Neonatal exposure to pathogens : determining key virulence factors

The neonatal stage is the most critical period for infections, with mortality rate of up to 45% amongst children under five years. The genus Cronobacter has been involved in many outbreaks in neonatal intensive care units, with recorded meningitis, bacteraemia, and necrotizing enterocolitis (NEC). Although this genus was deeply investigated in vitro with different cell lines, until now, few researches have been focused on the use of H4 cell line, which is supposed to be more representative of neonatal response. Therefore, the present PhD study aimed to investigate the interaction of selected clinical isolates and the seven-type species of the genus Cronobacter, as well as one E. coli K1 isolate with H4 cells compared with Caco-2 cell line. Physiological analyses revealed most of the strains to be motile, and capsule and biofilm producers. A link between capsule production and serum resistance was shown by some strains. Strains were examined regarding their attachment, invasion, translocation and cytotoxicity as well as for the role of host cytoskeleton in the invasion process to both cell lines. All strains, especially C. sakazakii ST12 (696 and 703) were significantly more adhesive to the H4 than Caco-2 cells; this sequence type has previously been associated with neonatal NEC. Importantly, this study indicated that some clinical strains were more invasive to H4 than Caco-2 cells such as C. sakazakii 767 (meningitis) and 701 (NECIII). Moreover, attachment and invasion of E.coli were higher in H4 than Caco-2 cells. High attachment and invasion is potentially linked with excessive inflammatory response and NEC development in neonates. Most studied strains were able to translocate human cell lines, causing necrotic damage in the polarized monolayer. More importantly, translocation of blood or meningitic isolates such as C. sakazakii 709, C. malonaticus 1569, C.turicensis 564 was higher via H4 compared with Caco-2 cells. However, translocation of E. coli K1 strain 939 was similar in both cell lines. Most of the bacterial strains were cytotoxic to both cell lines and C. sakazakii ST3 strain 798 showed an ability to kill H4 cells up to 90-fold of blank, and about 70-fold when co-cultured with Caco-2 cells. Results suggests that intact bacterial cells that able to produce new proteins while in direct contact with the host cells is essential for the cytotoxicity, indicating the potential involvement of an active secretion system in cytotoxicity. Cytochalasin D and Colchicine mostly inhibited invasion to the H4 cells and enhanced the invasion of some strains to Caco-2 up to five-fold. Only Nocodazole significantly enhanced the invasion of some strains to H4 cell, and variably effected strains’ invasion to Caco-2. Data obtained from human cytoskeleton inhibitors experiments suggested the possible strain specific role of inhibitors on both cell lines, and strains may encode different pathways for uptake, with the possible involvement of eukaryotic receptors that recognize the invading bacteria. This study indicated that the response of the H4 cells to bacterial challenge and production of inflammatory cytokines was higher than Caco-2 cells. The H4 cells produced more IL-1-β, IL-4, IL-6, IL-8, IP-10, MCP-1, and EGF-α. Furthermore, tested strains (n=12) variably affected the expression of human Toll-like receptors (TLRs) and NF-kB subunits 1 and 2 in both cell lines, only C. sakazakii strain 709 upregulated expression of TLR1-4 in H4 cells and was the strongest inducer of receptor gene expression in both cell lines. More importantly, the upregulation of NF-kB subunits 1 is more likely related to the increased inflammatory cytokine production, while no link with subunit 2. However, TLR-1 was not expressed in Caco-2 in response to these strains, which needs further investigation. The effect of the E. coli K1 strain 939 on the TLRs expression was varied and no specific pattern was detected, but in some cases it showed similarity to the effect of C. sakazakii 767 which is also linked with neonatal meningitis. As most of the investigated bacterial strains displayed virulence factors with H4 cells closely related to their clinical pathology, the present study provides an important initial work to introduce H4 cells as a new modle for neonatal cell lines to analyse neonatal infections.