Antimicrobial and cytotoxic synergism of biocides and quorum-sensing inhibitors against uropathogenic Escherichiacoli.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) are a primary cause of catheter-associated urinary tract infections (CAUTIs), often forming mature recalcitrant biofilms on the catheter surface. Anti-infective catheter coatings containing single biocides have been developed but display limited antimicrobial activity due to the selection of biocide-resistant bacterial populations. Furthermore, biocides often display cytotoxicity at concentrations required to eradicate biofilms, limiting their antiseptic potential. Quorum-sensing inhibitors (QSIs) provide a novel anti-infective approach to disrupt biofilm formation on the catheter surface and help prevent CAUTIs. AIM: To evaluate the combinatorial impact of biocides and QSIs at bacteriostatic, bactericidal and biofilm eradication concentrations in parallel to assessing cytotoxicity in a bladder smooth muscle (BSM) cell line. METHODS: Checkerboard assays were performed to determine fractional inhibitory, bactericidal, and biofilm eradication concentrations of test combinations in UPEC and combined cytotoxic effects in BSM cells. FINDINGS: Synergistic antimicrobial activity was observed between polyhexamethylene biguanide, benzalkonium chloride or silver nitrate in combination with either cinnamaldehyde or furanone-C30 against UPEC biofilms. However, furanone-C30 was cytotoxic at concentrations below those required even for bacteriostatic activity. A dose-dependent cytotoxicity profile was observed for cinnamaldehyde when in combination with BAC, PHMB or silver nitrate. Both PHMB and silver nitrate displayed combined bacteriostatic and bactericidal activity below the half-maximum inhibitory concentration (IC50). Triclosan in combination with both QSIs displayed antagonistic activity in both UPEC and BSM cells. CONCLUSION: PHMB and silver in combination with cinnamaldehyde display synergistic antimicrobial activity in UPEC at non-cytotoxic concentrations, suggesting potential as anti-infective catheter-coating agents.

Impact of long-term quorum sensing inhibition on uropathogenic Escherichia coli.

BACKGROUND: Quorum sensing is an extracellular bacterial communication system used in the density-dependent regulation of gene expression and development of biofilms. Biofilm formation has been implicated in the establishment of catheter-associated urinary tract infections and therefore quorum sensing inhibitors (QSIs) have been suggested as anti-biofilm catheter coating agents. The long-term effects of QSIs in uropathogens is, however, not clearly understood. OBJECTIVES: We evaluated the effects of repeated exposure to the QSIs cinnamaldehyde, (Z)-4-bromo-5(bromomethylene)-2(5H)-furanone-C30 (furanone-C30) and 4-fluoro-5-hydroxypentane-2,3-dione (F-DPD) on antimicrobial susceptibility, biofilm formation and relative pathogenicity in eight uropathogenic Escherichia coli (UPEC) isolates. METHODS: MICs, MBCs and minimum biofilm eradication concentrations and antibiotic susceptibility were determined. Biofilm formation was quantified using crystal violet. Relative pathogenicity was assessed in a Galleria mellonella model. To correlate changes in phenotype to gene expression, transcriptomic profiles were created through RNA sequencing and variant analysis of genomes was performed in strain EC958. RESULTS: Cinnamaldehyde and furanone-C30 led to increases in susceptibility in planktonic and biofilm-associated UPEC. Relative pathogenicity increased after cinnamaldehyde exposure (4/8 isolates), decreased after furanone-C30 exposure (6/8 isolates) and varied after F-DPD exposure (one increased and one decreased). A total of 9/96 cases of putative antibiotic cross-resistance were generated. Exposure to cinnamaldehyde or F-DPD reduced expression of genes associated with locomotion, whilst cinnamaldehyde caused an increase in genes encoding fimbrial and afimbrial-like adhesins. Furanone-C30 caused a reduction in genes involved in cellular biosynthetic processes, likely though impaired ribonucleoprotein assembly. CONCLUSIONS: The multiple phenotypic adaptations induced during QSI exposure in UPEC should be considered when selecting an anti-infective catheter coating agent.

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

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.