated microbial community has the potential to take on a more prominent role.

Deep-water or cold-water reefs have recently become an important topic in research and conservation, as more industries attempt to exploit fisheries, pharmaceutical, and mineral resources from deep ocean waters (Roberts & Hirshfield, 2004; Davies et al., 2007; Lumsden et al., 2007; Synnes, 2007). Deep-water reefs mirror the impressive levels of biodiversity commonly associated with tropical euphotic reefs (Reed, 2002a, b); however, the environmental conditions that support their growth are very different. Therefore, studies of deep-water reefs and their associated microbial communities are important additions to the field of coral ecology. Lophelia pertusa grows in benthic waters that remain between 4 and 13 °C; reefs in the Gulf of Mexico are limited to deep waters (>300 m) to find this temperature range, while L. pertusa reefs in Norway can be found in waters <50 m deep due to low surface water temperatures (Freiwald, 2002).

Molecular investigations have been performed on L. pertusa from the Atlantic, Gulf of Mexico, and Norwegian fjords, in order to characterize the associated microbiota and determine whether species-specific associations exist (Yakimov et al., 2006; Neulinger et al., 2008; Kellogg et al., 2009; Schöttner et al., 2009). These studies have shown that the bacterial communities found on L. pertusa are unique and distinct from the surrounding seawater and rubble, implying that the coral influences its microbial community. Neulinger et al. (2008) and Kellogg et al. (2009) found high numbers of L. pertusa bacteria that were closely related to bacteria associated with other coral species, both deep and shallow. In addition, L. pertusa-specific mycoplasma-like bacteria (i.e. Candidatus Mycoplasma corallicola; Kellogg et al., 2009; Neulinger et al., 2009) and thiotrophic bacteria were identified through culture-independent methods (Neulinger et al., 2008; Kellogg et al., 2009). The authors suggest a general class of coral-associated bacteria shared by both shallow- and deep-water corals, with ecological niches filled by a variety of bacterial phylotypes.

It is important to acknowledge that culture-based methods underestimate the true diversity of coral-associated bacteria; however, culturing is necessary to biochemically classify and analyze the bacterial portion of the coral holobiont. Culturing marine microorganisms presents many challenges, as studies have shown that there is minimal overlap between bacteria characterized using molecular and culture-based methods (Rohwer et al., 2001). To date, all L. pertusa bacterial-community studies have been based solely on molecular methods (Yakimov et al., 2006; Neulinger et al., 2008; Hansson et al., 2009; Kellogg et al., 2009; Schöttner et al., 2009), although culture-based studies of cold-water gorgonians have been performed (Brück et al., 2007; Hall-Spencer et al., 2007; Gray et al., 2011). This study is the first to examine bacteria cultured from L. pertusa, classify them by partial 16S rRNA gene sequences and characterize them by their antibiotic susceptibility patterns.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Sampling sites

Samples of the cold-water coral L. pertusa were collected in the northeastern Gulf of Mexico for the cultivation of associated bacteria (Fig. 1). At sample site Viosca Knoll 826 (VK 826), the corals were at a depth of 500 m, with the temperature ranging from 7 to 9 °C (8 °C during collection). The average pH was 7.79 ± 0.01, and the average oxygen saturation was 6.65 ± 0.02 mL L−1. Abundant Lophelia thickets were observed with heavily calcified skeletons, and minor hydrocarbon seepage was noted. Isolates with the prefix 4753 were collected and cultured in 2004, and isolates with the prefixes 4878 and 4881 were collected and cultured in 2005 on two separate dives 2 days apart. The corals at sample site Viosca Knoll 906/862 (VK 906/862) were located at 315 m depth, with temperatures ranging from 9 to 13 °C (11 °C during collection), possibly indicating thermal stress on L. pertusa. The average pH was 7.86 ± 0.00 and the average oxygen saturation was 6.22 ± 0.02 mL L−1. The coral skeletons at this site were more delicate and fragile than those at VK 826. Bacterial isolates with the prefix 4746 were collected and cultured in 2004 and isolates with the prefix 4873 were collected and cultured in 2005. Both sites are located on the upper continental shelf and had a salinity of 35 psu. Although two color morphs of L. pertusa have been described (white and orange/red; Neulinger et al., 2008), only the white type has been observed at these sites in the Gulf of Mexico.

image

Figure 1.  Location of the sampling sites in the northern Gulf of Mexico.

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Bacterial isolation

To retrieve deep-sea samples of L. pertusa without exposing them to extreme temperature gradients, the Kellogg sampler was used to maintain individual coral branches in separate, insulated compartments (see Kellogg et al. 2009 for a full description). Samples were processed immediately after the dive. Using sterile procedures, a homogenate of coral tissue, mucus, and skeleton was plated onto glycerol artificial seawater agar (GASWA; Smith & Hayasaka, 1982) and allowed to grow up to 14 days at 4 °C. Colonies with unique morphologies were picked and isolated. Over 200 bacterial colonies were isolated for characterization.

Bacterial identification by 16S rRNA gene amplification

DNA from each bacterial isolate was extracted using the Qiagen DNeasy kit, following the manufacturer's protocols, and then amplified with the primers Eco8F (5′-AGAGTTTGATCCTGGCTCAG) (Edwards et al., 1989) and 1492R (5′-GGTTACCTTGTTACGACTT) (Stackebrandt & Liesack, 1993) by a PCR. PCR reagents consisted of 25 μL of Amplitaq Gold master mix plus enzyme, 21 μL DI water, 1 μL (10 pmol) of each primer, and 2 μL of template. Thermal cycling conditions were 15 min at 95 °C; 35 cycles of 1 min at 95 °C, 1 min at 50 °C, and 2 min at 72 °C; and 10 min at 72 °C for a final extension and a 4 °C hold. The PCR product was purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA). DNA was either sequenced directly from the 16S rDNA or cloned using a Qiagen PCR cloning kit. Clone inserts were amplified using M13/pUC forward primers. Amplification products were sequenced by Northwoods DNA Inc. (Bemidji, MN) unidirectionally using Eco8F. Isolates were archived in duplicate in 75% glycerol/25% GASW and stored at −80 °C.

Kirby–Bauer antibiotic resistance profiling

Frozen isolates were regrown on GASWA plates and in 3 mL of GASW at 7 °C for further characterization. Using the BBL Prompt Inoculation System, an inoculum containing approximately 1.5 × 108 CFU mL−1 was made from each bacterial isolate and spread onto individual Petri dishes containing Mueller–Hinton II agar (amended with 20.8 g NaCl L−1 to allow halophilic bacterial growth). Antibiotic discs containing standardized concentrations of penicillin (10 IU/IE/UI), tetracycline (30 μg), clindamycin (2 μg), polymixin B (300 IU/IE/UI), chloramphenicol (30 μg), and novobiocin (30 μg) were pressed into the agar. Plates were incubated at 4–7 °C until a lawn of bacteria was observed, up to 30 days. Control plates were made using Staphylococcus aureus ATCC 25923 to confirm proper bacterial growth on the medium and standard antibiotic activities, and were incubated at 37 °C for 24 h. The diameters of the circular zones of bacterial growth inhibition were measured; the diameter of the antibiotic disc was subtracted from the total, leaving the zone of inhibition.

Phylogenetic analysis

Partial 16S rRNA gene sequences were processed using the base-calling software phred (Ewing & Green, 1998; Ewing et al., 1998) to add quality scores. greengenes (DeSantis et al., 2006) was used to trim the sequences based on their quality scores. Dereplication of the sequences was accomplished using fastgroupii (Yu et al., 2006), with matches of 97% or higher grouped together.

Nucleotide sequence accession numbers

Partial 16S rRNA gene sequences of all isolates have been archived in GenBank under accession numbers HQ640746 through HQ640941.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Bacterial isolation and growth

Of the 196 isolates with 16S rRNA gene sequence data (Tables 1–3), 26 (13.3%) could not be regrown on GASWA and are considered lost, 47 (24.0%) were grown on GASWA, but would not grow at all on Muller–Hinton II agar, and 45 (23.0%) grew poorly on Muller–Hinton II agar. Bacterial isolates from site VK 826, which contained large, healthy colonies of L. pertusa, all belonged to the Gammaproteobacteria and were dominated by Pseudoalteromonas spp. and Photobacterium spp. (Table 1). Bacterial isolates from all three dives on VK 826 were phylogenetically very similar; several isolates from the different dives had the same top match in GenBank (e.g. Pseudoalteromonas sp. B149). All except one isolate had >97% similarity to their top matches in GenBank.

Table 1.   Phylogenetic affiliations of bacterial isolates from site VK 826 in the northern Gulf of Mexico
VK 826      
IsolateTop GenBank matchSimilarity (%)Accession no.20042005Repeats
  • 16S rRNA gene sequences were compared with sequences in GenBank. Isolates were clustered using fastgroupii, with the number of isolates clustering together noted in the Repeats column. The top phylogenetic match for each isolate is indicated by the strain name, accession number, and similarity.

  • *

    Group includes isolates from both sites. These groups are expanded in Table 3.

Gammaproteobacteria
 Halomonadaceae
  4881K6-B5Cobetia marina strain D7065100FJ161356 X 
  4878K4-B1Uncultured bacterium clone CB-14100HM100916 X 
  4878K4-B11Uncultured bacterium clone CB-1499HM100916 X 
  4878K4-B13Uncultured bacterium clone CB-14100HM100916 X2
  4878K4-B4Uncultured bacterium clone CB-14100HM100916 X 
  4881K6-B4Uncultured bacterium clone CB-14100HM100916 X 
  4881K6-B2LHalomonadaceae bacterium SCSWA2299FJ461432 X 
  4878K4-B2Halomonas sp. mp199AJ551115 X 
  4881K2-B2Halomonas sp. mp199AJ551115 X 
  4881K6-B3Halomonas sp. mp199AJ551115 X 
  4878K4-B5Halomonas sp. ‘SK halo 2’100EU373441 X 
 Pseudoaltermonas
  4753K4-B14Bacterium QM3599DQ822528X  
  4878K2-B58Pseudoalteromonas elyakovii clone 1399EU770411 X 
  4878K4-B12Pseudoalteromonas elyakovii clone 1399EU770411 X 
  4881K6-B6Pseudoalteromonas elyakovii clone 1399EU770411 X 
  4878K4-B6Pseudoalteromonas elyakovii clone 1399EU770411 X 
  4878K4-B8Pseudoalteromonas elyakovii clone 1399EU770411 X 
  4881K6-B7Pseudoalteromonas elyakovii clone 1398EU770411 X 
  4881K4-B1Pseudoalteromonas sp. 139Z-1099GU584150 X 
  4881K6-B2BPseudoalteromonas sp. 159Xa299EU440060 X 
  4881K2-B1Pseudoalteromonas sp. ArcB8453012100GU120031 X 
  4753K10-B1Pseudoalteromonas sp. B149100FN295744X 3*
  4753K4-B10Pseudoalteromonas sp. B149100FN295744X  
  4753K4-B15Pseudoalteromonas sp. B149100FN295744X 2
  4753K4-B17Pseudoalteromonas sp. B149100FN295744X  
  4753K4-B20Pseudoalteromonas sp. B149100FN295744XX7*
  4753K4-B6Pseudoalteromonas sp. B149100FN295744X  
  4878K4-B9Pseudoalteromonas sp. B149100FN295744 X 
  4881K4-B2Pseudoalteromonas sp. B149100FN295744 X 
  4753K4-B2Pseudoalteromonas sp. B19399FN295768X  
  4753K4-B13Pseudoalteromonas sp. BSs20055100EU365473X  
  4878K2-B59Pseudoalteromonas sp. S1191100FJ457147 X 
  4878K2-B56Pseudoalteromonas sp. SCSWD1399FJ461453 X 
  4753K4-B8Pseudoalteromonas tetraodonis99AB563179X 2
  4878K4-B7Pseudoalteromonas tetraodonis99AB563179 X 
  4881K6-B1Uncultured bacterium clone O2899GQ377771 X 
  4753K4-B11Pseudoalteromonas sp. M71_D34100FM992789X  
 Psychrobacter
  4753K4-B3Psychrobacter sp. 4Dc99HM771256X  
  4753K4-B24Psychrobacter sp. JT05100AB554726X  
 Vibrionaceae
  4878K2-B30Photobacterium leiognathi strain LN10199AY292944 X 
  4878K2-B5Photobacterium leiognathi strain SN2B99AY292951 X 
  4881K8-B1Photobacterium phosphoreum strain CECT 417299FJ971860 X 
  4878K2-B12Photobacterium sp. 42X-1a4100EU440051 X2
  4878K2-B13Photobacterium sp. 42X-1a4100EU440051 X 
  4878K2-B17Photobacterium sp. 42X-1a4100EU440051 X 
  4878K2-B18Photobacterium sp. 42X-1a499EU440051 X 
  4878K2-B20Photobacterium sp. 42X-1a4100EU440051 X2
  4878K2-B21Photobacterium sp. 42X-1a4100EU440051 X2
  4878K2-B24Photobacterium sp. 42X-1a4100EU440051 X4
  4878K2-B25Photobacterium sp. 42X-1a4100EU440051 X 
  4878K2-B26Photobacterium sp. 42X-1a499EU440051 X 
  4878K2-B28Photobacterium sp. 42X-1a499EU440051 X4
  4878K2-B33Photobacterium sp. 42X-1a499EU440051 X 
  4878K2-B34Photobacterium sp. 42X-1a499EU440051 X 
  4878K2-B35Photobacterium sp. 42X-1a499EU440051 X 
  4878K2-B4Photobacterium sp. 42X-1a4100EU440051 X 
  4878K2-B40Photobacterium sp. 42X-1a4100EU440051 X 
  4878K2-B42Photobacterium sp. 42X-1a499EU440051 X 
  4878K2-B43Photobacterium sp. 42X-1a499EU440051 X 
  4878K2-B44Photobacterium sp. 42X-1a4100EU440051 X4*
  4878K2-B45Photobacterium sp. 42X-1a499EU440051 X 
  4878K2-B47Photobacterium sp. 42X-1a4100EU440051 X3
  4878K2-B48Photobacterium sp. 42X-1a4100EU440051 X2
  4878K2-B50Photobacterium sp. 42X-1a499EU440051 X2
  4878K2-B51Photobacterium sp. 42X-1a499EU440051 X 
  4878K2-B53Photobacterium sp. 42X-1a499EU440051 X 
  4878K2-B55Photobacterium sp. 42X-1a4100EU440051 X3
  4878K2-B63Photobacterium sp. 42X-1a4100EU440051 X 
  4878K2-B8Photobacterium sp. 42X-1a499EU440051 X 
  4878K2-B9Photobacterium sp. 42X-1a4100EU440051 X4
  4878K2-B10Photobacterium sp. Asur-199AB055784 X 
  4878K2-B60Photobacterium sp. OSar299DQ317688 X 
  4878K2-B23Photobacterium sp. S370499FJ457541 X7*
  4878K2-B2Uncultured marine microorganism clone 4035AA_87100EU188016 X 
  4753K6-B1Vibrio sp. 61S-28100GU371703X 3
Table 2.   Phylogenetic affiliations of bacterial isolates from site VK 906/862 in the northern Gulf of Mexico.
VK 906/862      
IsolateStrainSimilarity (%)Accession no.20042005Repeats
  • 16S rRNA gene sequences were compared with sequences in GenBank. Isolates were clustered using fastgroupii, with the number of isolates clustering together noted in the Repeats column. The top phylogenetic match for each isolate is indicated by the strain name, accession number, similarity.

  • *

    Group includes isolates from both sites. These groups are expanded in Table 3.

Gammaproteobacteria
 Colwellia
  4873K4-B8Uncultured bacterium clone ER_0.2_16S_394FJ875446 X 
 Pseudoalteromonas
  4873K4-B5Arctic seawater bacterium Bsw2044999DQ064619 X 
  4746K8-B15Pseudoalteromonas arctica strain A 37-1-299DQ787199X  
  4873K2-B19Pseudoalteromonas denitrificans strain MAR_121_B08_2010-01-2098HM003113 X 
  4873K2-B27Pseudoalteromonas denitrificans strain MAR_121_B08_2010-01-2099HM003113 X 
  4873K2-B30Pseudoalteromonas denitrificans strain MAR_121_B08_2010-01-2098HM003113 X 
  4873K2-B31Pseudoalteromonas denitrificans strain MAR_121_B08_2010-01-2098HM003113 X2
  4873K2-B33Pseudoalteromonas denitrificans strain MAR_121_B08_2010-01-2099HM003113 X 
  4873K2-B34Pseudoalteromonas denitrificans strain MAR_121_B08_2010-01-2098HM003113 X 
  4873K2-B35Pseudoalteromonas denitrificans strain MAR_121_B08_2010-01-2098HM003113 X 
  4873K2-B38Pseudoalteromonas denitrificans strain MAR_121_B08_2010-01-2098HM003113 X 
  4873K4-B15Pseudoalteromonas denitrificans strain MAR_121_B08_2010-01-2099HM003113 X 
  4746K2-B1Pseudoalteromonas elyakovii clone 13100EU770411X  
  4746K8-B11Pseudoalteromonas elyakovii clone 13100EU770411X  
  4873K2-B37Pseudoalteromonas elyakovii clone 13100EU770411 X 
  4746K6-B13Pseudoalteromonas haloplanktis strain HK599EU939699X  
  4873K4-B13Pseudoalteromonas sp. ArcB845301299GU120031 X 
  4873K4-B3Pseudoalteromonas sp. ArcB845301299GU120031 X 
  4746K6-B14Pseudoalteromonas sp. B149100FN295744X 2
  4746K6-B15Pseudoalteromonas sp. B149100FN295744X  
  4746K6-B5Pseudoalteromonas sp. B14999FN295744X 3*
  4746K8-B12Pseudoalteromonas sp. B149100FN295744X 2*
  4746K8-B4Pseudoalteromonas sp. B149100FN295744X  
  4746K8-B5Pseudoalteromonas sp. B49100FN295814X  
  4746K8-B13Pseudoalteromonas sp. BSw2056499EF635229X  
  4746K10-B1Pseudoalteromonas sp. P2299EU935098X  
  4746K8-B1Pseudoalteromonas sp. P58100EU935093X  
  4873K4-B4Pseudoalteromonas sp. QY202100GQ202280XX3*
  4746K6-B9Pseudoalteromonas sp. QY20299GQ202280X  
  4746K8-B14Pseudoalteromonas sp. QY202100GQ202280X  
  4746K6-B11Uncultured Pseudoalteromonas sp. clone CI3599FJ695595X  
  4746K6-B8Uncultured Pseudoalteromonas sp. clone CI35100FJ695595X  
  4746K8-B10Uncultured Pseudoalteromonas sp. clone CI35100FJ695595X  
  4746K8-B9Uncultured Pseudoalteromonas sp. clone CI35100FJ695595X 2
  4746K6-B2Pseudoalteromonas sp. M71_D34100FM992789X  
  4746K6-B10Pseudoalteromonas sp. M71_D3499FM992789X  
 Pseudomonas
  4746K6-B17Pseudomonas sp. AM04100GQ483506X  
 Psychrobacter
  4746K6-B16Psychrobacter sp. JT05100AB554726X  
  4746K8-B2Psychrobacter sp. JT05100AB554726X  
  4746K8-B7Psychrobacter sp. JT0599AB554726X  
  4746K8-B6Psychrobacter sp. OW20100GU434161X 2*
 Shewanella
  4873K4-B16Shewanella sediminis HAW-EB397CP000821 X 
  4873K4-B23Shewanella sediminis HAW-EB397CP000821 X 
  4873K4-B7Shewanella sp. B225100FN295775 X 
  4873K4-B24Shewanella sp. J32799AY369989 X 
  4873K4-B10Shewanella sp. J32799AY369989 X 
  4873K4-B22Shewanella sp. KCCM 4293698GQ869534 X 
  4873K4-B28Shewanella sp. KCCM 4293698GQ869534 X 
 Vibrionaceae
  4873K2-B18Aliivibrio logei isolate AV02/200799EU257755 X 
  4873K4-B14Aliivibrio wodanis strain SR699EU185827 X 
  4873K4-B26Aliivibrio wodanis strain SR699EU185827 X 
  4873K4-B20Photobacterium phosphoreum isolate PHPH99AY780009 X 
  4873K4-B21Uncultured bacterium clone A201_NCI98FJ456798 X 
  4873K2-B32Uncultured bacterium clone CA-2399HM100897 X 
  4873K4-B2Uncultured bacterium clone surface_16S_15100FJ875439 X 
  4873K4-B19Uncultured gammaproteobacterium clone D13W_6199HM057759 X 
  4873K4-B29Uncultured Photobacterium sp. clone 8B_137100AM501622 X 
  4873K4-B12Vibrio ichthyoenteri98AM181657 X 
  4873K2-B36Vibrio sp. HM12-37100AB525428 X3
  4873K2-B42Vibrio sp. HM12-37100AB525428 X 
  4873K2-B22Vibrio sp. W02799EF114129 X 
  4873K2-B26Vibrio sp. W027100EF114129 X 
  4873K2-B29Vibrio sp. W02799EF114129 X 
  4873K2-B40Vibrio sp. W027100EF114129 X 
  4873K2-B44Vibrio splendidus strain LMG 4042, clone a99AJ515229 X 
 Betaproteobacteria
  4746K6-B18Uncultured Achromobacter sp. clone Bfa115100GU472956X  
 Actinobacteria
  4746K6-B20Kocuria sp. Z21zhy99AM418389X  
Table 3.   Groups that contain isolates from both sites and years are expanded to show each isolate
Isolates grouped from multiple sites     
IsolateStrainSimilarity (%)Accession no.20042005Site
  1. Isolates in bold are the group representatives listed in Tables 1 and 2, followed by the other clones included in each group.

Gammaproteobacteria
 Pseudoalteromonas
  4753K10-B1Pseudoalteromonas sp. B149100FN295744X VK 826
  4753K4-B1   X VK 826
  4746K6-B3   X VK 906/862
  4753K4-B20Pseudoalteromonas sp. B149100FN295744X VK 826
  4753K4-B18   X VK 826
  4753K4-B5   X VK 826
  4746K6-B4   X VK 906/862
  4753K4-B9   X VK 826
  4873K4-B1    XVK 906/862
  4746K8-B3   X VK 906/862
  4746K6-B5Pseudoalteromonas sp. B14999FN295744X VK 906/862
  4746K6-B1   X VK 906/862
  4753K4-B19   X VK 826
  4746K8-B12Pseudoalteromonas sp. B149100FN295744X VK 906/862
  4753K4-B16   X VK 826
  4873K4-B4Pseudoalteromonas sp. QY202100GQ202280 XVK 906/862
  4873K4-B11    XVK 906/862
  4753K4-B23   X VK 826
 Psychrobacter
  4746K8-B6Psychrobacter sp. OW20100GU434161X VK 906/862
  4753K4-B4   X VK 826
 Vibrionaceae
  4878K2-B44Photobacterium sp. 42X-1a4100EU440051 XVK 826
  4878K2-B54    XVK 826
  4873K2-B1    XVK 906/862
  4878K2-B46    XVK 826
  4878K2-B23Photobacterium sp. S370499FJ457541 XVK 826
  4878K2-B7    XVK 826
  4878K2-B19    XVK 826
  4878K2-B32    XVK 826
  4878K2-B62    XVK 826
  4873K4-B9    XVK 906/862
  4878K2-B39    XVK 826

Site VK 906/862 contained sparse L. pertusa colonies, and the coral exhibited weaker skeletons with thinner calcification of branches. The water temperature ranged from 9 to 13 °C, possibly indicating thermal stress on L. pertusa. The cultured bacteria from VK 906/862 were slightly more diverse; while still dominated by Gammaproteobacteria, there was also one isolate each from Betaproteobacteria and Actinobacteria (Table 2). Isolate 4746K6-B18 was 100% identical to an uncultured Achromobacter sp. that was originally isolated from the rhizosphere of a potato. This was the only nonmarine GenBank match for all of the L. pertusa isolates. Overall, Pseudoalteromonas spp. and Vibrio spp. were the dominant groups cultured at the potentially heat-stressed reef.

Phylogenetic analysis

Partial 16S rRNA genes were amplified and sequenced for each of the isolates in order to identify them phylogenetically. The sequenced isolates were sorted by site and by year (Tables 1 and 2) and separated into broad phylogenetic groups. The majority of the cultured isolates had high similarity to previously described sequences in GenBank. Groups of isolates were formed by processing all 16S rRNA gene sequences through fastgroupii (Yu et al., 2006). Isolates that were >97% similar were grouped by fastgroupii, using a representative sequence to identify the group. These groups are indicated by a number in the ‘Repeats’ column of Tables 1 and 2, indicating how many isolate sequences are represented. If these groups contained isolates from both sites, they were further expanded in Table 3, which shows all the sequences within the group, along with the site and the year cultured. Isolates with the same top GenBank match, but that are not grouped together, were <97% similar to each other, and so are listed individually.

While only the top GenBank match is presented for each isolate, the top five to 10 matches were evaluated to better ascertain the phylogenetic relationships of the isolates. Bacterial isolates were grouped together by their top GenBank matches, first by a specific strain and then by species or genus. Isolates with a top hit to an uncultured organism were further vetted by running the 16S rRNA gene sequence through the Ribosomal Database Project Classifier program. All isolates with ‘uncultured’ top matches (e.g. uncultured bacterium clone CB-14) were found to be closely related to cultured bacteria, with only one isolate showing <97% (>400 bp sequence size fragment) similarity to a cultured representative.

Kirby–Bauer antibiotic susceptibility testing

Of the 196 total bacterial isolates, only 78 isolates (39.8%) could be read for Kirby–Bauer testing, and all were Gammaproteobacteria (Fig. 2). Certain isolates, notably many Vibrio spp., would not grow on Muller–Hinton II agar. Many cultures remained uncultivable on Muller–Hinton II agar even when it was amended with NaCl to a salinity identical to the isolation medium.

image

Figure 2.  Heat map of the Kirby–Bauer antibiotic susceptibility ranges for 76 isolates. The top GenBank matches for each isolate are listed and all isolates are grouped by species. Levels of susceptibility to each of the six antibiotics (penicillin, P10; tetracycline, TE30; clindamycin, CC2; polymixin B, PB300; chloramphenicol, C30; novobiocin, NB30) are indicated by colors. Black, totally resistant; red, clinically resistant; yellow, intermediate susceptibility; blue, susceptible; with each classification dependent on antibiotic-specific ranges. Isolates that group together phylogenetically have different patterns of susceptibility to the antibiotics.

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The results of Kirby–Bauer testing on the 78 isolates are presented in Fig. 2. Isolates are grouped by phylogenetic characterization, based on the top GenBank match as listed in Tables 1 and 2. The level of resistance or susceptibility to the six antibiotics is denoted by the different colors, with one bacterial isolate per row. Based on the manufacturer's instructions from BD BBL Sensi-Disc Antimicrobial Susceptibility Test Discs, the levels of susceptibility to each antibiotic were broken down into categories using their zone diameter interpretive standards for gram-negative bacteria. Completely resistant bacteria had no zones of inhibition to the antibiotic (0 mm). The ranges for clinically resistant, intermediate, and susceptible zones of inhibition were unique for each antibiotic and are listed in Fig. 2.

The most common antibiotic resistance was to clindamycin, which has a bacteriostatic effect characterized by binding to the 50S rRNA gene molecule of the bacterial ribosome subunit and preventing protein synthesis. Clindamycin is most active against anaerobic bacteria; hence, resistance was not unexpected because the bacterial isolates were grown under aerobic culture conditions (Weingarten-Arams & Adam, 2002). Only two isolates (2.6%) exhibited susceptibility to clindamycin, a Halomonas sp. and a Pseudoalteromonas sp., both from site VK 826.

There was consistent susceptibility to polymixin B, an antibiotic that affects the cell-membrane permeability of gram-negative bacteria. All bacteria tested using the Kirby–Bauer method were gram-negative; hence, these results are not unexpected, although the uniformity is surprising.

All isolates exhibited varying levels of susceptibility to the four other antibiotics chosen. Chloramphenicol was active against all isolates, with two isolates exhibiting intermediate susceptibility, while the rest were completely susceptible. Only one isolate was clinically resistant to tetracycline, with all others classified as susceptible. The levels of resistance to penicillin and novobiocin varied widely. Six of the 78 (7.7%) isolates were intermediately susceptible to penicillin, while 10 (12.8%) were clinically resistant. Novobiocin had some effect on all isolates, although they ranged from clinically resistant to susceptible. As seen in Fig. 2, isolates that would be considered the same species based on their partial 16S rRNA gene sequences have different antibiotic resistance profiles.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Culturing environmental isolates

The microorganisms cultured from L. pertusa had 16S rRNA gene sequences that were closely related to bacteria previously isolated and cultured from a variety of marine environments. The growth medium used in this study (GASWA) is nonspecific and nutrient rich, selecting for bacteria that grow quickly on solid agar. GASWA is commonly used in culture-based studies of shallow-water coral microbial communities (Ritchie & Smith, 1995; Ritchie, 2006; Mao-Jones et al., 2010) and has also been used to culture bacteria associated with deep-sea gorgonians (Gray et al, 2011). The majority of isolates was related to Gammaproteobacteria, with a heavy representation of Vibrionaceae and Pseudoalteromonas (Tables 1 and 2). Culture-independent studies of bacteria associated with the white color morph of L. pertusa have found a higher diversity of bacterial 16S rRNA genes, with large numbers of clones representing the Alphaproteobacteria, Bacteroidetes, Tenericutes, and Actinobacteria in addition to Gammaproteobacteria (Neulinger et al., 2008; Kellogg et al., 2009). No overlap was observed between cultured isolates and clones from previous Lophelia investigations.

Isolates phylogenetically designated as Halomonadaceae (including Halomonas sp. and Cobetia sp.) were only cultured from corals at VK 826 and only from the two dives in 2005 (Table 1). All isolates were 99% identical to their top GenBank match. All of the GenBank matches were marine in origin, with some existing as free-living (planktonic) or sediment-associated bacteria. The top match for six Halomonadaceae isolates was ‘uncultured bacterium clone CB-14’ (accession #HM100916), which is potentially symbiotic or commensal with a marine sponge; the closest cultured GenBank matches for these isolates were all Cobetia spp.

Bacteria with high similarity (≥98%) to marine Pseudoalteromonas spp. were isolated from all dives (Tables 1–3). Many had GenBank matches that could be considered potentially symbiotic, with hosts including euphasiids, anemones, red algae, shallow-water coral mucus, and multiple bryozoan species. The majority of these GenBank matches come from cultured Pseudoalteromonas spp., not clones. Overlap between the two sample sites was observed, with isolates from both sites and both years matching the same GenBank top hit (i.e. Pseudoalteromonas sp. B149).

Psychrobacter-like bacteria were isolated from both sites, but only in 2004. Six bacterial isolates were identified as Psychrobacter spp., although they were most similar to only two sequences in GenBank, indicating the low culturable diversity of this genus on Lophelia. The GenBank matches were cultured from seawater and deep-sea sediment.

There was a high abundance of Vibrionaceae represented in the culture samples. Photobacterium, Aliivibrio, and Vibrio were all isolated from L. pertusa. All except three Vibrio-related VK 826 isolates and all VK 906/862 isolates were cultured in 2005, which could be an artifact of the culturing procedures (e.g. heterogeneity of mucus/tissue slurry) or an indication of a temporal variation in the coral-associated bacterial communities. Bacterial isolates from VK 826 were dominated by Photobacterium spp., particularly Photobacterium sp. 42X-1a4 (accession #EU440051), which was originally isolated from infected krill tissue, indicating potential pathogenicity. However, other Photobacterium-like isolates (e.g. 4878K2-B30 and 4878K2-B5) were most similar to bacteria that had been cultured from squid light organs, a symbiotic lifestyle. More diversity was seen among the Vibrionaceae at VK 906/862, with the L. pertusa isolates matching to sequences in GenBank that include microorganisms associated with fish intestines (FJ456798, AM181657), sediments (AM501622, EF114129), and sponges (HM100897).

Shewanella-like bacteria were only isolated in 2005 from corals at VK 906/862. The L. pertusa isolates were 98–100% identical to their closest blast matches in GenBank, all of which were cultured. Two isolates were most similar to Shewanella-like sequences that are potentially symbiotic: Shewanella sp. B225 (FN295775) was isolated from a bryozoan and Shewanella sp. J327 (AY369989) was isolated from a deep-water sponge. A single bacterium from VK 906/862 was most closely related to Pseudomonas sp. AM04 (GQ483506), which produces a biosurfactant.

The drastic differences between culturable bacteria and bacterial sequences recovered using culture-independent methods from the same samples have been documented previously (Rohwer et al., 2001). Bacterial isolates from dive 4753 (Tables 1 and 3) can be compared with the 16S rRNA genes cloned from the same corals in an experiment conducted by Kellogg et al. (2009). Branches from the same coral were either preserved at depth using DMSO/EDTA/salt buffer or brought to the surface without being preserved. The branch that was brought to the surface live was subdivided: DNA extraction was performed on several polyps (see Kellogg et al. 2009 for full details) and the rest were homogenized into a slurry to spread-plate on growth media. Comparison of the clone libraries from the DNA extractions and the number of cultured bacteria shows large differences. The clone library of one coral colony (4753K4) that was preserved at depth had representatives of Alpha- and Gammaproteobacteria, Tenericutes, and Bacteroidetes. The unpreserved sample from the same coral (brought to the surface live and immediately DNA extracted) was dominated by Tenericutes, with small fractions of Alpha- and Gammaproteobacteria and Bacteroidetes. The cultured representatives from that coral (Table 1) are only from the Gammaproteobacteria, comprised of Pseudoalteromonas spp. and Psychrobacter spp. Recognizing the inherent biases in culturing bacteria from the environment is important, but culturing and isolation of bacteria is necessary for detailed studies of physiology and ecological function. The use of an assortment of media types and growth condition variables can aid in increasing the diversity of microorganisms recovered by culturing, an ongoing experimental endeavor.

Potential function of cultured bacteria

Pseudoalteromonas spp. and Vibrio spp. are commonly recovered when culturing bacteria from corals (Rohwer et al., 2001; Dobretsov & Qian, 2004; Lampert et al., 2006; Ritchie, 2006; Bally & Garrabou, 2007; Brück et al., 2007; Chimetto et al., 2008; Raina et al., 2009) and sponges (Lee et al., 2007; Mangano et al., 2009; Menezes et al., 2009), indicating their ubiquity in the marine environment, as well as their possible symbiosis with marine organisms. Hypotheses on the ecological or the metabolic function of these community members are varied. Pseudoalteromonas spp. are known to secrete bioactive compounds with antifouling functions (Dobretsov & Qian, 2004), and both Vibrio spp. and Pseudoalteromonas spp. can produce antibiotics (Ritchie, 2006). These findings may suggest a protective role, aiding L. pertusa in clearing or preventing the settlement of epibiotic organisms. Additionally, Vibrio spp. associated with a Brazilian coral have been shown to fix nitrogen (Chimetto et al., 2008). Vibrio spp. have also been shown to break down recalcitrant carbon sources, such as cellulose and lignin (Neulinger et al., 2008) and dimethyl-sulfoniopropionate (a sulfur-organic compound released by phytoplankton, Raina et al., 2009). Unlike zooxanthellate shallow-water corals, L. pertusa relies completely on capture-feeding for nutrition (Freiwald & Roberts, 2005; Roberts et al., 2006) and could supplement feeding by maintaining a community of microorganisms that cycle necessary nutrients such as nitrogen or break down carbon sources unusable by the coral.

While Vibrio spp. are often implicated in diseases of corals (Kushmaro et al., 1997; Ben-Haim et al., 2003; Hall-Spencer et al., 2007; Thurber et al., 2009), the corals collected for this study were apparently healthy at the time of collection. No diseases affecting L. pertusa have been identified through sampling, video footage, or still photographs. In addition, the prevalence of culturable Vibrio spp. on healthy corals indicates that they are normal members of coral microbiomes (Dobretsov & Qian, 2004; Lampert et al., 2006; Ritchie, 2006; Brück et al., 2007; Chimetto et al., 2008; Raina et al., 2009). This suggests that Vibrio spp. are playing a symbiotic role that may be disrupted in times of stress for the coral, leading to a population explosion that results in physical signs of disease and over-representation in microbial studies of disease.

All bacteria in this study were isolated and cultured from plates that were maintained between 4 and 10 °C, in an effort to maintain in situ temperatures. As noted by Gray et al. (2011), temperature can bias the number and diversity of bacteria recovered from cold-water coral samples. They found that fewer bacteria were recovered from plates incubated at 22 °C than at 4 °C, and of those bacteria, there was a higher percentage of Vibrio spp. isolated at the warmer incubation temperature (Gray et al., 2011). This finding also indicates that high-temperature stress on in situ Lophelia colonies could cause the community of microorganisms to shift, allowing more Vibrio-like growth and inhibiting the growth of other microorganisms.

Kirby–Bauer antibiotic susceptibility testing

Effect of incubation length on the zone of inhibition size

In order to apply the Kirby–Bauer test to bacteria isolated from the cold-water coral L. pertusa, the procedure was altered so that the plates were incubated at 4–7 °C for up to 30 days, instead of 37 °C for 24–48 h. Maintaining the bacterial cultures under conditions as close as possible to their natural environment was deemed important to obtain environmentally relevant data, and long incubation times were required to observe sufficient bacterial growth. However, concerns were raised that increasing the incubation time could lead to antibiotic breakdown (leading to smaller zones of inhibition) or greater antibiotic diffusion into the agar (leading to larger zones of inhibition). To address these concerns, incubation length was measured in hours and plotted against the zone of inhibition diameter. An extremely low correlation was observed (maximum r2=0.19) for all regression lines, and the low significance (Fs<1.0 for all, anova) of these regression lines suggests that the size of the zones of inhibition could not be well explained by the length of incubation (Supporting Information, Fig. S1).

Variability of Kirby–Bauer profile among grouped/closely related bacteria

Discussion of antibiotic susceptibility as measured using the Kirby–Bauer test is limited first by the number of cultivable bacteria isolated out of the total bacterial community associated with the coral holobiont, and secondly, by the subset of those bacteria that can grow on Muller–Hinton II agar amended with salt. Surprisingly, very few Vibrionaceae grew on the Mueller–Hinton II agar, indicating that some essential nutrient was missing, was present in the wrong amount, or that an inhibitor specific to that family was in the medium. The purpose of this test was to examine antibiotic sensitivity differences between bacterial isolates classified as the same species based on their 16S rRNA gene sequences. Lampert et al. (2006) used the same six antibiotics to examine strain-level differences between bacterial cultures isolated from mucus of the shallow-water coral Fungia scutaria.

Overall patterns of susceptibility to the six antibiotics were represented across the different phylogenetic groups: the majority of isolates were susceptible to three or four antibiotics, while five isolates were susceptible to five of the antibiotics, and a single isolate (4746K8-B6) was susceptible to only two antibiotics (Fig. 2). Individual patterns of susceptibility could vary within the phylogenetic groups, but were repeated between groups (e.g. 4878K4-B1, 4746K6-B13, and 4753K4-B4 share the same profile, but are in different phylogenetic groups). A study of culturable chloramphenicol-resistant bacteria from coastal waters in Jiaozhou Bay, China, revealed that the majority was identified as Pseudoalteromonadaceae sp. (Dang et al., 2008). The single bacterial isolate from the current study that was intermediately susceptible, indicating slight resistance to chloramphenicol, is also a Pseudoalteromonas sp.; however, all of the remaining bacteria were susceptible (Fig. 2).

Differing patterns of antibiotic susceptibility would suggest strain-level differences in accessory or antibiotic-resistance genes either within the genome or encoded on plasmids, integrons, or transposons in the bacterial isolates (Thomas & Nielsen, 2005; Allen et al., 2010). It has been shown that while bacteria maintain a core set of ‘housekeeping genes’ necessary for function (i.e. 16S rRNA genes), there is continuous swapping of accessory genes (e.g. antibiotic resistance genes; Staley, 2006). The definition of bacterial species is almost solely anchored on phylogenies constructed with core genes; however, the ecological role of the bacteria is highly dependent on the suite of accessory genes (Tettelin et al., 2005; Staley, 2006). Testing bacterial function within the coral holobiont, as well as phylogenetic identity, will aid researchers in deciphering the role of bacterial communities associated with corals.

Isolates with shared GenBank matches, which putatively identified them as the same bacterial species, showed differences in their antibiotic resistance profiles. For example, bacteria related to Pseudoalteromonas sp. B149 were isolated from both sites during both 2004 and 2005, indicating that this bacterium is temporally stable at the two sites. However, the antibiotic resistance profiles between and within sites and years were variable, with differing susceptibilities to penicillin, chloramphenicol, and novobiocin (Fig. 2). Analogous variability can be seen among isolates related to ‘uncultured bacterium clone CB-14,’ a Halomonas species. These isolates were cultured from site VK 826 in 2005 on two different dives and show variable antibiotic resistance profiles. Notably, one of the isolates (4881K6-B4) was susceptible to clindamycin, one of only two strains to exhibit this trait.

Differing antibiotic resistance profiles among bacterial strains that would be grouped together based on their 16S rRNA gene phylogenies illustrate the difficulty of assigning species identifications anchored solely by a single gene. For example, the identification of Vibrio spp. to the species or the strain level requires the use of multilocus sequencing (Thompson et al., 2005; Pollock et al., 2010). The variability in antibiotic resistance profiles could indicate important strain-level differences. At the minimum, it cautions against relying too heavily on identifications based on single genes in order predict ecological roles.

Natural antibiotic resistance

Antibiotic resistance in nonclinical or environmental settings is not necessarily an indication of an anthropogenic influence, although pharmaceutical waste has been implicated in influencing antibiotic resistance in natural microbial populations (Allen et al., 2010). Rather, there seems to be a constant, low-level existence of antibiotic resistance genes flowing throughout natural populations (Yim et al., 2007; Allen et al., 2010). In addition, antibiotics are found at subinhibitory or sublethal concentrations in the natural environment (Yim et al., 2007) and are commonly produced by microorganisms during the stationary growth phase (Fajardo et al., 2009). Gene expression studies have shown that sublethal doses of antibiotics can induce phenotype changes (e.g. biofilm production) or transcription patterns shifts (Davies et al., 2006; Fajardo et al., 2009). The results of these studies suggest that the natural molecules from which clinical antibiotics are derived can function as cell-to-cell signals in the environment, an important ability for microorganisms existing in a community structure (Davies et al., 2006; Yim et al., 2007; Fajardo et al., 2009). The concentrations of antibiotics used in Kirby–Bauer testing are meant to be bacteriostatic or bactericidal. An interesting experiment would be to observe possible phenotype changes when bacterial isolates are exposed to sublethal doses of the same antibiotics.

Initial microbiological studies of bacteria associated with the cold-water coral L. pertusa have all relied on culture-independent methods (Yakimov et al., 2006; Neulinger et al., 2008; Kellogg et al., 2009; Schöttner et al., 2009). This paper is the first to present the results from culture-based bacterial surveys. Culture-based experiments are significantly limited in the bacterial diversity recovered (Fuhrman & Campbell, 1998); however, they provide important information about the physiological capabilities of the microorganisms. Our results show that bacterial function is not necessarily tied to phylogeny, hinting at a cryptic functional potential in these bacterial isolates.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by the U.S. Geological Survey (USGS) Terrestrial, Freshwater, and Marine Ecosystem Program and was sponsored and facilitated by the Bureau of Ocean Energy Management, Regulation and Enforcement (formerly Minerals Management Service). We appreciate the patience and assistance of the rest of the USGS DISCOVRE team in the collection of these critical samples. Thanks are also due to the Harbor Branch Oceanographic Institute, the captain and crew of the R/V Seward Johnson, and especially the pilots and technicians of the submersible Johnson-Sea-Link, without whom this research could not have been conducted. Any use of trade names is for descriptive purposes only and does not imply endorsement by the U.S. government.

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  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Fig. S1. Comparison of hours of incubation and size of zones of inhibition for all bacterial isolates tested using the Kirby–Bauer antibiotic susceptibility protocol.

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