Comparative Analysis of Genome Sequences Covering the Seven Cronobacter Species

Background

Species of Cronobacter are widespread in the environment and are occasional food-borne pathogens associated with serious neonatal diseases, including bacteraemia, meningitis, and necrotising enterocolitis. The genus is composed of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. dublinensis, C. muytjensii, C. universalis, and C. condimenti. Clinical cases are associated with three species, C. malonaticus, C. turicensis and, in particular, with C. sakazakii multilocus sequence type 4. Thus, it is plausible that virulence determinants have evolved in certain lineages.

Methodology/Principal Findings

We generated high quality sequence drafts for eleven Cronobacter genomes representing the seven Cronobacter species, including an ST4 strain of C. sakazakii. Comparative analysis of these genomes together with the two publicly available genomes revealed Cronobacter has over 6,000 genes in one or more strains and over 2,000 genes shared by all Cronobacter. Considerable variation in the presence of traits such as type six secretion systems, metal resistance (tellurite, copper and silver), and adhesins were found. C. sakazakii is unique in the Cronobacter genus in encoding genes enabling the utilization of exogenous sialic acid which may have clinical significance. The C. sakazakii ST4 strain 701 contained additional genes as compared to other C. sakazakii but none of them were known specific virulence-related genes.

Conclusions/Significance

Genome comparison revealed that pair-wise DNA sequence identity varies between 89 and 97% in the seven Cronobacter species, and also suggested various degrees of divergence. Sets of universal core genes and accessory genes unique to each strain were identified. These gene sequences can be used for designing genus/species specific detection assays. Genes encoding adhesins, T6SS, and metal resistance genes as well as prophages are found in only subsets of genomes and have contributed considerably to the variation of genomic content. Differences in gene content likely contribute to differences in the clinical and environmental distribution of species and sequence types.     

Genotyping and Source Tracking of Cronobacter sakazakii and C. malonaticus Isolates from Powdered Infant Formula and an Infant Formula Production Factory in China

Cronobacter spp. (formerly defined as Enterobacter sakazakii) are opportunistic bacterial pathogens of both infants and adults. In this study, we analyzed 70 Cronobacter isolates from powdered infant formula (PIF) and an infant formula production facility in China to determine possible contamination routes. The strains were profiled by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR-based O-antigen serotyping, and ompA and rpoB sequence analyses. The isolates were primarily Cronobacter sakazakii (66/70) or Cronobacter malonaticus (4/70). The strains were divided into 38 pulsotypes (PTs) using PFGE and 19 sequence types (STs) by MLST. In contrast, rpoB and ompA sequence analyses divided the strains into 10 overlapping clusters each. PCR serotyping of the 66 C. sakazakii and 4 C. malonaticus strains resulted in the identification of four C. sakazakii serotypes (O1, O2, O4, and O7) and a single C. malonaticus serotype, O2. The dominant C. sakazakii sequence types from PIF and an infant formula production factory in China were C. sakazakii clonal complex 4 (CC4) (n = 19), ST1 (n = 14), and ST64 (n = 11). C. sakazakii CC4 is a clonal lineage strongly associated with neonatal meningitis. In the process of manufacturing PIF, the spray-drying, fluidized-bed-drying, and packing areas were the main areas with Cronobacter contamination. C. sakazakii strains with the same pulsotypes (PT3 and PT2) and sequence types (ST1 and ST64) were isolated both from processing equipment and from the PIF finished product.     

Immunoproteomic identification of immunogenic proteins in Cronobacter sakazakii strain BAA-894

Cronobacter spp. are emerging opportunistic pathogens. Cronobacter sakazakii is considered as the predominant species in all infections. So far, our understanding of the species’ immunogens and potential virulence factors of Cronobacter spp. remains limited. In this study, an immunoproteomic approach was used to investigate soluble and insoluble proteins from the genome-sequenced strain C. sakazakii ATCC BAA-894. Proteins were separated using two-dimensional electrophoresis, detected by Western blotting with polyclonal antibodies of C. sakazakii BAA-894, and identified using tandem mass spectrometry (MALDI-MS and MALDI-MS/MS, MS/MSMS). A total of 11 immunoreactive proteins were initially identified in C. sakazakii BAA-894, including two outer membrane proteins, four periplasmic proteins, and five cytoplasmic proteins. In silico functional analysis of the 11 identified proteins indicated three proteins that were initially described as immunogens of pathogenic bacteria. For the remaining eight proteins, one protein was categorized as a potential virulence factor involved in protection against reactive oxygen species, and seven proteins were considered to play potential roles in adhesion, invasion, and biofilm formation. To our knowledge, this is the first time that immunogenic proteins of C. sakazakii BAA-894 have been identified as immunogens and potential virulence factors by an immunoproteomics approach. Future studies should investigate the roles of these proteins in bacterial pathogenesis and modulation of host immune responses during infection to identify their potential as molecular therapeutic targets.     

The Genotypic Characterization of Cronobacter spp. Isolated in China

Cronobacter spp. (Enterobacter sakazakii) is an important pathogen contaminating powdered infant formula (PIF). To describe the genotypic diversity of Cronobacter isolated in China, we identified the isolates using fusA allele sequencing, and subtyped all of the isolates using pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and multiple-locus variable-number tandem-repeat analysis (MLVA). A total of 105 isolates were identified, which included C. sakazakii (58 isolates), C. malonaticus (30 isolates), C. dublinensis (11 isolates), C. turicensis (5 isolates), and C. muytjensii (1 isolate). These isolates were showed to have 85 PFGE-patterns, 71 sequence types (STs), and 55 MLVA-patterns. Comparisons among the three molecular subtyping methods revealed that the PFGE method was the most distinguishable tool in identifying clusters of Cronobacter spp. through DNA fingerprinting, and MLST method came second. However, ESTR-1, ESTR-2, ESTR-3, and ESTR-4 were not effective loci for subtyping Cronobacter spp. such that the MLVA method requires further improvement.     

Surveillance and characterisation of Cronobacter spp. in Czech retail food and environmental samples

Cronobacter spp. (formerly Enterobacter sakazakii) are emerging, opportunistic pathogens that are linked with food-borne infections in neonates and infants. In the present study, 291 samples of food, 36 samples from a dairy farm and 140 samples of dust from vacuum cleaners were examined for the presence of Cronobacter spp. using chromogenic media and biochemical tests. Altogether, 72 Cronobacter spp. strains were isolated in accordance with the reference standard ?SN P ISO/TS 22964 (2006). No Cronobacter spp. strains were detected in 10 samples of infant milk formula or in samples from a dairy farm. Twelve out of 20 positive food samples were dry products. The incidence of Cronobacter spp. in instant and powdered products and spices (12 positive isolates out of 82 samples) was significantly higher than that in other foods (P?=?0.002), but lower than that in samples of dust (52 isolates; P?<?0.001). The incidence of Cronobacter spp. in dust from restaurants, bars and hotels (13 positive isolates in 20 samples) was significantly higher than that in dust from households (P?=?0.010). The polymerase chain reaction assay for the species-specific detection of the rpoB gene was performed in 49 isolates. Thirty-four Cronobacter spp. isolates were identified as Cronobacter sakazakii, nine isolates as Cronobacter malonaticus and one isolate as Cronobacter turicensis.     

Multilocus sequence typing of Cronobacter sakazakii and Cronobacter malonaticus reveals stable clonal structures with clinical significance which do not correlate with biotypes

Background  

The Cronobacter genus (Enterobacter sakazakii) has come to prominence due to its association with infant infections, and the ingestion of contaminated reconstituted infant formula. C. sakazakii and C. malonaticus are closely related, and are defined according their biotype. Due to the ubiquitous nature of the organism, and the high severity of infection for the immunocompromised, a multilocus sequence typing (MLST) scheme has been developed for the fast and reliable identification and discrimination of C. sakazakii and C. malonaticus strains. It was applied to 60 strains of C. sakazakii and 16 strains of C. malonaticus, including the index strains used to define the biotypes. The strains were from clinical and non-clinical sources between 1951 and 2008 in USA, Canada, Europe, New Zealand and the Far East.     

Occurrence of Cronobacter spp. in retail foods

Aims: To study the occurrence of Cronobacter spp. in foods and to investigate the phenotypic properties of the strains isolated. Methods and Results: A total of 53 strains of Cronobacter spp. isolated from 399 food samples were identified using conventional biochemical methods and MALDI‐TOF mass spectrometry. Foods of plant origin were the most frequently contaminated samples. No Cronobacter spp. were found in infant milk formula, wheat‐based infant food, pasteurized and raw cow milk, mincemeat, chicken, chickpea and potato dumpling powder. The individual species were identified as Cronobacter sakazakii (54·7%), Cronobacter malonaticus (28·4%), Cronobacter dublinensis (7·5%), Cronobacter muytjensii (7·5%) and Cronobacter turicensis (1·9%). Cronobacter sakazakii and C. malonaticus belong to biotype 1, 2, 2a, 3, 4 and 5, 5a, respectively. Cronobacter dublinensis strains were subdivided into biotypes 6 and 12. All strains were resistant to erythromycin and two of them were resistant to both erythromycin and tetracycline. Conclusions: Cronobacter spp. were isolated from various food samples pre‐eminently of plant origin and dried food ingredients. Significance and Impact of the Study: These findings will increase and detail our knowledge of the presence and diversity of Cronobacter spp. in foods.     

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.     

Cronobacter,the emergent bacterial pathogen Enterobacter sakazakii comes of age; MLST and whole genome sequence analysis

Background

Following the association of Cronobacter spp. to several publicized fatal outbreaks in neonatal intensive care units of meningitis and necrotising enterocolitis, the World Health Organization (WHO) in 2004 requested the establishment of a molecular typing scheme to enable the international control of the organism. This paper presents the application of Next Generation Sequencing (NGS) to Cronobacter which has led to the establishment of the Cronobacter PubMLST genome and sequence definition database (http://pubmlst.org/cronobacter/) containing over 1000 isolates with metadata along with the recognition of specific clonal lineages linked to neonatal meningitis and adult infections

Results

Whole genome sequencing and multilocus sequence typing (MLST) has supports the formal recognition of the genus Cronobacter composed of seven species to replace the former single species Enterobacter sakazakii. Applying the 7-loci MLST scheme to 1007 strains revealed 298 definable sequence types, yet only C. sakazakii clonal complex 4 (CC4) was principally associated with neonatal meningitis. This clonal lineage has been confirmed using ribosomal-MLST (51-loci) and whole genome-MLST (1865 loci) to analyse 107 whole genomes via the Cronobacter PubMLST database. This database has enabled the retrospective analysis of historic cases and outbreaks following re-identification of those strains.

Conclusions

The Cronobacter PubMLST database offers a central, open access, reliable sequence-based repository for researchers. It has the capacity to create new analysis schemes ‘on the fly’, and to integrate metadata (source, geographic distribution, clinical presentation). It is also expandable and adaptable to changes in taxonomy, and able to support the development of reliable detection methods of use to industry and regulatory authorities. Therefore it meets the WHO (2004) request for the establishment of a typing scheme for this emergent bacterial pathogen. Whole genome sequencing has additionally shown a range of potential virulence and environmental fitness traits which may account for the association of C. sakazakii CC4 pathogenicity, and propensity for neonatal CNS.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1121) contains supplementary material, which is available to authorized users.     

Genes Involved in Cronobacter sakazakii Biofilm Formation

Cronobacter spp. are opportunistic food-borne pathogens that can cause severe and sometimes lethal infections in neonates. In some outbreaks, the sources of infection were traced to contaminated powdered infant formula (PIF) or contaminated utensils used for PIF reconstitution. In this study, we investigated biofilm formation in Cronobacter sakazakii strain ES5. To investigate the genetic basis of biofilm formation in Cronobacter on abiotic surfaces, we screened a library of random transposon mutants of strain ES5 for reduced biofilm formation using a polystyrene microtiter assay. Genetic characterization of the mutants led to identification of genes that are associated with cellulose biosynthesis and flagellar structure and biosynthesis and genes involved in basic cellular processes and virulence, as well as several genes whose functions are currently unknown. In two of the mutants, hypothetical proteins ESA_00281 and ESA_00282 had a strong impact on flow cell biofilm architecture, and their contribution to biofilm formation was confirmed by genetic complementation. In addition, adhesion of selected biofilm formation mutants to Caco-2 intestinal epithelial cells was investigated. Our findings suggest that flagella and hypothetical proteins ESA_00281 and ESA_00282, but not cellulose, contribute to adhesion of Cronobacter to this biotic surface.Biofilms are interface-associated consortia of microorganisms that are typically embedded in an endogenous slimy matrix referred to as extracellular polymeric substance (EPS). It is generally accepted that growth as a biofilm is the predominant microbial lifestyle in nature. Biofilms have several phenotypic characteristics that clearly set them apart from planktonic cultures, most notably increased resistance to a variety of environmental influences (16), which makes their eradication more difficult. Microbial biofilms are of special concern to the food industry, as biofilms on raw materials or food contact surfaces represent possible sources of product contamination with spoilage or pathogenic microorganisms (for a recent review, see reference 4).Cronobacter spp. are opportunistic food-borne pathogens that can cause severe disease in neonates which may present as septicemia, meningitis, or necrotizing enterocolitis (NEC). In several outbreaks, the source of infection was traced to contaminated powdered infant formula (PIF) or to spoons and blenders used in preparation of PIF (8, 10). The genus Cronobacter currently comprises six species: Cronobacter sakazakii, Cronobacter dublinensis, Cronobacter turicensis, Cronobacter malonaticus, Cronobacter muytjensii, and Cronobacter genomospecies 1 (20). Cronobacter spp. display remarkable resistance to desiccation compared to other Enterobacteriaceae (7), which may contribute to their long-term survival in PIF and on surfaces. Few studies of biofilm formation by Cronobacter spp. have been conducted so far. It has been observed that some strains are able to form biofilms on glass, stainless steel, polyvinyl chloride (PVC), polycarbonate, silicone, and enteral feeding tubes in different media (19, 24, 28). Like biofilm formation in other bacteria, biofilm formation is different for different strains and is highly dependent on the medium and surface used. Furthermore, the survival of C. sakazakii in biofilms under different environmental conditions has been investigated (23), and increased resistance of Cronobacter biofilms to disinfectants has been demonstrated (25). Cellulose has been described as a component of the Cronobacter extracellular matrix (15, 28, 51).In this study, we performed a genetic analysis of biofilm formation by Cronobacter sakazakii strain ES5, a clinical isolate, by using random transposon mutagenesis and subsequent screening of a mutant library for altered biofilm phenotype using a microtiter assay system. In addition, the biofilm structure of the wild type and selected mutants in a continuous-culture flow cell system was investigated by using confocal laser scanning microscopy (CLSM). Finally, we tested whether for selected mutants the defects in biofilm formation observed on the abiotic surface had an influence on the capacity of C. sakazakii to adhere to Caco-2 intestinal epithelial cells.     

Reconstruction of the Carotenoid Biosynthetic Pathway of Cronobacter sakazakii BAA894 in Escherichia coli

Cronobacter sakazakii could form yellow-pigmented colonies. However, the chemical structure and the biosynthetic pathway of the yellow pigments have not been identified. In this study, the yellow pigments of C. sakazakii BAA894 were purified and analyzed. The major components of the yellow pigments were confirmed as zeaxanthin-monoglycoside and zeaxanthin-diglycoside. A gene cluster containing seven genes responsible for the yellow pigmentation in C. sakazakii BAA894 was identified. The seven genes of C. sakazakii BAA894 or parts of them were reconstructed in a heterologous host Escherichia coli DH5α. The pigments formed in these E. coli strains were isolated and analyzed by thin layer chromatography, UV-visible spectroscopy, high performance liquid chromatography, and electron spray ionization-mass spectrometry. These redesigned E. coli strains could produce different carotenoids. E. coli strain expressing all the seven genes could produce zeaxanthin-monoglycoside and zeaxanthin-diglycoside; E. coli strains expressing parts of the seven genes could produce lycopene, β-carotene, cryptoxanthin or zeaxanthin. This study identified the gene cluster responsible for the yellow pigmentation in C. sakazakii BAA894.     

Stress tolerant virulent strains of Cronobacter sakazakii from food

Background

Cronobacter sakazakii is considered as an emerging foodborne pathogen. The aim of this study was to isolate and characterize virulent strains of Cronobacter sakazakii from food samples of Bangladesh.

Result

Six (6) Cronobacter sakazakii was isolated and identified from 54 food samples on the basis of biochemical characteristics, sugar fermentation, SDS-PAGE of whole cell protein, plasmid profile and PCR of Cronobacter spp. specific genes (esak, gluA, zpx, ompA, ERIC, BOX-AIR) and sequencing. These strains were found to have moderately high antibiotic resistance against common antibiotics and some are ESBL producer. Most of the C. sakazakii isolates were capable of producing biofilm (strong biofilm producer), extracellular protease and siderophores, curli expression, haemolysin, haemagglutinin, mannose resistant haemagglutinin, had high cell surface hydrophobicity, significant resistance to human serum, can tolerate high concentration of salt, bile and DNase production. Most of them produced enterotoxins of different molecular weight. The isolates pose significant serological cross-reactivity with other gram negative pathogens such as serotypes of Salmonella spp., Shigella boydii, Shigella sonnei, Shigella flexneri and Vibrio cholerae. They had significant tolerance to high temperature, low pH, dryness and osmotic stress.

Conclusion

Special attention should be given in ensuring hygiene in production and post-processing to prevent contamination of food with such stress-tolerant virulent Cronobacter sakazakii.

Electronic supplementary material

The online version of this article (doi:10.1186/0717-6287-47-63) contains supplementary material, which is available to authorized users.     

Flagella from Five Cronobacter Species Induce Pro-Inflammatory Cytokines in Macrophage Derivatives from Human Monocytes

Cronobacter spp. are opportunistic pathogens linked to lie-threatening infections in neonates and contaminated powdered infant formula that has been epidemiologically associated with these cases. Clinical symptoms of Cronobacter include necrotizing enterocolitis, bacteremia, and meningitis. Flagella from C. sakazakii are involved in biofilm formation and its adhesion to epithelial cells. We investigated the role of flagella from C. sakazakii ST1 and ST4, C. malonaticus, C. muytjensii, C. turicensis and C. dublinensis during the activation of cytokines (IL-8, TNF-α, and IL-10) in macrophage derivatives from human monocytes, which has not been extensively studied. The production and identity of flagella from the five Cronobacter species were visualized and recognized with anti-flagella antibodies by immunogold labeling through transmission electron microscopy. Purified flagella were dissociated into monomers in 12% SDS-PAGE Coomassie blue-stained gels showing a band of ∼28 kDa and, in addition, mass spectrometry revealed the presence of several peptides that correspond to flagellin. Flagella (100 ng) induced the release of IL-8 (3314–6025 pg/ml), TNF-α (39–359 pg/ml), and IL-10 (2–96 pg/ml), in macrophage isolates from human monocytes and similar results were obtained when flagella were dissociated into monomers. Inhibition assays using three dilutions of anti-flagella antibodies (1∶10, 1∶100, and 1∶200) suppressed the secretion of IL-8, TNF-α, and IL-10 between 95–100% using 100 ng of protein. A transfection assay using 293-hTLR5 cells showed IL-8 release of 197 pg/ml and suppression in the secretion of IL-8 when anti-hTLR5-IgA antibodies were used at different concentrations. These observations suggest that flagella and flagellin are involved in an inflammatory response dependent on TLR5 recognition, which could contribute to the pathogenesis of the bacteria.     

Outer Membrane Proteins A (OmpA) and X (OmpX) Are Essential for Basolateral Invasion of Cronobacter sakazakii

Cronobacter sakazakii is an opportunistic pathogen that actively invades host eukaryotic cells. To identify invasion factors responsible for the intestinal translocation of C. sakazakii, we constructed for the first time outer membrane protein X (OmpX) and A (OmpA) deletion mutants using the lambda Red recombination system. The ompX and ompA deletion mutants showed significantly reduced invasion of human enterocyte-like epithelial Caco-2 and human intestinal epithelial INT-407 cells, and significantly fewer mutant cells were recovered from the livers and spleens of rat pups. Furthermore, compared with intact target cells, the invasion and initial association potentials of the mutants increased at a rate similar to that of the wild type in tight-junction-disrupted target cells, suggesting that OmpX and OmpA are involved in basolateral invasion by C. sakazakii. This is the first report of C. sakazakii virulence determinants that are essential for basolateral invasion and that may be critical for the virulence of C. sakazakii.Enterobacter sakazakii is an emerging pathogen associated with several outbreaks of meningitis and local necrotizing enterocolitis in premature infants (2, 28, 37). There was considerable diversity among E. sakazakii isolates (13, 14), and the original taxonomic name of E. sakazakii was reclassified as Cronobacter spp., which included Cronobacter sakazakii (13, 14). Therefore, C. sakazakii will be used throughout this paper. Although the incidence of Cronobacter infection is rare, the mortality rate is as high as 33 to 80% (11, 27, 32, 39). Even when infants survive Cronobacter infection, they often experience serious sequelae, including brain abscesses, developmental delay, and impairment of sight and hearing (8). Premature infants, whose immune systems are not fully developed, may be at high risk for Cronobacter infection (26).Very little is known about the mechanisms of pathogenicity and the virulence determinants of the genus Cronobacter. Adhesion of Cronobacter spp. to eukaryotic cells showed two distinct patterns, i.e., a diffuse pattern and the formation of localized clusters, which was nonfimbrial (21). Pagotto et al. (29) reported that the genus Cronobacter produced enterotoxins and was lethal on intraperitoneal injection into suckling mice at levels as low as 10⁵ CFU per mouse. The genus Cronobacter interacts with and damages intestinal epithelial cells, which results in intestinal injury and villus disruption (12). In addition, the cell-bound zinc-containing metalloprotease encoded by zpx caused rounding of Chinese hamster ovary (CHO) cells (19), which may be important in dissemination of the pathogen into the systemic circulation. Furthermore, Townsend et al. (36) showed that Cronobacter can persist within rat macrophages.As an oral pathogen causing a systemic infection, C. sakazakii must translocate from the intestinal lumen into the blood circulation. The genus Cronobacter is capable of actively invading various epithelial and endothelial cells of human and animal origin (17, 25, 31). Kim and Loessner (17) reported that the active invasion of human intestinal Caco-2 cells by C. sakazakii requires de novo bacterial protein synthesis and the host cell cytoskeleton and that the invasion efficiency of C. sakazakii was enhanced in the absence of cellular tight junctions. With regard to the virulence determinants related to Cronobacter penetration of the host cells, Mohan Nair and Venkitanarayanan (25) and Singamsetty et al. (31) reported that outer membrane protein A (OmpA) of Cronobacter plays an important role in the invasion of human intestinal epithelial INT-407 cells and human brain microvascular endothelial cells (HBMECs); invasion was dependent on both microfilaments and microtubules in INT-407 cells but only on microtubule condensation in HBMECs. Obviously, bacterial translocation in the intestines is multifactorial, and more detailed studies are needed to gain a better understanding of C. sakazakii pathogenesis.Outer membrane protein X (OmpX) of C. sakazakii was identified in this study. Previously, OmpX in other bacteria was shown to be involved in the invasion of host cells (7, 18), neutralizing host defense mechanisms, and bacterial defense against the complement systems of the host (10, 38).In this study, we report for the first time a successful application of the lambda Red recombination system to construct in-frame OmpX and/or OmpA deletion mutants in C. sakazakii. We further report that both outer membrane proteins (OMPs) of C. sakazakii, OmpX and OmpA, play critical roles in its invasion through not only the apical side, but also the basolateral side, of the host cells. We also show that OmpX and OmpA are responsible for C. sakazakii translocation into the deeper organs (i.e., liver and spleen).     

Genes Involved in Yellow Pigmentation of Cronobacter sakazakii ES5 and Influence of Pigmentation on Persistence and Growth under Environmental Stress

Cronobacter spp. are opportunistic food-borne pathogens that are responsible for rare but highly fatal cases of meningitis and necrotizing enterocolitis in neonates. While the operon responsible for yellow pigmentation in Cronobacter sakazakii strain ES5 was described recently, the involvement of additional genes in pigment expression and the influence of pigmentation on the fitness of Cronobacter spp. have not been investigated. Thus, the aim of this study was to identify further genes involved in pigment expression in Cronobacter sakazakii ES5 and to assess the influence of pigmentation on growth and persistence under conditions of environmental stress. A knockout library was created using random transposon mutagenesis. The screening of 9,500 mutants for decreased pigment production identified 30 colorless mutants. The mapping of transposon insertion sites revealed insertions in not only the carotenoid operon but also in various other genes involved in signal transduction, inorganic ions, and energy metabolism. To determine the effect of pigmentation on fitness, colorless mutants (ΔcrtE, ΔcrtX, and ΔcrtY) were compared to the yellow wild type using growth and inactivation experiments, a macrophage assay, and a phenotype array. Among other findings, the colorless mutants grew at significantly increased rates under osmotic stress compared to that of the yellow wild type while showing increased susceptibility to desiccation. Moreover, ΔcrtE and ΔcrtY exhibited increased sensitivity to UVB irradiation.Cronobacter spp. (formerly Enterobacter sakazakii) are opportunistic food-borne pathogens that cause rare but life-threatening cases of meningitis, necrotizing enterocolitis, and septicemia in neonates (7, 30, 39, 40). While the pathogen appears to be ubiquitous, powdered infant formula (PIF) has been implicated as the main source of Cronobacter infection, necessitating effective means of both detecting this organism and preventing contamination in the PIF production environment (14, 26, 40).Although white strains have been observed occasionally, the production of yellow pigment on tryptic soy agar (TSA) is still one of the key discriminative criteria in the identification of presumptive Cronobacter spp. isolates via the ISO/TS 22964 standard protocol (3, 6, 11, 25). Studies of which colorless or cream-white strains of Cronobacter spp. (formerly Enterobacter sakazakii) were identified have reported prevalence rates of 8, 13, and 21.4% (6, 11, 24).The pigment''s carotenogenic nature recently was identified in Cronobacter strain ES5 on a molecular and chemical level (31). Carotenoids are known to stabilize cellular membranes and influence membrane fluidity (13, 22, 48). Functioning as antioxidants, carotenoids scavenge reactive oxygen species (37, 54, 55). Moreover, pigments play a role in the survival of bacteria in harmful environments and have been found to increase the virulence of pathogens such as Staphylococcus aureus and Erwinia chrysanthemi (32, 33, 44, 55). In Cronobacter strain ES5, a gene cluster comprised of seven genes (crtE-idi- crtXYIBZ) was found to be responsible for carotenoid biosynthesis (31). While the study mentioned above identified the operon responsible for carotenoid production, the involvement of other genes in pigment expression has not been investigated.Because no research exists on the influence of pigmentation on the fitness and persistence of Cronobacter spp., the potential implications of failing to detect colorless strains of this organism in the PIF production environment are difficult to assess. Thus, the aim of this study was to further describe the genetic basis of the pigmented phenotype of Cronobacter strain ES5 by isolating and characterizing isogenic white mutants via random transposon mutagenesis and subsequent sequencing, and to identify the impact of pigmentation on persistence and growth under conditions of environmental stress by comparing white mutants to the yellow wild type in a variety of growth and inactivation experiments, a macrophage assay, and a phenotype array.     

Comparative Genotypic and Phenotypic Analysis of Cronobacter Species Cultured from Four Powdered Infant Formula Production Facilities: Indication of Pathoadaptation along the Food Chain

Cronobacter species are opportunistic pathogens commonly found in the environment. Among the seven Cronobacter species, Cronobacter sakazakii sequence type 4 (ST-4) is predominantly associated with recorded cases of infantile meningitis. This study reports on a 26-month powdered infant formula (PIF) surveillance program in four production facilities located in distinct geographic regions. The objective was to identify the ST(s) in PIF production environments and to investigate the phenotypic features that support their survival. Of all 168 Cronobacter isolates, 133 were recovered from a PIF production environment, 31 were of clinical origin, and 4 were laboratory type strains. Sequence type 1 (n = 84 isolates; 63.9%) was the dominant type in PIF production environments. The majority of these isolates clustered with an indistinguishable pulsotype and persisted for at least an 18-month period. Moreover, DNA microarray results identified two phylogenetic lineages among ST-4 strains tested. Thereafter, the ST-1 and -4 isolates were phenotypically compared. Differences were noted based on the phenotypes expressed by these isolates. The ST-1 PIF isolates produced stronger biofilms at both 28°C and 37°C, while the ST-4 clinical isolates exhibited greater swimming activity and increased binding to Congo red dye. Given the fact that PIF is a low-moisture environment and that the clinical environment provides for an interaction between the pathogen and its host, these differences may be consistent with a form of pathoadaptation. These findings help to extend our current understanding of the epidemiology and ecology of Cronobacter species in PIF production environments.     

Computational identification and analysis of arsenate reductase protein in Cronobacter sakazakii ATCC BAA-894 suggests potential microorganism for reducing arsenate

This study focuses a bioinformatics-based prediction of arsC gene product arsenate reductase (ArsC) protein in Cronobacter sakazakii BAA-894 strain. A protein structure-based study encloses three-dimensional structural modeling of target ArsC protein, was carried out by homology modeling method. Ultimately, the detection of active binding regions was carried out for characterization of functional sites in protein. The ten probable ligand binding sites were predicted for target protein structure and highlighted the common binding residues between target and template protein. It has been first time identified that modeled ArsC protein structure in C. sakazakii was structurally and functionally similar to well-characterized ArsC protein of Escherichia coli because of having same structural motifs and fold with similar protein topology and function. Investigation revealed that ArsC from C. sakazakii can play significant role during arsenic resistance and potential microorganism for bioremediation of arsenic toxicity.     

Analysis of major band of Enterobacter sakazakii by ERIC-PCR and development of a species-specific PCR for detection of Ent. sakazakii in dry food samples

ERIC (Enterobacterial Repetitive Intergenic Consensus)-PCR was employed to generate stable and reproductive ERIC-PCR fingerprints of Ent. sakazakii ATCC51329. Moreover, this study also cloned and sequenced a major band of Ent. sakazakii (ATCC51329) ERIC-PCR fingerprints. The major band was amplified with primer ERIC2 and sequences extending primer ERIC 2 showed poor similarity with ERIC elements. A comparison of the nucleotide acid with other sequences available in the GenBank revealed 90% of identity with Ent. sakazakii ATCC BAA-894, and 73%–74% of identity with oligopeptiase gene or proteaseⅡgene of some species from the Enterobacteriaceae family. Two primers were synthesized to develop and optimize an Enterobacter sakazakii-specific PCR based on regions of major band unique to Ent. sakazakii. The expected fragment was amplified from all of Ent. sakazkaii but not from the negative controls. As few as 10² CFU/ml of Ent. sakazakii of PCR were directly detected in the infant formulas. This was the case even in the presence of other bacteria. A comparison of traditional methods and new developed PCR in commercial foods suggested that without using API20-E test, the DFI chromogenic medium and FDA method showed 46.15% and 50% false positive respectively. Moreover, one false negative was observed with FDA method. In contrast, PCR was highly sensitive and specific to Ent. sakazakii. A high heterogeneity between Ent. sakazakii and the other microorganisms was found on expected fragment sequence. In addition, Ent. sakazakii ATCC51329 formed a separate branch with > 5% divergence from the type strain ATCC BAA-894 and major strains.     

Prevalence and Relative Risk of Cronobacter spp., Salmonella spp., and Listeria monocytogenes Associated with the Body Surfaces and Guts of Individual Filth Flies

Although flies are important vectors of food-borne pathogens, there is little information to accurately assess the food-related health risk of the presence of individual flies, especially in urban areas. This study quantifies the prevalence and the relative risk of food-borne pathogens associated with the body surfaces and guts of individual wild flies. One hundred flies were collected from the dumpsters of 10 randomly selected urban restaurants. Flies were identified using taxonomic keys before being individually dissected. Cronobacter spp., Salmonella spp., and Listeria monocytogenes were detected using the PCR-based BAX system Q7. Positive samples were confirmed by culture on specific media and through PCR amplification and sequencing or ribotyping. Among collected flies were the housefly, Musca domestica (47%), the blowflies, Lucilia cuprina (33%) and Lucilia sericata (14%), and others (6%). Cronobacter species were detected in 14% of flies, including C. sakazakii, C. turicensis, and C. universalis, leading to the proposal of flies as a natural reservoir of this food-borne pathogen. Six percent of flies carried Salmonella enterica, including the serovars Poona, Hadar, Schwarzengrund, Senftenberg, and Brackenridge. L. monocytogenes was detected in 3% of flies. Overall, the prevalence of food-borne pathogens was three times greater in the guts than on the body surfaces of the flies. The relative risk of flies carrying any of the three pathogens was associated with the type of pathogen, the body part of the fly, and the ambient temperature. These data enhance the ability to predict the microbiological risk associated with the presence of individual flies in food and food facilities.