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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. Farmer et al. (20) established the taxonomic position of Enterobacter sakazakii, which was previously identified as yellow-pigmented Enterobacter cloacae based on DNA-DNA hybridization studies in combination with phenotypic

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°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.

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.

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 ϳ131-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 ␣2-antiplasmin (␣2-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 DH5␣ 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 ␣2-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.

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-AspTyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.Vibrio tubiashii is a marine organism that causes bacillary necrosis in larval and juvenile bivalve mollusks (24,25). The disease is characterized by a rapid onset of symptoms, such as a generalized reduction in larval motility, an increase in larval quiescence, and extensive soft tissue necrosis. The pathogen has been isolated from hard clam larvae, juvenile hard clams, and Eastern oyster spat and larvae (7,12,25). V. tubiashii has been isolated from diseased mollusks in the United States and United Kingdom and has been associated with red tides caused by Mesodinum rubrum along the northwest coast of Spain (12,23,24,25). In spite of the economic importance of V. tubiashii in the cultivation of bivalve mollusks, nothing is known about the virulence mechanisms of this pathogen. Romalde et al. (23) reported that culture supernatants of the pathogen exhibited cytotoxicity towards fathead minnow peduncle cells and mouse lung fibroblasts in tissue culture. We describe the purification and properties of a cytolysin that lyses various types of erythrocytes and Chinese hamster ovary (CHO) cells and is cytotoxic to human intestinal (Caco-2) cells and fish (Atlantic menhaden liver [AML]) cells in tissue culture.Cytolysin production and purification. Two V. tubiashii strains (ATCC 19105 and ATCC 19109) were obtained from the American Type Culture Collection (Manassas, Va). Both strains were confirmed to be V. tubiashii using biochemical tests and were stored at Ϫ70°C. The ATCC 19105 frozen culture was rapidly thawed and inoculated onto two plates containing Trypticase soy agar (BBL, Cockeysville, Md.) supplemented with 1% NaCl. The plates were incubated at 30°C for 16 to 18 h, and the bacteria were harvested in 5 ml of Casamino Acids-yeast extract broth (3% Casamino Acids, 0.4% yeast extract, 0.05% K 2 HPO 4 [pH 7.4] supplemented with 1% NaCl). A 2-liter flask containing 500 ml of Casamino Acids-yeast extract broth was inoculated with the seed culture suspension (25 optical density units at 650 nm; ca. 10 10 CFU), and the culture was incubated for 7 h at 37°C on a rotary shaker at 100 rpm. Culture supernatant fluids (stage 1) were recovered by centrifugation at 16,000 ϫ g (20 min). Disodium hydrogen phosphate and sodium chloride were dissolved in the sta...

The halophilic bacterium Vibrio fluvialis is an enteric pathogen that produces an extracellular hemolysin. This hemolysin was purified to homogeneity by using sequential hydrophobic-interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. It has a molecular weight of 63,000 and an isoelectric point of 4.6, and its hemolytic activity is sensitive to heat, proteases, and preincubation with zinc ions. The hemolysin lyses erythrocytes of the eight different animal species that we tested, is cytotoxic against Chinese hamster ovary cells in tissue culture, and elicits fluid accumulation in suckling mice. Lysis of erythrocytes occurs by a temperature-dependent binding step followed by a temperature-and pH-dependent lytic step. Fourteen of the first 20 N-terminal amino acid residues (Val-Ser-Gly-Gly-Glu-Ala-Asn-Thr-LeuPro-His-Val-Ala-Phe-Tyr-Ile-Asn-Val-Asn-Arg) are identical to those of the El Tor hemolysin of Vibrio cholerae and the heat-labile hemolysin of Vibrio mimicus. This homology was further confirmed by PCR analysis using a 5 primer derived from the amino-terminal sequence of the hemolysin and a 3 primer derived from the El Tor hemolysin structural gene. The hemolysin also reacts with antibodies to the El Tor-like hemolysin of non-O1 V. cholerae.Vibrio fluvialis is a halophilic bacterial pathogen that has been isolated from humans with diarrhea and from river and estuarine water, marine mollusks, crustaceans, and fish. It has been implicated in outbreaks and sporadic cases of diarrhea, and gastrointestinal illness caused by this pathogen is usually associated with consumption of raw or improperly cooked seafood. In the United States, recent data show that it was responsible for 82 of the 1,584 Vibrio infections reported to the Centers for Disease Control and Prevention since 1997 (M. C. Evans, P. M. Griffin, and R. V. Tauxe, CDC report: Vibrio surveillance system, summary data, 1997-2000 [http://www.cdc.gov /ncidod/dbmd/diseaseinfo/files/CSTE_Vibrio_2000.pdf]). Clinical symptoms of gastroenteritis caused by V. fluvialis and Vibrio cholerae are very similar, and therefore the ability of V. fluvialis to cause diarrhea has been examined in various animal models originally designed for V. cholerae. V. fluvialis elicits intestinal fluid when fed to suckling mice (18, 23) and in rabbit ileal loops (5, 25). It produces a toxin that elongates Chinese hamster ovary (CHO) cells, a nonhemolytic CHO cell-killing cytotoxin, a protease, and a cytolysin active against rabbit erythrocytes (5, 17). Lockwood et al. (18) showed that partially purified preparations of the elongation factor, protease, and cytolysin/hemolysin induced fluid accumulation in suckling mice. However, it is not clear what roles these products play in diarrheal disease since none of them have been purified. In their research with the V. fluvialis hemolysin (VFH), Rahim and Aziz (26) reported that of the six different growth media tested, brain heart infusion broth yielded maximal amounts of hemolysin. In order to ha...

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