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 three 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 × 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, could produce heat-labile cytotoxic compounds in cell filtrates. SIGNIFICANCE AND IMPACT OF THE 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.

Rutile nano-bio-interactions mediate dissimilar intracellular destiny in human skin cells.

The use of nanoparticles (NPs) in the healthcare market is growing exponentially, due to their unique physicochemical properties. Titanium dioxide nanoparticles (TiO2 NPs) are used in the formulation of sunscreens, due to their photoprotective capacity, but interactions of these particles with skin cells on the nanoscale are still unexplored. In the present study we aimed to determine whether the initial nano-biological interactions, namely the formation of a nano-bio-complex (other than the protein corona), can predict rutile internalization and intracellular trafficking in primary human fibroblasts and keratinocytes. Results showed no significant effect of NPs on fibroblast and keratinocyte viability, but cell proliferation was possibly compromised due to nano-bio-interactions. The bio-complex formation is dependent upon the chemistry of the biological media and NPs' physicochemical properties, facilitating NP internalization and triggering autophagy in both cell types. For the first time, we observed that the intracellular traffic of NPs is different when comparing the two skin cell models, and we detected NPs within multivesicular bodies (MVBs) of keratinocytes. These structures grant selected input of molecules involved in the biogenesis of exosomes, responsible for cell communication and, potentially, structural equilibrium in human tissues. Nanoparticle-mediated alterations of exosome quality, quantity and function can be another major source of nanotoxicity.

The C-terminal tail of tetraspanin proteins regulates their intracellular distribution in the parasite Trichomonas vaginalis.

The parasite Trichomonas vaginalis is the causative agent of trichomoniasis, a prevalent sexually transmitted infection. Here, we report the cellular analysis of T.vaginalis tetraspanin family (TvTSPs). This family of membrane proteins has been implicated in cell adhesion, migration and proliferation in vertebrates. We found that the expression of several members of the family is up-regulated upon contact with vaginal ectocervical cells. We demonstrate that most TvTSPs are localized on the surface and intracellular vesicles and that the C-terminal intracellular tails of surface TvTSPs are necessary for proper localization. Analyses of full-length TvTSP8 and a mutant that lacks the C-terminal tail indicates that surface-localized TvTSP8 is involved in parasite aggregation, suggesting a role for this protein in parasite : parasite interaction.

Trichomonas vaginalis kills and eats--evidence for phagocytic activity as a cytopathic effect.

This study reports that the cytopathic effect of Trichomonas vaginalis, an important human parasite of the urogenital tract, occurs due to mechanical stress and subsequent phagocytosis of the necrotic cells. The investigation was done using a primary culture of bovine oviduct epithelial cells (BOECs), grown either in monolayers or as floating cells. Trophozoites displaying different virulence levels were co-incubated with BOECs for times varying between 1 min and 48 h. Analyses were performed using videomicroscopy, scanning and transmission electron microscopy, colourimetric assays and cytochemistry. Injury was observed as early as 1 h after incubation, while after 12 h the host cells were severely damaged when a fresh trichomonad isolate was used. Trichomonads attack the host cells by clustering around them. Mechanical stress on the microvilli of the host cells was observed and appeared to induce plasma membrane damage and cell death. After membrane injury and lysis, fragments of the necrotic cells were ingested by trichomonads. Phagocytosis occurred by trichomonads avidly eating large portions of epithelial cells containing the nucleus and other organelles, but living or intact cells were not ingested. Necrotic fragments were rapidly digested in lysosomes, as shown by acid phosphatase and ruthenium red assays where only the BOECs were labelled. The lytic capacity of the trichomonads was more pronounced in host cell suspensions.

Cytopathic effects of Tritrichomonas foetus on bovine oviduct cells.

Tritrichomonas foetus is an extracellular parasite of the reproductive tract in cattle. To investigate the cytopathic effects of T. foetus in deeper parts of the reproductive tract, a bovine primary oviduct epithelial cell system (BOECs) was developed. Reproductive tracts were obtained from cows and the effect of co-incubating T. foetus with BOECs was analyzed by scanning electron, transmission electron and fluorescence microscopy. Viability tests were performed using colorimetric methods, TUNEL (Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling), fluorescein diacetate, propidium iodide, JC-1 and annexin-V. The results demonstrate that: (1) the in vitro oviduct epithelium is useful for interaction experiments with T. foetus; (2) T. foetus adheres to the BOECs as single separate cells, and later on the cells aggregate as large clusters; (3) the posterior region of the cell initiates the process of adhesion and forms filopodia and digitopodia; (4) T. foetus severely damages BOECs leaving imprints in the epithelial cells, wide intercellular spaces, and large lesions in the epithelium; and (5) T. foetus provokes bovine oviduct cell death by apoptosis and secondary necrosis. Our observations indicate the possibility that T. foetus can move through the reproductive tract to the oviduct and that infertility in cows can be mediated by an attack on the oviduct cells by T. foetus.