High-risk clones of *Pseudomonas aeruginosa* contaminate the drinking water networks of French cities

Pseudomonas aeruginosa is ubiquitous in moist environments, including hospital and community drinking water networks (DWNs), and can contaminate distal water system components (sinks, showers, U-bends), causing waterborne nosocomial infections. It causes severe infections (respiratory, bloodstream, wound) particularly in immunocompromised patients and can contaminate devices such as nebulizers used by cystic fibrosis patients. European regulations mandate monitoring for P. aeruginosa by immediate filtration and culture, but this approach fails to detect viable but non-culturable persister cells that survive under nutrient-poor and inhibitor-rich conditions (e.g., copper ions, chlorine) and can resuscitate and regain full virulence under suitable conditions. Prior work using a resuscitation culture method detected high-risk clones (e.g., ST308) in DWNs; ST308 tolerance to copper has been linked to a genomic island (G7-1) likely acquired from environmental Pseudomonas spp. The source of DWN contamination remains unclear: while reservoirs can be contaminated, treatment usually removes the organism before distribution, whereas contamination of plumbing components (e.g., water meters) prior to installation has been documented. This study investigated eight independent, geographically distant French DWNs (hospital and community) to determine the presence of P. aeruginosa persister cells and to characterize population structure and shared genetic elements to infer potential contamination sources.

The paper situates its work within evidence that DWNs and hospital plumbing can harbor P. aeruginosa and serve as reservoirs for healthcare-associated infections. Standard monitoring often misses persister (viable but non-culturable) cells that can resuscitate and retain virulence. Previous outbreaks have implicated water as a reservoir, and high-risk clones such as ST308 and ST395 have been reported in hospitals. Copper exposure in plumbing can select for tolerant strains; a 37-kb genomic island (G7-1) encoding copper transporters has been noted in ST308/ST395. Technical reports have found brand-new water meters contaminated with P. aeruginosa prior to installation, suggesting a potential dissemination route. The study builds on genome-based typing approaches (cgMLST/wgMLST) used to resolve clonal relationships among environmental and clinical isolates.

Setting and sampling: Between August 2019 and November 2020, 239 water samples were collected from hospital and community DWNs across eight French cities (Amiens, Angers, Besançon, Bordeaux, Nantes, Nîmes, Tours, Trévenans). Sampling followed EN ISO 19458 guidelines. For hospitals, faucets away from patient care areas (technical spaces) were sampled to avoid clinical/environmental contamination; sites included main water inlets and 3–5 distal points per building. Cold-water samples were collected monthly over three months per DWN. Procedures included aerator removal, outlet disinfection (gas burner or disinfectant swab), and a 1-minute pre-flush. Samples were collected in sterile polypropylene bottles containing 20 mL sodium thiosulfate and 100 μg EDTA to chelate copper and aid resuscitation of copper-stressed cells. Bottled sterile water served as negative control. Transport and culture/resuscitation: Samples were shipped to the Infection Control Laboratory of University Hospital of Besançon within 24 hours at room temperature and stored at 22 °C in the dark to prevent premature resuscitation. Each sample was filtered sequentially through 250 μm and then 0.45 μm membranes. Filtrates were cultured on (i) R2A agar and (ii) Columbia agar with 5% sheep blood, each incubated 48 h at 37 °C. Colonies of P. aeruginosa were identified by MALDI-TOF MS. Isolates were stored in brain heart infusion with 30% glycerol at −80 °C. Antimicrobial susceptibility testing: Disk diffusion on Mueller–Hinton agar was performed for clinically relevant antibiotics: ceftazidime, ceftazidime–avibactam, imipenem, meropenem, aztreonam, amikacin, tobramycin, and ciprofloxacin; results interpreted per EUCAST. Genome sequencing and typing: Isolates were typed by MLST (PCR/Sanger per a modified protocol). Genomic DNA was extracted (QIAamp). Whole-genome sequencing used Illumina NextSeq high-output v2.5 (paired-end 150 bp), with coverage >240×; reads were subsampled to ~300× and assembled with SPAdes v3.13 (optimized mode). Species-level phylogeny: from 5,415 P. aeruginosa genomes in NCBI (as of Apr 25, 2021), one complete genome per ST was randomly retained (753 genomes). A cgMLST scheme with 18,367 genes was constructed; a phylogeny was inferred from 3,786 genes present in >95% of genomes using FastTree 2.1.10 with GTR+G. ST308-specific analysis: 95 ST308 genomes from NCBI (Apr 25, 2021) were analyzed with a wgMLST based on Pa58 reference, selecting 2,937 genes present in 95% of ST308 genomes with at least one variant. Bayesian phylogeny used MrBayes v3.2.7a with GTR+G+I; a minimum spanning tree for study ST308 isolates was generated with GrapeTree v2.2. Data availability: Raw reads deposited under BioProject PRJNA680760 (NCBI). Additional data available from the corresponding author or via GitHub (github.com/bvlot/pMLST).

- Using a culture-based resuscitation method, P. aeruginosa was detected in 5 of 8 (62.5%) DWNs sampled across France, including both hospital and community networks. - Four of the five positive DWNs harbored high-risk clones, notably ST308 and ST395. - In a subset where 12 isolates underwent initial ST determination, five were ST235, three ST175, two ST106, and one ST329; additional detections included ST308 (retrieved during a 2017 sampling in a hospital DWN) and ST395 (detected in a community DWN in Besançon), both recognized as high-risk lineages implicated in hospital outbreaks. - ST308 was the most represented ST in the 2019–2020 campaign and was repeatedly found in multiple DWNs (Nantes, Nîmes, Besançon, Bordeaux). Comparative genomics showed French DWN ST308 isolates clustered together with fewer than nine wgMLST allele differences, indicating very close relatedness and suggesting potential common-source contamination. - Genomic island G7-1 (≈37 kb) encoding copper transporters—associated with copper tolerance—was present in 24 of 28 (86%) DWN isolates analyzed and enriched among ST308/ST309/ST395, supporting adaptation to copper-containing plumbing environments. - No hospital outbreaks due to these high-risk clones were reported during the sampling period, and DWN ST395 isolates were not clonally related to historical clinical ST395 outbreak isolates at UHB by wgMLST. - A 2016 technical report cited in the discussion found 23% of brand-new water meters from major suppliers contaminated with P. aeruginosa prior to installation, aligning with the observed pattern and supporting a hypothesis of plumbing component–mediated seeding of DWNs.

The study addressed whether persister-form P. aeruginosa contaminates DWNs across multiple cities and whether such contamination involves specific, potentially shared, high-risk clones with adaptive traits. Detection in 62.5% of DWNs using a resuscitation-based culture method, and the predominance of high-risk lineages (ST308, ST395), demonstrate that conventional monitoring likely underestimates true contamination. The close phylogenetic clustering of ST308 isolates from four distant DWNs (≤9 wgMLST allele differences) suggests a recent common source or repeated introductions from a shared reservoir rather than independent local evolution. Given independent water sources across cities, attention shifts to plumbing supplies (e.g., water meters), which are produced by few manufacturers and calibrated in facilities where contamination has been documented; these could disseminate P. aeruginosa into DWNs where persister cells can persist and seed distal sites. The frequent presence of the copper tolerance-associated island G7-1 suggests niche specialization: clones harboring copper-resistance determinants likely gain a selective advantage in copper-containing plumbing, promoting survival and persistence under biocidal metal stress and other DWN pressures (nutrient limitation, disinfectants, amoebal predation). These findings have public health implications: persister cells not recovered by standard protocols may act as reservoirs, potentially contributing to sporadic patient exposure and outbreaks, underscoring the need for monitoring methods that recover VBNC/persister states and for interventions targeting contamination points along the plumbing supply chain.

This multicity survey using a persister cell–resuscitating culture method revealed that P. aeruginosa contaminates a majority of tested DWNs in France, with high-risk clones (especially ST308 and ST395) present in most positive networks. Genomic analyses show strikingly close relatedness among ST308 isolates from geographically distant DWNs, pointing toward a common-source contamination pathway, plausibly via shared plumbing components such as water meters. The high prevalence of the copper tolerance island G7-1 supports niche adaptation facilitating persistence in copper-containing plumbing. Future work should: (i) systematically sample and culture plumbing components (e.g., water meters) pre-installation, (ii) expand geographically and across seasons to better quantify prevalence and dynamics, (iii) incorporate culture-independent high-throughput short sequence typing to detect high-risk clones in VBNC states, and (iv) investigate material-specific and operational factors in DWNs that select for or sustain these clones.

- Sampling covered only eight cities within one country, with unequal numbers of samples per DWN (n=239 total), limiting generalizability. - Seasonal variability was not controlled; water temperature at sampling was not measured, and winter sampling may have reduced detection (e.g., absence of ST308 in some winter-sampled DWNs). - DWNs were composed of mixed materials (copper, PVC, galvanized steel, cross-linked polyethylene), precluding correlation between materials and P. aeruginosa presence. - The resuscitation method might introduce selection biases for specific strains. - While the study posits plumbing components (e.g., water meters) as a possible common source, direct collections from pre-installation meters were unavailable for testing. - Strengths include a culture-based method enabling recovery of persister cells for genome sequencing and centralized, standardized processing of all samples and isolates.