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 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). 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 (gram-neg- as the exposure time needed to achieve a 5-log reduction (for ative ); 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. multocida), and 4-log reduction for the ATCC fungi (C. albicans and A. niger). †Distilled water was used without organic material according to AFNOR standard NF T 72–151. ‡0.3% bacteriological milk as organic material was used according to AFNOR standard NF T 72–171. §Synthetic phenols (0.07% 2-phenylphenol and 0.06% p-tertiary amylphenol). ||70% isopropyl alcohol. ¶3% hydrogen peroxide.

4.0

3.5 * † 3.0

2.5

2.0

* 1.5 *

Index of disinfectant strength disinfectant of Index 1.0

0.5

0 Hard water Synthetic Isopropyl Hydrogen (negative phenols alcohol 70% peroxide 3% control) Fig. 1. Results of the zone inhibition test against Pasteurella multocida. The Fig. 2. Representative result of a zone inhibition test against Pasteurella inhibition zone sizes for each disinfectant were measured with vernier cali- multocida. a, hard water (negative control); b, synthetic phenol (0.07% pers. *Significant difference vs. negative control p( <0.001). †The index of 2-phenylphenol and 0.06% p-tertiary amylphenol); c, 70% isopropyl alcohol; disinfectant strength was calculated as the ratio between the mean inhibi- d, 3% hydrogen peroxide. tion zone size (mm) for each disinfectant and that of hard water (negative control). ceptible only to 3% HP. whereas P. multocida did not show a 4- to 5-log reduction, Zone inhibition tests were performed to confirm the effec- even after 5 h of exposure, at which time the tests were dis- tiveness of 3% HP against P. multocida. The size of the zone continued. Similar results were obtained with B. subtilis of inhibition relative to that of the negative control (hard wa- spores. In contrast, when 3% HP was used, a 4- or 5-log vi- ter) was taken as an index of disinfectant strength (Fig. 1). ability reduction was achieved in 30 min for P. multocida The inhibition effect of each disinfectant against P. multoci- and in 1.5--2 h for B. subtilis spores. These results were not da is presented in Fig. 2. The zone of inhibition for 3% HP affected by the addition of milk. These data indicate that P. was significantly larger than that for the other disinfectants. multocida isolated from the environment exhibits stronger The relative zone sizes were 3.17±0.08 for 3% HP, 1.49± disinfectant resistance than the ATCC microorganisms used 0.05 for SP, and 1.57±0.04 for IA 70% (p<0.001 vs. nega- in the study, both in the presence and absence of organic tive control). These results confirmed the susceptibility of material. Furthermore, P. multocida was only slightly less P. multocida to 3% HP. disinfectant-resistant than B. subtilis spores and was sus- The appropriate use of effective disinfectants is very im-

1154 Yonsei Med J http://www.eymj.org Volume 55 Number 4 July 2014 Disinfectant Effects of HP on P. multocida portant for preventing the transmission of pathogenic bacte- for the spore-forming B. subtilis. On the basis of these re- ria commonly present in the environment. Particularly at risk sults, it is recommended that future studies on the efficacy of infection are elderly individuals, neonates, and immuno- of disinfectants use isolates that are extracted directly from compromised individuals. P. multocida is an example of a the environment rather than ATCC strains. From our re- pathogenic environmental bacterium, and because no vac- sults, it is clear that 3% HP is more effective than SP and cine is yet available against it, the use of appropriate disinfec- 70% IA for preventing growth of the pathogenic P. multoci- tants is currently the most effective way to prevent infections. da in the environment. Several steps were taken to make this study relevant for It is clear that the above-mentioned WHO warning con- practical applications. First, several readily available disin- cerning the increase in pasteurellosis is more important to- fectants with different modes of action were tested: 3% HP day as a consequence of changing patterns in human be- (oxidant), 70% IA (dehydrant), and SP (permeable denatur- havior. Specifically, the acceptance of pets as members of ant). The disinfectant that was found to be most effective the family has resulted in closer human contact with pets against P. multocida, 3% HP, is inexpensive; therefore, it that may be carrying human pathogens. In addition, P. mul- does not create an economic barrier to P. multocida elimi- tocida infections in the past were mostly due to animal bites nation. Second, a variety of representative microorganisms or scratches, but now predominantly involve local infec- were used as controls: B. subtilis (spores) and M. luteus tions and, less commonly, respiratory infections. P. multoci- (gram-positive bacteria), P. aeruginosa (gram-negative bac- da is now one of the most important human pathogens. teria), C. albicans (yeast), and A. niger (mold). Among the In conclusion, the results of this study suggest that until an microorganisms tested, P. multocida showed the strongest effective vaccine or antibiotic against P. multocida is avail- resistance to disinfectants. Third, 0.3% bacteriological milk able, cleaning of hospitals and other public places with hy- was included in the tests as an organic material to simulate drogen peroxide as a disinfectant is the most effective and more realistic conditions for practical applications. Log via- economical way to prevent pasteurellosis. bility reduction times were compared with and without the addition of milk; however, no difference was observed. Thus, under realistic conditions, P. multocida was shown to possess REFERENCES the strongest disinfectant resistance among the microorgan- isms tested; however, it was effectively inhibited by 3% HP. 1. Brogden KA, Lehmkuhl HD, Cutlip RC. Pasteurella haemolytica complicated respiratory infections in sheep and goats. Vet Res Bacterial resistance may originate from endogenous genes 1998;29:233-54. or may be acquired by mutation or the acquisition of plas- 2. Weber DJ, Wolfson JS, Swartz MN, Hooper DC. Pasteurella mul- mids or transposons.9 Bacterial resistance to disinfectants tocida infections. Report of 34 cases and review of the literature. typically results from cellular changes that impact biocide Medicine (Baltimore) 1984;63:133-54. 3. Hubbert WT, Rosen MN. Pasteurella multocida infections. II. Pas- 13 accumulation. 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