Microbiological survey and genomic analysis of Cronobacter sakazakii strains isolated from US households and retail foods.

Cronobacter species are opportunistic pathogens that are capable of causing morbidity and mortality, particularly in infants. Although the transmission dynamics involved in Cronobacter infections remain largely unknown, contaminated powdered infant formula (PIF) has been linked to 30% of Cronobacter sakazakii cases involving invasive illness in infants. As several lines of evidence have implicated the domestic environment in PIF contamination, we undertook a microbiological survey of homes (N = 263) across the US. Cronobacter spp. and C. sakazakii were isolated from 36.1% and 24.7% of US homes, respectively, with higher recovery rates observed for floor and kitchen surfaces. Multi-locus sequence typing indicated that the dominant strain was C. sakazakii ST4, the sequence type most commonly associated with neonatal meningitis. For comparison purposes, retail foods (N = 4,009) were also surveyed, with the highest contamination frequencies (10.1%-26.3%) seen for nut products, seeds, and grains/baked goods/flours. The sequence type profile of isolates recovered from homes mirrored that of isolates recovered from retail foods, with increased representation of ST1, ST4, ST13, ST17, and ST40. Analysis of 386 whole genomic sequences revealed significant diversity. Redundancies were only observed for isolates recovered from within the same domicile, and there were no identical matches with sequences archived at the NCBI pathogen database. Genes coding for putative virulence and antibiotic resistance factors did not segregate with clinically significant sequence types. Collectively, these findings support the possibility that contamination events occurring within the home should not be overlooked as a contributor to community-onset Cronobacter infections. IMPORTANCE: Cronobacter sakazakii is an opportunistic pathogen that can cause significant morbidity and mortality in neonates. Its transmission dynamics are poorly understood, though powered infant formula (PIF) is thought to be the major transmission vehicle. How the PIF becomes contaminated remains unknown. Our survey shows that roughly 1/4 of US homes are contaminated with Cronobacter sakazakii, particularly in the kitchen setting. Our analyses suggest that the domestic environment may contribute to contamination of PIF and provides insights into mitigating the risk of transmission.

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.

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.

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.

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.

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.

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.

Fatal Necrotizing Enterocolitis in Neonate Caused by Cronobacter sakazakii Sequence Type 64 Strain of CRISPR Sublineage b.

We report fatal neonatal necrotizing enterocolitis in China caused by Cronobacter sakazakii capsular profile K1:CA1, sequence type 64, and CRISPR type 197. Phylodynamic analyses indicated that the strain originated from the ancient, widespread, and antimicrobial drug-sensitive CRISPR sublineage b. Enhanced surveillance and pathogenesis research on this organism are required.

Phage-Displayed Nanobody as a Sensitive Nanoprobe to Enhance Chemiluminescent Immunoassay for Cronobacter sakazakii Detection in Dairy Products.

The exploitation of stable, high-affinity, and low-cost nanoprobes is essential to develop immunoassays for real-time monitoring of foodborne pathogens, so as to safeguard human health. The possible interaction of the Fc fragment of antibodies with spA protein on Staphylococcus aureus will result in unexpected interference. To address this consideration, we described herein for the first time the development of nanobodies that by definition are devoid of the Fc fraction. These nanobodies directed against Cronobacter sakazakii (C. sakazakii) were retrieved from a dedicated immune phage-displayed nanobody library. The binders showed superiority of low cost, strong stability, high binding affinity, and adequate load capacity. Thereafter, a phage-mediated sandwich enzyme-linked immunosorbent assay (ELISA) was constructed by using Cs-Nb2 as an antigen-capturing antibody and phage-displayed Cs-Nb1 as a detection probe. To further enhance the sensitivity, a chemiluminescent enzyme immunoassay (CISA) was established by replacing the substrate from 3,3',5,5'-tetramethylbenzidine (TMB) to luminol, providing a limit of detection of 1.04 × 104 CFU/mL, with a recovery of 98.15-114.63% for the detection of C. sakazakii in dairy products. The proposed nanobody-based phage-mediated sandwich CLISA shows various advantages, including high sensitivity, cost effectiveness, enhanced loading capacity of the enzyme, and high resistance to the matrix effect, providing a strategy for the design of immunoassays toward foodborne pathogens.

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.

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.

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.

Gut microbiota dysbiosis is associated with sepsis-induced cardiomyopathy in patients: A case-control study.

BACKGROUND: Myocardial injury is a major complication of sepsis and a key factor affecting prognosis. Therefore, early and accurate diagnosis and timely management of sepsis-induced cardiomyopathy (SICM) are of great significance for the prevention and treatment of sepsis. The gut microbiota has been shown to be closely associated with sepsis or myocardial injury, but the association between the gut microbiota and SICM is not fully understood. This study aimed to explore the link between gut microbiota composition and SICM. METHODS: A case-control and single-center study of clinical features and gut microbiota profiles by Metagenome and Virome was conducted in SICM patients (n = 15) and sepsis-uninduced cardiomyopathy patients (SNICM, n = 16). RESULTS: Compared with SNICM patients, SICM patients showed significant myocardial injury and higher 28-day mortality, SOFA scores, lactate levels, and infection levels on admission. Meanwhile, differences in the composition of gut bacteria, archaea, fungi, and viruses were analyzed between the two groups. Differential gut bacteria or viruses were found to have a good predictive effect on SICM. Furthermore, gut bacteria and viruses that differed between the two groups were strongly related. The abundance of Cronobacter and Cronobacter phage was higher in the SICM group than in the SNICM group, and the receiver operating characteristic curve showed that Cronobacter and Cronobacter phage both had a good predictive effect on SICM. CONCLUSIONS: SICM patients may have specific gut microbiota signatures, and Cronobacter and Cronobacter phages have a good ability to identify and diagnose SICM.

Effect of in vitro gastrointestinal digestion on the antibacterial activity of bioactive dairy formulas supplemented with lactoferrin against Cronobacter sakazakii.

Milk is a source of proteins with high nutritional value and relevant biological activities. Bioactive milk proteins, like lactoferrin, are important for newborn development and can also be used as ingredients in functional products to improve health. Lactoferrin is essential in infant's diet, since protects against infections and promotes immune system maturation. Bovine lactoferrin is used to supplement formula milk in order to strengthen baby's defences against some pathogenic bacteria. Thus, lactoferrin supplemented formula can be a barrier against emergent pathogens, such as Cronobacter sakazakii, which has caused great concern in the last few years. Milk proteins generate bioactive peptides in the digestion process, and it is known that industrial processing can modify their susceptibility to digestion. Treatments such as heating have been shown to denature whey proteins and make them more easily digestible. Therefore, the aim of this study was to analyze the effect of technological treatments and gastrointestinal digestion on the antibacterial activity against C. sakazakii of proteins present in dairy formulas supplemented with lactoferrin. Commercial bovine lactoferrin has been shown to have antibacterial activity against C. sakazakii, both in the native state and after static in vitro gastrointestinal digestion. In addition, the digests obtained from dairy formulas subjected to technological treatments, either homogenization or pasteurization, have higher antibacterial activity than non-treated formulas. The release of low molecular weight peptides during the in vitro gastric digestion is probably the cause that would explain the enhanced antibacterial activity of the digested dairy formulas.

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.

Lack of correlation between growth rate and sequence type among Cronobacter sakazakii.

Species identification and growth rates for a collection of Cronobacter strains from clinical and non-clinical sources have been previously reported. However, advancements in DNA sequencing-based identification methods now allow for more accurate identification. Here we report the sequence types (STs) for 24 strains of Cronobacter sakazakii and examine any possible correlation between sequence type and growth rate, which could influence risk through greater pathogen multiplication and reach of infectious doses during time between formula preparation and feeding. The most common clonal complexes (CCs) identified were C. sakazakii CC1 and CC4. CC1 strains belonged to ST1 (n = 8) and ST391 (n = 1), while CC4 included ST4 (n = 4), ST255 (n = 1) and ST295 (n = 1). Three strains were found to belong to CC100 and two were found to belong to ST64. The remaining STs identified were represented by single strains. CC4 strains have a slightly not significant tendency for faster growth rates at 25 °C; however, the small sample size suggests that more strains need to be analysed to determine if this is a true result. In conclusion, the growth rates of C. sakazakii strains do not appear to be strongly correlated to ST.

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.