Characterization of Putative Virulence Genes on the Related RepFIB Plasmids Harbored by Cronobacter spp

Abstract

Cronobacter spp. are emerging neonatal pathogens that cause meningitis, sepsis, and necrotizing enterocolitis. The genus Chronobacter consists of six species: C. sakazakii, C. malonaticus, C. muytjensii, C. turicensis, C. dublinensis, and Cronobacter genomospecies group 1. Whole-genome sequencing of C. sakazakii BAA-894 and C. turicensis z3032 revealed that they harbor similarly sized plasmids identified as pESA3 (131 kb) and pCTU1 (138 kb), respectively. In silico analysis showed that both plasmids encode a single RepFIB-like origin of replication gene, repA, as well as two iron acquisition systems (eitCBAD and iucABCD/iutA). In a chrome azurol S agar diffusion assay, it was demonstrated that siderophore activity was associated with the presence of pESA3 or pCTU1. Additionally, pESA3 contains a cpa (Cronobacter plasminogen activator) gene and a 17-kb type 6 secretion system (T6SS) locus, while pCTU1 contains a 27-kb region encoding a filamentous hemagglutinin gene (fhaB), its specifc transporter gene (fhaC), and associated putative adhesins (FHA locus), suggesting that these are virulence plasmids. In a repA-targeted PCR assay, 97% of 229 Cronobacter species isolates were found to possess a homologous RepFIB plasmid. All repA PCR-positive strains were also positive for the eitCBAD and iucABCD/iutA iron acquisition systems. However, the presence of cpa, T6SS, and FHA loci depended on species, demonstrating a strong correlation with the presence of virulence traits, plasmid type, and species. These results support the hypothesis that these plasmids have evolved from a single archetypical plasmid backbone through the cointegration, or deletion, of specific virulence traits in each species. Farmer et al. (20) established the taxonomic position of Enterobacter sakazakii, which was previously identified as yellow-pigmented Enterobacter cloacae based on DNA-DNA hybridization studies in combination with phenotypic

Cronobacter spp. – opportunistic food-borne pathogens. A review of their virulence and environmental-adaptive traits

“…Secretion systems, referred to as type IV secretion systems, that can transfer proteins and nucleoprotein complexes are considered to be important virulence factors as they were identified in C. sakazakii and C. turicensis as a plasmid-borne (pESA2/pCTU2) gene cluster (Franco et al, 2011b). In contrast, several newly identified type VI secretion systems (T6SSs) were found in the chromosomes of most Cronobacter spp.…”

“…The ability to acquire iron is crucial for most pathogens in establishing infection after entering a host cell. Franco et al (2011b) showed that the iucABCD/iutA siderophore (cronobactin) is the only functional siderophore possessed by Cronobacter. However, Grim et al (2013) demonstrated that cronobactin -a hydroxamate-type, aerobactin-like siderophore -was not the only iron acquisition system possessed among Cronobacter.…”

Insights into virulence factors determining the pathogenicity ofCronobacter sakazakii

“…This protease has an ability to render serum resistance by cleaving complement components, activating plasminogen and inactivating the plasmin inhibitor a2-AP. 76 Franco et al and Cruz et al also portray the prevalence and distribution of plasmid-encoded virulence genes cpa, a type 6 secretion system (T6SS, also encoded on pESA3) and a filamentous haemaggltunin/adhesion (FHA) gene locus (located on pCTU1) among 231 Cronobacter strains. 77,78 Sialic acid utilization Sialic acid is found in human milk and in infant formulae in the form of sialyloligosaccharides.…”

Plasmid-Encoded Iron Uptake Systems

“…They could constitute a virulence factor-replication unit that ensures perpetuation of the aerobactin genes even in the event of a deletion (37). The aerobactin system was also found in close proximity to the repFIB replicon in Chronobacter virulence plasmid and in a plasmid isolated from a sewage treatment plant (64)(65)(66).…”

“…The shiF gene has been found to be conserved upstream of the aerobactin cluster, in some cases in association with another gene, viuB (51,(64)(65)(66). Based on their homology, the products of these two genes have been proposed to be part of the aerobactin iron uptake system (64)(65)(66)93).…”