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

SUSCEPTIBILITY crossm Biocide Exposure Induces Changes in Susceptibility, Pathogenicity, and Biofilm Formation in Uropathogenic Escherichia coli E. L. Henly,a J. A. R. Dowling,a J. B. Maingay,a M. M. Lacey,a T. J. Smith,a S. Forbesa a Biomolecular Sciences Research Centre, Sheffield Hallam University, Sheffield, United Kingdom ABSTRACT Uropathogenic Escherichia coli (UPEC) is a frequent cause of catheterassociated 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 antiinfective catheter-coating agent. KEYWORDS biocide, biofilm, susceptibility C atheter-associated urinary tract infections (CAUTI) are among the most commonly acquired health care-associated infections, contributing considerably to patient morbidity and posing an economic burden on health care service providers (1). Complications associated with catheterization often arise due to contamination of the catheter surface with uropathogenic Escherichia coli (UPEC) during catheter insertion, leading to the formation of bacterial biofilms and subsequent infection. Patients undergoing long-term catheterization are at a particular risk of acquiring CAUTI, with studies indicating a 5% to 8% increase in the risk of developing bacteriuria for every day that the catheter remains inserted (2). The majority of patients exhibit bacteriuria after 4 weeks of catheterization, potentially leading to further complications, such as pyelonephritis and septicemia (2, 3). Bacterial biofilms are often recalcitrant to antimicrobial chemotherapy and to the March 2019 Volume 63 Issue 3 e01892-18 Antimicrobial Agents and Chemotherapy Citation Henly EL, Dowling JAR, Maingay JB, Lacey MM, Smith TJ, Forbes S. 2019. Biocide exposure induces changes in susceptibility, pathogenicity, and biofilm formation in uropathogenic Escherichia coli. Antimicrob Agents Chemother 63:e01892-18. https://doi .org/10.1128/AAC.01892-18. Copyright © 2019 Henly et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to S. Forbes, . Received 6 September 2018 Returned for modification 3 October 2018 Accepted 23 December 2018 Accepted manuscript posted online 14 January 2019 Published 26 February 2019 aac.asm.org 1 Henly et al. Antimicrobial Agents and Chemotherapy actions of the host immune system, making biofilm-associated infections, such as CAUTIs, difficult to treat (4). Biofilms show decreased susceptibility to antibiotics, partially due to the shielding effect of the extracellular polymeric substance (EPS) encasing the bacterial cells (5), the low metabolic activity of the cells within the biofilm (6), and the activity of membrane-bound efflux pumps that actively expel antimicrobial compounds from the bacterial cell (4). Furthermore, antibiotic resistance genes are frequently transferred between bacteria within a biofilm by horizontal gene transfer, allowing the dissemination of resistance through a bacterial population (7). Antibiotic treatment of CAUTIs is therefore often ineffective due to the recalcitrance of the biofilm, in addition to the increasing prevalence of antibiotic-resistant uropathogens (8). There is considerable interest in developing anti-infective catheter coatings that are refractory to microbial colonization and subsequent biofilm formation in an attempt to prevent the establishment of CAUTIs. Biocides are broad-spectrum antimicrobial chemicals that inhibit the growth of or that kill microorganisms (9). Biocide-coated urinary catheters incorporating biocides, such as silver nitrate and nitrofurazone, that are eluted from the surface of the catheter, providing an antimicrobial gradient and a potential selective pressure for biocideresistant populations of bacteria, have been developed (10). Current clinical trial data have highlighted the limited antimicrobial efficacy of silver-impregnated catheters compared to that of catheters without an antimicrobial coating, while nitrofurazonecontaining coatings have been shown to exhibit only short-term antimicrobial activity and may therefore be ineffective in patients undergoing long-term catheterization (11, 12). This has fueled the search for further anti-infective coating agents that display broad-spectrum activity which is maintained after prolonged use. Long-term exposure of certain bacterial species to biocides may cause the induction of biocide insusceptibility either through the selection of intrinsically resistant mutants or through induced phenotypic adaptations, bringing into question the long-term antimicrobial activity of various biocide-containing coatings (13). Concerns have also been raised that long-term biocide exposure may promote cross-resistance to antibiotics through the acquisition of mutations in shared target sites or through the activation of broad-range defense mechanisms (14), such as increased cellular efflux activity (15) or decreased cell permeability (16). It can, however, be argued that while long-term biocide exposure may lead to reductions in biocide or antibiotic susceptibility in bacteria, these reductions are small and would not impact the susce (...truncated)