Antimicrobial properties of resident coral mucus bacteria of Oculina patagonica

RESEARCH LETTER

Antimicrobial properties of resident coral mucus bacteria of Oculina patagonica Jozef Nissimov1, Eugene Rosenberg2 & Colin B. Munn1 1

School of Biological Sciences, University of Plymouth, Plymouth, UK; and 2Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel

Correspondence: Colin B. Munn, School of Biological Sciences, University of Plymouth, Plymouth PL4 8AA, UK. Tel.: 144 1752 233549; fax: 144 1752 232970; e-mail: [email protected] Received 6 August 2008; accepted 14 December 2008. First published online 21 January 2009. DOI:10.1111/j.1574-6968.2009.01490.x Editor: Skorn Mongkolsuk Keywords antimicrobial; coral bleaching; Pseudoalteromonas ; Roseobacter ; Vibrio shiloi ; Oculina patagonica.

Abstract The inhibitory properties of the microbial community of the coral mucus from the Mediterranean coral Oculina patagonica were examined. Out of 156 different colony morphotypes that were isolated from the coral mucus, nine inhibited the growth of Vibrio shiloi, a species previously shown to be a pathogen of this coral. An isolate identified as Pseudoalteromonas sp. was the strongest inhibitor of V. shiloi. Several isolates, especially one identified as Roseobacter sp., also showed a broad spectrum of action against the coral pathogens Vibrio coralliilyticus and Thallassomonas loyana, plus nine other selected Gram-positive and Gram-negative bacteria. Inoculation of a previously established biofilm of the Roseobacter strain with V. shiloi led to a 5-log reduction in the viable count of the pathogen within 3 h, while inoculation of a Pseudoalteromonas biofilm led to complete loss of viability of V. shiloi after 3 h. These results support the concept of a probiotic effect on microbial communities associated with the coral holobiont.

Introduction Coral bleaching is caused by disturbance of the mutual symbiotic relationship between algae (zooxanthellae) within the tissues of the coral animal (Rosenberg & Loya, 2004). The symbiosis can be disrupted due to a range of external environmental physical and toxic stressors, which can act either alone or together (Rosenberg & Loya, 2004). In addition to these widely accepted factors in coral bleaching, the hypothesis that bacterial infection may also trigger bleaching was developed as a result of the study of interactions observed since 1997 between the coral Oculina patagonica and the bacterium Vibrio shiloi (review, Rosenberg & Falkowitz, 2004). Oculina patagonica is an invasive species of the eastern Mediterranean Sea, first recorded in 1993 (Fine & Loya, 1995). The infection and bleaching of O. patagonica by V. shiloi was first described by Kushmaro et al. (1996, 1997) and shown to be temperature dependent; it does not occur at 16–20 1C, but is stimulated at temperatures above 25 1C (Kushmaro et al., 1998). Once attracted to the coral mucus, V. shiloi was shown to adhere to a b-galactoside receptor on the coral surface, but only at higher temperatures and only to corals that had photosynthetically active 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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zooxanthellae (Ben-Haim et al., 1999). Once in the coral tissue, the pathogen multiplies and produces extracellular toxins that block photosynthesis, bleach and lyse the zooxanthellae (Banin et al., 2000, 2001a, b). In addition to the zooxanthellae, the tissue of healthy corals and their secreted mucus layer supports a diverse community of other microorganisms, including bacteria (e.g. Rohwer et al., 2002; Bourne & Munn, 2005; Koren & Rosenberg, 2006; Ritchie, 2006), archaea (Kellogg, 2004; Wegley et al., 2004), fungi (Bentis et al., 2000) and viruses (Wilson et al., 2005; Marhaver et al., 2008). Since 2004, it has not been possible to recover V. shiloi from healthy or diseased corals (Reshef et al., 2006). Ainsworth et al. (2008) confirmed the absence of V. shiloi during the annual bleaching event in 2005. It was shown previously that microbial communities associated with tissue and mucus of O. patagonica differ between the seasons when sampled in 2005–2006 (Koren & Rosenberg, 2006). Reshef et al. (2006) showed that V. shiloi still adheres to O. patagonica tissue, but its population in the coral declines and eventually disappears. A possible explanation for this disappearance was suggested in the Coral Probiotic Hypothesis proposed by Reshef et al. (2006) and developed by Rosenberg et al. (2007). This proposes that the FEMS Microbiol Lett 292 (2009) 210–215

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abundance and types of microorganisms associated with corals change in response to global environmental changes such as temperature, allowing the coral to adapt to new conditions by altering its population of specific symbiotic bacteria. In the study reported here, we screened culturable members of the microbial community currently associated with the mucus of O. patagonica in order to determine their activity against V. shiloi and other pathogens.

Materials and methods Isolation, growth and identification of bacteria from coral mucus Two healthy fragments of O. patagonica were collected from 4 m depth at the reef of Sdot Yam (eastern Mediterranean Sea, 3212926.6500 N, 3415310.6200 E) on 25 June 2007, and transferred to the laboratory in cool bags within 2 h. Two 2  2 cm pieces of coral were detached from the rocky fragments and centrifuged for 7 min at 2675 g in order to separate mucus from the coral tissue. The mucus was then centrifuged again at 2675 g for a further 2 min in order to remove any particulate matter from the coral fragments, followed by centrifugation at 13 000 g for 2 min in order to pellet the bacteria. Tenfold serial dilutions (to 104) of the resuspended pellet were prepared in filtered (0.22 mm) artificial seawater and spread on marine agar (MA) plates (per litre; Difco Marine Broth 18 g, NaCl 9 g and agar 18 g) and grown at 30 1C for 48 h. Colony morphotypes were distinguished by size, shape and pigmentation and subcultured to fresh MA plates to obtain pure cultures. As shown in Table 1, nine isolates showing activity against V. shiloi were identified using PCR amplification and sequencing of 16S rRNA genes, as described by Koren & Rosenberg (2006).

Table 1. Identification by 16S rRNA gene analysis of bacteria isolated from Oculina patagonica showing inhibition of Vibrio shiloi Isolate

Closest match in GenBank

% Similarity

JNM 1 JNM 3 JNM 8 JNM 11 JNM 12 JNM 14 JNM 106 JNM 119 JNM 149

Ferrimonas sp. A3B-64-2 Alphaproteobacterium JE066 Pseudovibrio sp. MKT 94 Vibrio agarivorans 351A Pseudoalteromonas sp. LS3 Roseobacter gallaeciensis X129 Alphaproteobacterium JE064 Alphaproteobacterium JE062 Pelagiobacter variabilis

98.6 100 100 100 99.8 100 99.9 99.8 99.8

Sequences ( 4 400 base pairs) were aligned with

CLUSTALX, and a DNA distance matrix was created with BIOEDIT; sequences were compared with existing sequences in the GenBank using BLASTN.

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Detection of interactions of isolates with other bacteria Initial screening of the isolates was carried out to detect inhibition of growth of V. shiloi; strains showing activity were then tested against various target marine and terrestrial bacterial species obtained from the Tel Aviv laboratory culture collection (Table 2). Overnight cultures (100 mL) of each target bacterium were added to 3 mL of soft MA (formula as above, but with 0.7% w/v agar) and poured onto the surface of MA plates (15 mL). Cultures (40 mL) of the isolates from Oculina were added to 5-mm-diameter wells punched with a glass pipette, and plates were incubated at 30 1C up to 72 h. Zones of clear inhibition were measured from the edge of the well.

Inhibition of V. shiloi in liquid cultures Vibrio shiloi 18-h cultures (200 mL diluted to c. 105 CFU mL1) were inoculated into flasks containing 20 mL marine broth. Coral isolates were added to yield concentrations of c. 104, 106 or 108 CFU mL1 and incubated with shaking at 30 1C. Viable counts of V. shiloi were determined by performing colony counts using a surface-plating method (triplicate 10-mL drops of serial 10-fold dilutions) on selective thiosulphate citrate bile salts sucrose (TCBS) agar (Oxoid) incubated for 18 h at 30 1C. In order to facilitate analysis at a later date, cultures of strains JNM12 and JNM14 were centrifuged (10 000 g, 15 min) after 12 and 48 h of incubation, representing the late logarithmic phase and the stationary phase, respectively; viable counts showed that these contained c. 108 CFU mL1. The cells and the supernatants were frozen separately at  20 1C for 3 days, before thawing at room temperature and testing for inhibition of V. shiloi.

Inhibition of the settlement and viability of V. shiloi by established biofilms Biofilms were prepared by inoculating 96-well microtitre plates with 100-mL aliquots of individual cultures of coral isolates JNM12 and JNM14 and incubating for 18 h at 30 1C under static conditions. After carefully removing the liquid, 100-mL aliquots of V. shiloi broth culture (18 h, 30 1C) containing 104–105 CFU mL1 were added; the same was done with the control wells. The plate was incubated for a further 3 h, after which time replicate 10-mL samples were removed (a) by gentle pipetting of the surface liquid and (b) by vigorously mixing the entire contents of the well with a pipettor. Viable counts on TCBS were performed as described previously and analysed using a two-sample t-test (Minitab). 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Table 2. Inhibition of test bacteria by strains isolated from the mucus of coral Oculina patagonica Coral isolate

Test bacterium Zone of inhibition (mm) Vibrio shiloi Vibrio coralliilyticus Thalassomonas loyana Agrobacterium tumefaciens Bacillus subtilis Burkholderia cepacia Escherichia coli Micrococcus luteus Pseudomonas aeruginosa Paenibacillus dendritiformis Staphylococcus albus Staphylococcus aureus

JNM 1

JNM 3

JNM 8

JNM 11

JNM 12

JNM 14

JNM 106

JNM 119

JNM 149

10 – – – – – – – –

8 8 6 – 4 – – – – – – –

4 2 3 – – 4 – – – 1

2 – – – – 1 – – 3 – – –

10 8 7 – – 1 – – – – – –

2 2 6 10 12 14w 10 12 8w 8w 14 12

1 – – – – – – – – – – –

4 – –

4 2 3 – 4 4 – – – 1 – –

– –

1

– – – – – – – –

Coral isolate grown in marine broth with tryptone for 72 h. w

Isolate grown in minimal medium for 72 h; all other isolates grown in marine broth for 48 h.

Results

Inhibition of growth of V. shiloi by cells and supernatants of mucus bacteria cultures

Isolation and identification of bacteria from coral mucus and their antimicrobial activities

The viable count of V. shiloi was reduced 4 104-fold when grown with resuspended 12-h cells of strain JNM12 and 4 103-fold with 48-h cells. The supernatant fluid from strain JNM12 led to a 105-fold (12 h) or a 103-fold (48 h) reduction in viability (Fig. 2a). Both the resuspended cells and the supernatant fluid from strain JNM14 (obtained by centrifugation of cultures grown for either 12 or 48 h) resulted in a 102–103-fold decrease in the number of viable V. shiloi (Fig. 2b).

Out of 156 colonies tested, nine (5.8%) showed inhibition of growth of V. shiloi. The spectrum of the antibiotic activity of these nine isolates was then tested against other coral pathogens and common Gram-negative and Gram-positive bacteria. As shown in Table 2, strain JNM12 showed the highest activity against the coral pathogens V. shiloi, Vibrio coralliilyticus and Thallassomonas loyana, while strain JNM14 showed a broad spectrum of activity and inhibited the growth of all target species. These strains were, therefore, selected for further investigation.

Inhibition of growth of V. shiloi in mixed cultures The inhibition of V. shiloi in mixed cultures was dependent on the initial density of the coral isolates. Figure 1a shows that when the isolate JNM12 was present at 108 CFU mL1, no viable V. shiloi could be detected after 4 h, whereas at an initial density of 106 CFU mL1,. Vibrio shiloi was not recoverable after 6 h. At a starting density of 104 CFU mL1, the viability of V. shiloi was eliminated after 24 h (intermediate times were not sampled). With strain JNM14, clear inhibition was seen only at the highest initial concentration of 108 CFU mL1, with V. shiloi undetectable after 4 h (Fig. 1b). The control culture, containing only V. shiloi, showed typical bacterial growth and reached the expected concentration of c. 109 CFU mL1 after 24 h. 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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Interactions between V. shiloi and biofilms of the coral isolates Strains JNM12 and JNM14 formed biofilms in microtitre tray wells after a 18-h incubation; these remained adhered to the bottom of the wells and were only removed by vigorous pipetting. However, the numbers and density of bacteria were not determined. There was a small, but statistically significant, difference between the counts of V. shiloi in control wells and in wells containing a biofilm of strain JNM14. Viable counts of V. shiloi from the upper layer of the mixed culture showed five times fewer cells (2  103 CFU mL1) than the control (1.06  104 CFU mL1) containing only V. shiloi [two-sample t(10) =  21.11, P o 0.05]. Similarly, after vigorous mixing, the viable counts of V. shiloi showed a decrease of 4.5 times less V. shiloi (2.36  103 CFU mL1) in the presence of the biofilm than in the control sample (1.1  104 CFU mL1) [two-sample t(12) =  27.46, P o 0.05]. However, there was no difference between the amount of V. shiloi recovered FEMS Microbiol Lett 292 (2009) 210–215

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Fig. 1. Viable counts of Vibrio shiloi in marine broth at 30 1C in a mixed culture with (a) Pseudoalteromonas strain JNM12 or (b) Roseobacter strain JNM14 at initial densities of c. 104 CFU mL1 (’), 106 CFU mL1 (m) or 108 CFU mL1 ( ). The control culture (~) contained V. shiloi only at an initial density of c. 104 CFU mL1. Data points are the means of three determinations (  SD).



from the vigorously mixed culture, containing both cells from the bottom of the plastic well and those in the top layer [two-sample t(14) = 0.88, P 4 0.05]. When V. shiloi was added to a previously established biofilm of strain JNM12, no viable V. shiloi were recovered after 3 h either from the upper layer or from the vigorously mixed culture.

Discussion Several strains of bacteria cultured from O. patagonica showed antagonistic activities towards a range of marine and terrestrial pathogens, with 5.8% of the isolates active against V. shiloi, the former bleaching pathogen of this coral. Previous studies have shown similar Alpha- and Betaproteobacteria to be present in healthy O. patagonica (Koren & Rosenberg, 2006; Sharon & Rosenberg, 2008). Similarly, FEMS Microbiol Lett 292 (2009) 210–215

Fig. 2. Viable counts of Vibrio shiloi in marine broth at 30 1C when cultured alone (control) or with cells or supernatant obtained by centrifugation of 12-h (unshaded) or 48-h (shaded) cultures of (a) Pseudoalteromonas strain JNM12 or (b) Roseobacter strain JNM14. Data points are the means of three determinations (  SD).

Ritchie (2006) found that almost 20% of the cultured bacteria from the Acropora palmata coral in the Caribbean displayed antibiotic activity, including towards the causative agent of white pox disease. Not surprisingly, when the antibiotic producers that were isolated from O. patagonica mucus were tested against each other, no inhibition occurred, suggesting that the strains isolated could be resistant to these antagonistic mechanisms, and therefore may be better adapted to life in the mucus. Reshef et al. (2006) observed that although V. shiloi can no longer infect and bleach O. patagonica, it still penetrates the coral tissue, but is undetectable after 4 days. While it is obviously uncertain how the behaviour of isolated bacteria in artificial culture reflects natural interactions, our findings suggest a possible explanation for this observation, which warrants further investigation. Isolates from the mucus inhibited the 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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pathogen within 24 h, and in the case of strains JNM12 and JNM14 (presumptively identified as Pseudoalteromonas sp. and Roseobacter sp., respectively), inhibition occurred rapidly at high cell densities. To provide further evidence that such antagonistic interactions are relevant in the coral in its natural habitat, it will be necessary to quantify the abundance of specific bacterial types, for example using FISH with targeted probes. Other isolates (especially JNM1 and JNM3) also showed strong activity against V. shiloi and the other coral pathogens, and these warrant investigation in future studies. The bacteria had different strengths and spectra of activity against the various test bacteria, suggesting that the microbial interactions in the mucus are diverse and complicated. The Roseobacter isolate had the broadest range of activity and inhibited a range of terrestrial and marine pathogens. There are numerous reports of antibiotic production by bacteria belonging to the Roseobacter clade. Bruhn et al. (2005) and Rao et al. (2005, 2006) demonstrated the selective advantages that members of the Roseobacter clade have in colonizing the surface of algae and outcompeting previously established biofilms. In addition, Bruhn et al. (2007) demonstrated the fact that members of the Roseobacter lineage possess N-acyl homoserine lactone compounds that are important for the control of the production of antibiotics via quorum sensing. This might explain why, in our study, the Roseobacter showed reduction of V. shiloi viability only at high cell densities. Our results suggest that maximal activity may occur in the stationary phase, although Brinkhoff et al. (2004) showed that a Roseobacter sp., with characteristics very similar to the strain isolated here, showed antibiotic production throughout its complete growth cycle. The strongest inhibitor of V. shiloi and the other coral pathogens tested was the strain JNM12, identified as Pseudoalteromonas. This genus is known to produce a range of bioactive compounds (Bowman, 2007) and strain JNM12 shows red-brown pigmentation of its colonies, which has previously been associated with antibiotic production (Holmstrom et al., 1996). Rao et al. (2005) showed that Pseudoalteromonas tunicata produced several extracellular inhibitory products that allowed it to establish a biofilm on the surface of algae at the cost of other competitive microorganisms. In addition, the antibiotic was released from the cells in culture during its stationary phase. In our study, when Pseudoalteromonas was incubated in a mixed culture with V. shiloi, complete inhibition of the pathogen was also observed with stationary-phase cultures at low cell densities. Both cells and culture supernatants of Pseudoalteromonas JNM12 and Roseobacter JNM14 inhibited the growth of V. shiloi. Inhibition by both the supernatants and the cell pellets confirms the results of the initial agar screening method, indicating the production of a diffusible antibiotic 2009 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved

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compound as well as the possible production of cellassociated antibacterial activity. This should be investigated further. Previously established biofilms of both Pseudoalteromonas JNM12 and Roseobacter JNM14 showed rapid reduction of the viability of the V. shiloi pathogen compared with the control, suggesting the possible production of an antibiotic. Further experiments are required to determine the nature of this interaction. It is possible that similar effects could occur in the mucus on the surface of coral tissue, although considerable caution is obviously needed in extrapolating from our in vitro model system using the artificial substrate of a plastic surface to the complex interactions that may occur on the coral surface. Our results support the conclusions of Ritchie (2006) that coral mucus and its associated microorganisms play an important role in promoting beneficial microbial communities. From the experiments performed here, it is clear that different coral bacteria may contribute differently to the protection of the coral. It is unlikely that one or two coral mucus isolates can fully explain the development of immunity to a disease, but a ‘cocktail’ of bacteria with different antibiotic properties could together prevent infection by a pathogen such as V. shiloi.

Acknowledgements We thank Omry Koren and Gil Sharon from Tel Aviv University, and Michele Kiernan, Luke Peakman and Sarah Jamieson at the University of Plymouth for their help and patience during this work.

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