Biocide Exposure Induces Changes in Susceptibility, Pathogenicity, and Biofilm Formation in Uropathogenic Escherichia coli

Abstract

Uropathogenic Escherichia coli (UPEC) is a frequent cause of catheter-associated urinary tract infection (CAUTI). Biocides have been incorporated into catheter coatings to inhibit bacterial colonization while, ideally, exhibiting low cytotoxicity and mitigating the selection of resistant bacterial populations. We compared the effects of long-term biocide exposure on susceptibility, biofilm formation, and relative pathogenicity in eight UPEC isolates. MICs, minimum bactericidal concentrations (MBCs), minimum biofilm eradication concentrations (MBECs), and antibiotic susceptibilities were determined before and after long-term exposure to triclosan, polyhexamethylene biguanide (PHMB), benzalkonium chloride (BAC), and silver nitrate. Biofilm formation was quantified using a crystal violet assay, and relative pathogenicity was assessed via a Galleria mellonella waxworm model. Cytotoxicity and the resulting biocompatibility index values were determined by use of an L929 murine fibroblast cell line. Biocide exposure resulted in multiple decreases in biocide susceptibility in planktonic and biofilm-associated UPEC. Triclosan exposure induced the largest frequency and magnitude of susceptibility decreases at the MIC, MBC, and MBEC, which correlated with an increase in biofilm biomass in all isolates. Induction of antibiotic cross-resistance occurred in 6/84 possible combinations of bacteria, biocide, and antibiotic. Relative pathogenicity significantly decreased after triclosan exposure (5/8 isolates), increased after silver nitrate exposure (2/8 isolates), and varied between isolates for PHMB and BAC. The biocompatibility index ranked the antiseptic potential as PHMB > triclosan > BAC > silver nitrate. Biocide exposure in UPEC may lead to reductions in biocide and antibiotic susceptibility, changes in biofilm formation, and alterations in relative pathogenicity. These data indicate the multiple consequences of biocide adaptation that should be considered when selecting an anti-infective catheter-coating agent.

Keywords: biocide; biofilm; susceptibility.

FIG 1

Biofilm formation in biocide-adapted UPEC. The results of a crystal violet biofilm assay indicating the effect of previous biocide exposure on biofilm formation in eight isolates of UPEC are shown. The data show the mean absorbance (A₆₀₀), representative of biofilm formation for individual bacteria before and after long-term exposure to PHMB, triclosan, BAC, or silver nitrate or after passage on a biocide-free medium (Control). The data represent those for samples taken from two separate experiments, each with four technical replicates. For data that varied between replicates, SDs are given as error bars. Significance was determined using ANOVA. *, P ≤ 0.05.

FIG 2

Relative pathogenicity of biocide-adapted UPEC. G. mellonella survival curves for larvae injected with unexposed and biocide-exposed UPEC are shown. Data represent those for 24 biological replicates. Data from noninjected larvae, larvae injected with PBS alone, and larvae injected with control isolates passaged on a biocide-free medium (Control; C12) are also shown. *, a significant difference in pathogenicity when comparing biocide-adapted isolates to the respective control strain (P ≤ 0.05, log-rank reduction test).