Development of heat-shock resistance in <i>Legionella pneumophila</i> modeled by experimental evolution

Abstract

Because it can grow in buildings with complex hot water distribution systems (HWDS), healthcare facilities recognize the waterborne bacterium <i>Legionella pneumophila</i> 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 <i>in situ</i> 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 <i>L. pneumophila</i> 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 <i>htpG</i> and protease <i>clpX</i>. 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 <i>in situ</i> observations of adaptation to heat stress and demonstrates the potential of <i>L. pneumophila</i> to develop resistance to control measures. IMPORTANCE As a bacterium that thrives in warm water ecosystems, <i>Legionella pneumophila</i> is a key factor motivating regulations on hot water systems. Two major measures to control <i>Legionella</i> 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 <i>Legionella</i>. 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 <i>L. pneumophila</i> 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.