Bacterial viability and oxidative stress
2- E. coli recovery on plate, oxidative stress and VBNC phenome
Potentially pathogenic bacteria, such as Escherichia coli, Salmonella typhimurium and Vibrio cholerae, become Viable But Non Culturable (VBNC) during stress (stasis, oxidative stress, heat shock…). The existence of pathogenic VBNC cells is a public health concern since they could constitute unrecognized sources of infection if they retain their pathogenicity. The physiological significance of this phenotype is unclear : some authors have proposed that it is part of an adaptive response aimed at long-term survival under adverse conditions ; others argue that it is a consequence of stochastic cellular deterioration and that VBNC cells are on their way to death. Moreover, to date, many studies have addressed the ability of VBNC cells to remain infectious, but their conclusions are conflicting. In previous works, using a density-gradient centrifugation technique to separate culturable and VBNC cells, we have proposed that E. coli VBNC cells (generated during stasis) are produced due to stochastic deterioration, rather than an adaptive programme, and pinpoint oxidation management as the ’Achilles heel’ of these cells (Desnues et al., 2003). Moreover, we have found that future VBNC cells form a sub-population within the culture in entrance of stationary phase (Cuny et al., 2005).
Survival rates after phosphate buffer starvation, hydrogen peroxide exposure, and heat shock exposure of the two subpopulations isolated from an LB medium culture of E. coli incubated for 10 h. Bacteria were separated by ultracentrifugation in the radioselectan solution. (A) Ten milliliters of phosphate buffer was initially inoculated with 107 bacteria/ml. The experiments were repeated six times. (B and C) Ten milliliters of phosphate buffer was initially inoculated with 108 bacteria/ml and exposed to 60°C (B) or 8.8 mM H2O2 (C). Culturable bacteria were assayed by plating samples from suspensions onto LB agar plates after serial dilution in cold phosphate buffer. Colonies were counted after 48 h of incubation at 37°C. Symbols : ■, HD bacteria ; , LD bacteria.
Radioselectan solution centrifugation of E. coli cells at various stages of the transition from exponential growth to the stationary phase. E. coli was grown in LB medium. Cultures were harvested at different times and immediately subjected to centrifugation. The number of culturable cells and the pH of the medium were measured for each time. In the final tube the bacterial population was separated into two bands, the LD bacteria and the HD bacteria.
Plating conditions generate an oxidative stress
We have investigated the first events that occur when exponentially grown cells are transferred from a liquid medium (Luria-Bertani [LB]) to a solid medium (LB agar [LBA]). We observed an initial lag phase of 180 min for the wild type MG1655 without any apparent growth. This lack of growth was independent of the bacterial physiological state (either the stationary or the exponential phase), the solid medium composition, or the number of cells on the plate, but it was dependent on the bacterial genotype. Using lacZ-reporter fusions and two-dimensional electrophoresis analysis, we observed that when cells from exponential-phase cultures were plated on LBA, several global regulons, like heat shock regulons (RpoH, RpoE, CpxAR) and oxidative-stress regulons (SoxRS, OxyR, Fur), were immediately induced. Our results indicate that in order to grow on plates, bacteria must not only adapt to new conditions but also perceive a real stress (Cuny et al., 2007). This stress might have deleterious effects on cells and could explain at least in part the phenomenon of VBNC cells. Results support the idea that TiO2 photocatalysis generates damage which later becomes deleterious during recovery on plates. We found this to be partly due to DNA attack via hydroxyl radicals generated by the Fenton reaction during recovery (Gogniat and Dukan, 2007). In summary, oxidative stress performed by cells during the recovery period may explain partially the presence of VBNC cells after hypochlorous acid exposure (Dukan et al., 1997 ; 1999), TiO2 photocatalysis exposure (Gogniat et al., 2006) or the unability to form colonies on plate of the oxyR mutant during stasis (Dukan and Nystrom, 1999).
Global expression monitoring by 2D gel analysis. MC4100 cells were grown in M9 + 2% glucose and sampled in exponential phase (OD600=0.5) and plate on M9A 2 % glucose containing methionine 35S on the surface. After 10 minutes of incorporation in liquid (A) or on plate (B) cells were collected and 2D analysis performed. (A) repressed proteins in liquid compared to solid “circle”, (B) induced proteins in solid compare to liquid “circle”.
Protein aggregation and recovery on plate
Protein aggregation is a phenomenon observed in all organisms and has often been linked with cell disorders. We demonstrated the presence of protein aggregates in an exponentially grown E. coli culture. Our results led us to speculate that protein aggregates may function as a temporary “trash organelle” for cellular detoxification (Maisonneuve et al., 2008a). Silver-stained 2D gel analysis of proteins in pellets and supernatant. Panels show protein patterns in LP, SP, ILP, ISP, and supernatant obtained after 2D gel electrophoresis and visualized by silver staining.
In light of these observations, protein aggregates could be considered damage to cells that is able to pass from one generation to the next. Based on the assumption that the amount of aggregate protein could represent an aging factor, we monitored this amount in a bacterial culture during senescence. In doing so, we observed (i) a significant increase in the amount of aggregate protein over time, Aggregate protein accumulates over time. (A) Coomassie-stained SDS/PAGE gel showing the relative amount of aggregate protein (per mg of soluble protein) at three time points during exponential (2, 4 and 6 hours) or stationary phase (10, 24 and 48 hours). At each point, samples were prepared from equal OD amounts of cells. Panel (B) shows representative results from relative aggregate content for each time during exponential (2, 4 and 6 hours) or stationary phase (10, 24, 30, 36 and 48 hours) quantified using Quantity One software (BioRad).
(ii) a proportional relationship between the amount of aggregate protein and the level of VBNC cells, (iii) a larger amount in VBNC cells than in culturable cells, (iv) a heterogeneous distribution of different amounts within a homogenous population of culturable cells entering stasis, and (v) that the initial amount of aggregate protein within a culturable population conditioned the VBNC cell rate of the culture. Together, the results presented in this study suggest that protein aggregates represent one aging factor leading to VBNC cell formation (Maisonneuve et al., 2008b). For this study, we got a highlight in Microbes (nov. 2008).
(A) Determination of reproductive ability (CFU) and cell integrity in the Low Density (LD, shaded bar) and High Density (HD, unshaded bar) cell populations from a 48 hours stationary-phase culture. (B) Coomassie-stained SDS/PAGE gel showing the relative amount of aggregate protein (per mg of soluble protein) from LD and HD cells prepared from an equal OD amount of cells. (C) Relative aggregate amount from LD an HD cells quantified using Quantity One software (BioRad). (D) Determination of reproductive ability (CFU) and cell integrity in the Low Density (LD, shaded bar) and High Density (HD, unshaded bar) cell populations from a 10 hours culture. (E) Coomassie-stained SDS/PAGE gel showing the relative amount of aggregate protein (per mg of soluble protein) from LD and HD cells prepared from an equal OD amount of cells. (F) Relative aggregate amount from LD an HD cells quantified using Quantity One software (BioRad).