Whole Genome Sequencing-Based Comparison of Food Isolates of Cronobacter sakazakii.
ABSTRACT: Cronobacter sakazakii is an emerging foodborne pathogen, which is linked to life-threatening infections causing septicemia, meningitis, and necrotizing enterocolitis. These infections have been epidemiologically connected to ingestion of contaminated reconstituted powder infant formula. Even at low water activity C. sakazakii can survive for a long time; it is capable of protective biofilm formation and occasionally shows high virulence and pathogenicity even following stressful environmental conditions. Hence it is a challenging task for the food industry to control contamination of food ingredients and products through the entire production chain, since an increasing number of severe food-related outbreaks of C. sakazakii infections has been observed. The seemingly great capability of C. sakazakii to survive even strict countermeasures combined with its prevalence in many food ingredients requires a greater in depth understanding of its virulence factors to master the food safety issues related to this organism. In this context, we present the whole genome sequence (WGS) of two different C. sakazakii isolated from skimmed milk powder (C7) and ready-to-eat salad mix (C8), respectively. These are compared to other, already sequenced, C. sakazakii genomes. Sequencing of the fusA allele revealed that both isolates were C. sakazakii. We investigated the molecular characteristics of both isolates relevant for genes associated with pathogenesis and virulence factors, resistance to stressful environmental conditions (e.g., osmotic and heat), survival in desiccation as well as conducted a comparative genomic analysis. By using multi-locus sequence typing (MLST), the genetic type of both isolates is assessed and the number of unique genes is determined. DNA of C. sakazakii C8 is shown to hold a novel and unique sequence type; the number of unique genes identified in the genomic sequence of C. sakazakii C7 and C8 were 109 and 188, respectively. Some of the determined unique genes such as the rhs and VgrG genes are linked to the Type VI Secretion System cluster, which is associated with pathogenicity and virulence factors. Moreover, seven genes encoding for multi-drug resistance were found in both isolates. The finding of a number of genes linked to producing capsules and biofilm are likely related to the observed resistance to desiccation.
Project description:Strong desiccation tolerance is an outstanding feature of Cronobacter sakazakii and can enable the bacterium to survive in a dry food matrix (such as milk powder) for a long time. Therefore, contamination of food possessing low water activity with C. sakazakii can increase the risk of infection in human beings, particularly in neonates and infants. However, the mechanism underlying the desiccation tolerance property of C. sakazakii is largely unknown. In this study, the desiccation tolerance characteristics of 42 C. sakazakii strains were analyzed. Simultaneously, the sequence types and biofilm formation abilities of the strains were investigated, and their correlations with desiccation tolerance were analyzed. The results showed no significant correlation between desiccation tolerance and sequence type. However, there was a positive correlation between biofilm formation ability and desiccation tolerance. Raman spectroscopy was employed to investigate the biofilm formed by strains with distinct desiccation tolerance levels, and the results showed that the levels of polysaccharide, proteins and carotenoid might play important roles in the resistance to dry environments. In addition, 10 genes involved in osmoprotectant synthesis or transport were selected, and their differential expression in strains with diverse desiccation tolerance levels was compared to investigate whether these genes were responsible for cytoprotection in the dry environment. The results revealed a great difference in gene expression among strains with different desiccation tolerance levels, suggesting that these genes play a regulatory role in the resistance of C. sakazakii to dry environments. Our study provides a useful reference for follow-up studies investigating the mechanism of desiccation tolerance in C. sakazakii.
Project description:Cronobacter sakazakii is a xerotolerant neonatal pathogen epidemiologically linked to powdered infant food formula, often resulting in high mortality rates. Here, we used transcriptome sequencing (RNA-seq) to provide transcriptional insights into the survival of C. sakazakii in desiccated conditions. Our RNA-seq data show that about 22% of the total C. sakazakii genes were significantly upregulated and 9% were downregulated during desiccation survival. When reverse transcription-quantitative PCR (qRT-PCR) was used to validate the RNA-seq data, we found that the primary desiccation response was gradually downregulated during the tested 4 hours of desiccation, while the secondary response remained constitutively upregulated. The 4-hour desiccation tolerance of C. sakazakii was dependent on the immediate microenvironment surrounding the bacterial cell. The removal of Trypticase soy broth (TSB) salts and the introduction of sterile infant formula residues in the microenvironment enhanced the desiccation survival of C. sakazakii SP291. The trehalose biosynthetic pathway encoded by otsA and otsB, a prominent secondary bacterial desiccation response, was highly upregulated in desiccated C. sakazakii C. sakazakii SP291 ?otsAB was significantly inhibited compared with the isogenic wild type in an 8-hour desiccation survival assay, confirming the physiological importance of trehalose in desiccation survival. Overall, we provide a comprehensive RNA-seq-based transcriptional overview along with confirmation of the phenotypic importance of trehalose metabolism in Cronobacter sakazakii during desiccation.IMPORTANCE Cronobacter sakazakii is a pathogen of importance to neonatal health and is known to persist in dry food matrices, such as powdered infant formula (PIF) and its associated production environment. When infections are reported in neonates, mortality rates can be high. The success of this bacterium in surviving these low-moisture environments suggests that Cronobacter species can respond to a variety of environmental signals. Therefore, understanding those signals that aid the persistence of this pathogen in these ecological niches is an important step toward the development of strategies to reduce the risk of contamination of PIF. This research led to the identification of candidate genes that play a role in the persistence of this pathogen in desiccated conditions and, thereby, serve as a model target to design future strategies to mitigate PIF-associated survival of C. sakazakii.
Project description:Cronobacter spp. in powdered infant formula has been etiologically linked to meningitis and necrotizing enterocolitis in certain groups of infants. This study aimed to determine whether C. sakazakii Yrt2a strain experiencing desiccation stress could enter viable but nonculturable (VBNC) state as well as to examine the expression of genes associated with stress and virulence during the above states. Stress and VBNC conditions were determined based on viability and culturability assays. Expression of genes related to stress (rpoS) and virulence (hfq and ompA) was evaluated by real-time PCR. The results showed that C. sakazakii Yrt2a entered VBNC 24 days post exposure to 2 h of desiccation treatment. The expression of rpoS, hfq and ompA genes was up-regulated during stress conditions, suggesting that Cronobacter successfully managed stress to maintain its culturability while maintaining its virulence. The expression of the target genes decreased at VBNC state but remained higher than that of a normal state. These findings reinforce the assumption that C. sakazakii undergoing VBNC state maintains its pathogenicity.
Project description:Cronobacter sakazakii is a xerotolerant neonatal pathogen epidemiologically linked to powdered infant food formula, often leading to high mortality rates. Here, we used RNA-seq to provide physiological and transcriptional insights into the survival of C. sakazakii in desiccated conditions. Our RNA-seq data shows that about 22% of the total C. sakazakii genes were significantly up-regulated and 9% were down-regulated during desiccation survival. When qRT-PCR was used to validate the RNA-seq data, we found that the primary desiccation response was gradually down-regulated during the tested four hours of desiccation while the secondary response remained constitutively up-regulated. The desiccation tolerance of C. sakazakii was dependent on the immediate microenvironment surrounding the bacterial cell. Removal of TSB salts and that the introduction of sterile infant formula residues in the microenvironment enhanced the desiccation survival of C. sakazakii SP291. The trehalose biosynthetic pathway encoded by otsA and otsB, a prominent secondary bacterial desiccation response, was highly up-regulated in desiccated C. sakazakii. C. sakazakii SP291 ∆otsAB was significantly inhibited compared to the wild type in an eight hour desiccation survival assay confirming the physiological importance of trehalose in desiccation survival. Overall, we provide a comprehensive RNA-seq based transcriptional overview and the phenotypic importance of trehalose metabolism in Cronobacter sakazakii during desiccation. Overall design: Bacterial transcriptomic profile of desiccated and early stationary phase (ESP; WT) conditions were generated by deep sequencing, in triplicates, using Illumina HiSeq2500.
Project description:Cronobacter sakazakii is well-known for its desiccation tolerance in the powdered infant formula (PIF) food production environment and the bacterium has been linked with high fatality rates in neonates who consume contaminated product. In this study, using deep-level RNA-sequencing, differentially expressed genes were studied in C. sakazakii ATCCTM29544 grown in simulated low-moisture environment designed to mimic the PIF production environment. Desiccation of bacteria was carried out on stainless steel coupons from which total RNA was subsequently recovered and sequenced. During 4 h of desiccation from the early stationary phase (ESP) grown culture, an approximately 3 log10 reduction was recorded for C. sakazakii viable cell count, with the largest change in viable cells occurring between desiccation hour 1 and 2 during which the culture medium was completely dried. Transcriptomic data obtained after 4 h of desiccation highlighted several highly-up regulated osmotolerance-related genes which were associated with the secondary response mechanism. These actively expressed genes mainly modulate pathways that synthetize glycine betaine and trehalose as well as the transport of these two and other compatible solutes. Understanding the activities of these genes and pathways will assist the development of technologies that mitigate the survival of C. sakazakii in the PIF production process. Overall design: RNA-seq was carried out for C. sakazakii ATCCTM29544 at 7 different growth/desiccation/rehydration stages, duplicate samples were sequenced for each stage Developmental stages used in this study: EEP (early exponential phase) MEP (medium exponential phase) LEP (late exponential phase) ESP (early stationary phase) DP (desiccation phase) RP (rehydration phase) LSP (late stationary phase)
Project description:Cronobacter spp. (formerly Enterobacter sakazakii) and Salmonella spp. are increasingly implicated internationally as important microbiological contaminants in low-moisture food products, including powdered infant formula. Estimates indicate that 40 to 80% of infants infected with Cronobacter sakazakii and/or Salmonella in the United States may not survive the illness. A systematic approach, combining literature-based data mining, comparative genome analysis, and the direct sequencing of PCR products of specific biomarker genes, was used to construct an initial collection of genes to be targeted. These targeted genes, particularly genes encoding virulence factors and genes responsible for unique phenotypes, have the potential to function as biomarker genes for the identification and differentiation of Cronobacter spp. and Salmonella from other food-borne pathogens in low-moisture food products. In this paper, a total of 58 unique Salmonella gene clusters and 126 unique potential Cronobacter biomarkers and putative virulence factors were identified. A chitinase gene, a well-studied virulence factor in fungi, plants, and bacteria, was used to confirm this approach. We found that the chitinase gene has very low sequence variability and/or polymorphism among Cronobacter, Citrobacter, and Salmonella, while differing significantly in other food-borne pathogens, either by sequence blasting or experimental testing, including PCR amplification and direct sequencing. This computational analysis for Cronobacter and Salmonella biomarker identification and the preliminary laboratory studies are only a starting point; thus, PCR and array-based biomarker verification studies of these and other food-borne pathogens are currently being conducted.
Project description:Cronobacter sakazakii and C. malonaticus can cause serious diseases especially in infants where they are associated with rare but fatal neonatal infections such as meningitis and necrotising enterocolitis.This study used 104 whole genome sequenced strains, covering all seven species in the genus, to analyse capsule associated clusters of genes involved in the biosynthesis of the O-antigen, colanic acid, bacterial cellulose, enterobacterial common antigen (ECA), and a previously uncharacterised K-antigen.Phylogeny of the gnd and galF genes flanking the O-antigen region enabled the defining of 38 subgroups which are potential serotypes. Two variants of the colanic acid synthesis gene cluster (CA1 and CA2) were found which differed with the absence of galE in CA2. Cellulose (bcs genes) were present in all species, but were absent in C. sakazakii sequence type (ST) 13 and clonal complex (CC) 100 strains. The ECA locus was found in all strains. The K-antigen capsular polysaccharide Region 1 (kpsEDCS) and Region 3 (kpsMT) genes were found in all Cronobacter strains. The highly variable Region 2 genes were assigned to 2 homology groups (K1 and K2). C. sakazakii and C. malonaticus isolates with capsular type [K2:CA2:Cell(+)] were associated with neonatal meningitis and necrotizing enterocolitis. Other capsular types were less associated with clinical infections.This study proposes a new capsular typing scheme which identifies a possible important virulence trait associated with severe neonatal infections. The various capsular polysaccharide structures warrant further investigation as they could be relevant to macrophage survival, desiccation resistance, environmental survival, and biofilm formation in the hospital environment, including neonatal enteral feeding tubes.
Project description:<h4>Background</h4>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.<h4>Methodology/principal findings</h4>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.<h4>Conclusions/significance</h4>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.
Project description:BACKGROUND: E. sakazakii is considered to be an opportunistic pathogen, implicated in food borne diseases causing meningitis or enteritis especially in neonates and infants. Cultural standard identification procedures for E. sakazakii include the observation of yellow pigmentation of colonies and a positive glucosidase activity. Up to now, only one PCR system based on a single available 16S rRNA gene sequence has been published for E. sakazakii identification. However, in our hands a preliminary evaluation of this system to a number of target and non-target strains showed significant specificity problems of this system. In this study full-length 16S rRNA genes of thirteen E. sakazakii strains from food, environment and human origin as well as the type strain ATCC 51329 were sequenced. Based on this sequence data a new specific PCR system for E. sakazakii was developed and evaluated. RESULTS: By phylogenetic analysis of the new full-length 16S rRNA gene sequence data obtained we could show the presence of a second phylogenetic distinct lineage within the E. sakazakii species. The newly developed 16S rRNA gene targeting PCR system allows identification of E. sakazakii strains from both lineages. The assay's ability to correctly identify different E. sakazakii isolates as well as to differentiate E. sakazakii from other closely related Enterobacteriaceae species and other microorganisms was shown on 75 target and non-target strains. CONCLUSION: By this study we are presenting a specific and reliable PCR identification system, which is able to correctly identify E. sakazakii isolates from both phylogenetic distinct lines within the E. sakazakii species. The impact of this second newly described phylogenetic line within the E. sakazakii species in view of clinical and food safety aspects need further investigation.
Project description: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.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.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.