Identification of the virulence determinants of the neonatal meningitic bacterium Cronobacter sakazakii
The Cronobacter genus is a member of the family Enterobacteriaceae, comprising of seven species C. sakazakii, C. malonaticus, C. turicensis, C. dublinensis, C. muytjensii, C. universalis, and C. condimenti. Cronobacter is gaining importance as a pathogen due to the severity of the infection caused such as septicaemia, necrotizing entercolitis and severe infantile meningitis, and the numerous outbreaks reported. Clinical cases are associated with the three species C. malonaticus, C. turicensis and, in particular, with C. sakazakii multilocus sequence type 4. The understanding of its pathogenicity is still not fully understood despite the clinical evidence, resulting in the concern the FAO-WHO. Therefore, this study aimed to apply the Multiple Locus Sequence Typing scheme (MLST) to three collections of clinical strains which had not been previously profiled by MLST. These were from USA (2011), Israel (2000), and the Czech Republic (2007-2012). The strains from the latter two collections had only been identified as E. sakazakii, at that point. Among the Israeli strain collection, isolates from infants ranging from 2 to 36 weeks old, 7/9 strains were identified as belonging to the ST4 clonal complex. Similarly, 10/15 of the C.sakazakii strains isolated from US infant cases were found to belong to the ST4 clonal complex. Whereas 11 strains of the Czech isolates were from various age groups and were identified as C.malonaticus especially ST7, which is also the most clinically predominant ST of that species among non-infant infections and 6 strains were found to belong ST4. This research reported the first meningitis case by Cronobacter malonaticus (CC112) which was from an infant (age <1 month) with severe brain damage which led to their death. Of particular interest in this research was the finding that C. sakazakii and some strains of C. turicensis were unique in the Cronobacter genus in utilization of exogenous sialic acid as a carbon source which may have a role in the organism’s virulence. The presence of sialic acid utilization genes could be relatively recent evolution, as high levels of sialic acid are accessible to bacteria in intestinal mucosa and the brain. Another important finding was, the presence of a number of key virulence associated genes assessed by laboratory studies in Cronobacter sakazakii strains (in particular with ST4). In this study, 36 clinical isolates were analysed that included: two iron acquisition system gene clusters (eitA and iucC), a pla- like homologue named Cronobacter plasminogen activator (Cpa) ,and type VI secretion (T6SS) gene cluster. The majority of C. sakazakii strains were serum resistant (32/36), and thus, they had the ability to survive in blood by preventing serum-mediated killing. Also, different iron acquisition systems were encoded by C. sakazakii 97% to attain iron from the host. Some C. sakazakii strains encoding T6SS patterns were from of clinical cases such as NECII and severe meningitis strains Plasmid profiling experiments were carried out on the Cronobacter sakazakii strains sequenced in a parallel study. The high-size plasmid (molecular weight between 138 and 118 kb) was observed as common in (27/34) of all strains which known to encode an assortment of virulence factors. Also, plasmid DNA analysis publicised that there was no specific plasmid profiling among clinical strains. Furthermore, it shown there is no correlation observed between sequence type and present or absent the plasmid. Furthermore, it was of interest whether the presence of plasmid pESA3 is linked with virulence in C. sakakzaii. This research work developed a tool by inserting the plasmid pESA3 into a plasmid-less strain and observeing the significance changes in the phenotypic and virulence associated behaviour. Stains encoding large plasmid were able to invade human intestinal epithelial cells Caco-2, brain endothelial cells HBMEC and rBCEC4. Also have been reported significant observation in siderophore production and serum survival values whereas plasmid less strain and not shown less significant associated virulence.
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