Transforming the untransformable with knockout minicircles: High-efficiency transformation and vector-free allelic exchange knockout in the fish pathogen Photobacterium damselae.

Gene inactivation studies are critical in pathogenic bacteria, where insights into species biology can guide the development of vaccines and treatments. Allelic exchange via homologous recombination is a generic method of targeted gene editing in bacteria. However, generally applicable protocols are lacking, and suboptimal approaches are often used for nonstandard but epidemiologically important species. Photobacterium damselae subsp. piscicida (Pdp) is a primary pathogen of fish in aquaculture and has been considered hard to transform since the mid-1990s. Consequently, conjugative transfer of RK2/RP4 suicide vectors from Escherichia coli S17-1/SM10 donor strains, a system prone to off-target mutagenesis, was used to deliver the allelic exchange DNA in previous studies. Here we have achieved efficient electrotransformation in Pdp using a salt-free highly concentrated sucrose solution, which performs as a hypertonic wash buffer, cryoprotectant, and electroporation buffer. High-efficiency transformation has enabled vector-free mutagenesis for which we have employed circular minimalistic constructs (knockout minicircles) containing only allelic exchange essentials that were generated by Gibson assembly. Preparation of competent cells using sucrose and electroporation/integration of minicircles had virtually no detectable off-target promutagenic effect. In contrast, a downstream sacB selection apparently induced several large deletions via mobilization of transposable elements. Electroporation of minicircles into sucrose-treated cells is a versatile broadly applicable approach that may facilitate allelic exchange in a wide range of microbial species. The method permitted inactivation of a primary virulence factor unique to Pdp, apoptogenic toxin AIP56, demonstrating the efficacy of minicircles for difficult KO targets located on the high copy number of small plasmids.

The Evolution of a Specialized, Highly Virulent Fish Pathogen through Gene Loss and Acquisition of Host-Specific Survival Mechanisms.

Photobacterium damselae comprises two subspecies, P. damselae subsp. damselae and P. damselae subsp. piscicida, that contrast remarkably despite their taxonomic relationship. The former is opportunistic and free-living but can cause disease in compromised individuals from a broad diversity of taxa, while the latter is a highly specialized, primary fish pathogen. Here, we employ new closed curated genome assemblies from Australia to estimate the global phylogenetic structure of the species P. damselae. We identify genes responsible for the shift from an opportunist to a host-adapted fish pathogen, potentially via an arthropod vector as fish-to-fish transmission was not achieved in repeated cohabitation challenges despite high virulence for Seriola lalandi. Acquisition of ShdA adhesin and of thiol peroxidase may have allowed the environmental, generalist ancestor to colonize zooplankton and to occasionally enter in fish host sentinel cells. As dependence on the host has increased, P. damselae has lost nonessential genes, such as those related to nitrite and sulfite reduction, urea degradation, a type 6 secretion system (T6SS) and several toxin-antitoxin (TA) systems. Similar to the evolution of Yersinia pestis, the loss of urease may be the crucial event that allowed the pathogen to stably colonize zooplankton vectors. Acquisition of host-specific genes, such as those required to form a sialic acid capsule, was likely necessary for the emergent P. damselae subsp. piscicida to become a highly specialized, facultative intracellular fish pathogen. Processes that have shaped P. damselae subsp. piscicida from subsp. damselae are similar to those underlying evolution of Yersinia pestis from Y. pseudotuberculosis. IMPORTANCE Photobacterium damselae subsp. damselae is a ubiquitous marine bacterium and opportunistic pathogen of compromised hosts of diverse taxa. In contrast, its sister subspecies P. damselae subsp. piscicida (Pdp) is highly virulent in fish. Pdp has evolved from a single subclade of Pdd through gene loss and acquisition. We show that fish-to-fish transmission does not occur in repeated infection models in the primary host, Seriola lalandi, and present genomic evidence for vector-borne transmission, potentially via zooplankton. The broad genomic changes from generalist Pdd to specialist Pdp parallel those of the environmental opportunist Yersinia pseudotuberculosis to vector-borne plague bacterium Y. pestis and demonstrate that evolutionary processes in bacterial pathogens are universal between the terrestrial and marine biosphere.

Complete, closed and curated genome sequences of Photobacterium damselae subsp. piscicida isolates from Australia indicate mobilome-driven localized evolution and novel pathogenicity determinants.

Despite the recent advances in sequencing technologies, the complete assembly of multi-chromosome genomes of the Vibrionaceae, often containing several plasmids, remains challenging. Using a combination of Oxford Nanopore MinION long reads and short Illumina reads, we fully sequenced, closed and curated the genomes of two strains of a primary aquatic pathogen Photobacterium damselae subsp. piscicida isolated in Australia. These are also the first genome sequences of P. damselae subsp. piscicida isolated in Oceania and, to our knowledge, in the Southern hemisphere. We also investigated the phylogenetic relationships between Australian and overseas isolates, revealing that Australian P. damselae subsp. piscicida are more closely related to the Asian and American strains rather than to the European ones. We investigated the mobilome and present new evidence showing that a host specialization process and progressive adaptive evolution to fish are ongoing in P. damselae subsp. piscicida, and are largely mediated by transposable elements, predominantly in chromosome 2, and by plasmids. Finally, we identified two novel potential virulence determinants in P. damselae subsp. piscicida - a chorismate mutase gene, which is ubiquitously retained and co-localized with the AIP56 apoptogenic toxin-encoding gene on the pPHDP10 plasmid, and transfer-messenger RNA gene ssrA located on the main chromosome, homologous to a critical-to-virulence determinant in Yersinia pseudotuberculosis. Our study describes, to our knowledge, the only fully closed and manually curated genomes of P. damselae subsp. piscicida available to date, offering new insights into this important fish pathogen and its evolution.

[Support Strategies and provincial survey on nursing research]. / Strategie di promozione ed indagine provinciale sulla ricerca infermieristica.

The Board of Directors of the Nurses College of Venice has started a promotional strategy in order to stimulate the nursing research that will be developed in the next two years. There will be five different actions. Among them, an annual meeting on this subject. During the first of these meetings, a questionnaire has been given to the participants, to evaluate their interest for specific topics and investigate on their previous research experiences. The results show a variety of interests. Nevertheless the main topics are sanitary management and work safety. Furthermore, by splitting the preferences by subjects, it has been observed a greatest interest for organizational activities, the quality of assistance, the clinical nursing and the personal relationship with the customers. The collected data show also that only a small amount of participants has already managed some research projects that ended up with a scientific publication.

[Survey on quality of life of patients undergoing dialytic treatment]. / Indagine sulla Qualità di Vita di persone in trattamento dialitico.

The Nottingham Health Profile (NHP) was administered to 85 dialysis patients treated at Mestre Hospital, to assess their quality of life and compare any differences between haemodialysis (HD) and peritoneal dialysis (PD) patients. The two groups were similar for the main characteristics. Overall dialysis patients do not enjoy a good quality of life. Comparing baseline demographic and clinical scores, 72.9% patients have problems with energy and 69.4 with mobility, 47.1% refer pain and 54.1% sleep disorders. The main differences between HD and PD patients concern psychological reactions, where 42.9% of HD patients report "at risk/compromised" scores compared to 17.2% of PD patients. NHP showed to be an easy to administer and efficient instrument in identifying areas of concern for dialysis patients.