Alterations in the Transcriptional Landscape Allow Differential Desiccation Tolerance in Clinical Cronobacter sakazakii

Abstract

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, Cronobacter sakazakii ATCC™29544^T, and compared the desiccation tolerance with that of 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 with that of the environmental strain. Transcriptome sequencing (RNA-seq)-based deep transcriptomic characterization identified a unique transcriptional profile in the clinical strain compared with what was already known for the environmental strain. As RNA-seq was also carried out under 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 quantitative reverse transcription-PCR (qRT-PCR) determined that, though the secondary desiccation response genes were upregulated in C. sakazakii ATCC™29544^T, the level of upregulation was lower than that in C. sakazakii SP291. All these factors may collectively contribute to the compromised desiccation tolerance in the clinical strain. IMPORTANCE Cronobacter sakazakii has led to outbreaks in the past, particularly associated with foods 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 with that of a highly desiccation-tolerant environmental strain. We found that, even though the clinical strain is desiccation-tolerant, the rate of tolerance was compromised compared with that of 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.

Keywords: Cronobacter sakazakii; RNA sequencing; desiccation; production environment.

FIG 1

Desiccation tolerance curves of C. sakazakii ATCC™29544^T. (a) The 48-h growth depicting all the different growth phases and 4-h desiccation tolerance curve of C. sakazakii ATCC™29544^T. (b) The 4-h desiccation and 30-min rehydration curve of C. sakazakii ATCC™29544^T and SP291 showing the compromised desiccation of the former. (c) Comparative genomics of SP291 and ATCC™29544 focusing on the yqiQ region where the fec operon is inserted in the ATCC™29544 genome.

FIG 2

The transcriptional landscape of desiccated C. sakazakii ATCC™29544^T. (a) An overview of the differentially expressed C. sakazakii ATCC™29544^T genes during desiccation survival. (b) The differential expression of important primary and secondary desiccation response genes. The differential expression of each C. sakazakii ATCC™29544^T gene in each condition mentioned above is color-coded based on the color key given below. The differential expression of the same gene in the transcriptome of desiccated C. sakazakii SP291 (4) is also shown for comparison. The genes fitting the definition of desiccation responsive genes (DRGs) are marked. (c) The differential expression of all the DRG transcription factors in C. sakazakii color-coded as above with the expression in C. sakazakii SP291 for comparison.

FIG 3

The difference in transcript levels of osmotolerant genes confirmed using qRT-PCR. The experiment was carried out on biological replicates (n = 3). The error bars represent the standard deviation of gene expression (from triplicate data) associated with each gene for both strains—C. sakazakii SP291 and C. sakazakii ATCC™29544^T.