Global transcriptomic analysis of Cronobacter sakazakii CICC 21544 by RNA-seq under inorganic acid and organic acid stresses
Graphical abstract
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.
Section snippets
Culture conditions of strains and assessment of growth ability in acids
C. sakazakii CICC 21544 was obtained from Guangdong Culture Collection Center. It was stocked at −80 °C in Tryptic Soy Broth (TSB, HuanKai, Guangzhou, China) with 30% glycerol and activated after inoculated twice onto Tryptic Soy Agar (TSA, HuanKai, Guangzhou, China) plate and incubated at 37 °C for 24 h. Cells were collected by centrifugation at 5000g for 5 min when grown to exponential phase (OD600 = 0.5). The cells were washed three times with phosphate buffer and then resuspended to a final
Effect of acid stress on C. sakazakii growth
The ability of C. sakazakii CICC 21544 to tolerate acidic conditions was analyzed in TSB broth acidified with HCl and citric acid. The lowest tolerable pH values of C. sakazakii with HCl and citric acid were 4.0 and 4.2, respectively. The ability of C. sakazakii to grow under acidic conditions strongly depends on the type of acid and the pH value (Fig. 1). By comparing the growth curves of the two graphs, it is not difficult to find that the growth of C. sakazakii in hydrochloric acid and
Discussion
With more and more reports of foodborne infection events associated with acidic foods, the mechanism of survival of Enterobacteriaceae at low pH has attracted increased attention. According to previous reports, 85 out of 107 genes were identified as important genes associated with acid stress response (Table 2). Combining the identified DEGs with functional analysis such as KEGG and GO annotations (Fig. 4), we analyzed the underlying mechanism under acid stress in C. sakazakii from the
Conclusion
Here we systematically analyzed the acid tolerant mechanism of Cronobacter sakazakii CICC 21544 by using high throughput transcriptome sequencing technology. 85 of the 107 DEGs identified were functionally associated with acid tolerance response. In order to reduce excessive intracellular protons, C. sakazakii pumps them out through trans-membrane proteins or consumes them through metabolic reactions. For oxidative damage caused by acid stress, C. sakazakii scavenged reactive oxygen species and
CRediT authorship contribution statement
Ailian Zhou: Data curation, Formal analysis, Validation, Visualization, Writing - original draft, Writing - review & editing. Yifang Cao: Data curation, Formal analysis, Validation, Visualization, Writing - review & editing. Donggen Zhou: Resources. Shuangfang Hu: Writing - review & editing. Wanjing Tan: Validation, Visualization. Xinglong Xiao: Conceptualization. Yigang Yu: Writing - review & editing. Xiaofeng Li: Writing - review & editing.
Declaration of Competing Interest
The authors declared that there is no conflict of interest.
Acknowledgements
This work was supported by the National Key Research and Development Program of China (2018YFC1602201), Science and Technology Program Foundation of Guangzhou, China (201904010077) and Natural Science Fund of Zhejiang (No. LY16H260004). We thank Guangzhou Gene Denovo Biotechnology Co., Ltd., China, for technical assistance.
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These authors contributed equally to this work.