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.

Pseudoalteromonas spp. serve as initial bacterial attractants in mesocosms of coastal waters but have subsequent antifouling capacity in mesocosms and when embedded in paint.

The purpose of the present study was to determine if the monoculture antifouling effect of several pigmented pseudoalteromonads was retained in in vitro mesocosm systems using natural coastal seawater and when the bacteria were embedded in paint used on surfaces submerged in coastal waters. Pseudoalteromonas piscicida survived on a steel surface and retained antifouling activity for at least 53 days in sterile seawater, whereas P. tunicata survived and had antifouling activity for only 1 week. However, during the first week, all Pseudoalteromonas strains facilitated rather than prevented bacterial attachment when used to coat stainless steel surfaces and submerged in mesocosms with natural seawater. The bacterial density on surfaces coated with sterile growth medium was 10(5) cells/cm(2) after 7 days, whereas counts on surfaces precoated with Pseudoalteromonas were significantly higher, at 10(6) to 10(8) cells/cm(2). However, after 53 days, seven of eight Pseudoalteromonas strains had reduced total bacterial adhesion compared to the control. P. piscicida, P. antarctica, and P. ulvae remained on the surface, at levels similar to those in the initial coating, whereas P. tunicata could not be detected. Larger fouling organisms were observed on all plates precoated with Pseudoalteromonas; however, plates coated only with sterile growth medium were dominated by a bacterial biofilm. Suspensions of a P. piscicida strain and a P. tunicata strain were incorporated into ship paints (Hempasil x3 87500 and Hempasil 77500) used on plates that were placed at the Hempel A/S test site in Jyllinge Harbor. For the first 4 months, no differences were observed between control plates and treated plates, but after 5 to 6 months, the control plates were more fouled than the plates with pseudoalteromonad-based paint. Our study demonstrates that no single laboratory assay can predict antifouling effects and that a combination of laboratory and real-life methods must be used to determine the potential antifouling capability of new agents or organisms.

Marine bacteria from Danish coastal waters show antifouling activity against the marine fouling bacterium Pseudoalteromonas sp. strain S91 and zoospores of the green alga Ulva australis independent of bacteriocidal activity.

The aims of this study were to determine if marine bacteria from Danish coastal waters produce antifouling compounds and if antifouling bacteria could be ascribed to specific niches or seasons. We further assess if antibacterial effect is a good proxy for antifouling activity. We isolated 110 bacteria with anti-Vibrio activity from different sample types and locations during a 1-year sampling from Danish coastal waters. The strains were identified as Pseudoalteromonas, Phaeobacter, and Vibrionaceae based on phenotypic tests and partial 16S rRNA gene sequence similarity. The numbers of bioactive bacteria were significantly higher in warmer than in colder months. While some species were isolated at all sampling locations, others were niche specific. We repeatedly isolated Phaeobacter gallaeciensis at surfaces from one site and Pseudoalteromonas tunicata at two others. Twenty-two strains, representing the major taxonomic groups, different seasons, and isolation strategies, were tested for antiadhesive effect against the marine biofilm-forming bacterium Pseudoalteromonas sp. strain S91 and zoospores of the green alga Ulva australis. The antiadhesive effects were assessed by quantifying the number of strain S91 or Ulva spores attaching to a preformed biofilm of each of the 22 strains. The strongest antifouling activity was found in Pseudoalteromonas strains. Biofilms of Pseudoalteromonas piscicida, Pseudoalteromonas tunicata, and Pseudoalteromonas ulvae prevented Pseudoalteromonas S91 from attaching to steel surfaces. P. piscicida killed S91 bacteria in the suspension cultures, whereas P. tunicata and P. ulvae did not; however, they did prevent adhesion by nonbactericidal mechanism(s). Seven Pseudoalteromonas species, including P. piscicida and P. tunicata, reduced the number of settling Ulva zoospores to less than 10% of the number settling on control surfaces. The antifouling alpP gene was detected only in P. tunicata strains (with purple and yellow pigmentation), so other compounds/mechanisms must be present in the other Pseudoalteromonas strains with antifouling activity.

Survival and growth of Salmonella and Vibrio in som-fak, a Thai low-salt garlic containing fermented fish product.

Fermentation of raw fish is a common process in Asia for improvement of shelf life and safety, however, little is known about the survival of pathogenic bacteria in these products. Raw fish may be contaminated with Salmonella and Vibrio species. The purpose of this study was to determine survival and potential growth of Salmonella enterica serovar Weltevreden, S. enterica serovar Enteritidis, Vibrio cholerae and V. parahaemolyticus as influenced by the preservation parameters (sodium chloride, garlic and lactic acid) present in the Thai fermented fish product som-fak. The inhibitory effects of sodium chloride (0-4%), garlic (0-10%) and lactic acid (pH levels as in som-fak) were measured in modified brain heart infusion (BHI) broth at 30 degrees C. All bacteria were inhibited by 8-10% sodium chloride. Salmonella grew in all concentrations of garlic whereas Vibrio spp. were inhibited by 1.0-1.5%. Lactic acid was inhibitory at levels above 1.5%. The combinations of sodium chloride, lactic acid and garlic showed a distinct hurdle effect in the broth system. Neither S. Enteritidis, V. cholerae nor V. parahaemolyticus grew in garlic (0.5-1%), regardless of the level of sodium chloride (0.5-4% (w/v)), when lactic acid (0.5-2%) was present. S. Weltevreden was the least inhibited of the four bacteria and grew in the combination of 0.5% garlic and 0.5% lactic acid regardless of the NaCl level (0.5-4% (w/v)). Som-fak with 0 to 10% garlic or 2% glucose was inoculated with either (i) 10(3) CFU/g Salmonella Weltevreden, (ii) 10(6) CFU/g garlic fermenting Lactobacillus plantarum strain 509 or (iii) a combination of the two strains and stored at 30 degrees C. The Salmonella count increased to >10(8) CFU/g (>10(6) CFU/g for 10% garlic) in all types of som-fak inoculated with S. Weltevreden within the first day. Only a combination of at least 6% garlic and L. plantarum 509 was enough to prevent growth of the inoculated Salmonella whereas adding the Lactobacillus strain alone or in combination with glucose was insufficient to prevent growth. Our results show that Salmonella Weltevreden can grow in som-fak independently of the inhibitory substances normally present in this type of product, emphasising the importance of preventing contamination. However, our results also suggest that the use of garlic fermenting starter cultures in combination with garlic could improve safety of fermented fish products.

Potassium lactate combined with sodium diacetate can inhibit growth of Listeria monocytogenes in vacuum-packed cold-smoked salmon and has no adverse sensory effects.

Growth of Listeria monocytogenes in ready-to-eat fish products such as cold-smoked salmon is an important food safety issue. The objective of this study was to evaluate the antilisterial activity of potassium lactate (PL) in combination with sodium acetate (SA) or sodium diacetate (SDA) in cold-smoked salmon and to determine whether these compounds could be incorporated easily into the formulations and technology currently used by processors. A commercial brine injector was used to inject salmon filets with either saturated saline brine or saturated saline brine supplemented with combinations of PL and SA (PURASAL Opti. Form PA 4) or PL and SDA (PURASAL Opti. Form PD 4). In the brine-injected cold-smoked salmon, 2.1% (water phase) PL and 0.12% (water phase) SDA delayed the growth of L. monocytogenes for up to 42 days of vacuum-packaged storage at 10 degrees C. Storage at 25 degrees C for 6 h resulted in only a 1-log CFU/g increase in L. monocytogenes. Treatments with lower concentrations of PL and SDA or similar concentrations of PL and SA resulted in an extended lag phase and slower growth of L. monocytogenes. It was not possible to incorporate more than 2% (water phase) PL while ensuring a minimum of 3% (water phase) NaCl in the finished product because PL decreased the solubility of NaCl. Sensory analyses revealed that the preservatives did not negatively affect flavor or odor. The combination of PL and SDA is therefore a viable technology for preventing L. monocytogenes growth on cold-smoked salmon.