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Molecular typing techniques are utilized to determine genetic similarities between bacterial isolates. However, the use of environmental DNA profiling to assess epidemiologic links between patients and their environment has not been fully explored. This work reports the development and validation of two high-throughput short sequence typing (HiSST) schemes targeting the opportunistic pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia, along with a modified SM2I selective medium for the specific isolation of S. maltophilia. These HiSST schemes are based on four discriminative loci for each species and demonstrate high discriminating power, comparable to pairwise whole-genome comparisons. Each scheme includes species-specific PCR primers for precise differentiation from closely related taxa, without the need for upstream culture-dependent methods. For example, the primers targeting the bvgS locus make it possible to distinguish P. aeruginosa from the very closely related Pseudomonas paraeruginosa sp. nov. The selected loci included in the schemes are adapted to massive parallel amplicon sequencing technology. An R-based script implemented in the DADA2 pipeline was assembled to facilitate HiSST analyses for efficient and accurate genotyping of P. aeruginosa and S. maltophilia. We demonstrate the performance of both schemes through in silico validations, assessments against reference culture collections, and a case study involving environmental samples.

Shock chlorination and remedial flushing are suggested to address Legionella pneumophila (Lp) contamination in buildings or during their (re)commissioning. However, data on general microbial measurements (adenosine tri-phosphate [ATP], total cell counts [TCC]), and the abundance of Lp are lacking to support their temporary implementation with variable water demands. In this study, the weekly short-term (3-week) impact of shock chlorination (20–25 mg/L free chlorine, 16 h) or remedial flushing (5-min flush) combined with distinct flushing regimes (daily, weekly, stagnant) was investigated in duplicates of showerheads in two shower systems. Results showed that the combination of stagnation and shock chlorination prompted biomass regrowth, with ATP and TCC in the first draws reaching large regrowth factors of 4.31–7.07-fold and 3.51–5.68-fold, respectively, from baseline values. Contrastingly, remedial flushing followed by stagnation generally resulted in complete or larger regrowth in Lp culturability and gene copies (gc). Irrespective of the intervention, daily flushed showerheads resulted in significantly (p < 0.05) lower ATP and TCC, as well as lower Lp concentrations than weekly flushes, in general. Nonetheless, Lp persisted at concentrations ranging from 11 to 223 as the most probable number per liter (MPN/L) and in the same order of magnitude (103–104 gc/L) than baseline values after remedial flushing, despite daily/weekly flushing, unlike shock chlorination which suppressed Lp culturability (down 3-log) for two weeks and gene copies by 1-log. This study provides insights on the most optimal short-term combination of remedial and preventative strategies that can be considered pending the implementation of suitable engineering controls or building-wide treatment.

Intermittent reduction of temperature set-points and periodic shutdowns of water heaters have been proposed to reduce energy consumption in buildings. However, the consequences of such measures on the occurrence and proliferation of Legionella pneumophila (Lp) in hot water systems have not been documented. The impact of single and repeated heat shocks was investigated using an environmental strain of L. pneumophila and a reference strain of V. vermiformis. Heat shocks at temperatures ranging from 50 °C to 70 °C were applied for 1 h and 4 h in water and water heaters loose deposits (sludge). The regrowth potential of heat-treated culturable L. pneumophila in presence of V. vermiformis in water heaters sludges was evaluated. A 2.5-log loss of culturability of L. pneumophila was observed in simulated drinking water at 60 °C while a 4-log reduction was reached in water heaters loose deposits. Persistence of Lp after 4 h at 55 °C was shown and the presence of V. vermiformis in water heater’s loose deposits resulted in a drastic amplification (5-log). Results show that thermal inactivation by heat shock is only efficient at elevated temperatures (50 °C) in both water and loose deposits. The few remaining organisms can rapidly proliferate during storage at lower temperature in the presence of hosts.