Acute hepatopancreatic necrosis disease (AHPND) is a severe disease affecting recently stocked cultured shrimps. The disease is mainly caused by V. parahaemolyticus that harbors the pVA1 plasmid; this plasmid contains the pirA and pirB genes, which encode a delta-endotoxin. AHPND originated in China in 2009 and has since spread to several other Asian countries and recently to Latin America (2013). Many Asian strains have been sequenced, and their sequences are publicly accessible in scientific databases, but only four strains from Latin America have been reported. In this study, we analyzed nine pVA1-harboring V. parahaemolyticus sequences from strains isolated in Mexico along with the 38 previously available pVA1-harboring V. parahaemolyticus sequences and the reference strain RIMD 2210633. The studied sequences were clustered into three phylogenetic clades (Latin American, Malaysian, and Cosmopolitan) through pangenomic and phylogenomic analysis. The nucleotide sequence alignment of the pVA1 plasmids harbored by the Asian and Latin American strains confirmed that the main structural difference in the plasmid between the Asian and Latin American strains is the absence of the Tn3 transposon in the Asian strains; in addition, some deletions in the pirAB region were found in two of the Latin American strains. Our study represents the most robust and inclusive phylogenomic analysis of pVA1-harboring V. parahaemolyticus conducted to date and provides insight into the epidemiology of AHPND. In addition, this study highlights that disease diagnosis through the detection of the pirA and pirB genes is an inadequate approach due to the instability of these genes.
Two phylogenetically distinct Vibrionaceae strains C4II189Tand C4V358Tisolated from reef seawater off Ishigaki Island, Japan, in 2014 were studied with advanced genome-based taxonomy approaches. All aspects of phylogenetic (16S rRNA phylogeny, MLSA), phenotypic and genetic (ANI, DDH, AAI, and the number of core genes) cohesions between the two identified species were high enough to propose them as members of a new genus within the family Vibrionaceae. Consequently, an eighth genus Thaumasiovibrio gen. nov. is proposed that contains two new species Thaumasiovibrio occultus sp. nov. strain C4II189T(=DSM 101554T=JCM 31629T) (type species) and Thaumasiovibrio subtropicus sp. nov. strain C4V358T(=DSM 101555T=JCM 31630T). Thaumasiovibrio species were phylogenetically distinct from the other Vibrionaceae species based on pyrH gene sequences. The combination of catalase negative, sensitivity to vibriostatic agent O/129, and green colony formation on TCBS for the phylogenetically affiliated strains was the diagnostic features for the current tentative identification of this genus.
Biosensors based on graphene field effect transistors (GFETs) have the potential to enable the development of point-of-care diagnostic tools for early stage disease detection. However, issues with reproducibility and manufacturing yields of graphene sensors, but also with Debye screening and unwanted detection of nonspecific species, have prevented the wider clinical use of graphene technology. Here, we demonstrate that our wafer-scalable GFETs array platform enables meaningful clinical results. As a case study of high clinical relevance, we demonstrate an accurate and robust portable GFET array biosensor platform for the detection of pancreatic ductal adenocarcinoma (PDAC) in patients' plasma through specific exosomes (GPC-1 expression) within 45 min. In order to facilitate reproducible detection in blood plasma, we optimized the analytical performance of GFET biosensors via the application of an internal control channel and the development of an optimized test protocol. Based on samples from 18 PDAC patients and 8 healthy controls, the GFET biosensor arrays could accurately discriminate between the two groups while being able to detect early cancer stages including stages 1 and 2. Furthermore, we confirmed the higher expression of GPC-1 and found that the concentration in PDAC plasma was on average more than 1 order of magnitude higher than in healthy samples. We found that these characteristics of GPC-1 cancerous exosomes are responsible for an increase in the number of target exosomes on the surface of graphene, leading to an improved signal response of the GFET biosensors. This GFET biosensor platform holds great promise for the development of an accurate tool for the rapid diagnosis of pancreatic cancer.