An immunoliposome-based immunochromatographic strip assay for the rapid detection of Cronobacter species.

Cronobacter species are foodborne pathogens that pose a high risk in infant formula and can cause fatality rates of 40-80% in infected infants. To develop a rapid and easy detection method for Cronobacter species, especially in powdered infant formula (PIF), an immunoliposome-based immunochromatographic strip assay was developed using an anti-Cronobacter immunoglobulin G (IgG)-conjugated liposome and an anti-Cronobacter IgG-coated nitrocellulose membrane. The developed assay could detect Cronobacter species in both pure culture and artificially contaminated PIF. The detection limits of the developed assay were 106-107 colony forming units (CFU)/mL in pure culture and 107-108 CFU/g in PIF by visual judgment, respectively. When the immunoliposome-based immunochromatographic strip assay results were analyzed using QuantiScan, the detection limit decreased to 105-107 CFU/mL in pure culture and 106-108 CFU/g in PIF, except for Cronobacter malonaticus. Furthermore, visual judgment showed that the developed immunochromatographic strip could not detect Cronobacter malonaticus in pure culture or PIF. However, Cronobacter malonaticus could be detected after QuantiScan analysis, and the detection limits were 108 CFU/mL and 108 CFU/g in both pure culture and PIF. This developed immunoliposome-based immunochromatographic strip assay is simple, easy, and effective method to detect Cronobacter species and thus could be widely applied in the food industry, research institutes, and even for onsite detection.

Genes involved in tolerance to osmotic stress by random mutagenesis in Cronobacter malonaticus.

Cronobacter malonaticus is one of the opportunistic food-borne pathogens in powdered infant formula and has unusual abilities to survive under environmental stresses such as osmotic conditions. However, the genes involved in osmotic stress have received little attention in C. malonaticus. Here, genes involved in osmotic stress were determined in C. malonaticus using a transposon mutagenesis approach. According to the growth of mutants (n = 215) under 5.0% NaCl concentration, the survival of 5 mutants under osmotic stress was significantly decreased compared with that of the wild type strain. Five mutating sites, including potassium efflux protein KefA, inner membrane protein YqjF, peptidylprolyl isomerase, Cys-tRNA(Pro)/Cys-tRNA(Cys) deacylase, and oligogalacturonate lyase were successfully identified. In addition, the biofilm formation of 5 mutants was determined using crystal violet staining, scanning electron microscopy, and confocal laser scanning microscopy, and the biofilms of 5 mutants significantly decreased within 72 h compared with that of wild type strain. This is the first report to determine the genes involved in osmotic tolerance in C. malonaticus. The findings provided valuable information for deep understanding of the mechanism of survival of C. malonaticus under osmotic stress, and a possible relationship between biofilm formation and tolerance to osmotic stress was also demonstrated in C. malonaticus.

Novel Method for Reliable Identification of Siccibacter and Franconibacter Strains: from "Pseudo-Cronobacter" to New Enterobacteriaceae Genera.

In the last decade, strains of the genera Franconibacter and Siccibacter have been misclassified as first Enterobacter and later Cronobacter Because Cronobacter is a serious foodborne pathogen that affects premature neonates and elderly individuals, such misidentification may not only falsify epidemiological statistics but also lead to tests of powdered infant formula or other foods giving false results. Currently, the main ways of identifying Franconibacter and Siccibacter strains are by biochemical testing or by sequencing of the fusA gene as part of Cronobacter multilocus sequence typing (MLST), but in relation to these strains the former is generally highly difficult and unreliable while the latter remains expensive. To address this, we developed a fast, simple, and most importantly, reliable method for Franconibacter and Siccibacter identification based on intact-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Our method integrates the following steps: data preprocessing using mMass software; principal-component analysis (PCA) for the selection of mass spectrum fingerprints of Franconibacter and Siccibacter strains; optimization of the Biotyper database settings for the creation of main spectrum projections (MSPs). This methodology enabled us to create an in-house MALDI MS database that extends the current MALDI Biotyper database by including Franconibacter and Siccibacter strains. Finally, we verified our approach using seven previously unclassified strains, all of which were correctly identified, thereby validating our method.IMPORTANCE We show that the majority of methods currently used for the identification of Franconibacter and Siccibacter bacteria are not able to properly distinguish these strains from those of Cronobacter While sequencing of the fusA gene as part of Cronobacter MLST remains the most reliable such method, it is highly expensive and time-consuming. Here, we demonstrate a cost-effective and reliable alternative that correctly distinguishes between Franconibacter, Siccibacter, and Cronobacter bacteria and identifies Franconibacter and Siccibacter at the species level. Using intact-cell MALDI-TOF MS, we extend the current MALDI Biotyper database with 11 Franconibacter and Siccibacter MSPs. In addition, the use of our approach is likely to lead to a more reliable identification scheme for Franconibacter and Siccibacter strains and, consequently, a more trustworthy epidemiological picture of their involvement in disease.

Determination of single cell lag times of Cronobacter spp. strains exposed to different stress conditions: Impact on detection.

The variability of stress resistance and lag time of single cells can have a big impact on their growth and therefore on the probability of their detection in food. In this study, six strains of Cronobacter spp. were subjected to heat, acid and desiccation stress and single cell lag times were determined using optical density measurements. The duration of lag time was highest after acid stress and did not correlate to stress resistance. The effect that the inactivation caused by stress and an extended lag time had on the projected cfu level reached after enrichment was simulated in different scenarios. For most strains, an enrichment time of 18h was sufficient for stressed cells to reach the suggested minimum level of cell inoculum for the Cronobacter screening broth detection. Particular strains may require longer recovery periods. Further, probability calculations showed that the number of samples taken from a batch may have an important effect on detection probability, especially at low contamination rates. Therefore, in addition to increasing the recovery period, increasing the number of samples is a suitable strategy to improve detection.

A novel mechanism for direct real-time polymerase chain reaction that does not require DNA isolation from prokaryotic cells.

Typically, polymerase chain reaction (PCR) is performed after DNA isolation. Real-time PCR (qPCR), also known as direct qPCR in mammalian cells with weak membranes, is a common technique using crude samples subjected to preliminary boiling to elute DNA. However, applying this methodology to prokaryotic cells, which have solid cell walls, in contrast to mammalian cells which immediately burst in water, can result in poor detection. We successfully achieved PCR elongation with the addition of 1.3 cfu of Cronobacter muytjensii to a newly developed direct qPCR master mix without performing any crude DNA extraction (detection limit of 1.6 × 10(0) cfu/ml for the test sample compared with a detection limit of 1.6 × 10(3) cfu/ml primarily for crude (boiling) or classical DNA isolation). We revealed that the chromosomal DNA retained in prokaryotic cells can function as a PCR template, similarly to the mechanism in in situ PCR. Elucidating this reaction mechanism may contribute to the development of an innovative master mix for direct qPCR to detect genes in a single bacterium with solid cell walls and might lead to numerous novel findings in prokaryotic genomics research.

Diversity of O Antigens within the Genus Cronobacter: from Disorder to Order.

Cronobacter species are Gram-negative opportunistic pathogens that can cause serious infections in neonates. The lipopolysaccharides (LPSs) that form part of the outer membrane of such bacteria are possibly related to the virulence of particular bacterial strains. However, currently there is no clear overview of O-antigen diversity within the various Cronobacter strains and links with virulence. In this study, we tested a total of 82 strains, covering each of the Cronobacter species. The nucleotide variability of the O-antigen gene cluster was determined by restriction fragment length polymorphism (RFLP) analysis. As a result, the 82 strains were distributed into 11 previously published serotypes and 6 new serotypes, each defined by its characteristic restriction profile. These new serotypes were confirmed using genomic analysis of strains available in public databases: GenBank and PubMLST Cronobacter. Laboratory strains were then tested using the current serotype-specific PCR probes. The results show that the current PCR probes did not always correspond to genomic O-antigen gene cluster variation. In addition, we analyzed the LPS phenotype of the reference strains of all distinguishable serotypes. The identified serotypes were compared with data from the literature and the MLST database (www.pubmlst.org/cronobacter/). Based on the findings, we systematically classified a total of 24 serotypes for the Cronobacter genus. Moreover, we evaluated the clinical history of these strains and show that Cronobacter sakazakii O2, O1, and O4, C. turicensis O1, and C. malonaticus O2 serotypes are particularly predominant in clinical cases.

Effect of heat shock and recovery temperature on variability of single cell lag time of Cronobacter turicensis.

The effect of heat stress and subsequent recovery temperature on the individual cellular lag of Cronobacter turicensis was analysed using optical density measurements. Low numbers of cells were obtained through serial dilution and the time to reach an optical density of 0.035 was determined. Assuming the lag of a single cell follows a shifted Gamma distribution with a fixed shape parameter, the effect of recovery temperature on the individual lag of untreated and sublethally heat treated cells of Cr. turicensis were modelled. It was found that the shift parameter (Tshift) increased asymptotically as the temperature decreased while the logarithm of the scale parameter (θ) decreased linearly with recovery temperature. To test the validity of the model in food, growth of low numbers of untreated and heat treated Cr. turicensis in artificially contaminated infant first milk was measured experimentally and compared with predictions obtained by Monte Carlo simulations. Although the model for untreated cells slightly underestimated the actual growth in first milk at low temperatures, the model for heat treated cells was in agreement with the data derived from the challenge tests and provides a basis for reliable quantitative microbiological risk assessments for Cronobacter spp. in infant milk.

A Cronobacter turicensis O1 antigen-specific monoclonal antibody inhibits bacterial motility and entry into epithelial cells.

Cronobacter turicensis is an opportunistic foodborne pathogen that can cause a rare but sometimes lethal infection in neonates. Little is known about the virulence mechanisms and intracellular lifestyle of this pathogen. In this study, we developed an IgG monoclonal antibody (MAb; MAb 2G4) that specifically recognizes the O1 antigen of C. turicensis cells. The antilipopolysaccharide antibody bound predominantly monovalently to the O antigen and reduced bacterial growth without causing cell agglutination. Furthermore, binding of the antibody to the O1 antigen of C. turicensis cells caused a significant reduction of the membrane potential which is required to energize flagellar rotation, accompanied by a decreased flagellum-based motility. These results indicate that binding of IgG to the O antigen of C. turicensis causes a direct antimicrobial effect. In addition, this feature of the antibody enabled new insight into the pathogenicity of C. turicensis. In a tissue culture infection model, pretreatment of C. turicensis with MAb 2G4 showed no difference in adhesion to human epithelial cells, whereas invasion of bacteria into Caco-2 cells was significantly inhibited.

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

The O-polysaccharide (OPS) was isolated from Cronobacter universalis NCTC 9529(T), 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 ((1)H, 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 9529(T). [structure: see text]. OPS isolated from C. universalis was structurally characterized for the first time.

Taxonomic evaluation of the genus Enterobacter based on multilocus sequence analysis (MLSA): proposal to reclassify E. nimipressuralis and E. amnigenus into Lelliottia gen. nov. as Lelliottia nimipressuralis comb. nov. and Lelliottia amnigena comb. nov., respectively, E. gergoviae and E. pyrinus into Pluralibacter gen. nov. as Pluralibacter gergoviae comb. nov. and Pluralibacter pyrinus comb. nov., respectively, E. cowanii, E. radicincitans, E. oryzae and E. arachidis into Kosakonia gen. nov. as Kosakonia cowanii comb. nov., Kosakonia radicincitans comb. nov., Kosakonia oryzae comb. nov. and Kosakonia arachidis comb. nov., respectively, and E. turicensis, E. helveticus and E. pulveris into Cronobacter as Cronobacter zurichensis nom. nov., Cronobacter helveticus comb. nov. and Cronobacter pulveris comb. nov., respectively, and emended description of the genera Enterobacter and Cronobacter.

The taxonomy of Enterobacter has a complicated history, with several species transferred to and from this genus. Classification of strains is difficult owing to its polyphyletic nature, based on 16S rRNA gene sequences. It has been previously acknowledged that Enterobacter contains species which should be transferred to other genera. In an attempt to resolve the taxonomy of Enterobacter, MLSA based on partial sequencing of protein-encoding genes (gyrB, rpoB, infB and atpD) was performed on the type strains and reference strains of Enterobacter, Cronobacter and Serratia species, as well as members of the closely related genera Citrobacter, Klebsiella, Kluyvera, Leclercia, Mangrovibacter, Raoultella and Yokenella. Phylogenetic analyses of the concatenated nucleotide sequences revealed that Enterobacter can be divided into five strongly supported MLSA groups, suggesting that the species should be reclassified into five different genera. Further support for this was provided by a concatenated amino acid tree, phenotypic characteristics and fatty acid profiles, enabling differentiation of the MLSA groups. Three novel genera are proposed: Lelliottia gen. nov., Pluralibacter gen. nov. and Kosakonia gen. nov. and the following new combinations: Lelliottia nimipressuralis comb. nov., Lelliottia amnigena comb. nov., Pluralibacter gergoviae comb. nov., Pluralibacter pyrinus comb. nov., Kosakonia cowanii comb. nov., Kosakonia radicincitans comb. nov., Kosakonia oryzae comb. nov., Kosakonia arachidis comb. nov., Cronobacter helveticus comb. nov. and Cronobacter pulveris comb. nov. Additionally, the novel epithet Cronobacter zurichensis nom. nov. is proposed for the reclassification of Enterobacter turicensis into the genus Cronobacter, as Cronobacter turicensis (Iversen et al., 2008) is already in use.

Chemical structure of the O-polysaccharides isolated from Cronobacter turicensis sequence type 5 strains 57, 564, and 566.

The Cronobacter spp. are Gram-negative bacterial pathogens that can cause infections in all age groups, and have a high mortality rate in neonates due to necrotizing enterocolitis and meningitis. Recent genotyping studies have revealed a strong clonal lineage in the genus, but this has not been compared with physiological traits. The O-polysaccharides (OPS) were isolated from three C. turicensis sequence type 5 strains (57, 564, and 566) and structurally characterized using (1)H and (13)C NMR spectroscopy, including two-dimensional DQF-COSY, TOCSY, ROESY, and HSQC analysis. Further compositional determination was undertaken using classical chemical methods followed by GLC, and GLC-MS analysis. The repeating unit of the isolated O-polysaccharides consists of GlcNAc, Rha, Glc, and had the structure shown below and therefore complemented the sequence type. [structure: see text].

Structure and genetics of the O-antigen of Cronobacter turicensis G3882 from a new serotype, C. turicensis O2, and identification of a serotype-specific gene.

Lipopolysaccharides on the cell surface of Gram-negative bacteria are an important factor in pathogenicity, and the O-specific polysaccharide chain (O-polysaccharide, O-antigen) defines the immunospecificity of different bacterial strains. Cronobacter turicensis is an emerging foodborne pathogen which causes severe invasive infections in neonates. In this study, a new O serotype, C. turicensis O2, was established, the structure and genetics of the O-antigen were investigated, and a serotype-specific gene was identified. Sugar and methylation analyses, and nuclear magnetic resonance spectroscopy indicated that the O-polysaccharide contains D-galactose (D-Gal), N-acetyl-D-glucosamine (D-GlcNAc), L-rhamnose (L-Rha) and 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (di-N-acetyllegionaminic acid, Leg5Ac7Ac). The structure of the tetrasaccharide repeat of the O-polysaccharide was established as: [Formula: see text]. The O-antigen gene cluster of C. turicensis O2 was sequenced and compared with related gene clusters from available databases. Putative genes for the synthesis of L-Rha and Leg5Ac7Ac, and genes encoding sugar transferases and O-antigen processing genes (wzx and wzy) were found. The tentatively assigned functions of the O-antigen genes were in agreement with the structure of the O-polysaccharide. In addition, primers based on the wzy gene were shown to be specific for C. turicensis O2 in a screen against 145 strains.

Structure and genetics of the O-antigen of Cronobacter sakazakii G2726 (serotype O3) closely related to the O-antigen of C. muytjensii 3270.

The O-specific polysaccharide (O-antigen) was isolated from the lipopolysaccharide of Cronobacter sakazakii G2726 (serotype O3) and studied by sugar analysis, Smith degradation, and (1)H and (13)C NMR spectroscopy. The following structure of the acidic O-polysaccharide was established: [structure: see text]. This structure is closely related to that of the O-polysaccharide of Cronobacter muytjensii 3270, which has the same main chain and differs only in the lack of glucosylation. The O-antigen gene cluster of C. sakazakii G2726 found between the gnd and galF genes was sequenced, and the gene functions were tentatively assigned by similarity to related genes from available databases and taking into account the O-polysaccharide structure.

Identification of six species in the new genus Cronobacter (Enterobacter sakazakii) from culture using gas chromatographic analysis of fatty acid methyl esters.

Capillary GC with flame ionization detection (FID) was used to determine the cellular fatty acid (CFA) profiles of six species in the new genus Cronobacter (Enterobacter sakazakii). The six different species are C. sakazakii, C. malonaticus, C. dublinensis, C. muytjensii, C. turicensis, and C. genomospecies. For GC-FID analysis, whole cell fatty acid methyl esters (FAMEs) from cells cultured on brain heart infusion (BHI) agar at 35 degrees C for 24 h were obtained by saponification, methylation, and extraction into hexane-methyl tert-butyl ether. A data set for 57 strains of Cronobacter species was prepared using fatty acid profiles from two or three replicates prepared on different days. Major fatty acids of the Cronobacter strains evaluated in this study were straight-chain C12:0, C14:0, C16:0, and unsaturated C18:1, omega7c, summed C16:1 omega7c/C16:1 omega6c, and summed C14:0 3-OH/iso-C16:1, and C17:0 omega cyclo 7-8. The CFA profiles for the Cronobacter species are similar, but there are several fatty acids-C12:0, C14:0, C16:0, C18:1 omega7c, and summed C16:1 omega7c/ C16:1 omega6c--that differ significantly among these six species. Analysis of FAMEs from Cronobacter strains grown on BHI agar by a rapid GC-FID method is a sensitive procedure for the identification of these organisms, and this analytical method provides a procedure for the differentiation of strains from closely related Cronobacter species.

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

BACKGROUND: Cronobacter, formerly known as Enterobacter sakazakii, is a food-borne pathogen known to cause neonatal meningitis, septicaemia and death. Current diagnostic tests for identification of Cronobacter 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 Cronobacter 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. RESULTS: Here we report the use of Expectation Maximization clustering to categorise 98 strains of Cronobacter 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 Cronobacter sakazakii and C. turicensis revealed the gene for inositol monophosphatase is associated with putative virulence factors in pathogenic strains of Cronobacter. CONCLUSIONS: We demonstrated a computational approach allowing existing diagnostic kits to be used to identify pathogenic strains of Cronobacter. The resulting clusters correlated well with MLST sequence types and revealed new information about the pathogenicity of Cronobacter species.