Gut dysbiosis induced by florfenicol increases susceptibility to Aeromonas hydrophila infection in Zebrafish Danio rerio after the recommended withdrawal period.

OBJECTIVE: Florfenicol (FFC) is a broad-spectrum antibiotic approved by the U.S. Food and Drug Administration to treat both systemic and external bacterial infections in food fish. The objective of this study was to evaluate the effect of FFC-medicated feed on the gut microbiota of Zebrafish Danio danio to determine (1) if the therapeutic dose of FFC-medicated feed induces dysbiosis and (2) if fish with altered gut microbiota were more susceptible to subsequent infection by the common opportunistic fish pathogen Aeromonas hydrophila. METHODS: Zebrafish that were treated with regular and FFC-medicated feeds were artificially challenged with A. hydrophila at the end of the recommended 15-day antibiotic withdrawal period. The gut microbiota of the Zebrafish at different stages was analyzed using 16S ribosomal RNA gene sequencing. RESULT: Our results found that FFC-medicated feed induced disruption of the gut microbiota. Dysbiosis was observed in all treated groups, with a significant increase in bacterial diversity, and was characterized by a remarkable bloom of Proteobacteria and a drastic decline of Mycoplasma and Cetobacterium in treated animals but without noticeable clinical signs or mortalities. In addition, the increase of Proteobacteria was not significantly reduced after the recommended 15-day withdrawal period, and the Zebrafish treated with FFC-medicated feed exhibited a significantly higher mortality rate when they were subsequently challenged with A. hydrophila compared to the control (regular feed) groups. Interestingly, the most dramatic changes in the gut microbiome composition occurred at the transition time between the late stage of the medicated treatment and the beginning of the withdrawal period instead of the time during the Aeromonas infection. CONCLUSION: The administration of FFC-medicated feed at the recommended dose induced gut dysbiosis in Zebrafish, and fish did not recover to the baseline after the recommended withdrawal period. Our findings suggest that the use of antibiotics in fish elicits a response similar to those previously described in mammals and possibly makes the host more susceptible to subsequent infections of opportunistic pathogens. This study using a controlled model system suggests that antibiotics in aquaculture may have long-term effects on the general well-being of the fish.

Deciphering the Molecular Basis for Attenuation of Flavobacterium columnare Strain Fc1723 Used as Modified Live Vaccine against Columnaris Disease.

Vaccines are widely employed in aquaculture to prevent bacterial infections, but their use by the U.S. catfish industry is very limited. One of the main diseases affecting catfish aquaculture is columnaris disease, caused by the bacterial pathogen Flavobacterium columnare. In 2011, a modified-live vaccine against columnaris disease was developed by selecting mutants that were resistant to rifampin. The previous study has suggested that this vaccine is stable, safe, and effective, but the mechanisms that resulted in attenuation remained uncharacterized. To understand the molecular basis for attenuation, a comparative genomic analysis was conducted to identify specific point mutations. The PacBio RS long-read sequencing platform was used to obtain draft genomes of the mutant attenuated strain (Fc1723) and the parent virulent strain (FcB27). Sequence-based genome comparison identified 16 single nucleotide polymorphisms (SNP) unique to the mutant. Genes that contained mutations were involved in rifampin resistance, gliding motility, DNA transcription, toxin secretion, and extracellular protease synthesis. The results also found that the vaccine strain formed biofilm at a significantly lower rate than the parent strain. These observations suggested that the rifampin-resistant phenotype and the associated attenuation of the vaccine strain result from the altered activity of RNA polymerase (RpoB) and possible disrupted protein secretion systems.

Draft Genome Sequences of Cetobacterium somerae 2G Large and Two Novel Cetobacterium Isolates from Intestines of Channel Catfish (Ictalurus punctatus).

We report the draft genome sequences of Cetobacterium somerae 2G Large and two Cetobacterium isolates, 2A and 8H, which may represent novel species. The isolates were recovered from the intestines of catfish, and the genomes will assist in research to understand their potential use as probiotics in aquaculture.

Characterization of the normal equine conjunctival bacterial community using culture-independent methods.

BACKGROUND: The equine conjunctival microbiota has often been reported to be dominated by Gram-positive species such as Staphylococcus sp., Bacillus sp., and Corynebacterium sp. However, traditional culture-based methods can only recover a fraction of the bacterial species present in the sample. OBJECTIVES: This pilot study aimed at exploring the diversity of the equine conjunctival microbiota using culture-independent methods. STUDY DESIGN: Eight horses were included in this study, and only eyes with normal ophthalmic examination (n = 15 eyes) were sampled. METHODS: Conjunctival biopsies (culture-independent) were collected, and DNA was extracted from the tissues. Bacterial communities in conjunctival biopsies were characterized by next-generation sequencing of the 16S rRNA genes. Individual reads were ascribed to operational taxonomic units (OTUs) using BLASTn and Greengenes databases. Species richness, evenness, and Good's coverage were determined for each conjunctiva-associated microbial community. RESULTS: Culture-independent samples produced a total of 329 bacterial OTUs. The main OTUs identified in the study belonged to the Gram-negative species Ralstonia mannitolilytica (88.0%), Nicoletella semolina (3.3%), and Pseudomonas tolaasii (1.5%). CONCLUSIONS: Contrary to previously published data based on culture-dependent methods, the horse eye microbial community was dominated by Gram-negative bacteria of the phylum Proteobacteria.

Transcriptome analysis of the fish pathogen Flavobacterium columnare in biofilm suggests calcium role in pathogenesis.

BACKGROUND: Flavobacterium columnare is the causative agent of columnaris disease that affects cultured freshwater fishes worldwide. F. columnare easily colonizes surfaces by forming biofilm, which helps the pathogen resist antibiotic and disinfectant treatments. Previously, we had shown that increasing concentrations of calcium (Ca2+) promoted biofilm formation by F. columnare. The objective of this study was to further characterize the role of Ca2+ on biofilm formation and to compare the transcriptome profiles of planktonic and biofilm cells. RESULTS: RNA-Seq analysis was conducted to identify genes that were differentially expressed between the following states: i) planktonic cells in control medium (P), ii) planktonic cells in calcium-enriched medium (P/Ca), and iii) biofilm cells in calcium-enriched medium (B/Ca). Overall, we identified 441 significant (FDR-adjusted p < 0.05, fold change > 2) differentially expressed genes (DEGs) between P and B/Ca samples; 112 significant DEGs between P/Ca and B/Ca samples, and 175 significant DEGs between P/Ca and P samples, corresponding to 15.87, 4.03 and 6.30% of the total protein-coding sequences, respectively. The significant DEGs fell into different functional categories including iron acquisition, oxidative stress response, extracellular protein secretion, and respiratory metabolism. CONCLUSIONS: Our results posit Ca2+ as a critical signal in regulating bacterial surface adhesion and biofilm formation in F. columnare. Living in biofilm elicited a shift in several metabolic pathways that allowed the cells to cope with oxidative stress and nutrient starvation. In addition, Ca2+ supplementation induced the expression of putative virulence factors in F. columnare, such as extracellular protein secretion and iron acquisition.

Bacterial diversity and community structure of the intestinal microbiome of Channel Catfish (Ictalurus punctatus) during ontogenesis.

The acquisition of gut microbes does not occur randomly and is highly dependent on host factors, environmental cues, and self-assembly rules exerted by the microbes themselves. The main objective of this project was to characterize how the gut microbiome develops during the early life stages of Channel Catfish and to identify i) which bacteria are the main constituents of the gut microbiome at different ontogenesis stages, and ii) at which time point(s) the gut microbiome stabilizes. High-throughput Illumina Miseq DNA sequencing of the V4 domain of the 16S rRNA gene was used to assess the microbial community composition during the life stages of Channel Catfish along with water and feed samples. Microbiomes from fertilized eggs, sac fry, swim up fry, pre-fingerlings, and fingerlings were all significantly distinct. OTUs analyses showed that the phylum Proteobacteria, Firmicutes, Fusobacteria and Cyanobacteria dominated the Channel Catfish gut microbiome. During the early stages of ontogenesis, the fish microbiome was dynamic and highly diverse, with significant shifts occurring between fertilized eggs to sac fry (6dph), and from sac fry to swim up fry (15dph). The gut microbiome stabilized between the pre-fingerlings and fingerlings stage (≤90dph) with an observed reduction in species richness. Feed had a more significantly contribution to the microbial colonization of the gut than water. We have identified the period in which the gut microbiome changes rapidly from 15dph until 21dph before stabilizing after 90dph.

More than just antibodies: Protective mechanisms of a mucosal vaccine against fish pathogen Flavobacterium columnare.

A recently developed attenuated vaccine for Flavobacterium columnare has been demonstrated to provide superior protection for channel catfish, Ictalurus punctatus, against genetically diverse columnaris isolates. We were interested in examining the mechanisms of this protection by comparing transcriptional responses to F. columnare challenge in vaccinated and unvaccinated juvenile catfish. Accordingly, 58 day old fingerling catfish (28 days post-vaccination or unvaccinated control) were challenged with a highly virulent F. columnare isolate (BGSF-27) and gill tissues collected pre-challenge (0 h), and 1 h and 2 h post infection, time points previously demonstrated to be critical in early host-pathogen interactions. Following RNA-sequencing and transcriptome assembly, differential expression (DE) analysis within and between treatments revealed several patterns and pathways potentially underlying improved survival of vaccinated fish. Most striking was a pattern of dramatically higher basal expression of an array of neuropeptides (e.g. somatostatin), hormones, complement factors, and proteases at 0 h in vaccinated fish. Previous studies indicate these are likely the preformed mediators of neuroendocrine cells and/or eosinophilic granular (mast-like) cells within the fish gill. Following challenge, these elements fell to almost undetectable levels (>100-fold downregulated) by 1 h in vaccinated fish, suggesting their rapid release and/or cessation of synthesis following degranulation. Concomitantly, levels of pro-inflammatory cytokines (IL-1b, IL-8, IL-17) were induced in unvaccinated fish. In contrast, in vaccinated catfish, we observed widespread induction of genes needed for collagen deposition and tissue remodeling. Taken together, our results indicate an important component of vaccine protection in fish mucosal tissues may be the sensitization, proliferation and arming of resident secretory cells in the period between primary and secondary challenge.

Effects of Desiccation Practices of Cultured Atlantic Oysters (Crassostrea virginica) on Vibrio spp. in Portersville Bay, Alabama, USA.

The expansion of off-bottom aquaculture to the Gulf of Mexico has raised public health concerns for human health officials. High temperatures in the Gulf of Mexico are associated with high levels of Vibrio parahaemolyticus and Vibrio vulnificus. Routine desiccation practices associated with off-bottom aquaculture expose oysters to ambient air, allowing Vibrio spp. to proliferate in the closed oyster. Currently, there is limited research on the length of time needed for Vibrio spp. levels in desiccated oysters to return to background levels, defined as the levels found in oysters that remain continually submersed and not exposed to ambient air. This study determined the time needed to return V. parahaemolyticus, V. vulnificus, and Vibrio cholerae levels to background levels in oysters exposed to the following desiccation practices: 3-h freshwater dip followed by 24-h ambient air exposure, 27-h ambient air exposure, and control. All oysters were submerged at least 2 weeks prior to the beginning of each trial, with the control samples remaining submerged for the duration of each trial. Vibrio spp. levels were enumerated from samples collected on days 0, 1, 2, 3, 7, 10, and 14 after resubmersion using a three-tube most-probable-number enrichment followed by BAX PCR. V. cholerae levels were frequently (92%) below the limit of detection at all times, so they were not statistically analyzed. V. parahaemolyticus and V. vulnificus levels in the 27-h ambient air exposure and the 3-h freshwater dip followed by 24-h ambient air exposure samples were significantly elevated compared with background samples. In most cases, the Vibrio spp. levels in oysters in both desiccation treatments remained elevated compared with background levels until 2 or 3 days post-resubmersion. However, there was one trial in which the Vibrio spp. levels did not return to background levels until day 7. The results of this study provide scientific support that oyster farmers should be required to implement a minimum 7-day resubmersion regimen. This length of time allowed the Vibrio spp. levels to become not significantly different across all treatments.

Biofilm Formation on Aquaculture Substrates by Selected Bacterial Fish Pathogens.

The objective of this study was to determine whether common bacterial catfish pathogens could attach and colonize surfaces commonly found in aquaculture facilities. In addition, we evaluated the role of calcium in biofilm formation. Attachment to polystyrene plates was used to quantify biofilm formation by five bacterial pathogens (i.e., Flavobacterium columnare, Aeromonas hydrophila, Edwardsiella ictaluri, E. tarda, and E. piscicida). Flavobacterium columnare and A. hydrophila formed thick biofilms that were enhanced by calcium supplementation. Biofilm formation was significantly lower in all Edwardsiella species tested and calcium had little to no effect on Edwardsiella biofilm formation. Attachment to natural and artificial surfaces was quantified by a standard plate count method. Scanning electron microscopy (SEM) was used to confirm biofilm formation on the substrates. Flavobacterium columnare formed biofilm on the liner, flexible PVC, and nets. Bamboo prevented F. columnare attachment and inhibited cell growth. Aeromonas hydrophila and E. ictaluri formed biofilm on all materials tested, although significant differences were found among substrates. While E. ictaluri failed to form biofilm on microtiter polystyrene plates, it was able to colonize and multiply on all aquaculture materials tested. Our results demonstrated that common bacterial pathogens had the potential of colonizing surfaces and may use biofilm as reservoirs in fish farms. Received July 19, 2016; accepted January 19, 2017.

Microbiota of wild-caught Red Snapper Lutjanus campechanus.

BACKGROUND: The microbiota plays an essential role in host health, particularly through competition with opportunistic pathogens. Changes in total bacterial load and microbiota structure can indicate early stages of disease, and information on the composition of bacterial communities is essential to understanding fish health. Although Red Snapper (Lutjanus campechanus) is an economically important species in recreational fisheries and a primary aquaculture candidate, no information is available on the microbial communities of this species. The aim of this study was to survey the microbiota of apparently healthy, wild-caught Red Snapper from the Gulf of Mexico. Sampled Red Snapper showed no physical signs of disease. Tissues that are either primary entry routes for pathogens (feces, gill) or essential to disease diagnosis (blood) were sampled. Bacteria were enumerated using culture-based techniques and characterized by pyrosequencing. RESULTS: Aerobic counts of feces and gill samples were 107 and 104 CFU g-1, respectively. All individuals had positive blood cultures with counts up to 23 CFU g-1. Gammaproteobacteria dominated the microbiota of all sample types, including the genera Pseudoalteromonas and Photobacterium in feces and Pseudomonas in blood and gill. Gill samples were also dominated by Vibrio while blood samples had high abundances of Nevskia. High variability in microbiota composition was observed between individuals, with percent differences in community composition ranging from 6 to 76 % in feces, 10 to 58 % in gill, and 52 to 64 % in blood. CONCLUSIONS: This study provides the first characterization of the microbiota of the economically significant Red Snapper via pyrosequencing. Its role in fish health highlights the importance of understanding microbiota composition for future work on disease prevention using microbial manipulation.

Potassium permanganate elicits a shift of the external fish microbiome and increases host susceptibility to columnaris disease.

The external microbiome of fish is thought to benefit the host by hindering the invasion of opportunistic pathogens and/or stimulating the immune system. Disruption of those microbial communities could increase susceptibility to diseases. Traditional aquaculture practices include the use of potent surface-acting disinfectants such as potassium permanganate (PP, KMnO4) to treat external infections. This study evaluated the effect of PP on the external microbiome of channel catfish and investigated if dysbiosis leads to an increase in disease susceptibility. Columnaris disease, caused by Flavobacterium columnare, was used as disease model. Four treatments were compared in the study: (I) negative control (not treated with PP nor challenged with F. columnare), (II) treated but not challenged, (III) not treated but challenged, and (IV) treated and challenged. Ribosomal intergenic spacer analysis (RISA) and pyrosequencing were used to analyze changes in the external microbiome during the experiment. Exposure to PP significantly disturbed the external microbiomes and increased catfish mortality following the experimental challenge. Analysis of similarities of RISA profiles showed statistically significant changes in the skin and gill microbiomes based on treatment and sampling time. Characterization of the microbiomes using 16S rRNA gene pyrosequencing confirmed the disruption of the skin microbiome by PP at different phylogenetic levels. Loss of diversity occurred during the study, even in the control group, but was more noticeable in fish subjected to PP than in those challenged with F. columnare. Fish treated with PP and challenged with the pathogen exhibited the least diverse microbiome at the end of the study.

Comparison of DNA extraction protocols for the analysis of gut microbiota in fishes.

This study investigated the impacts of bacterial DNA extraction methodology on downstream analysis of fish gut microbiota. Feces and intestine samples were taken from three sympatric freshwater fish species with varying diets. Samples were processed immediately (approximately 4 h after capture; fresh), stored at -20 °C for 15 days or preserved in RNAlater® reagent for 15 days. DNA was then extracted using two commercial kits: one designed for animal tissues and one specifically formulated for stool samples. Microbial community fingerprints were generated using ribosomal intergenic spacer analysis. Factors including diversity as depicted by band number, band intensity, repeatability and practicalities such as cost and time were considered. Despite significant differences in microbiota structure, results were similar between feces and intestine samples. Frozen samples were consistently outperformed by other storage methods and the stool kit typically outperformed the tissue kit. Overall, we recommend extraction of bacterial DNA from fresh samples using the stool kit for both sample types. If samples cannot be processed immediately, preservation in RNAlater® is preferred to freezing. Choice of DNA extraction method significantly influences the results of downstream microbial community analysis and thus should be taken into consideration for metadata analysis.

Community Structure of Skin Microbiome of Gulf Killifish, Fundulus grandis, Is Driven by Seasonality and Not Exposure to Oiled Sediments in a Louisiana Salt Marsh.

Mucus of fish skin harbors complex bacterial communities that likely contribute to fish homeostasis. When the equilibrium between the host and its external bacterial symbionts is disrupted, bacterial diversity decreases while opportunistic pathogen prevalence increases, making the onset of pathogenic bacterial infection more likely. Because of that relationship, documenting temporal and spatial microbial community changes may be predictive of fish health status. The 2010 Deepwater Horizon oil spill was a potential stressor to the Gulf of Mexico's coastal ecosystem. Ribosomal intergenic spacer analysis (RISA) and pyrosequencing were used to analyze the bacterial communities (microbiome) associated with the skin and mucus of Gulf killifish (Fundulus grandis) that were collected from oiled and non-oiled salt marsh sites in Barataria Bay, LA. Water samples and fin clips were collected to examine microbiome structure. The microbiome of Gulf killifish was significantly different from that of the surrounding water, mainly attributable to shifts in abundances of Cyanobacteria and Proteobacteria. The Gulf killifish's microbiome was dominated by Gammaproteobacteria, specifically members of Pseudomonas. No significant difference was found between microbiomes of fish collected from oiled and non-oiled sites suggesting little impact of oil contamination on fish bacterial assemblages. Conversely, seasonality significantly influenced microbiome structure. Overall, the high similarity observed between the microbiomes of individual fish observed during this study posits that skin and mucus of Gulf killifish have a resilient core microbiome.

Epidemiology of columnaris disease affecting fishes within the same watershed.

In the southeastern USA, columnaris disease (caused by Flavobacterium columnare) typically affects catfish raised in earthen ponds from early spring until late summer. Recently, unusually severe outbreaks of columnaris disease occurred at the E. W. Shell Fisheries Center located in Auburn, AL, USA. During these outbreaks, catfish and other aquaculture and sport fish species that were in ponds located within the same watershed were affected. Our objective was to investigate the genetic diversity among F. columnare isolates recovered from different sites, sources, and dates to clarify the origin of these outbreaks and, ultimately, to better understand the epidemiology of columnaris disease. A total of 102 F. columnare isolates were recovered from catfishes (channel catfish Ictalurus puntactus, blue catfish I. furcatus, and their hybrid), bluegill Lepomis microchirus, Nile tilapia Oreochromis niloticus, largemouth bass Micropterus salmoides, egg masses, and water during columnaris outbreaks (from spring 2010 to summer 2012). Putative F. columnare colonies were identified following standard protocols. All isolates were ascribed to Genomovar II following restriction fragment length polymorphism analysis of the 16S rRNA gene. Genetic variability among the isolates was revealed by amplified fragment length polymorphism. Date of isolation explained most of the variability among our isolates, while host was the least influential parameter, denoting a lack of host specificity within Genomovar II isolates. The susceptibility of each of the isolates against commonly used antibiotics was tested by antibiogram. Our data showed that 19.6 and 12.7% of the isolates were resistant to oxytetracycline and kanamycin, respectively.

Predominant bacteria associated with red snapper from the Northern Gulf of Mexico.

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.

New attenuated vaccine against columnaris disease in fish: choosing the right parental strain is critical for vaccine efficacy.

Flavobacterium columnare, the causative agent of columnaris disease, is a highly diverse species comprised by three genomovars. Genomovar II strains are more virulent toward catfishes than genomovar I isolates. The objective of this study was to compare the vaccine efficacy of avirulent mutants derived from genomovars I and II using a rifampicin-resistance strategy. First, we compared the efficacy of 13 genomovar II mutants in channel catfish (Ictalurus punctatus) fingerlings and identified mutant 17-23 as the best vaccine candidate based on their relative percent survival (RPS) against a highly virulent genomovar II strain (BGFS-27). In the second experiment, we vaccinated zebrafish (Danio rerio) with two genomovar II mutants (17-23 and 16-534) and FCRR (genomovar I mutant) followed by exposure to BGFS-27 strain. RPS values were 28.4, 20.3 and 8.1% for 17-23, 16-534, and FCRR, respectively. For experiments 3 and 4, we tested both 17-23 and FCRR in channel catfish fry and Nile tilapia (Oreochromis niloticus). In both experiments, vaccinated fish were divided in two groups and each challenged with either a genomovar I (ARS-1) or a II (BGFS-27) strain. Channel catfish fry vaccinated with 17-23 and FCRR followed by challenge with BGFS-27 resulted in RPS values of 37.0% and 4.4%. When fish were challenged with ARS-1, RPS values were 90.9% and 72.7% for fish vaccinated with 17-23 and FCRR, respectively. Nile tilapia vaccinated with 17-23 and FCRR followed by challenged with BGFS-27 had RPS values of 82.1% and 16.1%, respectively. When fish were challenged with strain ARS-1, RPS values were 86.9% and 75.5%. Overall, our results demonstrated that vaccination with genomovar II mutant 17-23 confers better protection in channel catfish and Nile tilapia than FCRR against columnaris disease caused by genomovar II. Both mutants were equally protective against columnaris caused by genomovar I showing that 17-23 mutant cross-protected against both genomovars.

Biofilm formation by the fish pathogen Flavobacterium columnare: development and parameters affecting surface attachment.

Flavobacterium columnare is a bacterial fish pathogen that affects many freshwater species worldwide. The natural reservoir of this pathogen is unknown, but its resilience in closed aquaculture systems posits biofilm as the source of contagion for farmed fish. The objectives of this study were (i) to characterize the dynamics of biofilm formation and morphology under static and flow conditions and (ii) to evaluate the effects of temperature, pH, salinity, hardness, and carbohydrates on biofilm formation. Nineteen F. columnare strains, including representatives of all of the defined genetic groups (genomovars), were compared in this study. The structure of biofilm was characterized by light microscopy, confocal laser scanning microscopy, and scanning electron microscopy. F. columnare was able to attach to and colonize inert surfaces by producing biofilm. Surface colonization started within 6 h postinoculation, and microcolonies were observed within 24 h. Extracellular polysaccharide substances and water channels were observed in mature biofilms (24 to 48 h). A similar time course was observed when F. columnare formed biofilm in microfluidic chambers under flow conditions. The virulence potential of biofilm was confirmed by cutaneous inoculation of channel catfish fingerlings with mature biofilm. Several physicochemical parameters modulate attachment to surfaces, with the largest influence being exerted by hardness, salinity, and the presence of mannose. Maintenance of hardness and salinity values within certain ranges could prevent biofilm formation by F. columnare in aquaculture systems.

Diversity of the skin microbiota of fishes: evidence for host species specificity.

Skin microbiota of Gulf of Mexico fishes were investigated by ribosomal internal spacer analysis (RISA) and 16S rRNA gene sequencing. A total of 102 fish specimens representing six species (Mugil cephalus, Lutjanus campechanus, Cynoscion nebulosus, Cynoscion arenarius, Micropogonias undulatus, and Lagodon rhomboides) were sampled at regular intervals throughout a year. The skin microbiota from each individual fish was analyzed by RISA and produced complex profiles with 23 bands on average. Similarities between RISA profiles ranged from 97.5% to 4.0%. At 70% similarity, 11 clusters were defined, each grouping individuals from the same fish species. Multidimensional scaling and analysis of similarity correlated the RISA-defined clusters with geographic locality, date, and fish species. Global R values indicated that fish species was the most indicative variable for group separation. Analysis of 16S rRNA gene sequences (from pooled samples of 10 individual fish for each fish species) showed that the Proteobacteria was the predominant phylum in skin microbiota, followed by the Firmicutes and the Actinobacteria. The distribution and abundance of bacterial sequences were different among all species analyzed. Aeribacillus was found in all fish species representing 19% of all clones sequenced, while some genera were fish species-specific (Neorickettsia in M. cephalus and Microbacterium in L. campechanus). Our data provide evidence for the existence of specific skin microbiota associated with particular fish species.

Effective reduction of Vibrio vulnificus in the Eastern oyster (Crassostrea virginica) using high salinity depuration.

Depuration under different salinities was used to reduce the human pathogen Vibrio vulnificus from Eastern oysters (Crassostrea virginica). Individual recirculating systems were used to test the efficacy of depuration at three salinities (15, 25, and 35 psu) in four independent trials during a 14 day period. Initial loads of V. vulnificus were higher than 10,000 MPN/g of oyster meat in all trials. Data showed that 25 and 35 psu treatments were more efficient in reducing V. vulnificus numbers than 15 psu with an overall reduction of >3 logs. A significant decrease in MPN/g was observed as early as day 6 and further reductions were observed at day 10, while longer depurations did not improve efficacy. Only the highest salinity (35 psu) was capable of reducing V. vulnificus numbers to the FDA recommended level of <30 MPN/g in two of the four trials. Oysters survived well in the depuration systems with minimal mortality (<1%) but their condition index (meat quality and yield) decreased during the 14 day period in all treatments. The data presented in this study suggests that high salinity depuration is a promising method to reduce V. vulnificus in oysters.

Adaptive response to starvation in the fish pathogen Flavobacterium columnare: cell viability and ultrastructural changes.

BACKGROUND: The ecology of columnaris disease, caused by Flavobacterium columnare, is poorly understood despite the economic losses that this disease inflicts on aquaculture farms worldwide. Currently, the natural reservoir for this pathogen is unknown but limited data have shown its ability to survive in water for extended periods of time. The objective of this study was to describe the ultrastructural changes that F. columnare cells undergo under starvation conditions. Four genetically distinct strains of this pathogen were monitored for 14 days in media without nutrients. Culturability and cell viability was assessed throughout the study. In addition, cell morphology and ultrastructure was analyzed using light microscopy, scanning electron microscopy, and transmission electron microscopy. Revival of starved cells under different nutrient conditions and the virulence potential of the starved cells were also investigated. RESULTS: Starvation induced unique and consistent morphological changes in all strains studied. Cells maintained their length and did not transition into a shortened, coccus shape as observed in many other Gram negative bacteria. Flavobacterium columnare cells modified their shape by morphing into coiled forms that comprised more than 80% of all the cells after 2 weeks of starvation. Coiled cells remained culturable as determined by using a dilution to extinction strategy. Statistically significant differences in cell viability were found between strains although all were able to survive in absence of nutrients for at least 14 days. In later stages of starvation, an extracellular matrix was observed covering the coiled cells. A difference in growth curves between fresh and starved cultures was evident when cultures were 3-months old but not when cultures were starved for only 1 month. Revival of starved cultures under different nutrients revealed that cells return back to their original elongated rod shape upon encountering nutrients. Challenge experiments shown that starved cells were avirulent for a fish host model. CONCLUSIONS: Specific morphological and ultrastructural changes allowed F. columnare cells to remain viable under adverse conditions. Those changes were reversed by the addition of nutrients. This bacterium can survive in water without nutrients for extended periods of time although long-term starvation appears to decrease cell fitness and resulted in loss of virulence.