Interactions between chaperone and energy storage networks during the evolution of Legionella pneumophila under heat shock.

Waterborne transmission of the bacterium Legionella pneumophila has emerged as a major cause of severe nosocomial infections of major public health impact. The major route of transmission involves the uptake of aerosolized bacteria, often from the contaminated hot water systems of large buildings. Public health regulations aimed at controlling the mesophilic pathogen are generally concerned with acute pasteurization and maintaining high temperatures at the heating systems and throughout the plumbing of hot water systems, but L. pneumophila is often able to survive these treatments due to both bacterium-intrinsic and environmental factors. Previous work has established an experimental evolution system to model the observations of increased heat resistance in repeatedly but unsuccessfully pasteurized L. pneumophila populations. Here, we show rapid fixation of novel alleles in lineages selected for resistance to heat shock and shifts in mutational profile related to increases in the temperature of selection. Gene-level and nucleotide-level parallelisms between independently-evolving lineages show the centrality of the DnaJ/DnaK chaperone system in the heat resistance of L. pneumophila. Inference of epistatic interactions through reverse genetics shows an unexpected interaction between DnaJ/DnaK and the polyhydroxybutyrate-accumulation energy storage mechanism used by the species to survive long-term starvation in low-nutrient environments.

Development of heat-shock resistance in Legionella 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 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 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°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 htpG and protease clpX. 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 in situ observations of adaptation to heat stress and demonstrates the potential of L. pneumophila to develop resistance to control measures. IMPORTANCE As a bacterium that thrives in warm water ecosystems, Legionella pneumophila is a key factor motivating regulations on hot water systems. Two major measures to control Legionella 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 Legionella. 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 L. pneumophila 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.

Local Adaptation of Legionella pneumophila within a Hospital Hot Water System Increases Tolerance to Copper.

In large-building water systems, Legionella pneumophila is exposed to common environmental stressors such as copper. The aim of this study was to evaluate the susceptibility to copper of L. pneumophila isolates recovered from various sites: two clinical and seven environmental isolates from hot water system biofilm and water and from cooling tower water. After a 1-week acclimation in simulated drinking water, strains were exposed to various copper concentrations (0.8 to 5 mg/liter) for over 672 h. Complete loss of culturability was observed for three isolates following copper exposure to 5 mg/liter for 672 h. Two sequence type 1427 (ST1427)-like isolates were highly sensitive to copper, while the other two, isolated from biofilm samples, maintained higher culturability. The expression of the copper resistance gene copA evaluated by reverse transcription-quantitative PCR (RT-qPCR) was significantly higher for the biofilm isolates. All four ST1427-like isolates were recovered from the same water system during an outbreak. Whole-genome sequencing results confirmed that the four isolates are very close phylogenetically, differing by only 29 single nucleotide polymorphisms, suggesting in situ adaptation to microenvironmental conditions, possibly due to epigenetic regulation. These results indicate that the immediate environment within a building water distribution system influences the tolerance of L. pneumophila to copper. Increased contact of L. pneumophila biofilm strains with copper piping or copper alloys in the heat exchanger might lead to local adaptation. The phenotypic differences observed between water and biofilm isolates from the hot water system of a health care facility warrants further investigation to assess the relevance of evaluating disinfection performances based on water sampling alone.IMPORTANCELegionella pneumophila is a pathogen indigenous to natural and large building water systems in the bulk and the biofilm phases. The immediate environment within a system can impact the tolerance of L. pneumophila to environmental stressors, including copper. In health care facilities, copper levels in water can vary, depending on water quality, plumbing materials, and age. This study evaluated the impact of the isolation site (water versus biofilm, hot water system versus cooling tower) within building water systems. Closely related strains isolated from a health care facility hot water system exhibited variable tolerance to copper stress, shown by differential expression of copA, with biofilm isolates displaying highest expression and tolerance. Relying on the detection of L. pneumophila in water samples following exposure to environmental stressors such as copper may underestimate the prevalence of L. pneumophila, leading to inappropriate risk management strategies and increasing the risk of exposure for vulnerable patients.

Social Interaction Skill Intervention for Autistic Adults with Intellectual Disability and Limited Language: A Pilot of the SKILL Program.

There is a dearth of research that focuses on social intervention efforts for adults on the autism spectrum with intellectual disability and limited conversational language. Using a multiple baseline experimental design, this pilot investigation of the Socialization Knowledge for Individuals with Limited Language (SKILL) program evaluated a novel peer-facilitated group program specifically designed to target social interaction skills for this population. Findings from five pilot participants yielded evidence of social improvements across specific verbal skills (on-topic conversational contributions and responses) and nonverbal behaviors (eye-contact, active listening), as evidenced by coded social conversation probes and parent-report measures. These findings demonstrate the promise of a socialization intervention for a population that has historically been neglected in the social intervention research literature.