Evaluation of in Vitro Bactericidal Activity of 1.5% Olanexidine Gluconate, a Novel Biguanide Antiseptic Agent

Abstract

Recently, 1.5% olanexidine gluconate, a biguanide compounds, was launched as a new antiseptic agent in Japan. However, the comprehensive bactericidal spectrum of olanexidine gluconate is still unknown. In this study, we evaluated in vitro bactericidal activity of olanexidine gluconate using time-kill assay against various bacteria, mycobacteria, and fungi. With the exception of Burkholderia cepacia and Mycobacterium spp., 1.5% olanexidine gluconate exhibited fast-acting (≤60 s) bactericidal activity against all tested Gram-positive and Gram-negative bacteria, including vancomycin-resistant Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, extended spectrum β-lactamase producing Klebsiella pneumoniae, and multidrug-resistant Pseudomonas aeruginosa. Furthermore, 1.5% olanexidine gluconate eradicated Candida albicans, Microsporum canis, and Malassezia furfur within 3 min. Our findings indicate that olanexidine gluconate has broad spectrum bactericidal activity; therefore, it may be useful for the prevention of a wide range of infectious diseases.

INTRODUCTION

To prevent contact infections, blood stream infections, and surgical site infections (SSIs), such as surgical management and catheter insertion site infections, the proper use of antiseptic agents is critical. Antiseptic agents are classified predominantly into three levels: high, middle, and low. For the antisepsis of the human body, including skin, middle- or low-level antiseptics are generally used. For example, povidone-iodine (PI), ethanol, isopropyl alcohol, and chlorhexidine gluconate (CHG) are recommended as skin antiseptics. Recently, in skin antiseptics used for preoperative surgical sites, it was reported that the use of a chlorhexidine gluconate-including alcohol preparation (CHG-AL) led to better clinical outcomes than the use of PI; however, the antiseptic effects varied by surgical site.^1,2) In addition, PI has the potential to cause iodide allergy and a long time is required to achieve the bactericidal effect.^3,4) In contrast, it was also reported that CHG led to mucosal damage.^5,6) Therefore, PI and CHG are selected based on the surgical site and/or characteristics of the patients. Gram-positive bacteria, such as Staphylococcus aureus and Enterococcus faecalis (and Enterococcus faecium), and Gram-negative bacteria, such as Pseudomonas aeruginosa and Escherichia coli, are usually causative agents of SSIs. Moreover, these bacteria often acquire antimicrobial resistance, leading to intractable infections. Among these bacteria, emergence of CHG tolerant-bacteria has become a worldwide healthcare concern.^7,8)

Olanexidine gluconate, a novel biguanide antiseptic agent, was launched in 2015 in Japan for use as skin disinfectant for surgical sites. Olanexidine gluconate exerts strong and fast-acting bactericidal activity against wide range of bacteria.⁹⁾ The bactericidal mechanism has been reported to occur via an interaction with the bacterial surface molecules, such as lipopolysaccharide and lipoteichoic acid, which subsequently disrupts cell membranes and degrades proteins.⁹⁾ Therefore, the bactericidal effect of olanexidine gluconate is irreversible owing to the loss of intracellular components. This mechanism differs slightly from the most common biguanide compound, CHG.⁹⁾ However, the comprehensive bactericidal spectrum of olanexidine gluconate, including any resistant strains, is still unknown.¹⁰⁾ In this study, we evaluated the in vitro bactericidal activity of olanexidine gluconate by time-kill assay against various bacteria, mycobacteria, and fungi.

MATERIALS AND METHODS

Bacterial Strains

Thirty-nine microorganisms, containing clinical isolates, were tested (Table 1). ATC C (American Type Culture Collection) strains were purchased via Summit Pharmaceuticals International Corporation (Tokyo, Japan). JCM (Japan Collection of Microorganisms) strains were purchased from RIKEN BioResource Center (Ibaraki, Japan). NBRC strains were purchased from the National Institute of Technology and Evaluation (Tokyo, Japan). Vancomycin-resistant Enterococcus faecalis (VRE) NCTC12201 and methicillin-resistant Staphylococcus aureus (MRSA) N315 were kindly provided from Prof. Hiramatsu and Dr. Ito (Department of Bacteriology, Faculty of Medicine, Juntendo University).^11,12) Multidrug-resistant Pseudomonas aeruginosa (MDRP) clinical isolate TP1254 was obtained from our own laboratory stocks.

Table 1. Bactericidal Activity of 1.5% Olanexidine Gluconate by Time-Kill Assay

Strain	Viable bacterial count (CFU/mL)
	0 s	15 s	30 s	1 min
Gram-positive bacterium
Bacillus subtilis ATCC6633	3.1 × 106	<10	<10	<10
Enterococcus faecalis ATCC29212a)	4.6 × 107	<10	<10	<10
Vancomycin-resistant enterococci (VRE) NCTC12201	8.3 × 107	<10	<10	<10
Kokuria rizophila ATCC9341a)	1.1 × 107	<10	<10	<10
Staphylococcus aureus ATCC29213a)	2.4 × 108	3.3 × 102	1.3 × 102	<10
Methicillin-resistant Staphylococcus aureus (MRSA) N315	1.2 × 108	1.2 × 106	6.7 × 102	<10
Staphylococcus epidermidis ATCC14990b)	1.6 × 108	<10	<10	<10
Methicillin-resistant Staphylococcus epidermidis (MRSE) ATCC35984	7.1 × 107	<10	<10	<10
Streptococcus pyogenes ATCC12344b)	5.8 × 107	<10	<10	<10
Cutibacterium acnes ATCC6919b)	5.2 × 107	<10	<10	<10
Clostridioides difficile ATCC9689b)	5.7 × 107	<10	<10	<10
Peptostreptococcus anaerobius JCM6477	6.2 × 107	<10	<10	<10
Gram-negative bacterium
Acinetobacter baumannii ATCC19606b)	3.2 × 107	<10	<10	<10
Burkholderia cepacia JCM5506a)	1.3 × 108	1.3 × 105	5.6 × 104	5.0 × 104
Citrobacter freundii ATCC13316b)	4.8 × 107	<10	<10	<10
Enterobacter cloacae ATCC13047b)	3.5 × 107	<10	<10	<10
Escherichia coli ATCC25922a)	7.8 × 106	<10	<10	<10
Haemophilus influenzae ATCC49247a)	5.0 × 107	<10	<10	<10
Extended spectrum β-lactamase (ESBL) producing Klebsiella pneumoniae ATCC700603	3.4 × 107	<10	<10	<10
Proteus mirabilis ATCC29906b)	1.0 × 108	<10	<10	<10
Proteus hauseri ATCC13315a)	5.0 × 107	<10	<10	<10
Pseudomonas aeruginosa ATCC27853a)	4.0 × 107	<10	<10	<10
Multidrug-resistant Pseudomonas aeruginosa (MDRP) TP1254	7.1 × 107	<10	<10	<10
Salmonella Typhimurium ATCC14028	4.1 × 107	<10	<10	<10
Serratia marcescens ATCC13880b)	7.0 × 107	<10	<10	<10
Stenotrophomonas maltophilia ATCC13637b)	1.6 × 107	<10	<10	<10
Bacteroides fragilis ATCC25285b)	5.8 × 107	<10	<10	<10
Bacteroides ovatus ATCC8483b)	5.3 × 107	<10	<10	<10

<10, detection limit. a) Control strain for antimicrobial susceptibility test. b) Type strain.

Growth Condition

Bacillus subtilis, Kocuria rizophila, S. aureus, Staphylococcus epidermidis, Acinetobacter baumannii, Burkholderia cepacia, Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Proteus hauseri, P. aeruginosa, Salmonella Typhimurium, Serratia marcescens, and Stenotrophomonas maltophilia were grown for 24 h on Tryptone Soya agar (Oxoid Ltd., Hampshire, U.K.) at 35°C under aerobic conditions. E. faecalis and VRE were grown for 24 h on Brain Heart Infusion agar (Oxoid) at 35°C under aerobic condition. Rapidly growing mycobacteria (Mycobacterium fortuitum, Mycobacterium chelonae and Mycobacterium abscessus) were grown for 4 d and slow growing mycobacteria (Mycobacterium kansasii, Mycobacterium avium and Mycobacterium intracellulare) were grown for 14 d on Middlebrook 7H10 agar (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) at 35°C under aerobic conditions. Streptococcus pyogenes was grown for 24 h on sheep blood agar HEM (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) at 35°C in an atmosphere of 5% CO₂. Haemophilus influenzae was grown for 24 h on chocolate agar EX-II (Nissui Pharmaceutical) at 35°C in an atmosphere of 5% CO₂. Proteus mirabilis was grown for 24 h on MacConkey agar (Eiken Chemical Co., Ltd., Tokyo, Japan) at 35°C under aerobic conditions. Candida albicans and filamentous fungi were grown for 48 h and 7 d, respectively, on Sabouraud agar (Eiken Chemical) at 30°C under aerobic conditions. Malassezia furfur was grown for 72 h on 1% olive oil (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) covered Yeast Mold agar (Becton, Dickinson and Company, NJ, U.S.A.) at 30°C under aerobic conditions. Cutibacterium acnes (formerly Propionibacterium acnes), Bacteoides fragilis, Bacteroides ovatus, Peptostreptococcus anaerobius and Clostridioides difficile (formerly Clostridium difficile) were grown for 48 h on modified GAM agar (Nissui Pharmaceutical) under anaerobic conditions.

Evaluation of Bactericidal Effect of 1.5% Olanexidine Gluconate

The evaluation of bactericidal effect of 1.5% olanexidine gluconate (Otsuka Pharmaceutical Factory, Inc., Tokushima, Japan) was performed by time-kill assay.¹³⁾ Bacteria and Mycobacterium spp. were precultured in each medium and suspended into 3 mL phosphate buffered saline (PBS) or distilled water in the presence and absence of 1.5% olanexidine gluconate. To prepare the samples, filamentous fungi were first homogenized and all fungi were suspended into 3 mL distilled water and filtered through a 40 µm cell strainer (Corning Incorporated, NY, U.S.A.). After incubation for several timepoints, the test solutions were neutralized with 10 volumes of neutralization solution [10% Polysorbate (Sigma-Aldrich Co. LLC., Tokyo, Japan), 1.2% Lecithin (Nisshin Oillio Group, Ltd., Tokyo, Japan), 0.5% Sodium thiosulfate (FUJIFILM Wako), 0.04% Potassium dihydrogen phosphate (FUJIFILM Wako), 0.1% TritonX-100 (FUJIFILM Wako), 1.0% Disodium hydrogen phosphate anhydrous (FUJIFILM Wako) and 1.0% Tamol NN8906 (BASF Japan, Tokyo, Japan)]. Subsequently, the test solutions were diluted and plated onto each culture medium plate. The inactivation of olanexidine gluconate after neutralization was validated by using a disk diffusion assay. In addition, the same experiments were performed by using the base solution in the absence of olanexidine gluconate. All experiments were performed at least twice on independent occasions.

RESULTS AND DISCUSSION

We evaluated the bactericidal activity of 1.5% olanexidine gluconate against 39 strains of 35 species, including antimicrobial-resistant strains, by time-kill assay (Table 1). Gram-positive bacteria, excluding S. aureus, and Gram-negative bacteria, excluding B. cepacia, were completely eradicated (<10 colony forming unit (CFU)/mL, detection limit) within 15 s (Table 1). The viable counts of S. aureus were substantially decreased at 15 s, but 60 s was required for complete eradication. B. cepacia, which is known as an inherent antiseptic-tolerant bacteria,¹⁴⁾ was not completely eradicated by 1.5% olanexidine gluconate, even after 30 min exposure (data not shown). Hence, the bactericidal activity of olanexidine gluconate against B. cepacia appeared similar to other low-level antiseptic agents.¹⁴⁾ The 1.5% olanexidine gluconate was found to exert a bactericidal effect against B. subtilis and C. difficile. It was predicted that most of the spore-forming bacteria used in this study were in a vegetative form, because we used their fresh cultures. However, 1.5% olanexidine gluconate could not eradicate B. subtilis spore solution, even after 10 min exposure (data not shown).

Furthermore, we evaluated the bactericidal activity against VRE, MRSA, methicillin-resistant S. epidermidis (MRSE), extended spectrum β-lactamase (ESBL) producing K. pneumoniae, and MDRP, which are the major causative pathogens of nosocomial infections (Table 1). Notably, 1.5% olanexidine gluconate exhibited fast-acting (≤60 s) bactericidal activity against all tested antimicrobial-resistant bacteria (more than 6.5 log₁₀ reduction).

In mycobacterium, olanexidine gluconate did not exert sufficient bactericidal activity against all tested Mycobacterium strains (Table 2), including Mycobacterium tuberculosis H37Rv (data not shown), even after 60 min exposure. Hence, the bactericidal activity of olanexidine gluconate against mycobacterium is low, similar to chlorhexidine gluconate.¹⁵⁾ The mycobacterial cell wall is a highly hydrophobic structure with a mycoylarabinogalactan–peptidoglycan skeleton.¹⁶⁾ The peptidoglycan is covalently linked to the polysaccharide copolymer (arabinogalactan) made up of arabinose and galactose esterified to mycolic acids. Therefore, the most likely mechanism for the high resistance to antiseptics of mycobacteria is associated with their complex cell walls that provide an effective barrier to the entry of these agents.

Table 2. Bactericidal Activity of 1.5% Olanexidine Gluconate against Mycobacteria and Fungi by Time-Kill Assay

Strain	Viable bacterial count (CFU/mL)
	0 s	30 s	3 min	10 min	60 min
Mycobacterium
Mycobacterium kansasii ATCC12478a)	1.3 × 106	NT	8.3 × 105	9.1 × 105	9.9 × 105
Mycobacterium intracellulare ATCC13950	4.0 × 106	NT	3.4 × 106	2.6 × 106	2.2 × 106
Mycobacterium fortuitum ATCC6841	4.5 × 106	NT	1.3 × 106	8.9 × 104	1.3 × 103
Mycobacterium chelonae ATCC35752a)	4.5 × 105	NT	2.2 × 105	9.1 × 104	2.6 × 103
Mycobacterium abscessus ATCC19977	6.7 × 106	NT	5.4 × 106	4.4 × 106	3.7 × 106
Mycobacterium avium JCM15429	1.6 × 107	NT	6.7 × 106	2.5 × 106	1.5 × 106
Fungus
Candida albicans ATCC10231a)	2.8 × 105	<10	<10	<10	NT
Malassezia furfur ATCC14521	7.8 × 105	<10	<10	<10	NT
Aspergillus brasiliensis ATCC16404b)	1.8 × 105	4.1 × 104	1.5 × 104	9.1 × 103	NT
Trichophyton rubrum NBRC6203	9.9 × 105	1.3 × 105	8.3 × 102	<10	NT
Microsporum canis NBRC32464	1.8 × 104	6.8 × 103	<10	<10	NT

<10, detection limit. NT, not tested. a) Control strain for antimicrobial susceptibility test. b) Formerly Aspergillus niger.

In contrast, in fungi, C. albicans and M. furfur exhibited high susceptibility to 1.5% olanexidine gluconate and were eradicated within 30 s. In addition, 1.5% olanexidine gluconate could eradicate Microsporum canis (≤3 min) and Trichophyton rubrum (≤10 min). However, Aspergillus brasiliensis (formerly Aspergillus niger) survived for at least 10 min in 1.5% olanexidine gluconate. These data were consistent with previous reports on the activity of chlorhexidine gluconate.^17,18)

Our data showed that olanexidine gluconate has strong bactericidal activity and broad antibacterial and antifungal spectra. Hagi et al. reported that bactericidal activity of olanexidine gluconate against various bacterial species was compared to CHG and PI by determination of minimum bactericidal concentration.⁹⁾ They used 100 species including Enterococcus faecalis, VRE, Staphylococcus aureus, MRSA, Staphylococcus epidermidis, Streptococcus pyogenes, Cutibacterium acnes, Acinetobacter baumannii, Burkholderia cepacia, Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Proteus mirabilis, Proteus hauseri, Pseudomonas aeruginosa, Salmonella Typhimurium, Serratia marcescens, Stenotrophomonas maltophilia, and Bacteroides fragilis, which were also used in this study. The data showed that olanexidine gluconate had high bactericidal activity as well or better than CHG and PI. Our data were consistent with the previous data. The bactericidal activity of olanexidine gluconate was not affected by organic substances, which often reduce the efficacy of antiseptics and lead to problems in the antisepsis of contaminated surfaces.¹⁹⁾ Therefore, olanexidine gluconate is considered useful for the prevention of contact infections and surgical site infections from various pathogenic microorganisms including antimicrobial-resistant bacteria. In contrast, Nagai et al. recently reported that allergic contact dermatitis was caused by olanexidine gluconate.²⁰⁾ Hence, further clinical studies are necessary to collect more data on not only the antiseptic effects, but also any adverse events arising from the use of olanexidine gluconate.

Acknowledgments

This work was supported by Otsuka Pharmaceutical Factory, Inc.

Conflict of Interest

Hisae Nishioka and Akifumi Hagi are employees of Otsuka Pharmaceutical Factory, Inc.

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