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In vitro evaluation of the antimicrobial activity of HM-242, a novel antiseptic compound

Abstract

The antimicrobial activities of N4-octyl-6,6-dimethyl-N2-(4-methylbenzyl)-1,6-dihydro-1,3,5-triazine-2,4-diamine (HM-242), a novel synthetic compound, were compared with those of chlorhexidine gluconate (CHG). HM-242 was a more potent microbicide than CHG in vitro; however, its minimal inhibitory concentrations were similar. In particular, HM-242 killed various Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecalis, both efficiently and rapidly. HM-242 also showed potent virucidal activity against enveloped viruses such as influenza virus and herpes simplex virus. These characteristics suggest that HM-242 may well be useful as an antiseptic.

Introduction

Antiseptics have a number of important roles in infection control in clinical settings, including hand hygiene and skin surface disinfection of surgical fields and catheter insertion sites. Antiseptics prevent infection by decreasing the number of microbes and thereby decreasing the transmission of pathogens.1, 2, 3, 4, 5, 6

Currently, healthcare-associated infections caused by multidrug-resistant organisms (including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE) and certain Gram-negative bacilli4, 7, 8, 9, 10) are a significant problem. The presence of MRSA and VRE in US hospitals has been steadily increasing since the 1990s. As multidrug-resistant organisms become more prevalent, it becomes more important to prevent infection by using antiseptics besides administration of antibiotics to cure infectious diseases.8, 11 Several investigations have found that various antibiotic-resistant pathogenic microorganisms were as susceptible to disinfectants as antibiotic-sensitive strains, and the Center for Disease Control and Prevention (CDC) does not recommend any special strategies or germicides with higher potencies against multidrug-resistant organisms.4, 5, 8, 12

Chlorhexidine gluconate (CHG) has been widely used for many years. CHG is one of the best antiseptics because it has not only broad-spectrum antibacterial activity, persistent efficacy and residual activity, but is also compatible with most materials, and is safe for humans.1, 3, 7, 13, 14, 15, 16, 17, 18 Therefore, the CDC specifically recommends CHG as the preferred agent for cutaneous antisepsis and surgical hand antisepsis in its ‘Guideline for the Prevention of Intravascular Catheter-Related Infections’ and its ‘Guideline for Hand Hygiene in Health-Care Settings.’17, 19

Methodologies such as time-kill studies, minimal bactericidal concentration (MBC) studies, minimal inhibitory concentration (MIC) studies, carrier tests and phenol coefficient tests are ordinarily used to evaluate the efficacy of antiseptics in vitro. One of the most important characteristics of an antiseptic is that it has microbicidal activity rather than just microbistatic activity because an antiseptic agent needs to kill microorganisms quickly.1, 3 According to some in vitro time-kill studies, chlorhexidine does not kill Gram-positive cocci any faster than it kills Gram-negative bacilli.3, 20 It is important to develop a new antiseptic agent that is fast-acting, persistent, safe and has broad-spectrum virucidal efficacy because Gram-positive cocci such as MRSA and VRE cause healthcare-associated infections. HM-242 is a novel candidate antiseptic that was synthesized recently.21 We show here the results of in vitro efficacy studies that compare HM-242 with CHG. We found that HM-242 is a fast-acting, broad-spectrum microbicide with characteristics that make it suitable for use as an antiseptic.

Materials and Methods

Test substance and antiseptic

HM-242 was synthesized by Hamari Chemicals Ltd, Osaka, Japan (Figure 1). Maskin Solution (20% (w/v) CHG aqueous solution) was provided by Maruishi Pharmaceutical Co. Ltd, Osaka, Japan.

Figure 1
figure1

Chemical structure of HM-242.

Test organisms

The following microorganisms except for clinical isolate were obtained from American Type Culture Collection (Manassas, VA, USA) and used in this study:

Test bacteria and fungi for in vitro evaluations

Acinetobacter calcoaceticus (ATCC no. 23055), Acinetobacter baumannii (ATCC no. 15308), Bacteroides fragilis (ATCC no. 25285), Candida albicans (ATCC no. 10231), C. tropicalis (ATCC no. 750), Citrobacter koseri (ATCC no. 27028), Enterobacter aerogenes (ATCC no. 13048), E. faecalis VSE (ATCC no. 29212), E. faecalis MDR (ATCC no. 51299), E faecalis VRE (ATCC no. 51575), E. faecium VSE (ATCC no. 6569), E. faecium VRE (ATCC no. 51559), Escherichia coli (ATCC nos. 25922 and 11229), E. coli O157:H7 (ATCC no. 43895), Haemophilus influenzae (ATCC no. 19418), Klebsiella pneumoniae pneumoniae (ATCC no. 29995), Micrococcus luteus (ATCC no. 7468), Proteus mirabilis (ATCC no. 7002), Proteus vulgaris (ATCC no. 13315), Providencia rettgeri (ATCC no. 35565), Pseudomonas aeruginosa (ATCC nos. 27853, 15442, BAA-47; PAO1), P. aeruginosa MDR (no. 112905Pa16 Clinical isolate), Salmonella enterica enterica serovar Typhimurium (ATCC no. 14028), Serratia marcescens (ATCC no. 14756), S. aureus aureus MSSA (ATCC no. 29213, ATCC no. 6538), S. aureus aureus MRSA (ATCC no. 33591), S. epidermidis MSSE (ATCC no. 12228), S. epidermidis MRSE (ATCC no. 51625), S. haemolyticus (ATCC no. 29970), S. hominis hominis (ATCC no. 27844), S. saprophyticus (ATCC no. 15305), S. pneumoniae PSSP (ATCC no. 6303), S. pneumoniae PRSP (ATCC no. 700904), S. pyogenes (ATCC no. 19615), Stenotrophomonas maltophilia (ATCC no. 13637).

Viruses and cells used for in vitro evaluations

Herpes simplex virus (HSV) type 1 was passaged and cultured in Vero cells (African green monkey kidney cells). Human influenza virus A H3N2 (FluV) was passaged and cultured in MDCK cells (Madin–Darby canine kidney epithelial cells).

In vitro evaluation of antimicrobial activity

In vitro antimicrobial activity against 37 bacterial strains and 2 yeast strains was evaluated by microdilution in accordance with the CLSI method of MIC evaluation.22 The drug was serially diluted to obtain concentrations ranging from 256 to 0.12 μg ml−1. Next, the challenge strain was added to the assay solution. Finally, the reaction mixture was incubated under ordinary culture conditions, and MICs were determined visually.

In vitro bactericidal activity

The MBCs were determined for 37 bacterial strains and 2 yeast strains. Test solutions were prepared containing 12 different concentrations (0.25–512 μg ml−1) of each drug in water. A 10 μl aliquot of each challenge strain containing approximately 5 × 106 CFU ml−1 was exposed to 190 μl of each concentration of each drug for 15, 30 and 60 s. After the exposure, 30 μl of the reaction mixture was transferred to 30 μl of neutralizer (3% lecithin, 10% Tween-80). Next, 20 μl of the neutralization mixture was transferred to 180 μl of the growth medium and incubated at 37 °C for 24 h. After the incubations were complete, the MBC of each product at each time of exposure was determined visually on the basis of the turbidity of the growth media.

In vitro time-kill evaluation using bacteria and yeast

A time-kill study was performed using the same bacteria and yeast strains that were used in the MIC and MBC evaluations. Test solutions containing three concentrations (0.005, 0.05 and 0.5%) of each drug in water were prepared. Challenge strain solutions containing approximately 1 × 109 CFU ml−1 of each organism in sterile peptone-saline solution were exposed to 9.5 ml of each test solution at room temperature for 15, 30 and 60 s. After the exposure, a 500 μl aliquot of each test mixture was transferred to 9.5 ml of neutralizer (3% lecithin, 10% Tween-80). The neutralization mixture was serially diluted and inoculated onto appropriate agar plates. The plates were incubated under conditions appropriate for each species. After incubation, the number of colonies was counted, and the logarithm decrement (log10 reduction) was calculated.

In vitro virucidal activity

In vitro virucidal evaluations were conducted similar to the time-kill studies, except that viruses were used instead of bacteria and yeasts. Exposure times, neutralization solutions and drug concentrations also varied. In detail, viral titers were determined by calculating 50% tissue culture infectious doses (TCID50) from titration end points in 96-well microtiter plates with preformed cell culture monolayers. The virucidal reaction was carried out with drug concentrations of 0.1 and 0.5% for 0.5, 1, 5 and 10 min. After each exposure, a portion of the mixture was diluted more than 500-fold with the appropriate neutralization solution. Log10 reduction values were calculated as (log10TCID50 of control−log10TCID50 of test sample).

Results

In vitro antimicrobistatic effects of HM-242 on bacteria and yeasts

The susceptibility of 37 bacterial strains and 2 yeast strains to HM-242 was evaluated with the microdilution method (Table 1). HM-242's MIC values were similar to those of CHG. The MIC values of HM-242 ranged from <0.125 to 64 μg ml−1, and those of CHG ranged from <0.125 to 128 μg ml−1. The MIC90 values of HM-242 and CHG were 64 and 32 μg ml−1, respectively.

Table 1 Minimal inhibitory concentrations of HM-242 and CHG

In vitro antimicrobicidal activity

The MBC of HM-242 in the 60-s exposure protocol was less than 8 μg ml−1 for eight of the strains, and it ranged from 16 to 128 μg ml−1 for the other 30 strains (Table 2). The most resistant test organism, C. tropicalis, was killed by 256 μg ml−1 of HM-242. By contrast, the 60-s MBC of CHG for 26 strains was higher than the highest concentration used in this assay (512 μg ml−1), and was therefore not determined. The 60-s MBC of CHG for three strains was less than 8 μg ml−1, and it ranged from 256 to 16 μg ml−1 for seven strains. The other three strains were killed by 512 μg ml−1 of CHG. The 60-s MBC90 of HM-242 was 64 μg ml−1, and that of CHG was not determined (>512 μg ml−1).

Table 2 Minimum bactericidal concentrations of HM-242 and CHG

The results of the time-kill studies using bacteria and fungi are shown in Table 3. Exposure for 15 s to 0.5, 0.05 or 0.005% HM-242 reduced the populations of 38, 32 and 8 challenge strains, respectively, by more than 5.0 log10 (Table 3). Exposure for 15 s to 0.5 and 0.05% CHG reduced the populations of eight and one challenge strains, respectively, by more than 5.0 log10. Exposure for 60 s to 0.05% CHG failed to reduce the population of any of the strains by 5.0 log10.

Table 3 Distribution of test strains in the time-kill study of the bactericidal activities HM-242 and CHG

The results of the time-kill studies using HSV and FluV are shown in Figure 2. Within 0.5 min, 0.5 and 0.1% HM-242 reduced the viability titer of the test viruses below the detection limit of the assay. CHG at concentrations of 0.5 and 0.1% failed to detectably kill FluV at any exposure time less than 5 min. CHG killed only 45.1% of FluV after 10 min of exposure. Against HSV, 0.5% CHG reduced the viability titer to below the detection limit after just 1 min. The cytotoxicities of HM-242 and CHG to Vero cells and MDCK cells were assayed by the same procedure. A 1:256 dilution of a 0.5% solution of either drug was toxic to Vero cells, and a 1:512 dilution of either drug was toxic to MDCK cells.

Figure 2
figure2

Time-kill study of activity against herpes simplex virus (HSV) and FluV. Symbols: closed circles, 0.5% HM-242; closed squares, 0.1% HM-242; open circles, 0.5% chlorhexidine gluconate (CHG); open squares, 0.1% CHG.

Discussion

Antiseptic hand washing has been used by medical personnel to control infections ever since Semmelweis discovered that it reduces patient mortality from hand-borne pathogens.1, 16, 23, 24 Today, antiseptic hand washing has moved beyond the medical professions and is now common practice in food preparation, public facilities and domestic life. CHG has long been used in clinical settings and is an excellent antiseptic. Various reports and guidelines recommend CHG because of its broad activity spectrum, persistence, consistent efficacy and safety.1, 3, 7, 12, 13, 14, 15, 16, 17 However, some problems with CHG have been noted, including its suboptimal bactericidal kinetics (CHG was slower than another antiseptic) and rare anaphylactic reactions to it in humans.25, 26, 27, 28, 29, 30 Recently, a new compound, HM-242, was synthesized as a novel disinfectant. We found that HM-242 has broad-spectrum and fast-acting microbicidal activity in vitro. All of the bacterial and yeast strains tested, including the antibiotic-resistant strains MRSA, VRE, MDRP and PRSP, were sensitive to HM-242, and were significantly inhibited by the presence of HM-242 (64 μg ml−1). Although HM-242's antimicrobial activity is similar to that of CHG, the MBC study revealed that HM-242 is a more effective microbicide for a wide variety of microorganisms. In the time-kill study, HM-242 showed excellent bactericidal and fungicidal efficiency at lower concentrations and shorter exposure times than did CHG. The viability counts of all the microorganisms tested were reduced by exposure to 0.05% HM-242 for 15 s. By contrast, nearly half of the test strains treated with 0.05% CHG were unaffected. In general, MIC data are used to estimate the susceptibility patterns of test strains. A compound that has shown effectiveness in MIC studies and with good time-kill kinetics against both nosocomial flora and resident flora would be effective in the health-care settings where these pathogens are encountered. One important characteristic of antiseptics used by health-care professionals is that they act as microbiologically lethal agents. Time-kill kinetic studies allow the rate of kill of an antiseptic to be assessed provided certain variables are controlled.1, 5 The results of the time-kill studies with HM-242 revealed that it possesses fast-acting and potent bactericidal activity against all of the tested strains, including the multidrug-resistant organisms.

Furthermore, HM-242 had potent virucidal activity that reduced HSV and FluV titers to a level below the detection limit of our assay within 30 s. By contrast, CHG was ineffective against FluV even after 300 s. To be appropriate for use in health-care settings, an antiseptic must have a broad microbial spectrum that includes the nosocomial pathogens. In this respect, HM-242's virucidal activity makes it superior to CHG.

To be an effective antiseptic agent, a drug cannot be merely bacteriostatic; it must be a fast-acting bactericide. The results of the MBC and time-kill studies indicate that HM-242 kills pathogenic bacteria, yeasts and viruses both efficiently and rapidly. Antiseptics with broad-spectrum activity that includes virucidal activity are vital to preventing the spread of infections. The cytotoxicity data indicate that HM-242 is as safe as CHG, which is well known for its low irritability to human skin. Thus, HM-242 has many of the properties required of an antiseptic substance, and it may be as suitable as CHG for controlling the spread of infections.

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Correspondence to Junji Okunishi.

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Okunishi, J., Nishihara, Y., Maeda, S. et al. In vitro evaluation of the antimicrobial activity of HM-242, a novel antiseptic compound. J Antibiot 62, 489–493 (2009). https://doi.org/10.1038/ja.2009.56

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Keywords

  • bactericidal
  • chlorhexidine gluconate
  • disinfection
  • microbicidal
  • virucidal

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