Yersinia ruckeri is the cause of hemorrhagic septicemia, known as enteric redmouth disease, in salmonid fish species. This bacterial pathogen can form biofilms on abiotic surfaces of aquaculture settings or even on the surfaces of the fish themselves, contributing to their persistence in the aquatic environment. Detection methods for this and other fish pathogens can be time-consuming and lack specificity and sensitivity, limiting timely monitoring, the treatment of microbial infections, and effective control of their transmission in aquaculture settings. Rapid and sensitive detection methods for nucleic acids can be crucial for an appropriate surveillance of bacterial pathogens, and the CRISPR/Cas-based assays have emerged as a good alternative since it has been proven to be a useful tool for the rapid, specific, and sensitive detection of viruses and some bacteria. In this study, we explored the capability of the CRISPR/Cas13a system (SHERLOCK) to specifically detect both DNA and RNA (gene transcripts) from planktonic and biofilm samples of the bacterial fish pathogen Y. ruckeri. The assay was designed to detect the gyrA gene and the small noncoding RNAs (sRNAs) MicA and RprA from planktonic cultures and biofilm samples prepared in marine broth. The specific crRNA designed for these gene targets included a 28 nt specific gene sequence, and a scaffold sequence necessary for Cas13-binding. For all the assays, the nucleic acids obtained from samples were previously subjected to isothermal amplification with the recombinase polymerase amplification (RPA) method and the subsequent T7 transcription of the RPA amplicons. Finally, the detection of nucleic acids of Y. ruckeri was by means of a reporter signal released by the Cas13a collateral RNA cleavage triggered upon target recognition, measured by fluorescence- or lateral-flow-based readouts. This CRISPR/Cas13a-based assay was able to specifically detect both DNA and sRNAs from the Y. ruckeri samples, and the sensitivity was comparable to that obtained with qPCR analysis, highlighting the potential applicability of this CRISPR/Cas13a-based assay for fish pathogen surveillance.

The fish pathogen Yersinia ruckeri forms biofilms on abiotic surfaces, contributing to recurrent infections in aquaculture. Increasing evidence suggests that the RNA chaperone Hfq and small non-coding RNAs (sRNAs) are key regulators of bacterial biofilm formation. However, the regulatory mechanisms mediated by these factors remain largely unexplored in Y. ruckeri. In this study, we investigated the roles of Hfq and the Hfq-dependent sRNAs RprA, ArcZ, and RybB in the biofilm formation of Y. ruckeri. We first characterized the sRNAome of biofilm-forming cells, identifying the conserved RprA, ArcZ, and RybB, among the upregulated sRNAs. We then evaluated motility, biofilm formation, and architecture in strains lacking either hfq (Δhfq) or these sRNAs (ΔsRNA). Our results reveal that both Δhfq and ΔsRNA strains exhibit significant alterations in biofilm and motility phenotypes, including changes in bacterial morphology and extracellular matrix. Furthermore, expression analyses indicate that these sRNAs modulate the transcription of key regulatory factors, flagellar and phosphodiesterase genes, ultimately influencing intracellular cyclic di-GMP levels, a key second messenger in biofilm formation. Together, our findings demonstrate that Hfq and its associated sRNAs play critical regulatory roles in Y. ruckeri biofilm formation by controlling the expression of genes involved in motility, bacterial envelope proteins, and c-di-GMP metabolism.

Biofilm formation by bacterial pathogens represents a major challenge in aquaculture, since biofilms promote persistence and increase tolerance to antimicrobial treatments. Yersinia ruckeri, the etiological agent of enteric redmouth disease in salmonids, contributes to recurrent infections in aquaculture. However, the molecular mechanisms governing biofilm development in this pathogen remain poorly understood. In this study, we investigated the role of the global transcriptional regulator Fur in the biofilm formation of Y. ruckeri. To this aim, a fur deletion mutant (Δfur) was phenotypically, structurally, and transcriptionally characterized under biofilm-forming conditions. Fur deletion resulted in impaired motility, reduced flagellar synthesis, and a marked decrease in mature biofilm formation. These changes were accompanied by alterations in three-dimensional architecture, cellular organization, and extracellular matrix production, as revealed by scanning electron and confocal microscopy analyses. Furthermore, transcriptomic profiling via RNA-seq demonstrated that the absence of Fur leads to extensive transcriptional reprogramming. Genes associated with the biosynthesis of flagella and oxidative stress response were significantly down-regulated, whereas genes associated with SOS responses, non-ribosomal peptide biosynthesis, and those related to iron acquisition and siderophore systems exhibited significant up-regulation in the absence of Fur. Interestingly, the strong up-regulation of P2-type prophage genes was also observed in the Δfur strain. Collectively, our findings identify Fur as an important contributor to the normal progression toward mature and spatially organized Y. ruckeri biofilms. Likewise, the Fur-dependent pathways and factors identified here provide candidates for future mechanistic studies and may ultimately help identify strategies to interfere with Y. ruckeri persistence in aquaculture environments.