A proposed harmonized LPS molecular-subtyping scheme for Cronobacter species.

Cronobacter are opportunistic pathogens, which cause infections in all age groups. To aid the characterization of Cronobacter in foods and environments a harmonized LPS identification scheme for molecular serotyping is needed. To this end, we studied 409 Cronobacter isolates representing the seven Cronobacter species using two previously reported molecular serotyping schemes, described here as Mullane-Jarvis (M-J) and Sun schemes. PCR analysis revealed many overlapping results that were obtained when independently applying the two serotyping schemes. There were complete agreements between the two PCR schemes for Cronobacter sakazakii (Csak) O:1, Csak O:3, and Csak O:7 serotypes. However, only thirty-five of 41 Csak O:4 strains, identified using the M-J scheme, were PCR-positive with the Sun scheme primers. Also the Sun scheme Csak O:5 primers failed to identify this serotype in any of the C. sakazakii strains tested, but did recognize seven Cronobacter turicensis strains, which were identified as Ctur O:3 using the M-J scheme. Similarly, the Sun scheme Csak O:6 primers recognized 30 Cronobacter malonaticus O:2 strains identified with the M-J scheme, but failed to identify this serotype in any C. sakazakii strain investigated. In this report, these findings are summarized and a harmonized molecular-serotyping scheme is proposed which is predicated on the correct identification of Cronobacter species, prior to serotype determination. In summary, fourteen serotypes were identified using the combined protocol, which consists of Csak O:1-O:4, and Csak O:7; Cmal O:1-O:2; Cdub O:1-O:2, Cmuy O:1-O:2, Cuni O:1, as well as Ctur O:1 and Ctur O:3.

Pan-genome analysis of the emerging foodborne pathogen Cronobacter spp. suggests a species-level bidirectional divergence driven by niche adaptation.

BACKGROUND: Members of the genus Cronobacter are causes of rare but severe illness in neonates and preterm infants following the ingestion of contaminated infant formula. Seven species have been described and two of the species genomes were subsequently published. In this study, we performed comparative genomics on eight strains of Cronobacter, including six that we sequenced (representing six of the seven species) and two previously published, closed genomes. RESULTS: We identified and characterized the features associated with the core and pan genome of the genus Cronobacter in an attempt to understand the evolution of these bacteria and the genetic content of each species. We identified 84 genomic regions that are present in two or more Cronobacter genomes, along with 45 unique genomic regions. Many potentially horizontally transferred genes, such as lysogenic prophages, were also identified. Most notable among these were several type six secretion system gene clusters, transposons that carried tellurium, copper and/or silver resistance genes, and a novel integrative conjugative element. CONCLUSIONS: Cronobacter have diverged into two clusters, one consisting of C. dublinensis and C. muytjensii (Cdub-Cmuy) and the other comprised of C. sakazakii, C. malonaticus, C. universalis, and C. turicensis, (Csak-Cmal-Cuni-Ctur) from the most recent common ancestral species. While several genetic determinants for plant-association and human virulence could be found in the core genome of Cronobacter, the four Cdub-Cmuy clade genomes contained several accessory genomic regions important for survival in a plant-associated environmental niche, while the Csak-Cmal-Cuni-Ctur clade genomes harbored numerous virulence-related genetic traits.

Efficacy of biocides used in the modern food industry to control salmonella enterica, and links between biocide tolerance and resistance to clinically relevant antimicrobial compounds.

Biocides play an essential role in limiting the spread of infectious disease. The food industry is dependent on these agents, and their increasing use is a matter for concern. Specifically, the emergence of bacteria demonstrating increased tolerance to biocides, coupled with the potential for the development of a phenotype of cross-resistance to clinically important antimicrobial compounds, needs to be assessed. In this study, we investigated the tolerance of a collection of susceptible and multidrug-resistant (MDR) Salmonella enterica strains to a panel of seven commercially available food-grade biocide formulations. We explored their abilities to adapt to these formulations and their active biocidal agents, i.e., triclosan, chlorhexidine, hydrogen peroxide, and benzalkonium chloride, after sequential rounds of in vitro selection. Finally, cross-tolerance of different categories of biocidal formulations, their active agents, and the potential for coselection of resistance to clinically important antibiotics were investigated. Six of seven food-grade biocide formulations were bactericidal at their recommended working concentrations. All showed a reduced activity against both surface-dried and biofilm cultures. A stable phenotype of tolerance to biocide formulations could not be selected. Upon exposure of Salmonella strains to an active biocidal compound, a high-level of tolerance was selected for a number of Salmonella serotypes. No cross-tolerance to the different biocidal agents or food-grade biocide formulations was observed. Most tolerant isolates displayed changes in their patterns of susceptibility to antimicrobial compounds. Food industry biocides are effective against planktonic Salmonella. When exposed to sublethal concentrations of individual active biocidal agents, tolerant isolates may emerge. This emergence was associated with changes in antimicrobial susceptibilities.

Human isolates of Cronobacter sakazakii bind efficiently to intestinal epithelial cells in vitro to induce monolayer permeability and apoptosis.

BACKGROUND: Cronobacter sakazakii (CS) is an emerging opportunistic pathogen that causes life-threatening infections in infants. This pathogen has been implicated in the outbreaks of necrotizing enterocolitis (NEC) with associated rates of high mortality and morbidity. In this study, we compared the abilities of CS strains isolated from human and environmental sources to bind to intestinal epithelial cells and trigger apoptosis. MATERIALS AND METHODS: CS strains were isolated from human and environmental sources and their abilities to bind to intestinal epithelial cells were determined. Monolayer permeability was determined by transepithelial electrical resistance (TEER) and horseradish peroxidase (HRP) leakage. Apoptosis was examined by ApoTag and AnnexinV-7AAD staining. PKC activation was evaluated by non-radioactive PepTag assay. RESULTS: Human isolates of CS bind to rat and human enterocytes more efficiently than environmental strains. Additionally, these strains induced increased enterocyte monolayer permeability as indicated by a decrease in TEER and an increase in transcellular leakage of exogenously added HRP. Human isolates also caused tight junction disruption and significant apoptosis of enterocytes compared with environmental strains due to increased production of inducible nitric oxide. We also observed that human CS isolates caused 2-fold increase in the activation of phosphokinase C (PKC) than environmental strains. Blocking the PKC activity in enterocytes by an inhibitor, Gö 6983, suppressed CS-mediated tight junction disruption, monolayer permeability, and apoptosis of the cells. CONCLUSION: These results suggest that human isolates of CS more efficiently bind to and cause damage to intestinal epithelial cells compared with environmental strains.

Development and validation of a PulseNet standardized protocol for subtyping isolates of Cronobacter species.

Cronobacter (formerly known as Enterobacter sakazakii) is a genus comprising seven species regarded as opportunistic pathogens that can be found in a wide variety of environments and foods, including powdered infant formula (PIF). Cronobacter sakazakii, the major species of this genus, has been epidemiologically linked to cases of bacteremia, meningitis in neonates, and necrotizing enterocolitis, and contaminated PIF has been identified as an important source of infection. Robust and reproducible subtyping methods are required to aid in the detection and investigation, of foodborne outbreaks. In this study, a pulsed-field gel electrophoresis (PFGE) protocol was developed and validated for subtyping Cronobacter species. It was derived from an existing modified PulseNet protocol, wherein XbaI and SpeI were the primary and secondary restriction enzymes used, generating an average of 14.7 and 20.3 bands, respectively. The PFGE method developed was both reproducible and discriminatory for subtyping Cronobacter species.

Pantoea gaviniae sp. nov. and Pantoea calida sp. nov., isolated from infant formula and an infant formula production environment.

Five Gram-negative, facultatively anaerobic, non-spore-forming, coccoid rod-shaped bacterial isolates were obtained from infant formula and an infant formula production environment and were investigated by use of a polyphasic taxonomic study. Biochemical tests and partial rpoB gene sequence analysis of the five isolates revealed that they formed two distinct groups in the family Enterobacteriaceae, closely related to several species of the genera Pantoea and Erwinia, which indicated a phylogenetic position within the genus Pantoea or the genus Erwinia. Multilocus sequence analysis of concatenated partial atpD, gyrB, infB and rpoB gene sequences of two of the isolates suggested that they represented two novel species of the genus Pantoea, phylogenetically related most closely to Pantoea septica. The five isolates had general characteristics consistent with those of the genus Pantoea, and DNA-DNA hybridizations between two representatives and the type strains of their phylogenetically closest relatives based on comparative 16S rRNA gene sequence analysis showed that the isolates represented two novel genospecies. These two genospecies could be differentiated from each other based on fermentation of galacturonate, sorbitol and potassium 5-ketogluconate. They could be differentiated from phylogenetically related Pantoea species based on their ability to ferment lactose and to utilize ß-gentiobiose and raffinose, their inability to ferment or utilize d-arabitol, and their inability to produce indole. On the basis of the results obtained, the five isolates are considered to represent two novel species of the genus Pantoea, for which the names Pantoea gaviniae sp. nov. (type strain A18/07(T) =LMG 25382(T) =DSM 22758(T)) and Pantoea calida sp. nov. (type strain 1400/07(T) =LMG 25383(T) =DSM 22759(T)) are proposed.

Cronobacter (Enterobacter sakazakii): an opportunistic foodborne pathogen.

Cronobacter spp. (Enterobacter sakazakii) are a recently described genus that is comprised of six genomospecies. The classification of these organisms was revised based on a detailed polyphasic taxonomic study. Cronobacter spp. are regarded as ubiquitous organisms having been isolated from a wide variety of foods. These bacteria are opportunistic pathogens and are linked with life-threatening infections in neonates. Clinical symptoms of Cronobacter infection include necrotizing enterocolitis, bacteremia, and meningitis, with case fatality rates of 50-80% being reported. Contaminated powdered infant formula has been epidemiologically linked with infections. Recently, infections among immunocompromised adults, mainly the elderly, have also been reported. A high tolerance to osmotic stress and elevated temperatures contribute to the survival of Cronobacter spp. in dried foods such as powdered infant formula. Controlling the organism in the production environment, thereby reducing dissemination, necessitates the provision of suitable diagnostic tools. Studies demonstrated that a high degree of variability exists amongst the phenotypic-based methods used to identify Cronobacter spp. However, advances in molecular detection and subtyping techniques have significantly improved the identification and characterization of Cronobacter spp. The dose required to induce infection has yet to be determined. In vitro virulence studies have shown that Cronobacter spp. may survive in macrophage cells and efficiently attach to and invade epithelial cell lines. The production of exopolysaccharide may contribute to the formation of biofilm and active efflux pumps promote resistance to antimicrobial agents such as bile salts and disinfectants. A holistic approach combining techniques such as comparative genome analysis, proteomics, and in vivo challenges could help unravel the complex interactions between this pathogen and its host. These data would help identify those properties in Cronobacter spp. which enable the bacterium to survive in the production environment and infect vulnerable neonates via the food chain.

Analysis of the Molecular Diversity Among Cronobacter Species Isolated From Filth Flies Using Targeted PCR, Pan Genomic DNA Microarray, and Whole Genome Sequencing Analyses.

Cronobacter species are opportunistic pathogens capable of causing life-threatening infections in humans, with serious complications arising in neonates, infants, immuno-compromised individuals, and elderly adults. The genus is comprised of seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti. Despite a multiplicity of genomic data for the genus, little is known about likely transmission vectors. Using DNA microarray analysis, in parallel with whole genome sequencing, and targeted PCR analyses, the total gene content of two C. malonaticus, three C. turicensis, and 14 C. sakazaki isolated from various filth flies was assessed. Phylogenetic relatedness among these and other strains obtained during surveillance and outbreak investigations were comparatively assessed. Specifically, microarray analysis (MA) demonstrated its utility to cluster strains according to species-specific and sequence type (ST) phylogenetic relatedness, and that the fly strains clustered among strains obtained from clinical, food and environmental sources from United States, Europe, and Southeast Asia. This combinatorial approach was useful in data mining for virulence factor genes, and phage genes and gene clusters. In addition, results of plasmidotyping were in agreement with the species identity for each strain as determined by species-specific PCR assays, MA, and whole genome sequencing. Microarray and BLAST analyses of Cronobacter fly sequence datasets were corroborative and showed that the presence and absence of virulence factors followed species and ST evolutionary lines even though such genes were orthologous. Additionally, zebrafish infectivity studies showed that these pathotypes were as virulent to zebrafish embryos as other clinical strains. In summary, these findings support a striking phylogeny amongst fly, clinical, and surveillance strains isolated during 2010-2015, suggesting that flies are capable vectors for transmission of virulent Cronobacter spp.; they continue to circulate among United States and European populations, environments, and that this "pattern of circulation" has continued over decades.

Characterization of Cronobacter recovered from dried milk and related products.

BACKGROUND: Cronobacter is a recently proposed genus consisting of six genomospecies that encompass the organisms previously identified as Enterobacter sakazakii. Cronobacter are opportunistic pathogens and are known to cause serious infections in infants, particularly neonates. High case fatality rates have been associated with infections and acute sequelae can occur in survivors with severe ramifications on neurological development. Infant formula has been identified as one route of transmission for infection in infants. However, the primary reservoirs for subsequent contamination of foods with Cronobacter remain undefined due to the ubiquitous nature of these organisms. More recently, infections in adults have been reported, especially amongst the elderly and patients who are immunocompromised. To help prevent the transmission of infection, it is important to identify the main food sources for Cronobacter. The aim of this study was to identify and characterize Cronobacter isolated from dried-milk and related products available in an Egyptian food market. RESULTS: In total sixteen Cronobacter strains were isolated from 152 dairy-based products. These were identified and characterized using pheno- and genotyping experiments. Real-time PCR confirmed the detection of Cronobacter. Following antibiotic susceptibility tests, 3 strains showed resistance to trimethoprim and/or neomycin. Phenotype profiles were generated based on key biochemical distinguishing tests. Pulsed-field gel electrophoresis (PFGE) identified 8 PFGE types amongst the collection of strains. Repetitive sequence based PCR (rep-PCR) analysis identified 3 rep-PCR types amongst the collection of strains. Sequencing of the recN gene was used to differentiate among the recently described species of Cronobacter. CONCLUSION: This study identified the presence of Cronobacter in dried milk and related products sourced from the Nile-Delta region of Egypt. Although the majority of the strains were susceptible to the antibiotics tested, resistance was observed in three isolates, highlighting the risks associated with Cronobacter contamination in foods. Phenotype and genotype analysis should be applied to further characterize Cronobacter spp. and prevent its transmission into food products.

Evaluation of a new one-step enrichment in conjunction with a chromogenic medium for the detection of Cronobacter spp. (Enterobacter sakazakii) in powdered infant formula.

The aim of the present study was to evaluate a new one-step enrichment protocol, consisting of a combined preenrichment and enrichment broth (Cronobacter Enrichment Broth [CEB]) used in conjunction with selective-differential agar ChromID Sakazakii, to facilitate a shortened 2-day cultural method for detection of Cronobacter spp. (Enterobacter sakazakii) in powdered infant formula (PIF). The CEB was evaluated using samples artificially inoculated with low concentrations of 10 lyophilized strains, representative of the genus Cronobacter. The detection of strains was compared in parallel with the enrichment medium from ISO/TS 22964 and a recently proposed differential screening broth for the detection of Cronobacter. All of the Cronobacter strains were recovered using the CEB, and a significantly higher final bacterial concentration was obtained with the CEB than with the other enrichment broths (P < 0.01). There was no significant difference between the cell concentrations for cultures grown in CEB at 37 degrees C and those grown at 41.5 degrees C. Cronobacter was recovered from both 1/10 (50 g:450 ml) and 1/5.5 (100 g:450 ml) sample-to-broth ratios, with no significant difference observed between the final bacterial concentrations obtained from the two ratios. Further studies on a wider range of PIFs, including naturally contaminated samples, are warranted to determine if the use of this protocol may facilitate the rapid release (within 40 to 48 h) of PIF.

Improving the enrichment procedure for Enterobacteriaceae detection.

The current ISO standard method for detection of Enterobacteriaceae (21528-1:2004) includes enrichment in EE broth which has been shown to be inhibitory to some members of this family, notably Cronobacter spp. A shortened procedure omitting the EE broth has been proposed, however competition from Gram-positive flora may be detrimental to the effective recovery of low levels of target organisms in some sample matrices. In this study we investigated novel cost effective modifications, designed to improve ISO 21528-1:2004 for the detection of Enterobacteriaceae. Initial experiments used a worse-case scenario involving stressed Enterobacteriaceae strains known to grow poorly in laboratory media as well as representative background competitors from powdered milk. The interaction between the Enterobacteriaceae and their competitors was characterised and additives to enhance the growth of target strains over non-target strains were investigated. Supplementation of BPW with 40 microM 8-hydroxyquinoline, 0.5 gL(-1) ammonium iron(III) citrate, 0.1 gL(-1) sodium deoxycholate and 0.1 gL(-1) sodium pyruvate (BPW-S) improved the recovery of Enterobacteriaceae from artificially and naturally contaminated samples. This improvement of the pre-enrichment broth may also be of interest for methods designed to detect specific foodborne pathogens belonging to the Enterobacteriaceae (e.g. Salmonella spp., Cronobacter spp.) that require a pre-enrichment step in BPW.

Development of a novel screening method for the isolation of "Cronobacter" spp. (Enterobacter sakazakii).

A differential medium, "Cronobacter" screening broth, has been designed to complement agars based on hydrolysis of chromogenic alpha-glucopyranoside substrates. The broth was evaluated using 329 Enterobacteriaceae strains (229 target isolates), spiked/naturally contaminated samples, and a parallel comparison with current methods for raw materials, line/end products, and factory environment samples.

A rapid and reliable alternative to ISO 21528-1:2004 for detection of Enterobacteriaceae.

Current legislation in Europe uses the Enterobacteriaceae as a parameter in process hygiene criteria for various food products and refers to the corresponding ISO standard (ISO 21528-1:2004) as mandatory analytical method for this purpose. The ISO procedure includes an enrichment step in EE ("Enterobacteriaceae Enrichment") broth, but it has been reported recently that some isolates of Enterobacteriaceae do not grow well or will even die off in this broth, which could lead to false negative results. To determine if this trait is common among the Enterobacteriaceae, a collection of 95 strains was screened for growth in EE broth. Inhibition was observed with 9 strains (7 Cronobacter sakazakii, 1 Cronobacter malonaticus and 1 Enterobacter amnigenus). Factors affecting cell death were found to be related mainly to the inclusion of bile salts and dyes in this medium. In a second step, an alternative method omitting the EE broth was evaluated using 326 samples, comprising 8 different food matrices and environmental samples from the corresponding manufacturing sites. Positive results were obtained for 235 samples using the ISO standard method and 232 samples using the alternative shortened method. No significant difference was found between the results for the two methods. It is proposed that the standard method for detection of Enterobacteriaceae is revised accordingly.

Cellulose as an extracellular matrix component present in Enterobacter sakazakii biofilms.

Cellulose was identified and characterized as an extracellular matrix component present in the biofilm of an Enterobacter sakazakii clinical isolate grown in nutrient-deficient (M9) medium. Using a bacterial artificial cloning approach in Escherichia coli and subsequent screening of transformants for fluorescence on calcofluor plates, nine genes organized in two operons were identified as putatively responsible for the biosynthesis of cellulose. In addition to the genes already described for cellulose production, two more genes were identified, putatively transcribed together with the genes from the first operon. Putative cellulose in E. sakazakii ES5 biofilm grown on glass coverslips was visualized by calcofluor staining and confocal fluorescence laser scanning microscopy. For the first time, the presence of cellulose in biofilms produced by E. sakazakii was confirmed by methylation analysis.

Genomic characterization of malonate positive Cronobacter sakazakii serotype O:2, sequence type 64 strains, isolated from clinical, food, and environment samples.

BACKGROUND: Malonate utilization, an important differential trait, well recognized as being possessed by six of the seven Cronobacter species is thought to be largely absent in Cronobacter sakazakii (Csak). The current study provides experimental evidence that confirms the presence of a malonate utilization operon in 24 strains of sequence type (ST) 64, obtained from Europe, Middle East, China, and USA; it offers explanations regarding the genomic diversity and phylogenetic relatedness among these strains, and that of other C. sakazakii strains. RESULTS: In this study, the presence of a malonate utilization operon in these strains was initially identified by DNA microarray analysis (MA) out of a pool of 347 strains obtained from various surveillance studies involving clinical, spices, milk powder sources and powdered infant formula production facilities in Ireland and Germany, and dried dairy powder manufacturing facilities in the USA. All ST64 C. sakazakii strains tested could utilize malonate. Zebrafish embryo infection studies showed that C. sakazakii ST64 strains are as virulent as other Cronobacter species. Parallel whole genome sequencing (WGS) and MA showed that the strains phylogenetically grouped as a separate clade among the Csak species cluster. Additionally, these strains possessed the Csak O:2 serotype. The nine-gene, ~ 7.7 kbp malonate utilization operon was located in these strains between two conserved flanking genes, gyrB and katG. Plasmidotyping results showed that these strains possessed the virulence plasmid pESA3, but in contrast to the USA ST64 Csak strains, ST64 Csak strains isolated from sources in Europe and the Middle East, did not possess the type six secretion system effector vgrG gene. CONCLUSIONS: Until this investigation, the presence of malonate-positive Csak strains, which are associated with foods and clinical cases, was under appreciated. If this trait was used solely to identify Cronobacter strains, many strains would likely be misidentified. Parallel WGS and MA were useful in characterizing the total genome content of these Csak O:2, ST64, malonate-positive strains and further provides an understanding of their phylogenetic relatedness among other virulent C. sakazakii strains.

The taxonomy of Enterobacter sakazakii: proposal of a new genus Cronobacter gen. nov. and descriptions of Cronobacter sakazakii comb. nov. Cronobacter sakazakii subsp. sakazakii, comb. nov., Cronobacter sakazakii subsp. malonaticus subsp. nov., Cronobacter turicensis sp. nov., Cronobacter muytjensii sp. nov., Cronobacter dublinensis sp. nov. and Cronobacter genomospecies 1.

BACKGROUND: Enterobacter sakazakii is an opportunistic pathogen that can cause infections such as necrotizing enterocolitis, bacteraemia, meningitis and brain abscess/lesions. When the species was defined in 1980, 15 biogroups were described and it was suggested that these could represent multiple species. In this study the taxonomic relationship of strains described as E. sakazakii was further investigated. RESULTS: Strains identified as E. sakazakii were divided into separate groups on the basis of f-AFLP fingerprints, ribopatterns and full-length 16S rRNA gene sequences. DNA-DNA hybridizations revealed five genomospecies. The phenotypic profiles of the genomospecies were determined and biochemical markers identified. CONCLUSION: This study clarifies the taxonomy of E. sakazakii and proposes a reclassification of these organisms.

Corrigendum: Molecular Characterization of Salmonella Serovars Anatum and Ealing Associated with Two Historical Outbreaks, Linked to Contaminated Powdered Infant Formula.

[This corrects the article on p. 1664 in vol. 7, PMID: 27818652.].

Use of a Pan-Genomic DNA Microarray in Determination of the Phylogenetic Relatedness among Cronobacter spp. and Its Use as a Data Mining Tool to Understand Cronobacter Biology.

Cronobacter (previously known as Enterobacter sakazakii) is a genus of Gram-negative, facultatively anaerobic, oxidase-negative, catalase-positive, rod-shaped bacteria of the family Enterobacteriaceae. These organisms cause a variety of illnesses such as meningitis, necrotizing enterocolitis, and septicemia in neonates and infants, and urinary tract, wound, abscesses or surgical site infections, septicemia, and pneumonia in adults. The total gene content of 379 strains of Cronobacter spp. and taxonomically-related isolates was determined using a recently reported DNA microarray. The Cronobacter microarray as a genotyping tool gives the global food safety community a rapid method to identify and capture the total genomic content of outbreak isolates for food safety, environmental, and clinical surveillance purposes. It was able to differentiate the seven Cronobacter species from one another and from non-Cronobacter species. The microarray was also able to cluster strains within each species into well-defined subgroups. These results also support previous studies on the phylogenic separation of species members of the genus and clearly highlight the evolutionary sequence divergence among each species of the genus compared to phylogenetically-related species. This review extends these studies and illustrates how the microarray can also be used as an investigational tool to mine genomic data sets from strains. Three case studies describing the use of the microarray are shown and include: (1) the determination of allelic differences among Cronobacter sakazakii strains possessing the virulence plasmid pESA3; (2) mining of malonate and myo-inositol alleles among subspecies of Cronobacter dublinensis strains to determine subspecies identity; and (3) lastly using the microarray to demonstrate sequence divergence and phylogenetic relatedness trends for 13 outer-membrane protein alleles among 240 Cronobacter and phylogenetically-related strains. The goal of this review is to describe microarrays as a robust tool for genomics research of this assorted and important genus, a criterion toward the development of future preventative measures to eliminate this foodborne pathogen from the global food supply.

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 alpha-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.