Global transcriptomic analysis of Cronobacter sakazakii CICC 21544 by RNA-seq under inorganic acid and organic acid stresses

Highlights

• Proton reduction, cell protection, energy-saving are main mechanisms to acid stress.

• Arginine deaminase pathway and 2,3-butanediol pathway consume protons.

• Che, mcp, flg up-regulated in inorganic acid while down-regulated in organic acid.

Abstract

Cronobacter sakazakii is a common foodborne pathogen that can tolerate various stress conditions. Acidic environment is a common stress condition encountered by bacteria in food processing and gastrointestinal digestion, including both inorganic and organic acids. In order to elucidate the Acid Tolerance Response (ATR) of C. sakazakii, we performed high-throughput RNA-seq to compare gene expression under hydrochloric acid and citric acid stresses. In this study, 107 differentially expressed genes (DEGs) were identified in both acids, of which 85 DEGs were functionally related to the regulation of acid tolerance. Multiple layers of mechanisms may be applied by C. sakazakii in response to acid stress: Firstly, in order to reduce excessive intracellular protons, C. sakazakii pumps them out through trans-membrane proteins or consumes them through metabolic reactions. Secondly, under acidic conditions, a large amount of reactive oxygen species and hydroxyl radicals accumulate in the cells, resulting in oxidative damage. C. sakazakii protects cells by up-regulating the antioxidant stress genes such as soxS and madB. Thirdly, C. sakazakii chooses energy efficient metabolic pathways to reduce energy consumption and maintain necessary processes. Finally, genes involved in chemotaxis and motility were differentially expressed to respond to different acidic conditions. This study systematically analyzed the acid-resistant mechanism of C. sakazakii under the stress of organic and inorganic acids, and provided a theoretical basis for better control of its contamination in food.

Introduction

Cronobacter sakazakii is a Gram-negative, non-spore-forming bacterium that has been associated with rare but life-threatening infections especially in premature infants and immunocompromised adults, with a reported mortality rate of 27% (Friedemann, 2009).

C. sakazakii has been frequently isolated from acidic foods such as fresh-cut fruits, yogurt and cheese products, suggesting that C. sakazakii is relatively resistant to acidic growth conditions (Jaradat et al., 2014, Ueda, 2017). It was reported that C. sakazakii is similar to Salmonella spp. but less than Escherichia coli in tolerance to acid, which also varies with different types of acids (Edelson-Mammel, Porteous, & Buchanan, 2006). A series of experiments were conducted to measure the survival curves of C. sakazakii in nine fruit and vegetable juices. Results showed that most fruit and vegetable juices support the growth of C. sakazakii (Beuchat et al., 2009). It was reported that the antibacterial activity of Kefir is mainly attributed to the metabolites produced during the fermentation process instead of pH and acidity. Further analysis showed that organic acids play the key role (Kim et al., 2018). Similar results were obtained in another study comparing the antibacterial effects of Kefir and common fermented yoghurt. Even though the pH of ordinary fermented yogurt is lower than that of Kefir, ordinary fermented yogurt cannot completely inactivate pathogens like Kefir (Chang et al., 2018). Therefore, we can guess that the antibacterial effects of organic acids and inorganic acids are quite different. It is necessary to study the differences between them.

Up to now, the response of C. sakazakii exposed to various stresses and the resistance mechanisms have been well documented, mainly including desiccation stress, cold or heat shock and osmotic stress (Burgess et al., 2016, Fei et al., 2017, Hu et al., 2018). Moreover, studies of virulence related genes showed that they also contribute to the environmental stress resistance of C. sakazakii (Aly et al., 2019, Bao et al., 2017). However, there are few studies on the acid response of C. sakazakii and the underlying mechanisms still remain unclear. To assess the role of yellow pigmentation on the growth of C. sakazakii under acid stress, experiments were conducted to compare colorless mutants with wild-type strains. These strains exhibited similar tolerance to acid stress at pH 4.5, but mutants became more sensitive when incubated at pH 2.0 (Johler, Stephan, Hartmann, Kuehner, & Lehner, 2010). The analyses on the ompR defective mutant demonstrated that this gene is a key player in the response of C. sakazakii to acid stress (Alvarez-Ordonez et al., 2014). Ling et al. (2018) concluded that gene grxB seems to contribute to the survival and biofilm formation of C. sakazakii under acidic growth conditions by investigating cellular morphology, surface hydrophobicity and motility.

Acids can be classified into organic acids and inorganic acids according to their constituent elements. Organic acids are generally recognized as safe and have been widely used in reducing pathogens in foods. According to reports, the fact that organic acids inactivate microorganism more effectively is due to the undissociated form (Cherrington, Hinton, Mead, & Chopra, 1991). They penetrate bacterial cell membranes more easily and dissociate into anions and protons within cells to decrease cytoplasmic pH, thus affecting cell metabolic activity. According to several researches, the order of inhibition of acids against C. sakazakii was acetic acid ≈ butyric acid ≈ propionic acid > lactic acid > citric acid > malic acid > formic acid > hydrochloric acid (Kim and Park, 2018, Oshima et al., 2012).

Obviously, C. sakazakii may have different resistant mechanisms for inorganic acids and organic acids. However, there has been no systematic analysis about the tolerant mechanisms of C. sakazakii under acidic stress so far, let alone the differences between organic acids and inorganic acids. We compared the transcriptomic differentiations between control group and acid treatment groups by using high throughout RNA-sequencing (RNA-seq). The aim of our study is to elucidate the major transcriptomic features of acid tolerance response (ATR) in C. sakazakii under different acidic environments.