Genetic and phenotypic diversity of Flavobacterium psychrophilum isolates from Czech salmonid fish farms.

BACKGROUND: The salmonid pathogen Flavobacterium psychrophilum poses a significant economic threat to global aquaculture, yet our understanding of its genetic and phenotypic diversity remains incomplete across much of its geographic range. In this study, we characterise the genetic and phenotypic diversity of 70 isolates collected from rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta m. fario) from fish farms in the Czech Republic between 2012 and 2019 to compare their genomic content with all draft or complete genomes present in the NCBI database (n = 187). RESULTS: The Czech isolates underwent comprehensive evaluation, including multiplex PCR-based serotyping, genetic analysis, antimicrobial resistance testing, and assessment of selected virulence factors. Multiplex PCR serotyping revealed 43 isolates as Type 1, 23 as Type 2, with sporadic cases of Types 3 and 4. Multi-locus sequence typing unveiled 12 sequence types (ST), including seven newly described ones. Notably, 24 isolates were identified as ST329, a novel sequence type, while 22 were classified as the globally-distributed ST2. Phylogenetic analysis demonstrated clonal distribution of ST329 in the Czech Republic, with these isolates lacking a phage sequence in their genomes. Antimicrobial susceptibility testing revealed a high proportion of isolates classified as non-wild type with reduced susceptibility to oxolinic acid, oxytetracycline, flumequine, and enrofloxacin, while most isolates were classified as wild type for florfenicol, sulfamethoxazole-trimethoprim, and erythromycin. However, 31 isolates classified as wild type for florfenicol exhibited minimum inhibitory concentrations at the susceptibility breakpoint. CONCLUSION: The prevalence of the Czech F. psychrophilum serotypes has evolved over time, likely influenced by the introduction of new isolates through international trade. Thus, it is crucial to monitor F. psychrophilum clones within and across countries using advanced methods such as MLST, serotyping, and genome sequencing. Given the open nature of the pan-genome, further sequencing of strains promises exciting discoveries in F. psychrophilum genomics.

Effect of two insect meals on the gut commensal microbiome of healthy sea trout (Salmo trutta vr. trutta).

BACKGROUND: The balance of the intestinal commensal microbiome of fish and other animals plays an important role in the physiological processes of healthy animals, contributes to the defense against pathogens, stimulates the immune system and facilitates nutrient metabolism. In the last decade, the interest in the application of the insects in fish nutrition increased, although little is known regarding the effects of insect meals on the gastrointenstinal tract microbiome of the sea trout fingerlings. The aim of this study was to evaluate the effect of two diets containing mealworm (MW) and superworm (SW) on the microbiome of the digesta of sea trout fingerlings and the relative abundances of different taxa among communities under controlled conditions. RESULTS: The insect meals produced a similar weight gain and survival rate to sea trout fed fishmeal. The most abundant bacterial phylum in all the treatment groups was Firmicutes followed by Proteobacteria and Actinobacteria, and significant differences in the amount of Cyanobacteria were observed in the SW group. CONCLUSIONS: The insect meals did not produce differences in the three most abundant phyla in the sea trout digesta. However, the effect of each type of meal on the lower taxonomic levels was evident, particularly in the case of the superworm meal. These microbiome differences indicated that mealworm meal was more related to fishmeal than superworm meal. Our results highlight the potential effects of insect meals, such as mealworm and superworm meals, on the microbiota of sea trout.

Comparative genomics of Flavobacterium columnare unveils novel insights in virulence and antimicrobial resistance mechanisms.

This study reports the comparative analyses of four Flavobacterium columnare isolates that have different virulence and antimicrobial resistance patterns. The main research goal was to reveal new insights into possible virulence genes by comparing the genomes of bacterial isolates that could induce tissue damage and mortality versus the genome of a non-virulent isolate. The results indicated that only the genomes of the virulent isolates possessed unique genes encoding amongst others a methyl-accepting chemotaxis protein possibly involved in the initial colonization of tissue, and several VgrG proteins engaged in interbacterial competition. Furthermore, comparisons of genes unique for the genomes of the highly virulent (HV) carp and trout isolates versus the, respectively, low and non-virulent carp and trout isolates were performed. An important part of the identified unique virulence genes of the HV-trout isolate was located in one particular gene region identified as a genomic island. This region contained araC and nodT genes, both linked to pathogenic and multidrug-resistance, and a luxR-gene, functional in bacterial cell-to-cell communication. Furthermore, the genome of the HV-trout isolate possessed unique sugar-transferases possibly important in bacterial adhesion. The second research goal was to obtain insights into the genetic basis of acquired antimicrobial resistance. Several point-mutations were discovered in gyrase-genes of an isolate showing phenotypic resistance towards first and second-generation quinolones, which were absent in isolates susceptible to quinolones. Tetracycline-resistance gene tetA was found in an isolate displaying acquired phenotypic resistance towards oxytetracycline. Although not localized on a prophage, several flanking genes were indicative of the gene's mobile character.

Genetic diversity and phenotypic characterization of Iodobacter limnosediminis associated with skin lesions in freshwater fish.

The relatively unknown genus Iodobacter sp. has been repeatedly isolated from skin ulcers and saprolegniosis on freshwater fish in Finland, especially farmed salmonids. Genetic characterization verified that all 23 bacterial isolates studied here belonged to the species Iodobacter limnosediminis, previously undescribed from the fish microbiota. Whole-genome pulsed-field gel electrophoresis revealed variability between the I. limnosediminis strains, suggesting that they were most likely of environmental origin. Two I. limnosediminis strains caused lesions in 27%-53% of brown trout (Salmo trutta) injected intramuscularly (p ≤ .05). The lesions represented moderate to severe tissue damage, but for most fish, the tissues had been repaired by the end of the experiment through the accumulation of fibrocytes and macrophages at the site of the lesion. I. limnosediminis was reisolated from some lesions and/or internal organs. Phenotypically and biochemically, I. limnosediminis resembles several common bacterial species found in the aquatic environment, as it grows well on several media as whitish medium-sized colonies, is Gram negative and rod-shaped. Here, we characterized I. limnosediminis strains with several methods, including MALDI-TOF. This characterization will help in further investigations into the occurrence and possible involvement of I. limnosediminis in skin lesions of freshwater fish.

Seasonal habitat drives intestinal microbiome composition in anadromous Arctic char (Salvelinus alpinus).

Intestinal microbial communities from 362 anadromous Arctic char (Salvelinus alpinus) from the high Arctic Kitikmeot region, Nunavut, Canada, were characterized using high-throughput 16S rRNA gene sequencing. The resulting bacterial communities were compared across four seasonal habitats that correspond to different stages of annual migration. Arctic char intestinal communities differed by sampling site, salinity and stages of freshwater residence. Although microbiota from fish sampled in brackish water were broadly consistent with taxa seen in other anadromous salmonids, they were enriched with putative psychrophiles, including the nonluminous gut symbiont Photobacterium iliopiscarium that was detected in >90% of intestinal samples from these waters. Microbiota from freshwater-associated fish were less consistent with results reported for other salmonids, and highly variable, possibly reflecting winter fasting behaviour of these char. We identified microbiota links to age for those fish sampled during the autumn upriver migration, but little impact of the intestinal content and water microbiota on the intestinal community. The strongest driver of intestinal community composition was seasonal habitat, and this finding combined with identification of psychrophiles suggested that water temperature and migratory behaviour are key to understanding the relationship between Arctic char and their symbionts.

Renibacterium salmoninarum and Mycobacterium spp.: two bacterial pathogens present at low levels in wild brown trout (Salmo trutta fario) populations in Austrian rivers.

BACKGROUND: Renibacterium salmoninarum and Mycobacterium sp. are important bacterial pathogens of fish. R. salmoninarum is the causative agent of bacterial kidney disease, a Gram-positive bacterium mostly known for causing chronic infections in salmonid fish, while multiple species belonging to the Mycobacterium genus have been associated with mycobacteriosis in fish as well as in human. The objective of this study was to determine the prevalence of these two bacterial pathogens in populations of wild brown trout (Salmo trutta fario) in four rivers (Kamp, Wulka, Traun and Ybbs) in Austria. RESULTS: A total of 457 kidney samples were examined for both bacterial agents using nested and conventional PCR as well as bacterial cultivation on KDM-2, histological examination and immunohistochemistry. Molecular evidence showed an estimated prevalence level of 0.94% for R. salmoninarum in 2017 while the bacterium could not be detected in 2018 and histology showed signs consistent with a low-level chronic inflammation in the kidney of infected fish. Similarly, no fish were found positive for Mycobacterium in 2017 but in 2018, the prevalence was found to be 37.03% in the Kamp river (4.08% across all rivers). The sequencing data confirmed that these fish carried Mycobacterium sp. although the precise species of Mycobacterium could not be ascertained. CONCLUSIONS: This survey constitutes the first insight into the prevalence rate of R. salmoninarum and Mycobacterium sp. in wild brown trout (Salmo trutta fario) populations in Austria. Both of these pathogens were only detected in the summer months (June and July), which might suggest that the stress linked to increased water temperature could act as stressor factor and contribute to the outbreak of these diseases. The age of the fish might also play a role, especially in the case of Mycobacterium sp. as all the infected fish were in their first summer (June).

Whole-Genome Sequencing-Based Characterization of Listeria monocytogenes from Fish and Fish Production Environments in Poland.

Listeria monocytogenes, an important foodborne pathogen, may be present in different kinds of food and in food processing environments where it can persist for a long time. In this study, 28 L. monocytogenes isolates from fish and fish manufactures were characterized by whole genome sequencing (WGS). Core genome multilocus sequence typing (cgMLST) analysis was applied to compare the present isolates with publicly available genomes of L. monocytogenes strains recovered worldwide from food and from humans with listeriosis. All but one (96.4%) of the examined isolates belonged to molecular serogroup IIa, and one isolate (3.6%) was classified to serogroup IVb. The isolates of group IIa were mainly of MLST sequence types ST121 (13 strains) and ST8 (four strains) whereas the isolate of serogroup IVb was classified to ST1. Strains of serogroup IIa were further subtyped into eight different sublineages with the most numerous being SL121 (13; 48.1% strains) which belonged to six cgMLST types. The majority of strains, irrespective of the genotypic subtype, had the same antimicrobial resistance profile. The cluster analysis identified several molecular clones typical for L. monocytogenes isolated from similar sources in other countries; however, novel molecular cgMLST types not present in the Listeria database were also identified.

Spleen and head kidney differential gene expression patterns in trout infected with Lactococcus garvieae correlate with spleen granulomas.

Lactococcus garvieae is a significant pathogen in aquaculture with a potential zoonotic risk. To begin to characterize the late immune response of trout to lactococcosis, we selected infected individuals showing clinical signs of lactococcosis. At the time lactococcosis clinical signs appeared, infection by L. garvieae induced a robust inflammatory response in the spleen of rainbow trout, which correlated with abundant granulomatous lesions. The response in kidney goes in parallel with that of spleen, and most of the gene regulations are similar in both organs. A correlation existed between the early inflammatory granulomas in spleen (containing macrophages with internalized L. garvieae) and up-regulated gene sets, which defined the presence of macrophages and neutrophils. This is the first analysis of the immune transcriptome of rainbow trout following L. garvieae infection during the initiation of adaptive immune mechanisms and shows a transcriptome induction of antibody response by both IgM (+) and IgT (+) spleen B cells to respond to systemic infection. These results increase our understanding of lactococcosis and pave the way for future research to improve control measures of lactococcosis on fish farms.

Effects of dietary b-glucan, mannan oligosaccharide, Lactobacillus plantarum and their combinations on growth performance, immunity and immune related gene expression of Caspian trout, Salmo trutta caspius (Kessler, 1877).

This study investigated the effect of individual and combination of dietary pre- and probiotics (ß-glucan, 3 mg/g; mannan oligosaccharide (MOS), 4 mg/g; and Lactobacillus plantarum; 108 CFU/mg diet) on growth performance, blood immune parameters, expression of immune related genes, and intestinal microbial of Caspian trout (Salmo trutta caspius). On the basis of feeding with immunostimulant diets, the fish were assigned into eight groups denoted as: control (basal diet), bß (basal diet + ß-glucan), bM (basal diet + MOS), bLp (basal diet + L. plantarum), bßLp (basal diet + ß-glucan + L. plantarum), bMLp (basal diet + MOS + L. plantarum), bMß (basal diet + MOS + ß-glucan), and bMßLp (basal diet + MOS + ß-glucan + L. plantarum). All of the immunostimulant diets, in general, reduced feed intake (FI) and food conversion ratio (FCR) and increased WG, PER, and final weight. Condition factor (CF) demonstrated the lowest level in the experimental group received bMßLp. Total lipid increased in the fish received the additives, especially bM and bMß. Ash content demonstrated significant increase in the fish fed on bß and bMßLp, whereas moisture content was reduced in the group fed with L. plantarum-supplemented diet. All immunostimulant diets enhanced the activity and levels of lysozyme, Immunoglobulin M (IgM), and serum alternative complement activity (ACH50); the highest value for these indices was observed in the groups fed with bMß, bMßLp, and bßLp. bMß-treated fish group displayed the highest cortisol and glucose levels. bM diet induced the highest mRNA transcription of TNF-α1 in head kidney, whereas bLp, bMß, and bMßLp showed no effect. IL1ß exhibited the greatest up-regulation, about 8.75 fold change, in response to the diet supplemented only with ß-glucan. bßLp and bß significantly enhanced the relative IL-8 mRNA expression in the head kidney (about 2.75 and 1.9 folds, respectively), yet in response to bMßLp treatment it showed a decrease of about 5.7 times lower than the control group. In addition, intestinal population of L. plantarum showed the highest loads in the groups fed on the diets which were treated with the probiotic. Taken together, combinational use of these immunostimulants enhanced humoral innate immune system, whereas their individual and combinational application could increase and decrease the transcription of inflammation-related genes, respectively.

Effects of Enterococcus faecalis UGRA10 and the enterocin AS-48 against the fish pathogen Lactococcus garvieae. Studies in vitro and in vivo.

The aim of this study was to evaluate the effects of Enterococcus faecalis UGRA10 and its enterocin AS-48 against the fish pathogen Lactococcus garvieae. The minimum bactericidal concentrations of AS-48 against L. garvieae CECT 5807, 5806, and 5274 were 15.62, 15.62, and 7.81 µg/ml respectively. In broth cultures, enterocin at 100, 50, and 25 µg/ml reduced 108 CFU/ml lactococci after 2, 5, and 10 h, respectively. In co-cultures of UGRA10/L. garvieae at a 1/10 CFU/ml ratio, lactococci were eliminated after 24 h. Studies on UGRA10 biosafety and AS-48 toxicity in R1 cells and in rainbow trout have shown a lack of adverse effects from both the strain and bacteriocin. Trout challenged with L. garvieae and UGRA10 administered in diet 30 days before infection had a cumulative survival rate of 50% compared with 0% for control fish. Trout inoculated with the pathogen and treated by regular dipping in AS-48 baths had a survival rate of 60% after 20 days compared with that of untreated fish (0%). These results indicate the protective effect of the UGRA10 strain and the bacteriocin AS-48 against L. garvieae and the potential of these natural products as alternatives to antibiotics for controlling diseases in aquaculture.

Activity of Metabolic Enzymes in Farmed Rainbow Trout Oncorhynchus mykiss Walb. Affected by Bacterial Septicemia: The Effect of Food Additives.

The effect of food additive including antioxidant dihydroquercetin and polysaccharide arabinogalactan on the activity of metabolic enzymes in muscles and liver of artificially grown rainbow trout Oncorhynchus mykiss affected by bacterial infection was investigated. The results of the study indicated an increase in the resistance of trout to the action of bacterial infection with the enrichment of the diet with the studied bioactive components, apparently mediated, among other factors, by the activation of metabolic pathways of synthesis of energy and reducing equivalents.

Effects of host genetics and environment on egg-associated microbiotas in brown trout (Salmo trutta).

Recent studies found fish egg-specific bacterial communities that changed over the course of embryogenesis, suggesting an interaction between the developing host and its microbiota. Indeed, single-strain infections demonstrated that the virulence of opportunistic bacteria is influenced by environmental factors and host immune genes. However, the interplay between a fish embryo host and its microbiota has not been studied yet at the community level. To test whether host genetics affects the assemblage of egg-associated bacteria, adult brown trout (Salmo trutta) were sampled from a natural population. Their gametes were used for full-factorial in vitro fertilizations to separate sire from dam effects. In total, 2520 embryos were singly raised under experimental conditions that differently support microbial growth. High-throughput 16S rRNA amplicon sequencing was applied to characterize bacterial communities on milt and fertilized eggs across treatments. Dam and sire identity influenced embryo mortality, time until hatching and composition of egg-associated microbiotas, but no link between bacterial communities on milt and on fertilized eggs could be found. Elevated resources increased embryo mortality and modified bacterial communities with a shift in their putative functional potential. Resource availability did not significantly affect any parental effects on embryo performance. Sire identity affected bacterial diversity that turned out to be a significant predictor of hatching time: embryos associated with high bacterial diversity hatched later. We conclude that both host genetics and the availability of resources define diversity and composition of egg-associated bacterial communities that then affect the life history of their hosts.

Epitheliocystis in lake trout Salvelinus namaycush (Walbaum) is associated with a ß-proteobacteria.

Lake trout Salvelinus namaycush (Walbaum) raised for stocking experienced yearly (2011-13) winter epizootics of epitheliocystis. Affected fish were dispersed on the bottom of the tank, had decreased feed and fright response, and mortality often reached 40%. Peak mortality occurred within 3 weeks of the appearance of clinical signs, and outbreaks typically lasted 6 weeks. Affected fish had no gross lesions but histologically had branchial epithelial necrosis and lamellar hyperplasia, with small to large numbers of scattered epithelial cells containing 10- to 20-µm inclusions. A longitudinal study was undertaken of one annual outbreak, and lamellar hyperplasia was most closely associated with mortality. The number of inclusions was statistically greater (P < 0.05) before and during peak mortality, but inclusions were present in low numbers before clinical signs occurred. Results of histochemical staining, immunohistochemistry and transmission electron microscopy supported the presence of a ß-proteobacteria rather than a Chlamydiales bacterium within inclusions. PCR primers to identify Chlamydiales did not give consistent results. However, the use of universal 16S rDNA bacterial primers in conjunction with laser capture microdissection of inclusions demonstrated that a ß-proteobacteria was consistently associated with affected gills and is more likely the cause of the disease in lake trout.

Dietary peppermint (Mentha piperita) extracts promote growth performance and increase the main humoral immune parameters (both at mucosal and systemic level) of Caspian brown trout (Salmo trutta caspius Kessler, 1877).

The effects of dietary administration of peppermint (Mentha piperita L.) on Caspian brown trout fish (Salmo trutta caspius) were studied. Fish were divided into 4 groups before being fed diets supplemented with 0% (control), 1%, 2% and 3% of peppermint extracts for 8 weeks. Dose-dependent increases in growth, immune (both in skin mucus and blood serum) and hematological parameters (number of white cells, hematocrit and hemoglobin content), as well as in amylase activity and in the number of lactic acid bacteria on intestine were recorded in fish fed supplemented diets compared to control fish. However, the dietary peppermint supplements have different effects on the number of blood leucocytes depending on the leukocyte cell type. While no significant differences were observed in the number of blood monocytes and eosinophils, the number of lymphocytes was decreased, respectively, on fish fed peppermint enriched diets, respect to the values found in control fish. Furthermore, dietary peppermint supplements have no significant effect on blood biochemical parameters, enzymatic activities of liver determined in serum and total viable aerobic bacterial count on intestine of Caspian brown trout. Present results support that dietary administration of peppermint promotes growth performance and increases the main humoral immune parameters (both at mucosal and systemic level) and the number of the endogenous lactic acid bacteria of Caspian brown trout. This study underlying several positive effects of dietary administration of peppermint to farmed fish.

Occurrence of antibiotic resistance genes in culturable bacteria isolated from Turkish trout farms and their local aquatic environment.

Antibiotic resistance and presence of the resistance genes were investigated in the bacteria isolated from water, sediment, and fish in trout farms. A total of 9 bacterial species, particularly Escherichia coli, were isolated from the water and sediment samples, and 12 species were isolated from fish. The antimicrobial test indicated the highest resistance against sulfamethoxazole and ampicillin in coliform bacteria, and against sulfamethoxazole, imipenem, and aztreonam in known pathogenic bacteria isolated from fish. The most effective antibiotics were rifampicin, chloramphenicol, and tetracycline. The multiple antibiotic resistance index was above the critical limit for almost all of the bacteria isolated. The most common antibiotic resistance gene was ampC, followed by tetA, sul2, blaCTX-M1, and blaTEM in the coliform bacteria. At least one resistance gene was found in 70.8% of the bacteria, and 66.6% of the bacteria had 2 or more resistance genes. Approximately 36.54% of the bacteria that contain plasmids were able to transfer them to other bacteria. The plasmid-mediated transferable resistance genes were ampC, blaCTX-M1, tetA, sul2, and blaTEM. These results indicate that the aquatic environment could play an important role in the development of antibiotic resistance and the dissemination of resistance genes among bacteria.

Chitosan coating incorporated with the lactoperoxidase system: an active edible coating for fish preservation.

BACKGROUND: As a result of consumers' concerns about chemicals there is a particular interest in the food industry to use natural bio-preservatives such as antimicrobial enzymes for antimicrobial packaging. Based on the antimicrobial activity of the lactoperoxidase system (LPOS), the present study evaluated the coating effect of LPOS incorporated into chitosan solution (CH) on the quality and shelf life extension of rainbow trout during refrigerated storage (4 ± 1 °C), for a period of 16 days. RESULTS: The results indicated that samples of the CH+LPOS group had significantly lower numbers of Shewanella putrefaciens, Pseudomonas fluorescens, and psychrotrophic and mesophilic bacteria than did the CH and control group during the entire storage period. Total volatile basic nitrogen (TVB-N) levels for the CH+LPOS samples (22.07 mg 100 g(-1)) did not exceed the limit of consumption (30-35 mg N 100 g(-1)), while the CH (31.03 mg 100 g(-1) ) and control groups (37.78 mg 100 g(-1) ) reached this level at days 12 and 16, respectively. Thiobarbituric acid values of the CH and CH+LPOS samples, ranged between 0.49 and 0.51 on day 0 and 4.59-4.66 mg kg(-1) on day 16, were significantly lower (P < 0.05) than the corresponding values of the control samples (0.47 on day 0 to 4.78 mg kg(-1) on day 16 of storage) during the chilled storage period. CONCLUSION: The coating treatments (CH and CH+LPOS) extended the shelf life of trout fillets by at least 4 days as compared to the control samples, so that they showed moderate to high acceptability in all investigated sensory attributes even on the 16th day of storage.

Chryseobacterium aahli sp. nov., isolated from lake trout (Salvelinus namaycush) and brown trout (Salmo trutta), and emended descriptions of Chryseobacterium ginsenosidimutans and Chryseobacterium gregarium.

Two strains (T68T and T62) of a Gram-reaction-negative, yellow-pigmented bacterium containing flexirubin-type pigments were recovered from the kidney of a cultured lake trout (Salvelinus namaycush) and necrotic fins of a brown trout (Salmo trutta) during disease surveillance in 2009. Both isolates possessed catalase and cytochrome oxidase activities and degraded multiple substrates (e.g. gelatin, casein, elastin and Tweens 20 and 80). The mean DNA G+C content of strain T68T was 34.1 mol%. 16S rRNA gene sequencing demonstrated that strains T68T and T62 had nearly identical sequences (≥99 % similarity) and placed the bacterium within the genus Chryseobacterium, where Chryseobacterium ginsenosidimutans THG 15T (97.8%), C. gregarium DSM 19109T (97.7%) and C. soldanellicola PSD1-4T (97.6%) were its closest relatives. Subsequent phylogenetic analyses using neighbour-joining, maximum-parsimony and Bayesian methodologies demonstrated that strains T68T and T62 formed a well-supported clade (bootstrap values of 100 and 97%; posterior probability 0.99) that was distinct from other species of the genus Chryseobacterium. The major fatty acids of strains T68T and T62 were characteristic of the genus Chryseobacterium and included iso-C15:0, summed feature 3 (C16:1ω6c and/or C16:1ω7c), iso-C17:0 3-OH, C16:0 and C16:0 3-OH. The mean DNA-DNA relatedness of strain T68T to C. ginsenosidimutans JCM 16719T and C. gregarium LMG 24952T was 24 and 21%, respectively. Based on the results from our polyphasic characterization, strains T68T and T62 represent a novel species of the genus Chryseobacterium, for which the name Chryseobacterium aahli sp. nov. is proposed. The type strain is T68T (=LMG 27338T=ATCC BAA-2540T). Emended descriptions of Chryseobacterium ginsenosidimutans and Chryseobacterium gregarium are also proposed.

Probiotic treatment by indigenous bacteria decreases mortality without disturbing the natural microbiota of Salvelinus fontinalis.

Next-generation sequencing is revealing the complex interactive networks of host-bacteria interactions, as it is now possible to screen in detail the microbiota harbored by a host. This study investigated the influence of a probiotic treatment on the survival and microbiota of brook charr (Salvelinus fontinalis), focusing on its disturbance of the natural microbiota (dysbiosis). The results indicated that an indigenous probiotic strain (identified as Rhodococcus sp.) colonized neither the fish skin mucus nor the water following the probiotic treatment. Instead, the probiotic strain was detected only in the biofilm of the test tank. Nevertheless, a substantial beneficial effect of the probiotic treatment was observed: the population of the pathogen Flavobacterium psychrophilum decreased in the treated tank water. This study clearly shows that the indigenous strain chosen for the probiotic treatment did not disturb the natural fish skin mucus microbiota but acted directly through the production system to control the growth of the pathogen and, as a consequence, to enhance fish survival.

Absence of Staphylococcus aureus in Wild Populations of Fish Supports a Spillover Hypothesis.

Staphylococcus aureus is a human commensal and opportunistic pathogen that also infects other animals. In humans and livestock, where S. aureus is most studied, strains are specialized for different host species. Recent studies have also found S. aureus in diverse wild animals. However, it remains unclear whether these isolates are also specialized for their hosts or whether their presence is due to repeated spillovers from source populations. This study focuses on S. aureus in fish, testing the spillover hypothesis in two ways. First, we examined 12 S. aureus isolates obtained from the internal and external organs of a farmed fish. While all isolates were from clonal complex 45, genomic diversity indicates repeated acquisition. The presence of a φSa3 prophage containing human immune evasion genes suggests that the source was originally human. Second, we tested for S. aureus in wild fish that were isolated from likely sources. In particular, we sampled 123 brown trout and their environment at 16 sites in the remote Scottish Highlands with variable levels of exposure to humans, birds, and livestock. This screen found no S. aureus infection in any of the wild populations or their environment. Together, these results support that the presence of S. aureus in fish and aquaculture is due to spillover from humans rather than specialization. Given the trends of increasing fish consumption, a better understanding of the dynamics of S. aureus spillover in aquaculture will mitigate future risks to fish and human health. IMPORTANCE Staphylococcus aureus is a human and livestock commensal but also an important pathogen responsible for high human mortality rates and economic losses in farming. Recent studies show that S. aureus is common in wild animals, including fish. However, we do not know whether these animals are part of the normal host range of S. aureus or whether infection is due to repeated spillover events from true S. aureus hosts. Answering this question has implications for public health and conservation. We find support for the spillover hypothesis by combining genome sequencing of S. aureus isolates from farmed fish and screens for S. aureus in isolated wild populations. The results imply that fish are unlikely to be a source of novel emergent S. aureus strains but highlight the prominence of the spillover of antibiotic-resistant bacteria from humans and livestock. This may affect both future fish disease potential and the risk of human food poisoning.