Effects of the husky oil spill on gut microbiota of native fishes in the North Saskatchewan River, Canada.

In July 2016, a Husky Energy pipeline spilled 225,000 L of diluted heavy crude oil, with a portion of the oil entering the North Saskatchewan River near Maidstone, SK, Canada. This event provided a unique opportunity to assess potential effects of a crude oil constituent (namely polycyclic aromatic hydrocarbons, PAHs) on a possible sensitive indicator of freshwater ecosystem health, the gut microbiota of native fishes. In summer 2017, goldeye (Hiodon alosoides), walleye (Sander vitreus), northern pike (Esox lucius), and shorthead redhorse (Moxostoma macrolepidotum) were collected at six locations upstream and downstream of the spill. Muscle and bile were collected from individual fish for quantification of PAHs and intestinal contents were collected for characterization of the microbial community of the gut. Results suggested that host species is a significant determinant of gut microbiota, with significant differences among the species across sites. Concentrations of PAHs in dorsal muscle were significantly correlated with gut community compositions of walleye, but not of the other fishes. Concentrations of PAHs in muscle were also correlated with abundances of several families of bacteria among fishes. This study represents one of the first to investigate the response of the gut microbiome of wild fishes to chemical stressors.

Biogenic amines formation in high-pressure processed pike flesh (Esox lucius) during storage.

The effects of vacuum packaging followed by high pressure processing on the shelf-life of fillets of pike (Esox lucius) were examined. Samples were pressure-treated at 300 and 500 MPa and stored at 3.5 and 12 °C for up to 70 days. The content of eight biogenic amines (putrescine, cadaverine, spermidine, spermine, histamine, tyramine, tryptamine and phenylethylamine) were determined. Putrescine showed very good correspondence with the level of applied pressure and organoleptic properties. Polyamines spermidine and spermine did not show statistically significant changes with the level of applied pressure and the time of storage. Increased cadaverine and tyramine contents were found in samples with good sensory signs, stored for longer time and/or kept at 12 °C, thus indicating the loss of freshness. Tryptamine and phenylethylamine were not detected in pressure-treated samples kept at 3.5 °C. Histamine was not detected in samples of good quality.

Microbiological properties and biogenic amines of whole pike-perch (Sander lucioperca, linnaeus 1758): a perspective on fish safety during postharvest handling practices and frozen storage.

The biogenic amines (tyramine, histamine, cadaverine, and puterscine) and microbiological properties (mesophilic, psychrotrophic, and Pseudomonas spp.) of whole pike-perch (Sander lucioperca) was investigated during 2 d prestorage icing and 90 d frozen storage (-24 °C). At the end of ice storage, a noticeable increase only was found for puterscine level (P < 0.05), and microbial loads of fish increased in comparison with fresh fish (P < 0.05). During the frozen storage, as time passed, a continuous increase of biogenic amines and decrease of bacterial load (except for Pseudomonas spp. at the last 30 d) was detected (P < 0.05). The total contents of biogenic amines ranged from 6.24 to 91.76 µg/g during the investigated period. Puterscine was the major amine detected in pike-perch and its concentration varied between 1.75 and 56.95 µg/g; due to a more step-wise increase it was a good quality indicator. At the end of storage, all of the obtained values are below the tolerable maximum amounts based on available regulations. Based on biogenic amines content and microbial load, it could be concluded that pike-perch can be consumed without any health risks after 2 d icing condition and 90 d frozen storage.

[Taxonomic characteristics and physiological properties of microorganisms from the gut of pike (Esox lucius)].

The taxonomic composition and distribution of microorganisms differing in the degree of association with the intestinal mucosa of the pike (Lucius lucius) has been studied. Microorgansism of the families Enterobacteriaceae, Aeromonadaceae, and Vibrionaceae dominate in the gut microflora. Numerically prevailing bacterial species are characterized by high proteolytic and amylolytic enzyme activities as well as by high persistence accounted for by antilysozyme and antihistone activities. The results of this study show that Hafnia alvei, Yersinia ruckeri, Vibrio vulnificus, V. furnissii, Aeromonas salmonicida, and Shewanella putrefaciens may be regarded as normal components of the pike gut microflora.

[Indigenous microflora associated with the tegument of Triaenophorus nodulosus (Cestoda) and the intestine of its pike host].

Investigations of the indigenous microflora associated with the mucous intestines of fish and its cestode parasites have been for the first time carried out using the methods of transmission and scanning electron microscopy. New data on the bacterial biodiversity in the cestode and its fish host are obtained. Nanobacteria and spirochaetes are for the first time revealed in a fish host together with the previously known bacteria forming the intestinal microflora of fish. Spirochaetes were shown to be associated with the intestines of a pike host only, while nanobacteria cover abundantly the surface of the apical parts of the intestinal microvilli and the apical parts of the microtriches in the cestode tegument. The similarity of the bacterial floras associated with the apical surface of the parasite tegument and the intestine of the host should be noted. At the same time, deeper bacterial communities represented by obligate symbionts are specific. Thus, there is a normal indigenous microflora in cestodes, associated with the tegumental surface. This symbiotic microflora has specific morphological features and provides the balance of relations in the parasite-host system.

Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case.

AIMS: To evaluate the public health significance of representative strains of two Aeromonas spp., mainly from freshwater fish, on the basis of production of virulence-associated factors and presence of the haemolytic genes aerA and hlyA. METHODS AND RESULTS: Eleven strains of Aer. hydrophila, three strains of Aer. veronii biovar sobria (all from freshwater fish) and one strain of Aer. hydrophila from human diarrhoea were tested for potential virulence traits and for the presence of the haemolytic genes aerA and hlyA. Ten Aer. hydrophila isolates were aerA(+)hlyA(+) and two aerA(+)hlyA(-). Aeromonas veronii biovar sobria isolates were aerA(-)hlyA(-). Strains from the three genotypes showed enterotoxic activity in the suckling mouse assay. At 28 degrees C, four Aer. hydrophila fish strains could be considered as potentially virulent (possessing at least two of these characteristics: haemolytic, cytotoxic and enterotoxic). One Aer. veronii biovar sobria strain and the clinical isolate were cytotoxic on Vero cells. When grown at 4 degrees C, these six isolates fulfilled virulence criterion, but at 37 degrees C, only one fish strain, an Aer. hydrophila, did. CONCLUSIONS: The potential health risk derived from the presence of Aer. hydrophila and Aer. veronii biovar sobria in ice-stored freshwater fish should not be underestimated. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of virulence factors is affected by temperature incubation and not always related to the presence of haemolytic genes.

Mesophilic aeromonads in wild and aquacultured freshwater fish.

Numbers and species of motile Aeromonas were determined in freshly caught freshwater fish, in the surrounding environment, and also during iced chilled storage of fish specimens. Although no significant differences were observed in water samples, initial levels for skin, gill, and intestines were significantly lower in farmed rainbow trout (Oncorhynchus mykiss) than in wild brown trout (Salmo trutta) and pike (Esox lucius). During storage of wild specimens, naturally occurring aeromonads grew fairly well on the surfaces of skin and body cavity. Of 171 strains assigned to the genus Aeromonas, 88% were identified to phenospecies and putative genospecies level by using comprehensive biochemical schemes. The isolates were allocated to putative hybridization groups (HGs) 1 and 3 Aeromonas hydrophila (29%); putative HG 8 Aeromonas veronii biovar sobria (19%); putative HG 2 Aeromonas bestiarum (18%); putative HG 9 Aeromonas jandaei (16%); putative HGs 4 and 5a Aeromonas caviae (2%); putative HG 12 Aeromonas schubertii (2%); and putative HG 11 (unnamed, 0.6%). The remaining 20 isolates (12%) resembled A. schubertii but could not be allocated to currently recognized phenospecies or to putative HGs. Although cultured rainbow trout yielded strains of putative HGs 1, 4, and 8, which appear to be of major clinical importance, most isolates assigned to putative HGs 1 and 8 were recovered from pike. Differences among HGs found in wild animals could be related to their origin (unpolluted rivers for brown trout and urban rivers for pike). The recovery of these aeromonads species was not related to sampling site. The initial levels of motile aeromonads, their behavior during storage, and the strong potential spoilage activity of most isolates confirm that these bacteria can contribute to deterioration of iced wild freshwater fish. Although adequate cooking would inactivate motile aeromonads, the high incidence of isolates belonging to gastroenteritis-associated HGs should be regarded as a potential health concern, particularly for susceptible populations when there is a possibility of cross-contamination.

Psychrobacters and related bacteria in freshwater fish.

Three phenotypic identification systems were employed to identify 106 strains of gram-negative, nonmotile, aerobic bacteria obtained during iced storage of wild (Salmo trutta and Esox lucius) and farmed (Oncorhynchus mykiss) freshwater fish. Using diagnostic tables and computer-assisted identification, the isolates were Psychrobacter (64 strains), Acinetobacter (24 strains), Moraxella (6 strains), Chryseobacterium (5 strains), Myroides odoratus (2 strains), Flavobacterium (1 strain), Empedobacter (1 strain), and unidentified (3 strains). Overall similarities of all strains were determined for 108 characters by numerical analysis (simple matching coefficient of similarity [S] and clustering by unweighted pair group average linkage [UPGMA]). At the 77% similarity level, 92 strains formed nine major clusters (3 or more strains) and four small clusters (2 strains). Cluster 1 (25 isolates divided into two main subclusters) could be assigned to Psychrobacter phenylpyruvicus, clusters 2 and 3 (26 isolates) were designated as Psychrobacter immobilis, and clusters 4 (3 isolates) and 7 (4 isolates) were identified as Psychrobacter urativorans and Psychrobacter spp., respectively. Clusters 5 (five isolates), 6 (three isolates), and 9 (five isolates) were labeled as Acinetobacter spp., Acinetobacter johnsonii, and Acinetobacter lwoffii, respectively. Cluster 8 (12 isolates), with a high resemblance to Thornley's phenon 4 (a heterogeneous group of bacteria isolated from poultry and related to Acinetobacter), remained unnamed. The restriction pattern was identical for strains grouped into clusters 2 and 3 (P. immobilis) but was different for the remaining Psychrobacter isolates. A large proportion of isolates belonging to the family Moraxellaceae were closely related. Psychrobacters and A. johnsonii were present in freshly caught fish and river water. In the latter stages of storage, P. phenylpyruvicus and acinetobacters tended to decrease, whereas P. immobilis increased.

Hemolytic activity and siderophore production in different Aeromonas species isolated from fish.

The hemolytic activity and siderophore production of several strains of motile aeromonads were determined. The hemolytic activity of Aeromonas caviae and Aeromonas eucrenophila was enhanced after trypsinization of the samples. The enhancement of hemolysis was observed in strains that carried an aerolysin-like gene, detected by a PCR procedure. Siderophore production was demonstrated in all but one strain of Aeromonas jandaei. No apparent relationship was observed between the presence of plasmid DNA and hemolysis or siderophore production.

Bacterial microflora of wild brown trout (Salmo trutta), wild pike (Esox lucius), and aquacultured rainbow trout (Oncorhynchus mykiss).

Initial numbers of bacteria associated with wild (brown trout and pike) and cultured (rainbow trout) freshwater fish as well as with the water in which they were caught were determined. Subsequently, a total of 979 randomly selected isolates were characterized and identified to the genus level. For all counts performed (aerobes, psychrotrophs, anaerobes, Enterobacteriaceae, and enterococci), no significant differences were observed in water samples, the highest level corresponding to psychrotrophs in pike environments (4.23 X 10(3) CFU/ml). Overall, the skin and intestinal content of brown trout were the most contaminated, while rainbow trout specimens (gills and gut) yielded the lowest numbers. For all bacterial groups, pike gills had the highest numbers. Counts for all of the sampling sites compare well with findings in other temperate geographical environments. Biological characteristics (feeding and skin properties) and the use of antimicrobials in aquaculture might have influenced these results. Motile and nonmotile aerobic gram-negative bacteria together with Enterobacteriaceae accounted for 50 to 70% of the psychrotrophs isolated from water. Micrococcaceae, lactic acid bacteria, Bacillus, and coryneforms were also found. The groups represented in psychrotrophic isolates from the outer surfaces do not reflected those detected in water, so it was common that those organisms recovered in significant numbers from fish were not detected in surrounding habitat of the fish. Motile aeromonads and Carnobacterium were the dominant psychrotrophs in the guts of pike and brown trout, respectively. The intestinal content of reared fish gave a high incidence of Bacillus and coryneforms, while Enterobacteriaceae was absent. Again, rearing practices could have influenced this finding. Listeria monocytogenes was not detected in any of the examined samples. Two strains of Salmonella, which belonged to the same serovar and lysotype, were recovered from pond-water samples taken from one facility on different sampling days. From the gut of a pike specimen and from the pike's environment, two Plesiomonas shigelloides strains of different serovars were recovered. These latter four strains were resistant to a considerable number of antimicrobial compounds (multiple antibiotic resistance indices > 0.2).