Introduction

Multidrug-resistant (MR) Gram-negative (GN) bacteria pose a major and growing threat for healthcare systems, given the paucity of available and efficient antibiotics drastically complicating the treatment of infections [1, 2]. MR Enterobacteriaceae, Pseudomonas, and Acinetobacter strains have emerged as particularly serious concerns [3, 4].

Antiseptic molecules may help to reduce the dissemination of MR bacteria, especially in high-risk areas, such as intensive care units, when used in patient decolonization procedures [5, 6]. However, reduced susceptibility of some MRGN isolates to some frequently used biocides (i.e., chlorhexidine) has been reported [7,8,9]. A significant bactericidal activity of octenidine dihydrochloride (OCT) was demonstrated against mupirocin-resistant methicillin-resistant Staphylococcus aureus (MRSA) strains [10]. However, information about its efficacy against MRGN remains poorly investigated [11, 12]. In the present study, we aimed to assess the in vitro bactericidal activity of OCT against the most relevant GN species responsible for hospital-acquired infections, including isolates exhibiting MR phenotypes.

Methods

We selected bacterial isolates from five different clinically relevant GN species (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii, and Pseudomonas aeruginosa) from the Culture Collection of the Emerging Antibiotic Resistance Unit (University of Fribourg, Switzerland). Five clonally unrelated isolates were chosen for each species, including, in each case, a single susceptible wild-type strain, and four MR isolates previously characterized for their sequence type and resistance mechanisms, including the production of extended-spectrum β-lactamases (ESBLs) and carbapenemases (Table 1). Susceptibility to antibiotics was determined by disk diffusion, according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints [13]. All the clinical isolates corresponded to either 3MRGN (resistance to three out of the following antibiotic substances: penicillins, cephalosporins, quinolones, and carbapenems) or 4MRGN (resistance to all four pre-cited classes), according to the Robert Koch Institute definition of multidrug resistance [14].

Table 1 Bacterial isolates tested, their resistance mechanisms, and susceptibility profile
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Evaluation of the minimum inhibitory concentrations (MICs) of chlorhexidine digluconate (CHG; Sigma-Aldrich, St. Louis, MO, USA) were carried out, following the standards of the Clinical and Laboratory Standards Institute (CLSI) [15]. The range of concentrations tested was 0.3–312.8 mg/L, as used previously [10].

The efficacy of OCT was determined in accordance with BS EN 13727:2012+A1 [16], (a) without organic load, (b) under “clean conditions” (0.3 g/L BSA, Sigma-Aldrich, St. Louis, MO, USA), and (c) under “dirty conditions” (3 g/L BSA + 3 mL/L defibrinated sheep blood, Oxoid, Pratteln, Switzerland). Samples were incubated for different contact times (30 s, 1, 2.5, or 5 min) with OCT (Schülke & Mayr GmbH, Germany) diluted to the final test concentrations ranging from 0.00001% to 0.01%. After the given contact times, the activity of OCT was neutralized using a combination of 0.1% tryptone, 0.85% NaCl, 3% Tween 80, 0.3% lecithin, 3% saponin, and 0.1% histidine, without interfering with bacterial growth. Subsequently, serial dilutions of the final mixture were spread onto neutralizing agar plates and incubated for 24 h and 48 h at 37 °C. Colonies were counted and the reduction factor (RF) was determined as the difference between the log10 number of cells in the test solution at the beginning of the contact time and the log10 number of recovered colonies in the test solution. A 5 log10 reduction within ≤5 min was considered effective according to the test norm EN13727.

Results and discussion

Although all E. coli isolates had low MICs (ranging from 1 to 4 mg/L), all other isolates had MICs of CHG ranging from 32 to 128 mg/L, thus showing poor efficacy of the molecule against most MRGN isolates (Table 1). Noteworthy, to date, there is no breakpoint consensus to define biocide-reduced susceptibility, including for CHG. Based on the epidemiological cutoff (ECOFF) proposed by Morrissey et al. [17], we might consider most of our isolates as non-susceptible to CHG.

A reduction of >5 log10 was obtained for all wild-type strains after 30-s contact with OCT at a concentration of 0.01% (=100 ppm), 0.001% (=10 ppm), and 0.0005% (=5 ppm). OCT at a concentration of 0.0001% (=1 ppm) showed a time-dependent activity, achieving a reduction of >5 log10 after a contact time of 2.5 min. Finally, a concentration of 0.00001% (=0.01 ppm) was not effective for any given time lapse (Fig. 1).

Fig. 1
figure 1

Time-dependent efficacy of different concentrations of octenidine against Acinetobacter baumannii wild type. These data are representative of the efficacy observed for the other strains tested

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All strains showed sensitivity against 0.01% OCT at a contact time of only 1 min, even in the presence of 0.3 g/L BSA (“clean conditions”) or of 3 g/L BSA + 3 mL/L erythrocytes (“dirty conditions”), resulting in a reduction factor of >5 log10 (Table 2).

Table 2 Log10 reduction factor obtained with octenidine 0.01% at a contact time of 1 min for all test isolates, with or without organic load
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Conclusions

The present study showed that octenidine dihydrochloride (OCT) is highly effective against multidrug-resistant (MR) Gram-negative (GN) pathogens within a very short period of time, under either non-organic or organic conditions, independent of increased minimum inhibitory concentrations (MICs) towards chlorhexidine digluconate (CHG) or of the overall susceptibility to antibiotics. Among the tested isolates, some produced the most threatening resistance mechanisms that may be encountered worldwide in MRGN pathogens, namely carbapenemases.

The results obtained in this study encourage considering OCT, which is well tolerated and without resistances reported so far [18], as an alternative antiseptic for controlling the spread of MR bacteria, either being Gram-positive or Gram-negative. Further studies are required to better evaluate the impact of OCT in clinical practice for preventing infections caused by MRGN, such as that recently performed by Gastmeier et al. [19], who showed no significant impact on the prevention of MRGN acquisition in intensive care units.