Cytotoxicity profile of Cronobacter species isolated from food and clinical specimens in Brazil

AIMS The objective of this study was to evaluate the cytotoxic activity of Cronobacter strains isolated from foods (n=50) and clinical samples (n=6) in Brazil and genotype selected strains (n=18) using multi-locus sequence typing (MLST). METHODS AND RESULTS The cytotoxic activity of C. sakazakii (n=29), C. dublinensis (n=13), C. malonaticus (n=6), C. turicensis (n=6), and C. muytjensii (n=2) was screened using Vero, RK13, Hep2c, NCTC clone 929, and BHK-21 cell lines. Selected Cronobacter strains were assigned to C. sakazakii ST 21, C. turicensis ST 252, C. sakazakii ST 647, and 3 newly assigned STs; C. turicensis STs 738-740. The maximum death caused by non-heat treated filtrates was 20.4, 86.2, 47.0, and 84.0%, in Vero, RK13, Hep2c, and NCTC clone 929 cells, respectively. These were caused by C. sakazakii strains C291 and C292 (ST 494) which had been isolated during neonatal Cronobacter meningitis infection, and C110 (ST 395) isolated from flaxseed flour. Thermal treatment (100°C/20 min) significantly reduced the cytotoxicity activity in NCTC clone 929 and Vero cells (p ≤ 2 x 10-6), but not in RK13 (p = 0.12) and Hep2c (p = 0.85), indicating the cytotoxin(s) were probably proteinaceous. Electron microscopy revealed that cytotoxic compounds from C. sakazakii induced several cell death characteristics, including loss of cell-cell contact, microvilli reduction and cellular lysis. Autophagic vacuoles and mitochondrial damage were the most common ultrastructural features observed. CONCLUSIONS It was concluded that Cronobacter strains, especially C. sakazakii, can produce heat-labile cytotoxic compounds in cell filtrates. SIGNIFICANCE AND IMPACT OF STUDY This study providing insights into the pathogenesis of the Cronobacter genus. Cytotoxins were identified in excreted filtrates of C. sakazakii strains isolated from food and clinical specimens. The presence of Cronobacter strains that can produce cytotoxins in foods can be a potential threat to human health and highlight the need for high levels of hygiene.