Abstract
Cronobacter sakazakii is an emerging foodborne pathogen, which is linked to life-threatening infections causing septicemia, meningitis, and necrotizing enterocolitis. These infections have been epidemiologically connected to ingestion of contaminated reconstituted powder infant formula. Even at low water activity C. sakazakii can survive for a long time; it is capable of protective biofilm formation and occasionally shows high virulence and pathogenicity even following stressful environmental conditions. Hence it is a challenging task for the food industry to control contamination of food ingredients and products through the entire production chain, since an increasing number of severe food-related outbreaks of C. sakazakii infections has been observed. The seemingly great capability of C. sakazakii to survive even strict countermeasures combined with its prevalence in many food ingredients requires a greater in depth understanding of its virulence factors to master the food safety issues related to this organism. In this context, we present the whole genome sequence (WGS) of two different C. sakazakii isolated from skimmed milk powder (C7) and ready-to-eat salad mix (C8), respectively. These are compared to other, already sequenced, C. sakazakii genomes. Sequencing of the fusA allele revealed that both isolates were C. sakazakii. We investigated the molecular characteristics of both isolates relevant for genes associated with pathogenesis and virulence factors, resistance to stressful environmental conditions (e.g., osmotic and heat), survival in desiccation as well as conducted a comparative genomic analysis. By using multi-locus sequence typing (MLST), the genetic type of both isolates is assessed and the number of unique genes is determined. DNA of C. sakazakii C8 is shown to hold a novel and unique sequence type; the number of unique genes identified in the genomic sequence of C. sakazakii C7 and C8 were 109 and 188, respectively. Some of the determined unique genes such as the rhs and VgrG genes are linked to the Type VI Secretion System cluster, which is associated with pathogenicity and virulence factors. Moreover, seven genes encoding for multi-drug resistance were found in both isolates. The finding of a number of genes linked to producing capsules and biofilm are likely related to the observed resistance to desiccation.
Highlights
Cronobacter sakazakii is regarded as an opportunistic foodborne pathogen, which can cause meningitis, bacteremia and necrotizing enterocolitis, in infants (Drudy et al, 2006; Aly et al, 2018)
whole genome sequence (WGS) offers a more detailed resolution of how closely bacterial isolates are related than Pulsed-field gel electrophoresis (PFGE) does; it provides insights concerning the multi-locus sequence typing (MLST), pathogenicity genes, as well as a complete molecular characterization of strains (Kozyreva et al, 2016; Forsythe, 2018)
We focus our study on parts of the genome that support our understanding of previous observations of pathogenicity and long-term persistence of C. sakazakii
Summary
Cronobacter sakazakii is regarded as an opportunistic foodborne pathogen, which can cause meningitis, bacteremia and necrotizing enterocolitis, in infants (Drudy et al, 2006; Aly et al, 2018). It is known that after humans are infected by C. sakazakii, it can infect intestinal epithelial cells; from there, it invades the brain to infect human brain microvascular endothelial cells (HBMEC), confirming a route by which to cause meningitis (Giri et al, 2012) In this context, the outer membrane proteins A (OmpA) and X (OmpX) play important roles to explain how Cronobacter efficiently can adhere to and invade HBMEC, Caco-2, and INT-407 cells (Townsend et al, 2008; Kim et al, 2010; Holý et al, 2019). WGS offers a more detailed resolution of how closely bacterial isolates are related than PFGE does; it provides insights concerning the MLST, pathogenicity genes, as well as a complete molecular characterization of strains (Kozyreva et al, 2016; Forsythe, 2018). We investigate and characterize some of the unique genes detected and associated with virulence and pathogenicity mechanisms, resistance to stressful conditions, biofilm formation, and multidrug-resistance
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