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.

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 ¼ 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.

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.

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.

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: