Audrey Jeanvoine1, Alexandre Meunier2, Hélène Puja3, Xavier Bertrand1, Benoît Valot3, Didier Hocquet4. 1. Laboratoire d'Hygiène Hospitalière, Centre Hospitalier Régional Universitaire, Besançon, France; UMR CNRS 6249, Chrono-environnement, Université de Bourgogne Franche-Comté, Besançon, France. 2. Laboratoire d'Hygiène Hospitalière, Centre Hospitalier Régional Universitaire, Besançon, France. 3. UMR CNRS 6249, Chrono-environnement, Université de Bourgogne Franche-Comté, Besançon, France. 4. Laboratoire d'Hygiène Hospitalière, Centre Hospitalier Régional Universitaire, Besançon, France; UMR CNRS 6249, Chrono-environnement, Université de Bourgogne Franche-Comté, Besançon, France; Centre de Ressources Biologiques - Filière Microbiologique de Besançon, Centre Hospitalier Régional Universitaire, Besançon, France. Electronic address: dhocquet@chu-besancon.fr.
Abstract
BACKGROUND: Pseudomonas aeruginosa (PA) is an important opportunistic pathogen that thrives best in the distal elements of plumbing and waste-water systems. Although nosocomial outbreaks of PA have been associated with water sources, the role of the plumbing system of healthcare premises as a reservoir for this pathogen is still unclear. MATERIALS AND METHODS: We collected water samples from 12 technical areas, distant from any medical activity, in a teaching hospital in France once a week for 11 weeks. We used a method that resuscitates persister cells because of the nutrient-poor conditions and the presence of inhibitors (e.g. chlorine and copper ions). Briefly, water was sampled in sterile bottles containing 100 μM of the copper-ion chelating agent diethyldithiocarbamate (DDTC). A portion of the samples was immediately filtered through 0.45-μm membranes, deposited on R2A agar plates, and incubated seven days at 22 °C (following European recommendations). The remaining water was incubated 14 days at 22 °C and then filtered and cultured on R2A, blood-, or cetrimide-containing agar plates. PA isolates were identified by MS MALDI-TOF, genotyped by PFGE and WGS, and tested for survival in a 150 μg/L copper (II) sulphate solution. RESULTS: Although the 12 water sampling points always tested negative with the recommended method, 67% were positive at least once for PA with the adapted method (i.e. with DDTC). The 14 PA persister isolates found throughout the plumbing system were clonal and belong to the high-risk clone ST308. Their genome harbours a 37-kb genomic island (GI-7) containing 13 genes linked to copper resistance. ST308 survived better in the copper solution than comparators that did not harbour GI-7 (P. aeruginosa strains PAO1, PA14, and ST235). The deletion of GI-7 in ST308 abrogated its tolerance to copper. The GI-7 nucleotide sequence shares 98% and 72% identity with sequences from the environmental species Pseudomonas putida and the phytopathogenic species Pseudomonas syringae, respectively. CONCLUSION: We report the contamination of the plumbing system of a healthcare premises by persister cells of the high-risk clone P. aeruginosa ST308. New recommendations for the monitoring of water contamination should consider persister cells. The genomic island GI-7, which confers tolerance to copper, probably originates from Pseudomonas species found in copper-contaminated soils and plants. Agricultural practices may have an unexpected consequence, allowing copper-tolerant pathogens to survive in the hospital environment and contaminate fragile patients.
BACKGROUND:Pseudomonas aeruginosa (PA) is an important opportunistic pathogen that thrives best in the distal elements of plumbing and waste-water systems. Although nosocomial outbreaks of PA have been associated with water sources, the role of the plumbing system of healthcare premises as a reservoir for this pathogen is still unclear. MATERIALS AND METHODS: We collected water samples from 12 technical areas, distant from any medical activity, in a teaching hospital in France once a week for 11 weeks. We used a method that resuscitates persister cells because of the nutrient-poor conditions and the presence of inhibitors (e.g. chlorine and copper ions). Briefly, water was sampled in sterile bottles containing 100 μM of the copper-ion chelating agent diethyldithiocarbamate (DDTC). A portion of the samples was immediately filtered through 0.45-μm membranes, deposited on R2A agar plates, and incubated seven days at 22 °C (following European recommendations). The remaining water was incubated 14 days at 22 °C and then filtered and cultured on R2A, blood-, or cetrimide-containing agar plates. PA isolates were identified by MS MALDI-TOF, genotyped by PFGE and WGS, and tested for survival in a 150 μg/L copper (II) sulphate solution. RESULTS: Although the 12 water sampling points always tested negative with the recommended method, 67% were positive at least once for PA with the adapted method (i.e. with DDTC). The 14 PA persister isolates found throughout the plumbing system were clonal and belong to the high-risk clone ST308. Their genome harbours a 37-kb genomic island (GI-7) containing 13 genes linked to copper resistance. ST308 survived better in the copper solution than comparators that did not harbour GI-7 (P. aeruginosa strains PAO1, PA14, and ST235). The deletion of GI-7 in ST308 abrogated its tolerance to copper. The GI-7 nucleotide sequence shares 98% and 72% identity with sequences from the environmental species Pseudomonas putida and the phytopathogenic species Pseudomonas syringae, respectively. CONCLUSION: We report the contamination of the plumbing system of a healthcare premises by persister cells of the high-risk clone P. aeruginosa ST308. New recommendations for the monitoring of water contamination should consider persister cells. The genomic island GI-7, which confers tolerance to copper, probably originates from Pseudomonas species found in copper-contaminated soils and plants. Agricultural practices may have an unexpected consequence, allowing copper-tolerant pathogens to survive in the hospital environment and contaminate fragilepatients.