RNA Sequencing-Based Transcriptional Overview of Xerotolerance in Cronobacter sakazakii SP291

Abstract

Cronobacter sakazakii is a xerotolerant neonatal pathogen epidemiologically linked to powdered infant food formula, often resulting in high mortality rates. Here, we used transcriptome sequencing (RNA-seq) to provide transcriptional insights into the survival of C. sakazakii in desiccated conditions. Our RNA-seq data show that about 22% of the total C. sakazakii genes were significantly upregulated and 9% were downregulated during desiccation survival. When reverse transcription-quantitative PCR (qRT-PCR) was used to validate the RNA-seq data, we found that the primary desiccation response was gradually downregulated during the tested 4 hours of desiccation, while the secondary response remained constitutively upregulated. The 4-hour desiccation tolerance of C. sakazakii was dependent on the immediate microenvironment surrounding the bacterial cell. The removal of Trypticase soy broth (TSB) salts and the introduction of sterile infant formula residues in the microenvironment enhanced the desiccation survival of C. sakazakii SP291. The trehalose biosynthetic pathway encoded by otsA and otsB, a prominent secondary bacterial desiccation response, was highly upregulated in desiccated C. sakazakiiC. sakazakii SP291 ΔotsAB was significantly inhibited compared with the isogenic wild type in an 8-hour desiccation survival assay, confirming the physiological importance of trehalose in desiccation survival. Overall, we provide a comprehensive RNA-seq-based transcriptional overview along with confirmation of the phenotypic importance of trehalose metabolism in Cronobacter sakazakii during desiccation.IMPORTANCECronobacter sakazakii is a pathogen of importance to neonatal health and is known to persist in dry food matrices, such as powdered infant formula (PIF) and its associated production environment. When infections are reported in neonates, mortality rates can be high. The success of this bacterium in surviving these low-moisture environments suggests that Cronobacter species can respond to a variety of environmental signals. Therefore, understanding those signals that aid the persistence of this pathogen in these ecological niches is an important step toward the development of strategies to reduce the risk of contamination of PIF. This research led to the identification of candidate genes that play a role in the persistence of this pathogen in desiccated conditions and, thereby, serve as a model target to design future strategies to mitigate PIF-associated survival of C. sakazakii.

Keywords: Cronobacter sakazakii; RNA-seq; desiccation; transcriptome; xerotolerance.

FIG 1

Progression of the 32-month desiccation tolerance assay conducted on the members of Enterobacteriaceae isolated from a PIF production facility under surveillance. C. sakazakii SP291 survived desiccation for a period of 32 months, which was greater than other strains; therefore, it was taken as a model organism for downstream desiccation analysis.

FIG 2

The desiccation-associated growth curve of C. sakazakii SP291. The blue curve represents the static growth of C. sakazakii SP291 in TSB at 25°C. TSB-grown cells were directly subjected to desiccation, and the survival for 4 h is shown in the green curve. TSB-grown cells were resuspended in TSB, sterile water, sterile infant formula, Luria-Bertani broth (LB), LB plus 0.25% glucose, and LB plus 0.25% lactose and then subjected to desiccation on sterile stainless steel coupons. The viability of bacterial cells associated with desiccation in the presence of different media are depicted in different colored curves, and the representation of each color is described in the key.

FIG 3

(A) A figure summarizing the experimental strategy deployed for the RNA-seq experiment. ESP-grown C. sakazakii SP291 cells were subjected to desiccation for 4 h on sterile stainless steel coupons, and total RNA was purified from both ESP-grown and desiccated cells. The graphs represent the RNA-seq reads mapped uniquely against the C. sakazakii SP291 genome in the respective condition, as visualized in Integrated Genome Browser. The genes on the C. sakazakii SP291 genome were categorized as upregulated (green arrows) or downregulated (pink arrows) based on the ratio of gene expression values, desiccation versus ESP. (B) Number of differentially regulated genes in desiccated C. sakazakii SP291 (Table S1, WS2). The differential expression was calculated from the ratio of TPM-based gene expression data, desiccation versus ESP. The thresholds for the selection of upregulated and downregulated genes are mentioned in Materials and Methods. TPM, transcripts per million approach.

FIG 4

Differential expression of selected genes associated with osmoprotection in C. sakazakii SP291. The list of genes was obtained from a previous publication (41). The locations of genes are shown according to the order as they appear in the C. sakazakii SP291 genome. Each gene is in scale with each other. The tracks up and below each gene represent the RNA transcripts that map against the location as visualized in IGB (60).

FIG 5

Differential expression of functionally categorized genes in desiccated C. sakazakii SP291. The list of genes belonging to each category was obtained online (www.patricbrc.org). The genes were arranged according to their differential expression desiccation/ESP and expressed as percentage. The list of genes belonging to each category can be obtained from Table S1, WS3. The gene expression values can be obtained from Table S1, WS2.

FIG 6

qRT-PCR validation of RNA-seq-based differential expression data. The trkH gene represents the primary desiccation response, while the proP and otsB represent the secondary desiccation response.

FIG 7

Physiological importance of trehalose accumulation in desiccated C. sakazakii. (A) Growth of C. sakazakii NalR and C. sakazakii NalR ΔotsBA in TSB. (B) Desiccation survival of C. sakazakii NalR and C. sakazakii NalR ΔotsBA on desiccated stainless steel coupons. All experiments were conducted in independent triplicates, and error bars were generated from standard deviations. Statistical significance was calculated from Student’s t test.

FIG 8

The transcriptional model on the desiccation of C. sakazakii SP291. All the arrows depict the genes encoding the enzyme needed in the conversion step. The color of each arrow depicts the expression pattern of the gene in desiccated C. sakazakii SP291 based on the key given below.