## 4 Discussion  
The goal of this study was to investigate whether non-growing, carbon-starved _S. marcescens_ cells are more resistant to heat (50 \({}^{\circ}\)C), acid (pH 2.8), and H\({}_{2}\)O\({}_{2}\) (15 mM) by performing cross-resistance challenges and directly comparing carbon-starved (CS) cells to growing, log-phase (LP) cells. To investigate the influence of growth temperature, experiments were carried out at either 30 \({}^{\circ}\)C or 37 \({}^{\circ}\)C. Cultures starved of glucose in minimal media for a period of 24 h, developed high levels of cross-resistance. The difference between LP and CS was greatest in terms of starvation-induced thermal resistance and was similar for the two pre-growth temperatures. Overall, CS cells were more \begin{table}
\begin{tabular}{c c c} \hline \hline
**Strains** & **Type of Challenge** & **SIC Phenotypes** \\ \hline \multirow{3}{*}{ATCC 13880} & \multirow{3}{*}{Acid challenge} & pigmentation only in the center of some colonies \\  & & (“fish eye” phenotype) \\  & Long-term carbon-starvation & non-pigmented, translucent colonies \\ \hline \multirow{3}{*}{UWG6} & Thermal challenge & non-pigmented (white) colonies \\  & Acid challenge & non-pigmented (white to translucent) colonies \\ \cline{1-1}  & Long-term carbon-starvation & translucent colonies; some with pink centers \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Stress-induced colony (SIC) phenotypes of pigmented _Serratia marcescens_ strains. Figure 2: Long-term carbon-starvation survival of _Serratia marcescens_ strains UWG6, ATCC 13880, and Db10. Cultures were grown and then starved of glucose in minimal media for a total of 21 days at either 30 \({}^{\circ}\)C or 37 \({}^{\circ}\)C as described in Materials and Methods. Survival is shown as a percentage of the highest viability (_i.e._, CFU/mL) reached for that culture. Results are shown as the Mean \(\pm\) SEM calculated from at least three independent experiments. resistant to acid stress than LP cells, but the level of acid resistance was temperature-dependent. At 30 \({}^{\circ}\)C, LP cells of the pigmented strains ATCC 13880 and UWG6 were highly resistant to acid pH, with survival values of 30% and 50%, respectively, compared to around 2% for type strain ATCC 13880. These survival values for _S. marcescens_ can be compared to \(<\) 0.1% survival for LP cells of _S. enterica_ serovar Typhimurium following an identical type of acid challenge experiment (data not shown). At 37 \({}^{\circ}\)C, both LP and CS cells of _S. marcescens_ exhibited a much lower level of acid tolerance. The buffering capacity of prodigiosin is one possible explanation for this difference in acid tolerance, but it is also possible that other strain-specific differences play a role [33]. Furthermore, growth temperature is known to have an effect on prodigiosin biosynthesis but it also influences many other physiological processes (e.g., biosurfactant production) and has previously been shown to affect both stress resistance and virulence [20,34]. In terms of H\({}_{2}\)O\({}_{2}\) resistance, CS cells survived much better than LP cells at both temperatures, although LP cells appeared to possess a certain level of inherent resistance, perhaps due to the constitutive expression of catalases and other protective factors specific to _S. marcescens_. Previous studies have reported that _S. marcescens_ possesses its own collection of catalase enzymes and that H\({}_{2}\)O\({}_{2}\) sensitivity is strain-dependent [35, 36, 37, 38]. The long-term carbon-starvation survival of _S. marcescens_ ATCC 13880 was appreciably higher at 30 \({}^{\circ}\)C compared to 37 \({}^{\circ}\)C (Figure 2), perhaps suggesting that the cellular damage caused by carbon-starvation is more severe at the elevated temperature. Interestingly, in thermal and acid cross-resistance challenges and in long-term carbon-starvation experiments, colonies, which grew on LB agar following stress exposure, frequently had aberrant morphologies.