Cisse Hedegaard Porsby

Phaeobacter and Ruegeria Species of the Roseobacter Clade Colonize Separate Niches in a Danish Turbot (Scophthalmus maximus)-Rearing Farm and Antagonize Vibrio anguillarum under Different Growth Conditions

ABSTRACT Members of the Roseobacter clade colonize a Spanish turbot larval unit, and one isolate (Phaeobacter strain 27-4) is capable of disease suppression in in vivo challenge trials. Here, we demonstrate that roseobacters with antagonistic activity against Vibrio anguillarum also colonize a Danish turbot larval farm that relies on a very different water source (the Danish fiord Limfjorden as opposed to the Galician Atlantic Ocean). Phylogenetic analyses based on 16S rRNA and gyrase B gene sequences revealed that different species colonized different niches in the larval unit. Phaeobacter inhibens- and Phaeobacter gallaeciensis-like strains were primarily found in the production sites, whereas strains identified as Ruegeria mobilis or Ruegeria pelagia were found only in the algal cultures. Phaeobacter spp. were more inhibitory against the general microbiota from the Danish turbot larval unit than were the Ruegeria spp. Phaeobacter spp. produced tropodithietic acid (TDA) and brown pigment and antagonized V. anguillarum when grown under shaking (200 rpm) and stagnant (0 rpm) conditions, whereas Ruegeria spp. behaved similarly to Phaeobacter strain 27-4 and expressed these three phenotypes only during stagnant growth. Both genera attached to an inert surface and grew in multicellular rosettes after stagnant growth, whereas shaking conditions led to single cells with low attachment capacity. Bacteria from the Roseobacter clade appear to be universal colonizers of marine larval rearing units, and since the Danish Phaeobacter spp. displayed antibacterial activity under a broader range of growth conditions than did Phaeobacter strain 27-4, these organisms may hold greater promise as fish probiotic organisms.

Inactivation of Vibrio anguillarum by Attached and Planktonic Roseobacter Cells

ABSTRACT The purpose of the present study was to investigate the inhibition of Vibrio by Roseobacter in a combined liquid-surface system. Exposure of Vibrio anguillarum to surface-attached roseobacters (107 CFU/cm2) resulted in significant reduction or complete killing of the pathogen inoculated at 102 to 104 CFU/ml. The effect was likely associated with the production of tropodithietic acid (TDA), as a TDA-negative mutant did not affect survival or growth of V. anguillarum.

Resistance and Tolerance to Tropodithietic Acid, an Antimicrobial in Aquaculture, is Hard to Select

ABSTRACT The antibacterial compound tropodithietic acid (TDA) is produced by bacteria of the marine Roseobacter clade and is thought to explain the fish probiotic properties of some roseobacters. The aim of the present study was to determine the antibacterial spectrum of TDA and the likelihood of development of TDA resistance. A bacterial extract containing 95% TDA was effective against a range of human-pathogenic bacteria, including both Gram-negative and Gram-positive bacteria. TDA was bactericidal against Salmonella enterica serovar Typhimurium SL1344 and Staphylococcus aureus NCTC 12493 and killed both growing and nongrowing cells. Several experimental approaches were used to select mutants resistant to TDA or subpopulations of strains with enhanced tolerance to TDA. No approach (single exposures to TDA extract administered via different methods, screening of a transposon library for resistant mutants, or prolonged exposure to incremental concentrations of TDA) resulted in resistant or tolerant strains. After more than 300 generations exposed to sub-MIC and MIC concentrations of a TDA-containing extract, strains tolerant to 2× the MIC of TDA for wild-type strains were selected, but the tolerance disappeared after one passage in medium without TDA extract. S. Typhimurium mutants with nonfunctional efflux pump and porin genes had the same TDA susceptibility as wild-type strains, suggesting that efflux pumps and porins are not involved in innate tolerance to TDA. TDA is a promising broad-spectrum antimicrobial in part due to the fact that enhanced tolerance is difficult to gain and that the TDA-tolerant phenotype appears to confer only low-level resistance and is very unstable.

Influence of processing steps in cold-smoked salmon production on survival and growth of persistent and presumed non-persistent Listeria monocytogenes.

Cold-smoked salmon is a ready-to-eat product in which Listeria monocytogenes sometimes can grow to high numbers. The bacterium can colonize the processing environment and it is believed to survive or even grow during the processing steps. The purpose of the present study was to determine if the steps in the processing of cold-smoked salmon affect survival and subsequent growth of a persistent strain of L. monocytogenes to a lesser degree than presumed non-persistent strains. We used a sequence of experiments increasing in complexity: (i) small salmon blocks salted, smoked or dried under model conditions, (ii) fillets of salmon cold-smoked in a pilot plant and finally, (iii) assessment of the bacterial levels before and after processing during commercial scale production. L. monocytogenes proliferated on salmon blocks that were brined or dipped in liquid smoke and left at 25 degrees C in a humidity chamber for 24 h. However, combining brining and liquid smoke with a drying (25 degrees C) step reduced the bacterium 10-100 fold over a 24 h period. Non-salted, brine injected or dry salted salmon fillets were surface inoculated with L. monocytogenes and cold-smoked in a pilot plant. L. monocytogenes was reduced from 10(3) to 10-10(2) CFU/cm(2) immediately after cold-smoking. The greatest reductions were observed in dry salted and brine injected fillets as compared to cold-smoking of non-salted fresh fillets. Levels of L. monocytogenes decreased further when the cold-smoked fish was vacuum-packed and stored at 5 degrees C. A similar decline was seen when inoculating brine injected fillets after cold-smoking. High phenol concentrations are a likely cause of this marked growth inhibition. In a commercial production facility, the total viable count of salmon fillets was reduced 10-1000 fold by salting, cold-smoking and process-freezing (a freezing step after smoking and before slicing). The prevalence of L. monocytogenes in the commercial production facility was too low to determine any quantitative effects, however, one of nine samples was positive before processing and none after. Taken together, the processing steps involved in cold-smoking of salmon are bactericidal and reduce, but do not eliminate L. monocytogenes. A persistent strain was no less sensitive to the processing steps than a clinical strain or strain EGD.

Phaeobacter inhibens from the Roseobacter clade has an environmental niche as a surface colonizer in harbors

Phaeobacter inhibens belongs to the marine Roseobacter clade and is important as a carbon and sulfur metabolizer, a biofilm former and producer of the antibiotic tropodithietic acid (TDA). The majority of cultured strains have been isolated from marine aquaculture sites, however, their niche in the environment is to date unknown. Here, we report on the repeated isolation of Phaeobacter inhibens strains from a marine environment (harbors) not related to aquaculture. Based on phenotype and 16S rRNA gene sequence similarity, a total of 64 P. inhibens strains were identified from 35 samples (eukaryotic organisms or biofilms on inert surfaces) in Jyllinge Harbor during late summer and autumn, but not during winter and spring in 2009, 2011, and 2012. P. inhibens strains were also isolated from biofilms at three other Danish harbors (in 2012), but not from the surrounding seawater. Ten of the 14 samples from which P. inhibens was cultured contained bryozoans. DNA was extracted (in 2012) from 55 out of 74 Jyllinge Harbor samples, and 35 were positive for Phaeobacter using a genus-specific PCR. P. inhibens strains were isolated from nine of these samples. DNA and RNA were isolated from 13 random samples and used for amplification of 16S rRNA. P. inhibens was detected in five of these samples, all of which were biofilm samples, by pyrotag-sequencing at a prevalence of 0.02-0.68% of the prokaryotic community. The results indicated that P. inhibens had a niche in biofilms of fouled surfaces in harbor areas and that the population followed a seasonal fluctuation.

Global occurrence and heterogeneity of the Roseobacter -clade species Ruegeria mobilis

Tropodithietic acid (TDA)-producing Ruegeria mobilis strains of the Roseobacter clade have primarily been isolated from marine aquaculture and have probiotic potential due to inhibition of fish pathogens. We hypothesized that TDA producers with additional novel features are present in the oceanic environment. We isolated 42 TDA-producing R. mobilis strains during a global marine research cruise. While highly similar on the 16S ribosomal RNA gene level (99–100% identity), the strains separated into four sub-clusters in a multilocus sequence analysis. They were further differentiated to the strain level by average nucleotide identity using pairwise genome comparison. The four sub-clusters could not be associated with a specific environmental niche, however, correlated with the pattern of sub-typing using co-isolated phages, the number of prophages in the genomes and the distribution in ocean provinces. Major genomic differences within the sub-clusters include prophages and toxin-antitoxin systems. In general, the genome of R. mobilis revealed adaptation to a particle-associated life style and querying TARA ocean data confirmed that R. mobilis is more abundant in the particle-associated fraction than in the free-living fraction occurring in 40% and 6% of the samples, respectively. Our data and the TARA data, although lacking sufficient data from the polar regions, demonstrate that R. mobilis is a globally distributed marine bacterial species found primarily in the upper open oceans. It has preserved key phenotypic behaviors such as the production of TDA, but contains diverse sub-clusters, which could provide new capabilities for utilization in aquaculture.