White feces syndrome (WFS) is a multifactorial disease that affects global shrimp production. The diagnostic approach to identify WFS involves traditional and molecular scientific methods by examining histopathology, bioassays, PCR (polymerase chain reaction), and calorimetric estimation. The pathogenesis of WFS is closely associated with Vibrio spp., intestinal microbiota (IM) dysbiosis, and Enterocytozoon hepatopenaei (EHP). It also has caused over 10-15 % loss in the aquaculture industry and is also known to cause retardation, lethargy and slowly leading to high mortality in shrimp farms. Therefore, it is necessary to understand the molecular mechanisms processed under the association of IM dysbiosis, Vibrio spp., and EHP to analyze the impact of disease on the innate immune system of shrimp. However, only very few reviews have described the molecular pathways involved in WFS. Hence, this review aims to elucidate an in-depth analysis of molecular pathways involved in the innate immune system of shrimp and their response to pathogens. The analysis and understanding of the impact of shrimp\'s innate immune system on WFS would help in developing treatments to prevent the spread of disease, thereby improving the economic condition of shrimp farms worldwide.

This study aimed to determine the incidence and virulence factor profiling of Vibrio species from hospital wastewater (HWW) and community wastewater effluents. Wastewater samples from selected sites were collected, processed, and analysed presumptively by the culture dependent methods and molecular techniques. A total of 270 isolates were confirmed as Vibrio genus delineating into V. cholerae (27%), V. parahaemolyticus (9.1%), V. vulnificus (4.1%), and V. fluvialis (3%). The remainder (>50%) may account for other Vibrio species not identified in the study. The four Vibrio species were isolated from secondary hospital wastewater effluent (SHWE), while V. cholerae was the sole specie isolated from Limbede community wastewater effluent (LCWE) and none of the four Vibrio species was recovered from tertiary hospital wastewater effluent (THWE). However, several virulence genes were identified among V. cholerae isolates from SHWE: ToxR (88%), hylA (81%), tcpA (64%), VPI (58%), ctx (44%), and ompU (34%). Virulence genes factors among V. cholerae isolates from LCWE were: ToxR (78%), ctx (67%), tcpA (44%), and hylA (44%). Two different genes (vfh and hupO) were identified in all confirmed V. fluvialis isolates. Among V. vulnificus, vcgA (50%) and vcgB (67%) were detected. In V. parahaemolyticus, tdh (56%) and tlh (100%) were also identified. This finding reveals that the studied aquatic niches pose serious potential health risk with Vibrio species harbouring virulence signatures. The distribution of virulence genes is valuable for ecological site quality, as well as epidemiological marker in the control and management of diseases caused by Vibrio species. Regular monitoring of HWW and communal wastewater effluent would allow relevant establishments to forecast, detect, and mitigate any public health threats in advance.

Virulence determinants are crucial to the risk assessment of pathogens in an environment. This study investigated the presence of eleven key virulence-associated genes in Vibrio cholerae (n = 111) and Vibrio mimicus (n = 22) and eight virulence determinants in Vibrio alginolyticus (n = 65) and Vibrio parahaemolyticus (n = 17) isolated from six important water resources in Eastern Cape, South Africa, using PCR techniques. The multiple virulence gene indexes (MVGI) for sampling sites and isolates as well as hotspots for potential vibriosis outbreaks among sampling sites were determined statistically based on the comparison of MVGI.
The PCR assay showed that all the V. cholerae isolates belong to non-O1/non-O139 serogroups. Of the isolates, Vibrio Cholera (84%), V. mimicus (73%), V. alginolyticus (91%) and V. parahaemolyticus (100%) isolates harboured at least one of the virulence-associated genes investigated. The virulence gene combinations detected in isolates varied at sampling site and across sites. Typical virulence-associated determinants of V. cholerae were detected in V. mimicus while that of V. parahaemolyticus were detected in V. alginolyticus. The isolates with the highest MVGI were recovered from three estuaries (Sunday river, Swartkopps river, buffalo river) and a freshwater resource (Lashinton river). The cumulative MVGI for V. cholerae, V. mimicus, V. alginolyticus and V. parahaemolyticus isolates were 0.34, 0.20, 0.45, and 0.40 respectively. The targeted Vibrio spp. in increasing order of the public health risk posed in our study areas based on the MVGI is V. alginolyticus > V. parahaemolyticus > V. cholerae > V. mimicus. Five (sites SR, PA5, PA6, EL4 and EL6) out of the seventeen sampling sites were detected as the hotspots for potential cholera-like infection and vibriosis outbreaks.
Our findings suggest that humans having contact with water resources in our study areas are exposed to potential public health risks owing to the detection of virulent determinants in human pathogenic Vibrio spp. recovered from the water resources. The study affirms the relevancy of environmental Vibrio species to the epidemiology of vibriosis, cholera and cholera-like infections. Hence we suggest a monitoring program for human pathogenic Vibrio spp. in the environment most especially surface water that humans have contact with regularly.

BACKGROUND: Vibrio species are among the autochthonous bacterial  populations found in surface waters and associated with various life-threatening extraintestinal diseases, especially in human populations with underlying illnesses and wound infections. Presently, very diminutive information exists regarding these species\' mutational diversity of virulence and resistance genes. This study evaluated variations in endonucleases and mutational diversity of the virulence and resistance genes of Vibrio isolates, harboring virulence-correlated gene (vcgCPI), dihydropteroate synthase type 1 and type II genes (Sul 1 and 11), (aadA) aminoglycoside (3\'\') (9) adenylyltransferase gene, (aac(3)-IIa, (aacC2)a, aminoglycoside N(3)-acetyltransferase III, and (strA) aminoglycoside 3\'-phosphotransferase resistance genes.
METHODS: Using combinations of molecular biology techniques, bioinformatics tools, and sequence analysis.
RESULTS: Our result revealed various nucleotide variations in virulence determinants of V. vulnificus (vcgCPI) at nucleotide positions (codon) 73-75 (A → G) and 300-302 (N → S). The aminoglycosides resistance gene (aadA) of Vibrio species depicts a nucleotide difference at position 482 (A → G), while the aminoglycosides resistance gene (sul 1 and 11) showed two variable regions of nucleotide polymorphism (102 and 140). The amino acid differences exist with the nucleotide polymorphism at position 140 (A → E). The banding patterns produced by the restriction enzymes HinP1I, MwoI, and StyD4I showed significant variations. Also, the restriction enzyme digestion of protein dihydropteroate synthase type 1 and type II genes (Sul 1 and 11) differed significantly, while enzymes DpnI and Hinf1 indicate no significant differences. The restriction enzyme NlaIV showed no band compared to reference isolates from the GenBank. However, the resistant determinants show significant point nucleotide mutation, which does not produce any amino acid change with diverse polymorphic regions, as revealed in the restriction digest profile.
CONCLUSIONS: The described virulence and resistance determinants possess specific polymorphic locus relevant to pathogenomics studies, pharmacogenomic, and control of such water-associated strains.

BACKGROUND: Vibrio species bloodstream infections have been associated with significant mortality and morbidity. Limited information is available regarding the epidemiology of bloodstream infections because of Vibrio species in the Australian context.
OBJECTIVE: The objective of this study was to define the incidence and risk factors for developing Vibrio species bloodstream infections and compare differences between different species.
METHODS: All patients with Vibrio spp. isolated from positive blood cultures between 1 January 2000 and 31 December 2019 were identified by the state-wide Pathology Queensland laboratory. Demographics, clinical foci of infections and comorbid conditions were collected in addition to antimicrobial susceptibility results.
RESULTS: About 100 cases were identified between 2000 and 2019 with an incidence of 1.2 cases/1 million person-years. Seasonal and geographical variation occurred with the highest incidence in the summer months and in the tropical north. Increasing age, male sex and multiple comorbidities were identified as risk factors. Vibrio vulnificus was isolated most frequently and associated with the most severe disease. Overall case fatality was 19%.
CONCLUSIONS: There is potential for increasing cases of Vibrio species infections globally with ageing populations and climate change. Ongoing clinical awareness is required to ensure optimal patient outcomes.

BACKGROUND: Vibrio spp. are a diverse group of ecologically important marine bacteria responsible for several foodborne outbreaks of gastroenteritis around the world. Their detection and characterization are moving away from conventional culture-based methods towards next generation sequencing (NGS)-based approaches. However, genomic methods are relative in nature and suffer from technical biases arising from library preparation and sequencing. Here, we introduce a quantitative NGS-based method that enables the quantitation of Vibrio spp. at the limit of quantification (LOQ) through artificial DNA standards and their absolute quantification via digital PCR (dPCR).
RESULTS: We developed six DNA standards, called Vibrio-Sequins, together with optimized TaqMan assays for their quantification in individually sequenced DNA libraries via dPCR. To enable Vibrio-Sequin quantification, we validated three duplex dPCR methods to quantify the six targets. LOQs were ranging from 20 to 120 cp/µl for the six standards, whereas the limit of detection (LOD) was ~ 10 cp/µl for all six assays. Subsequently, a quantitative genomics approach was applied to quantify Vibrio-DNA in a pooled DNA mixture derived from several Vibrio species in a proof-of-concept study, demonstrating the increased power of our quantitative genomic pipeline through the coupling of NGS and dPCR.
CONCLUSIONS: We significantly advance existing quantitative (meta)genomic methods by ensuring metrological traceability of NGS-based DNA quantification. Our method represents a useful tool for future metagenomic studies aiming at quantifying microbial DNA in an absolute manner. The inclusion of dPCR into sequencing-based methods supports the development of statistical approaches for the estimation of measurement uncertainties (MU) for NGS, which is still in its infancy.

The outbreak of waterborne diseases such as cholera and non-cholera (vibriosis) is continuously increasing in the environment due to fecal and sewage discharge in water sources. Cholera and vibriosis are caused by different species of Vibrio genus which are responsible for acute diarrheal disease and soft tissue damage. Although incidences of cholera and vibriosis have been reported from the Vaishali district of Bihar, India, clinical or environmental strains have not been characterized in this region. Out of fifty environmental water samples, twelve different biochemical test results confirmed the presence of twenty Vibrio isolates. The isolates were found to belong to five different Vibrio species, namely V. proteolyticus, V. campbellii, V. nereis, V. cincinnatiensis, and V. harveyi. From the identified isolates, 65% and 45% isolates were found to be resistant to ampicillin and cephalexin, respectively. Additionally, two isolates were found to be resistant against six and four separately selected antibiotics. Furthermore, virulent hlyA and ompW genes were detected by PCR in two different isolates. Additionally, phage induction was also noticed in two different isolates which carry lysogenic phage in their genome. Overall, the results reported the identification of five different Vibrio species in environmental water samples. The isolates showed multiple antibacterial resistance, phage induction, and virulence gene profile in their genome.

The main goal of the present study was to evaluate the influence of thermal exposure on Vibrio population and HSP genes expression (HSP 90, HSP70, and HSP20) in rayed pearl oyster (P. radiata). To this end, the oysters were reared for 30 days at temperatures of 22 °C (control), 25 °C, 27 °C, and 29 °C. The results showed that five dominate Vibrio strains including Vibrio hepatarius, V. harveyi, V. alginolyticus, V. parahaemolyticus, and V. rotiferianus were identified. The highest population of V. parahaemolyticus, V. alginolyticus, and V. harveyi, was found in 29οC group. According to real-time PCR, mantle exhibited the highest expression levels of HSP20, HSP70, and HSP90 genes. A higher level of HSP20 expression was observed at high temperatures (25 °C, 27 °C, and 29 °C) in the gonad and mantle compared to the control group (22 °C) while decrease in HSP90 expression level was recorded in 25 °C, 27 °C, and 29 °C groups. HSP20 expression level in adductor muscle was remarkably down-regulated in 27 °C and 29 °C groups. In this tissue, HSP70 was detected at highest levels in the 29οC group. In mantle, HSP90 gene expression was lowest at 22 °C water temperature. Several Vibrio strains have been identified from pearl Gulf oyster that haven\'t been previously reported. The identification of dominant Vibrio species is essential for epidemiological management strategies to control and prevent Vibrio outbreaks in pearl oyster farms. The expression pattern of HSP genes differs in rayed pearl oyster tissues due to differences in their thermal tolerance capability and physiological and biological characteristics. The present study provides useful molecular information for the ecological adaptation of rayed pearl oysters after exposure to different temperature levels.

The Greenland shark (Somniosus microcephalus) is a large species of shark found in the North Atlantic and Arctic Oceans and is believed to be the longest living vertebrate. Relatively little is known about its biology, abundance, health or diseases. In March 2022, only the third reported UK stranding of this species occurred and it was the first to undergo post-mortem examination. The animal was a sexually immature female, measuring 3.96 m in length and 285 kg in weight, and was in poor nutritional state. Gross findings included haemorrhages in the skin and soft tissues, particularly of the head, and silt in the stomach suggestive of live stranding, bilateral corneal opacity, slightly turbid cerebrospinal fluid (CSF) and patchy congestion of the brain. Histopathological findings included keratitis and anterior uveitis, fibrinonecrotic and lymphohistiocytic meningitis of the brain and proximal spinal cord and fibrinonecrotizing choroid plexitis. A near pure growth of a Vibrio organism was isolated from CSF. This is believed to be the first report of meningitis in this species.

The aim of the study was to understand the genetic basis of resistance of five β-lactam resistant Vibrio anguillarum isolates obtained from the gut content of Atlantic mackerel (Scomber scomberus), using whole genome sequencing and to characterize a novel β-lactamase (VAN-1) from these isolates.
Antibiotic sensitivity pattern was determined using Sensititre™ plates and whole genome sequencing was carried out using Illumina MiSeq-based sequencing. The blaVAN-1 gene was synthesized and expressed in Escherichia coli Top10 cells.
Five isolates obtained (out of 73) from the gut content of Atlantic mackerel were identified as Vibrio anguillarum. Whole genome assemblies ranged from 3.894 to 3.906 million bases in length with an average of 50 contigs. A novel β-lactamase blaVAN-1, sharing 77.7% nucleotide identity with a known mobile β-lactamase from Vibrio species was detected. The blaVAN-1 gene in these isolates is flanked by a truncated IS5 family transposase on one end and a hypothetical protein and outer membrane protein followed by another IS5 family transposase on the other end, suggesting its potential for mobility. The blaVAN-1 gene was absent in V. anguillarum type strain (ATCC 14181) and V. anguillarum isolates from bivalves and sea water in Norway. VAN-1 conferred ampicillin resistance when expressed in E. coli, thus confirming the functionality of this gene.
Our study highlights the importance of the marine environment as a reservoir of new antibiotic resistance genes. Our results suggest that migratory fish may transport novel antibiotic resistance determinants over long distances.