http://dx.doi.org/10.3349/ymj.2014.55.4.1152 Brief Communication pISSN: 0513-5796, eISSN: 1976-2437 Yonsei Med J 55(4):1152-1156, 2014 Hydrogen Peroxide as an Effective Disinfectant for Pasteurella multocida In-Soo Jung, Hyun-Jung Kim, Won-Yong Jung, and Chan-Wha Kim College of Life Sciences and Biotechnology, Korea University, Seoul, Korea. Received: March 6, 2014 Pasteurella multocida (P. multocida) infections vary widely, from local infections Revised: April 10, 2014 resulting from animal bites and scratches to general infections. As of yet, no vac- Accepted: April 11, 2014 cine against P. multocida has been developed, and the most effective way to pre- Corresponding author: Dr. Chan-Wha Kim, vent pathogenic transmission is to clean the host environment using disinfectants. College of Life Sciences and Biotechnology, Korea University, 145 Anam-ro, Seongbuk-gu, In this study, we identified which disinfectants most effectively inhibited environ- Seoul 136-701, Korea. mental isolates of P. multocida. Three readily available disinfectants were com- Tel: 82-2-3290-3439, Fax: 82-2-3290-3957 pared: 3% hydrogen peroxide (HP), 70% isopropyl alcohol, and synthetic phenol. E-mail: [email protected] In suspension tests and zone inhibition tests, 3% HP was the most promising disin- fectant against P. multocida. ∙ The authors have no financial conflicts of interest. Key Words: Pasteurella multocida, human pathogen, environmental isolate, dis- infectant, 3% hydrogen peroxide Pasteurella multocida (P. multocida) has been deemed as a zoonotic disease agent by the World Health Organization (WHO 1959). Pasteurellosis is caused by P. multocida and Mannheimia haemolytica and frequently manifests as respiratory infections in small farmed ruminants, which has major worldwide economic and welfare implications.1 It has long been known that P. multocida is a frequent cause of infection following animal bites or scratches.2,3 Recently reported cases of P. multocida infection in Japan have predominantly involved local infections; respi- ratory infections from sputum of human contact with animals are less common.4,5 No vaccine against this bacterium has been developed thus far.6 Therefore, regu- lar cleaning of the host environment with disinfectants that are inhibitory to micro- organism transmission is, currently, the best way to prevent disease induced by this pathogen.7 Traditionally, disinfectants have been classified into five agent types: permeable, dehydrating, sulfhydryl-combining, alkylating, and oxidizing. In this study, three of © Copyright: these were considered “challenged disinfectants”: hydrogen peroxide (an oxidizing Yonsei University College of Medicine 2014 agent);8,9 alcohol (a dehydrating agent);10,11 and phenol (a permeable agent). This is an Open Access article distributed under the The objective of this study was to determine the most effective disinfectant for terms of the Creative Commons Attribution Non- Commercial License (http://creativecommons.org/ the prevention of pathogenic infections by P. multocida. To address this, three licenses/by-nc/3.0) which permits unrestricted non- commercial use, distribution, and reproduction in any readily available disinfectants were tested: 3% hydrogen peroxide (HP), 70% iso- medium, provided the original work is properly cited. propyl alcohol (IA), and synthetic phenol (SP) (0.07% 2-phenylphenol, 0.06% p- 1152 Yonsei Med J http://www.eymj.org Volume 55 Number 4 July 2014 Disinfectant Effects of HP on P. multocida tertiary amylphenol). An environmental isolate of P. multoci- Mod. Agar (Scharlau) for fungi. Colonies were then counted. da was obtained from Konkuk University and the following Counts were recorded for exposure to disinfectant from a microorganisms were used as representative controls: Bacil- minimum of 5 min to a maximum of 5 h. Bactericidal activi- lus subtilis (spore-forming, gram-positive bacterium); Mi- ty, sporicidal activity, and fungicidal activity were expressed crococcus luteus and Pseudomonas aeruginosa (gram-neg- as the exposure time needed to achieve a 5-log reduction (for ative bacteria); Candida albicans (yeast); and Aspergillus bactericidal and sporicidal activity) or 4-log reduction (for niger (mold). fungicidal activity) in viability, in accordance with AFNOR For the disinfectant efficacy test, three commercial disin- guidelines. fectants were selected: 3% HP (Sanosil-S006; Sanosil Ltd., For the zone inhibition test, 0.1 mL of the P. multocida CH-8634 Hombrechtikon, Switzerland), SP, and 70% IA. SP suspension (1×107 CFU/mL) was spread onto Tryptone was composed of 0.07% 2-phenylphenol and 0.06% p-tertia- Soya Agar plates. Next, filter paper discs (Whatman, Mid- ry amylphenol. In the suspension test, disinfectants at double dlesex, UK) with 6-mm diameters were placed on the plates, concentration (for HP and SP) or original concentration (for and then 0.01 mL disinfectant solution was carefully dis- IA) were diluted with microbial suspensions or tryptone wa- pensed onto the discs. As a negative control, 0.01 mL hard ter (Scharlau, Barcelona, Spain) to the final disinfectant con- water was substituted for the disinfectant. The plates were centrations. In the zone inhibition test, each disinfectant was incubated at 32.5°C for 3--5 days, and the zones of inhibi- adjusted to its final concentration with sterile hard water tion were measured with vernier calipers (Mitutoyo, Kana- (0.442 g/L CaCl2·2H2O) before experimental use. All disin- gawa, Japan). Each test was repeated three times. fectants were prepared and used on the same day. In the zone inhibition test, statistical analyses for suscepti- For the suspension test, AFNOR standard NF T 72--151 bility to disinfectants were conducted using one-way ANO- (in distilled water) and AFNOR standard NF T 72--171 (in VA with SPSS 12.0.1 for windows (SPSS Inc., Chicago, IL, the presence of protein) were used as references to measure USA). p-values <0.05 were considered statistically signifi- bactericidal activity, and AFNOR standards NF T 72--201 cant. and NF T 72--231 were used as references for fungicidal To identify the disinfectant resistance levels of P. multo- and sporicidal activity, respectively.12 The concentrations of cida, its viability reduction time in the suspension test was microbial suspensions cultured in Tryptone Soya Broth or compared with that of other microorganisms. Microorgan- Sabouraud Chloramphenicol Broth were determined by se- ism suspensions were incubated with each disinfectant so- rial dilution in tryptone water. In the absence of the added lution for 5, 15, and 30 min, and 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, protein, 1 mL of the microbial suspension (diluted to 1×108 and 5 h at room temperature (20.0°C) and then filtered. The CFU/mL in distilled water) was added to a tube containing filtered membrane was cultured on Tryptone Soya Agar or 4 mL of tryptone water, followed by 5 mL of 2×-concentrat- Sabouraud Chloram Mod. Agar plates for 3--5 days, and the ed disinfectant solution. For 70% IA, the volumes of the mi- number of colonies was counted in order to find the expo- croorganisms and disinfectants that were added differed be- sure time required for a 5-log reduction (for vegetative bac- cause of the different original concentration (99%). For tests teria, bacterial spores and the environmental isolate) or a performed in the presence of protein, 1 mL of the microbial 4-log reduction (for fungi) of each microorganism, in accor- suspension containing 1×108 CFU/mL with 0.3% bacterio- dance with AFNOR guidelines. Mixed solution with tryp- logical milk (Scharlau, Barcelona, Spain) was used. The final tone water and microorganisms without disinfectants were cell concentration was 1×107 CFU/mL. Cultures were incu- employed as a positive control, and tryptone water without bated for 5, 15, and 30 min and 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, microorganisms was used as a negative control. To simu- and 5 h at room temperature (20°C). At each time point, 1 late environmental conditions, another experiment was con- mL of the mixed suspension was dispensed into 100 mL ster- ducted in which 0.3% bacteriological milk was added as an ile water, filtered using a membrane filtration system (MF organic material. The suspension test results are summarized System, Millipore, Bedford, MA, USA), and washed three in Table 1. Four- or 5-log viability reduction times were re- times with sterile water. After filtration, the membrane was corded for the ATCC-derived bacteria and fungi, including incubated for 3--5 days at either 32.5°C on Tryptone Soya P. aeruginosa, M. luteus, C. albicans, and A. niger. When Agar (Scharlau) for vegetative bacteria, bacterial spores, and SP, 70% IA, or 3% HP were used, the reduction times for environmental isolates or at 22.5°C on Sabouraud Chloram the ATCC microorganisms ranged from 5 min to 15 min, Yonsei Med J http://www.eymj.org Volume 55 Number 4 July 2014 1153 In-Soo Jung, et al. Table 1. Summary of the Results of the Suspension Test Exposure time for a 4- or 5-log reduction* Microorganism No organic material† Organic material present‡ SP§ 70% IA|| 3% HP¶ SP§ 70% IA|| 3% HP¶ P. multocida >5 hrs >5 hrs 30 min >5 hrs >5 hrs 30 min B. subtilis >5 hrs >5 hrs 1 hr 30 min >5 hrs >5 hrs 2 hrs P. aeruginosa 5 min 5 min 5 min 5 min 5 min 5 min M. luteus 5 min 5 min 5 min 5 min 5 min 5 min C. albicans 5 min 5 min 5 min 5 min 5 min 15 min A. niger 5 min 5 min 15 min 5 min 5 min 15 min SP, synthetic phenol; HP, hydrogen peroxide; IA, isopropyl alcohol. *5-log reduction for the ATCC bacteria (B. subtilis, P. aeruginosa, and M. luteus) and the environmental isolate (P.