A hybrid reference-guided de novo assembly approach for generating Cyclospora mitochondrion genomes.

Cyclospora cayetanensis is a coccidian parasite associated with large and complex foodborne outbreaks worldwide. Linking samples from cyclosporiasis patients during foodborne outbreaks with suspected contaminated food sources, using conventional epidemiological methods, has been a persistent challenge. To address this issue, development of new methods based on potential genomically-derived markers for strain-level identification has been a priority for the food safety research community. The absence of reference genomes to identify nucleotide and structural variants with a high degree of confidence has limited the application of using sequencing data for source tracking during outbreak investigations. In this work, we determined the quality of a high resolution, curated, public mitochondrial genome assembly to be used as a reference genome by applying bioinformatic analyses. Using this reference genome, three new mitochondrial genome assemblies were built starting with metagenomic reads generated by sequencing DNA extracted from oocysts present in stool samples from cyclosporiasis patients. Nucleotide variants were identified in the new and other publicly available genomes in comparison with the mitochondrial reference genome. A consolidated workflow, presented here, to generate new mitochondrion genomes using our reference-guided de novo assembly approach could be useful in facilitating the generation of other mitochondrion sequences, and in their application for subtyping C. cayetanensis strains during foodborne outbreak investigations.

Genome Sequences of Malonate-Positive Cronobacter sakazakii Serogroup O:2, Sequence Type 64 Strains CDC 1121-73 and GK1025, Isolated from Human Bronchial Wash and a Powdered Infant Formula Manufacturing Plant.

We introduce draft genome sequences of strains CDC1121-73 (human bronchial wash isolate) and GK1025 (powdered infant formula manufacturing facility isolate), which are both malonate-positive Cronobacter sakazakii serogroup O:2, sequence type 64. Assemblies for these strains have sizes of 4,442,307 and 4,599,266 bp and % G+C contents of 56.9 and 56.7, respectively.

The Pathogen-annotated Tracking Resource Network (PATRN) system: a web-based resource to aid food safety, regulatory science, and investigations of foodborne pathogens and disease.

Investigation of foodborne diseases requires the capture and analysis of time-sensitive information on microbial pathogens that is derived from multiple analytical methods and sources. The web-based Pathogen-annotated Tracking Resource Network (PATRN) system (www.patrn.net) was developed to address the data aggregation, analysis, and communication needs important to the global food safety community for the investigation of foodborne disease. PATRN incorporates a standard vocabulary for describing isolate metadata and provides a representational schema for a prototypic data exchange standard using a novel data loading wizard for aggregation of assay and attribution information. PATRN currently houses expert-curated, high-quality "foundational datasets" consisting of published experimental results from conventional assays and next generation analysis platforms for isolates of Escherichia coli, Listeria monocytogenes, and Salmonella, Shigella, Vibrio and Cronobacter species. A suite of computational tools for data mining, clustering, and graphical representation is available. Within PATRN, the public curated data repository is complemented by a secure private workspace for user-driven analyses, and for sharing data among collaborators. To demonstrate the data curation, loading wizard features, and analytical capabilities of PATRN, three use-case scenarios are presented. Use-case scenario one is a comparison of the distribution and prevalence of plasmid-encoded virulence factor genes among 249 Cronobacter strains with similar attributes to that of nine Cronobacter isolates from recent cases obtained between March and October, 2010-2011. To highlight PATRN's data management and trend finding tools, analysis of datasets, stored in PATRN as part of an ongoing surveillance project to identify the predominant molecular serogroups among Cronobacter sakazakii isolates observed in the USA is shown. Use-case scenario two demonstrates the secure workspace available for private users to upload and analyze sensitive data, and for collating cross-platform datasets to identify and validate congruent datapoints. SNP datasets from WGS assemblies and pan-genome microarrays are analyzed in a combinatorial fashion to determine relatedness of 33 Salmonella enterica strains to six strains collected as part of an outbreak investigation. Use-case scenario three utilizes published surveillance results that describe the incidence and sources of O157:H7 E. coli isolates associated with a produce pre-harvest surveillance study that occurred during 2002-2006. In summary, PATRN is a web-based integrated platform containing tools for the management, analysis and visualization of data about foodborne pathogens.

Multiplex PCR assay targeting a diguanylate cyclase-encoding gene, cgcA, to differentiate species within the genus Cronobacter.

In a comparison to the widely used Cronobacter rpoB PCR assay, a highly specific multiplexed PCR assay based on cgcA, a diguanylate cyclase gene, that identified all of the targeted six species among 305 Cronobacter isolates was designed. This assay will be a valuable tool for identifying suspected Cronobacter isolates from food-borne investigations.

Identification and characterization of Cronobacter iron acquisition systems.

Cronobacter spp. are emerging pathogens that cause severe infantile meningitis, septicemia, or necrotizing enterocolitis. Contaminated powdered infant formula has been implicated as the source of Cronobacter spp. in most cases, but questions still remain regarding the natural habitat and virulence potential for each strain. The iron acquisition systems in 231 Cronobacter strains isolated from different sources were identified and characterized. All Cronobacter spp. have both the Feo and Efe systems for acquisition of ferrous iron, and all plasmid-harboring strains (98%) have the aerobactin-like siderophore, cronobactin, for transport of ferric iron. All Cronobacter spp. have the genes encoding an enterobactin-like siderophore, although it was not functional under the conditions tested. Furthermore, all Cronobacter spp. have genes encoding five receptors for heterologous siderophores. A ferric dicitrate transport system (fec system) is encoded specifically by a subset of Cronobacter sakazakii and C. malonaticus strains, of which a high percentage were isolated from clinical samples. Phylogenetic analysis confirmed that the fec system is most closely related to orthologous genes present in human-pathogenic bacterial strains. Moreover, all strains of C. dublinensis and C. muytjensii encode two receptors, FcuA and Fct, for heterologous siderophores produced by plant pathogens. Identification of putative Fur boxes and expression of the genes under iron-depleted conditions revealed which genes and operons are components of the Fur regulon. Taken together, these results support the proposition that C. sakazakii and C. malonaticus may be more associated with the human host and C. dublinensis and C. muytjensii with plants.

Cronobacter species (formerly known as Enterobacter sakazakii) in powdered infant formula: a review of our current understanding of the biology of this bacterium.

Cronobacter species (formerly known as Enterobacter sakazakii) are opportunistic pathogens that can cause necrotizing enterocolitis, bacteraemia and meningitis, predominantly in neonates. Infection in these vulnerable infants has been linked to the consumption of contaminated powdered infant formula (PIF). Considerable research has been undertaken on this organism in the past number of years which has enhanced our understanding of this neonatal pathogen leading to improvements in its control within the PIF production environment. The taxonomy of the organism resulted in the recognition of a new genus, Cronobacter, which consists of seven species. This paper presents an up-to-date review of our current knowledge of Cronobacter species. Taxonomy, genome sequencing, current detection protocols and epidemiology are all discussed. In addition, consideration is given to the control of this organism in the manufacturing environment, as a first step towards reducing the occurrence of this pathogen in PIF.

Molecular characterization of Cronobacter lipopolysaccharide O-antigen gene clusters and development of serotype-specific PCR assays.

Cronobacter (formerly Enterobacter sakazakii) is a recently defined genus consisting of six species, C. sakazakii, C. malonaticus, C. dublinensis, C. muytjensii, C. turicensis, and Cronobacter genomospecies 1. In this study, MboII restriction fragment length polymorphism (RFLP) patterns of O-antigen gene clusters, located between galF and gnd, were used to identify serotypes in Cronobacter spp. Seven O-antigen RFLP clusters were generated, including three C. sakazakii clusters, previously identified as serotypes O1, O2, and O3. The O-antigen regions of six strains with unique RFLP patterns, including two C. sakazakii strains, two C. malonaticus strains, one C. turicensis strain, and one C. muytjensii strain, revealed three O-antigen gene clusters shared among Cronobacter species. PCR assays were developed, targeting the wzx O-antigen polymerase gene, and used to screen 231 Cronobacter strains to determine the frequency of these newly identified serotypes.

Characterization of putative virulence genes on the related RepFIB plasmids harbored by Cronobacter spp.

Cronobacter spp. are emerging neonatal pathogens that cause meningitis, sepsis, and necrotizing enterocolitis. The genus Chronobacter consists of six species: C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, and Cronobacter genomospecies group 1. Whole-genome sequencing of C. sakazakii BAA-894 and C. turicensis z3032 revealed that they harbor similarly sized plasmids identified as pESA3 (131 kb) and pCTU1 (138 kb), respectively. In silico analysis showed that both plasmids encode a single RepFIB-like origin of replication gene, repA, as well as two iron acquisition systems (eitCBAD and iucABCD/iutA). In a chrome azurol S agar diffusion assay, it was demonstrated that siderophore activity was associated with the presence of pESA3 or pCTU1. Additionally, pESA3 contains a cpa (Cronobacter plasminogen activator) gene and a 17-kb type 6 secretion system (T6SS) locus, while pCTU1 contains a 27-kb region encoding a filamentous hemagglutinin gene (fhaB), its specifc transporter gene (fhaC), and associated putative adhesins (FHA locus), suggesting that these are virulence plasmids. In a repA-targeted PCR assay, 97% of 229 Cronobacter species isolates were found to possess a homologous RepFIB plasmid. All repA PCR-positive strains were also positive for the eitCBAD and iucABCD/iutA iron acquisition systems. However, the presence of cpa, T6SS, and FHA loci depended on species, demonstrating a strong correlation with the presence of virulence traits, plasmid type, and species. These results support the hypothesis that these plasmids have evolved from a single archetypical plasmid backbone through the cointegration, or deletion, of specific virulence traits in each species.

Cpa, the outer membrane protease of Cronobacter sakazakii, activates plasminogen and mediates resistance to serum bactericidal activity.

Cronobacter spp. are emerging neonatal pathogens in humans, associated with outbreaks of meningitis and sepsis. To cause disease, they must survive in blood and invade the central nervous system by penetrating the blood-brain barrier. C. sakazakii BAA-894 possesses an ~131-kb plasmid (pESA3) that encodes an outer membrane protease (Cpa) that has significant identity to proteins that belong to the Pla subfamily of omptins. Members of this subfamily of proteins degrade a number of serum proteins, including circulating complement, providing protection from the complement-dependent serum killing. Moreover, proteins of the Pla subfamily can cause uncontrolled plasmin activity by converting plasminogen to plasmin and inactivating the plasmin inhibitor α2-antiplasmin (α2-AP). These reactions enhance the spread and invasion of bacteria in the host. In this study, we found that an isogenic cpa mutant showed reduced resistance to serum in comparison to its parent C. sakazakii BAA-894 strain. Overexpression of Cpa in C. sakazakii or Escherichia coli DH5α showed that Cpa proteolytically cleaved complement components C3, C3a, and C4b. Furthermore, a strain of C. sakazakii overexpressing Cpa caused a rapid activation of plasminogen and inactivation of α2-AP. These results strongly suggest that Cpa may be an important virulence factor involved in serum resistance, as well as in the spread and invasion of C. sakazakii.

Isolation and characterization of Vibrio tubiashii outer membrane proteins and determination of a toxR homolog.

Outer membrane proteins (OMPs) expressed by Vibrio tubiashii under different environmental growth conditions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and PCR analyses. Results showed the presence of a 38- to 40-kDa OmpU-like protein and ompU gene, a maltoporin-like protein, several novel OMPs, and a regulatory toxR homolog.

Characterization of the zinc-containing metalloprotease encoded by zpx and development of a species-specific detection method for Enterobacter sakazakii.

Enterobacter sakazakii causes a severe form of neonatal meningitis that occurs as sporadic cases as well as outbreaks. The disease has been epidemiologically associated with consumption of reconstituted, dried infant formulas. Very little information is available regarding pathogenicity of the organism and production of virulence factors. Clinical and environmental strains were screened for production of factors which have activity against Chinese hamster ovary (CHO) cells in tissue culture. Polymyxin B lysate and sonicate preparations but not culture supernatants from the strains caused "rounding" of CHO cells. Subsequent studies showed that the CHO cell-rounding factor is a proteolytic enzyme that has activity against azocasein. The cell-bound protease was isolated by using a combination of polymyxin B lysis, followed by sonication of cells harvested from tryptone broth. The protease was purified to homogeneity by sequential ammonium sulfate precipitation, gel filtration chromatography with Sephadex G-100, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, and a second gel filtration with Sephadex G-100. In addition to activity against azocasein, the purified protease also exhibits activity against azocoll and insoluble casein but not elastin. The protease has a molecular weight of 38,000 and an isoelectric point of 4.4. It is heat labile and for maximal activity against azocasein has an optimum temperature of 37 degrees C and a pH range of 5 to 7. Proteolytic activity is inhibited by ortho-phenanthroline and Zincov but is not affected by phenylmethylsulfonyl fluoride, N-ethylmaleimide, and trypsin inhibitors, which demonstrates that the protease is a zinc-containing metalloprotease. The metalloprotease does not hemagglutinate chicken or sheep erythrocytes. Twenty-three to 27 of the first 42 N-terminal amino acid residues of the metalloprotease are identical to proteases produced by Serratia proteamaculans, Pectobacterium carotovorum, and Anabaena sp. PCR analysis using primers designed from a consensus nucleotide sequence showed that 135 E. sakazakii strains possessed the metalloprotease gene, zpx, and 25 non-E. sakazakii strains did not. The cloned zpx gene of strain 29544 consists of 1,026 nucleotides, and the deduced amino acid sequence of the metalloprotease has 341 amino acid residues, which corresponds to a theoretical protein size of 37,782 with a theoretical pI of 5.23. The sequence possesses three well-characterized zinc-binding and active-site motifs present in other bacterial zinc metalloproteases.

Rugosity in Grimontia hollisae.

Grimontia hollisae, formerly Vibrio hollisae, produces both smooth and rugose colonial variants. The rugose colony phenotype is characterized by wrinkled colonies producing copious amounts of exopolysaccharide. Cells from a rugose colony grown at 30 degrees C form rugose colonies, while the same cells grown at 37 degrees C form smooth colonies, which are characterized by a nonwrinkled, uncrannied appearance. Stress response studies revealed that after exposure to bleach for 30 min, rugose survivors outnumbered smooth survivors. Light scatter information obtained by flow cytometry indicated that rugose cells clumped into clusters of three or more cells (average, five cells) and formed two major clusters, while smooth cells formed only one cluster of single cells or doublets. Fluorescent lectin-binding flow cytometry studies revealed that the percentages of rugose cells that bound either wheat germ agglutinin (WGA) or Galanthus nivalis lectin (GNL) were greater than the percentages of smooth cells that bound the same lectins (WGA, 35% versus 3.5%; GNL, 67% versus 0.21%). These results indicate that the rugose exopolysaccharide consists partially of N-acetylglucosamine and mannose. Rugose colonies produced significantly more biofilm material than did smooth colonies, and rugose colonies grown at 30 degrees C produced more biofilm material than rugose colonies grown at 37 degrees C. Ultrastructurally, rugose colonies show regional cellular differentiation, with apical and lateral colonial regions containing cells embedded in a matrix stained by Alcian Blue. The cells touching the agar surface are packed tightly together in a palisade-like manner. The central region of the colony contains irregularly arranged, fluid-filled spaces and loosely packed chains or arrays of coccoid and vibrioid cells. Smooth colonies, in contrast, are flattened, composed of vibrioid cells, and lack distinct regional cellular differences. Results from suckling mouse studies showed that both orally fed rugose and smooth variants elicited significant, but similar, amounts of fluid accumulated in the stomach and intestines. These observations comprise the first report of expression and characterization of rugosity by G. hollisae and raise the possibility that expression of rugose exopolysaccharide in this organism is regulated at least in part by growth temperature.

Characterization of Vibrio fluvialis-like strains implicated in limp lobster disease.

Studies were undertaken to characterize and determine the pathogenic mechanisms involved in a newly described systemic disease in Homarus americanus (American lobster) caused by a Vibrio fluvialis-like microorganism. Nineteen isolates were obtained from eight of nine lobsters sampled. Biochemically, the isolates resembled V. fluvialis, and the isolates grew optimally at 20 degrees C; none could grow at temperatures above 23 degrees C. The type strain (1AMA) displayed a thermal reduction time (D value) of 5.77 min at 37 degrees C. All of the isolates required at least 1% NaCl for growth. Collectively, the data suggest that these isolates may embody a new biotype. Pulsed-field gel electrophoresis (PFGE) analysis of the isolates revealed five closely related subgroups. Some isolates produced a sheep hemagglutinin that was neither an outer membrane protein nor a metalloprotease. Several isolates possessed capsules. The isolates were highly susceptible to a variety of antibiotics tested. However, six isolates were resistant to erythromycin. Seventeen isolates harbored plasmids. Lobster challenge studies revealed that the 50% lethal dose of a plasmid-positive strain was 100-fold lower than that of a plasmid-negative strain, suggesting that the plasmid may enhance the pathogenicity of these microorganisms in lobsters. Microorganisms that were recovered from experimentally infected lobsters exhibited biochemical and PFGE profiles that were indistinguishable from those of the challenge strain. Tissue affinity studies demonstrated that the challenge microorganisms accumulated in heart and midgut tissues as well as in the hemolymph. Culture supernatants and polymyxin B lysates of the strains caused elongation of CHO cells in tissue culture, suggesting the presence of a hitherto unknown enterotoxin. Both plasmid-positive and plasmid-negative strains caused significant dose-related intestinal fluid accumulations in suckling mice. Absence of viable organisms in the intestinal contents of mice suggests that these microorganisms cause diarrhea in mice by intoxication rather than by an infectious process. Further, these results support the thermal reduction data at 37 degrees C and suggest that the mechanism(s) that led to fluid accumulation in mice differs from the disease process observed in lobsters by requiring neither the persistence of viable microorganisms nor the presence of plasmids. In summary, results of lobster studies satisfy Koch's postulates at the organismal and molecular levels; the findings support the hypothesis that these V. fluvialis-like organisms were responsible for the originally described systemic disease, which is now called limp lobster disease.

Isolation and characterization of a zinc-containing metalloprotease expressed by Vibrio tubiashii.

A Vibrio tubiashii hemagglutinin, a protease, was purified by ammonium sulfate precipitation, gel filtration, and hydrophobic interaction chromatography. It agglutinates sheep, chicken, bovine, rabbit, guinea pig, and human erythrocytes. It has a molecular mass of 35 kDa, isoelectric points of 3.5 and 3.7, and is inhibited by ortho-phenanthro line, phosphoramidon, and Zincov. The N-terminal amino acid sequence (Ala-Gln-Ala-Thr-Gly-Thr-Gly- Pro-Gly-Gly-Asn-Gln-Lys-Thr-Gly-Gln- Tyr-Asn-Phe-Gly) has strong homology to other Vibrio proteases.

Identification of foodborne bacteria by infrared spectroscopy using cellular fatty acid methyl esters.

Identification of bacterial species by profiling fatty acid methyl esters (FAMEs) has commonly been carried out by using a 20-min capillary gas chromatographic procedure followed by library matching of FAME profiles using commercial MIDI databases and proprietary pattern recognition software. Fast GC (5 min) FAME procedures and mass spectrometric methodologies that require no lipid separation have also been reported. In this study, bacterial identification based on the rapid (2 min) infrared measurement of FAME mixtures was demonstrated. The microorganisms investigated included Gram positive bacteria Staphylococcus aureus, Listeria monocytogenes, Bacillus anthracis, and Bacillus cereus, and Gram negative bacteria from the family Enterobacteriacae: Yersinia enterocolitica, Salmonella typhimurium, Shigella sonnei, and Escherichia coli (four strains of E. coli), and non-Enterobacteriacae: Vibrio cholerae, Vibrio vulnificus, and Vibrio parahemolyticus. Foodborne bacterial mixtures of FAMEs were measured by using an attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectroscopic procedure and discriminated by multivariate analysis. Results showed that the Enterobacteriacae could be discriminated from the vibrios. The identification was at the level of species (for the Bacillus and Vibrio genera) or strains (for the E. coli species). A series of bacterial FAME test samples were prepared and analyzed for accuracy of identification, and all were correctly identified. Our results suggest that this infrared strategy could be used to identify foodborne pathogens.

Purification and characterization of a vulnificolysin-like cytolysin produced by Vibrio tubiashii.

An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.

Zoosporulation of a new Perkinsus species isolated from the gills of the softshell clam Mya arenaria.

A gill-associated Perkinsus sp. isolated from the softshell clam (Mya arenaria) is described as a new species, P. chesapeaki sp. nov. Examination of the parasite in seawater cultures revealed life cycle stages and zoosporulation processes similar to those described for other species of the genus Perkinsus. Prezoosporangia developed thickened cell walls upon contraction of the cytoplasm and development of a distinctive clear area between the cell wall and the protoplast. Successive bipartition of the protoplast led to the formation of hundred's of zoospores within mature sporangia. Zoospores were released into seawater through one or more discharge tubes. Ultrastructural studies revealed an oblong zoospore possessing two flagella that arose from a concave side located in the upper third of the zoospore body. The anterior flagellum possessed a unilateral array of hair-like structures. A large anterior vacuole and basolateral nucleus dominated the cytoplasm of the zoospore body. The presence of a rudimentary apical complex including an open-sided conoid, rhoptries, micronemes, and subpellicular microtubules were also discerned. Differences in zoospore morphology, and sequence analyses of two genes previously reported, support the designation of the gill-associated Perkinsus from the softshell clam as a new species.

Shigella flexneri IpaH(7.8) facilitates escape of virulent bacteria from the endocytic vacuoles of mouse and human macrophages.

The behavior of Shigella flexneri ipaH mutants was studied in human monocyte-derived macrophages (HMDM), in 1-day-old human monocytes, and in J774 mouse macrophage cell line. In HMDM, strain pWR700, an ipaH(7.8) deletion mutant of S. flexneri 2a strain 2457T, behaved like the wild-type strain 2457T. This strain caused rapid host cell death by oncosis, and few bacterial CFU were recovered after incubation in the presence of gentamicin as previously described for 2457T-infected HMDM. However, analysis of bacterial compartmentalization within endocytic vacuoles with gentamicin and chloroquine indicated that more pWR700 than 2457T was present within the endocytic vacuoles of HMDM, suggesting that ipaH(7.8) deletion mutant transited more slowly from the vacuoles to the cytoplasm. In contrast to findings with HMDM, CFU recovered from pWR700-infected mouse J774 cells were 2 to 3 logs higher than CFU from 2457T-infected J774 cells. These values exceeded CFU recovered after infection of J774 cells with plasmid-cured avirulent strain M4243A1. Incubation with gentamicin and chloroquine clearly showed that pWR700 within J774 cells was mostly present within the endocytic vacuoles. This distribution pattern was similar to that seen with M4243A1 and contrasted with the pattern seen with 2457T. Complementation of pWR700 with a recombinant clone expressing ipaH(7. 8) restored the intracellular distribution of bacteria to that seen with the wild-type strain. Strains with deletions in ipaH(4.5) or ipaH(9.8), however, behaved like 2457T in both HMDM and J774 cells. The distribution profile of pWR700 in 1-day-old monocytes was similar to that seen in J774 cells. Like infected J774 cells, 1-day-old human monocytes demonstrated apoptosis upon infection with virulent Shigella. These results suggest that a role of the ipaH(7. 8) gene product is to facilitate the escape of the virulent bacteria from the phagocytic vacuole of monocytes and macrophages.

Perkinsus marinus extracellular protease modulates survival of Vibrio vulnificus in Eastern oyster (Crassostrea virginica) hemocytes.

The in vitro effects of the Perkinsus marinus serine protease on the intracellular survival of Vibrio vulnificus in oyster hemocytes were examined by using a time-course gentamicin internalization assay. Results showed that protease-treated hemocytes were initially slower to internalize V. vulnificus than untreated hemocytes. After 1 h, the elimination of V. vulnificus by treated hemocytes was significantly suppressed compared with hemocytes infected with invasive and noninvasive controls. Our data suggest that the serine protease produced by P. marinus suppresses the vibriocidal activity of oyster hemocytes to effectively eliminate V. vulnificus, potentially leading to conditions favoring higher numbers of vibrios in oyster tissues.

Hemolysin-positive enteroaggregative and cell-detaching Escherichia coli strains cause oncosis of human monocyte-derived macrophages and apoptosis of murine J774 cells.

Infection of human monocyte-derived macrophages (HMDM) and J774 cells (murine macrophage cell line) with several enteroaggregative and cytodetaching Escherichia coli (EAggEC and CDEC, respectively) strains demonstrated that some strains could induce macrophage cell death accompanied by release of lactate dehydrogenase activity and interleukin 1beta (IL-1beta) into culture supernatants. The mode of cell death differed in the two types of macrophages. Damage to macrophage plasma membrane integrity without changes in nuclear morphology resulted in cytolysis of HMDM. This mechanism of cell death has been previously described for virulent Shigella infection of HMDM and is termed oncosis. In contrast, infection of J774 cells by EAggEC and CDEC strains resulted in apoptosis. The presence of alpha-hemolysin (Hly) in EAggEC and CDEC strains appears to be critical for both oncosis in HMDM and apoptosis in J774 cells. Bacteria lacking Hly, including Hly- EAggEC strains as well as enterotoxigenic, enteropathogenic, and enterohemorrhagic E. coli strains, behaved like avirulent Shigella flexneri in that the macrophage monolayers were intact, with no release of lactate dehydrogenase activity or IL-1beta into the culture supernatants.