Gold Nanoparticle Paper Immunoassays for Sensing the Presence of Vibrio parahaemolyticus in Oyster Hemolymph.

Seafood contamination with Vibrio bacteria is a problem for aquaculture, especially with oysters, which are often consumed raw. Current methods for diagnosing bacterial pathogens in seafood involve lab-based assays such as polymerase chain reaction or culturing, which are time consuming and must occur in a centralized location. Detection of Vibrio in a point-of-care assay would be a significant tool for food safety control measures. We report here a paper immunoassay that can detect the presence of Vibrio parahaemolyticus (Vp) in buffer and oyster hemolymph. The test uses gold nanoparticles conjugated to polyclonal anti-Vibrio antibodies in a paper-based sandwich immunoassay. A sample is added to the strip and wicked through by capillary action. If Vp is present, it results in a visible color at the test area that can be read out by eyes or a standard mobile phone camera. The assay has a limit of detection of 6.05 × 105 cfu/mL and a cost estimate of $5 per test. Receiver operating characteristic curves with validated environmental samples showed a test sensitivity of 0.96 and a specificity of 1.00. Because the assay is inexpensive and can be used on Vp directly without the requirement for culturing, or sophisticated equipment, it has the potential to be used in fieldable settings.

Draft Genome Sequence of Enterococcus faecalis AS003, a Strain Possessing All Three Type II-a CRISPR Loci.

Enterococcus faecalis is a clinically significant member of the human microbiome. Three CRISPR-Cas loci are located in conserved locations. Previous studies provide evidence that E. faecalis strains with functional CRISPR-Cas genes are negatively correlated with antibiotic resistance. Here, we report the genome sequence of an unusual strain possessing all three CRISPR-Cas loci.

Optimization of paper-based nanoparticle immunoassays for direct detection of the bacterial pathogen V. parahaemolyticus in oyster hemolymph.

The detection of foodborne pathogens is critical for disease control and infection prevention, especially in seafood consumed raw or undercooked. Paper-based diagnostic tools are promising for rapid fieldable detection and provide a readout by eye due to the use of gold nanoparticle immunoprobes. Here we study different strategies to overcome these challenges in a real biological matrix, oyster hemolymph, for the detection of the pathogenic bacteria Vibrio parahaemolyticus (Vp). Nanoparticle surface chemistry, nitrocellulose speed and blocking, running steps, and antibody concentrations on the NP and nitrocellulose were all studied. Their effect on paper immunoassay signal intensity was quantified to determine optimal conditions, which enabled the detection of Vp directly from hemolymph below pathogenic concentrations.

Incidence of Type II CRISPR1-Cas Systems in Enterococcus Is Species-Dependent.

CRISPR-Cas systems, which obstruct both viral infection and incorporation of mobile genetic elements by horizontal transfer, are a specific immune response common to prokaryotes. Antiviral protection by CRISPR-Cas comes at a cost, as horizontally-acquired genes may increase fitness and provide rapid adaptation to habitat change. To date, investigations into the prevalence of CRISPR have primarily focused on pathogenic and clinical bacteria, while less is known about CRISPR dynamics in commensal and environmental species. We designed PCR primers and coupled these with DNA sequencing of products to detect and characterize the presence of cas1, a universal CRISPR-associated gene and proxy for the Type II CRISPR1-Cas system, in environmental and non-clinical Enterococcus isolates. CRISPR1-cas1 was detected in approximately 33% of the 275 strains examined, and differences in CRISPR1 carriage between species was significant. Incidence of cas1 in E. hirae was 73%, nearly three times that of E. faecalis (23.6%) and 10 times more frequent than in E. durans (7.1%). Also, this is the first report of CRISPR1 presence in E. durans, as well as in the plant-associated species E. casseliflavus and E. sulfureus. Significant differences in CRISPR1-cas1 incidence among Enterococcus species support the hypothesis that there is a tradeoff between protection and adaptability. The differences in the habitats of enterococcal species may exert varying selective pressure that results in a species-dependent distribution of CRISPR-Cas systems.

Induction of resistance to Staphylococcus aureus in an environmental marine biofilm.

The study of environmental biofilms is complicated by the difficulty of working with them under lab conditions. Nonetheless, knowledge of cellular activity and interactions within environmental biofilms could lead to novel biomedical applications. As a first step in this direction we propose a novel technique for inducing resistance to Staphylococcus aureus (S. aureus) in an intact environmental biofilm. Agar plates were prepared with or without the addition of 20% S. aureus spent culture media and immersed in coastal seawater (Boston Harbor, Massachusetts, USA) for four days to grow up an environmental biofilm. Nucleopore filters inoculated with an overnight culture of S. aureus were then applied to the surface of the agar plates with the environmental biofilms, incubated 4h at 37°C, removed and subsequently stained and analyzed. Marine environmental biofilms grown on agar containing S. aureus spent culture media were significantly more inhibitory of S. aureus growth than were marine environmental biofilms grown on plain agar.

Plant age and genotype impact the progression of bacterial community succession in the Arabidopsis rhizosphere.

The rhizosphere is strongly influenced by plant-derived phytochemicals exuded by roots and plant species exert a major selective force for bacteria colonizing the root-soil interface. We have previously shown that rhizobacterial recruitment is tightly regulated by plant genetics, by showing that natural variants of Arabidopsis thaliana support genotype-specific rhizobacterial communities while also releasing a unique blend of exudates at six weeks post-germination. To further understand how exudate release is controlled by plants, changes in rhizobacterial assemblages of two Arabidopsis accessions, Cvi and Ler where monitored throughout the plants' life cycle. Denaturing gradient gel electrophoresis (DGGE) fingerprints revealed that bacterial communities respond to plant derived factors immediately upon germination in an accession-specific manner. Rhizobacterial succession progresses differently in the two accessions in a reproducible manner. However, as plants age, rhizobacterial and control bulk soil communities converge, indicative of an attenuated rhizosphere effect, which coincides with the expected slow down in the active release of root exudates as plants reach the end of their life cycle. These data strongly suggest that exudation changes during plant development are highly genotype-specific, possibly reflecting the unique, local co-evolutionary communication processes that developed between Arabidopsis accessions and their indigenous microbiota.

Influence of Arabidopsis thaliana accessions on rhizobacterial communities and natural variation in root exudates.

Plant species is considered to be one of the most important factors in shaping rhizobacterial communities, but specific plant-microbe interactions in the rhizosphere are still not fully understood. Arabidopsis thaliana, for which a large number of naturally occurring ecotype accessions exist, lacks mycorrhizal associations and is hence an ideal model for rhizobacterial studies. Eight Arabidopsis accessions were found to exert a marked selective influence on bacteria associated with their roots, as determined by terminal-restriction fragment length polymorphism (T-RFLP) and ribosomal intergenic spacer analysis (RISA). Community differences in species composition and relative abundance were both significant (P <0.001). The eight distinct and reproducible accession-dependent community profiles also differed from control bulk soil. Root exudates of these variants were analysed by high performance liquid chromatography (HPLC) to try to establish whether the unique rhizobacterial assemblages among accessions could be attributed to plant-regulated chemical changes in the rhizosphere. Natural variation in root exudation patterns was clearly exhibited, suggesting that differences in exudation patterns among accessions could be influencing bacterial assemblages. Other factors such as root system architecture are also probably involved. Finally, to investigate the Arabidopsis rhizosphere further, the phylogenetic diversity of rhizobacteria from accession Cvi-0 is described.

Differentiation of bacterial strains by thermal gradient gel electrophoresis using non-GC-clamped PCR primers for the 16S-23S rDNA intergenic spacer region.

The method for DNA fingerprinting of the 16S-23S rDNA intergenic spacer region was modified to increase resolution of bacterial strains by thermal gradient gel electrophoresis (TGGE) analysis. By utilizing the high melting temperature region of the tRNA gene located in the middle of the 16S-23S rDNA intergenic spacer region as an internal clamp for TGGE, multiple melting domain problems were solved. PCR primers lacking a stretch of GC-rich sequences (GC-clamp) amplified the intergenic spacer region more efficiently than GC-clamped primers. Therefore, PCR artifacts were avoided by using low, 17-cycle, PCR. The method was successfully applied to diverse bacterial species for strain differentiation by TGGE without requiring a special PCR primer set.