Metabolic characteristics and markers in viable but nonculturable state of Pseudomonas aeruginosa induced by chlorine stress

Many Gram-negative pathogens enter the viable but nonculturable (VBNC) state to resist external environmental stress (such as disinfection). However, little is known about the metabolic properties, especially for the metabolic markers, of VBNC bacteria, which impedes the development of efficient disinfection technologies and causes more potential health risks. In this study, we analyzed the metabolic characteristics of chlorine stress-induced VBNC Pseudomonas aeruginosa at the population and single-cell levels. The overall metabolic activity of VBNC bacteria showed a downward trend, but the glyoxylate cycle, fatty acid and glycerophospholipid metabolism pathways were up-regulated. Based on the metabolic profiles of VBNC bacteria, nine metabolic markers (pyruvate, glyoxylate, guanine, glutamate, sn glycero-3-phos-phocholine, fatty acid, D-alanine, glutathione, N-Butanoyl-D-homoserine lactone) were determined. The results of single-cell Raman spectroscopy showed that the metabolic activity of VBNC bacteria was significantly reduced, but showed more significant metabolic heterogeneity. The redshift of the Raman peaks of 15N and 13C labeled VBNC bacteria was significantly weaker than that of the culturable bacteria, suggesting that the VBNC bacteria have a reduced ability to synthesize proteins, nucleotides, phospholipids, and carbohydrates. The result of this study can help to better understand the metabolic mechanisms and energy management strategy of VBNC bacteria, to achieve precise identification and effective control of VBNC bacteria.