This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus , with ompU , toxR , tcpI , and hlyA for V. cholerae , and with tlh , tdh , trh , and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50°C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10 ² to 10 ³ CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.

In this paper we describe optimization of SYBR Green I-based real-time PCR parameters and testing of a large number of microbial species withvvh-specific oligonucleotide primers to establish a rapid, specific, and sensitive method for detection ofVibrio vulnificusin oyster tissue homogenate and Gulf of Mexico water (gulf water). Selected oligonucleotide primers for thevvhgene were tested for PCR amplification of a 205-bp DNA fragment with a melting temperature of approximately 87°C for 84 clinical and environmental strains ofV. vulnificus. No amplification was observed with other vibrios or nonvibrio strains with these primers. The minimum level of detection by the real-time PCR method was 1 pg of purified genomic DNA or 10²V. vulnificuscells in 1 g of unenriched oyster tissue homogenate or 10 ml of gulf water. It was possible to improve the level of detection to oneV. vulnificuscell in samples that were enriched for 5 h. The standard curves prepared from the real-time PCR cycle threshold values revealed that there was a strong correlation between the number of cells in unenriched samples and the number of cells in enriched samples. Detection of a single cell ofV. vulnificusin 1 g of enriched oyster tissue homogenate is in compliance with the recent Interstate Shellfish Sanitation Conference guidelines. The entire detection method, including sample processing, enrichment, and real-time PCR amplification, was completed within 8 h, making it a rapid single-day assay. Rapid and sensitive detection ofV. vulnificuswould ensure a steady supply of postharvest treated oysters to consumers, which should help decrease the number of illnesses or outbreaks caused by this pathogen.

In this paper we describe the microbial composition and their predictive metabolic profile in the sea urchin Lytechinus variegatus gut ecosystem along with samples from its habitat by using NextGen amplicon sequencing and downstream bioinformatics analyses. The microbial communities of the gut tissue revealed a near exclusive abundance of Campylobacteraceae, whereas the pharynx tissue consisted of Tenericutes, followed by Gamma-, Alpha-, and Epsilonproteobacteria at approximately equal capacities. The gut digesta and egested fecal pellets exhibited a microbial profile comprised of Gammaproteobacteria, mainly Vibrio , and Bacteroidetes. Both the seagrass and surrounding sea water revealed Alpha- and Betaproteobacteria. Bray-Curtis distances of microbial communities indicated a clustering profile with low intra-sample variation. Predictive metagenomics performed on the microbial communities revealed that the gut tissue had high relative abundances of metabolisms assigned to the KEGG-Level-2 designation of energy metabolisms compared to the gut digesta, which had higher carbohydrate, amino acid, and lipid metabolisms. Overall, the results of this study elaborate the spatial distribution of microbial communities in the gut ecosystem of L. variegatus , and specifically a selective attribute for Campylobacteraceae in the gut tissue. Also, the predictive functional significance of bacterial communities in uniquely compartmentalized gut ecosystems of L. variegatus has been described.

We developed a multiplexed real-time PCR assay using four sets of gene-specific oligonucleotide primers and four TaqMan probes labeled with four different fluorophores in a single reaction for detection of total and pathogenicVibrio parahaemolyticus, including the pandemic O3:K6 serotype in oysters.V. parahaemolyticushas been associated with outbreaks of food-borne gastroenteritis caused by the consumption of raw or undercooked seafood and therefore is a concern to the seafood industry and consumers. We selected specific primers and probes targeting the thermostable direct hemolysin gene (tdh) andtdh-related hemolysin gene (trh) that have been reported to be associated with pathogenesis in this organism. In addition, we targeted open reading frame 8 of phage f237 (ORF8), which is associated with a newly emerged virulent pandemic serotype ofV. parahameolyticusO3:K6. TotalV. parahaemolyticuswas targeted using the thermolabile hemolysin gene (tlh). The sensitivity of the combined four-locus multiplexed TaqMan PCR was found to be 200 pg of purified genomic DNA and 10⁴CFU per ml for pure cultures. Detection of an initial inoculum of 1 CFUV. parahaemolyticusper g of oyster tissue homogenate was possible after overnight enrichment, which resulted in a concentration of 3.3 × 10⁹CFU per ml. Use of this method with natural oysters resulted in 17/33 samples that were positive fortlhand 4/33 samples that were positive fortdh. This assay specifically and sensitively detected total and pathogenicV. parahaemolyticusand is expected to provide a rapid and reliable alternative to conventional detection methods by reducing the analysis time and obviating the need for multiple assays.

We compared three sets of oligonucleotide primers and two probes designed for Vibrio vulnificus hemolysin A gene ( vvhA ) for TaqMan-based real-time PCR method enabling specific detection of Vibrio vulnificus in oysters. Two of three sets of primers with a probe were specific for the detection of all 81 V. vulnificus isolates by TaqMan PCR. The 25 nonvibrio and 12 other vibrio isolates tested were negative. However, the third set of primers, F-vvh1059 and R-vvh1159, with the P-vvh1109 probe, although positive for all V. vulnificus isolates, also exhibited positive cycle threshold ( C _T ) values for other Vibrio spp. Optimization of the TaqMan PCR assay using F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and the P-vvh874 probe detected 1 pg of purified DNA and 10 ³ V. vulnificus CFU/ml in pure cultures. The enriched oyster tissue homogenate did not exhibit detectable inhibition to the TaqMan PCR amplification of vvhA . Detection of 3 × 10 ³ CFU V. vulnificus , resulting from a 5-h enrichment of an initial inoculum of 1 CFU/g of oyster tissue homogenate, was achieved with F-vvh785/R-vvh990 or F-vvh731/R-vvh1113 primers and P-vvh875 probe. The application of the TaqMan PCR using these primers and probe, exhibited detection of V. vulnificus on 5-h-enriched natural oysters harvested from the Gulf of Mexico. Selection of appropriate primers and a probe on vvhA for TaqMan-PCR-based detection of V. vulnificus in post-harvest-treated oysters would help avoid false-positive results, thus ensuring a steady supply of safe oysters to consumers and reducing V. vulnificus -related illnesses and deaths.