Plasmid dynamics in Vibrio parahaemolyticus strains related to shrimp Acute Hepatopancreatic Necrosis Syndrome (AHPNS).

Vibrio parahaemolyticus is a causative agent of acute hapatopancreatic necrosis syndrome (AHPNS) which causes early mortality in white shrimp. Emergence of AHPNS has caused tremendous economic loss for aquaculture industry particularly in Asia since 2010. Previous studies reported that strains causing AHPNS harbor a 69-kb plasmid with possession of virulence genes, pirA and pirB. However, genetic variation of the 69-kb plasmid among AHPNS related strains has not been investigated. This study aimed to analyze genetic composition and diversity of the 69-kb plasmid in strains isolated from shrimps affected by AHPNS. Plasmids recovered from V. parahaemolyticus strain VPE61 which represented typical AHPNS pathogenicity, strain VP2HP which did not represent AHPNS pathogenicity but was isolated from AHPNS affected shrimp and other AHPNS V. parahaemolyticus isolates in Genbank were investigated. Protein coding genes of the 69-kb plasmid from the strain VPE61 were identical to that of AHPNS strain from Vietnam except the inverted complement 3.4-kb transposon covering pirA and pirB. The strain VP2HP possessed remarkable large 183-kb plasmid which shared similar protein coding genes to those of the 69-kb plasmid from strain VPE61. However, the 3.4-kb transposon covering pirA and pirB was absent from the 183-kb plasmid in strain VP2HP. A number of protein coding genes from the 183-kb plasmid were also detected in other AHPNS strains. In summary, this study identified a novel 183-kb plasmid that is related to AHPNS causing strains. Homologous recombination of the 69-kb AHPNS plasmid and other naturally occurring plasmids together with loss and gain of AHPNS virulence genes in V. parahaemolyticus were observed. The outcome of this research enables understanding of plasmid dynamics that possibly affect variable degrees of AHPNS pathogenicity.

Characterization and Genetic Variation of Vibrio cholerae Isolated from Clinical and Environmental Sources in Thailand.

Cholera is still an important public health problem in several countries, including Thailand. In this study, a collection of clinical and environmental V. cholerae serogroup O1, O139, and non-O1/non-O139 strains originating from Thailand (1983 to 2013) was characterized to determine phenotypic and genotypic traits and to investigate the genetic relatedness. Using a combination of conventional methods and whole genome sequencing (WGS), 78 V. cholerae strains were identified. WGS was used to determine the serogroup, biotype, virulence, mobile genetic elements, and antimicrobial resistance genes using online bioinformatics tools. In addition, phenotypic antimicrobial resistance was determined by the minimal inhibitory concentration (MIC) test. The 78 V. cholerae strains belonged to the following serogroups O1: (n = 44), O139 (n = 16) and non-O1/non-O139 (n = 18). Interestingly, we found that the typical El Tor O1 strains were the major cause of clinical cholera during 1983-2000 with two Classical O1 strains detected in 2000. In 2004-2010, the El Tor variant strains revealed genotypes of the Classical biotype possessing either only ctxB or both ctxB and rstR while they harbored tcpA of the El Tor biotype. Thirty O1 and eleven O139 clinical strains carried CTXϕ (Cholera toxin) and tcpA as well four different pathogenic islands (PAIs). Beside non-O1/non-O139, the O1 environmental strains also presented chxA and Type Three Secretion System (TTSS). The in silico MultiLocus Sequence Typing (MLST) discriminated the O1 and O139 clinical strains from other serogroups and environmental strains. ST69 was dominant in the clinical strains belonging to the 7th pandemic clone. Non-O1/non-O139 and environmental strains showed various novel STs indicating genetic variation. Multidrug-resistant (MDR) strains were observed and conferred resistance to ampicillin, azithromycin, nalidixic acid, sulfamethoxazole, tetracycline, and trimethoprim and harboured variants of the SXT elements. For the first time since 1986, the presence of V. cholerae O1 Classical was reported causing cholera outbreaks in Thailand. In addition, we found that V. cholerae O1 El Tor variant and O139 were pre-dominating the pathogenic strains in Thailand. Using WGS and bioinformatic tools to analyze both historical and contemporary V. cholerae circulating in Thailand provided a more detailed understanding of the V. cholerae epidemiology, which ultimately could be applied for control measures and management of cholera in Thailand.

Genetic diversity of Vibrio parahaemolyticus strains isolated from farmed Pacific white shrimp and ambient pond water affected by acute hepatopancreatic necrosis disease outbreak in Thailand.

Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease that causes massive die-offs in farmed shrimps. Recent outbreaks of AHPND in Asia have been causing great losses for shrimp culture and have become a serious socioeconomic problem. The causative agent of AHPND is Vibrio parahaemolyticus, which is typically known to cause food-borne gastroenteritis in humans. However, there have been few reports of the epidemiology of V. parahaemolyticus AHPND strains, and the genetic relationship among AHPND strains is unclear. Here, we report the genetic characterization of V. parahaemolyticus strains isolated from AHPND outbreaks in Thailand. We found eight isolates from AHPND-suspected shrimps and pond water that were positive for AHPND markers AP1 and AP2. PCR analysis confirmed that none of these eight AP-positive AHPND strains possesses the genes for the conventional virulence factors affecting to humans, such as thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH) and type III secretion system 2. Phylogenetic analysis by multilocus sequence typing showed that the AHPND strains are genetically diverse, suggesting that AHPND strains were not derived from a single genetic lineage. Our study represents the first report of molecular epidemiology of AHPND-causing V. parahaemolyticus strains using multilocus sequence typing, and provides an insight into their evolutionary mechanisms.

Shelf life extension of whole Norway lobster Nephrops norvegicus using modified atmosphere packaging.

Once a nuisance by-catch, today the Norway lobster (Nephrops norvegicus) is a valuable UK fisheries commodity. Unfortunately, the species is very susceptible to quality deterioration post harvest as it quickly develops black spots and also spoils rapidly due to bacterial growth. Treatment with chemicals can stop the blackening and carefully monitored cold storage can result in a sensory shelf life of up to 6.5 days. The high susceptibility to spoilage greatly restricts the extent to which N. norvegicus can be distributed to retailers and displayed for sale. The application of modified atmosphere (MA) could be extremely beneficial, allowing the chilled product to stay fresh for a long period of time, thus ensuring higher sales. In the present study, we identified a gas mix for the MA packaging (MAP) of whole N. norvegicus lobster into 200 g retail packs. Our results show that a shelf life extension to 13 days can be achieved when retail packs are stored in MAP at 1 °C. Effectiveness of the MAP was evaluated by using a newly developed QIM for MA-packaged whole N. norvegicus and also by analyzing bacterial plate counts. Changes in the microflora and effects of different storage temperatures on the quality of the MA packs are also presented. The main specific spoilage organism (SSO) of modified atmosphere packaged Norway lobster is Photobacterium phosphoreum.

Genetic relationships of Vibrio parahaemolyticus isolates from clinical, human carrier, and environmental sources in Thailand, determined by multilocus sequence analysis.

Vibrio parahaemolyticus is a seafood-borne pathogenic bacterium that is a major cause of gastroenteritis worldwide. We investigated the genetic and evolutionary relationships of 101 V. parahaemolyticus isolates originating from clinical, human carrier, and various environmental and seafood production sources in Thailand using multilocus sequence analysis. The isolates were recovered from clinical samples (n = 15), healthy human carriers (n = 18), various types of fresh seafood (n = 18), frozen shrimp (n = 16), fresh-farmed shrimp tissue (n = 18), and shrimp farm water (n = 16). Phylogenetic analysis revealed a high degree of genetic diversity within the V. parahaemolyticus population, although isolates recovered from clinical samples and from farmed shrimp and water samples represented distinct clusters. The tight clustering of the clinical isolates suggests that disease-causing isolates are not a random sample of the environmental reservoir, although the source of infection remains unclear. Extensive serotypic diversity occurred among isolates representing the same sequence types and recovered from the same source at the same time. These findings suggest that the O- and K-antigen-encoding loci are subject to exceptionally high rates of recombination. There was also strong evidence of interspecies horizontal gene transfer and intragenic recombination involving the recA locus in a large proportion of isolates. As the majority of the intragenic recombinational exchanges involving recA occurred among clinical and carrier isolates, it is possible that the human intestinal tract serves as a potential reservoir of donor and recipient strains that is promoting horizontal DNA transfer, driving evolutionary change, and leading to the emergence of new, potentially pathogenic strains.