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.

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.

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.

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.

Two-step purification and partial characterization of a variant of the Vibrio cholerae non-O1 hemolysin.

A two-step purification method using ammonium sulfate precipitation and gel filtration was developed for the purification of a variant of the El Tor hemolysin/cytolysin from supernatant fluids of a Vibrio cholerae non-O1 human isolate (strain 2194c). The toxin displayed delayed elution from a Sephacryl gel filtration column, eluting at between two and three column volumes. The molecular mass and isoelectric point of the purified 2194c toxin were 60 kDa and 5. 3, respectively. The N-terminal amino acid sequence was ASPAPANSETNTLPHVAFYI. Purified toxin was cytolytic for Chinese hamster ovary cells and erythrocytes from several animal species.

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.

DNA homology and immunological cross-reactivity between Aeromonas hydrophila cytotonic toxin and cholera toxin.

DNA colony hybridization with three 18- to 20-base-long synthetic oligonucleotide probes for cholera toxin (CT) was used to screen 12 clinical isolates of Aeromonas hydrophila. Under stringent hybridizing (overnight at 40 degrees C) and washing (1 h at 50 degrees C) conditions, nine strains reacted with the 32P-labeled CT probes. Concentrated (10x) cell-free supernatants or lysates from eight cultures, heated at 56 degrees C for 20 min, produced cytotonic effects in Y-1 mouse adrenal cells and Chinese hamster ovary (CHO) cells and caused a 1.5- to 22-fold increase in production of cyclic AMP in CHO cells. Preincubation with anti-CT reduced the CHO cell titer of cell lysates by 10-fold. In the GM1 ganglioside enzyme-linked immunosorbent assay, heated supernatants and lysates gave readings equivalent to 3.5 to 100 ng of CT. Three proteins with molecular weights of 89,900, 37,000, and 11,000 reacted with anti-CT on immunoblots of cell lysates from sodium dodecyl sulfate-polyacrylamide gels. These results suggest that there is DNA homology and immunological cross-reactivity between CT and the A. hydrophila cytotonic toxin.

Chemical nature of agrocin 84 and its effect on a virulent strain of Agrobacterium tumefaciens.

Agrocin 84, produced by Agrobacterium radiobacter K84, inhibited ribonucleic acid, deoxyribonucleic acid, and protein synthesis and amino acid transport in a susceptible, virulent strain of A. tumefaciens H-38-9. Cell motility was immediately stopped by action of the agrocin, 50% of the cells were killed within 15 min of contact, and the remainder were inhibited. Agrocin 84 is trypsin and pepsin resistant, but chemical analysis indicated a small peptide with a molecular weight of 2,500 containing six different amino acids, including nine molecules of glutamine or glutamic acid and seven molecules of serine.

Development of Defined, Minimal, and Complete Media for the Growth of Hyphomicrobium neptunium.

A complete synthetic medium containing 15 amino acids, a minimal synthetic medium (GAMS) containing 4 amino acids, and a supplemented minimal medium (GAMS + calcium pantothenate) have been developed for the cultivation of Hyphomicrobium neptunium ATCC 15444. Depending on the complexity of the synthetic media, generation times were approximately 2 to 3 times longer, and maximum cell densities were 0.3 to 0.9 log(10) lower than in ZoBell marine broth 2216. The fates of C-labeled amino acids in GAMS were monitored. Results suggested that H. neptunium was auxotrophic for methionine, utilized glutamic acid as a primary energy source, and readily anabolized and catabolized serine and aspartic acid. Individual amino acid concentrations above 125 mM induced prolonged lag periods, whereas only methionine was not growth limiting at a concentration as low as 2 mM.

Isolation and characterization of a cytolysin produced by Vibrio cholerae serogroup non-O1.

A thermolabile toxin (molecular weight, 52 711; isoelectric point, 8.65) produced by a clinical isolate of Vibrio cholerae serogroup non-O1 was cytotoxic for Y-1 mouse adrenal cells and Chinese hamster ovary cells. The toxin lysed rabbit red blood cells and produced a hemorrhagic zone in rabbit skin. When injected intravenously into adult mice, the cytolysin was rapidly lethal and caused fluid accumulation in both 5- and 18-h rabbit ileal loops. Strains of V. cholerae that produced cytolysin but no cholerae enterotoxin were able to cause fluid accumulation in rabbit intestinal loops.

Campylobacter diarrhea in an adult mouse model.

An adult mouse (18-20 g) model was developed for studying the pathogenesis of Campylobacter isolates. Iron-loaded BALB/c mice given 10(8)-10(9) Campylobacter colony forming units by intraperitoneal injection developed a severe mucoid diarrhea within 4 h. Severe diarrhea, consisting of unformed stools containing blood, mucus, and fecal leukocytes, persisted for 24 h. Diarrheal symptoms in surviving mice resolved gradually; no diarrhea was observed 5 days after inoculation. Mice not pretreated with iron developed no diarrheal symptoms, and no severe diarrhea was produced in mice inoculated orally. A transient (less than 24 h) bacteremia occurred in mice inoculated either orally or intraperitoneally. Liver, spleen, and kidney were positive for Campylobacter for 48 h; intestinal contents were positive for 5-7 days. Mice given greater than or equal to 10(10) colony forming units showed symptoms of endotoxemia (ruffled fur, inactivity, shaking, tearing, and hypothermia) and died without diarrheal symptoms. Mice given nonpathogenic Escherichia coli strain HB101, heat-killed C. jejuni cells (greater than 10(10)), C. jejuni lipopolysaccharide extract, or purified lipopolysaccharide from either Vibrio cholerae 569B or Salmonella typhimurium showed no diarrheal symptoms.

Effect of iron concentration on toxin production in Campylobacter jejuni and Campylobacter coli.

The effect of iron concentrations in culture media on supernatant yields of campylobacter cytotonic toxin (CCT) was studied. Of the 118 Campylobacter spp. strains surveyed, 78.8% produced toxin in brucella broth or in casamino acids--yeast extract (CYE) broth. When the iron concentration of CYE was increased from 0.44 microgram/mL (7.9 microM) to 0.65 microgram/mL (11.6 microM) by the addition of ferric chloride, 94.9% of the strains were positive for toxin in a ganglioside GM1 based, enzyme-linked immunosorbent assay, using antibody to affinity-purified CCT. The addition of iron as ferrous sulfate was less effective. When four toxin-positive strains were grown in a deferrated medium of conalbumin-treated CYE with 0.04-0.08 microgram iron/mL (0.72-1.43 microM), two of the culture supernatants became negative (absorbance at 410 nm, less than 0.1 and less than 10 ng CCT/mL), and two produced about 90% less CCT but were still classified as positive (absorbance, greater than or equal to 0.1 and greater than or equal to 10 ng CCT/mL). It was therefore concluded that the production of CCT by Campylobacter spp. is influenced by iron concentration.

Identification of a CHO cell-elongating factor produced by Vibrio cholerae O1.

Vibrio cholerae strains with all known toxin genes deleted or inactivated still cause diarrhoea in some volunteers, suggesting the presence of an unknown virulence factor or factors. Lysozyme-EDTA treated cells of JBK70, a genetically manipulated cholera toxin negative strain of Vibrio cholerae O1, biotype El Tor, release a factor that causes elongation of Chinese hamster ovary (CHO) cells. CHO cell-elongating toxin (Cef) was purified by FPLC chromatography (anion exchange; Q Sepharose High Performance) followed by 2D electrophoresis (isoelectric focusing gel, IEF; pH 3-9 and SDS-PAGE, 8-25% gradient gel). Partly purified toxin (anion exchange or IEF-eluted concentrate) caused fluid accumulation in sealed infant mice suggesting that Cef shows some properties of an enterotoxin. On SDS-PAGE (8-25%) and IEF (pH 2.5-5.0) gels, CHO cell activity was associated with a single band at 85 kDa and a pI of 3.8, respectively. A unique amino terminal sequence, XGDETNSSGASTEVVYESYIQQ, was determined by automated Edman degradation of gel-purified protein. The unique molecular mass, N-terminal sequence and activity on CHO cells indicate that this factor is not zonula occludens toxin (Zot) or accessory cholera enterotoxin (Ace) or the Hly A haemolysin. Partly purified Cef did not increase cyclic AMP or prostaglandin E(2)levels in CHO cells which suggests that its mechanism of action differs from that of cholera toxin.

Purification and characterization of a CHO cell-elongating toxin produced by Aeromonas hydrophila.

A Chinese hamster ovary (CHO) cell-elongating toxin produced by Aeromonas hydrophila was purified from cell-free supernatant fluids by ammonium sulfate precipitation and fast protein liquid chromatography. The purified toxin had an isolelectric point (pl) of 3.7 and a molecular weight of 70,000 in a single band on isoelectric focusing (IEF) gels and SDS-PAGE gels, respectively. The N-terminal sequence, amino acids 1-20, and the amino acid content were determined from Western blots of the 70 kDa band. No homology with any known microbial toxin was found. CHO cell activity was not neutralized by antiserum to cholera toxin (anti-CT), and the toxin did not react with anti-CT on Western blots. The toxin did not increase cyclic AMP, cyclic GMP, or prostaglandin E2 levels in CHO cells. No cytotoxic activity was observed. Intragastric administration of purified toxin (5 x 10(4) and 5 x 10(8) CHO cell units) induced intestinal fluid accumulation in infant mice. These results suggest that this toxin may be a novel cytotonic toxin distinct from previously described toxins produced by A. hydrophila or A. sobria.

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.

Cloning, expression and characterization of the CHO cell elongating factor (Cef) from Vibrio cholerae O1.

CHO cell-elongating factor (Cef) is a recently identified putative virulence factor of Vibrio cholerae. Our previous studies show that this 85 kDa protein elongates CHO cells, causes fluid accumulation in suckling mice and has esterase activity. In this study, the cef gene was cloned in Escherichia coli using a yeast vector and subsequently expressed in the yeast Pichia pastoris. The cef genes from V. cholerae candidate vaccine strains JBK 70 and CVD 103-HgR were sequenced and found to be nearly identical (100 and 99.9% respectively) with an open reading frame (ORF) from the published sequence of V. cholerae N16961. Cloned toxin was purified to homogeneity in 3 steps using anion exchange, hydrophobic interaction and gel filtration chromatography. The size of cloned Cef on SDS-PAGE gels was 114 kDa. The increased size was probably due to glycosylation by the yeast since cloned protein reacted strongly with a glycoprotein stain. The cloned protein could not be directly sequenced, but when treated with trypsin, yielded a protein fragment with an amino acid sequence that matched the sequence predicted for the Cef protein. The purified cloned protein had esterase and CHO cell activity, but no suckling mouse activity.

Binding of cholera toxin to Giardia lamblia.

Binding of cholera toxin to Giardia lamblia was demonstrated by two slightly different methods: an immunofluorescence technique using antibody to cholera toxin and anti-rabbit immunoglobulin G conjugated to fluorescein isothiocyanate, and a one-step fluorescence method in which G. lamblia was incubated with the B subunit of cholera toxin conjugated to fluorescein isothiocyanate.

Purification and characterization of a cytotonic protein expressed In vitro by the live cholera vaccine candidate CVD 103-HgR.

Cholera vaccines developed by the deletion of CTX genes from Vibrio cholerae induce a residual reactogenicity in up to 10% of vaccinees. A novel cytotonic agent named secreted CHO cell elongating protein (S-CEP) was purified from culture supernatants of CVD 103-HgR (Levine et al., Lancet ii:467-470, 1988). Five fractionation steps yielded electrophoretically pure S-CEP with an M(r) of 79,000. A partially purified preparation caused fluid accumulation in the sealed infant mouse model. The amino terminus bore a unique sequence with strong homology to a cytotonic toxin of El Tor V. cholerae.

Virulence of three clinical isolates of Vibrio cholerae non-O-1 serogroup in experimental enteric infections in rabbits.

Three Vibrio cholerae, [ill] isolates, pathogenic in both adult rabbit ligated ileal loop and infant rabbit assays, produced no heat-labile or heat-stable toxins. Their pathogenicity did not correlate with the presence of plasmids.

Foodborne enterotoxigenic Escherichia coli: detection and enumeration by DNA colony hybridization.

Four methods were compared for detecting heat-labile toxin production by Escherichia coli: DNA colony hybridization, two enzyme-linked immunosorbent assays, and the mouse Y-1 adrenal cell reaction. Although results of the methods were in general agreement, there were some differences in specificity and sensitivity. DNA colony hybridization was used to detect and enumerate enterotoxigenic E. coli isolates in artificially contaminated food without enrichment. Sensitivity level was 100 cells per g.