Abstract
The viable but nonculturable (VBNC) state is a survival strategy adopted by many pathogens when exposed to harsh environmental stresses. In this study, we investigated for the first time that whether high pressure CO2 (HPCD), one of the nonthermal pasteurization techniques, can induce Escherichia coli O157:H7 into the VBNC state. By measuring plate counts, viable cell counts and total cell counts, E. coli O157:H7 in 0.85% NaCl solution (pH 7.0) was able to enter the VBNC state by HPCD treatment at 5 MPa and four temperatures (25°C, 31°C, 34°C and 37°C). Meanwhile, with the improvement of treatment temperature, the time required for E. coli O157:H7 to enter VBNC state would shorten. Enzymatic activities in these VBNC cells were lower than those in the exponential-phase cells by using API ZYM kit, which were also reduced with increasing the treatment temperature, but the mechanical resistance of the VBNC cells to sonication was enhanced. These results further confirmed VBNC state was a self-protection mechanism for some bacteria, which minimized cellular energetic requirements and increased the cell resistance. When incubated in tryptic soy broth at 37°C, the VBNC cells induced by HPCD treatment at 25°C, 31°C and 34°C achieved resuscitation, but their resuscitation capabilities decreased with increasing the treatment temperature. Furthermore, electron microscopy revealed changes in the morphology and interior structure of the VBNC cells and the resuscitated cells. These results demonstrated that HPCD could induce E. coli O157:H7 into the VBNC state. Therefore, it is necessary to detect if there exist VBNC microorganisms in HPCD-treated products by molecular-based methods for food safety.
Highlights
Escherichia coli O157:H7, which was first recognized as a human pathogen in 1982 [1], can cause haemorrhagic colitis and haemolytic uremic syndrome (HUS) in humans [2]
In order to explain this phenomenon, Makino et al [8] re-estimated the number of E. coli O157:H7 cells in the implicated salted salmon roe from their membrane integrity, cell elongation and pathogenicity for mice, and found that almost all of E. coli O157:H7 cells had entered a viable but nonculturable (VBNC) state, the authors suggested that VBNC cells of E. coli O157:H7 in the food should be the source of the outbreak
These results indicated that all the four HPCD treatments in this study could induce the entry of E. coli O157:H7 cells into the VBNC state
Summary
Escherichia coli O157:H7, which was first recognized as a human pathogen in 1982 [1], can cause haemorrhagic colitis and haemolytic uremic syndrome (HUS) in humans [2]. This pathogen has been implicated in large food-borne outbreaks all over the world, such as Canada, UK, Japan and Scotland [3]. Asai et al [7] found that only 0.75 to 1.5 viable cells of E. coli O157:H7 in salted salmon roe could cause the infection. In order to explain this phenomenon, Makino et al [8] re-estimated the number of E. coli O157:H7 cells in the implicated salted salmon roe from their membrane integrity, cell elongation and pathogenicity for mice, and found that almost all of E. coli O157:H7 cells had entered a viable but nonculturable (VBNC) state, the authors suggested that VBNC cells of E. coli O157:H7 in the food should be the source of the outbreak
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