Incorporation of Pseudomonas aeruginosa into drinking-water biofilms on elastomeric materialBressler, D., Balzer, M., Dannehl, A., Flemming, H.-C., Wingender, J.

Biofilm Centre, Aquatic MicrobiologyUniversity 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. aeruginosato 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 forP. 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 drivedfrom PAO1

PATI5

n. d.+LecB mutant derived from PAO1

PATI2n. d.+Wild-typePAO1n. d.+Mucoid clinical strainFRD1

n. d.+Non-mucoid revertant, derived from SG81

SG81/R1

++Mucoid (alginate-producing) environmental strain

SG81

++Type strainDSM 50071

Persistence under flow conditions (28 d)

Persistence under stagnant conditions (7 d)

Genotype/phenotypeStrain

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 biofilmsgrowth for 14 d; flow-rate 50 mL/min,

flow-through reactor (Fig. 1)

EPDM Paraffin

Stagnant Flow-through50 mL/min

Flow-through50 mL/min

Inoculation with P. aeruginosa:stagnant conditions, ~106 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

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beforeinoculation

after inoculation 7d

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cent

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n (c

ells

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fu/c

m²)

total cell count [cells/cm²]

viable cell count (HPC) [cfu/cm²]

P. aeruginosa [cfu/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

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beforeinoculation

1d 7d 14d 28d

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cent

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ells

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fu/c

m²)

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

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cent

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ells

or c

fu/c

m²)

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 108 to 109 cells/cm2

- 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%

denaturinggradient

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).