Microbiological analysis of skin lesions of cod (Gadus morhua) from the southern part of the Baltic Sea.

Introduction: Since the middle of the 1980s, severe skin disorders have been observed in Baltic cod (Gadus morhua) each year. Available data on the spectrum of bacteria isolated from the clinical cases being limited, and evaluation of the microbial background of fish skin lesions being useful, a bacteriological examination has been undertaken. Material and Methods: A total of 1,381 cod were caught during two voyages of the Baltica research vessel in the Polish exclusive economic zone of the southern Baltic Sea. After an examination which found lesions in 164 of the fish, a microbiological analysis was performed to isolate bacteria from them. The collected strains were phenotyped and genotyped, and their antimicrobial resistance was analysed by minimum inhibitory concentration (MIC) techniques. Results: Bacteriological examinations provided 850 isolates. The dominant microorganisms were mesophilic Aeromonas spp., Pseudomonas spp. and Shewanella baltica. Opportunistic bacteria potentially hazardous to human health were also isolated, e.g. Alcaligenes faecalis, Staphylococcus epidermidis, Stenotrophomonas maltophilia and Vibrio sp. The MIC analysis determined the highest number of bacteria to resist sulphamethoxazole and amoxicillin and clavulanic acid. Conclusion: Most of the collected bacteria were opportunistic pathogens for fish, widespread in the aquatic environment, and potentially threatening to humans.

Immunochemical studies and gene cluster relationships of closely related O-antigens of Aeromonas hydrophila Pt679, Aeromonas popoffii A4, and Aeromonas sobria K928 strains classified into the PGO1 serogroup dominant in Polish aquaculture of carp and rainbow trout.

The present study included three Aeromonas sp. strains isolated from fish tissues during Motile Aeromonas Infection/Motile Aeromonas Septicaemia disease outbreaks on commercial farms, i.e.: Aeromonas hydrophila Pt679 obtained from rainbow trout as well as Aeromonas popoffii A4 (formerly Aeromonas encheleia) and Aeromonas sobria K928 both isolated from carp, which were classified into the new provisional PGO1 serogroup prevailing among aeromonads in Polish aquaculture. The structure of the O-specific polysaccharides of A4 and K928 has been previously established. Here, immunochemical studies of the O-specific polysaccharide of A. hydrophila Pt679 were undertaken. The O-specific polysaccharide was obtained from the lipopolysaccharide of A. hydrophila Pt679 after mild acid hydrolysis and separation by gel-permeation chromatography. The high-molecular-mass fraction was studied using chemical methods and 1H and 13C NMR spectroscopy, including 1H,1H NOESY, and 1H,13C HMBC experiments. The following structure of the branched repeating unit of the O-polysaccharide from A. hydrophila Pt679 was determined: [Formula: see text] The studies indicated that O-polysaccharides from A. hydrophila Pt679, A. popoffii A4 and A. sobria K928 share similarities but they also contain unique characteristics. Western blotting and an enzyme-linked immunosorbent assay revealed that the cross-reactivity of the related O-antigens is caused by the occurrence of common structural elements, whereas additional epitopes define the specificity of the O-serotypes. For genetic relationship studies, the O-antigen gene cluster was characterized in the genome of the A. hydrophila Pt679 strain and compared with the corresponding sequences of A. popoffii A4 and A. sobria K928 and with sequences available in the databases. The composition of the regions was found to be consistent with the O-antigen structures of Aeromonas strains classified into the same PGO1 serogroup.

Phylogenetic analysis of Shewanella spp. isolated from fish.

Different species of Shewanella spp. widely inhabit freshwater and marine environments. Some of them are opportunistic fish pathogens. The application of high-throughput sequencing enabled the characterization and taxonomic reclassification of many Shewanella spp. species. Still, some strains collected from fish need to be better recognized. The aim of the present study was to classify and determine the phylogenetic relationships of Shewanella spp. collected from fish. The complete genomes of 94 strains of Shewanella spp. from different fish species were sequenced using Illumina platform (MiSeq). The 16S rRNA gene, genomic features and whole-genome relationships of those bacteria were comprehensively analysed in comparison to reference strains. Whole-genome analysis showed that the tested Shewanella spp. strains were clustered into six groups similar to reference strains of S. xiamenensis, S. oneidensis, S. glacialipiscicola, S. hafniensis, S. baltica and S. oncorhynchi. Our study indicates that the whole-genome sequence analysis enabled taxonomic classification and assessment of the diversity of the Shewanella spp. strains, as opposed to recently the gold standard method of 16S rRNA amplicon sequencing. The high genetic diversity and low similarity to the reference genome of S. oneidensis indicate that the group of strains may be a subspecies or even new species. Furthermore, we showed that the most frequent Shewanella spp. species occurring in freshwater fish in our study is the recently described species S. oncorhynchi.

Structure and gene cluster annotation of the O-antigen of Aeromonas sobria strain K928 isolated from common carp and classified into the new Aeromonas PGO1 serogroup.

Aeromonas sobria strain K928 was isolated from a common carp during a Motile Aeromonas Infection/Motile Aeromonas Septicaemia disease outbreak on a Polish fish farm and classified into the new provisional PGO1 serogroup. The lipopolysaccharide of A. sobria K928 was subjected to mild acid hydrolysis, and the O-specific polysaccharide, which was isolated by gel-permeation chromatography, was studied using sugar and methylation analyses and 1H and 13C NMR spectroscopy. The following structure of the branched O-specific polysaccharide repeating unit of A. sobria K928 was established. →2)[α-D-Fucp3NRHb-(1→3)]-α-L-Rhap-(1→3)-ß-L-Rhap-(1→4)-α-L-Rhap-(1→3)-ß-D-FucpNAc-(1→ The O-antigen gene cluster was identified and characterized in the genome of the A. sobria K928 strain after comparison with sequences in the available databases. The composition of the O-antigen genetic region was found to be consistent with the O-polysaccharide structure, and its organization was proposed.

Evaluation of Proteomic and Lipidomic Changes in Aeromonas-Infected Trout Kidney Tissue with the Use of FT-IR Spectroscopy and MALDI Mass Spectrometry Imaging.

Aeromonas species are opportunistic bacteria causing a vast spectrum of human diseases, including skin and soft tissue infections, meningitis, endocarditis, peritonitis, gastroenteritis, and finally hemorrhagic septicemia. The aim of our research was to indicate the molecular alterations in proteins and lipids profiles resulting from Aeromonas sobria and A. salmonicida subsp. salmonicida infection in trout kidney tissue samples. We successfully applied FT-IR (Fourier transform infrared) spectroscopy and MALDI-MSI (matrix-assisted laser desorption/ionization mass spectrometry imaging) to monitor changes in the structure and compositions of lipids, secondary conformation of proteins, and provide useful information concerning disease progression. Our findings indicate that the following spectral bands' absorbance ratios (spectral biomarkers) can be used to discriminate healthy tissue from pathologically altered tissue, for example, lipids (CH2/CH3), amide I/amide II, amide I/CH2 and amide I/CH3. Spectral data obtained from 10 single measurements of each specimen indicate numerous abnormalities concerning proteins, lipids, and phospholipids induced by Aeromonas infection, suggesting significant disruption of the cell membranes. Moreover, the increase in the content of lysolipids such as lysophosphosphatidylcholine was observed. The results of this study suggest the application of both methods MALDI-MSI and FT-IR as accurate methods for profiling biomolecules and identifying biochemical changes in kidney tissue during the progression of Aeromonas infection.

Studies on The Inhibitory Properties of Leaf Ethanolic Extracts Obtained from Ficus (Moraceae) Species Against Aeromonas Spp. Strains.

INTRODUCTION: The most frequently isolated bacteria in Polish aquaculture are of the Aeromonas genus; also pathogenic to human fish consumers, they cause substantial economic losses, and require antibiotic therapy to treat. Antibiotic residues in animal-derived food provoke concern. The aim of the study was to appraise the antimicrobial activity of ethanolic extracts of Ficus plant species against Aeromonas strains. MATERIAL AND METHODS: Leaves of 41 Ficus species were collected from two Ukrainian botanic gardens. They were crushed, washed, homogenized in ethanol and centrifuged, and the supernatants were applied in the Kirby-Bauer disc-diffusion method to assess the susceptibility to them of Aeromonas hydrophila, A. sobria, and A. salmonicia subsp. salmonicida isolates confirmed as K886, K825, and St30 strains. Analogous assessment was also made of these bacteria's susceptibility to sulfonamides, quinolones, tetracyclines, and one amphenicol. Data were analysed statistically. RESULTS: The majority of the extracts considerably inhibited bacterial growth, A. sobria being susceptible to 14 Ficus species, A. salmonicida subsp. salmonicida to 13, and A. hydrophila to 10. CONCLUSION: Treatment with plant extracts has promise as an alternative to antibiotic therapy. Botanic gardens may offer new sources of plant-derived agents with a broad spectrum of biological and antimicrobial action. Further research will be useful to broaden knowledge of Ficus' therapeutic potential.

Structural Studies of the Lipopolysaccharide of Aeromonas veronii bv. sobria Strain K133 Which Represents New Provisional Serogroup PGO1 Prevailing among Mesophilic Aeromonads on Polish Fish Farms.

In the present work, we performed immunochemical studies of LPS, especially the O-specific polysaccharide (O-PS) of Aeromonas veronii bv. sobria strain K133, which was isolated from the kidney of carp (Cyprinus carpio L.) during an outbreak of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) on a Polish fish farm. The structural characterization of the O-PS, which was obtained by mild acid degradation of the LPS, was performed with chemical methods, MALDI-TOF mass spectrometry, and 1H and 13C NMR spectroscopy. It was revealed that the O-PS has a unique composition of a linear tetrasaccharide repeating unit and contains a rarely occurring sugar 2,4-diamino-2,4,6-trideoxy-D-glucose (bacillosamine), which may determine the specificity of the serogroup. Western blotting and ELISA confirmed that A. veronii bv. sobria strain K133 belongs to the new serogroup PGO1, which is one of the most commonly represented immunotypes among carp and trout isolates of Aeromonas sp. in Polish aquacultures. Considering the increase in the MAI/MAS incidences and their impact on freshwater species, also with economic importance, and in the absence of an effective immunoprophylaxis, studies of the Aeromonas O-antigens are relevant in the light of epidemiological data and monitoring emergent pathogens representing unknown antigenic variants and serotypes.

Structure of the disaccharide repeating unit of O-specific polysaccharide isolated from Aeromonas veronii strain Bs8 pathogenic to common carp (Cyprinus carpio).

The O-specific polysaccharide (OPS) was isolated from the lipopolysaccharide of Aeromonas veronii strain Bs8, which is pathogenic to common carp (Cyprinus carpio), after mild acid hydrolysis followed by gel-permeation chromatography. The high-molecular-mass OPS fraction was investigated using chemical methods, mass spectrometry, and 1H and 13C NMR spectroscopy techniques, including 2D homonuclear 1H,1H TOCSY, DQF COSY, NOESY, and heteronuclear 1H-detected 1H,13C HSQC, and HMBC experiments. The analysis revealed that the O-specific polysaccharide contains sugars with the galacto configuration of the ring and is composed of a disaccharide repeating unit with the following structure.

17α-ethinylestradiol and 4-tert-octylphenol concurrently disrupt the immune response of common carp.

The aquatic environment is massively polluted with endocrine-disrupting compounds (EDCs) including synthetic estrogens (e.g. 17α-ethinylestradiol, EE2) and alkylphenols (e.g. 4-tert-octylphenol, 4t-OP). A major mechanism of action for estrogenic EDCs is their interaction with estrogen receptors and consequently their modulation of the action of enzymes involved in steroid conversion e.g. aromatase CYP19. We now studied the effects of EE2 and 4t-OP on the anti-bacterial immune response of common carp. We investigated effects on the number/composition of inflammatory leukocytes and on the gene expression of mediators that regulate inflammation and EDC binding. In vitro we found that high concentrations of both EE2 and 4t-OP down-regulated IFN-γ2 and IFN-γ-dependent immune responses in LPS-stimulated monocytes/macrophages. Similarly, during bacterial infection in fish, in vivo treated with EE2 and 4t-OP, decreased gene expression of il-12p35 and of ifn-γ2 was found in the focus of inflammation. Moreover, during A. salmonicida-induced infection in EE2-treated carp, but not in fish fed with 4t-OP-treated food, we found an enhanced inflammatory reaction manifested by high number of inflammatory peritoneal leukocytes, including phagocytes and higher expression of pro-inflammatory mediators (inos, il-1ß, cxcl8_l2). Furthermore, in the liver, EE2 down-regulated the expression of acute phase proteins: CRPs and C3. Importantly, both in vitro and in vivo, EDCs altered the expression of estrogen receptors: nuclear (erα and erß) and membrane (gpr30). EDCs also induced up-regulation of the cyp19b gene. Our findings reveal that contamination of the aquatic milieu with estrogenic EDCs, may considerably violate the subtle and particular allostatic interactions between the immune response and endogenous estrogens and this may have negative consequences for fish health.

17ß-Estradiol affects the innate immune response in common carp.

Inflammation is the evolutionary conserved immune response to harmful stimuli such as pathogens or damaged cells. This multistep process acts by removing injurious stimuli and initiating the healing process. Therefore, it must be tightly regulated by cytokines, chemokines, and enzymes, as well as neuroendocrine mediators. In the present work, we studied the immunoregulatory properties of 17ß-estradiol (E2) in common carp. We determined the in vitro effects of E2 on the activity/polarization of macrophages and the in vivo effects during Aeromonas salmonicida-induced inflammation. In vitro, E2 reduced the lipopolysaccharide (LPS)-stimulated expression of pro- and anti-inflammatory mediator genes but did not change the gene expression of the estrogen receptors and of aromatase CYP19. In contrast, in vivo in the head kidney of A. salmonicida-infected fish, E2-treated feeding induced an upregulation of gene expression of pro-inflammatory (il-12p35 and cxcb2) and anti-inflammatory (arginase 1, arginase 2, il-10, and mmp9) mediators. Moreover, in infected fish fed with E2-treated food, a higher gene expression of the estrogen receptors and of the aromatase CYP19 was found. Our results demonstrate that estrogens can modulate the carp innate immune response, though the in vitro and in vivo effects of this hormone are contrasting. This implies that estradiol not only induces a direct effect on macrophages but rather exerts immunomodulatory actions through indirect mechanisms involving other cellular targets.

Structure of the O-specific polysaccharide of Aeromonas veronii bv. sobria strain Pt393 isolated from rainbow trout (Oncorhynchus mykiss), which contains a rarely occurring sugar 4-acetamido-4,6-dideoxy-d-galactose, tomosamine.

The O-specific polysaccharide (OPS) was isolated from the lipopolysaccharide of Aeromonas veronii bv. sobria strain Pt393, which is pathogenic to the rainbow trout (Oncorhynchus mykiss), after mild acid hydrolysis followed by GPC. The high-molecular-weight OPS fraction was studied with chemical methods, mass spectrometry, and 1H and 13C NMR spectroscopy techniques, including 2D 1H,1H COSY, TOCSY, NOESY, 1H-detected heteronuclear 1H,13C HSQC, and HMBC experiments. It was found that the O-specific polysaccharide was built of a tetrasaccharide repeating unit composed of α-GalpNAc, α-FucpNAc, ß-QuipNAc, and α-Fucp4NAc (4-acetamido-4,6-dideoxy-d-galactose, tomosamine) residues. The following structure of the OPS of A. sobria strain Pt393 was established: →4)-α-d-GalpNAc-(1 â†’ 3)-α-l-FucpNAc-(1 â†’ 3)-ß-d-QuipNAc-(1 â†’ 3)-α-d-Fucp4NAc-(1→.

Could mycotoxigenic Fusarium sp. play a role in ulcerative dermal necrosis (UDN) of brown trout (Salmo trutta morpha trutta)?

Fusarium infections have been reported in aquatic animals, but are still poorly investigated in wild salmonids. The aim of the study was to determine the impact of the fungi and their toxins on the health status of brown trout (Salmo trutta morpha trutta) migrating from the Baltic Sea to the freshwater. Individuals from the wild brown trout population exhibiting ulcerative skin lesions were collected from the Slupia River in Poland and subjected to microbiological, histopathological, and hematological examinations, as well as toxicological analysis for a presence of mycotoxins. The results of microflora isolation from the brown trout skin samples revealed the presence of conditionally pathogenic bacteria and fungi classified by molecular techniques as Fusarium spp. Toxicological analysis allowed for detection of zearalenone (ZEN) in the liver, kidney, and gastrointestinal tract of the fish. In several cases, there was α-zearalenone (α-ZEL) identified at trace levels in the liver, as well as sterigmatocystin and enniatin B at low levels in the kidney and the liver. Histopathological examination revealed the presence of fungal hyphae disrupting the epidermis and penetrating into the necrotic dermis and hypodermis. The decreased values of the blood parameters, i.e., hemoglobin concentration (HGB), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and white blood cell count (WBC), were indicative of osmoregulation failure being a consequence of the skin damage. The results of the study provide new information regarding Fusarium sp. infection in brown trout and serve as the basis for further research on the potential impact of the fungi and their mycotoxins on the Baltic salmonid population, including their role in ulcerative dermal necrosis.

Phenotypic Diversity and Potential Virulence Factors of the Shewanella Putrefaciens Group Isolated from Freshwater Fish.

INTRODUCTION: The Shewanella putrefaciens group are ubiquitous microorganisms recently isolated from different freshwater fish species and causing serious health disorders. The purpose of the study was to characterise isolates of the S. putrefaciens group with special emphasis on elucidating serological diversity and determining putative virulence factors. MATERIAL AND METHODS: Isolates collected from freshwater fish (n = 44) and reference strains were used. The identification of bacteria was carried out using biochemical kits and 16S rRNA sequencing. Polyclonal antibodies were prepared against the S. putrefaciens group. The bacterium's susceptibility to antimicrobial agents, its enzymatic properties, and its adhesion ability to fish cell lines were also tested. Finally, selected isolates were used in challenge experiments in common carp and rainbow trout. RESULTS: Excluding six isolates undeterminable for species, the bacteria were classified to three species: S. putrefaciens, S. xiamenensis, and S. oneidensis, and showed some phenotypic diversity. Fourteen serological variants of the S. putrefaciens group were determined with the newly developed serotyping scheme. CONCLUSION: Serodiversity may play an important role in the virulence of particular isolates. Further, S. putrefaciens group members adhere to epithelial cells and produce enzymes which may contribute to their virulence. Challenge tests confirmed the pathogenicity of the S. putrefaciens group for fish.

Antibacterial Activity of Commercial Phytochemicals against Aeromonas Species Isolated from Fish.

Antimicrobial activities of phytochemicals-trans-cinnamaldehyde (TC), ferulic acid (FA), p-coumaric acid (p-CA), caffeic acid (CA), chlorogenic acid (CHA), Thymus vulgaris essential oil (TO), Eugenia caryophyllus essential oil (ECO), and Melaleuca alternifolia oil (TTO) against Aeromonas species-were assessed. Growth of all Aeromonas salmonicida subsp. salmonicida and almost all Aeromonas sobria strains was inhibited by TC at concentration 0.01 mg/mL, and for most Aeromonas hydrophila strains minimal inhibitory concentrations (MIC) ranged from 0.01 to 0.19 mg/mL. The inhibitory effect of TC against A. salmonicida subsp. salmonicida was comparable to the effect of oxytetracycline, and in the case of A. salmonicida subsp. salmonicida and A. sobria was higher compared to gentamicin. MIC of FA, p-CA, and CA for most strains ranged from 1.56 to 3.12 mg/mL, and MIC values of TO for most strains ranged from 0.39 to 0.78 mg/mL. TO and TC at the concentrations below ½ MIC values used in mixtures exhibited strong synergism. ECO and TC showed synergy in mixture of ⅛ MIC of ECO and » MIC of TC. TC and TO exhibited the strongest inhibitory and bactericidal effect against investigated Aeromonas species, and they are a promising alternative to the use of antibiotics in controlling the growth of these fish pathogens.

Structural and Serological Studies of the O6-Related Antigen of Aeromonas veronii bv. sobria Strain K557 Isolated from Cyprinus carpio on a Polish Fish Farm, which Contains L-perosamine (4-amino-4,6-dideoxy-L-mannose), a Unique Sugar Characteristic for Aeromonas Serogroup O6.

Amongst Aeromonas spp. strains that are pathogenic to fish in Polish aquacultures, serogroup O6 was one of the five most commonly identified immunotypes especially among carp isolates. Here, we report immunochemical studies of the lipopolysaccharide (LPS) including the O-specific polysaccharide (O-antigen) of A. veronii bv. sobria strain K557, serogroup O6, isolated from a common carp during an outbreak of motile aeromonad septicemia (MAS) on a Polish fish farm. The O-polysaccharide was obtained by mild acid degradation of the LPS and studied by chemical analyses, mass spectrometry, and 1H and 13C NMR spectroscopy. It was revealed that the O-antigen was composed of two O-polysaccharides, both containing a unique sugar 4-amino-4,6-dideoxy-L-mannose (N-acetyl-L-perosamine, L-Rhap4NAc). The following structures of the O-polysaccharides (O-PS 1 and O-PS 2) were established.

A Unique Sugar l-Perosamine (4-Amino-4,6-dideoxy-l-mannose) Is a Compound Building Two O-Chain Polysaccharides in the Lipopolysaccharide of Aeromonas hydrophila Strain JCM 3968, Serogroup O6.

Lipopolysaccharide (LPS) is the major glycolipid and virulence factor of Gram-negative bacteria, including Aeromonas spp. The O-specific polysaccharide (O-PS, O-chain, O-antigen), i.e., the surface-exposed part of LPS, which is a hetero- or homopolysaccharide, determines the serospecificity of bacterial strains. Here, chemical analyses, mass spectrometry, and 1H and 13C NMR spectroscopy techniques were employed to study the O-PS of Aeromonas hydrophila strain JCM 3968, serogroup O6. MALDI-TOF mass spectrometry revealed that the LPS of A. hydrophila JCM 3968 has a hexaacylated lipid A with conserved architecture of the backbone and a core oligosaccharide composed of Hep6Hex1HexN1HexNAc1Kdo1P1. To liberate the O-antigen, LPS was subjected to mild acid hydrolysis followed by gel-permeation-chromatography and revealed two O-polysaccharides that were found to contain a unique sugar 4-amino-4,6-dideoxy-l-mannose (N-acetyl-l-perosamine, l-Rhap4NAc), which may further determine the specificity of the serogroup. The first O-polysaccharide (O-PS1) was built up of trisaccharide repeating units composed of one α-d-GalpNAc and two α-l-Rhap4NAc residues, whereas the other one, O-PS2, is an α1→2 linked homopolymer of l-Rhap4NAc. The following structures of the O-polysaccharides were established: O-PS1 →3)-α-l-Rhap4NAc-(1→4)-α-d-GalpNAc-(1→3)-α-l-Rhap4NAc-(1→ O-PS2 →2)-α-l-Rhap4NAc-(1→ The present paper is the first work that reveals the occurrence of perosamine in the l-configuration as a component of bacterial O-chain polysaccharides.

Genotypic diversity among Shewanella spp. collected from freshwater fish.

Different Shewanella species are isolated both from healthy and from diseased fish. To date, contemporary methods do not provide sufficient insight to determine species and detail differentiation between tested strains. Bacteria isolated from cultured (n = 33), wild (n = 12) and ornamental (n = 6) fish, as well as several reference strains, were tested by 16S rRNA gene sequencing, ERIC-PCR and pulsed-field gel electrophoresis (PFGE) assays. Our study indicates that isolates collected from freshwater fish were genetically diverse. Based on 16S rRNA gene sequences, bacteria were clustered into groups S. putrefaciens, S. xiamenensis and S. oneidensis. Some isolates were classified only to genus Shewanella; thus, 16S rRNA gene analyses were not enough to determine the species. ERIC-PCR revealed 49 different genotype profiles indicating that the method might be useful for differentiation of Shewanella isolates irrespectively to species identification, contrary to PFGE which is not suitable for Shewanella typing.

Contemporary Threats of Bacterial Infections in Freshwater Fish.

Changes occurring in freshwater ecosystems seem to be fundamental in the development of all microorganisms, including those pathogenic to fish. This has been especially evident in recent years during which dynamic variations in bacterial fish pathology have been observed. Gram-negative bacteria commonly known to be pathogenic to fish, like Aeromonas spp., Flavobacterium spp., Pseudomonas spp., and Shewanella putrefaciens are replaced by other species, which until now have not been known to be virulent or even conditionally pathogenic to fish. Nowadays, among these other species Acinetobacter spp., Plesiomonas shigelloides, Sphingomonas paucimobilis, and Stenotrophomonas maltophilia are the most frequently isolated from fish exhibiting clinical signs of disease. Two Gram-positive bacteria have become pathogens of particular importance in fish pathology in Poland: Lactococcus garviae and Streptococcus iniae. In addition, infections caused by the Gram-positive bacterium Kocuria rhizophila have appeared in recent years. This bacterium has not been known until now to be pathogenic to fish. Therefore, this infection could be called an emergent disease.