Development of heat-shock resistance in modeled by experimental evolution.

Because it can grow in buildings with complex hot water distribution systems (HWDS), healthcare facilities recognize the waterborne bacterium as a major nosocomial infection threat and often try to clear the systems with a pasteurization process known as superheat-and-flush. After this treatment, many facilities find that the contaminating populations slowly recover, suggesting the possibility of evolution favoring increased survival in high-temperature conditions. To mimic this process in a controlled environment, an adaptive laboratory evolution model was used to select a wild-type strain of for survival to transient exposures to temperatures characteristic of routine hot water use or failed pasteurization processes in HWDS. Over their evolution, these populations became insensitive to exposure to 55°C and developed the ability to survive short exposures to 59°C heat shock. Heat-adapted lineages maintained a higher expression of heat-shock genes during low-temperature incubation in freshwater, suggesting a pre-adaptation to heat stress. Although there were distinct mutation profiles in each of the heat-adapted lineages, each acquired multiple mutations in the DnaJ/DnaK/ClpB disaggregase complex, as well as mutations in chaperone and protease . These mutations were specific to heat-shock survival and were not seen in control lineages included in the experimental model without exposure to heat shock. This study supports observations of adaptation to heat stress and demonstrates the potential of to develop resistance to control measures. IMPORTANCE As a bacterium that thrives in warm water ecosystems, is a key factor motivating regulations on hot water systems. Two major measures to control are high circulating temperatures intended to curtail growth and the use of superheat-and-flush pasteurization processes to eliminate established populations. Facilities often suffer recolonization of their hot water systems; hospitals are particularly at risk due to the severe nosocomial pneumoniae caused by . To understand these long-term survivors, we have used an adaptive laboratory evolution model to replicate this process. We find major differences between the mutational profiles of heat-adapted and heat-naïve populations including mutations in major heat-shock genes like chaperones and proteases. This model demonstrates that well-validated treatment protocols are needed to clear contaminated systems and-in an analog to antibiotic resistance-the importance of complete eradication of the resident population to prevent selection for more persistent bacteria.