Parahaemolyticus Organisms Research Articles

Transcriptome analysis of the biofilm formation mechanism of Vibrio parahaemolyticus under the sub-inhibitory concentrations of copper and carbenicillin

Biofilm formation of Vibrio parahaemolyticus enhanced its tolerance to the environment, but caused many serious problems to food safety and human health. In this paper, the effects of copper and carbenicillin (CARB) stress on the formation of the biofilms of V. parahaemolyticus organisms were studied, and RNA sequencing technology was used to compare the differences in transcriptome profiles of the biofilm-related genes of V. parahaemolyticus organisms under different sub-inhibitory stresses. The results proved that V. parahaemolyticus had a large growth difference under the two stresses, copper and CARB at 1/2 minimal inhibitory concentration (MIC), and it could form a stable biofilm under both stress conditions. The amount of biofilm formed under CARB stress was significantly higher than that of copper stress (p < 0.05). Based on the analysis of transcriptome sequencing results 323, 1,550, and 1,296 significantly differential expressed genes were identified in the three treatment groups namely 1/2 MIC CARB, Cu2+, and Cu2++CARB. Through COG annotation, KEGG metabolic pathway analysis and gene expression analysis related to biofilm formation, the functional pathways of transcriptome changes affecting V. parahaemolyticus were different in the three treatment groups, and the CARB treatment group was significantly different from the other two groups. These differences indicated that the ABC transport system, two-component system and quorum sensing were all involved in the biofilm formation of the V. parahaemolytic by regulating flagellar motility, extracellular polysaccharides and extracellular polymer synthesis. Exploring the effects of different stress conditions on the transcriptome of V. parahaemolyticus could provide a basis for future research on the complex network system that regulates the formation of bacterial biofilms.

Characterization of vibrio parahaemolyticus organisms isolated from shrimps and shrimp fields located in the coastal areas of Bangladesh

Background: In recent years, this group isolated trh positive Vibrio parahaemolyticus serotype 01:K56 from fresh water sediments around Dhaka city, Bangladesh. V. parahaemolyticus is a well known marine bacterium with pandemic effects of causing enteric diseases and the presence of trh positive V. parahaemolyticus serotype 01;K56 in the rivers of Bangladesh imply great public health concern. On the other hand, shrimp export sector is the second largest export earner for Bangladesh and contamination of shrimp with pathogenic V. parahaemolyticus could be a threat to the shrimp export sector. Therefore, this study was carried out to characterize V. parahaemolyticus isolates from shrimps and shrimp fields located in the coastal areas of Bangladesh. Methods and materials: Thirty four V. parahaemolyticus organisms were isolated and confirmed by standard biochemical, serological and molecular techniques. All these isolates belonged to 10 serogroups (O10:KUT, O1:K25, OUT:K61, O11:K33, O2:K28, O1:K33, O1:KUT, O5:KUT, O11:KUT and O8:KUT) confirmed by duplicate testing. Along with the occurrence of thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH), the incidence of two sets of gene clusters that encode the type 3 secretion system apparatus (T3SS1 & T3SS2α) which contribute in pathogenicity of V. parahaemolyticus were also investigated. Results: All V. parahaemolyticus isolates were found to be negative for both tdh and trh genes, which were confirmed by PCR. T3SS1 was found to be present in all V. parahaemolyticus strains as confirmed by the PCR. All these environmental isolates were found to be negative for T3SS2α, which confirmed that T3SS2α is not associated with non pathogenic environmental strains. We also phenotypically characterized the environmental isolates for the enterotoxicity test, which showed no fluid accumulation in rabbit ileal loop assay. All the above mentioned tests were done twice and the variances were less than 5 percent. Conclusion: All these findings clearly indicate that the V.parahaemolyticus organisms, which are associated with the shrimp and shrimp fields of the coastal areas are non pathogenic in nature and thus, would not be a threat for shrimp export sector, as long as the concerned bacterium is V. parahaemolyticus.

Long-term study of Vibrio parahaemolyticus prevalence and distribution in New Zealand shellfish.

The food-borne pathogen Vibrio parahaemolyticus has been reported as being present in New Zealand (NZ) seawaters, but there have been no reported outbreaks of food-borne infection from commercially grown NZ seafood. Our study determined the current incidence of V. parahaemolyticus in NZ oysters and Greenshell mussels and the prevalence of V. parahaemolyticus tdh and trh strains. Pacific (235) and dredge (21) oyster samples and mussel samples (55) were obtained from commercial shellfish-growing areas between December 2009 and June 2012. Total V. parahaemolyticus numbers and the presence of pathogenic genes tdh and trh were determined using the FDA most-probable-number (MPN) method and confirmed using PCR analysis. In samples from the North Island of NZ, V. parahaemolyticus was detected in 81% of Pacific oysters and 34% of mussel samples, while the numbers of V. parahaemolyticus tdh and trh strains were low, with just 3/215 Pacific oyster samples carrying the tdh gene. V. parahaemolyticus organisms carrying tdh and trh were not detected in South Island samples, and V. parahaemolyticus was detected in just 1/21 dredge oyster and 2/16 mussel samples. Numbers of V. parahaemolyticus organisms increased when seawater temperatures were high, the season when most commercial shellfish-growing areas are not harvested. The numbers of V. parahaemolyticus organisms in samples exceeded 1,000 MPN/g only when the seawater temperatures exceeded 19°C, so this environmental parameter could be used as a trigger warning of potential hazard. There is some evidence that the total V. parahaemolyticus numbers increased compared with those reported from a previous 1981 to 1984 study, but the analytical methods differed significantly.

Pre-Enrichment of Estuarine and Fresh Water Environmental Samples with Sodium Chloride Yields in Better Recovery of &amp;lt;i&amp;gt;Vibrio parahaemolyticus&amp;lt;/i&amp;gt;

Vibrio parahaemolyticus organisms cause acute gastroenteritis in humans. These bacteria are natural inhabitants of both marine and estuarine ecosystems. In the present study, we investigated the effectiveness of a non-selective enrichment of sediment samples with sodium chloride prior to selective enrichment with alkaline peptone water for a better recovery of V. parahaemolyticus. Sediment samples were collected with or without 1% NaCl from the river Buriganga, located besides Dhaka city and about 400 km away from the Bay of Bengal, and from the estuary of the river Karnaphuli which flows into the Bay of Bengal. Very small number of V. parahaemolyticus (V. parahaemolyticus increased to more than 40 times (1500 MPN/g) in the river and 32 times (960 MPN/g) in the estuary where NaCl were added. River sediment sample contained the serotype O9:K41 of V. parahaemolyticus and the estuarine sample contained O3:K41 and O3:KUT Our results suggest that a pre-enrichment of environmental samples with 1% NaCl helps V. parahaemolyticus to survive for at least 7 days until they are enriched with alkaline peptone water, for better recovery.

Development and evaluation of a loop-mediated isothermal amplification assay for rapid and sensitive detection of Vibrio parahaemolyticus.

Loop-mediated isothermal amplification (LAMP) assays targeting the rpoD and toxR genes were developed to detect Vibrio parahaemolyticus. All 78 tested V. parahaemolyticus strains yielded positive results within 40 min, while negative results were obtained for 69 strains of other organisms even at 60 min. For V. parahaemolyticus ATCC 17802 in pure culture, the detection limits of LAMP assays targeting rpoD and toxR were 3.7 and 450 CFU per test, respectively. Due to the higher sensitivity of rpoD-LAMP, it was further evaluated for the ability to detect V. parahaemolyticus in seafood samples. V. parahaemolyticus populations spiked in short-necked clams were enumerated by the most-probable-number (MPN) method combined with the rpoD-LAMP assay and the MPN method with a culture method using agar medium. The MPN-rpoD-LAMP method had better sensitivity and was more rapid than the conventional method. These results indicate that the MPN-LAMP assay targeting the rpoD gene is a specific, sensitive, and rapid method to enumerate V. parahaemolyticus organisms.

PandemicVibrio parahaemolyticusO3:K6 Spread, France

Pandemic<i>Vibrio parahaemolyticus</i>O3:K6 Spread, France

Density of Total and Pathogenic (tdh+) Vibrio parahaemolyticus in Atlantic and Gulf Coast Molluscan Shellfish at Harvest

The densities of total and pathogenic Vibrio parahaemolyticus in 671 samples of molluscan shellfish harvested in 1999 and 2000 from 14 sites in seven Gulf and Atlantic coast states were determined at 2-week intervals over a period of 12 to 16 months in each state. Changes in V. parahaemolyticus densities in shellfish between harvest and sample analysis were minimized with time and temperature controls. Densities were measured by direct plating techniques, and gene probes were used for identification. Total and pathogenic V. parahaemolyticus organisms were identified with probes for the thermolabile direct hemolysin (tlh) gene and the thermostable direct hemolysin (tdh) gene, respectively. An enrichment procedure involving 25 g of shellfish was also used for the recovery of pathogenic V. parahaemolyticus. The densities of V. parahaemolyticus in shellfish from all harvest sites were positively correlated with water temperature. Shellfish from the Gulf Coast typically had higher densities of V. parahaemolyticus than did shellfish harvested from the North Atlantic or mid-Atlantic coast. Vibrio parahaemolyticus counts exceeded 1,000 CFU/g for only 5% of all samples. Pathogenic (tdh+) V. parahaemolyticus was detected in approximately 6% of all samples by both procedures, and 61.5% of populations in the positive samples from the direct plating procedure were at the lower limit of detection (10 CFU/g). The frequency of detection of pathogenic V. parahaemolyticus was significantly related to water temperature and to the density of total V. parahaemolyticus. The failure to detect pathogenic V. parahaemolyticus in shellfish more frequently was attributed to the low numbers and uneven distribution of the organism.

Direct Plating Medium Procedure for Isolating and Enumerating Vibrio parahaemolyticus in Fish and Shellfish

A modified arabinose ammonium sulfate cholate agar (MAAC agar) plate procedure for isolating and enumerating Vibrio parahaemolyticus organisms in fish and shellfish was investigated. The medium consisted of 0.1% peptone, 0.1% yeast extract, 0.5% arabinose, 0.1% ammonium sulfate, 4.0% sodium chloride, 1.0% sodium thiosulfate, 0.1% sodium cholate, 0.004% bromothymol blue, 0.004% thymol blue, and 1.5% agar, with the pH adjusted to 8.6. Forty samples of shellfish and 30 samples of ice water used for soaking fish were collected from the Tokyo Central Wholesale Market in August 1977. Enumeration of V. parahaemolyticus counts in the samples was made by directly spreading each 0.1ml of the homogenates, the ice water samples and the appropriate dilutions on MAAC and TCBS plate media. The MAAC plates were incubated at 42°C for 18hr and TCBS plates at 35°C for 18hr. Presumptive V. parahaemolyticus counts, obtained from the numbers of yellow colonies formed on MAAC plates, ranged from 100 to 16, 000/g in the shellfish samples, and from 10 to 400/ml in the ice water samples. Out of 222 isolates from the MAAC media 185 (83.3%) were identified as V. parahaemolyticus, 26 (11.7%) as V. alginolyticus, and 11 (5.0%) as the allied organisms. Conversely, out of 121 isolates from TCBS media only 25 (20.7%) were identified as V. parahaemolyticus. MAAC agar plate procedure was found to be rapid and reliable for isolating and enumerating V. parahaemolyticus in various sea foods.