Does the freshwater mussel Anodonta anatina remove the fish pathogen Flavobacterium columnare from water?

Global decline of freshwater mussels (Unionoida) is threatening biodiversity and the essential ecosystem services that mussels provide. As filter-feeding organisms, freshwater mussels remove phytoplankton and suspended particles from the water. By filtering bacteria, freshwater mussels also decrease pathogen loads in the water. The objective of this study was to evaluate whether the common freshwater bivalve Anodonta anatina (duck mussel) could remove the bacterial fish pathogen Flavobacterium columnare from the water. Mussels reduced bacteria in both of the two experiments performed, so that the bacterial concentration at the end of the 96-h monitoring in mussel treatments was only 0.3–0.5 times that of the controls. Surprisingly, mussels did not reduce algal cell concentration statistically significantly. Mussel behavior (shell openness, foot position, and movement) was not affected by the presence of bacteria or algae, except for biodeposition formation, which was greatest in algal-fed treatments, followed by bacterial-fed treatments and controls, respectively. The intestines of bacteria-incubated A. anatina harbored F. columnare, suggesting that mussels ingested the bacteria. Present results suggest that freshwater mussels may also have a potential to mitigate aquaculture pathogen problems, as well as play a role in water quality management.

LuxS modulates motility and secretion of extracellular protease in fish pathogen Vibrio harveyi

In this study, an in-frame deletion of the luxS gene was constructed to reveal the role of LuxS in the physiology and virulence of V. harveyi. The statistical analysis showed no significant differences in the growth ability, biofilm formation, antibiotic susceptibility, virulence by intraperitoneal injection, and the ability of V. harveyi to colonize the spleen and liver of the pearl gentian grouper between the wild-type (WT) and the luxS mutant. However, the deletion of luxS decreased the secretion of extracellular protease, while increased the ability of swimming and swarming. Simultaneously, a luxS-deleted mutant showed overproduction of lateral flagella, and an intact luxS complemented the defect. Since motility is flagella dependent, 16 of V. harveyi flagella biogenesis related genes were selected for further analysis. Based on quantitative real-time reverse transcription-PCR (qRT-PCR), the expression levels of these genes, including the polar flagella genes flaB, flhA, flhF, flhB, flhF, fliS, and flrA and the lateral flagella genes flgA, flgB, fliE, fliF, lafA, lafK, and motY, were significantly up-regulated in the ΔluxS: pMMB207 (ΔluxS+) strain as compared with the V. harveyi 345: pMMB207 (WT+) and C-ΔluxS strains during the early, mid-exponential, and stationary growth phase.

Mucin induces CRISPR-Cas defence in an opportunistic pathogen

Parasitism by bacteriophages has led to the evolution of a variety of defense mechanisms in their host bacteria. However, it is unclear what factors lead to specific defenses being deployed upon phage infection. To explore this question, we exposed the bacterial fish pathogen Flavobacterium columnare to its virulent phage V156 in the presence of a eukaryotic host signal (mucin). All tested conditions led to some level of innate immunity, but the presence of mucin led to a dramatic increase in CRISPR spacer acquisition, especially in low nutrient conditions where over 60% of colonies had obtained at least one new spacer. Additionally, we show that the presence of a competitor bacterium further increases CRISPR spacer acquisition in F. columnare. These results suggest that ecological factors are important in determining defense strategies against phages, and that the concentration of phages on metazoan surfaces may select for the diversification of bacterial immune systems.

Potential Application of PCR Based Molecular Methods in Fish Pathogen Identification: A Review

Molecular biology developments have led to fast growth in new methods for fish disease diagnosis. Molecular diagnostic methods are rapid and more specific, more sensitive than the culture of pathogens, serology, histology, and biochemical methods which are traditionally utilized to identify causative agent fish disease. Molecular diagnostic methods are valuable for detecting specific pathogens that are difficult to culture in vitro or require a long cultivation period and it significantly more rapid in providing results compared to culture. It enables earlier informed decision-making and rapid diagnosis of bacteremia, particularly for low levels of bacteria in specimens. Molecular techniques which have the major significance are mainly PCR-based molecular diagnostic methods including Polymerase Chain Reaction (PCR), Real-Time Polymerase Chain Reaction (RT-PCR), Multiplex Polymerase Chain Reaction (multiplexPCR), and Random Amplified Polymorphic DNA (RAPD). These have been increasingly utilized to diagnose fish disease for the last recent years. Molecular diagnostic methods can detect pathogens from asymptomatic fish, so disease outbreaks could be prevented. As a consequence, antibiotic treatment can be reduced and the development of antibiotic-resistant bacteria can be eliminated. In this review paper, we attempt to summarize the potentiality of PCR-based molecular diagnostic methods and their application in fish pathogen identification.

The fish pathogen Aliivibrio salmonicida LFI1238 can degrade and metabolize chitin despite major gene loss in the chitinolytic pathway

The fish pathogen Aliivibrio (Vibrio) salmonicida LFI1238 is thought to be incapable of utilizing chitin as a nutrient source since approximately half of the genes representing the chitinolytic pathway are disrupted by insertion sequences. In the present study, we combined a broad set of analytical methods to investigate this hypothesis. Cultivation studies revealed that Al. salmonicida grew efficiently on N -acetylglucosamine (GlcNAc) and chitobiose ((GlcNAc) 2 ), the primary soluble products resulting from enzymatic chitin hydrolysis. The bacterium was also able to grow on chitin particles, albeit at a lower rate compared to the soluble substrates. The genome of the bacterium contains five disrupted chitinase genes (pseudogenes) and three intact genes encoding a glycoside hydrolase family 18 (GH18) chitinase and two auxiliary activity family 10 (AA10) lytic polysaccharide monooxygenases (LPMOs). Biochemical characterization showed that the chitinase and LPMOs were able to depolymerize both α- and β-chitin to (GlcNAc) 2 and oxidized chitooligosaccharides, respectively. Notably, the chitinase displayed up to 50-fold lower activity compared to other well-studied chitinases. Deletion of the genes encoding the intact chitinolytic enzymes showed that the chitinase was important for growth on β-chitin, whereas the LPMO gene-deletion variants only showed minor growth defects on this substrate. Finally, proteomic analysis of Al. salmonicida LFI1238 growth on β-chitin showed expression of all three chitinolytic enzymes, and intriguingly also three of the disrupted chitinases. In conclusion, our results show that Al. salmonicida LFI1238 can utilize chitin as a nutrient source and that the GH18 chitinase and the two LPMOs are needed for this ability. IMPORTANCE The ability to utilize chitin as a source of nutrients is important for the survival and spread of marine microbial pathogens in the environment. One such pathogen is Aliivibrio (Vibrio) salmonicida , the causative agent of cold water vibriosis. Due to extensive gene decay, many key enzymes in the chitinolytic pathway have been disrupted, putatively rendering this bacterium incapable of chitin degradation and utilization. In the present study we demonstrate that Al. salmonicida can degrade and metabolize chitin, the most abundant biopolymer in the ocean. Our findings shed new light on the environmental adaption of this fish pathogen.

Proteome analysis of the Gram-positive fish pathogen Renibacterium salmoninarum reveals putative role of membrane vesicles in virulence

The bacterial kidney disease (BKD) is a chronic bacterial disease affecting both wild and farmed salmonids. The causative agent for BKD is the Gram-positive fish pathogen Renibacterium salmoninarum. As treatment and prevention of BKD has proven to be difficult, it is important to know and identify the key bacterial proteins that interact with the host. We used subcellular fractionation to report semi-quantitative data for the cytosolic, membrane, extracellular and membrane vesicle (MV) proteome of R. salmoninarum. These data can aid as a backbone for more targeted experiments regarding the development of new drugs for the treatment of BKD. Further analysis was focused on the MV proteome, where both major immunosuppressive proteins P57/Msa and P22 and proteins involved in bacterial adhesion were found in high abundance. Interestingly, the P22 protein was enriched only in the extracellular and MV fraction, implicating that MVs may play a role in host-pathogen interaction. Compared to the other subcellular fractions, the MVs were also enriched in lipoproteins and all four cell wall hydrolases belonging to the New Lipoprotein C/Protein of 60 kDa (NlpC/P60) family were detected, suggesting an involvement in the formation of the MVs.