Search Results (68)

CRISPR/Cas systems have made significant progress in the field of molecular diagnostics in recent years. To overcome the aerosol contamination problem brought on by amplicon transfer in the common two-step procedure, the “one-pot method” has become a major research hotspot in this field. However, these methods usually rely on specially designed devices or additional chemical modifications. In this study, a novel “one-pot” strategy was developed to detect the foodborne pathogen Cronobacter sakazakii (C. sakazakii). A specific sequence was screened out from the virulence gene ompA of C. sakazakii as the detection target. Combining with the recombinase polymerase amplification (RPA), a rapid detection platform for C. sakazakii based on the CRISPR/Cas12a system was established for the first time. The sensitivity of this method was determined from three different levels, which are 10⁻⁴ ng/μL for genomic DNA (gDNA), 1.43 copies/μL for target DNA, and 6 CFU/mL for pure bacterial culture. Without any microbial enrichment, the detection limits for artificially contaminated cow and goat milk powder samples were 4.65 CFU/mL and 4.35 CFU/mL, respectively. To address the problem brought on by aerosol contamination in the common RPA-CRISPR/Cas12a two-step method, a novel pipette tip-in-tube (PTIT) method for simple and sensitive one-pot nucleic acid detection was further developed under the inspiration of the capillary principle. The RPA and CRISPR/Cas systems were isolated from each other by the force balance of the solution in a pipette tip before amplification. The detection limits of the PTIT method in pure bacterial culture and the spiked samples were exactly the same as that of the two-step method, but with no false positive cases caused by aerosol contamination at all. Compared with other existing one-pot methods, the PTIT method requires no additional or specially designed devices, or any chemical modifications on crRNA and nucleic acid probes. Therefore, the PTIT method developed in this study provides a novel strategy for realizing one-pot CRISPR/Cas detection easily and holds significant potential for the rapid point-on-care testing (POCT) application. Full article

Pathogenic bacteria have developed different ways to cause infections. One strategy involves using components from host cells. This study looks at the role of the cytoskeleton in the human colon adenocarcinoma Caco-2 and neonatal non-transformed epithelial H4 cell lines during bacterial invasion. The bacteria studied include Cronobacter malonaticus, Cronobacter sakazakii, and E. coli K1, as they are associated with known diseases. Salmonella enteritidis 358 served as a positive control and E. coli K12 as a negative control for the invasion experiments. Before the invasion experiments, cell lines were treated with microfilament inhibitors, specifically Cytochalasin D, and microtubule inhibitors, such as Colchicine, Nocodazole, Vinblastine, and Taxol. The results showed that Cytochalasin D reduced about 60–80% of Cronobacter invasion into H4 cells and 50% of E. coli K1 invasion. In contrast, Colchicine reduced the invasion of some strains to just 2% compared to untreated cells. Meanwhile, Nocodazole and Taxol increased the invasion of C. sakazakii 709 and C. malonaticus 1569 into H4 cells by about 140% and 160%, respectively, while slightly inhibiting other strains. In Caco-2 cells, certain strains exhibited increased invasion due to Cytochalasin D, Vinblastine, and Colchicine treatment. This led to increases of up to 500%, 227%, and 248% compared to untreated cells. However, Nocodazole and Taxol decreased invasion into Caco-2 cells, with only E. coli K1 showing an increase of about 150% in Taxol-treated cells. The findings with eukaryotic cytoskeleton inhibitors on neonatal H4 cells suggest that bacterial invasion mainly relies on microfilaments or microfilament-dependent. No specific dependence on the cytoskeleton was seen in Caco-2 cells. In conclusion, cytoskeletal inhibitors significantly affected bacterial invasion, specifically Cronobacter, compared to untreated cells. This suggests that invasion methods may vary by strain and are influenced by how each inhibitor alters cytoskeleton behavior. Therefore, the invasion process, both with and without cytoskeletal inhibitors, is crucial for understanding how bacteria manipulate cell components during infection. Full article

Cronobacter sakazakii is a foodborne pathogen in powdered infant formula, which poses a significant risk to susceptible populations such as infants and the elderly. This study aims to develop a visual detection method for viable C. sakazakii using the reverse transcription-polymerase spiral reaction and hydroxynaphthol blue indicator. Under the optimized conditions, the detection process could be completed within 55 min with low equipment dependence. It was evaluated to have high specificity and sensitivity with the detection limit low to 1.2 × 10¹ CFU/mL. The assay also showed 100% accuracy in artificially contaminated samples. Full article

Cronobacter sakazakii is a food-borne pathogen that can thrive in various environments, including the human body. The human body’s physiological temperature exceeds that of the environment (22–30 °C), necessitating adaptations to heat stress during this transition. Managing heat stress is crucial when transitioning from the environment to the human body. In this study, we explored the effect of human body temperature on the growth of planktonic C. sakazakii, as well as its acid resistance, osmotic stress resistance, autoaggregation, and cell surface hydrophobicity. Our study demonstrated that human body temperature facilitated the growth, acid resistance, and osmotic resistance of C. sakazakii, compared to 28 °C. The relationship between human body temperature and phenotypes was studied by comparing gene expression at human and environmental temperatures (37 to 28 °C) using high-throughput sequencing. The results revealed up-regulation in the expression of 626 genes, including genes involved in arginine and proline metabolism, carbon fixation pathways, and nitrogen metabolism. Further analysis showed that human body temperature is essential for the environmental stress resistance of C. sakazakii. It boosts denitrification, betaine transport, and universal stress proteins, supporting membrane integrity and osmoprotectant transport. This study enhances our understanding of the strategies employed by C. sakazakii during its adaptation to the human body. Full article

Cronobacter sakazakii is an opportunistic food-borne pathogen that causes severe infections with high morbidity and mortality rates in neonates, the elderly, and immunocompromised individuals. The plant extracts containing natural antibacterial compounds are currently under consideration as alternatives to synthetic artificial preservatives for the control of C. sakazakii. There has been increasing interest in using plant-derived antimicrobials in combination with mild heat to control pathogens in preservative-free foods. In this study, the individual and combined effects of four independent variables, i.e., polyphenol-rich haskap extract (HE) concentration (2–10%), tannic acid (TA) concentration (0.1–0.5), temperature (35–55 °C), and time (1–5 min), on C. sakazakii inactivation were investigated by response surface methodology (RSM) with a five-level four factor central composite design (CCD) and an optimal combination for maximum inhibition was determined. The statistic metrics of R², R²_adjusted, R²_predicted, coefficient of variation (CV), Predicted Residual Error Sum of Squares (PRESSs), adequate precision, and lack-of-fit were used to reveal the prediction performance. The results revealed that all the independent variables, except time, influenced C. sakazakii inactivation. Among the independent variables, the temperature was the most effective variable (p < 0.0001) as regards inactivation. The synergistic effects of HE with TA and temperature were observed. Many possible optimum conditions of mild heat treatment that maximized the inhibition of C. sakazakii were obtained. The findings indicated that two distinct combinations were identified as the most effective inhibition of C. sakazakii: high concentration at low temperature and high temperature at low concentration. It can be concluded that haskap polyphenol extract, alone or in combination with tannic acid, has the potential to be used as a natural preservative to reduce the risk of C. sakazakii. Full article

Cronobacter sakazakii is a foodborne pathogen characterized by its robust stress tolerance and ability to form biofilms, which facilitates its survival in powdered infant formula (PIF) processing environments for prolonged periods. Gamma-aminobutyric acid (GABA) is a kind of non-protein amino acid that acts as an osmoprotectant. This study aimed to elucidate the effects of the gabT gene on the survival of C. sakazakii, GABA accumulation, and biofilm formation under desiccation, osmotic stress, and acid exposure. A gabT knockout strain of C. sakazakii was developed using gene recombination techniques. The GABA content and survival rates of both the wild-type and knockout strains were compared under various stress conditions. Scanning electron microscopy (SEM) was used to observe cellular damage and biofilm formation. Statistical analysis was performed using a one-way analysis of variance (ANOVA). The deletion of gabT resulted in enhanced GABA accumulation under different stress conditions, improving the bacterium’s tolerance to desiccation, osmotic pressure, and acid treatment. SEM images revealed that under identical stress conditions, the gabT knockout strain exhibited less cellular damage compared to the wild-type strain. Both strains were capable of biofilm formation under low osmotic pressure stress, but the gabT knockout strain showed higher GABA content, denser biofilm formation, and increased biofilm quantity. Similar trends were observed under acid stress conditions. The gabT gene plays a key role in modulating GABA accumulation, which enhances the stress tolerance and biofilm formation of C. sakazakii. These findings provide new insights into the role of GABA in bacterial survival mechanisms and highlight the potential for targeting GABA pathways to control C. sakazakii in food processing environments. Full article

The opportunistic pathogenic bacterium C. sakazakii poses a significant infection risk to infants through contaminated powdered infant formulae (PIFs) when proper hygiene and temperature control are neglected during reconstitution. This study aimed to investigate whether the acid-buffering capacity (ABC) of commercially available PIFs enriched with the probiotic strain Bifidobacterium animalis subsp. lactis (BB-12^®) could influence the growth of C. sakazakii. Two PIFs differing in their ABC were reconstituted (RIF), inoculated, and incubated in monoculture and co-culture at 22 °C and 37 °C for 24 h. After 24 h of incubation at 22 °C, regardless of the ABC type of PIF, the population of C. sakazakii in the monoculture was approx. 1.4 log cycles higher than the inoculum, while, in the co-culture, the C. sakazakii count was approx. 0.34 log cycles lower. In contrast, at 37 °C during the 24 h co-culture in the lower ABC infant formula, C. sakazakii was <10 CFU/mL. In all co-culture samples, the pH was significantly lower (p < 0.05) in the PIF with the lower ABC. An analysis of the weak organic acids at 12 and 24 h of incubation revealed that the antimicrobial activity is significantly affected by the final pH value, the type of the weak organic acids, and their ionic–non-ionic ratio, which is formed through the common ion effect. Full article

Cronobacter sakazakii, an opportunistic foodborne pathogen, has a strong resistance to osmotic stress and desiccation stress, but the current studies cannot elucidate this resistance mechanism absolutely. A mechanosensitive channel MscM was suspected of involving to desiccation resistance mechanism of C. sakazakii. To investigate the specific molecular mechanism, the mscM mutant strain (ΔmscM) was constructed using the homologous recombination method, and the cpmscM complementary strain was obtained by gene complementation, followed by the analysis of the difference between the wild-type (WT), mutant, and complementary strains. Compared to the wild-type bacteria (WT), the inactivation rate of the ΔmscM strain decreased by 15.83% (p < 0.01) after desiccation stress. The absence of the mscM gene led to an increase in the membrane permeability of mutant strains. Through turbidity assay, it was found that the intracellular content of potassium ion (K⁺) of the ΔmscM strain increased by 2.2-fold (p < 0.05) compared to the WT strain, while other metal ion contents, including sodium ion (Na⁺), calcium ion (Ca²⁺), and magnesium ion (Mg²⁺), decreased by 48.45% (p < 0.001), 24.29% (p < 0.001), and 26.11% (p < 0.0001), respectively. These findings indicate that the MscM channel primarily regulates cell membrane permeability by controlling K⁺ efflux to maintain the homeostasis of intracellular osmotic pressure and affect the desiccation tolerance of bacteria. Additionally, the deletion of the mscM gene did not affect bacterial growth and motility but impaired surface hydrophobicity (reduced 20.52% compared to the WT strain, p < 0.001), adhesion/invasion capability (reduced 26.03% compared to the WT strain, p < 0.001), and biofilm formation ability (reduced 30.19% compared to the WT strain, p < 0.05) of the bacteria. This study provides a reference for the role of the mscM gene in the desiccation resistance and biofilm formation of C. sakazakii. Full article

Cronobacter sakazakii is a dangerous pathogen easily found in powdered infant formula (PIF), causing severe infections and even death in infants. Herein, a bacteriophage-immobilized magnetic nanozyme (Fe₃O₄@EspYZU13) was prepared for C. sakazakii detection. Bacteriophage EspYZU13 isolated and identified by our group exhibits specific lytic capacity. Fe₃O₄@EspYZU13 possesses remarkable enrichment capacity towards C. sakazakii, efficiently enriching different concentrations of C. sakazakii from a mixed bacterial solution. Furthermore, Fe₃O₄@EspYZU13 shows peroxidase-like activity, which can catalyze the 3,3′,5,5′-tetramethylbenzidine (TMB) chromogenic reaction in the presence of H₂O₂. Upon introduction of C. sakazakii, it can be specifically captured by Fe₃O₄@EspYZU13, inhibiting its peroxidase-like activity. Based on it, C. sakazakii ranging from 3.2 × 10¹ to 3.2 × 10⁷ CFU mL⁻¹ can be determined, offering a detection limit (LOD) of 26 CFU mL⁻¹. Moreover, this reaction system keeps high specificity towards C. sakazakii, which can resist interferences of other possible coexisting bacteria. C. sakazakii in the artificially contaminated PIF can be detected, offering good recoveries (96.76% to 103.13%). These results indicate that our proposed reaction system demonstrates its practical application potential for efficient enrichment of C. sakazakii from complex samples and accurate determination of C. sakazakii in PIF. Full article

Gram-negative bacteria possess an asymmetric outer membrane, where the outer leaflet consists of LPSs and the inner leaflet comprises phospholipids. Cronobacter sakazakii, an opportunistic milk-borne pathogen that causes severe neonatal meningitis and bacteremia, displays diverse lipopolysaccharide (LPS) structures. As a barrier of the bacterial cell, LPSs likely influenced C. sakazakii resistance to environment stresses; however, there are no research reports on this aspect, hindering the development of novel bactericidal strategies overcoming the pathogen’s resilience. In the present study, therefore, C. sakazakii BAA894 and two LPS mutants (ΔlpxM and ΔwaaC) were employed to investigate its influences. The ΔwaaC mutant showed lower resistance to acidic, alkali, oxidative, and osmotic stresses compared to the wild-type strain BAA894, and the ΔlpxM mutant exhibited lower desiccation resistance but higher osmotic resistance. To uncover potential reasons for these differences, comparative lipidomics was conducted. The results showed that compared to BAA894, both mutants showed drastic changes in lipid quantity, and many changed lipids were unsaturated. Additionally, eleven lipid classes exhibited significant variation in the relative content. In particular, the polyunsaturated TGs with double bonds at 5, 7, 12, and 14 displayed significant variation between the wild type and two mutants. Our study is the first to reveal that the changes in the LPS structure of C. sakazakii resulted in altered lipid profiles and intensities, which may be a critical biochemical basis for bacterial resistance to harsh stresses. Full article

Precision metagenomic approaches using Oxford Nanopore Technology (ONT) sequencing has been shown to allow recovery of complete genomes of foodborne bacteria from overnight enrichments of agricultural waters. This study tests the applicability of a similar approach for Cronobacter genome recovery from powdered infant formula (PIF) overnight enrichments, where Cronobacter typically dominates the overall microbiome (>90%). This study aimed to test whether using ONT sequencing of overnight PIF enrichments could recover a completely closed Cronobacter genome for further genomic characterization. Ten PIF samples, each inoculated with different Cronobacter strains, covering Cronobacter sakazakii, C. muytjensii, C. dublinensis, C. turicensis, and C. universalis, were processed according to the Bacteriological Analytical Manual (BAM) protocol. Real-time quantitative PCR (qPCR) was used for initial screening (detection and quantification) of the overnight enrichments and confirmed that the inoculated PIF samples after the overnight enrichment had high levels of Cronobacter (10⁷ to 10⁹ CFU/mL). DNA from overnight PIF enrichments was extracted from the enrichment broth and sequenced using ONT. Results showed that ONT sequencing could accurately identify, characterize, and close the genomes of Cronobacter strains from overnight PIF enrichments in 3 days, much faster than the nearly 2 weeks required by the current BAM method. Complete genome recovery and species differentiation were achieved. This suggests that combining qPCR with ONT sequencing provides a rapid, cost-effective alternative for detecting and characterizing Cronobacter in PIF, enabling timely corrective actions during outbreaks. Full article

Following an outbreak of Cronobacter sakazakii infection in infant formula, the US Food and Drug Administration commissioned a series of reports and then undertook a major reorganization of its food program. This article describes the changes that went into effect on 1 October 2024, why those changes are likely to improve the new Human Foods Program, and additional work that must be undertaken to enhance the agency’s impact upon public health. Full article

The Chilean Technical Manual for Milk Dietary Service (SEDILE) was evaluated to determine its effectiveness in ensuring the microbiological safety and vitamin D adequacy of a commercially available powdered infant formula (PIF) used in SEDILE. The evaluation focused on whether adherence to the manual’s guidelines positively influenced these factors. Both the PIF and the reconstituted PIF (RIF) were found to be free from Cronobacter sakazakii and Salmonella enterica contamination, with levels of total coliforms and Escherichia coli within acceptable limits. Moreover, the vitamin D content in the formula was within the expected range. These findings suggest that following the Chilean Technical Manual for SEDILE contributes to the microbiological safety and nutritional adequacy of RIF in dietary services. Full article

Cronobacter sakazakii is associated with the ingestion of contaminated reconstituted powdered infant formula (PIF), resulting in necrotizing enterocolitis, sepsis and meningitis in neonatal infants. Potential virulence determinants include the variable capsular polysaccharides; K-antigen and colanic acid (CA). Strains encoding for the capsule variant K2:CA2 have been strongly associated with neonatal meningitis cases. This study aimed to develop and apply a multiplex PCR assay to determine C. sakazakii K-antigen and colanic acid types. Twenty-six strains of C. sakazakii which had previously been isolated from food and environmental sources were used. These cover 18 multilocus sequence types and four serotypes. Based on our research findings, we have identified two K-antigen types present. Specifically, the K1-antigen was observed in sequence types ST1, ST8, ST20, ST23, ST64, ST198, ST263, ST264 and ST406, while the K2-antigen was present in ST4, ST9, ST12, ST13, ST136, ST233, ST245 and ST405. Additionally, we detected colanic acid (CA) type 1 in sequence types ST1, ST8, ST9, ST20, ST245 and ST405, and colanic acid (CA) type 2 in ST4, ST12, ST13, ST23, and ST64. We compared the predicted K-antigen and colanic acid types with the entire genome sequences of the strains. The comparison showed complete agreement between the PCR amplification results and the genomic analysis of the K-antigen and colanic acid-encoding regions. This assay is a useful tool for rapid identification of C. sakazakii, K-antigen and colanic acid types, in routine diagnoses and foodborne investigations. In addition, it will contribute to our knowledge of virulence factors associated with life-threatening neonatal meningitis. Full article

Cronobacter sakazakii (C. sakazakii) is a foodborne pathogen capable of causing severe infections in newborns. The PhoP/PhoQ two-component system exerts a significant influence on bacterial virulence. This study aimed to investigate the impact of the PhoP/PhoQ system on intestinal inflammation in neonatal mice induced by C. sakazakii. Neonatal mice were infected orally by C. sakazakii BAA-894 (WT), a phoPQ-gene-deletion strain (ΔphoPQ), and a complementation strain (ΔphoPQ^C), and the intestinal inflammation in the mice was monitored. Deletion of the phoPQ gene reduced the viable count of C. sakazakii in the ileum and alleviated intestinal tissue damage. Moreover, caspase-3 activity in the ileum of the WT- and ΔphoPQ^C-infected mice was significantly elevated compared to that of the ΔphoPQ and control groups. ELISA results showed elevated levels of TNF-α and IL-6 in the ileum of the mice infected with WT and ΔphoPQ^C. In addition, deletion of the phoPQ gene in C. sakazakii resulted in a down-regulation of inflammatory genes (IL-1β, TNF-α, IL-6, NF-κB p65, TLR4) within the ileum and decreased inflammation by modulating the TLR4/NF-κB pathway. It is suggested that targeting the PhoP/PhoQ two-component system could be a potential strategy for mitigating C. sakazakii-induced neonatal infections. Full article