Induction of Viable but Non-culturable (VBNC) State in <i>Salmonella</i> Cultured in M9 Minimal Medium Containing High Glucose

Abstract

An environmental isolate of Salmonella enterica serovar Enteritidis (SE) clone, SE Cl#15-1, loses its culturability during 72-h culture in M9 minimal medium containing 0.8% glucose, a concentration twice higher than that in normal M9 medium, whereas the bacterium retains its culturability in normal M9 medium. Live/dead analysis using the 5-cyano-2,3-di( p-tolyl) tetrazolium chloride (CTC)-reduction assay revealed that SE cells cultured in M9 medium containing 0.8% glucose died with time when in the "viable but nonculturable" (VBNC) state. Assay of the culturability of SE cells in the used supernatant (0.4 spent M9 or 0.8 spent M9) also indicated that 0.8 spent M9 soon showed a lethal effect on intact SE cells. These results suggest that large amounts of glucose metabolites might have been responsible for the toxicity. Analysis of the 0.8 spent M9 revealed that formate rapidly accumulated in the medium. The pH of the medium rapidly dropped to 4.7, leading to conversion of formate to formic acid, which might have damaged the bacterial cell membrane. These results suggest that the excessive amount of glucose in the M9 medium might have injured SE cells in the VBNC state by being metabolized to formic acid and other acidic compounds.Key words viable but non-culturable (VBNC) induction; Salmonella; formate; glucose; prolonged culture Salmonella is one of the most causative of food-borne diseases in the world; and it is detected not only in food but also in natural environments including river water, soil, manure, and air-borne dust.1-5) Among Salmonella spp., Salmonella enterica serovar Enteritidis (SE) is one of the dominant species, and has sometimes caused mass food poisoning in many countries. [6][7][8][9][10] In consideration of the strong infectivity of Salmonella, a better understanding of the characteristics of environmental Salmonella isolates is necessary for the control of spreading and contamination of foods by Salmonella as well as for the risk management for food safety in general.We previously reported that SEp22, [11][12][13][14] which is a novel pathogenicity-related factor of S. Enteritidis, and is identical to Salmonella Dps, 15) plays a very important role in the acquisition of the dry-resistance of S. Enteritidis. 16) In the course of studying the characteristics of SEp22, we happened to find that the prolonged batch culturing of SE cells in M9 minimal medium containing 0.8% glucose (a concentration twice higher than that used in the standard culture) significantly reduced their culturability. Analysis of the resultant culture supernatant revealed that this two-fold higher dose of glucose induced a sudden decrease in pH and the accumulation of large amounts of acetate, formate, and pyruvate. It was also remarkable that pyruvate was produced to a much higher extent in the medium with 0.8% glucose than in that with 0.4% glucose in the same time-course studies.Recent findings revealed that many pathogens could enter a viable but non-culturable (VBNC) state 17,18) after exposure to adverse environm...

Analysis of the Resuscitation-Availability of Viable-But-Nonculturable Cells ofVibrio parahaemolyticusupon Exposure to the Refrigerator Temperature

“…Major food-borne pathogens, including Vibrio parahaemolyticus , Vibrio vulnificus , Camphylobacter jejuni , Escherichia coli O157:H7, Salmonella enterica serovar Enteritidis, and Shigella dysenteriae , are known to become the viable-but-nonculturable (VBNC) state when challenged by various environmental stresses such as low temperatures (≤15°C), starvation, copper, and CO 2 (1-3). It should be noted that VBNC cells of these pathogens are incapable of producing their own colonies on culture media on which these organisms can grow routinely, thereby escaping from the cultivation-based surveillances and diagnosis tools.…”