Validation of AKACID Plus as a Room Disinfectant in the Hospital Setting

Abstract

AKACID Plus, a novel polymeric guanidine with broad antimicrobial activity against multiantibiotic-resistant bacterial strains, was used in the present study as a room disinfectant. Disinfection of closed rooms experimentally contaminated with antibiotic-susceptible and multiresistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Escherichia coli was performed using AKACID Plus at concentrations of 0.1, 0.25, and 0.5% for 100 min. Bacterial suspensions were distributed on plastic and stainless steel plates and placed in a test room. Recovery of the test microorganisms was determined before nebulizing, 60 and 100 min after initiation, and 4 h after the end of room disinfection by a simple swab-rinse technique. The swab-rinse method demonstrated a dose- and time-dependent effectiveness of AKACID Plus in eradicating S. aureus, E. coli, and P. aeruginosa on plastic and stainless steel plates. Nebulized 0.5% AKACID Plus was successful in eliminating all hospital pathogens within 340 min. After the use of 0.25% AKACID Plus, MRSA was still detectable on microbial carrier plates. The test concentration of 0.1% AKACID Plus achieved a significant reduction of S. aureus and P. aeruginosa on plastic and stainless steel plates but was sufficient to eradicate only E. coli. These results suggest that nebulized AKACID Plus at a concentration of 0.5% is a potent substance for eradication of pathogenic organisms in the hospital setting.

Data from the World Health Organization show that in the United States some 14,000 individuals are infected and die each year as a result of drug-resistant microbes acquired in hospitals. In intensive care units, nosocomial infections increase the total costs by $3,306 and the length of stay by 18.2 days per patient (3). Methicillin-resistant Staphylococcus aureus (MRSA) and multiresistant gram-negative rods are wreaking havoc in hospital wards around the world (24). Herr et al. (12) determined additional costs of hygiene measures (barrier precautions, isolation, and decontamination) required for MRSA carriers in German hospitals by averaging 372 euros for one MRSA patient hospital-day and 9,263 euros per MRSA case. Already, MRSA and extended-spectrum-beta-lactamase-producing Enterobacteriaceae have spread outside hospitals. The epidemic spread of resistant bacteria and resistance genes is supported primarily by selection of resistant microorganisms by frequent application of antimicrobial agents, inadequate or inappropriate therapy, use of broad-spectrum antibiotics as growth promoters for animal foods and as pesticides for agriculture (16), and, because of lack of general hygiene measures, transmission of hospital strains to other patients and medical staff (2, 10, 18, 20).

Clearly, preventive measures for the termination of this mode of selection and transmission are of vital importance. In hospital and health care settings, antiseptics and disinfectants are essential tools for infection control and aid in prevention of nosocomial infections (5, 9). By acting rapidly, disinfectants can prevent the spread of antibiotic-resistant pathogens (4). It has been postulated that room disinfection in hospital settings is an important measure in the prevention of colonization and new infections.

The novel polymeric guanidine AKACID Plus is a new member of the cationic family of disinfectants. It shows high water solubility with broad in vitro activity against gram-positive and gram-negative bacteria and fungi. Recently, we have demonstrated bactericidal activity of AKACID Plus in basic and extended quantitative suspension tests against bacterial quality control strains (15). Moreover, similar MICs were determined for antibiotic-sensitive and multiantibiotic-resistant bacterial strains, whereas the MIC₉₀s of chlorhexidine and mupirocin showed 4-fold and 32-fold increases for MRSA, respectively, in comparison to methicillin-sensitive S. aureus (A. Buxbaum, C. Kratzer, W. Graninger, and A. Georgopoulos, submitted for publication). The aim of the present study was to evaluate the antimicrobial activities of different concentrations of AKACID Plus for disinfection of closed rooms, which had been experimentally contaminated by antibiotic-susceptible and multiresistant S. aureus, Pseudomonas aeruginosa, and Escherichia coli, by a simple swab-rinse technique.

MATERIALS AND METHODS

Disinfectant and neutralizing solutions.

A stock solution of AKACID Plus, a 3:1 mixture of poly(hexamethylen-guanidinium-chloride) and poly[2-(2-ethoxy)-ethoxyethyl]-guanidinium-chloride as a 25% (vol/vol) aqueous solution (Ch. 1007; POC, Vienna, Austria), was used and diluted with tap water to the desired concentrations of 0.1, 0.25, and 0.5%, vol/vol (pH 6.5). Sodium tryptone solution supplemented with neutralizing substances, i.e., 3% (wt/wt) saponin (VWR International, Fontenay sous Bois, France), 3% (wt/wt) polysorbate 80 (Merck, Hohenbrunn, Germany), 0.1% (wt/wt) histidine (Fluka, Buchs, Switzerland), and 0.1% (wt/wt) cysteine (Fluka), was used as a neutralizer as described previously (15).

Microorganisms.

For activity testing, S. aureus ATCC 6538, P. aeruginosa ATCC 15442, and E. coli ATCC 10536 were selected. The multiantibiotic-resistant clinical strains S. aureus 9892 (resistant to oxacillin, amoxicillin-clavulanic acid, cefazolin, gentamicin, erythromycin, clindamycin, ciprofloxacin, and mupirocin), P. aeruginosa A9726I (resistant to piperacillin-tazobactam, ceftazidime, cefepime, fosfomycin, tobramycin, and ciprofloxacin), and E. coli 1905 (resistant to mezlocillin, piperacillin, amoxicillin-clavulanic acid, cefazolin, cefotaxime, cefepime, gentamicin, trimethoprim, and ciprofloxacin) were utilized. These strains were isolated and identified in 2005 at the Department of Internal Medicine I, Division of Infectious Diseases and Chemotherapy, Medical University of Vienna, from wound, respiratory tract, and urinary tract infections.

Preparation of test plates.

Plastic boards (6.5 by 5 cm) (Fackelmann, Hersbruck, Germany) and stainless steel plates (5 by 5 cm) served as microbial carriers. Viable ATCC and multiresistant strains of S. aureus, P. aeruginosa, and E. coli were used as test organisms. Following the procedure of the European basic quantitative suspension test (6), bacteria were grown on tryptone soy agar (TSA) (Oxoid, Basingstoke, Hampshire, United Kingdom) for 24 h and transferred to TSA for another 24 h. The test bacteria were then suspended in sodium tryptone solution to an optical density at 620 nm corresponding to a concentration of (1.5 × 10⁸ to 5 × 10⁸ CFU/ml). One hundred microliters of the phase 1 standard test suspension was inoculated onto the hard surfaces and evenly disturbed with a sterile glass spatula. A single test plate was contaminated with the test suspension of only one test strain. Microbial carriers were dried for 1 h in a laminar airflow cabinet at a room temperature of 20 to 22°C and a relative humidity ranging from 45 to 60%.

Room disinfection in the test room.

In order to evaluate the activity of AKACID Plus as a room disinfectant, a test room of approximately 41 m³ was chosen. Medical devices and equipment were left in the room. The inlet and outlet vents of the air-conditioning system were sealed with adhesive tape, and the door and windows were closed. For each test strain, microbial carrier plates were placed in a floor corner, below the table, on the work space, and on the cupboard. After the bacterial carriers were placed, 5 liters of liquid containing 0.1, 0.25, or 0.5% AKACID Plus solution or 5 liters of liquid alone (AKACID Plus-free control) was poured in a FONTAN Portastar ULV aerosol applicator (Swingtec, Isny, Germany), which produces a droplet size of 2 to 20 μm. Nebulizing with gaseous AKACID Plus or water alone was performed for 100 min.

Recovery of the test bacteria.

To evaluate the antimicrobial activity of AKACID Plus, the survival of the test bacteria was determined before nebulizing (time point 0) and at 60 (time point 1) and 100 (time point 2) min after the initiation and 4 h after the end (time point 3) of room disinfection in the test room, using a simple swab-rinse technique with neutralizing solution. For this detection method, 1.5 ml neutralizing solution was transferred onto each test surface. With this fluid and a premoistened cotton swab, the test area was systematically abraded for 15 s, 0.5-ml amounts of the neutralizing solution were collected, and 10-fold dilutions in neutralizing solution were prepared and plated on TSA containing neutralizing substances. Swab-rinse cultures were incubated for 48 to 72 h at 37°C. Bacterial colonies on TSA were distinguished on the basis of different morphology (size and color of the colonies). To confirm the presence of S. aureus, E. coli, and P. aeruginosa, bacterial cells were cultured on blood agar and identified by biochemical tests (API Staph, API 20E, and API 20NE).

Data and statistical analysis.

The reduction of the number of viable bacterial cells (CFU/plate) was described by arithmetic means and standard deviations from three individual experiments for 0.1, 0.25, and 0.5% AKACID Plus in comparison to the biocide-free control at three different time points (60 and 100 min after the initiation and 4 h after the end of room disinfection) on plastic and stainless steel plates. Differences between the selected concentrations and the biocide-free control were assessed with Student's t test for independent samples. If significance was achieved, the multiple comparison of means was performed using the Bonferroni-Holm correction; the multiple-comparison significance level was ≤0.05.

RESULTS

Room disinfection with 0.1, 0.25, and 0.5% AKACID Plus.

Three room disinfection trials with 0.1, 0.25, and 0.5% AKACID Plus and with the biocide-free control were performed with antibiotic-sensitive and multiresistant strains of S. aureus (Fig. 1), E. coli (Fig. 2), and P. aeruginosa (Fig. 3) applied on stainless steel and plastic plates. In the presence and absence of AKACID Plus, the temperature and relative humidity in the test room increased from 21 to 23°C and 40 to 60% humidity to 24 to 25°C and 85 to 100% humidity during the nebulizing procedure. Four hours after the end of nebulizing, the relative humidity reached the initial value again, while the temperature was still elevated. Recovery of the tested strains from steel and plastic plates was evaluated before nebulizing and at 60 and 100 min after the initiation and 4 h after the end of room disinfection by a swab-rinse technique. At time point 0 (before room disinfection) 1.2 × 10⁶ to 1.0 × 10⁷ CFU of S. aureus, 6.0 × 10⁵ to 2.2 × 10⁶ CFU of E. coli, and 2.0 × 10⁵ to 1.5 × 10⁶ CFU of P. aeruginosa were detectable on stainless steel plates and 5.0 × 10⁵ to 4.8 × 10⁶ CFU of S. aureus, 2.5 × 10⁵ to 1.3 × 10⁶ CFU of E. coli, and 1.5 × 10⁵ to 1.0 × 10⁶ CFU of P. aeruginosa were detectable on plastic plates. In the absence of AKACID Plus, a moderate reduction of the bacterial count of <1 log₁₀ unit was seen for S. aureus (Fig. 1) at 4 h after the end of nebulizing, whereas a reduction of 1 to 3 log₁₀ units was detected for the gram-negative test organisms (Fig. 2 and 3) on plastic and stainless steel plates.

FIG. 1.

Time-dependent bacterial reduction of the ATCC strain (top) and the multiantibiotic-resistant strain 9892 (bottom) of S. aureus on stainless steel (m) and plastic (p) plates after the use of 0.5, 0.25, and 0.1% AKACID Plus (AP) in comparison to an AKACID Plus-free control on steel and plastic plates, determined by the swab-rinse technique. Results represent averages for four samples ± 1 standard deviation for three independent experiments.

FIG. 2.

Time-dependent bacterial reduction of the ATCC strain (top) and the multiantibiotic-resistant strain 1905 (bottom) of E. coli on stainless steel (m) and plastic (p) plates after the use of 0.5, 0.25, and 0.1% AKACID Plus (AP) in comparison to an AKACID Plus-free control on steel and plastic plates, determined by the swab-rinse technique. Results represent averages for four samples ± 1 standard deviation for three independent experiments.

FIG. 3.

Time-dependent bacterial reduction of the ATCC strain (top) and the multiantibiotic-resistant strain A9726I (bottom) of P. aeruginosa on stainless steel (m) and plastic (p) plates after the use of 0.5, 0.25, and 0.1% AKACID Plus (AP) in comparison to an AKACID Plus-free control on steel and plastic plates, determined by the swab-rinse technique. Results represent averages for four samples ± 1 standard deviation for three independent experiments.

Room disinfection with 0.5% AKACID Plus was successful in eliminating all tested pathogens (lower detection limit, 3 CFU/plate) on stainless steel and plastic plates within 340 min. On plastic plates, both strains of S. aureus (P = 0.006) and E. coli (P = 0.005 to 0.008) were killed within 60 min, while P. aeruginosa required a longer exposure for 340 min. On stainless steel plates, E. coli (P = 0.007) was eliminated within 60 min. In contrast, ATCC and antibiotic-resistant strains of P. aeruginosa (P = 0.005 to 0.007) and S. aureus (P < 0.001 to 0.002) were still detectable on steel plates after nebulization of AKACID Plus for 100 min (∼10¹ CFU/plate), but they were eradicated within 340 min.

Four hours after nebulization, 0.25% AKACID Plus stainless steel and plastic plates still yielded 6.0 × 10¹ to 1.2 × 10² bacterial cells of MRSA strain 9892 (P = 0.002 to 0.003), whereas the gram-negative microorganisms were not detectable on test materials regardless of their antibiotic susceptibility.

The test concentration of 0.1% AKACID Plus achieved a significant reduction of S. aureus (P = 0.002 to 0.005) and P. aeruginosa (P = 0.006 to 0.007) on bacterial carriers but was sufficient to eradicate only E. coli (P = 0.003 to 0.005) within 340 min.

DISCUSSION

AKACID Plus, a mixture of two different polymeric guanidine compounds (Chemical Abstracts Service registry no. 374572-91-5 and 57028-96-3), is a new commercial product of an Austrian company and is registered in the European Union. The present study demonstrates the activity of AKACID Plus as a room disinfectant of closed rooms contaminated with antibiotic-susceptible and multiresistant strains of S. aureus, E. coli, and P. aeruginosa. Disinfectants currently validated for room disinfection achieve high antimicrobial activity but also have high toxicity. Therefore, safety guidelines have to be considered. At present the only accepted method available for decontaminating a biological safety cabinet is to use formaldehyde gas (7, 17). Formaldehyde is highly effective against bacteria, viruses, bacterial toxins, and spores (21) but also is highly toxic (23). Formaldehyde gas has a pungent, irritating odor that is detectable even at very low concentrations (below 1 ppm) and can cause irritation of the eye, skin, and respiratory tract even at low levels for short periods. Its vapor is between 7% and 73% flammable at room temperature, and it is explosive in the presence of strong oxidizers (1). Hydrogen peroxide vapor, which is highly active as a room disinfectant against MRSA (8), Clostridium botulinum spores (13), and Mycobacterium tuberculosis (14), also requires exact surveillance of the gas concentration throughout the decontamination cycle due to its corrosive and toxic properties (22). In contrast, the well-known cationic antimicrobials such as benzalkonium chloride, chlorhexidine, and polyhexamethylene biguanide combine a broad antimicrobial activity and a low toxicity profile (11). Similarly, low toxicity of AKACID Plus was detected in toxicological animal experiments. In an acute oral toxicity study and an acute dermal toxicity study with rats, a median lethal dose of AKACID Plus of >2,000 mg/kg body weight was determined. An acute dermal irritation/corrosion study did not reveal any irritating or corrosive properties of the novel polymeric guanidine (Buxbaum et al., submitted). AKACID Plus is a safe, nonflammable, nonexplosive, and odorless substance. Patients in hospital side rooms in direct vicinity to the contaminated room are not disturbed or endangered during the disinfection process. Due to its low toxicity and noncorrosive properties, there was no need for preparations such as protection of medical devices and computer monitors or sealing of doors with adhesive tape when nebulizing was performed.

To evaluate the antimicrobial activity of AKACID Plus, quantitative cultures of experimentally contaminated stainless steel and plastic plates were performed by a simple swab-rinse technique with neutralizing solution. The detection method demonstrated a dose-dependent and time-dependent activity of nebulized AKACID Plus. All in-door controls of the bacterial pathogens applied on the stainless steel plates tended to reach higher bacterial counts than those on plastic plates. Although Neely (19) has shown a short survival time (only 1 to 7 h) for E. coli and P. aeruginosa on fabrics and plastics used in hospitals, in the present study viable bacterial cells on test materials were detectable during the whole nebulization and exposure to the AKACID Plus-free control. Nevertheless, the stainless steel and plastic plates yielded higher counts of S. aureus than of the gram-negative microorganisms.

The 0.5% AKACID Plus solution was active in eradicating most tested pathogens within 100 min; ∼10¹ CFU of S. aureus ATCC 6538 and MRSA strain 9892 were still detectable on stainless steel plates. Further exposure for 4 h was required to eliminate all bacterial strains. Complete room disinfection takes on average less than 6 h.

Due to its cationic nature, AKACID Plus can be inactivated by the presence of anionic soaps. Therefore, the conventional terminal cleaning must be performed not before the nebulization but following the complete disinfection procedure.