The Cronobacter genus is an emergent group of bacterial pathogens in the Enterobacteriaceae family. The majority of infections (bacteraemia, and urinary tract infections) are in the adult population, however the most publicized cases are severe, and frequently fatal infections in neonates and infants [1, 2]. In these highly vulnerable populations, the organism is associated with necrotizing enterocolitis and a highly destructive form of meningitis in which the bacterium crosses the blood–brain barrier and causes abscess formation in the brain cavity [3, 4]. The genus is composed of seven species, and multilocus sequence typing has been used to describe the diversity of the genus [5, 6]. Evolutionary analysis suggests that the C. sakazakii species separated from the rest of the Cronobacter genus 15–23 million years ago (MYA) . Recent whole genome studies have revealed that C. sakazakii is the only Cronobacter species that has the nanAKT gene cluster encoding for sialic acid utilization [7, 8]. Since sialic acid is found in breast milk, infant formula, mucin lining the intestinal tract and gangliosides in the brain , it is plausible that this metabolism may account for the predominance of C. sakazakii in neonatal and infant infections. However no laboratory studies investigating this trait have been undertaken to date.
Sialic acid can exist in nearly 50 different forms with the most studied being 2-keto-3-deoxy-5-acetamido-D-glycero-D-galacto-nonulosonic acid, often abbreviated as Neu5Ac. This sialic acid is generally found bound to sugars to form polysaccharides, and also can be bound to lipids or proteins to form sialo-glycoconjugates. With few exceptions, sialic acid conjugates are notably absent from many eukaryotic lineages, including most protostomes, plants, fungi, and protists. It is postulated that sialic acid synthesis evolved in animals and later emerged in bacterial pathogens and commensals either by convergent evolution or horizontal gene transfer. A number of microbial strategies have evolved which target host sialic acids for adherence, mimicry, and degradation [10, 11]. Some bacteria can produce sialidase (or neuraminidase), encoded by nanH, to cleave sialic acid from the glycoconjugate forms. This gene has low homology (<30%) across bacterial groups, and has not been described in many organisms [12, 13]. Although neonatal meningitic E. coli K1 is able to grow on sialic acid, it lacks the enzyme sialidase. However it is possibly able to obtain sufficient sialic acid from the activities of other sialidase-producing bacteria in the environment or from the host cells expressing the enzyme in conditions of inflammation [10, 13].
The uptake of sialic acid through the outer cell membrane of Gram negative bacteria is by an outer membrane porin, NanC. There are three different types of transporters for the inner membrane: NanT, a major facilitator superfamily (MFS) protein; TRAP, a tripartite ATP-independent periplasmic transport system; and an ATP-binding cassette (ABC) transporter. All members of the Enterobacteriaceae studied to date have shown the presence of the single-component NanT transport system [10, 11, 14]. Once transported into the cell, the Neu5Ac lyase (NanA) converts sialic acid (Neu5Ac) into N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP). NanK is an ATP-dependent kinase specific for ManNAc generating N-acetylmannosamine-6-phosphate (ManNAc-6-P). ManNAc-6-P epimerase (NanE) then converts the ManNAc-6-P into N-acetylglucosamine-6-phosphate (GlcNAc-6-P). GlcNAc-6-P deacetylase (NagA) and glucosamine-6-P deaminase (NagB) convert GlcNAc-6-P into fructose-6-phosphate, which is a substrate in the glycolytic pathway. NanR is the repressor that regulates the activity of these genes. The genes for the first three enzymes (nanA, nanK and nanE) are usually found clustered together forming the nan gene cluster . However, there have been a few exceptions such as Citrobacter freundii and Edwardsiella tarda where the nanE gene is located in a separate region from the rest of the operon . The genes encoding NagA and NagB are located adjacent to each other, but most often not necessarily in the vicinity of the nan gene cluster on the bacterial genomes [12–14]. The genes within the cluster show independent evolutionary histories. Several horizontal gene transfer events are noted in the phylogenetic trees for all three proteins. Most significantly, the NanA protein shows several possible horizontal gene transfer events between the Eukaryotes and Prokaryotes. Two examples are the clustering of Trichomonas vaginalis NanA protein sequences with Pasteurellaceae, and the Bacteroides, Yersinia and Vibrio branching closely with the Eukarya kingdom [11, 14].
The uptake of sialic acid into bacterial cells has been associated with a number of virulence factors. The bacterial glycolipid capsule is an example of molecular mimicry of the host as it aids the organism to overcome the immune responses of the host. Neonatal meningitic E. coli K1 uses sialic acid to decorate the cell surface, and Cronobacter does produce capsular material, especially when grown on milk . Sun et al.  reported the O-PS gene for C. turicensis G3882 included N-acetylneuraminic acid synthetase and CMP-N-acetylneuraminic acid synthetase. However the accurate identification of this strain is uncertain as the 16S rRNA gene sequence (Accession no. HQ880409) does not match other strains of C. turicensis.
As previously reported for the genome sequenced strain C. sakazakii BAA-894, the gene cluster encoding for a putative sugar isomerase (YhcH) and the nanKTAR genes (encoding N-acetylneuraminate and N-acetylmannosamine degradation) are located at ESA_03609-13 [7, 8]. However, the remaining nan genes for sialic acid metabolism have not been described in detail, and these previous bioinformatic studies did not identify any candidate sialidase (NanH) genes .
C. sakazakii is associated with infections of low birth weight neonates, and this could be linked to a number of opportunities for the organism to utilize sialic acid for growth. Human milk contains sialic acid in the form of sialyloligosaccharides which are highest in colostrum and decreases by nearly 80% over the following 3 months after birth [9, 17]. Human milk from mothers of preterm infants contains 13–23% more sialic acid than milk from mothers of full-term infants at 3 of the 4 lactation stages. Similarly infant formulas contain sialic acid which may be bound to glycoproteins. Although the nutritional significance of sialic acid is unknown, it is plausible that it contributes to sialic acid accumulation in the brain. Breast milk also contains a variety of sialoglycans; secretory IgA, lactoferrin, and oligosaccharides. Human milk oligosaccharides are poorly digested and are substrates for intestinal bacterial metabolism, promoting bacterial growth in the intestinal tract. As a site of bacterial attachment, the intestinal microvilli of neonates have increased sialic acid and N-acetylglucosamine residues whereas adults have increased mannose, glucose, and fucose residues [17, 18]. Finally, the brain is the major site of sialic acid display in the form of gangliosides (sialylated glycolipids) and sialic acid may have a role in the structural and functional establishment of synaptic pathways . It is possible that, like Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae which cause bacterial meningitis in children <5 years, that C. sakazakii has a developmental dependence on access to the central nervous system.
Given the common clinical association of C. sakazakii infection of neonates with necrotising enterocolitis and brain damage, an improved understanding of sialic acid utilisation by the organism was warranted. This paper describes the plausible link between the recent evolution of sialic acid metabolism by C. sakazakii and its pathogenicity as well as re-investigates the possible presence of sialidase activity.