In Silico identification of pathogenic strains of Cronobacter from Biochemical data reveals association of inositol fermentation with pathogenicity

<p>Abstract</p> <p>Background</p> <p><it>Cronobacter</it>, formerly known as <it>Enterobacter sakazakii</it>, is a food-borne pathogen known to cause neonatal meningitis, septicaemia and death. Current diagnostic tests for identification of <it>Cronobacter </it>do not differentiate between species, necessitating time consuming 16S rDNA gene sequencing or multilocus sequence typing (MLST). The organism is ubiquitous, being found in the environment and in a wide range of foods, although there is variation in pathogenicity between <it>Cronobacter </it>isolates and between species. Therefore to be able to differentiate between the pathogenic and non-pathogenic strains is of interest to the food industry and regulators.</p> <p>Results</p> <p>Here we report the use of Expectation Maximization clustering to categorise 98 strains of <it>Cronobacter </it>as pathogenic or non-pathogenic based on biochemical test results from standard diagnostic test kits. Pathogenicity of a strain was postulated on the basis of either pathogenic symptoms associated with strain source or corresponding MLST sequence types, allowing the clusters to be labelled as containing either pathogenic or non-pathogenic strains. The resulting clusters gave good differentiation of strains into pathogenic and non-pathogenic groups, corresponding well to isolate source and MLST sequence type. The results also revealed a potential association between pathogenicity and inositol fermentation. An investigation of the genomes of <it>Cronobacter sakazakii </it>and <it>C. turicensis </it>revealed the gene for inositol monophosphatase is associated with putative virulence factors in pathogenic strains of <it>Cronobacter</it>.</p> <p>Conclusions</p> <p>We demonstrated a computational approach allowing existing diagnostic kits to be used to identify pathogenic strains of <it>Cronobacter</it>. The resulting clusters correlated well with MLST sequence types and revealed new information about the pathogenicity of <it>Cronobacter </it>species.</p

Radio Telemetry and Post-Emergent Habitat Selection of Neonate Box Turtles (Emydidae: Terrapene Carolina) in Central Illinois

Although factors influencing turtle offspring prior to nest emergence have received considerable attention by researchers (Gutzke and Crews 1988; Janzen et al. 2000; Packard and Packard 1987), the activity of neonates following their emergence from the nest is poorly understood (but see Burger 1976; Butler and Graham 1995; Keller et al. 1997). Previous field research has produced valuable information on several aspects of neonate ecology for several species (Brewster and Brewster 1991; Butler and Sowell 1996; Janzen 1993). However, a thorough understanding of life history patterns for many species is absent, and some existing information is conflicting (e.g., Congdon et al. 1999; Janzen et al. 2000). The lack of knowledge is primarily due to the cryptic nature of neonates and various logistical problems associated with studying small animals in the field. Recent advances in radio telemetry technology such as decreased transmitter size and increased battery life facilitate tracking small neonate turtles for a longer duration. We studied nest dispersal and habitat use in neonate box turtles using a relatively new, very small radio transmitter. Few studies have been conducted using telemetry on neonate turtles (e.g., Butler et al. 1995), and none has focused on nest dispersal and habitat use of neonate box turtles. The study was conducted at Rhodes-France Boy Scout Reservation (RFBSR) located in western Shelby County, Illinois, USA (39°19\u27N; 89°02\u27W), from March to April 2002. Two nests were located by radio tracking gravid female turtles during summer 2001 (Flitz 2003). The nests were sited in relatively open areas next to a tree stump in a grassy field and at the edge of a fire trail (see Flitz 2003 for more description). Nest disturbance was prevented by using excluder devices, made of hardware cloth of 0.6 cm2 mesh and 30 cm diameter with walls buried 15 cm into the ground, around the nest until the end of the 2001 activity season. Upon hatching and emergence, neonate turtles from both nests (clutch sizes were 4 and 5, respectively) were collected, brought to our laboratory and allowed to overwinter in an outdoor enclosure (1.5 x 1.5 m) under ambient conditions. Each turtle was marked with a unique series of notches in the marginal scutes. This facilitated identification and placement back at the proper nest site the following spring

The biochemical differentiation of Enterobacter sakazakii genotypes

BACKGROUND: Enterobacter sakazakii is an emergent pathogen that has been associated with neonatal infections through contaminated powdered infant milk formula. The species was defined by Farmer et al. (1980) who described 15 biogroups according to the biochemical characterization of 57 strains. This present study compares genotypes (DNA cluster groups based on partial 16S rDNA sequence analysis) with the biochemical traits for 189 E. sakazakii strains. RESULTS: Analysis of partial 16S rDNA sequences gave 4 well defined phylogenetic groups. Cluster group 1 was composed of the majority of strains (170/189) and included Biogroups 1–5, 7–9, 11, 13 and 14. Cluster 3 corresponded with Biogroup 15 and cluster 4 with Biogroups 6, 10 and 12. Cluster group 2 comprised a new Biogroup 16. For the isolates in this study, the four DNA cluster groups can be distinguished using the inositol, dulcitol and indole tests. CONCLUSION: This study demonstrates an agreement between genotyping (partial 16S rDNA) and biotyping and describes a new biogroup of E. sakazakii

Identification of Enterobacter sakazakii from closely related species: The use of Artificial Neural Networks in the analysis of biochemical and 16S rDNA data

BACKGROUND: Enterobacter sakazakii is an emergent pathogen associated with ingestion of infant formula and accurate identification is important in both industrial and clinical settings. Bacterial species can be difficult to accurately characterise from complex biochemical datasets and computer algorithms can potentially simplify the process. RESULTS: Artificial Neural Networks were applied to biochemical and 16S rDNA data derived from 282 strains of Enterobacteriaceae, including 189 E. sakazakii isolates, in order to identify key characteristics which could improve the identification of E. sakazakii. The models developed resulted in a predictive performance for blind (validation) data of 99.3 % correct discrimination between E. sakazakii and closely related species for both phenotypic and genotypic data. Three main regions of the partial rDNA sequence were found to be key in discriminating the species. Comparison between E. sakazakii and other strains also constitutively positive for expression of the enzyme α-glucosidase resulted in a predictive performance of 98.7 % for 16S rDNA sequence data and 100% for phenotypic data. CONCLUSION: The computationally based methods developed here show a remarkable ability in reducing data dimensionality and complexity, in order to eliminate noise from the system in order to facilitate the speed and reliability of a potential strain identification system. Furthermore, the approaches described are also able to provide valuable information regarding the population structure and distribution of individual species thus providing the foundations for novel assays and diagnostic tests for rapid identification of pathogens

From Mott insulator to ferromagnetic metal: a pressure study of Ca2_{2}RuO4_{4}

We show that the pressure-temperature phase diagram of the Mott insulator Ca$_{2}$RuO$_{4}$ features a metal-insulator transition at 0.5GPa: at 300K from paramagnetic insulator to paramagnetic quasi-two-dimensional metal; at $T \leq$ 12K from antiferromagnetic insulator to ferromagnetic, highly anisotropic, three-dimensional metal. % We compare the metallic state to that of the structurally related p-wave superconductor Sr$_{2}$RuO$_{4}$, and discuss the importance of structural distortions, which are expected to couple strongly to pressure.Comment: 4 pages, 4figure

Lack of continuity between Cronobacter biotypes and species as determined using multilocus sequence typing

The accuracy of the Cronobacter biotyping scheme was compared with the 7-loci multilocus sequence typing scheme. Biotyping did not reliably assign species level identification, as only half (17/31) of the biotype variants were unique to any of the seven Cronobacter species and the remaining biotypes were shared across the genus

Comparative Outer Membrane Protein Analysis of High and Low-Invasive Strains of Cronobacter malonaticus

are an important group of foodborne pathogens that has been linked to life-threatening infections in both infants and adults. The major infections associated with species are neonatal meningitis, necrotizing enterocolitis, and septicaemia. There are seven species in the genus, of which only three are of clinical importance; , and . To date most studies have focussed on as it is the major species associated with neonatal infections. However, recently , in particular sequence type 7 (ST7), has been noted as being prevalent in adult infections and therefore warranting further investigation. In this study, eight strains of ST7, that had been isolated from a wide range of sources and varied in their virulence, were chosen for proteomic analysis of their outer membrane proteins (OMPs). One-dimensional gel analysis revealed a ~29 kDa size band that was only present in the highly invasive strains. Subsequent mass spectrometric analysis identified several peptides that matched the flagellin protein. The presence of flagellin protein was confirmed in 2D gel spot. Mass spectrometry analysis of total OMPs revealed that the four highly invasive strains expressed the main flagellum proteins that were absent from the four low invasive strains. These were the flagellar hook protein FlgE, flagellar hook-associated protein 1, flagellar hook-associated protein, flagellin, and flagellar hook-filament junction protein FlgL. This data indicates that flagellar proteins may have an important role in the organism's invasion properties

The structure of O-polysaccharide isolated from Cronobacter universalis NCTC 9529T

The O-polysaccharide (OPS) was isolated from Cronobacter universalis NCTC 9529T, a new species in the genus Cronobacter, which was created by the reclassification of the species Enterobacter sakazakii. Purified polysaccharide was analyzed by NMR spectroscopy (1H, COSY, TOCSY, ROESY, HSQC, and HSQC-TOCSY) and chemical methods. The monosaccharide derivatives were analyzed by gas chromatography, and gas chromatography-mass spectrometry. These experiments enabled the type and number of monosaccharides in the repeating unit of OPS, their positions of linkages, and absolute configuration to be determined. Together the chemical analysis established a structure of the OPS of C. universalis NCTC 9529T: →3)--L-FucpNAc-(1→4)--D-Manp-(1→3)--L-FucpNAc-(1→3)-β-D-GlcpNAc-(1→ [A, B, C, D] OPS isolated from C. universalis was structurally characterized for the first time

Cronobacter condimenti sp. nov., isolated from spiced meat, and Cronobacter universalis sp. nov., a species designation for Cronobacter sp. genomospecies 1, recovered from a leg infection, water, and food ingredients

A re-evaluation of the taxonomic position of five strains, one assigned to Cronobacter sakazakii (strain 1330T), two previously assigned to Cronobacter genomospecies 1 (strains NCTC 9529T and 731) and two as Cronobacter turicensis (strains 96 and 1435) was carried out. The analysis included a phenotypic characterization, sequencing of the 16S rRNA gene and multilocus sequence analysis (MLSA) of seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, ppsA; 3036 bp). The 16S rRNA gene sequence analysis and MLSA showed strain 1330T, isolated from spiced meat purchased in Slovakia, to form an independent phylogenetic line. Cronobacter dublinensis was the closest neighbour species on the basis of the MLSA. DNA–DNA reassociation and phenotypic analysis revealed that strain 1330T represented a novel species, for which the name Cronobacter condimenti sp. nov. is proposed, type strain 1330T = CECT 7863T, = LMG 26250T). The four bacterial strains NCTC 9529T, 731, 96 and 1435, isolated from water, a leg infectionand two food ingredients; onion powder and rye flour, repectively, showed on the phylogenetic tree to cluster together within an independent phylogenetic line, with Cronobacter turicensis as the closest species. The DNA–DNA hybridization data and the phenotypic characterization confirmed that these strains represented a novel species, for which the name Cronobacter universalis sp. nov. is proposed with type strain NCTC 9529T = CECT 7864T, = LMG 26249T