Cronobacter sakazakii: stress survival and virulence potential in an opportunistic foodborne pathogen

Abstract

A characteristic feature of the opportunistic foodborne pathogen Cronobacter sakazakii is its ability to survive in extremely arid environments, such as powdered infant formula, making it a dangerous opportunistic pathogen of individuals of all age groups, especially infants and neonates. Herein, we provide a brief overview of the pathogen; clinical manifestations, environmental reservoirs and our current understanding of stress response mechanisms and virulence factors which allow it to cause disease.

Keywords:

• Cronobacter sakazakii
• virulence
• stress
• osmotolerance
• reservoirs

The Genus Cronobacter

Cronobacter spp, previously known as Enterobacter sakazakii¹ are Gram-negative, rod-shaped, motile, facultative anaerobic bacteria.² The Cronobacter genus is genetically closely related to the Citrobacter (97.8%) and Enterobacter genera (97.0%)³ and until recently consisted of 10 species^2,4,5: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinesis, Cronobacter condiment, Cronobacter universalis, Cronobacter pulveris, Cronobacter helveticus, Cronobacter zurichensis. Three of which; Cronobacter sakazakii, Cronobacter malonaticus and Cronobacter turicensis, have been linked to neonatal infections.^6,7 However, recent studies proposed that Cronobacter species C. helveticus, C. pulveris and C zurichensis form their own sub-clades, genetically distinct from Cronobacter.⁸ The prototypical member of the Cronobacter family, C. sakazakii, is an opportunistic foodborne pathogen that has been linked with life-threatening conditions in neonates, immunocompromised infants, children (from 3 months to 4 y old) and elderly adults.^9-11

Clinical manifestations of C. sakazakii

While Cronobacter sakazakii is primarily a foodborne pathogen, its association with severe neonatal infections has significantly increased awareness and triggered a surge of interest in recent years.¹² C. sakazakii can cause bacteraemia and sepsis, cerebro-spinal and peritoneal fluid accumulation, brain abscesses, cyst formation, necrotizing enterocolitis (NEC), meningitis and intracerebral infarctions.^13,14 NEC follows colonisation of the intestine by C. sakazakii, with the incidence increasing in low birth weight or premature neonates.¹⁵ NEC is characterized by inflammation and death of intestinal tissue and is one of the most common gastrointestinal conditions that can arise in neonates.¹⁶ Indeed, this gastrointestinal pathogen is responsible for up to 80% of infant deaths associated with infection.¹¹ Infants that survive C. sakazakii infection often suffer delayed neurological symptoms, e.g., delayed brain development, brain abscesses or hydrocephalus.^17,18 Consequently, The International Commission on Microbiological Specification for Foods¹⁹ has classified C. sakazakii as a ‘severe hazard for restricted populations, life-threatening or with substantial chronic sequelae over long duration’. Furthermore, the World Health Organization and Food and Agriculture Organization in 2008 issued a joint call to the scientific community for more data on this organism (WHO/FAO).

Morbidity and mortality as a result of C. sakazakii infection are largely dependent on the immune status of the host. Neonates and infants, up to the age of 12 months, have an immunodeficiency as a result of an immature immune system that can be worsened by premature or traumatic delivery, maternal disease or certain drugs.²⁰ Furthermore, a innate immunity in the form of their competing intestinal microflora is not yet fully established. One of the most severe cases of C. sakazakii infections recorded to date took place in France in 1994 in a Neonatal Intensive Care Unit (NICU). 17 new-born children were infected, of which 7 resulted in necrotizing enterocolitis, 1 case of septicemia and 1 case of meningitis. No clinical symptoms were identified in 8 infants that were infected and colonized, yet 3 neonatal deaths occurred as a result of the infection. The cause of the outbreak was traced to the feeding practices employed in the NICU.²¹ This study found the same Cronobacter strains in the intestinal tracts of healthy infants, as those presenting with symptoms.²¹ Therefore, suggesting that the host immune response is a major factor in contributing to C. sakazakii associated disease. A further study which involved collecting faecal samples from both healthy neonates and adult carriers of C. sakazakii, found 3 C. sakazakii isolates in neonate faecal samples, whereas the adults’ faecal samples contained only one C. sakazakii strain and one C. malonaticus strain.²² This suggests that C. sakazakii might be more likely to colonize neonatal intestinal tracts, most likely as a consequence of their comparatively naive immune systems. C. malonaticus on the other hand, was only found in adults suggesting that this species is associated more with adult than neonatal infection.²²

Antibiotic Resistance and Alternative Means of Control

Originally C. sakazakii has been reported to be susceptible to a wide range of antibiotics including β-lactams,²³ however, several new strains have been described that were found to be resistant to tetracycline,²⁴ neomycin and trimethoprim²⁵ and cephalotin.²⁶ Furthermore, recent studies have described a strain carrying an unusual ampC gene, conferring resistance to cephalosporine²⁷ and found environmental isolates of C. sakazakii from domestic kitchens exhibiting resistance to penicillin, tetracycline, ciprofloxacin and nalidixic acid.²⁸ A comprehensive analysis by next generation whole genome sequencing and annotation of the C. sakazakii strain SP291 indicates that C. sakazakii possesses a substantial number of genes associated with antibiotic resistance, including ampC (cephalosporine), fosA (fosfomycine), gyrA, gyrB, parC and parB conferring resistance to fluoroquinolones and encoding a multitude of multi-drug resistance mechanisms, mainly drug efflux pumps.²⁹ Increasing drug resistance in bacteria is a general problem and consequently the need to identify alternative means to control bacterial infections is pressing.³⁰ The need for new therapeutics and disinfectants has re-kindled interest in bacteriophages as a method of controlling bacterial growth.^31,32 In this context C. sakazakii infecting bacteriophages have been isolated, including the second largest known bacteriophage; phage GAP32.³³ The applicability of bacteriophages to control C. sakazakii infections in various systems has been the subject of several studies. Two studies in particular have characterized the ability of bacteriophages to reduce and control growth of C. sakazakii in PIF.^34,35 However, the application of bacteriophages is not limited to environmental control but can also be used in vivo, as has been demonstrated in insect and mouse infection models, where bacteriophages were shown to protect insect larvae³⁶ or reduce bacterial load in urinary tract infections in mice.³⁷ Bacteriophages have furthermore been successfully used as immuno-stimulants to increase expression of pro-inflammatory host factors.³⁸

Environmental reservoirs

C. sakazakii is a ubiquitous bacterium found in a wide variety of reservoirs in the environment; from flies³⁹ to plants such as wheat, rice, herbs and spices⁴⁰ or meat and other common household foods.⁴¹ The wide variety of environments in which C. sakazakii is present, indicates that it has developed various traits that increase survival in difficult conditions, e.g., resistance to UV irradiation,⁴² the ability to adhere to various surfaces due to fimbriae formation,⁴² biofilm formation⁴³ and the ability to survive desiccation.^7,9,44 The ability of C. sakazakii to survive in low water environments is a unique stress survival strategy which is linked with survival and persistence of the pathogen in powdered infant formula (PIF), subject to an a_w of ∼0.2 (inhospitable to most micro-organisms). A 2004 study found that 8 out of 9 PIF factories investigated were contaminated with C. sakazakii.⁴⁵ The same study also identified C. sakazakii in households, where 5 isolates were found in 16 family homes. It is likely that contamination of PIF with C. sakazakii occurs during the manufacturing process. While C. sakazakii may not survive pasteurization it is likely that the PIF becomes contaminated after pasteurisation during the processing and addition of non-sterile ingredients to the PIF.^9,40,46 Furthermore, several reports have shown that C. sakazakii contamination can occur during the reconstitution of formula, both at home and in hospitals, and is therefore not limited to the production stages.⁴⁷ In an effort to prevent contamination at this stage the WHO/FAO issued guidelines to hospitals for the reconstitution and storage of PIF (WHO/FAO).

C. sakazakii osmotolerance

An improved understanding of C. sakazakii osmotolerance will provide valuable insights into the mechanisms which aid the survival of this neonatal pathogen as it transits the gastrointestinal tract, where it is subject to varying osmolality,⁴⁸ in addition to its survival in a dry environment such as PIF. Desiccation is an extreme form of osmotic stress⁴⁹ and the upregulation of osmotolerance genes is the first line of defense against drying for a bacterial cell.⁴⁴ It is also worth noting that while microorganisms acquire a state of tolerance to one type of stress, this state of acquired tolerance will also add resistance to other types of stress.^50-52 Trollmo et al demonstrated that Saccharomyces cerevisiae cells conditioned to osmotic dehydration were equally as thermotolerant as heat conditioned cells, however heat conditioned cells were not osmotolerant.⁵¹ More recently Hoffman et al demonstrated that a single point mutation in the Listeria monocytogenes betL gene leads to improved osmotolerance and chill tolerance in this foodborne pathogen.^52,53 Indeed, this link between osmotolerance and other environmental stress resistance mechanisms highlights the multiple benefits of an improved understanding of C. sakazakii osmotolerance in addition to providing an insight into the mechanisms which facilitate its survival in PIF.

While few studies have focused on C. sakazakii osmotolerance, the ability of this organism to survive in such an extreme hyperosmotic environment is a unique adaptation which is of fundamental importance to its pathogenic potential.⁵⁴ Most microorganisms require an a_w significantly higher than that of PIF for growth and survival, suggesting that C. sakazakii is armed with a vast array of osmotic stress survival mechanisms.⁵⁴ However, the growth phase of the organism has also been demonstrated to play a role in C. sakazakii osmotolerance, with stationary phase cells demonstrating a higher resistance to osmotic and dry stress in comparison to other known osmotolerant organisms such as E. coli and Salmonella.⁴⁴ Further elucidation of C. sakazakii osmotolerance is aided by the wealth of knowledge available on how other bacterial cells overcome hyperosmotic stress and it is believed that C. sakazakii responds in a similar way.^55-57

Bacterial cells in general need to maintain an intracellular osmotic pressure greater than that of the surrounding media in order to generate cell turgor and prevent plasmolysis and ultimately cell death.⁵⁸ Most bacteria survive osmotic stress through a biphasic response, which first involves the accumulation of potassium and its counter ion glutamate; representing the primary response.⁵⁵ However, given that high levels of potassium are detrimental to the normal functioning of the cell, a secondary response is triggered which stimulates the synthesis and/or uptake of osmoprotective compounds called compatible solutes,⁵⁵ so called because they are compatible with cellular physiology at high internal concentrations.^59-61 Compatible solutes are generally soluble molecules with no net charge at physiological pH and do not interact with proteins and other macromolecules, thus they do not disrupt vital cellular processes such as DNA repair, DNA-protein interactions or the cellular metabolic machinery.⁵⁵

There are a large variety of compatible solutes available in the environment which can be acquired by microorganisms under high osmotic stress. Most microorganisms harbour multiple osmoregulatory transporters with overlapping substrate specificity which allow them to overcome changes in the osmolality of the surrounding media.⁶² One of the most extensively studied Gram-positive foodborne pathogens in this respect is L. monocytogenes.⁵⁷ This pathogen uses the compatible solutes glycine betaine,⁶³ proline⁶⁴ and carnitine⁶⁵ to counteract the cytotoxic effects of elevated osmolality. Indeed, Gram-negative bacteria share similar preferences and mechanisms of compatible solute accumulation as those seen in Gram-positives. The compatible solute trehalose was previously demonstrated to play a role in the desiccation survival of C. sakazakii, illustrated by a more than five-fold increase in the trehalose concentration in dried stationary phase cells.⁶⁶ The mechanism of trehalose mediated osmoprotection is believed to play a significant role in the survival of microorganisms during dry stress as a result of the substitution of the water layer around the biomolecules by trehalose. This enables maintenance of the 3 dimensional structure of essential biological macromolecules; a mechanism referred to as the water replacement theory (Fig. 1).⁶⁷ However, experimental data have demonstrated that this substitution has an effect on the mobility of the macromolecule and further research is needed to determine the mechanism of stabilization by trehalose.⁶⁸ The existence of a trehalose biosynthesis pathway in C. sakazakii is demonstrated by the presence of a putative otsBA operon⁵⁴; suggesting that the mechanism of trehalose biosynthesis in C. sakazakii is similar to that of E. coli. Furthermore, in addition to trehalose, other potentially important C. sakazakii compatible solutes include proline, betaine and ectoine, accumulated via secondary transporters homologous to the ProP porter in E. coli. Indeed, while E. coli possesses only a single ProP homolog, multiple ProP homologues exist in the C. sakazakii genome; a feature which goes some way to explaining the increased osmotolerance of the pathogen in PIF compared to E. coli.⁶⁹

Figure 1. Structure stabilization of enzymes at elevated osmolarity. Preferential exclusion of compatible solutes (blue circles) from the protein surface helps to maintain enzyme structure at elevated osmolarity, while also helping to increase cell volume (adapted from Sleator and Hill ⁵⁵).

While relatively few studies have focused on the osmotolerance capacity of C. sakazakii at the physiological level, our knowledge of C. sakazakii osmotolerance has been significantly expanded by a recent in silico analysis of the C. sakazakii BAA-894 genome; revealing 53 putative osmotolerance loci, including both hypo- and hyper-osmotic stress response systems.⁵⁴ While homologues of all the principal E. coli osmotolerance loci were identified in the BAA-894 genome; multiple copies of certain osmotolerance loci; including 7 copies of the E. coli proP homolog were found. The ProP protein encoded by the gene locus tag ESA_02131 encodes a protein with 90% identity to E. coli ProP.⁵⁴ While the remaining 6 homologues encode proteins exhibiting features of classic secondary transporters, they were all 60–70 amino acids shorter than the E. coli ProP; lacking the extended carboxyl tail.⁵⁴ This structural inconsistency is unique and likely contributes to the increased osmotolerance of this pathogen compared to E. coli.

Gene expression analysis of each proP homolog demonstrated an increase in expression levels in C. sakazakii during an osmotic upshift, indicating that each homolog responds to osmotic stress conditions, at least at the level of transcription.⁶⁹ However, expression levels varied significantly between each of the proP homologues analyzed, with the ProP encoded by gene locus tag ESA_04214, a sequence homologous to the inner membrane transporter YhjE in E.coli, demonstrating the highest level of up-regulation when subject to hyperosmotic stress. Furthermore, functional analysis revealed that each of the 6 ProP homologues analyzed conferred increased osmotolerance when heterologously expressed against an osmotically sensitive E. coli host (E. coli MKH13), albeit to different degrees; suggesting that each of the 7 proteins might be tailored to specific osmotic conditions.

C. sakazakii virulence

While the unique osmotolerance phenotype associated of C. sakazakii likely aids the pathogenicity of the bacterium by providing access to a highly susceptible host via PIF, other factors influence the virulence of C. sakazakii. To cause systemic infection and some of the more severe clinical manifestations of Cronobacter infections, like NEC, sepsis or meningitis, C. sakazakii must either directly infect cells of the inner gut lining or cross this barrier to reach the bloodstream. Indeed, C. sakazakii has been shown to infect mucous membranes, gastric and intestinal epithelial, and endothelial tissues.^22,70,71 As such, the genes encoding C. sakazakii's ability to adhere to and invade cells of the inner gut lining represent major virulence factors.

Crossing the gut barrier–adhesion

The first step in the invasion of the gastrointestinal tract is adherence of bacteria to the surface of these tissues. Studying the invasion of internal epithelial barriers in situ is not trivial and several cell lines e.g., Caco-2 isolated from gut tissue have been established as laboratory models.^72-75 The first study to investigate the adherence of C. sakazakii to Caco-2 human epithelial cell lines involved 50 C. sakazakii strains from different environmental niches. Twenty-eight of these isolates were found to adhere to the surface of Caco-2 cells. Furthermore, this study outlined 3 basic adhesion patterns for C. sakazakii: diffuse adhesion, formation of localized clusters and a mixed phenotype.⁷¹ While the ability of C. sakazakii to adhere to the tested cell models was independent of bacterial fimbrae formation,⁷¹ Hartmann et al. investigated the adherence efficiency of selected mutants of C. sakazakii strain ES5 to Caco-2 cells and found that the absence of flagella reduced the adherence ability of the bacterium,⁴³ demonstrating that flagella are important for this process. Others investigated adherence of clinical isolates from an outbreak in France.¹³ This was the first in vitro study that used clinical samples with associated patient information and therefore could link genotypes to symptoms and severity of the infection. The clinical manifestations associated with these genotypes were necrotising enterocolitis (NEC), bacteriaemia, sepsis and meningitis and the study found a link between the virulence of the strains and their ability to adhere to and invade Caco-2 cells.¹³

To better understand the interactions on the cell surface that lead to invasion of C. sakazakii, significant efforts have been made to identify the host receptors for this pathogen-host interaction. Fibronectin, a glycoprotein which forms part of the extracellular matrix of eukaryotic tissue,⁷⁶ plays a role in host cell adhesion, movement, growth and differentiation, and is one of the primary targets in the adhesion process of several pathogenic organisms.⁷⁷ Studies identified binding of fibronectin as an important step in the adherence of C. sakazakii to the cells of the gastrointestinal tract.⁷¹ Reduction of fibronectin levels has been shown to impair C. sakazakii attachment to INT-407 cells - an in vitro model of embryonic intestinal cells.⁷⁶ It is likely that this is due to interaction between host receptors and adhesins on the surface of the bacterial cells. The C. sakazakii outer membrane protein A (OmpA) was identified as a major fibronectin-binding protein which plays a significant role in the adherence of this gastrointestinal pathogen to neonatal and immunocompromised hosts.^76,78 However, due to the high diversity of the genus, not all Cronobacter spp. possess this ability.

Crossing the gut barrier–invasion

In many pathogenic bacteria, adherence to host tissues is followed by invasion of the tissue, allowing the pathogen to breach this first line of defense. While gastrointestinal invasion is common with other pathogenic bacteria such as Salmonella typhimurium (relative invasion rate of 60% in 60 minutes), only moderate invasion rates have been observed in C. sakazakii (0.2% in 60 min). This is similar to the moderately invasive gastrointestinal pathogen Campylobacter jejuni (0.1%–0.4%).⁷⁹

The mechanism of invasion in C. sakazakii is not yet fully understood, however, several host factors and bacterial membrane proteins seem to be involved in this process.

On the host side, the intercellular tight-junctions of the gut epithelia play a role in preventing molecules and bacteria from bypassing the epithelial cells of healthy individuals. It is therefore not surprising that invasion by C. sakazakii strain ATCC 29544 is greatly improved by the disruption of tight junctions,⁷⁰ which can be caused by lipopolysaccharides (LPS) from Gram-negative bacteria.⁸⁰ Furthermore, epithelium cell tight-junctions and gut microbial flora of neonates are not yet fully developed and therefore would not be as effective in preventing invasion of pathogens such as C. sakazakii from entering the blood stream.⁸¹ Taken together with high levels of LPS found in PIF, and the contamination of PIF with C. sakazakii, this could provide an underlying mechanism for the observed tenfold higher incidence rate of NEC observed in infants fed with PIF compared to breast fed infants.⁸²

Outer membrane protein A (OmpA) of C. sakazakii has been shown to be necessary for invasion of Caco-2 and INT407 cells,^83-85 since the invasiveness of a ΔompA mutant decreased by 80% in Caco-2 and 87% in INT407 cells.⁸⁵ However, Kim and colleagues further demonstrated that another outer membrane protein, OmpX, also plays a major role in invasion of Caco-2 epithelial cells⁸⁵ and has been shown to be necessary for the invasion of Hep-2 cells by Yersinia pestis and C. sakazakii.^86,87 As a consequence, it was observed that ompA/X deletion mutants were significantly less efficient in the dissemination of the bacterium to other organs such as the liver and spleen in vivo.⁸⁵

C. sakazakii is a member of a unique subset of microorganisms that can penetrate the blood-brain barrier. This characteristic is facilitated by the organism's ability to invade human brain microvascular endothelial cells (HBMEC), an essential feature in meningitis causing organisms. Indeed, Townsend et al. demonstrated that invasion of HBMEC by C. sakazakii strains was equivalent or greater than that of the neonatal E. coli meningitis strain K1.¹³ A more recent study observed significant levels of transcytosis across a tight monolayer of HBMEC and included electron-microscopic analysis demonstrating the presence of Cronobacter within host vacuoles of HBMEC, in addition to being trancytosed at the basolateral surface.⁸⁸ While significant strain variation was noted, this study further contributes to our understanding of how this gastrointestinal pathogen penetrates the blood brain barrier (BBB) and ultimately causes meningitis. The previously mentioned OmpA protein, which plays a role in attachment of C. sakazakii to the gastrointestinal tract, has also been implicated in the invasion of HBMEC. Nair et al. demonstrated that a lack of this protein resulted in a reduction of HBMEC invasion by 83%.⁷⁸ While OmpA and OmpX likely play a role in the organism, penetrating the blood-brain barrier, the mechanism which leads to the death of the brain cells is as yet unknown and it is possible that the host's immune response may also play a role.⁶ Furthermore, C. sakazakii possess zpx, a gene which encodes a zinc containing metalloprotease which is responsible for the lysis of collagen and may allow the pathogen to cross the blood brain barrier.⁸⁹ It is important to note however that not all C. sakazakii strains have been shown to cause meningitis. An extensive multilocus sequence typing (MLST) method identified C. sakazakii sequence type 4 as the predominant sequence type found in cerebro-spinal fluid from cases of neonatal meningitis.⁶

Virulence factors beyond invasion – toxins, immune-evasion and immune-toxicity

The first putative virulence factors identified in Cronobacter were enterotoxins produced by 4 of the 18 isolates studied by Pagotto et al.⁹⁰ Purification and characterization identified a 66kDa protein which was most stable at pH 6. The ability of the toxin to withstand high temperatures (90°C for 30mins), coupled with its potent cell toxicity (LD50=56pg), represented a significant virulence factor for this foodbourne pathogen.⁹¹ However, to date there remains a dearth of information available on enterotoxin production by C. sakazakii and its role in pathogenesis as other factors have been studied in more detail.

The ability of some C. sakazakii strains to evade the innate immune response by surviving and even replicating in macrophages,¹⁷ represents another important virulence factor. While the survival of this organism in macrophages varies significantly between strains, the presence of putative sod genes, encoding a superoxide dismutase, is believed to aid survival in phagosomes, where the bacteria are subjected to acidic conditions and macrophage oxidase activity.¹⁷ Furthermore, C. sakazakii has been found to persist in macrophages for up to 48 hours, suggesting that this organism is well equipped to survive within this extreme environment¹³; a stress survival strategy possibly aided by compatible solute accumulation.⁵⁵

It has been demonstrated more recently that the flagella of C. sakazakii ST1 and ST4 strains stimulate the activation of pro- and anti-inflammatory cytokines in human monocytes, such as TNF, IL8 and IL10, and this response is dependent on TLR5 recognition.⁹² The role of these cytokines in the pathogenicity of C. sakazakii is not clear and as such further research is needed.

While the C. sakazakii genome encodes several virulence factors, the host also contributes to its pathogenicity through immune-related cell toxicity. Characterization of OmpA showed that disruption of the epithelium triggers release of various pro and anti-inflammatory chemokines, cytokines such as transforming growth factor β (TGF-β) and Nitric Oxide (NO). TGF-β is essential for proper immune functions, such as growth, repair and development, and is produced in high amounts by dendritic cells leading to tight junction disruption and cell death in the presence of the pathogen. Nitric Oxide has been associated with NEC as a result of intestinal barrier failure, caused by high localized concentrations of NO that trigger cell apoptosis or necrosis.⁸³

Conclusion and Future Prospects

The severity of disease coupled with a high mortality rate (up to 80%) in C. sakazakii infected infants and neonates, highlights the need for further research into the virulence and environmental stress survival mechanisms of this gastrointestinal pathogen. Since the reclassification of Enterobacter sakazakii in 2007 there has been a surge in research aimed at elucidating the virulence factors of C. sakazakii, however our understanding of the mechanisms of its pathogenesis is far from complete. In particular the identification of surface proteins on C. sakazakii which facilitate adherence and invasion of the neonatal host tissue has made a significant contribution to the field. OmpA, a fibronectin binding protein, has been identified as playing a major role in the invasion of host cells of the gastrointestinal tract and is also implicated in facilitating the invasion. However, further elucidation of the structure and mechanism of OmpA and other proteins involved in attachment to host cells would significantly contribute to our understanding of the virulence of this organism and may indeed aid the development of adequate control measures. Figure 2 gives an overview of the discussed mechanisms, virulence factors and clinical manifestations of C. sakazakii infections.

Figure 2. Schematic representation of the different stages and organs/tissues involved in a C. sakazakii infection. The different stages are represented by colored boxes, with important factors and consequences associated with these stages indicated. Main clinical manifestations (e.g., NEC) linked to C. sakazakii infection in different organs/tissues are shown.

The progress made in C. sakazakii virulence and osmotic stress survival research was largely aided by genome sequence analysis. To date 16 C. sakazakii draft genomes are available, however only 4 of these are gap-less chromosomal sequences. It is certainly important to note that increasing the number of available whole genome sequences would significantly benefit C. sakazakii research in general, and in particular would facilitate the comparative analysis of closely related strains. Previous taxonomic revision of Cronobacter was based on DNA hybridization, 16s rDNA sequence analysis and biotyping. However, with the increasing number of available C. sakazakii genome sequences, MLST is the most favorable typing method to date due to its higher resolution and promising clinical significance.⁹³ MLST data has demonstrated the C. sakazakii sequence type (ST) 4 is the sequence type implicated most often in cases of neonatal meningitis.⁹⁴ Indeed, 75% of ST4 isolates correlated with cases of meningitis over a 50 y period in a study spanning 6 countries.^12,94 Improved availability of genomic sequence information would therefore be beneficial to the future of C. sakazakii research and will undoubtedly help to direct future studies toward the most pathogenic sequence types.

While sequence analysis studies are improving our knowledge of C. sakazakii virulence and osmotolerance, “wet lab” information available on C. sakazakii osmotolerance is still very limited. The osmotic stress survival mechanisms of C. sakazakii represent a fundamental survival strategy; enabeling the pathogen to survive in environments of low a_w such as PIF, thus an improved knowledge of these mechanisms would have a significant impact on the manufacture of PIF and other dried food products. Furthermore, identification of the compatible solute uptake and synthesis systems utilized by pathogenic C. sakazakii strains may identify traits unique to pathogenic isolates, e.g., ST4, so that targeted inhibition analysis can be carried out. An improved understanding of the structures of the compatible solute transporters could provide valuable insights for the development of control measures using either small molecule research (known as smuggling technology), or protein-protein interactions which mimic transporter-compatible solute interactions but do not facilitate the uptake of compatible solutes and therefore do not have an osmoprotective effect. Such control measures may significantly reduce or prevent survival of C. sakazakii in low a_w environments and would therefore significantly improve the safety of dry foods such as PIF.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

RDS is Coordinator of the EU FP7 ClouDx-i project which funds KK. AF is funded by an IRCSET EMBARK Postgraduate Scholarship RS/2010/2300. RO’C was funded by the Biological Sciences Department at CIT.