Development of heat shock resistance in L. pneumophila modeled by experimental evolution

Because it can grow in buildings with complex hot water distribution systems (HWDS), healthcare facilities recognize the waterborne bacterium Legionella pneumophila 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 in situ evolution favouring increased survival in high temperature conditions. To mimic this process in a controlled environment, an adaptive laboratory evolution (ALE) model was used to select a wild-type strain of L. pneumophila 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 {degrees}C and innovated the ability to survive short exposures to 59 {degrees}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 htpG and protease clpX. These mutations were specific to heat shock survival and were not seen in control lineages included in the ALE without exposure to heat shock. This study supports in situ observations of adaptation to heat stress and demonstrate the potential of L. pneumophila to develop resistance to control measures. ImportanceAs a bacterium that thrives in warm water ecosystems, Legionella pneumophila is a key factor motivating regulations on hot water systems. Two major measures intended to control Legionella are the maintenance of high circulating temperatures to curtail growth and the use of superheat-and-flush pasteurization processes to eliminate established populations. Although hospitals are particularly vulnerable to nosocomial pneumoniae caused by Legionella, they recurrently experience recolonization of their hot water systems after treatment. To understand these long-term survivors, we have used an experimental evolution model to replicate this process. We find major differences between the mutational profiles of heat-adapted and heat-naive L. pneumophila populations, including mutations in major heat shock genes like chaperones and proteases. This model demonstrates the value of appropriate heat treatment of L. pneumophila contaminated systems and - in an analogue to antibiotic resistance - the importance of complete eradication of the resident population to prevent selection for more persistent bacteria.