MoWa: A Disinfectant for Hospital Surfaces Contaminated With Methicillin-Resistant Staphylococcus aureus (MRSA) and Other Nosocomial Pathogens

Abstract

Introduction: Staphylococcus aureus strains, including methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA), are a main cause of nosocomial infection in the world. The majority of nosocomial S. aureus-infection are traced back to a source of contaminated surfaces including surgery tables. We assessed the efficacy of a mixture of levulinic acid (LA) and sodium dodecyl sulfate (SDS), hereafter called MoWa, to eradicate nosocomial pathogens from contaminated surfaces.

Methods and results: A dose response study demonstrated that MoWa killed 24 h planktonic cultures of S. aureus strains starting at a concentration of (LA) 8.2/(SDS) 0.3 mM while 24 h preformed biofilms were eradicated with 32/1.3 mM. A time course study further showed that attached MRSA bacteria were eradicated within 4 h of incubation with 65/2 mM MoWa. Staphylococci were killed as confirmed by bacterial counts, and fluorescence micrographs that were stained with the live/dead bacterial assay. We then simulated contamination of hospital surfaces by inoculating bacteria on a surface prone to contamination. Once dried, contaminated surfaces were sprayed with MoWa or mock-treated, and treated contaminated surfaces were swabbed and bacteria counted. While bacteria in the mock-treated samples grew at a density of ~10⁴ cfu/cm², those treated for ~1 min with MoWa (1.0/0.04 M) had been eradicated below limit of detection. A similar eradication efficacy was obtained when surfaces were contaminated with other nosocomial pathogens, such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, or Staphylococcus epidermidis.

Conclusions: MoWa kills planktonic and biofilms made by MRSA and MSSA strains and showed great efficacy to disinfect MRSA-, and MSSA-contaminated, surfaces and surfaces contaminated with other important nosocomial pathogens.

Keywords: Staphylococcus aureus; contaminated surface; disinfectant; methicillin-resistant Staphylococcus aureus; nosocomial pathogens.

Figure 1

MoWa eradicates planktonic and biofilm cultures of MRSA strains. Bacteria were inoculated (~1 × 10⁶ cfu/ml) in 24-well plates containing BHI broth and left untreated (control) or treated with the indicated dose of a mixture of levulinic acid (LA) and SDS (MoWa). (A) Treated and untreated NRSA384 cultures were incubated at 37°C for 24 h and then planktonic staphylococci were harvested, diluted and plated to obtain the density (cfu/ml). (B) The indicated MRSA or MSSA strain was left untreated or challenged with MoWa 8.2/0.3 mM. (C) Strain NRS384 was inoculated as above and incubated from 24 h. Planktonic staphylococci were then removed, and biofilms were washed and added with BHI broth. These biofilms were treated with the indicated dose of a mixture of levulinic acid (LA) and SDS for 24 h and then biofilms were harvested to obtain colony counts (cfu/ml). Error bars represent the standard errors of the means calculated using data from at least three independent experiments. *p<0.05 compared to control. LOD, limit of detection. In panels (A, C) the median density is shown inside the bar of the untreated control.

Figure 2

MoWa kills preformed biofilm cultures of MRSA strain NRS384. (A) Bacteria were inoculated (1 × 10⁶ cfu/ml) in (A) 24-well plates, or (B) 8-well slides, containing BHI broth and incubated for 4 h. Planktonic staphylococci were then removed and biofilms were washed and added with BHI broth. These biofilms were left untreated or treated for (A) 24 h, 12 h, or 4 h, or (B) for 4 h with a mixture levulinic acid (LA) and SDS at a concentration of 65/2 mM, 32/1 mM, or 16/0.6 mM. Biofilms were (A) harvested to obtain colony counts (cfu/ml) or (B) stained with the LIVE/DEAD BacLight bacterial viability kit and photograph using a Nikon upright fluorescence microscope. Error bars in (A) represent the standard errors of the means calculated using data from at least three independent experiments. *p<0.05 compared to control. (C) Bacteria were inoculated as above in eight-well slides and incubated for 24 h after which biofilms were treated for 24 h with MoWa 65/2 mM or left untreated. Preparations were stained with the LIVE/DEAD BacLight bacterial viability kit and imaged using a confocal microscope. Panels show XY or YZ optical images.

Figure 3

Minimum inhibitory concentration of MoWa against MRSA and MSSA strains. S. aureus strain (A) NRS408, (B) NRS49, (C) NRS236, (D) NRS230, or (E) NRS242 was inoculated in a six-well plate and treated with a mixture levulinic acid (LA) and SDS at a concentration of 1/0.04 M, 0.52/0.02 M, or 0.26/0.01 M for 1 h. Bacteria were removed, diluted and plated onto BHI agar plates containing 7% of NaCl to obtain colony counts (cfu/ml). Error bars represent the standard errors of the means calculated using data from at least three independent experiments. *p<0.05 compared to control.

Figure 4

MoWa efficiently decontaminate surfaces containing MRSA and MSSA strains. (A) Sterilized surface of 10 cm2 was spiked with ~10⁶ cfu S. aureus strain and air-dried during 1 h. Contaminated surfaces were then sprayed with PBS (control) or with a mixture levulinic acid (LA) and SDS at a concentration of 1/0.04 M (MoWa). Bacteria were removed from MoWa treated, or mock-treated, surfaces 1 min post-treatment and immediately diluted and plated onto BHI agar plates containing 7% of NaCl to obtain colony counts (cfu/ml). Strains tested were (B) NRS384, (C) NRS408, (D) NRS49, (E) NRS236, (F) NRS230 or (G) NRS242. Error bars represent the standard errors of the means calculated using data from at least three independent experiments.

Figure 5

MoWa efficiently decontaminate surfaces containing nosocomial pathogens. The indicated bacterial strain was inoculated in a 10-cm2 sterile surface and let dry for ~1 h. Contaminated surfaces were sprayed with PBS (control) or with a mixture levulinic acid (LA) and SDS at a concentration of 1/0.04 M. Bacteria were removed from MoWa-treated or mock-treated surfaces after 1 min and immediately diluted and plated onto BHI agar plates to obtain colony counts (cfu/ml). Error bars represent the standard errors of the means calculated using data from at least three independent experiments. *p<0.05 compared to control.