Exposure of sink drain microcosms to triclosan: population dynamics and antimicrobial susceptibility

Abstract

Recent concern that the increased use of triclosan (TCS) in consumer products may contribute to the emergence of antibiotic resistance has led us to examine the effects of TCS dosing on domestic-drain biofilm microcosms. TCS-containing domestic detergent (TCSD) markedly lowered biofouling at 50% (wt/vol) but was poorly effective at use levels. Long-term microcosms were established and stabilized for 6 months before one was subjected to successive 3-month exposures to TCSD at sublethal concentrations (0.2 and 0.4% [wt/vol]). Culturable bacteria were identified by 16S rDNA sequence analysis, and their susceptibilities to four biocides and six antibiotics were determined. Microcosms harbored ca. 10 log(10) CFU/g of biofilm, representing at least 27 species, mainly gamma proteobacteria, and maintained dynamic stability. Viable cell counts were largely unaffected by TCSD exposure, but species diversity was decreased, as corroborated by denaturing gradient gel electrophoresis analysis. TCS susceptibilities ranged widely within bacterial groups, and TCS-tolerant strains (including aeromonads, pseudomonads, stenotrophomonads, and Alcaligenes spp.) were isolated before and after TCSD exposure. Several TCS-tolerant bacteria related to Achromobacter xylosoxidans became clonally expanded during dosing. TCSD addition did not significantly affect the community profiles of susceptibility to the test biocides or antibiotics. Several microcosm isolates, as well as reference bacteria, caused clearing of particulate TCS in solid media. Incubations of consortia and isolates with particulate TCS in liquid led to putative TCS degradation by the consortia and TCS solubilization by the reference strains. Our results support the view that low-level exposure of environmental microcosms to TCS does not affect antimicrobial susceptibility and that TCS is degradable by common domestic biofilms.

FIG. 1.

Bioburdens, direct counts, and viable cell counts of selected groups of drain bacteria in the simulator before and during product addition. Data are means ± standard deviations of results from two separate sample pans analyzed in triplicate. The schedule of TCSD addition is indicated. Graphs show numbers 5 days prior to dosing and then after successive 2-day exposures to 0.2, 1.0, 10, and 50% TCSD. (A) Bioburden. (B) Live-dead direct counts. Closed circles, total count; closed squares, live cell count; open circles, dead cell count. (C) Total culturable aerobes and facultative species. (D) Total culturable anaerobes. (E) Total culturable pseudomonads. (F) Culturable enteric species.

FIG. 2.

Phylogenetic trees representing drain microcosm consortia (culturable species) before (consortium A) and after (consortium B) 6 months of exposure to TCSD. Cluster 1 comprised bacteria able to degrade particulate TCS. Isolated strains are displayed in shaded boxes, and reference strains are unshaded. The tree is based on 540-bp sequences of 16S RNA genes and was constructed by using the neighbor-joining method of Jukes and Cantor (25). The scale bar indicates 0.10 estimated substitutions per nucleotide. TREECON for Windows (52) was used to construct the tree. Bootstrap values denote confidence limits on the phylogenies, based on percentages of 100 replications. Methanobacterium thermautotrophicum was used to root the trees.

FIG. 3.

Negative image of parallel DGGE gels showing community fingerprints for biofilm samples and pure cultures of reference strains. (A and B) Long-term drain microcosm samples taken 4 weeks apart from steady-state fermenters (before TCSD addition). (C) Drain microcosms after 3 months of exposure to TCSD (0.2%). (D) Drain microcosms following a further 3 months of exposure to TCSD (0.4%). (E) Pseudomonas nitroreducens MBRG 4.6. (F) Aeromonas sp. strain MBRG 4.2. (G) Stenotrophomonas maltophilia MBRG 4.17. (H) Pseudoxanthomonas sp. (I) Achromobacter xylosoxidans MBRG 4.31. (J) Citrobacter sp. (K) Eubacterium MBRG 7.1. (L) Microbacterium phyllosphaerae.

FIG. 4.

Clear zones produced on agar containing particulate TCS (2 g/liter). (a) Unidentified eubacterium MBRG 7.6 on R2A agar. (b) E. coli P2500 on MacConkey agar. Opacity is due to the presence of TCS at concentrations considerably higher than its aqueous solubility.