Chemotaxis and Shorter O-Antigen Chain Length Contribute to the Strong Desiccation Tolerance of a Food-Isolated Cronobacter sakazakii Strain

Cronobacter sakazakii is an opportunistic pathogen causing a lethality rate as high as 80% in infants. Desiccation tolerance ensures its survival in powdered infant formula (PIF) and contributes to the increased exposure to neonates, resulting in neonatal meningitis, septicemia, and necrotizing enterocolitis. This study showed that a food-isolated C. sakazakii G4023 strain exhibited a stronger desiccation tolerance than C. sakazakii ATCC 29544 strain. Considering the proven pathogenicity of G4023, it could be a big threat to infants. Transcriptome and proteome were performed to provide new insights into the desiccation adaptation mechanisms of G4023. Integrated analyses of these omics suggested that 331 genes were found regulated at both transcriptional and protein levels (≥2.0- and ≥1.5-fold, respectively). Deletion of chemotaxis system encoded genes cheA and cheW resulted in decreased tolerance in both short- and long-term desiccation. Reduced O-antigen chain length contributed to the biofilm formation and desiccation tolerance in the short term rather than the long term. In addition, biosynthesis of flagella, arginine and its transport system, and Fe/S cluster were also observed regulated in desiccated G4023. A better understanding of desiccation adaptation mechanisms of G4023 could in turn guide the operations during production and preservation of PIF or other food to reduce survival odds of G4023 and lower its exposure to get to infants.

Current and Future Perspectives on the Role of Probiotics, Prebiotics, and Synbiotics in Controlling Pathogenic Cronobacter Spp. in Infants

Cronobacter species, in particular C. sakazakii, is an opportunistic bacterial pathogen implicated in the development of potentially debilitating illnesses in infants (<12months old). The combination of a poorly developed immune system and gut microbiota put infants at a higher risk of infection compared to other age groups. Probiotics and prebiotics are incorporated in powdered infant formula and, in addition to strengthening gut physiology and stimulating the growth of commensal gut microbiota, have proven antimicrobial capabilities. Postbiotics in the cell-free supernatant of a microbial culture are derived from probiotics and can also exert health benefits. Synbiotics, a mixture of probiotics and prebiotics, may provide further advantages as probiotics and gut commensals degrade prebiotics into short-chain fatty acids that can provide benefits to the host. Cell-culture and animal models have been widely used to study foodborne pathogens, but sophisticated gut models have been recently developed to better mimic the gut conditions, thus giving a more accurate representation of how various treatments can affect the survival and pathogenicity of foodborne pathogens. This review aims to summarize the current understanding on the connection between Cronobacter infections and infants, as well as highlight the potential efficacy of probiotics, prebiotics, and synbiotics in reducing invasive Cronobacter infections during early infancy.

Alterations in the transcriptional landscape allows differential desiccation tolerance in different strains of Cronobacter sakazakii

Cronobacter sakazakii is a typical example of a xerotolerant bacterium. It is epidemiologically linked to low moisture foods like powdered infant formula (PIF) and is associated with high fatality rates among neonates. We characterized the xerotolerance in a clinically isolated strain, C. sakazakii ATCC™29544 T , and compared the desiccation tolerance with an environmental strain, C. sakazakii SP291, whose desiccation tolerance was previously characterized. We found that, although the clinical strain was desiccation-tolerant, the level of tolerance was compromised when compared to the environmental strain. RNA-seq based deep transcriptomic characterization identified a unique transcriptional profile in the clinical strain compared to what was already known for the environmental strain. As RNA-seq was also carried out in different TSB growth conditions, genes that were expressed specifically under desiccated conditions were identified and denoted as desiccation responsive genes (DRGs). Interestingly, these DRGs included transcriptomic factors like fnr , ramA, and genes associated with inositol metabolism, a phenotype as yet unreported in C. sakazakii . Further, the clinical strain did not express the proP gene, which was previously reported to be very important for desiccation survival and persistence. Interestingly, analysis of the plasmid genes showed that the iron metabolism in desiccated C. sakazakii ATCC™29544 T cells specifically involved the siderophore cronobactin encoded by the iucABCD genes. Confirmatory studies using qRT-PCR determined that, though the secondary desiccation response genes were upregulated in C. sakazakii ATCC™29544 T , the level of up-regulation was lower compared to that in C. sakazakii SP291. All these factors could collectively contribute to the compromised desiccation tolerance in the clinical strain. IMPORTANCE Cronobacter sakazakii has in past led to outbreaks, particularly associated with food that are low in moisture content. This species has adapted to survive in low water conditions and can survive in such environments for long periods. These characteristics have enabled the pathogen to contaminate powder infant formula, a food matrix with which the pathogen has been epidemiologically associated. Even though clinically adapted strains can also be isolated, there is no information on how the clinical strains adapt to low moisture environments. Our research assessed the adaptation of a clinically isolated strain to low moisture survival on sterile stainless steel coupons and compared the survival to a highly desiccation-tolerant environmental strain. We found that, even though the clinical strain is desiccation-tolerant, the rate of tolerance was compromised compared to the environmental strain. A deeper investigation using RNA-seq identified that the clinical strain used pathways different from that of the environmental strain to adapt to low moisture conditions. This shows that the adaptation to desiccation conditions, at least for C. sakazakii , is strain-specific and that different strains have used different evolutionary strategies for adaptation.

Infection of Cronobacter sakazakii ST1 Producing SHV-12 in a Premature Infant Born from Triplet Pregnancy

Cronobacter sakazakii can cause severe life-threatening invasive infections in neonates, with a high mortality rate mostly associated with powdered infant formula consumption. The study describes a fatal C. sakazakii infection in premature infant fed only with expressed human milk. Despite the identification of etiological factor from patient’s blood, the epidemiological investigation, including mother’s skin, hospital surfaces, milk expressing devices, and milk samples, did not show bacterial contamination. The infection was caused by C. sakazakii ST1, being one of the leading genotypes reported in invasive infections. The phylogenetic analysis of the international collection of the ST1 organisms allowed us to identify the isolate as a member of the main cluster. The pathogenic potential of the isolate was augmented by the presence of IncFIB-like molecule representing virulence plasmids of pESA-3 family. Isolate presented ESBL phenotype associated with blaSHV-12 gene harboured by IncX3 plasmid. The described case gave valuable information to genetics of Cronobacter, and also urges the need of wider whole-genome sequencing implementation as a part of diagnostic procedure.

Genomic Landscape and Phenotypic Assessment of Cronobacter sakazakii Isolated From Raw Material, Environment, and Production Facilities in Powdered Infant Formula Factories in China

Cronobacter is a foodborne pathogen associated with severe infections and high mortality in neonates. The bacterium may also cause gastroenteritis, septicemia, and urinary tract and wound infectious in adults. A total of 15 Cronobacter isolates collected from 617 raw materials and environment samples from Powdered Infant Formula manufacturing factories during 2016 in Shaanxi, China, were analyzed for antimicrobial susceptibilities, species identification, biofilm formation, and whole-genome sequencing. The results showed that all 15 isolates were Cronobacter sakazakii, while the antimicrobial susceptibility test showed that all 15 C. sakazakii were pan susceptible. Most isolates were able to produce a weak biofilm, and two isolates from soil samples produced a strong biofilm formation. All isolates were classified into seven STs including ST4, ST40, ST64, ST93, ST148, ST256, and ST494, with ST64 (4/15, 26.7%) being dominant, and most were clinically related. The isolates harbored at least 11 virulence genes and two plasmids, with one isolate being positive for all virulence genes. Phylogenetic and ANI analysis showed strong clustering by sequence types and isolates from different sources or regions with a similar genomic background. The fact that isolates were obtained from raw materials and environment samples of PIF facilities shared a close phylogeny with one another suggests that cross-contamination events may have occurred between the processing room and external environments, which may give rise to a recurring risk of a continuous contamination during production.