Activity of N-Chlorotaurine against Long-Term Biofilms of Bacteria and Yeasts

Background: N-chlorotaurine (NCT), an antiseptic that originates from the human defense system, has broad-spectrum microbicidal activity and is well tolerated by human tissue and applicable to sensitive body regions. Bacteria in short-term biofilms, too, have been shown to be killed by NCT. It was the aim of the present study to demonstrate the activity of NCT against bacteria and yeasts in longer-lasting biofilms, including their co-culture. Materials and methods: Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella variicola biofilms were grown for 14 weeks in MBEC^TM inoculator with 96 well base. Some pegs were pinched off weekly and incubated in 1% NCT in PBS (PBS only for controls) at pH 7.1 and 37 °C, for 30 and 60 min. Subsequently, bacteria were resuspended by ultrasonication and subjected to quantitative cultures. Similar tests were conducted with C. albicans biofilms grown on metal (A2-steel) discs for 4 weeks. Mixed co-cultures of C. albicans plus each of the three bacterial strains on metal discs were grown for 5-7 weeks and weekly evaluated, as mentioned above. Results: Single biofilms of S. aureus, P. aeruginosa, and K. variicola grew to approximately 1 × 10⁶ colony forming units (CFU)/mL and C. albicans to 1 × 10⁵ CFU/mL. In combined biofilms, the CFU count was about 1 log₁₀ lower. Viable counts of biofilms of single bacteria were reduced by 2.8 to 5.6 log₁₀ in 1% NCT after 60 min (0.9 to 4.7 log₁₀ after 30 min) with Gram-negative bacteria being more susceptible than S. aureus. Significant reduction of C. albicans by 2.0 to 2.9 log₁₀ occurred after 4 h incubation. In combined biofilms, viable counts of C. albicans were reduced by 1.1 to 2.4 log₁₀ after 4 h, while they reached the detection limit after 1 to 2 h with bacteria (2.0 to > 3.5 log₁₀ reduction). Remarkably, older biofilms demonstrated no increase in resistance but constant susceptibility to NCT. This was valid for all tested pathogens. In electron microscopy, morphological differences between NCT-treated and non-treated biofilms could be found. Conclusions: NCT is active against long-term biofilms of up to several months irrespective of their age. Combined biofilm cultures of yeasts and bacteria show a similar susceptibility pattern to NCT as single ones. These results contribute to the explanation of the clinical efficacy of NCT, for instance, in infected chronic wounds and purulently coated crural ulcerations.