Phenotypic fate differentiation and its mechanism in Cronobacter sakazakii under different chlorine concentrations

Cronobacter sakazakii is a significant foodborne pathogen commonly found in dairy processing environments. Under chlorine stress, C. sakazakii may survive by adopting stress-resistant phenotypes such as biofilm and viable but nonculturable (VBNC) state. The formation of these phenotypes is regulated by the stringent response. However, the mechanisms by which the stringent response directs phenotypic fate differentiation under different chlorine concentrations remain unclear. In this study, we exposed C. sakazakii to chlorine concentrations (0.1-100 μg/mL) for 24 h and quantified viable cells, 100S ribosome levels, extracellular polymeric substances, and stringent response signals. The results showed that C. sakazakii exhibited pronounced differentiation into biofilm and VBNC phenotypes under chlorine stress ranging from 0.1 to 100 μg/mL. At low chlorine concentration (0.1 μg/mL), biofilm viable cell counts reached 5.10 × 10⁵ cfu/mL, accompanied by significant increases in extracellular polysaccharides and proteins, promoting biofilm formation. In contrast, at high chlorine concentration (100 μg/mL), VBNC cell counts rose to 5.99 × 10⁵ cfu/mL, extracellular components declined, and 100S ribosome levels were significantly elevated, indicating deeper metabolic dormancy. Time-course analysis further revealed that the first 0.5 h after chlorine treatment represents a critical window for phenotypic differentiation. During this period, the stringent response alarmone guanosine tetraphosphate (ppGpp) levels increased by 1.55- to 5.16-fold compared with the control and regulated the formation of biofilm and VBNC phenotypes. Gene expression analysis revealed that in the relA-overexpressing strain, ppGpp ribosome dimerization genes rmf and hpf were upregulated by 5.51- and 2.82-fold, VBNC-related genes ppk and lon upregulated by 3.44- and 3.24-fold, whereas biofilm-related genes dgcQ and bcsA were downregulated by 2.86- and 3.98-fold. These changes guided the selection of phenotypic fate differentiation: Low ppGpp levels interacted with cyclic diguanylate monophosphate (c-di-GMP) to promote biofilm formation, whereas high ppGpp levels induced VBNC phenotype by activating the PPK/Lon cascade. This study not only deepens our understanding of the mechanisms governing bacterial fate determination under environmental stress but also provides a theoretical basis and potential strategies for optimizing dairy disinfection processes and reducing pathogen survival.