The role of DsbA and PepP genes in the environmental tolerance and virulence factors of Cronobacter sakazakii.

Cronobacter sakazakii, an opportunity foodborne pathogen, could contaminate a broad range of food materials and cause life-threatening symptoms in infants. The bacterial envelope structure contribute to bacterial environment tolerance, biofilm formation and virulence in various in Gram-negative bacteria. DsbA and PepP are two important genes related to the biogenesis and stability of bacterial envelope. In this study, the DsbA and PepP were deleted in C. sakazakii to evaluate their contribution to stress tolerance and virulence of the pathogen. The bacterial environment resistance assays showed DsbA and PepP are essential in controlling C. sakazakii resistance to heat and desiccation in different mediums, as well as acid, osmotic, oxidation and bile salt stresses. DsbA and PepP also played an important role in regulating biofilm formation and motility. Furthermore, DsbA and PepP deletion weaken C. sakazakii adhesion and invasion in Caco-2, intracellular survival and replication in RAW 264.7. qRT-PCR results showed that DsbA and PepP of C. sakazakii played roles in regulating the expression of several genes associated with environment stress tolerance, biofilm formation, bacterial motility and cellular invasion. These findings indicate that DsbA and PepP played an important regulatory role in the environment resisitance, biofilm formation and virulence of C. sakazakii, which enrich understanding of genetic determinants of adaptability and virulence of the pathogen.

DnaK-Mediated Protein Deamidation: a Potential Mechanism for Virulence and Stress Adaptation in Cronobacter sakazakii.

Cronobacter sakazakii is a Gram-negative bacterium that causes infections in individuals of all ages, with neonates being the most vulnerable group. The objective of this study was to explore the function of the dnaK gene in C. sakazakii and to elucidate the impact of alterations in the protein composition regulated by dnaK on virulence and stress adaptation. Our research demonstrates the critical role of the dnaK gene in various key virulence factors, including adhesion, invasion, and acid resistance in C. sakazakii. Through the use of proteomic analysis, we discovered that deletion of the dnaK gene in C. sakazakii leads to an upregulation of protein abundance and increased levels of deamidated posttranscriptional modifications, suggesting that DnaK may play a role in maintaining proper protein activity by reducing protein deamidation in bacteria. These findings indicate that DnaK-mediated protein deamidation may be a novel mechanism for virulence and stress adaptation in C. sakazakii. These findings suggest that targeting DnaK could be a promising strategy for developing drugs to treat C. sakazakii infections. IMPORTANCE Cronobacter sakazakii can cause disease in individuals of all ages, with infections in premature infants being particularly deadly and resulting in bacterial meningitis and sepsis with a high mortality rate. Our study demonstrates that dnaK in Cronobacter sakazakii plays a critical role in virulence, adhesion, invasion, and acid resistance. Using proteomic analysis to compare protein changes in response to dnaK knockout, we found that dnaK knockout significantly upregulates the abundance of some proteins but also results in the deamidation of many proteins. Our research has identified a connection between molecular chaperones and protein deamidation, which suggests a potential future drug development strategy of targeting DnaK as a drug target.

Crystal structures of YeiE from Cronobacter sakazakii and the role of sulfite tolerance in gram-negative bacteria.

SignificanceYeiE has been identified as a master virulence factor of Cronobacter sakazakii. In this study, we determined the crystal structures of the regulatory domain of YeiE in complex with its physiological ligand sulfite ion (SO32-). The structure provides the basis for the molecular mechanisms for sulfite sensing and the ligand-dependent conformational changes of the regulatory domain. The genes under the control of YeiE in response to sulfite were investigated to reveal the functional roles of YeiE in the sulfite tolerance of the bacteria. We propose the molecular mechanism underlying the ability of gram-negative pathogens to defend against the innate immune response involving sulfite, thus providing a strategy to control the pathogenesis of bacteria.

First case report of acute cholangitis secondary to Cronobacter sakazakii.

INTRODUCTION: Cronobacter sakazakii is an opportunistic Gram-negative, rod-shaped bacterium which may be a causative agent of meningitis in premature infants and enterocolitis and bacteremia in neonates and adults. While there have been multiple cases of C. sakazakii infections, there have been no acute cholangitis cases reported in humans. CASE PRESENTATION: An 81-year-old male with a past medical history of basal cell carcinoma, alcoholic liver cirrhosis, transjugular intrahepatic portosystemic shunt procedure, complicated by staphylococcus bacteremia, pituitary tumor, glaucoma, and hypothyroidism presented to the emergency room with the complaint of diffuse and generalized 10/10 abdominal pain of 1 day's duration. There was a concern for pancreatitis, acute cholangitis, and possible cholecystitis, and the patient underwent a percutaneous cholecystostomy tube placement. Blood cultures from admission and biliary fluid cultures both grew C. sakazakii. The patient was treated with a carbapenem and clinically improved. CONCLUSIONS: The case study described a patient with multiple medical comorbidities that presented with C. sakazakii bacteremia and cholangitis. While this bacterium has been implicated in other infections, we believe this is the first time the bacteria is being documented to have caused acute cholangitis.

Two Cases of Cronobacter Sakazakii Meningitis in Infants: The Importance Of Early Advanced Brain Imaging and Public Health Reporting.

We report 2 infants hospitalized with Cronobacter sakazakii meningitis. Each infant had exposure to powdered infant formula at home. Both infants survived, but 1 infant had a subdural empyema drained and developed left sensorineural hearing loss. Early advanced brain imaging is recommended in infants with C. sakazakii meningitis. Reporting to state and federal public health officials may help identify outbreaks.

The lipoprotein NlpD in Cronobacter sakazakii responds to acid stress and regulates macrophage resistance and virulence by maintaining membrane integrity.

Cronobacter sakazakii, an emerging opportunistic pathogen, is implicated in severe foodborne outbreak infections in premature and full-term infants. Generally, acid tolerance is vital for the pathogenesis of foodborne pathogens; however, its role in C. sakazakii virulence remains largely unknown. To screen out acid-tolerance determinants from transposon mutants, anovel counterselection method using gentamicin and acid was developed. Using the counterselection method and growth assay, we screened several acid-sensitive mutants and found that nlpD encodes an acid-resistance factor in C. sakazakii.  Compared to the wild-type strain, the nlpD mutant exhibited attenuated virulence in a rat model. Using macrophage THP-1 cells and a pH probe, we verified that nlpD enables bacteria to resist macrophages by resisting acidification. Finally, we confirmed that nlpD maintains C. sakazakii membrane integrity in acid using propidium iodide permeabilization assays via flow cytometry. Our results confirm that nlpD is a novel virulence factor that permits C. sakazakii to survive under acid stress conditions. Considering that NlpD is a conserved lipoprotein located in the bacterial outer membrane, NlpD could be used as a target for drug development.

Advances in our understanding and distribution of the Cronobacter genus in China.

This review considers how research in China has progressed our understanding and subsequent improved control of Cronobacter. This emergent bacterial pathogen is associated with neonatal infections through the ingestion of contaminated prepared feed. The review includes large-scale surveys of various sources of the organism, including infant formula production facilities. The analysis of over 20,000 samples is presented. Over 10,000 being from powdered infant formula and other infant foods as well as environmental sampling of production facilities, the remaining being from food, food ingredients, and human carriage. A major advance in China was adopting DNA-sequence-based methods (that is, multilocus sequence typing, clustered regularly interspaced short palindromic repeats-cas array profiling, and single-nucleotide polymorphism analysis) for the identification and genotyping of the organism. These methods have considerably advanced our understanding of the taxonomy, ecology, and virulence of this organism. In turn, this has improved source tracking of the organism both in infant formula production facilities and epidemiological investigations. Furthermore, whole-genome sequencing has revealed a range of virulence and persistence mechanisms as well as plasmid-borne multidrug resistance traits. China now has reliable and robust methods for accurate microbial source tracking of Cronobacter for use both in the food production environment and epidemiological analysis.

Cronobacter sakazakii Infection in Early Postnatal Rats Impaired Contextual-Associated Learning: a Putative Role of C5a-Mediated NF-κß and ASK1 Pathways.

This study was designed to test whether the Cronobacter sakazakii infection-impaired contextual learning and memory are mediated by the activation of the complement system; subsequent activation of inflammatory signals leads to alternations in serotonin transporter (SERT). To test this, rat pups (postnatal day, PND 15) were treated with either C. sakazakii (107 CFU) or Escherichia coli OP50 (107 CFU) or Luria bertani broth (100 µL) through oral gavage and allowed to stay with their mothers until PND 24. Experimental groups' rats were allowed to explore (PNDs 31-35) and then trained in contextual learning task (PNDs 36-43). Five days after training, individuals were tested for memory retention (PNDs 49-56). Observed behavioural data showed that C. sakazakii infection impaired contextual-associative learning and memory. Furthermore, our analysis showed that C. sakazakii infection activates complement system complement anaphylatoxin (C5a) (a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS1)) and mitogen-activated protein kinase kinase1 (MEKK1). Subsequently, MEKK1 induces pro-inflammatory signals possibly through apoptosis signal-regulating kinase-1 (ASK-1), c-Jun N-terminal kinase (JNK1/3) and protein kinase B gamma (AKT-3). In parallel, activated nuclear factor kappa-light-chain-enhancer B cells (NF-κB) induces interleukin-6 (IL-6) and IFNα-1, which may alter the level of serotonin transporter (SERT). Observed results suggest that impaired contextual learning and memory could be correlated with C5a-mediated NF-κß and ASK1 pathways.

Lactobacillus gastricus BTM 7 prevents intestinal colonization by biofilm forming Cronobacter sakazakii in Caenorhabditis elegans model host.

The study reports protective role of potential probiotic cultures against infection by biofilm forming Cronobacter sakazakii in Caenorhabditis elegans model system. Among the fifteen indigenous potential probiotics, the cell free supernatant of Lactobacillus gastricus BTM7 possessed highest antimicrobial action and biofilm inhibition against C. sakazakii. The competitive exclusion assays revealed that preconditioning with probiotics resulted in increased mean life span of the nematode to 12-13 days as compared to 5-6 days when the pathogen was administered alone. Enhanced expression of the marker genes (pmk-1, daf-16 and skn-1) was observed during the administration of probiotic cultures. The highest expression of pmk-1 (2.5 folds) was observed with administration of L. gastricus BTM7. The principal component analysis on selected variables revealed that L. gastricus BTM7 has the potential to limit the infection of C. sakazakii in C. elegans and enhance the expression of key genes involved in extending life span of the worm.

Pan-genome diversification and recombination in Cronobacter sakazakii, an opportunistic pathogen in neonates, and insights to its xerotolerant lifestyle.

BACKGROUND: Cronobacter sakazakii is an emerging opportunistic bacterial pathogen known to cause neonatal and pediatric infections, including meningitis, necrotizing enterocolitis, and bacteremia. Multiple disease outbreaks of C. sakazakii have been documented in the past few decades, yet little is known of its genomic diversity, adaptation, and evolution. Here, we analyzed the pan-genome characteristics and phylogenetic relationships of 237 genomes of C. sakazakii and 48 genomes of related Cronobacter species isolated from diverse sources. RESULTS: The C. sakazakii pan-genome contains 17,158 orthologous gene clusters, and approximately 19.5% of these constitute the core genome. Phylogenetic analyses reveal the presence of at least ten deep branching monophyletic lineages indicative of ancestral diversification. We detected enrichment of functions involved in proton transport and rotational mechanism in accessory genes exclusively found in human-derived strains. In environment-exclusive accessory genes, we detected enrichment for those involved in tryptophan biosynthesis and indole metabolism. However, we did not find significantly enriched gene functions for those genes exclusively found in food strains. The most frequently detected virulence genes are those that encode proteins associated with chemotaxis, enterobactin synthesis, ferrienterobactin transporter, type VI secretion system, galactose metabolism, and mannose metabolism. The genes fos which encodes resistance against fosfomycin, a broad-spectrum cell wall synthesis inhibitor, and mdf(A) which encodes a multidrug efflux transporter were found in nearly all genomes. We found that a total of 2991 genes in the pan-genome have had a history of recombination. Many of the most frequently recombined genes are associated with nutrient acquisition, metabolism and toxin production. CONCLUSIONS: Overall, our results indicate that the presence of a large accessory gene pool, ability to switch between ecological niches, a diverse suite of antibiotic resistance, virulence and niche-specific genes, and frequent recombination partly explain the remarkable adaptability of C. sakazakii within and outside the human host. These findings provide critical insights that can help define the development of effective disease surveillance and control strategies for Cronobacter-related diseases.

Novel Multidrug-Resistant Cronobacter sakazakii Causing Meningitis in Neonate, China, 2015.

We report a case of meningitis in a neonate in China, which was caused by a novel multidrug-resistant Cronobacter sakazakii strain, sequence type 256, capsular profile K1:CA1. We identified genetic factors associated with bacterial pathogenicity and antimicrobial drug resistance in the genome and plasmids. Enhanced surveillance of this organism is warranted.

Immunogold Nanoparticles for Rapid Plasmonic Detection of C. sakazakii.

Cronobacter sakazakii is a foodborne pathogen that can cause a rare, septicemia, life-threatening meningitis, and necrotizing enterocolitis in infants. In general, standard methods for pathogen detection rely on culture, plating, colony counting and polymerase chain reaction DNA-sequencing for identification, which are time, equipment and skill demanding. Recently, nanoparticle- and surface-based immunoassays have increasingly been explored for pathogen detection. We investigate the functionalization of gold nanoparticles optimized for irreversible and specific binding to C. sakazakii and their use for spectroscopic detection of the pathogen. We demonstrate how 40-nm gold nanoparticles grafted with a poly(ethylene glycol) brush and functionalized with polyclonal antibodies raised against C. sakazakii can be used to specifically target C. sakazakii. The strong extinction peak of the Au nanoparticle plasmon polariton resonance in the optical range is used as a label for detection of the pathogens. Individual binding of the nanoparticles to the C. sakazakii surface is also verified by transmission electron microscopy. We show that a high degree of surface functionalization with anti-C. sakazakii optimizes the detection and leads to a detection limit as low as 10 CFU/mL within 2 h using a simple cuvette-based UV-Vis spectrometric readout that has great potential for further optimization.

Electrochemical coupled immunosensing platform based on graphene oxide/gold nanocomposite for sensitive detection of Cronobacter sakazakii in powdered infant formula.

A sensitive electrochemical immunosensing platform for the detection of Cronobacter sakazakii was developed using a graphene oxide/gold (GO/Au) composite. Transmission electron microscopy showed that the Au nanoparticles, with an average size of < 30 nm, were well dispersed on the GO surface. For the detection of C. sakazakii, a polyclonal anti-C. sakazakii antibody (IgG) was covalently immobilized to the Au nanoparticles on the surface of the GO/Au composite coated glassy carbon electrode (GCE). The electrochemical sensing performance of immunofunctionalized GCE was characterized by cyclic voltammetry and differential pulse voltammetry. Under optimized conditions, in pure culture there was a linear relationship between electrical signal and C. sakazakii levels over the range 2.0 × 102-2.0 × 107 cfu/mL (R2 = 0.999), with a detection limit of 2.0 × 101 cfu/mL. The total analytical time was 15 min per sample. The C. sakazakii electrochemical immunosensing assay was able to successfully detect 2.0 × 101 cfu/mL of C. sakazakii in artificially contaminated powdered infant formula without any enrichment or pre-enrichment steps. Furthermore, the recovery rates of the C. sakazakii electrochemical immunosensing assay following spiking of powdered infant formula with different concentrations of C. sakazakii (cfu/mL) were 82.58% at 2.0 × 101 cfu/mL, 84.86% at 2.0 × 102 cfu/mL, and 95.40% at 2.0 × 103 cfu/mL. The C. sakazakii electrochemical immunosensing assay had good selectivity, reproducibility, and reactivity compared with other Cronobacter spp. and/or pathogens belonging to other genera, indicating its significant potential in the clinical diagnosis of C. sakazakii.

New virulence factor CSK29544_02616 as LpxA binding partner in Cronobacter sakazakii.

Cronobacter sakazakii is an opportunistic pathogen that can cause meningitis and necrotizing enterocolitis in premature infants, but its virulence determinants remain largely unknown. In this study, a transposon-mediated random-mutant library of C. sakazakii was used to identify new virulence factors. Compared to wild-type bacteria, a mutant lacking CSK29544_02616 (referred to as labp) was defective in invasion into intestinal epithelial cells (by at least 1000-fold) and showed less phagocytosis by macrophages (by at least 50-fold). The lack of labp in C. sakazakii changed the profile of outer membrane proteins, decreased the production of lipopolysaccharides, and increased the production of membrane phospholipids. Bacterial physiological characteristics including surface hydrophobicity and motility were also altered in the absence of labp, presumably because of changes in the bacterial-envelope structure. To systematically determine the role of labp, ligand fishing was conducted using Labp as a bait, which revealed LpxA as a binding partner of Labp. LpxA is UDP-N-acetylglucosamine (GlcNAc) acyltransferase, the first enzyme in the pathway of lipid A biosynthesis. Labp increased the enzymatic activity of LpxA without influencing lpxA expression. Considering multifaceted roles of lipopolysaccharides in virulence regulation, Labp is a novel virulence factor that promotes the production of lipid A by LpxA in Cronobacter.

Potential factors involved in virulence of Cronobacter sakazakii isolates by comparative transcriptome analysis.

Cronobacter species are important foodborne pathogens causing severe infections in neonates through consumption of contaminated powdered infant formula. However, the virulence-associated factors in Cronobacter are largely unknown. In this study, the transcriptome analysis between highly virulent Cronobacter sakazakii G362 and attenuated L3101 strains was used to reveal the potential factors involved in virulence. The total transcripts were grouped into 20 clusters of orthologous group categories and summarized in 3 gene ontology categories (biological process, cellular component, and molecular function). In addition, the differentially expressed genes (DEG) between these isolates were analyzed using Volcano plots and gene ontology enrichment. The predominant DEG were flagella-associated genes such as flhD, motA, flgM, flgB, and fliC. Furthermore, the expression abundance of outer membrane protein or lipoprotein genes (ompW, slyB, blc, tolC, and lolA), potential virulence-related factors (hlyIII and hha), and regulation factors (sdiA, cheY, Bss, fliZ) was also significantly different between G362 and L3101. Interestingly, 3 hypothetical protein genes (ESA_01022, ESA_01609, and ESA_00609) were found to be expressed only in G362. Our findings provide valuable transcriptomic information about potential virulence factor genes, which will be needed in future molecular biology studies designed to understand the pathogenic mechanism of Cronobacter.

Virulent and pathogenic features on the Cronobacter sakazakii polymyxin resistant pmr mutant strain s-3.

Cronobacter sakazakii is a well-known opportunistic pathogen responsible for necrotizing enterocolitis, meningitis and septicaemia in the premature, immunocompromised infants and neonates. This pathogen possesses various virulence factors and regulatory systems, and pmrA/pmrB regulatory system has been identified in a variety of bacterial species. The current study aims to investigate role of pmrA gene in the pathogenicity and virulence characteristics of Cronobacter sakazakii using whole genome sequencing and RNA-seq. Results demonstrated that the absence of pmrA has the potential to affect Cronobacter sakazakii on its pathogenicity, virulence and resistance abilities by regulating expression of numerous related genes, including CusB, CusC, CusR and ESA_pESA3p05434.

Analysis on pathogenic and virulent characteristics of the Cronobacter sakazakii strain BAA-894 by whole genome sequencing and its demonstration in basic biology science.

Cronobacter sakazakii is an opportunistic pathogen responsible for necrotizing enterocolitis, meningitis and septicaemia especially to infant and neonate, with high lethality ranging in 40%-80%. This strain is able to survive in infant milk formula and possesses capability of pathogenicity and virulence, biofilm formation, and high resistance to elevated osmotic, low pH, heat, oxidation, and desiccasion. This study is aims to investigate the molecular characteristics of Cronobacter sakazakii BAA 894, including mechanisms of its invasion and adherence, biofilm formation, unusual resistance to environmental stress employing whole genome sequencing and comparative genomics. Results in this study suggest that numerous genes and pathways, such as LysM, Cyx system, luxS, vancomycin resistance pathway, insulin resistance pathway, and sod encoding superoxide dismutase for the survival of C. sakazakii in macrophages, contribute to pathogenicity and resistance to stressful environment of C. sakazakii BAA 894.

Development and Application of a Probabilistic Risk-Benefit Assessment Model for Infant Feeding Integrating Microbiological, Nutritional, and Chemical Components.

A probabilistic and interdisciplinary risk-benefit assessment (RBA) model integrating microbiological, nutritional, and chemical components was developed for infant milk, with the objective of predicting the health impact of different scenarios of consumption. Infant feeding is a particular concern of interest in RBA as breast milk and powder infant formula have both been associated with risks and benefits related to chemicals, bacteria, and nutrients, hence the model considers these three facets. Cronobacter sakazakii, dioxin-like polychlorinated biphenyls (dl-PCB), and docosahexaenoic acid (DHA) were three risk/benefit factors selected as key issues in microbiology, chemistry, and nutrition, respectively. The present model was probabilistic with variability and uncertainty separated using a second-order Monte Carlo simulation process. In this study, advantages and limitations of undertaking probabilistic and interdisciplinary RBA are discussed. In particular, the probabilistic technique was found to be powerful in dealing with missing data and to translate assumptions into quantitative inputs while taking uncertainty into account. In addition, separation of variability and uncertainty strengthened the interpretation of the model outputs by enabling better consideration and distinction of natural heterogeneity from lack of knowledge. Interdisciplinary RBA is necessary to give more structured conclusions and avoid contradictory messages to policymakers and also to consumers, leading to more decisive food recommendations. This assessment provides a conceptual development of the RBA methodology and is a robust basis on which to build upon.

Profiling of Virulence Determinants in Cronobacter sakazakii Isolates from Different Plant and Environmental Commodities.

Cronobacter sakazakii is an emerging pathogen causing meningitis, sepsis and necrotizing enterocolitis in neonates and immune-compromised adults. The present study describes the profiling of different virulence factors associated with C. sakazakii isolates derived from plant-based materials and environmental samples (soil, water, and vacuum dust). All the isolates exhibited ß-hemolysis and chitinase activity, and were able to utilize inositol. Among the nine virulence-associated genes, hly gene coding for hemolysin was detected in all the isolates followed by ompA (outer membrane protein); however, plasmid-borne genes were detected at a level of 60% for both cpa (cronobacter plasminogen activator) and eitA (Ferric ion transporter protein) gene, respectively. Furthermore, the isolate C. sakazakii N81 showed cytotoxicity for Caco-2 cells. The presence of the virulence determinants investigated in this study indicates the pathogenic potential of C. sakazakii with their plausible connection with clinical manifestations.