Efficacy of octenidine against antibiotic-resistantStaphylococcus aureusepidemic clones

Sir, The increased prevalence of MRSA strains resistant to mupirocin and chlorhexidine1,2 has demonstrated the need for alternative substances for effective MRSA decolonization, without adverse reactions in patients during application. The bactericidal effect of octenidine has been previously shown.3,4 Here, we investigated for the first time, to our knowledge, the bactericidal activity of the antimicrobial octenidine against international epidemic clones of Staphylococcus aureus in vitro, including mupirocin-resistant strains. Bacterial isolates were selected from the Staphylococcal Culture Collection of the Molecular Genetics Laboratory (ITQB, Portugal) in order to include both MRSA and MSSA and different mupirocin-resistant phenotypes: (i) high-level mupirocin-resistant MRSA isolates HP002 and HP032; (ii) low-level mupirocin-resistant MRSA isolates 1A and 4A; (iii) mupirocin-susceptible MRSA isolates HDES57 and HU245; and (iv) mupirocin-susceptible MSSA isolates IPOP37 and HSA9. The selected isolates were originally recovered from inpatients and belonged to various international epidemic clones: MRSA isolates corresponded to USA300 (ST8-IVa) or related (ST8-IVc), ST22-IVh and ST5-II, while the MSSA isolates were characterized by ST30 and ST15 (Table 1). S. aureus test isolates: demographic data, mupirocin resistance and molecular characterization HL, high level; LL, low level; S, susceptible; SCCmec, staphylococcal cassette chromosome mec; PVL, Panton–Valentine leucocidin. S. aureus test isolates: demographic data, mupirocin resistance and molecular characterization HL, high level; LL, low level; S, susceptible; SCCmec, staphylococcal cassette chromosome mec; PVL, Panton–Valentine leucocidin. Mupirocin resistance was determined by the disc diffusion method according to EUCAST breakpoints (http://www.eucast.org/). High- and low-level mupirocin resistance were distinguished by comparison of 5 and 200 μg mupirocin disc diffusion halos5 and MICs were determined by Etest (bioMérieux, Marcy-l’Étoile, France). All isolates were tested for the presence of mupA and mupB genes.6,7 Two MRSA isolates showed high-level mupirocin resistance (MIC >1024 mg/L and presence of mupA), two MRSA isolates showed low-level mupirocin resistance (MICs = 24 and 32 mg/L), and the two remaining MRSA isolates and the two MSSA isolates were susceptible to mupirocin (MICs = 0.25–0.75 mg/L). Quantitative suspension tests were carried out according to European Standard Guidelines EN 13727:2012 + A1.8 Samples were incubated for different contact times (18 s, 30 s, 1 min, 2.5 min and 5 min) with octenidine dihydrochloride (Schülke & Mayr GmbH, Norderstedt, Germany) diluted to final test concentrations ranging from 0.00001% to 0.05% (w/w). After a given contact time, 1 mL of sample was added to 9 mL of neutralizing solution in order to terminate the activity of octenidine without interfering with bacterial growth, and spread onto neutralizing agar plates. Colonies were counted after incubation for 24 and 48 h at 37°C, and the reduction factor was determined. A 5 log10 reduction in ≤5 min was considered effective according to EN 13727.8 Octenidine was not sufficiently effective at a concentration of 0.00001% (=0.1 ppm), independently of given contact times, whereas a bacterial reduction of >6 log10 was obtained after 30 s for all eight isolates tested when 0.001% octenidine (=10 ppm) was used. When the active agent was tested at 0.0001% (=1 ppm), a time-dependent activity was demonstrated, resulting in full efficacy at a contact time of 5 min (Figure 1). Time-dependent efficacy of different octenidine concentrations against high-level mupirocin-resistant MRSA strain HP002. The data for the high-level mupirocin-resistant MRSA strain HP002 are representative of the time-dependent efficacy observed for the eight isolates tested. Serum albumin (Sigma–Aldrich, St Louis, MO, USA) at 0.6% was used to simulate high organic load at a clinically relevant concentration, which was determined in human nasal mucosa in a previous study.9 Use of 0.001% octenidine only resulted in >4 log10 reduction within 30 s when protein was added. Although a high albumin level delayed the antibacterial activity of octenidine, which has also been reported for polyhexanide–biguanide-based antiseptics,10 all strains showed susceptibility to 0.01% octenidine (=100 ppm) in the presence of organic load within a contact time of 30 s, resulting in a reduction factor >6 log10 (Table 2). Log10 reduction factor at a contact time of 30 s determined for eight different S. aureus strains with or without organic load (0.6% albumin) Colonies were counted at 24 h and confirmed at 48 h. The log10 reduction factor was calculated according to EN 13727. Log10 reduction factor at a contact time of 30 s determined for eight different S. aureus strains with or without organic load (0.6% albumin) Colonies were counted at 24 h and confirmed at 48 h. The log10 reduction factor was calculated according to EN 13727. Additionally, octenisept®, a registered product containing octenidine intended for use as an antiseptic for wounds and mucous membranes (0.1% octenidine + 2% phenoxyethanol; Schülke & Mayr GmbH, Norderstedt, Germany), was used in the same experimental set-up and was demonstrated to be highly effective, showing >6 log10 reduction within 30 s with and without albumin (Table 2). No significant differences were observed in the octenidine efficacy against the different S. aureus clonal lineages or against strains with different mupirocin resistance levels, suggesting results may be extrapolated to other clones. Susceptibility to octenidine might be different when S. aureus is growing under biofilm conditions. Here, we showed that 0.001% octenidine was fully effective after a short time against several worldwide spread epidemic clones, some of which were mupirocin resistant, such as the international epidemic MRSA USA300. Octenidine is known to be a safe and effective antimicrobial with a low risk of resistance development,4 and has been successfully used for MRSA decontamination.3 Although promising results have been reported in the past with polyhexanide,11 a recent double-blind, placebo-controlled superiority trial demonstrated that a single polyhexanide decolonization course is not effective in eliminating MRSA carriage.12 Therefore, octenidine appears to be a promising alternative for S. aureus eradication; all the more so since the tested concentrations of octenidine, which were effective, were significantly lower than those used in the clinical setting (0.05%–0.1%). This could be of particular value given that strains of S. aureus are becoming increasingly resistant to some commonly used antimicrobials, such as mupirocin. This study was partially supported by Schülke & Mayr GmbH, Vienna, Austria, and by Fundação para a Ciência e a Tecnologia, Portugal, through grant UID/CBQ/04612/2013 to Research Unit MOSTMICRO. T. C. received scholarship SFRH/BPD/72422/2010. The sponsors had no role in the design of the study or the collection, analysis and interpretation of data. None to declare. The active compounds were provided by Schülke & Mayr GmbH, Norderstedt, Germany. We thank Dr Decousser for providing low-level mupirocin-resistant MRSA isolates.