Effect of glutathione-transport-related gene gsiD on desiccation tolerance of Cronobacter sakazakii and its related regulatory mechanism.

The outstanding desiccation tolerance of Cronobacter sakazakii (C. sakazakii) enables long-term persistence in food products with low-water activity to increase the infection risk, especially in low-birth-weight, immuno-compromised neonates, and infants less than 4 weeks of age. In our previous study, the disruption of glutathione transport-related gene gsiD by transposon was found to significantly increase its inactivation rate under drying stress challenges. However, the mechanism underlying the association between glutathione transport and desiccation tolerance of C. sakazakii remains to be clarified. In this study, the mechanism underlying their association was investigated in detail by constructing the gsiD gene deletion mutant. gsiD gene deletion was found to cause the dysfunction of the glutathione transport system GsiABCD and the limitation of glutathione import. The resulting decrease in intracellular glutathione caused the decreased potassium ions uptake and increased potassium ions efflux, inhibited the proline synthesis process, limited extracellular glutathione utilization, increased oxidant stress, reduced biofilm formation, and increased outer membrane permeability, which may be the main reasons for the significant reduction of the desiccation tolerance of C. sakazakii.IMPORTANCEContributing to its superior environmental adaptability, Cronobacter sakazakii can survive under many abiotic stress conditions. The outstanding desiccation tolerance makes this species persist in low-water activity foods, which increases harm to humans. For decades, many studies have focused on the desiccation tolerance of C. sakazakii, but the existing research is still insufficient. Our study found that gsiD gene deletion inhibited glutathione uptake and further decreased intracellular glutathione content, causing a decrease in desiccation tolerance and biofilm formation and an increase in outer membrane permeability. Moreover, the expression level of relative genes verified that gsiD gene deletion made the mutant not conducive to surviving in dry conditions due to restricting potassium ions uptake and efflux, inhibiting the conversion of glutamate to compatible solute proline, and increasing the oxidative stress of C. sakazakii. The above results enrich our knowledge of the desiccation tolerance mechanism of C. sakazakii.

Isolation of Pathogenic Cronobacter Species as Bacteriological Risks Indicator in Powdered Infant Formula Available to Deprived Infants in Lagos Metropolis, Nigeria.

Powdered infant formula (PIF) is rich in nutrients that support the survival and growth of bacteria that trigger food safety disorders in deprived infants through life-threatening illnesses. The study aims to examine and identify the incidence of pathogenic bacteria of concern in PIF upon reconstitution in lukewarm water. A total of 172 samples consisting of 38 brands of PIF available in the Lagos metropolis were sampled, suspended in water (10 g in 100 mL), and bacteria strains were isolated using combinational enrichment and selective culture techniques. Pure bacterial strains were characterized and identified based on their physiology and 16S rRNA gene sequence homology. While 85 bacterial strains were isolated from the enriched culture system, 20 strains were selectively isolated based on tolerance to sodium deoxycholate. Approximately 13% of the selected bacteria were identified as Cronobacter spp., exhibiting virulence traits including extracellular protease production, coagulation and proteolysis of casein, haem-agglutination, and ß-haemolysis of human blood. Approximately 82% of the Cronobacter strains tolerated NaCl (10%) and bile salt; and exhibited resistance to cefotaxime, ceftriaxone, gentamicin, and Amoxicillin-clavulanic acid antibiotics. The presence of Cronobacter spp. in 13% of the PIF brands available to infants calls for concern about the safety of deprived infants that might be fed with such PIF. Consequently, PIF safety alerts need to be activate while further studies on critical points at which the pathogens get introduced to the PIFs need to be identified.

A universal mechanism on desiccation tolerance of Cronobacter based on intracellular trehalose accumulation regulated by EnvZ/OmpR.

Cronobacter (seven species) can survive in dry powdered infant formula for a long time, but the thorough molecular mechanism of resistance to desiccation remains elusive. Here we examine the regulation mechanism of Cronobacter's tolerance to desiccation by the typical two-component system (TCS) EnvZ/OmpR. When exposed to desiccation conditions, Cronobacter showed higher survival than other pathogens, as well as significantly up-regulated expression of ompR and otsAB genes with markedly decreased survival of their mutants, suggesting their relationship with desiccation tolerance. OmpR directly binds to the promoter of trehalose biosynthesis operon otsBA, significantly enhancing their expression, and boosting the trehalose levels. The ompR-deletion in other six species further confirmed its positive regulation in desiccation tolerance. Our data present a hypothesis that EnvZ/OmpR increases intracellular trehalose levels against damage to the cells, which prompts Cronobacter to survive in desiccation conditions. This study reveals a universal molecular mechanism for desiccation resistance in Cronobacter species.

Occurrence, genetic characterization, and antibiotic susceptibility of Cronobacter spp. isolated from low water activity functional foods in Brazil.

Cronobacter spp. are bacterial pathogens isolated from a wide variety of foods. This study aims at evaluating the occurrence of Cronobacter spp. in low water activity functional food samples, detect the presence of virulence genes, and determine the antibiotic susceptibility of strains. From 105 samples, 38 (36.2%) were contaminated with Cronobacter spp. The species identified by polymerase chain reaction (PCR) and sequencing analyses (rpoB and fusA genes, respectively) were C. sakazakii (60.3%), C. dublinensis (25.4%), C. turincensis (9.5%), and C. malonaticus (4.8%). Nineteen fusA alleles were identified, including four new alleles. The virulence genes were identified by PCR and all isolates were positive for ompX and sodA genes, 60.3% to cpa gene, and 58.7% to hly gene. Using the disk diffusion method, antibiotic susceptibility to twelve antibiotics was assessed twice, separated by a 19-month period. In the first test, the isolates showed diverse antibiotic susceptibility profiles, with nineteen isolates (30.2%) being multi-drug resistant (resistant to three or more antibiotic classes), in the second, the isolates were susceptible to all antibiotics. Cronobacter spp. in functional foods demonstrates the need for continued investigation of this pathogen in foods, and further research is needed to clarify the loss of resistance of Cronobacter strains.

Electrotransformation of Foodborne Pathogen Cronobacter sakazakii by a Simple Method.

Cronobacter sakazakii is an opportunistic foodborne pathogen that mainly infects infants and immunocompromised people, with a high mortality rate. However, the efficient transformation method of this bacterium has not been systematically reported. In this study, we developed a fast and efficient transformation method for C. sakazakii by cold sucrose treatment. Compared with CaCl2 or glycerol treatment, the transformation efficiency of this method is significantly high when bacteria were cultured overnight at 42°C before cold sucrose treatment. Furthermore, applying this method, we successfully knocked out the pppA gene by direct electroporation. Collectively, our study provides a simple, time-saving, and efficient method for competent cell preparation of C. sakazakii, which is conducive to the further research of C. sakazakii.

Genome Mining of Novel Targets and Construction of Ladder-shaped melting temperature isothermal amplification Assays for the Identification of Cronobacter sakazakii and Cronobacter malonaticus.

Cronobacter species are potential pathogens that can contaminate powdered infant formula. C. sakazakii and C. malonaticus are the most common species of Cronobacter associated with infections. This study mined new molecular targets for the detection of C. sakazakii and C. malonaticus by using comparative genome approaches. Specific target genes mngB and ompR were obtained and used to detect C. sakazakii and C. malonaticus, respectively. A novel detection method, termed ladder-shape melting temperature isothermal amplification (LMTIA), was developed and evaluated. The detection limit for pure C. sakazakii DNA was 1 pg per reaction and 1 fg per reaction for C. malonaticus. The C. sakazakii, C. malonaticus, and the reference stains were all correctly identified. The amplicons can be successfully visualized and identified by naked eyes when hydroxy naphthol blue dye (HNB dye) was used in the reaction. Therefore, the LMTIA assays developed in this study showed potential application for microorganism identification and detection.

Rosa roxburghii Tratt Pomace Crude Extract Inactivates Cronobacter sakazakii Isolated from Powdered Infant Formula.

Cronobacter sakazakii is an important foodborne pathogen in powder infant formula (PIF). The objective of this study was to evaluate the inactivation effect of Rosa roxburghii Tratt pomace crude extract (RRPCE) on C. sakazakii isolated from PIF and to reveal the mechanism of action. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were used to evaluate the inhibitory activity of RRPCE against C. sakazakii. The inhibitory mechanism was revealed from the perspective of effects of RRPCE on intracellular adenosine 5'-triphosphate (ATP), reactive oxygen species (ROS), membrane potential, protein and nucleic acid leakage, and cell morphology of C. sakazakii. The inactivation effects of RRPCE on C. sakazakii in biofilms on stainless steel, tinplate, glass, silica gel, polyethylene terephthalate, and polystyrene to evaluate its potential as a natural disinfectant. The results showed that the MIC and MBC of RRPCE against C. sakazakii were 7.5 and 15 mg/mL, respectively. After treatments with RRPCE, intracellular ATP content decreased significantly while intracellular ROS level increased significantly (p < 0.05). The cell membrane depolarization, large leakage of proteins and nucleic acids, and severely damaged cell morphology also occurred in C. sakazakii treated with RRPCE. In addition, a 20-minute treatment with 2 MIC (15 mg/mL) of RRPCE could inactivate all C. sakazakii (from 6.10 to 6.40 CFU/mL) in biofilms on all six contact surfaces. Our findings suggest that RRPCE is ideal for the inactivation of C. sakazakii and has the potential to be used as a natural disinfectant for the inactivation of PIF packaging materials and containers.

Inactivation of Carotenogenic-Biosynthesizing Genes Altered Lipids Composition and Intensity in Cronobacter sakazakii.

Cronobacter sakazakii, an opportunistic milk-borne pathogen responsible for severe neonatal meningitis and bacteremia, can synthesize yellow pigment (various carotenoids) benefiting for bacterial survival, while little literature was available about the influence of various carotenoids on bacterial resistance to a series of stresses and the characteristics of cell membrane, obstructing the development of novel bactericidal strategies overcoming the strong tolerance of C. sakazakii. Thus in this study, for the first time, five carotenogenic genes of C. sakazakii BAA-894 were inactivated, respectively, to construct a series of mutants producing various carotenoids and their effects on the cell membrane properties, and resistances to food- and host-related stresses, were investigated systematically. Furthermore, to explore its possible mode of action, comparative lipidomics analysis was performed to reveal the change of lipids that were mainly located at cell membranes. The results showed that five mutants (ΔcrtB, ΔcrtI, ΔcrtY, ΔcrtZ, and ΔcrtX) displayed negligible change in growth rate but higher permeability of the outer membrane and lower fluidity of cell membrane compared to the wild type. Besides, these mutants exhibited poorer ability of biofilm formation and lower resistances to acid, oxidative, osmotic, and desiccation stresses, indicating that different carotenoid composition significantly affected environmental tolerance of C. sakazakii. To discover the possible causes, lipidomics analysis of C. sakazakii was conducted and more than 500 lipid species belonging to 27 classes had been identified at first. Compared to that of BAA-894, the composition and relative intensity of lipid species in five mutants varied significantly, especially the monounsaturated and biunsaturated phosphatidylethanolamine. The evidence presented in this study demonstrated that the varied composition of carotenoids in C. sakazakii significantly altered the lipid profile and intensity, which maybe a crucial means to influencing the characteristics of cell membranes and resistance to environmental stresses.

High-affinity truncated aptamers for detection of Cronobacter spp with magnetic separation-assisted DNAzyme-driven 3D DNA walker.

After optimizing the original aptamer sequence by truncation strategy, a magnetic separation-assisted DNAzyme-driven 3D DNA walker fluorescent aptasensor was developed for detecting the food-borne pathogen Cronobacter species. Iron oxide magnetic nanoparticles (MNPs) modified with a hybrid of truncated aptamer probe and DNAzyme strand (AP-E1) denoted as MNPs@AP-E1, were employed as capture probes. Simultaneously, a DNAzyme-driven 3D-DNA walker was utilized as the signal amplification element. The substrate strand (Sub) was conjugated with the gold nanoparticles (AuNPs), resulting in the formation of AuNPs@Sub, which served as a 3D walking track. In the presence of the target bacteria and Mg2+, E1-DNAzyme was activated and moved along AuNPs@Sub, continuously releasing the signal probe. Under optimized conditions, a strong linear correlation was observed for Cronobacter sakazakii (C. sakazakii) in the concentration range 101 to 106 CFU mL-1, with a low detection limit of 2 CFU mL-1. The fluorescence signal responses for different Cronobacter species exhibited insignificant differences, with a relative standard deviation of 3.6%. Moreover, the aptasensor was successfully applied to determine  C. sakazakii in real samples with recoveries of 92.86%-108.33%. Therefore, the novel method could be a good candidate for ultra-sensitive and selective detection of Cronobacter species without complex manipulation.

Genomic Analysis of Cronobacter condimenti s37: Identification of Resistance and Virulence Genes and Comparison with Other Cronobacter and Closely Related Species.

Cronobacter condimenti are environmental commensals that have not been associated with any clinical infections. To date, they are the least understood and described Cronobacter species within the genus. The objective of this study was to use a draft genome sequence (DGS) of the Cronobacter condimenti strain s37 to screen for genes encoding for antibiotic resistance, virulence, response to environmental stress, and biofilm formation. The strain was isolated in Poland from commercial small radish sprouts. This is the second genome of this species available in the GenBank database. The comparative genome analysis (cgMLST) of C. condimenti s37 with other Cronobacter spp. including the pathogenic species C. sakazakii and the plant-associated closely related genera Franconibacter and Siccibacter was also performed. The assembled and annotated genome of the C. condimenti s37 genome was 4,590,991 bp in length, with a total gene number of 4384, and a GC content of 55.7%. The s 37 genome encoded for genes associated with resistance to stressful environmental conditions (metal resistance genes: zinc, copper, osmotic regulation, and desiccation stress), 17 antimicrobial resistance genes encoding resistance to various classes of antibiotics and 50 genes encoding for the virulence factors. The latter were mainly genes associated with adhesion, chemotaxis, hemolysis, and biofilm formation. Cg-MLST analysis (3991 genes) revealed a greater similarity of C. condimenti s37 to S. turicensis, F. pulveris, and C. dublinensis than to other species of the genus Cronobacter. Studies on the diversity, pathogenicity, and virulence of Cronobacter species isolated from different sources are still insufficient and should certainly be continued. Especially the analysis of rare strains such as s37 is very important because it provides new information on the evolution of these bacteria. Comparative cgMLST analysis of s37 with other Cronobacter species, as well as closely related genera Franconibacter and Siccibacter, complements the knowledge on their adaptability to specific environments such as desiccation.

Assessment of the Sensitivity of Foodborne Cronobacter spp. Strains and Other Enterobacteria to Temperature Stresses and Chlorine-Containing Biocides.

We examined the effects of elevated temperatures and biocides on survivability of food isolates of Cronobacter spp. (C. sakazakii) and concomitant enterobacteriaceae obtained in microbiological control of infant nutrition products. Increased resistance of certain strains of Cronobacter, Enterobacter cloacae, and Pantoea spp. to thermal processing was revealed. Salmonella, Pantoea, and Cronobacter bacteria were least sensitive to antimicrobial action of chlorine-containing agents. The above properties varied in the strains of the same species. Specifically, only two of three examined isolates of Cronobacter spp. demonstrated lower sensitivity to heat in comparison with the enterobacterial test-cultures of other species.

Cronobacter sakazakii Pyridoxal Kinase PdxY Mediated by TreR and pESA3 Is Essential for Vitamin B6 (PLP) Maintenance and Virulence.

Cronobacter sakazakii is an opportunistic pathogen capable of causing severe infections, particularly in neonates. Despite the bacterium's strong pathogenicity, the pathogenicity of C. sakazakii is not yet well understood. Using a comparative proteomic profiling approach, we successfully identified pdxY, encoding a pyridoxal kinase involved in the recycling of pyridoxal 5'-phosphate (PLP), as a gene essential for the successful pathogenesis of C. sakazakii. Knocking out the pdxY gene resulted in slower growth and reduced virulence. Our study sheds light on the fundamental importance of pyridoxal kinase for the survival and virulence of C. sakazakii. The identification of pdxY as gene essential for successful pathogenesis provides a potential target for the development of new antibiotic treatments. IMPORTANCE The opportunistic pathogen Cronobacter sakazakii is known to cause severe infections, particularly in neonates, and can result in high mortality rates. In this study, we used a comparative proteomic profiling approach to identify genes essential for the successful pathogenesis of C. sakazakii. We successfully identified pdxY, encoding a pyridoxal kinase involved in the salvage pathway of pyridoxal 5'-phosphate (PLP), as a gene essential for the successful pathogenesis of C. sakazakii. Knocking out the pdxY gene resulted in impaired growth and reduced virulence. This study sheds light on the fundamental importance of pyridoxal kinase for the survival and virulence of C. sakazakii, which can be a potential target for the development of new antibiotic treatments. This study highlights the importance of comparative proteomic profiling in identifying virulence factors that can be targeted for the development of new antibiotics.

Uncovering virulence factors in Cronobacter sakazakii: insights from genetic screening and proteomic profiling.

The increasing problem of antibiotic resistance has driven the search for virulence factors in pathogenic bacteria, which can serve as targets for the development of new antibiotics. Although whole-genome Tn5 transposon mutagenesis combined with phenotypic assays has been a widely used approach, its efficiency remains low due to labor-intensive processes. In this study, we aimed to identify specific genes and proteins associated with the virulence of Cronobacter sakazakii, a pathogenic bacterium known for causing severe infections, particularly in infants and immunocompromised individuals. By employing a combination of genetic screening, comparative proteomics, and in vivo validation using zebrafish and rat models, we rapidly screened highly virulent strains and identified two genes, rcsA and treR, as potential regulators of C. sakazakii toxicity toward zebrafish and rats. Proteomic profiling revealed upregulated proteins upon knockout of rcsA and treR, including FabH, GshA, GppA, GcvH, IhfB, RfaC, MsyB, and three unknown proteins. Knockout of their genes significantly weakened bacterial virulence, confirming their role as potential virulence factors. Our findings contribute to understanding the pathogenicity of C. sakazakii and provide insights into the development of targeted interventions and therapies against this bacterium.IMPORTANCEThe emergence of antibiotic resistance in pathogenic bacteria has become a critical global health concern, necessitating the identification of virulence factors as potential targets for the development of new antibiotics. This study addresses the limitations of conventional approaches by employing a combination of genetic screening, comparative proteomics, and in vivo validation to rapidly identify specific genes and proteins associated with the virulence of Cronobacter sakazakii, a highly pathogenic bacterium responsible for severe infections in vulnerable populations. The identification of two genes, rcsA and treR, as potential regulators of C. sakazakii toxicity toward zebrafish and rats and the proteomic profiling upon knockout of rcsA and treR provides novel insights into the mechanisms underlying bacterial virulence. The findings contribute to our understanding of C. sakazakii's pathogenicity, shed light on the regulatory pathways involved in bacterial virulence, and offer potential targets for the development of novel interventions against this highly virulent bacterium.

Genomic insights into Cronobacter spp. recovered from food and human clinical cases in Zhejiang province, China (2008-2021).

AIMS: Cronobacter spp. are emerging food-borne pathogens capable of causing life-threatening illness via several distinct routes. Although endeavors to reduce the incidence of Cronobacter infections are implemented, potential risk of these microorganisms on food safety remains poorly understood. Here, we evaluated the genomic features of clinical Cronobacter and the possible food reservoirs of these infections. METHODS AND RESULTS: Whole-genome sequencing (WGS) data of all human clinical cases (n = 15) during 2008-2021 in Zhejiang were used and compared to sequenced Cronobacter genomes (n = 76) representing various food products. Cronobacter strains exhibited a high degree of genetic diversity by WGS-based subtyping. A variety of serotypes (n = 12) and sequence types (n = 36) were identified, including six novel STs (ST762-ST765, ST798, and ST803) first-time described in this study. Nine clinical clusters representing 12/15 (80%) patients match a potential food source. Genomic insights into virulence genes revealed species/hosts specificity signatures associated with autochthonous populations. Resistance to streptomycin, azithromycin, sulfanilamide isoxazole, cefoxitin, amoxicillin, ampicillin, and chloramphenicol, as well as multidrug resistance, was noted. WGS data can be used to predict resistance phenotypes in amoxicillin, ampicillin, and chloramphenicol, which were extensively used in clinical treatment. CONCLUSIONS: The wide dissemination of pathogenic potential and antibiotic-resistant strains in multiple food sources emphasized the importance of rigorous food safety policies to reduce Cronobacter contamination in China.

Inactivation of Cronobacter sakazakii biofilms using high voltage atmospheric cold plasma on various food-contact surfaces-a preliminary study.

Cronobacter sakazakii is an opportunistic foodborne pathogen in powdered infant formula (PIF) associated with several foodborne outbreaks. Biofilms of C. sakazakii in the PIF manufacturing plant may be a source of contamination, but information on the treatment of these biofilms is limited. This study investigated the inactivation of C. sakazakii biofilms on three food contact surfaces (stainless steel, silicone, and PVC) using high voltage atmospheric cold plasma (HVACP) applied as a dielectric barrier discharge in a 10:90 air: helium modified atmosphere. After the 90 s  of cold plasma exposure at 40 kV, C. sakazakii was reduced by ∼3 log CFU/coupon comparing to without cold plasma treatments. HVACP treatment caused cell shrinkage, fragmentation of the cell membrane, and leakage of cytoplasm. The inactivation of the cells on the surface was confirmed by live/dead staining. These above-mentioned results indicate the antibiofilm efficacy of HVACP on C. sakazakii isolates on various food contact surfaces.

Depletion of reactive oxygen species induced by beetroot (Beta vulgaris) extract leads to apoptosis-like death in Cronobacter sakazakii.

This research aimed to disclose the antibacterial activity of beetroot extract (Beta vulgaris) against Cronobacter sakazakii and its possible mechanisms. We evaluated its antibacterial activity by measuring the minimum inhibitory concentration (MIC) and time-kill kinetics. We also evaluated the intracellular ATP levels, bacterial apoptosis-like death (ALD), and reactive oxygen species (ROS) levels to reveal the possible antibacterial mechanisms. Our results showed that the MIC of beetroot extract against C. sakazakii was 25 mg/mL and C. sakazakii (approximately 8 log cfu/mL) was completely inhibited after treatment with 2 MIC of beetroot extract for 3 h. Beetroot extract reduced intracellular ATP levels and facilitated characteristics of ALD in C. sakazakii, such as membrane depolarization, increased intracellular Ca2+ levels, phosphatidylserine externalization, caspase-like protein activation, and DNA fragmentation. Additionally, and different from most bacterial ALD caused by the accumulation of ROS, beetroot extract reduced the intracellular ROS levels in C. sakazakii. Our experimental data provide a rationale for further research of bacterial ALD and demonstrate that beetroot extract can inhibit C. sakazakii in food processing environments.

Cronobacter sakazakii Cue for the Attraction and Its Impact on the Immunity of Caenorhabditis elegans.

Cronobacter sakazakii, an opportunistic foodborne pathogen prevalently detected in contaminated powdered infant formula, is associated with different diseases, including meningitis. It can cross the blood-brain barrier and affects the CNS. The impact of C. sakazakii on host neuronal cells and behavior is largely unknown. Hence, detailed molecular data are required to understand its severity. Caenorhabditis elegans is a unique model for studying chemical communication, as it relies on chemosensation for searching nutritional supplements. Although, C. sakazakii is pathogenic to C. elegans, our analysis indicated that C. elegans was highly attracted toward C. sakazakii compared to its food source, E. coli OP50. To study the cue for the attraction, bioactive components (RNA/Protein/Lipopolysaccharides/Metabolites) of C. sakazakii were isolated and used for observing the chemotaxis behavior of C. elegans. The results signified that C. elegans was more attracted toward acid extracted metabolites than those of the other extraction methods. The combined action of acid extracted metabolites of C. sakazakii and a candidate pathogen drastically reduced the survival of C. elegans. In addition, qPCR analysis suggested that the exposure of isolated metabolites through acid extraction to C. elegans for 24 h modified the candidate immune regulatory genes involved in pathogen recognition and kinase activity such as clec-60, clec-87, lys-7, akt-2, pkc-1, and jnk-1.

Membrane Proteins and Proteomics of Cronobacter sakazakii Cells: Reliable Method for Identification and Subcellular Localization.

Members of the genus Cronobacter are responsible for severe infections in infants and immunosuppressed individuals. Although several virulence factors have been described, many proteins involved in the pathogenesis of such infections have not yet been mapped. This study is the first to fractionate Cronobacter sakazakii cells into outer membrane, inner membrane, periplasmic, and cytosolic fractions as the basis for improved proteome mapping. A novel method was designed to prepare the fractionated samples for protein identification. The identification was performed via one-dimensional electrophoresis-liquid chromatography electrospray ionization tandem mass spectrometry. To determine the subcellular localization of the identified proteins, we developed a novel Python-based script (Subcelloc) that combines three web-based tools, PSORTb 3.0.2, CELLO 2.5, and UniProtKB. Applying this approach enabled us to identify 1,243 C. sakazakii proteins, which constitutes 28% of all predicted proteins and 49% of all theoretically expressed outer membrane proteins. These results represent a significant improvement on previous attempts to map the C. sakazakii proteome and could provide a major step forward in the identification of Cronobacter virulence factors. IMPORTANCECronobacter spp. are opportunistic pathogens that can cause rare and, in many cases, life-threatening infections, such as meningitis, necrotizing enterocolitis, and sepsis. Such infections are mainly linked to the consumption of contaminated powdered infant formula, with Cronobacter sakazakii clonal complex 4 considered the most frequent agent of serious neonatal infection. However, the pathogenesis of diseases caused by these bacteria remains unclear; in particular, the proteins involved throughout the process have not yet been mapped. To help address this, we present an improved method for proteome mapping that emphasizes the isolation and identification of membrane proteins. Specific focus was placed on the identification of the outer membrane proteins, which, being exposed to the surface of the bacterium, directly participate in host-pathogen interaction.

Insights into the mechanisms of Cronobacter sakazakii virulence.

Cronobacter species have adapted to survive harsh conditions, particularly in the food manufacture environment, and can cause life-threatening infections in susceptible hosts. These opportunistic pathogens employ a multitude of mechanisms to aid their virulence throughout three key stages: environmental persistence, infection strategy, and systemic persistence in the human host. Environmental persistence is aided by the formation of biofilms, development of subpopulations, and high tolerance to environmental stressors. Successful infection in the human host involves several mechanisms such as protein secretion, motility, quorum sensing, colonisation, and translocation. Survival inside the host is achieved via competitive acquisition and utilization of minerals and metabolites respectively, coupled with host immune system evasion and antimicrobial resistance (AMR) mechanisms. Across the globe, Cronobacter sakazakii is associated with often fatal systemic infections in populations including neonates, infants, the elderly and the immunocompromised. By providing insight into the mechanisms of virulence utilised by this pathogen across these three stages, this review identifies current gaps in the literature. Further research into these virulence mechanisms is required to inform novel mitigation measures to improve global food safety with regards to this food-borne pathogen.

Plant growth-promoting rhizobacteria Shewanella putrefaciens and Cronobacter dublinensis enhance drought tolerance of pearl millet by modulating hormones and stress-responsive genes.

Drought is a major abiotic stress that affects crop productivity. Endophytic bacteria have been found to alleviate the adverse effects of drought on plants. In the present study, we evaluated the effects of two endophytic bacteria Shewanella putrefaciens strain MCL-1 and Cronobacter dublinensis strain MKS-1 on pearl millet (Pennisetum glaucum (L.) R. Br.) under drought stress conditions. Pearl millet plants were grown under three water levels: field capacity (FC), mild drought stress (MD), and severe drought stress (SD). The effects of inoculation on plant growth, physiological attributes, phytohormone content, and drought stress-responsive genes were assessed. The inoculation of pearl millet seeds with endophytes significantly improved shoot and root dry weight and root architecture of plants grown under FC and drought stress conditions. There was a significant increase in relative water content and proline accumulation in the inoculated plants. Among the phytohormones analyzed, the content of ABA and IAA was significantly higher in endophyte-treated plants under all moisture regimes than in uninoculated plants. C. dublinensis-inoculated plants had higher GA content than uninoculated plants under all moisture regimes. The expression level of genes involved in phytohormone biosynthesis (SbNCED, SbGA20oX, and SbYUC) and coding drought-responsive transcription factors (SbAP2, SbSNAC1 and PgDREB2A) was significantly higher under SD in endophyte-inoculated plants than in uninoculated plants. Thus, these endophytic bacteria presumably enhanced the tolerance of pearl millet to drought stress by modulating root growth, plant hormones, physiology and the expression of genes involved in drought tolerance.