Bacteriophages against Vibrio coralliilyticus and Vibrio tubiashii: Isolation, Characterization, and Remediation of Larval Oyster Mortalities.

Vibrio coralliilyticus and Vibrio tubiashii are pathogens responsible for high larval oyster mortality rates in shellfish hatcheries. Bacteriophage therapy was evaluated to determine its potential to remediate these mortalities. Sixteen phages against V. coralliilyticus and V. tubiashii were isolated and characterized from Hawaiian seawater. Fourteen isolates were members of the Myoviridae family, and two were members of the Siphoviridae In proof-of-principle trials, a cocktail of five phages reduced mortalities of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) by up to 91% 6 days after challenge with lethal doses of V. coralliilyticus Larval survival depended on the oyster species, the quantities of phages and vibrios applied, and the species and strain of Vibrio A later-generation cocktail, designated VCP300, was formulated with three lytic phages subsequently named Vibrio phages vB_VcorM-GR7B, vB_VcorM-GR11A, and vB_VcorM-GR28A (abbreviated 7B, 11A, and 28A, respectively). Together, these three phages displayed host specificity toward eight V. coralliilyticus strains and a V. tubiashii strain. Larval C. gigas mortalities from V. coralliilyticus strains RE98 and OCN008 were significantly reduced by >90% (P < 0.0001) over 6 days with phage treatment compared to those of untreated controls. Genomic sequencing of phages 7B, 11A, and 28A revealed 207,758-, 194,800-, and 154,046-bp linear DNA genomes, respectively, with the latter showing 92% similarity to V. coralliilyticus phage YC, a strain from the Great Barrier Reef, Australia. Phage 7B and 11A genomes showed little similarity to phages in the NCBI database. This study demonstrates the promising potential for phage therapy to reduce larval oyster mortalities in oyster hatcheries.IMPORTANCE Shellfish hatcheries encounter episodic outbreaks of larval oyster mortalities, jeopardizing the economic stability of hatcheries and the commercial shellfish industry. Shellfish pathogens like Vibrio coralliilyticus and Vibrio tubiashii have been recognized as major contributors of larval oyster mortalities in U.S. East and West Coast hatcheries for many years. This study isolated, identified, and characterized bacteriophages against these Vibrio species and demonstrated their ability to reduce mortalities from V. coralliilyticus in larval Pacific oysters and from both V. coralliilyticus and V. tubiashii in larval Eastern oysters. Phage therapy offers a promising approach for stimulating hatchery production to ensure the well-being of hatcheries and the commercial oyster trade.

Mechanisms for Pseudoalteromonas piscicida-Induced Killing of Vibrios and Other Bacterial Pathogens.

Pseudoalteromonas piscicida is a Gram-negative gammaproteobacterium found in the marine environment. Three strains of pigmented P. piscicida were isolated from seawater and partially characterized by inhibition studies, electron microscopy, and analysis for proteolytic enzymes. Growth inhibition and death occurred around colonies of P. piscicida on lawns of the naturally occurring marine pathogens Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio cholerae, Photobacterium damselae, and Shewanella algae Inhibition also occurred on lawns of Staphylococcus aureus but not on Escherichia coli O157:H7 or Salmonella enterica serovar Typhimurium. Inhibition was not pH associated, but it may have been related to the secretion of a cysteine protease with strong activity, as detected with a synthetic fluorogenic substrate. This diffusible enzyme was secreted from all three P. piscicida strains. Direct overlay of the Pseudoalteromonas colonies with synthetic fluorogenic substrates demonstrated the activity of two aminopeptidase Bs, a trypsin-like serine protease, and enzymes reactive against substrates for cathepsin G-like and caspase 1-like proteases. In seawater cultures, scanning electron microscopy revealed numerous vesicles tethered to the outer surface of P. piscicida and a novel mechanism of direct transfer of these vesicles to V. parahaemolyticus Vesicles digested holes in V. parahaemolyticus cells, while the P. piscicida congregated around the vibrios in a predatory fashion. This transfer of vesicles and vesicle-associated digestion of holes were not observed in other bacteria, suggesting that vesicle binding may be mediated by host-specific receptors. In conclusion, we show two mechanisms by which P. piscicida inhibits and/or kills competing bacteria, involving the secretion of antimicrobial substances and the direct transfer of digestive vesicles to competing bacteria.IMPORTANCEPseudoalteromonas species are widespread in nature and reduce competing microflora by the production of antimicrobial compounds. We isolated three strains of P. piscicida and characterized secreted and cell-associated proteolytic enzymes, which may have antimicrobial properties. We identified a second method by which P. piscicida kills V. parahaemolyticus It involves the direct transfer of apparently lytic vesicles from the surface of the Pseudoalteromonas strains to the surface of Vibrio cells, with subsequent digestion of holes in the Vibrio cell walls. Enzymes associated with these vesicles are likely responsible for the digestion of holes in the cell walls. Pseudoalteromonas piscicida has potential applications in aquaculture and food safety, in control of the formation of biofilms in the environment, and in food processing. These findings may facilitate the probiotic use of P. piscicida to inactivate pathogens and may lead to the isolation of enzymes and other antimicrobial compounds of pharmacological value.

Mortalities of Eastern and Pacific oyster Larvae caused by the pathogens Vibrio coralliilyticus and Vibrio tubiashii.

Vibrio tubiashii is reported to be a bacterial pathogen of larval Eastern oysters (Crassostrea virginica) and Pacific oysters (Crassostrea gigas) and has been associated with major hatchery crashes, causing shortages in seed oysters for commercial shellfish producers. Another bacterium, Vibrio coralliilyticus, a well-known coral pathogen, has recently been shown to elicit mortality in fish and shellfish. Several strains of V. coralliilyticus, such as ATCC 19105 and Pacific isolates RE22 and RE98, were misidentified as V. tubiashii until recently. We compared the mortalities caused by two V. tubiashii and four V. coralliilyticus strains in Eastern and Pacific oyster larvae. The 50% lethal dose (LD50) of V. coralliilyticus in Eastern oysters (defined here as the dose required to kill 50% of the population in 6 days) ranged from 1.1 × 10(4) to 3.0 × 10(4) CFU/ml seawater; strains RE98 and RE22 were the most virulent. This study shows that V. coralliilyticus causes mortality in Eastern oyster larvae. Results for Pacific oysters were similar, with LD50s between 1.2 × 10(4) and 4.0 × 10(4) CFU/ml. Vibrio tubiashii ATCC 19106 and ATCC 19109 were highly infectious toward Eastern oyster larvae but were essentially nonpathogenic toward healthy Pacific oyster larvae at dosages of ≥1.1 × 10(4) CFU/ml. These data, coupled with the fact that several isolates originally thought to be V. tubiashii are actually V. coralliilyticus, suggest that V. coralliilyticus has been a more significant pathogen for larval bivalve shellfish than V. tubiashii, particularly on the U.S. West Coast, contributing to substantial hatchery-associated morbidity and mortality in recent years.

DNA sequence and analysis of a 90.1-kb plasmid in Shiga toxin-producing Escherichia coli (STEC) O145:NM 83-75.

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O145 are important emerging food-borne pathogens responsible for sporadic cases and outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. A large plasmid carried by STEC O145:NM strain 83-75 and named pO145-NM was sequenced, and the genes were annotated. pO145-NM is 90,103bp in size and carries 89 open reading frames. Four genes/regions in pO145-NM encode for STEC virulence factors, including toxB (protein involved in adherence), espP (a serine protease), katP (catalase peroxidase), and the hly (hemolysin) gene cluster. These genes have also been identified in large virulence plasmids found in other STEC serogroups, including O26, O157, O111, and O103. pO145-NM carries the espPα subtype that is associated with STEC strains that cause more severe disease. Phylogenetic analyses of HlyB, EspP, and ToxB in various STEC strains showed a high degree of similarity of these proteins in E. coli serotypes O145:NM, O26:H11/H-, O111:NM/H-, and O157:H7 potentially placing these STEC into a related group.

Cloning, sequencing, and characterization of lipase genes from a polyhydroxyalkanoate (PHA)-synthesizing Pseudomonas resinovorans.

Lipase (lip) and lipase-specific foldase (lif) genes of a biodegradable polyhydroxyalkanoate (PHA)-synthesizing Pseudomonas resinovorans NRRL B-2649 were cloned using primers based on consensus sequences, followed by polymerase chain reaction-based genome walking. Sequence analyses showed a putative Lip gene product (314 amino acids, a.a.) with its catalytic active site (Ser(111), Asp(258), and His(280)) identified. The foldase lif gene that is located 55 bp downstream of lip codes for a putative Lif (345 a.a.). To verify the biological function of the cloned lip gene for lipase expression in P. resinovorans, we constructed a lip knock-out mutant (lip::Tn5) by transposon insertion. Complementation of the lip knock-out P. resinovorans mutant with a lipase expression plasmid (pBS29-P2-lip) was performed, and its effect on lipase expression was investigated. The wild-type P. resinovorans and the lip::Tn5[pBS29-P2-lip] recombinant (but not the lip::Tn5 mutant) showed fluorescence on rhodamine B plates indicative of lipase activity. The wild type exhibited extracellular lipase activity when grown on medium containing triacylglycerol substrates (tallow, olive oil, and tributyrin) as sole carbon sources, but the lip::Tn5 mutant did not show such activity. Lipase activity of various strains was also confirmed by TLC analysis of the composition of acylglycerols and free fatty acid in the extracts of the spent culture medium. We further found that tributyrin was more effective than olive oil in inducing lipase expression in P. resinovorans.

The complete DNA sequence and analysis of the virulence plasmid and of five additional plasmids carried by Shiga toxin-producing Escherichia coli O26:H11 strain H30.

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O26 have been associated with sporadic cases and outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. In addition to chromosomal virulence genes, STEC strains usually harbor a large plasmid that carries genes associated with pathogenicity. The complete nucleotide sequence and genetic organization of 6 plasmids carried by STEC O26:H11 strain H30 were determined. The large virulence plasmid (pO26-Vir) was approximately 168 kb in size and contained 196 open reading frames (ORFs). pO26-Vir possesses a mosaic structure and shows similarity to the virulence plasmids in locus of enterocyte effacement (LEE)-negative STEC O113:H21 EH41 (pO113), in E. coli clinical strain C1096 (pSERB1), and in E. coli O157:H7 RIMD 0509952 (pO157). Plasmid pO26-Vir shares several highly conserved regions with pO157 and carries important virulence genes, including toxB, katP, espP, and the hly gene cluster. In addition, pO26-Vir possesses genes encoding for type IV pili (pilL-V). The second largest plasmid, pO26-L (73 kb) contains 101 ORFs. pO26-L carries the tetracycline resistance gene and has regions that show similarity to the E. coli conjugative resistance plasmid NR1. The third largest plasmid, pO26-S4 (5.8 kb), is homologous to the ColE2 colicinogenic plasmid that encodes for colicin E2. The remaining 3 plasmids, pO26-S1 (1.5 kb), pO26-S2 (3.1 kb), and pO26-S3 (4.2 kb), carry very little genetic information except for putative proteins involved in plasmid replication and DNA maintenance. The data presented underscore the diversity among the STEC virulence plasmids and provide insights into the evolution of these plasmids in STEC strains that cause serious human illness.

From ontology selection and semantic web to an integrated information system for food-borne diseases and food safety.

Several factors have hindered effective use of information and resources related to food safety due to inconsistency among semantically heterogeneous data resources, lack of knowledge on profiling of food-borne pathogens, and knowledge gaps among research communities, government risk assessors/managers, and end-users of the information. This paper discusses technical aspects in the establishment of a comprehensive food safety information system consisting of the following steps: (a) computational collection and compiling publicly available information, including published pathogen genomic, proteomic, and metabolomic data; (b) development of ontology libraries on food-borne pathogens and design automatic algorithms with formal inference and fuzzy and probabilistic reasoning to address the consistency and accuracy of distributed information resources (e.g., PulseNet, FoodNet, OutbreakNet, PubMed, NCBI, EMBL, and other online genetic databases and information); (c) integration of collected pathogen profiling data, Foodrisk.org ( http://www.foodrisk.org ), PMP, Combase, and other relevant information into a user-friendly, searchable, "homogeneous" information system available to scientists in academia, the food industry, and government agencies; and (d) development of a computational model in semantic web for greater adaptability and robustness.

A Targeted Sequencing Assay for Serotyping Escherichia coli Using AgriSeq Technology.

The gold standard method for serotyping Escherichia coli has relied on antisera-based typing of the O- and H-antigens, which is labor intensive and often unreliable. In the post-genomic era, sequence-based assays are potentially faster to provide results, could combine O-serogrouping and H-typing in a single test, and could simultaneously screen for the presence of other genetic markers of interest such as virulence factors. Whole genome sequencing is one approach; however, this method has limited multiplexing capabilities, and only a small fraction of the sequence is informative for subtyping or identifying virulence potential. A targeted, sequence-based assay and accompanying software for data analysis would be a great improvement over the currently available methods for serotyping. The purpose of this study was to develop a high-throughput, molecular method for serotyping E. coli by sequencing the genes that are required for production of O- and H-antigens, as well as to develop software for data analysis and serotype identification. To expand the utility of the assay, targets for the virulence factors, Shiga toxins (stx 1, and stx 2) and intimin (eae) were included. To validate the assay, genomic DNA was extracted from O-serogroup and H-type standard strains and from Shiga toxin-producing E. coli, the targeted regions were amplified, and then sequencing libraries were prepared from the amplified products followed by sequencing of the libraries on the Ion S5™ sequencer. The resulting sequence files were analyzed via the SeroType Caller™ software for identification of O-serogroup, H-type, and presence of stx 1 , stx 2, and eae. We successfully identified 169 O-serogroups and 41 H-types. The assay also routinely detected the presence of stx 1a,c,d (3 of 3 strains), stx 2c-e,g (8 of 8 strains), stx 2f (1 strain), and eae (6 of 6 strains). Taken together, the high-throughput, sequence-based method presented here is a reliable alternative to antisera-based serotyping methods for E. coli.

Complete Genome Sequences of Bacteriocin-Producing Streptococcus thermophilus Strains ST106 and ST109.

Streptococcus thermophilus strains ST106 and ST109 produce broad-spectrum bacteriocins encoded within a bacteriocin-like peptide (blp) gene cluster. This study reports the complete genome sequences for both strains, with the ST109 chromosome containing 1,788,866 nucleotides (nt) and 1,572 predicted genes, and ST106 having 1,856,083 nt and 1,601 predicted genes.

Whole-Genome Sequences of Two Pseudoalteromonas piscicida Strains, DE1-A and DE2-A, with Strong Antibacterial Activity against Vibrio vulnificus.

Highly vesiculated Pseudoalteromonas piscicida strains DE1-A and DE2-A were isolated from seawater and show bactericidal properties toward Vibrio vulnificus and other Gram-positive and Gram-negative bacteria. Here, we report the complete genome sequences of these two P. piscicida strains and identify proteolytic enzymes potentially involved in their antibacterial properties.

Broiler Campylobacter contamination and human campylobacteriosis in Iceland.

To examine whether there is a relationship between the degree of Campylobacter contamination observed in product lots of retail Icelandic broiler chicken carcasses and the incidence of human disease, 1,617 isolates from 327 individual product lots were genetically matched (using the flaA short variable region [SVR[) to 289 isolates from cases of human campylobacteriosis whose onset was within approximately 2 weeks from the date of processing. When there was genetic identity between broiler isolates and human isolates within the appropriate time frame, a retail product lot was classified as implicated in human disease. According to the results of this analysis, there were multiple clusters of human disease linked to the same process lot or lots. Implicated and nonimplicated retail product lots were compared for four lot descriptors: lot size, prevalence, mean contamination, and maximum contamination (as characterized by direct rinse plating). For retail product distributed fresh, Mann-Whitney U tests showed that implicated product lots had significantly (P = 0.0055) higher mean contamination than nonimplicated lots. The corresponding median values were 3.56 log CFU/carcass for implicated lots and 2.72 log CFU/carcass for nonimplicated lots. For frozen retail product, implicated lots were significantly (P = 0.0281) larger than nonimplicated lots. When the time frame was removed, retail product lots containing Campylobacter flaA SVR genotypes also seen in human disease had significantly higher mean and maximum contamination numbers than lots containing no genotypes seen in human disease for both fresh and frozen product. Our results suggest that cases of broiler-borne campylobacteriosis may occur in clusters and that the differences in mean contamination levels may provide a basis for regulatory action that is more specific than a presence-absence standard.

Draft Genomic Sequencing of Six Potential Extraintestinal Pathogenic Escherichia coli Isolates from Retail Chicken Meat.

Potential extraintestinal pathogenic Escherichia coli strains DP254, WH333, WH398, F356, FEX675, and FEX725 were isolated from retail chicken meat products. Here, we report the draft genome sequences for these six E. coli isolates, which are currently being used in food safety research.

Draft Genome Sequences of Four Uropathogenic Escherichia coli Serotype O4:H5 Isolates (ATCC 700414, 700415, 700416, and 700417).

Uropathogenic Escherichia coli serotype O4:H5 isolates (ATCC 700414, 700415, 700416, and 700417) were recovered from women with first-time urinary tract infections. Here, we report the draft genome sequences for these four E. coli isolates, which are currently being used to validate food safety processing technologies.

Complete Genome Sequence of Vibrio coralliilyticus RE22, a Marine Bacterium Pathogenic toward Larval Shellfish.

Vibrio coralliilyticus RE22 is an indigenous marine pathogen that infects larval bivalve shellfish. This strain is particularly problematic in oyster hatcheries, where it causes high larval mortality. It contains two circular chromosomes and one megaplasmid. Annotation reveals multiple genes which may encode important virulence factors.

Complete Genome Sequence of Pseudoalteromonas piscicida Strain DE2-B, a Bacterium with Broad Inhibitory Activity toward Human and Fish Pathogens.

Pseudoalteromonas piscicida strain DE2-B is a halophilic bacterium which has broad inhibitory activity toward vibrios and other human and fish pathogens. We report the first closed genome sequence for this species, which consists of two chromosomes (4,128,210 and 1,188,838 bp). Annotation revealed multiple genes encoding proteases with potential antibacterial properties.

Complete Genome Sequence of Streptococcus thermophilus Strain B59671, Which Naturally Produces the Broad-Spectrum Bacteriocin Thermophilin 110.

Streptococcus thermophilus strain B59671 is a Gram-positive lactic acid bacterium that naturally produces a broad-spectrum bacteriocin, thermophilin 110, and is capable of producing gamma-aminobutyric acid (GABA). The complete genome sequence for this strain contains 1,821,173 nucleotides, 1,936 predicted genes, and an average G+C content of 39.1%.

Evaluation of methods for sequence analysis of highly repetitive DNA templates.

The DNA Sequencing Research Group (DSRG) of the ABRF conducted a study to assess the ability of DNA sequencing core facilities to successfully sequence a set of well-defined templates containing difficult repeats. The aim of this study was to determine whether repetitive templates could be sequenced accurately by using equipment and chemistries currently utilized in participating sequencing laboratories. The effects of primer and template concentrations, sequencing chemistries, additives, and instrument formats on the ability to successfully sequence repeat elements were examined. The first part of this study was an analysis of the results of 361 chromatograms from participants representing 40 different laboratories who attempted to sequence a panel of difficult-to-sequence templates using their best in-house protocols. The second part of this study was a smaller multi-laboratory evaluation of a single robust protocol with the same panel of templates. This study provides a measure of the potential success of different approaches to sequencing across homopolymer tracts and repetitive elements.

Advances in Molecular Serotyping and Subtyping of Escherichia coli.

Escherichia coli plays an important role as a member of the gut microbiota; however, pathogenic strains also exist, including various diarrheagenic E. coli pathotypes and extraintestinal pathogenic E. coli that cause illness outside of the GI-tract. E. coli have traditionally been serotyped using antisera against the ca. 186 O-antigens and 53 H-flagellar antigens. Phenotypic methods, including bacteriophage typing and O- and H- serotyping for differentiating and characterizing E. coli have been used for many years; however, these methods are generally time consuming and not always accurate. Advances in next generation sequencing technologies have made it possible to develop genetic-based subtyping and molecular serotyping methods for E. coli, which are more discriminatory compared to phenotypic typing methods. Furthermore, whole genome sequencing (WGS) of E. coli is replacing established subtyping methods such as pulsed-field gel electrophoresis, providing a major advancement in the ability to investigate food-borne disease outbreaks and for trace-back to sources. A variety of sequence analysis tools and bioinformatic pipelines are being developed to analyze the vast amount of data generated by WGS and to obtain specific information such as O- and H-group determination and the presence of virulence genes and other genetic markers.

Comparison of O-Antigen Gene Clusters of All O-Serogroups of Escherichia coli and Proposal for Adopting a New Nomenclature for O-Typing.

Escherichia coli strains are classified based on O-antigens that are components of the lipopolysaccharide (LPS) in the cell envelope. O-antigens are important virulence factors, targets of both the innate and adaptive immune system, and play a role in host-pathogen interactions. Because they are highly immunogenic and display antigenic specificity unique for each strain, O-antigens are the biomarkers for designating O-types. Immunologically, 185 O-serogroups and 11 OX-groups exist for classification. Conventional serotyping for O-typing entails agglutination reactions between the O-antigen and antisera generated against each O-group. The procedure is labor intensive, not always accurate, and exhibits equivocal results. In this report, we present the sequences of 71 O-antigen gene clusters (O-AGC) and a comparison of all 196 O- and OX-groups. Many of the designated O-types, applied for classification over several decades, exhibited similar nucleotide sequences of the O-AGCs and cross-reacted serologically. Some O-AGCs carried insertion sequences and others had only a few nucleotide differences between them. Thus, based on these findings, it is proposed that several of the E. coli O-groups may be merged. Knowledge of the O-AGC sequences facilitates the development of molecular diagnostic platforms that are rapid, accurate, and reliable that can replace conventional serotyping. Additionally, with the scientific knowledge presented, new frontiers in the discovery of biomarkers, understanding the roles of O-antigens in the innate and adaptive immune system and pathogenesis, the development of glycoconjugate vaccines, and other investigations, can be explored.

Correction: Comparison of O-Antigen Gene Clusters of All O-Serogroups of Escherichia coli and Proposal for Adopting a New Nomenclature for O-Typing.

[This corrects the article DOI: 10.1371/journal.pone.0147434.].