Alive and Well in Low-moisture Conditions - What We Can Learn about Cronobacter sakazakii Using RNA Sequencing (RNA-Seq) and Transposon-directed Insertion Site Sequencing (TraDIS)

Introduction: Cronobacter species are opportunistic pathogens, associated with serious illness in neonates.  Powdered infant formula (PIF) has been epidemiologically linked to infections.  Little is known about the mechanisms Cronobacter employ to survive and persist in low-moisture environments, including PIF production.

Purpose: To determine the genetic signals contributing to the survival and persistence phenotype in low-moisture environments.

Methods: Cronobacter sakazakii SP291, a persistent PIF factory isolate, was selected.  Early stationary phase cells were dried onto industrial grade stainless steel coupons for 4h at 24°C, to simulate a low-moisture environment.  Liquid culture was used as control.  Total RNA was purified for RNA sequencing (RNA-seq) and sequences were mapped to the reference genome.  A transposon-mutant library was constructed in C. sakazakiiSP291.  Pools of random insertion mutants were desiccated as before.  The mutant library was screened by transposon-directed insertion site sequencing (TraDIS) and compared against the original, to identify genes involved in low-moisture survival.

Results: Absolute and relative levels of gene expression were determined using the transcripts per million (TPM) method, applying a log₂-foldchange cut-off value of 3.  A total of 4,177 genes (99.9%) were expressed in the RNA pool, with 107 genes (2.56%) being upregulated and 22 genes (0.53%) downregulated in low-moisture conditions.  The upregulation genes included the osmotic stress response genes betA, betB, betI, betT, proX, proW, and opuCB; the heat-shock response gene rpoH; oxidative stress response gene osmC, among others.  Downregulation genes included the anti-RssB factor gene and several hypothetical genes of unknown function.  Comparative analysis of the un-desiccated and the desiccated mutant pools following TraDIS confirmed the RNAseq data.  Furthermore, qRT-PCR validated a selected sub-set of these gene targets, thereby confirming both approaches.  A model describing the transcriotomic response of C. sakazakii SP291 is presented.

Significance: This is the first report combining RNA-seq and TraDIS to study gene expression in Cronobacter.  Results show the bacterial response at the transcriptional level in low-moisture conditions.  These findings can be used to assist managers in developing guidance measures to reduce the risk of Cronobacter contamination in PIF and production environments.