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

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

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

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

Comparison of methods for the microbiological identification and profiling of cronobacter species from ingredients used in the preparation of infant formula

Cronobacter spp. (formerly Enterobacter sakazakii) can be isolated from a wide range of foods and environments, and its association with neonatal infections has drawn considerable attention from regulatory authorities. The principle route of neonatal infection has been identified as the ingestion of contaminated infant formula. A number of methods have been developed to identify Cronobacter spp., however these were before the most recent (2012 ) taxonomic revision of the genus into seven species. In this study, phenotyping, protein profiling and molecular methods were used to identify Cronobacter strains which had been recently isolated from ingredients used in the preparation of infant formula. Pulsed field gel electrophoresis revealed that different Cronobacter strains had been recovered from the same food products. All isolates were identified as C sakazakii according to four genus specific PCR-probes and protein profiling using MALDI-TOF analysis. However, 16S rDNA sequence analyses and fusA allele sequencing gave more accurate identification: four strains were C sakazakii, one strain was C malonaticus and the remaining strain was C universalis. Multilocus sequence typing showed the strains were different sequence types. These results demonstrate the presence of different Cronobacter species in food ingredients used in the preparation of infant formula, and also the need for molecular identification and profiling methods to be revised according to taxonomic revisions

The molecular characterisation of Escherichia coli K1 isolated from neonatal nasogastric feeding tubes

Background: The most common cause of Gram-negative bacterial neonatal meningitis is E. coli K1. It has a mortality rate of 10–15%, and neurological sequelae in 30– 50% of cases. Infections can be attributable to nosocomial sources, however the pre-colonisation of enteral feeding tubes has not been considered as a specific risk factor. Methods: Thirty E. coli strains, which had been isolated in an earlier study, from the residual lumen liquid and biofilms of neonatal nasogastric feeding tubes were genotyped using pulsed-field gel electrophoresis, and 7-loci multilocus sequence typing. Potential pathogenicity and biofilm associated traits were determined using specific PCR probes, genome analysis, and in vitro tissue culture assays. Results: The E. coli strains clustered into five pulsotypes, which were genotyped as sequence types (ST) 95, 73, 127, 394 and 2076 (Achman scheme). The extra-intestinal pathogenic E. coli (ExPEC) phylogenetic group B2 ST95 serotype O1:K1:NM strains had been isolated over a 2 week period from 11 neonates who were on different feeding regimes. The E. coli K1 ST95 strains encoded for various virulence traits associated with neonatal meningitis and extracellular matrix formation. These strains attached and invaded intestinal, and both human and rat brain cell lines, and persisted for 48 h in U937 macrophages. E. coli STs 73, 394 and 2076 also persisted in macrophages and invaded Caco-2 and human brain cells, but only ST394 invaded rat brain cells. E. coli ST127 was notable as it did not invade any cell lines. Conclusions: Routes by which E. coli K1 can be disseminated within a neonatal intensive care unit are uncertain, however the colonisation of neonatal enteral feeding tubes may be one reservoir source which could constitute a serious health risk to neonates following ingestion

Cronobacter sakazakii clinical isolates overcome host barriers and evade the immune response

Cronobacter sakazakii is the most frequently clinically isolated species of the Cronobacter genus. However the virulence factors of C. sakazakii including their ability to overcome host barriers remains poorly studied. In this study, ten clinical isolates of C. sakazakii were assessed for their ability to invade and translocate through human microvascular endothelial cells (HBMEC). Their ability to avoid phagocytosis in human macrophages U937 and human brain microglial cells was investigated. Additionally, they were tested for serum sensitivity and the presence of the Cronobacter plasminogen activation gene (cpa) gene, which is reported to confer serum resistance. Our data showed that the clinical C. sakazakii strains invaded and translocated through Caco-2 and HBMEC cell lines and some strains showed significantly higher levels of invasion and translocation. Moreover, C. sakazakii was able to persist and even multiply in phagocytic macrophage and microglial cells. All strains, except one, were able to withstand human serum exposure, the single serum sensitive strain was also the only one which did not encode for the cpa gene. These results demonstrate that C. sakazakii clinical host immune response indicating their capacity to cause diseases such as necrotizing enterocolitis (NEC) and meningitis. Our data showed for the first time the ability of C. sakazakii clinical isolates to survive and multiply within human microglial cells. Additionally, it was shown that C. sakazakii clinical strains have the capacity to translocate through the Caco-2 and HBMEC cell lines paracellularly