Incorporation of Pseudomonas aeruginosa into drinking-water biofilms on elastomeric material Bressler, D., Balzer, M., Dannehl, A., Flemming, H.-C., Wingender, J. Biofilm Centre, Aquatic Microbiology University of Duisburg-Essen, Germany Introduction Conclusions Methods - Biofilms in water distribution systems are supposed to be possible reservoirs for the opportunistic pathogen Pseudomonas aeruginosa. - The aim of the study was to investigate the potential of P. aeruginosa to become incorporated into drinking-water biofilms on ethylene propylene diene monomer (EPDM), which represents an elastomeric plumbing material with a tendency to support biofilm formation. - P. aeruginosa can colonize established mixed-population drinking- water biofilms and persist there for at least 7 days under stagnant and 4 weeks under flow conditions, respectively. - From a health perspective, these biofilms can provide a reservoir for P. aeruginosa and represent a source of contamination in water distribution systems. Influent Paraffin-coated glass slide Effluent EPDM Fig. 1: Laboratory-scale flow- through reactor n. d. + Lec AB mutant drived from PAO1 PATI5 n. d. + LecB mutant derived from PAO1 PATI2 n. d. + Wild-type PAO1 n. d. + Mucoid clinical strain FRD1 n. d. + Non-mucoid revertant, derived from SG81 SG81/R1 + + Mucoid (alginate- producing) environmental strain SG81 + + Type strain DSM 50071 Persistence under flow conditions (28 d) Persistence under stagnant conditions (7 d) Genotype/phenotype Strain Table: Persistence of different P. aeruginosa strains in drinking-water biofilms; n. d., not determined - Under flow conditions, P. aeruginosa became incorporated into the biofilms both on EPDM (Fig. 4) and paraffin surfaces (Fig. 5), persisted for 4 weeks (termination of experiments) in the biofilms with a slow decline in culturable numbers and could be detected in the water phase during this period. - Under stagnant conditions, P. aeruginosa became incorporated into the biofilms on EPDM and persisted for 7 days (termination of experiments) at high culturable levels in the biofilms (Fig. 6). sampling: before and after inoculation, 1 d, 7 d, 14 d, 28 d sampling: before and after inoculation, after 7 d Characterization of water and biofilms: - total cell count: microscopic enumeration of DAPI-staind bacteria - heterotrophic plate count: colony-forming units (cfu) on R2A medium, 20°C, 7 d - P. aeruginosa: cfu on CN or CFC ager, 36°C, 2d - Biofilm architecture: confocal laser-scanning microscopy (CLSM) - Bacterial diversity: 16S rDNA-directed polymerase chain reaction followed by denaturing gradient gel electrophoresis (PCR-DGGE) Drinking-water biofilms growth for 14 d; flow-rate 50 mL/min, flow-through reactor (Fig. 1) EPDM Paraffin Stagnant Flow-through 50 mL/min Flow-through 50 mL/min Inoculation with P. aeruginosa: stagnant conditions, ~10 6 cfu/mL, 1d Fig. 6: Persistence of type strain P. aeruginosa DSM 50071 in drinking-water biofilms on EPDM surfaces under stagnant conditions Biofilm on EPDM under stagnant conditions 1,00E+00 1,00E+01 1,00E+02 1,00E+03 1,00E+04 1,00E+05 1,00E+06 1,00E+07 1,00E+08 1,00E+09 1,00E+10 before inoculation after inoculation 7d Concentration (cells or cfu/cm²) total cell count [cells/cm²] viable cell count (HPC) [cfu/cm²] P. aerug ino sa [ cf u/ cm²] Fig. 4: Persistence of type strain P. aeruginosa DSM 50071 in drinking-water biofilms on EPDM surfaces under flow conditions Biofilm on EPDM under flow conditions 1,00E+00 1,00E+01 1,00E+02 1,00E+03 1,00E+04 1,00E+05 1,00E+06 1,00E+07 1,00E+08 1,00E+09 1,00E+10 before inoculation 1d 7d 14d 28d Concentration (cells or cfu/cm²) total cell count [cells/cm²] viable cell count (HPC) [cfu/cm²] P. aeruginosa [cfu/cm²] Fig. 5: Persistence of type strain P. aeruginosa DSM 50071 in drinking-water biofilms on paraffin-coated surfaces under flow conditions Biofilm on paraffin under flow conditions 1,00E+00 1,00E+01 1,00E+02 1,00E+03 1,00E+04 1,00E+05 1,00E+06 1,00E+07 1,00E+08 1,00E+09 before inoculation 1d 7d 14d 28d Concentration (cells or cfu/cm²) total cell count [cells/cm²] viable cell count (HPC) [cfu/cm²] P. aeruginosa [cfu/cm²] Fig. 2: 3D-CLSM image of 14 d-old biofilm on EPDM (1000x). Biofilms were staind using SYTO 9 (green) and propidium iodide (red). [Live/Dead-Kit, Molecular Probes] Drinking-water biofilms on EPDM and paraffin (material known to support biofilm growth) were in a quasi-stationary state after 14 d under flow conditions Characterisation of these biofilms: - Heterogenous architecture visualized by CLSM (Fig. 2) - Total cell counts: in the range of 10 8 to 10 9 cells/cm 2 - Viable cell count (HPC): approximately 1 log unit lower than total cell counts - Uniformity of coupon populations determined by PCR-DGGE: Almost identical in biofilms on EPDM couponds at different positions of the same reactor run, and an average similarity of 71 % in EPDM biofilms of separate reactor runs (Fig. 3) Results Fig. 3: DGGE-profiles of PCR-amplified 16S rDNA fragments derived from three 14 d-old biofilms on EPDM couponds at different positions in a flow-through reactor. Number of bands: 68 40% 60% denaturing gradient Effluent Influent Paraffin- coated glass slide EPDM Results - Incorporation and persistence was observed, independent of the genotype or phenotype of several P. aeruginosa strains: mucoid/non-mucoid, deficient in lectins LecA and LecB supposed to be involved in biofilm formation (Table).