Draft Genome Sequence of Lactococcus lactis Strain PrHT3, Isolated from Organic Basil.

Here, we report the draft genome sequence of Lactococcus lactis strain PrHT3, which was isolated from organic basil. This strain possesses one chromosome and two plasmids. This strain possesses potential probiotic characteristics.

Draft Genome Sequences of the Vibrio parahaemolyticus Strains VHT1 and VHT2, Pasteurization-Resistant Isolates from Environmental Seafood.

Two pasteurization-resistant strains, VHT1 and VHT2, of environmental, viable but nonculturable, pathogenic Vibrio parahaemolyticus were isolated from environmental oysters. Their whole-genome sequences were constructed. The genome sizes for VHT1 and VHT2 are 5.11 Mbp and 5.26 Mbp, respectively.

Recovery of Pasteurization-Resistant Vibrio parahaemolyticus from Seafoods Using a Modified, Two-Step Enrichment.

Persistent Vibrio-parahaemolyticus-associated vibriosis cases, attributed, in part, to the inefficient techniques for detecting viable-but-non-culturable (VBNC) Vibrio pathogens and the ingestion of undercooked seafood, is the leading cause of bacterial seafood-borne outbreaks, hospitalizations, and deaths in the United States. The effect of extreme heat processing on Vibrio biology and its potential food safety implication has been underexplored. In the present work, environmental samples from the wet market, lagoon, and estuarine environments were analyzed for V. parahaemolyticus recovery using a modified, temperature-dependent, two-step enrichment method followed by culture-based isolation, phenotype, and genotype characterizations. The work recovered novel strains (30% of 12 isolates) of V. parahaemolyticus from prolonged-heat-processing conditions (80 °C, 20 min), as confirmed by 16S rDNA bacterial identification. Select strains, VHT1 and VHT2, were determined to be hemolysis- and urease-positive pathogens. PCR analyses of chromosomal DNA implicated the tdh-independent, tlh-associated hemolysis in these strains. Both strains exhibited significant, diverse antibiotic profiles (p < 0.05). Turbidimetric and viable count assays revealed the pasteurization-resistant V. parahaemolyticus VHT1/VHT2 (62 °C, 8 h). These findings disclose the efficiency of Vibrio extremist recovery by the modified, two-step enrichment technique and improve knowledge of Vibrio biology essential to food safety reformation.

RT-qPCR Analysis of 15 Genes Encoding Putative Surface Proteins Involved in Adherence of Listeria monocytogenes.

L. monocytogenes adherence to food-associated abiotic surfaces and the development of biofilms as one of the underlying reasons for the contamination of ready-to-eat products is well known. The over-expression of internalins that improves adherence has been noted in cells growing as attached cells or at elevated incubation temperatures. However, the role of other internalin-independent surface proteins as adhesins has been uncharacterized to date. Using two strains each of weakly- and strongly-adherent L. monocytogenes as platforms for temperature-dependent adherence assays and targeted mRNA analyses, these observations (i.e., sessile- and/or temperature-dependent gene expression) were further investigated. Microplate fluorescence assays of both surface-adherent strains exhibited significant (P < 0.05) adherence at higher incubation temperature (42 °C). Of the 15 genes selected for RT-qPCR, at least ten gene transcripts recovered from cells (weakly-adherent strain CW35, strongly-adherent strain 99-38) subject to various growth conditions were over expressed [planktonic/30 °C (10), sessile/30 °C (12), planktonic/42 °C (10)] compared to their internal control (16SrRNA transcripts). Of four genes overexpressed in all three conditions tested, three and one were implicated as virulence factors and unknown function, respectively. PCR analysis of six unexpressed genes revealed that CW35 possessed an altered genome. The results suggest the presence of other internalin-independent adhesins (induced by growth temperature and/or substratum) and that a group of suspect protein members are worthy of further analysis for their potential role as surface adhesins. Analysis of the molecular basis of adherence properties of isolates of L. monocytogenes from food-associated facilities may help identify sanitation regimens to prevent cell attachment and biofilm formation on abiotic surfaces that could play a role in reducing foodborne illness resulting from Listeria biofilms.

Comparison of Surface Proteomes of Adherence Variants of Listeria Monocytogenes Using LC-MS/MS for Identification of Potential Surface Adhesins.

The ability of Listeria monocytogenes to adhere and form biofilms leads to persistence in food processing plants and food-associated listeriosis. The role of specific surface proteins as adhesins to attach Listeria cells to various contact surfaces has not been well characterized to date. In prior research comparing different methods for surface protein extraction, the Ghost urea method revealed cleaner protein content as verified by the least cytoplasmic protein detected in surface extracts using LC-MS/MS. The same technique was utilized to extract and detect surface proteins among two surface-adherent phenotypic strains of L. monocytogenes (i.e., strongly and weakly adherent). Of 640 total proteins detected among planktonic and sessile cells, 21 protein members were exclusively detected in the sessile cells. Relative LC-MS/MS detection and quantification of surface-extracted proteins from the planktonic weakly adherent (CW35) and strongly adherent strains (99-38) were examined by protein mass normalization of proteins. We found that L. monocytogenes 99-38 exhibited a total of 22 surface proteins that were over-expressed: 11 proteins were detected in surface extracts of both sessile and planktonic 99-38 that were ≥5-fold over-expressed while another 11 proteins were detected only in planktonic 99-38 cells that were ≥10-fold over-expressed. Our results suggest that these protein members are worthy of further investigation for their involvement as surface adhesins.

Comparison of five methods for direct extraction of surface proteins from Listeria monocytogenes for proteomic analysis by orbitrap mass spectrometry.

Extracts of surface proteins, with minimal artifacts from contaminating cytosolic components, are highly desirable for investigating surface factors involved in the attachment and formation of biofilms by bacteria that are problematic in commercial food processing facilities. In this study, we compared the protein profiles of the food pathogen, Listeria monocytogenes, recovered after applying different surface protein extraction methods compiled from the literature: trypsin-enzymatic shaving with BICAM/sucrose or Tris/sucrose buffers (Tryp B+S, Tryp T+S), Tris-buffered urea (UB), lithium chloride (LiCl) and Tris-buffered urea applied with hypotonic-stressed cells (UB-Ghost), and subjected them to liquid chromatography tandem mass spectrometry and protein identification. The data indicate that the UB-Ghost extraction method provides a cleaner extract of surface proteins including the predicted (this study and the literature) or validated members (literature) from L. monocytogenes. This was determined by an accumulative lower unique peptide number exhibited by mass spectrometry for total cytoplasmic proteins among different surface extracts, with a majority of proteins demonstrating hydrophilic properties. The extracted proteins were from different functional categories and have associations with the cell surface, intermediary metabolism, information pathways, or functionally unknown proteins as suggested by in silico analyses performed by other groups (Leger and ListiList). The utilization of an optimized method for surface protein extraction should greatly facilitate identification by LC-MS/MS that could be useful to anyone working on molecular proteomics of bacterial surfaces.