Phenotypic Variation in Clinical S. aureus Isolates Did Not Affect Disinfection Efficacy Using Short-Term UV-C Radiation

Abstract

Pigmentation, catalase activity and biofilm formation are virulence factors that cause resistance of Staphylococcus aureus to environmental stress factors including disinfectants. In recent years, automatic UV-C room disinfection gained greater importance in enhanced disinfection procedures to improve disinfection success in hospitals. In this study, we evaluated the effect of naturally occurring variations in the expression of virulence factors in clinical S. aureus isolates on tolerance against UV-C radiation. Quantification of staphyloxanthin expression, catalase activity and biofilm formation for nine genetically different clinical S. aureus isolates as well as reference strain S. aureus ATCC 6538 were performed using methanol extraction, a visual approach assay and a biofilm assay, respectively. Log₁₀ reduction values (LRV) were determined after irradiation of artificially contaminated ceramic tiles with 50 and 22 mJ/cm² UV-C using a commercial UV-C disinfection robot. A wide variety of virulence factor expression was observed, indicating differential regulation of global regulatory networks. However, no direct correlation with the strength of expression with UV-C tolerance was observed for either staphyloxanthin expression, catalase activity or biofilm formation. All isolates were effectively reduced with LRVs of 4.75 to 5.94. UV-C disinfection seems therefore effective against a wide spectrum of S. aureus strains independent of occurring variations in the expression of the investigated virulence factors. Due to only minor differences, the results of frequently used reference strains seem to be representative also for clinical isolates in S. aureus.

Keywords: UV-C; biofilm; catalase activity; disinfection; pigmentation; tolerance.

Figure 1

Quantification of pigmentation (A) catalase level (B) and biofilm formation (C) in different clinical S. aureus isolates. Staphyloxanthin levels (A) were determined by absorbance 465 nm and were normalized to the OD₆₀₀ of low-pigmented ST45. The dots represent the values (n = 6) determined in three independent experiments. The median value of each isolate is shown by red horizontal lines. Catalase activity (B) is illustrated by foam height determined in five independent experiments. The median value is represented by bar height, which is supplemented by error bars showing the interquartile range. Biofilm formation (C) was evaluated in three independent experiments, resulting in 48 values per condition and strain. The bars show the mean value of the A₅₇₀ value, supplemented by the standard deviation, which is represented as an error bar. The colouring of the bars represents the growth medium used (TSB: light grey; TSB + 4% NaCl: medium grey; TSB + 4% ethanol: dark grey). A₅₇₀ values < 0.1 were defined as biofilm-negative (dark grey dotted line) whereas values > 0.5 corresponded strong biofilm formation (light grey dotted line), respectively.

Figure 2

Log reduction values (LRV) of clinical S. aureus isolates after irradiation with a UV-C dose of 50 mJ/cm² (A) and 22 mJ/cm² (B). Boxplots represent LRV determined in five independent experiments. Scatter of the individual measured values is represented by the individual points. The unfilled boxplot display the standard test organism. Significant result (p < 0.001 = **) of statistical pairwise comparison in (A) is shown by the bracket.