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수의학 석사학위논문
Effect of Manuka essential oil on
Staphylococcus pseudintermedius
isolated from
canine skin and ears
개의 피부와 귀에서 분리한 Staphylococcus
pseudintermedius에대한 Manuka essential oil의 효과
2013 년 2 월
서울대학교 대학원
수의학과 수의내과학(피부과학)전공
ⅰ
Effect of Manuka essential oil on
Staphylococcus pseudintermedius
isolated from
canine skin and ears
Chi Youn Song
Department of Veterinary Internal Medicine
The Graduate School
Seoul National University
Abstract
Staphylococcus pseudintermedius is a primary pathogen of bacterial skin
infections in dogs. Recently, the emergence of methicillin-resistant S.
pseudintermedius (MRSP) has become a challenge because it shows resistance
toward various kinds of antibiotics. Thus, there is growing interest in novel
antimicrobial compounds and Manuka is one of these. The aim of this study was to
ⅱ
confirm the antimicrobial and biofilm formation inhibition activities of Manuka
essential oil against clinically isolated S. pseudintermedius, including MRSP. Fifty
S. pseudintermedius were isolated from clinical cases of canine pyoderma and otitis
externa and show a high MRSP rate (78%). To confirm the antimicrobial activity of
Manuka essential oil, the minimum inhibitory concentrations (MICs) of Manuka
essential oil for S.pseudintermedius isolates were determined using the agar
dilution method and the results were compared with cephalexin. The MICs of
Manuka essential oil and cephalexin against 50 S. pseudintermedius isolates ranged
from 2-6
to 2- 9
(v/v) and 0.125 to 128 mg/ml, respectively. Only 41 isolates (82%)
were susceptible to cephalexin; however, Manuka essential oil showed
antimicrobial activity against all clinically islolated S. pseudintermedius, including
MRSP (2-7
to 2-9
v/v). Biofilm formation was assessed by colorimetric assay with
crystal violet staining. Statistically significant (p<0.05) inhibition of biofilm
formation was found in S. pseudintermedius exposed to 0.01%, 0.1%, and 1% (v/v)
Manuka essential oil, dose-dependently. These results showed that Manuka
essential oil has antimicrobial and antibiofilm formation activity on S.
pseudintermedius and can be a potential treatment option for controlling S.
pseudintermedius infections in dogs.
Keywords: Manuka essential oil, S.pseudintermedius, Antimicrobial
activity, Biofilm, dogs Student Number : 2011-21672
`
ⅲ
CONTENTS
Introduction ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 1
Materials and Method
1. Sample collection and S. pseudintermedius identification∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 3
2. Detection of MRSP∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 4
3. Manuka essential oil ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 5
4. Determination of the MICs of Manuka essential oil on
S. pseudintermedius ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 6
5. Effect of Manuka essential oil on biofilm formation ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 7
6. Statistical analysis ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 8
Results
1. MIC of Manuka essential oil ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙9
2. Biofilm inhibition activity of Manuka essential oil∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙12
Discussion∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙14
References ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙18
국문 초록 ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙24
1
Introduction
Bacterial skin infections involving canine pyoderma and otitis externa
are some of the most common complaints from owners who visit
veterinary clinics. Of the numerous bacteria that can cause canine
bacterial skin infections, Staphylococcus pseudintermedius, formerly
known as Staphylococcus intermedius, is the most common bacterial
pathogen of canine skin and ear infections [3, 11, 23]. In veterinary
practice, bacterial skin infections caused by S. pseudintermedius are
usually treated with systemic antibiotics and a topical antimicrobial
compound [24]. Recently, however, therapeutic options have become
limited due to a considerable increase of methicillin-resistant S.
pseudintermedius (MRSP) [14, 16, 20-22, 29]. Thus, there has been
increasing interest in finding novel antimicrobial agents.
Essential oils obtained from plant materials, such as tea tree oil and
rosemary oil, have been traditionally used for a variety of medicinal
purposes because of their pharmacological effects, including
antimicrobial, antifungal, and anti-inflammatory activity [10, 26]. Due
to these characteristics, there has been a growing international interest
in the use of essential oils as a substitution for synthetic antimicrobials.
In previous studies, it was demonstrated that honey and varieties of the
essential oil of Manuka, derived from the Manuka tree (Leptospermum
2
scoparium) in New Zealand, had a remarkable effect on wound healing,
dental plaque and gingivitis, and dermatophytosis [1, 4, 7, 12, 28].
Recently, triketone-rich Manuka essential oil has become commercially
important because of its antimicrobial activity against gram-positive
bacteria, including antibiotic-resistant strains [6, 17].
To evaluate the potential use of Manuka essential oil as a natural
antimicrobial compound in veterinary medicine, this study investigated
the antimicrobial activity and biofilm formation inhibition activities of
Manuka essential oil against S. pseudintermedius isolated from dogs
with skin and ear infections.
3
Materials and Methods
1. Sample collection and S. pseudintermedius identification
A total of 50 clinical isolates were collected aseptically from dogs
suffering from pyoderma (n=34) or otitis externa (n=16). After
collection, isolates were cultured overnight at 37℃ on blood agar
plates. These isolates were presumptively identified as SIG (S.
intermedius group) by standard biochemical tests and the Vitek 2
system (Biomerieux, Lyon, France). To differentiate S.
pseudintermedius from the other SIG species, all the SIG isolates were
examined using the polymerase chain reaction (PCR)-restriction
fragment length polymorphism (RFLP). Staphylococcal genomic DNA
was extracted using a DNase tissue kit (Qiagen, CA, USA) according to
the manufacture’s recommendation. PCR amplification of the pta gene
(primer F, 5’-AAA GAC AAA CTT TCA GGT AA-3’; primer R, 5’-
GCA TAA ACA AGC ATT GTA CCG-3’) was conducted, and the 320
bp PCR products were digested with MboI restriction enzyme, into two
restriction fragments of 213 bp and 107 bp only in S. pseudintermedius,
as reported previously [2].
4
2.Detection of MRSP
For the identification of MRSP, determination of the MICs of oxacillin
was performed according to CLSI guideline [15] and detection of the
methicillin-resistance encoding gene mecA was determined using a
PCR assay. PCR primers were used to amplify the mecA gene fragment
of 310 bp (primer F, 5’-TGG CTA TCG TGT CAC AAT CG-3’; primer
R, 5’-CTG GAA CTT GTT GAG CAG AG-3’) and the conditions were
as described previously [31].
5
3.Manuka essential oil
Pure 100% Manuka essential oil was extracted using the steam
distillation method provided by Honey Collection, Marlborough, New
Zealand.
6
4. Determination of the MICs of Manuka essential oil on
S.pseudintermedius
Minimum inhibitory concentrations (MICs) of Manuka essential oil and
cephalexin against S. pseudintermedius isolates were determined using
the agar dilution method, according to the CLSI guideline [15]. Briefly,
a series of twofold dilutions of manuka essential oils (ranging from 2-
1 % to 2
-14 % v/v) and cephalexin (ranging from 256 to 0.125 mg/ml)
was prepared in Brain Heart Infusion (BHI; BD Diagnostic systems,
Sparks, MD, USA) agar plates. The agar plates were then inoculated
with a final inoculum of 5 x 107
CFU/ml of each isolate. Inoculated
BHI plates were incubated at 37°C for 24 h. The MICs were
determined as the lowest concentration of test agents inhibiting the
visual growth of bacteria on the agar plate. All determinations were
performed in duplicate.
7
5.Effect of Manuka essential oil on biofilm formation
The effect of different concentrations of Manuka essential oil (ranging
from 2-1
to 2-14
v/v) on the biofilm-forming ability was tested with a
colorimetric microtiter plate assay. All isolates were grown overnight at
37℃ in tryptic soy broth (TSB). An aliquot of 100 ㎕ from the
bacterial culture was inoculated into each well of a 96-well flat-
bottomed polystyrene microtiter plate (corning, NY, USA) in the
presence of 100 ㎕ of Manuka essential oil. The wells containing only
bacteria served as the control. After overnight incubation, media and
non-adherent bacteria were removed by gently washing the plates with
PBS. The remaining adherent bacteria were stained with crystal violet
and the wells were washed with PBS. The quantitative analysis of
biofilm production was performed by adding 200 ㎕ of 95% ethanol
to de-stain the wells. One hundred ㎕ from each well was transferred
to a new microtitier plate, and the stained bacteria and control wells
were read at 540 nm using a microtiter plate reader (Bio-Rad, Munich,
Germany). All experiments were performed in triplicate.
8
6.Statistical analysis
Student’s t-test was performed to compare the biofilm inhibition
activity between each concentration of Manuka essential oil and the
controls. P values of less than 0.05 were considered as statistically
significant.
9
Results
All 50 clinical isolates from canine skin and ear infections were
confirmed as S. pseudintermedius and 39 (78%) of the 50 isolates were
identified as MRSP through the detection of the methicillin-resistant
gene (mecA) and determination of the MICs of oxacillin.
1.MIC of Manuka essential oil
To confirm the antimicrobial activity of Manuka essential oil, the MICs
of Manuka essential oil for S. pseudintermedius isolates were
determined using the agar dilution method. These results were
compared with those of cephalexin, a commonly used antibiotic for
bacterial skin infections in dogs. Table 1 shows the ranges of MICs and
the concentrations necessary to inhibit 50% (MIC50) and 90% (MIC90)
of the isolates.
Table 1. Minimum inhibitory concentrations (MICs) of Manuka essential
oil and cephalexin against Staphylococcus pseudintermedius
MRSP: methicillin-resistant Staphylococcus pseudintermedius; MSSP:
methicillin-susceptible S. pseudintermedius
10
Total isolates (n=50) MRSP (n=39) MSSP (n=11)
Manuka cephalexin Manuka cephalexin Manuka cephalexin
MIC 2-9
~ 2-6
(v/v) 0.125 ~
128 mg/ml 2
-9 ~ 2
-6(v/v)
0.125 ~ 128
mg/ml 2
-9 ~ 2
-7(v/v)
0.125 ~ 1
mg/ml
MIC50 2-8
(v/v) 0.5 mg/ml 2-8
(v/v) 1 mg/ml 2-8
(v/v) 0.5 mg/ml
MIC90 2-7
(v/v) 16 mg/ml 2-7
(v/v) 16 mg/ml 2-7
(v/v) 0.5 mg/ml
The MICs of Manuka essential oil and cephalexin against 50 S.
pseudintermedius isolates ranged from 2-6
to 2- 9
v/v and 0.125 to 128
mg/ml, respectively. All 50 S. pseudintermedius isolates were
susceptible to Manuka essential oil, whereas 41 (82%) of the 50
isolates were susceptible to cephalexin. This study also confirmed the
antimicrobial activity of Manuka essential oil against MRSP, as the
MIC results were divided into MRSP and MSSP group. The
distribution of MIC results for Manuka essential oil were similar
between the MRSP (2-7
to 2-9
v/v) and MSSP (2-9
to 2-6
v/v) groups. In
contrast, the MICs of cephalexin were different between the MRSP
(0.125 to 128 mg/ml) and MSSP groups (0.125 to 1 mg/ml) (Figure 1).
These results showed the antimicrobial activity of cephalexin against S.
pseudintermedius, affected by presence of mecA gene whereas,
Manuka essential oil has antimicrobial activity regardless of the
presence of the mecA gene.
11
Figure 1. Distribution of the MICs of Manuka essential oil and cephalexin
against MRSP and MSSP
MRSP: methicillin-resistant Staphylococcus pseudintermedius; MSSP:
methicillin-susceptible S. pseudintermedius.
2-11
2-10
2-9
2-8
2-7
2-6
2-5
2-4
12
2.Biofilm inhibition activity of Manuka essential oil
To assess the ability of Manuka essential oil to inhibit the formation of
S. pseudintermedius biofilms, different concentrations (0.01%, 0.1%,
and 1% v/v) of Manuka essential oil were tested against biofilm-grown
S. pseudintermedius isolates. The biofilms produced by S.
pseudintermedius declined with increasing concentrations of Manuka
essential oil. The percentages of biofilm inhibition using 0.01%, 0.1%,
and 1% (v/v) of Manuka essential oil were 50%, 22%, and 12% of the
control containing only bacteria, respectively. There were significant
differences with respect to concentrations of Manuka essential oil as
compared with the control (p < 0.001) (Figure 2).
To confirm the biofilm formation inhibition activity of Manuka
essential oil against MRSP, biofilm formation inhibition results were
divided into two groups, MRSP and MSSP. The percentages of biofilm
inhibition by 0.01%, 0.1%, and 1% (v/v) of Manuka essential oil were
48%, 22%, and 11% in the MRSP group, and 60%, 25%, and 21% in
the MSSP group, respectively. The inhibition of biofilm formation by
Manuka essential oil was significantly increased in both the MRSP and
MSSP groups (p < 0.001 and p < 0.05, respectively) (Figure 2).
13
Figure 2. Results of colorimetric assay for quantification of S.
psedintermedius biofilm formation inhibition by Manuka essential oil
Absorbance was measured using a microtitier reader at optical density 540 nm.
Results are means ± standard error of triplicate assays. MRSP: methicillin
resistant S. pseudintermedius; MSSP: methicillin resistant S.
pseudintermedius (* p<0.05, *** p<0.001)
***
*** ***
***
*** *** *** ***
*
14
Discussion
S. intermedius has traditionally been identified as the primary pathogen
of bacterial skin infections in dogs. Recently, the isolates formerly
identified as S. intermedius were reclassified into SIG, including S.
intermedius, S. delphini, and S. pseudintermedius, and based on 16s
rRNA gene sequence analysis it was discovered that S.
pseudintermedius, not S. intermedius, is the primary pathogen of canine
pyoderma and otitis externa [3, 11]. In the past, S. pseudintermedius
was generally susceptible to penicillin-stable β-lactam antibiotics and
cephalosprins [16, 18, 19]. Recently, however, the increasing
prevalence of MRSP has became a problem in veterinary practice
because these strains are usually resistant to the various classes of
antibiotics, and thus only limited treatment options remain [14, 16, 20-
22, 29]. Because of this situation, interest in plants as sources of
biological active compounds was growing, and Manuka essential oil
from Leptospermum scoparium was considered as one of these
biological active compounds. Previous studies reported that Manuka
essential oil has antimicrobial activity, but there is a lack of knowledge
on its effectiveness on S. pseudintermedius from dogs [6, 17]. In this
study, we investigate the antimicrobial activity of Manuka essential oil
against clinically isolated S. pseudintermedius from dogs with bacterial
15
skin infections.
All of the 50 isolates from dogs with pyoderma and otitis externa were
identified as S. pseudintermedius and 39 of the 50 S. pseudintermedius
(78%) were MRSP. The results of this study show a higher MRSP rate
than previous studies in Japan, Canada, the US, and South Korea [13,
14, 22, 30].
To confirm the antimicirobial activity of Manuka essential oil, the
agar dilution test was performed against clinically isolated S.
pseudintermedius from dogs with pyoderma and otitits externa,
including 39 MRSP. Then, the results were compared with those of
cephalexin, one of the most commonly used antibiotics for bacterial
skin infections in dogs. The MICs of Manuka essential oil and
cephalexin against 50 isolated S. pseudintermedius ranged from 2-9
to 2-
6 (v/v) and 0.125 mg/ml to 128 mg/ml (CLSI cephalexin-susceptible
breakpoint; MIC ≤ 8 mg/ml), respectively. All 50 S. pseudintermedius
isolates were susceptible to Manuka essential oil at a concentration of
2-6
(v/v), whereas only 41 (82%) of the 50 isolates were susceptible to
cephalexin according to CLSI recommendation. For comparing
antimicrobial activity, MIC50 and MIC90 of the Manuka essential oil and
the cephalexin were determined. MIC50 and MIC90 of cephalexin against
the MSSP group were the same at a concentration of 0.5 mg/ml;
however, MIC50 and MIC90 against MRSP were 1 mg/ml and 128
16
mg/ml, respectively. In contrast, MIC50 and MIC90 of Manuka essential
oil against MSSP and MRSP were the same at a concentration of 2-7
(v/v). From these results, we can guess that cephalexin is less effective
against MRSP isolates and treatment failure can occur when used on
MRSP. On the other hand, Manuka essential oil inhibits the growth of
all isolates in a narrow range of concentration, regardless of the
existence of the mecA gene. Therefore, this plant-delivered compound
can be used effectively against cephalexin-resistant S. pseudintermedius
and MRSP from dogs.
Biofilm formation is associated with chronic and acute bacterial skin
infections. Biofilm can give rise to planktonic bacteria, and if the host
immune mechanism cannot kill these individual bacteria, biofilm can
act as the nidus of acute infection [8]. Chronic infection is also
associated with biofilm, as bacteria uses the biofilm as a defensive
shield against hostile environments and antibiotics. The defense
mechanism is mediated by the down-regulated rates of cell division of
the embedded bacteria [5]. This structure can prevent inward diffusion
of some antibiotics [27] and offer defense shields from reactive
oxidants produced by the host immune system [9]. Thus, the biofilm
inhibition activity of Manuka essential oil has clinical implications in
the treatment of persistent S. pseudintermedius infections. In this study,
the biofilm inhibition activity of Manuka essential oil on MSSP and
17
MRSP was demonstrated with various different concentrations (0.01,
0.1, and 1% v/v), and Manuka essential oil significantly inhibits biofilm
formation level dose-dependently, and statistically significant
differences were found in comparison with the control group (p < 0.05).
Based on these data, the antimicrobial and biofilm formation inhibition
activities of Manuka essential oil against S. pseudintermedius are
confirmed. Manuka essential oil was able to inhibit the growth of all S.
pseudintermedius isolates, regardless of mecA gene existence, at
concentrations of 2-6
(v/v). Although the specific mechanisms of the
antimicrobial activity of Manuka essential oil is poorly understood, the
lipophilic character of the essential oil may contribute to this
antimicrobial action [25]. Additionally, we found that Manuka essential
oil can inhibit biofilm formation dose-dependently. The limitation of
this study is that all experiments were performed only in vitro; therefore,
if further in vivo studies definitely show the safety and efficacy of
Manuka essential oil, this natural antimicrobial compound may
represent a valuable new treatment option against MSSP and MRSP
from dogs.
18
References
1. Allaker, R. P. and C. Douglas. 2009. Novel anti-microbial
therapies for dental plaque-related diseases. Int. J. Antimicrob.
Agents. 33: 8-13.
2. Bannoehr, J., A. Franco, M. Iurescia, A. Battisti, and J. R.
Fitzgerald. 2009. Molecular diagnostic identification of
Staphylococcus pseudintermedius. J. Clin. Microbiol. 47: 469-
471.
3. Bannoehr, J., N. L. B. Zakour, A. S. Waller, L. Guardabassi, K.
L. Thoday, A. H. M. van den Broek, and J. R. Fitzgerald. 2007.
Population genetic structure of the Staphylococcus intermedius
group: insights into agr diversification and the emergence of
methicillin-resistant strains. J. Bacteriol. 189: 8685-8692.
4. Brady, N., P. Molan, and C. Harfoot. 1996. The sensitivity of
dermatophytes to the antimicrobial activity of manuka honey
and other honey. Pharm. Pharmacol. Commun. 2: 471-473.
5. Brown, M. R. W., D. G. Allison, and P. Gilbert. 1988.
Resistance of bacterial biofilms to antibiotics a growth-rate
related effect? J. Antimicrob. Chemother. 22: 777-780.
6. Christoph, F., K. H. Kubeczka, and E. Stahl-Biskup. 1999. The
composition of commercial manuka oils from New Zealand. J.
19
Essent. Oil Res. 11: 705-710.
7. Cooper, R., P. Molan, L. Krishnamoorthy, and K. Harding. 2001.
Manuka honey used to heal a recalcitrant surgical wound. Eur. J.
Clin. Microbiol. 20: 758-759.
8. Costerton, J. W., P. S. Stewart, and E. P. Greenberg. 1999.
Bacterial biofilms: a common cause of persistent infections.
Science 284: 1318-1322.
9. De Beer, D., R. Srinivasan, and P. S. Stewart. 1994. Direct
measurement of chlorine penetration into biofilms during
disinfection. Appl. Environ. Microbiol. 60: 4339-4344.
10. Deans, S. and G. Ritchie. 1987. Antibacterial properties of plant
essential oils. Int. J. Food Microbiol. 5: 165-180.
11. Devriese, L. A., M. Vancanneyt, M. Baele, M. Vaneechoutte, E.
De Graef, C. Snauwaert, I. Cleenwerck, P. Dawyndt, J. Swings,
et al. 2005. Staphylococcus pseudintermedius sp. nov., a
coagulase-positive species from animals. Int. J. Syst. Evol. Micr.
55: 1569-1573.
12. English, H., A. Pack, and P. Molan. 2004. The effects of manuka
honey on plaque and gingivitis: a pilot study. J. Int. Acad.
Periodontol. 6: 63.
13. Griffeth, G. C., D. O. Morris, J. L. Abraham, F. S. Shofer, and S.
C. Rankin. 2008. Screening for skin carriage of
20
methicillin‐resistant coagulase‐positive staphylococci and
Staphylococcus schleiferi in dogs with healthy and inflamed
skin. Vet. Dermatol. 19: 142-149.
14. Hanselman, B. A., S. Kruth, and J. S. Weese. 2008. Methicillin-
resistant staphylococcal colonization in dogs entering a
veterinary teaching hospital. Vet. Microbiol. 126: 277-281.
15. Institute, C. A. L. S. 2011. Performance standards for
antimirobial susceptibility testing. Clinical and Laboatory
Standards Institute.
16. Kania, S. A., N. L. Williamson, L. A. Frank, R. P. Wilkes, R. D.
Jones, and D. A. Bemis. 2004. Methicillin resistance of
staphylococci isolated from the skin of dogs with pyoderma. Am.
J. Vet. Res. 65: 1265-1268.
17. Kim, E., S. J. Hwang, S. Park, S. Park, and G. Rhee. 2000.
Topical formulation and antimicrobial activity of ketonic
fraction from Leptospermum scoparium. J. Kor. Pharmaceut.
Sci. 30: 151-158.
18. Medleau, L., R. Long, J. Brown, and W. Miller. 1986.
Frequency and antimicrobial susceptibility of Staphylococcus
species isolated from canine pyodermas. Am. J. Vet. Res. 47:
229.
19. Pellerin, J., P. Bourdeau, and H. Sebbagand JM. 1998.
21
Epidemiosurveillance of antimicrobial compound resistance of
Staphylococcus intermedius clinical isolates from canine
pyodermas. Comp. Immunol. Microb. 21: 115-133.
20. Rowe, F., S. Vargas Superti, R. Machado Scheibe, and C. G.
Dias. 2002. Agar diffusion, agar dilution, Etest® , and agar
screening test in the detection of methicillin resistance in
staphylococci. Diagn. Microbiol. Infect. Dis. 43: 45-48.
21. Rutland, B. E., J. S. Weese, C. Bolin, J. Au, and A. N. Malani.
2009. Human-to-dog transmission of methicillin-resistant
Staphylococcus aureus. Emerg. Infect. Dis. 15: 1328.
22. Sasaki, T., K. Kikuchi, Y. Tanaka, N. Takahashi, S. Kamata, and
K. Hiramatsu. 2007. Methicillin-resistant Staphylococcus
pseudintermedius in a veterinary teaching hospital. J. Clin.
Microbiol. 45: 1118-1125.
23. Sasaki, T., K. Kikuchi, Y. Tanaka, N. Takahashi, S. Kamata, and
K. Hiramatsu. 2007. Reclassification of phenotypically
identified Staphylococcus intermedius strains. J. Clin. Microbiol.
45: 2770-2778.
24. Scott, D. W., W. H. Miller, and C. E. Griffin. 2001. Small
animal dermatology. Saunders 6th edition: 1203-1235.
25. Sikkema, J., J. De Bont, and B. Poolman. 1995. Mechanisms of
membrane toxicity of hydrocarbons. Microbiol. Rev. 59: 201-
22
222.
26. Smith-Palmer, A., J. Stewart, and L. Fyfe. 1998. Antimicrobial
properties of plant essential oils and essences against five
important food-borne pathogens. Lett. Appl. Microbiol. 26: 118-
122.
27. Stewart, P. S. 1996. Theoretical aspects of antibiotic diffusion
into microbial biofilms. Antimicrob. Agents. Chemother. 40:
2517-2522.
28. Visavadia, B. G., J. Honeysett, and M. H. Danford. 2008.
Manuka honey dressing: an effective treatment for chronic
wound infections. Brit. J. Oral. Max. Surg. 46: 55-56.
29. Weese, J. S. and E. Van Duijkeren. 2010. Methicillin-resistant
Staphylococcus aureus and Staphylococcus pseudintermedius in
veterinary medicine. Vet. Microbiol. 140: 418-429.
30. Won, Y., K. J. Lee, S. Y. Lee, M. J. Chae, J. K. Park, J. H. Yoo,
and H. M. Park. 2010. Antibiotic resistance profiles of
Staphylococcus pseudintermedius isolates from canine patients
in Korea. J. Microbiol. Biotechnol. 20: 1764-1768.
31. Zubeir, I. E. M. E., T. Kanbar, J. Alber, C. Lämmler, Ö .
Akineden, R. Weiss, and M. Zschöck. 2007. Phenotypic and
genotypic characteristics of methicillin/oxacillin-resistant
Staphylococcus intermedius isolated from clinical specimens
23
during routine veterinary microbiological examinations. Vet.
Microbiol. 121: 170-176.
24
국문 초록
개의 피부와 귀에서 분리한
Staphylococcus pseudintermedius에
대한 Manuka essential oil의 효과
지도교수: 황 철 용
서울대학교 대학원
수의학과 수의내과학(피부과학)전공
송 치 윤
Staphylococcus pseudintermedius는 개 피부감염증의 주 원인균이다. 최
근에, methicillin-resistant S.pseudintermedius (MRSP)가 다양한 항생제에
저항성을 보이면서 수의분야에서 새로운 도전과제가 되었다. 따라서
새로운 항생물질에 대한 관심도가 증가하고 있으며 Manuka는 이러
한 물질중에 하나이다. 본 연구의 목적은 MRSP를 포함한
S.pseudintermedius에 대한 Manuka essential oil의 항균력과 Biofilm 형
성억제효과를 확인하는 것이다. 개 농피증, 외이염환자에서 50개의
25
S.pseudintermedius가 분리되었으며 높은 methicillin-resistant 비율을 나
타냈다 (78%). Manuka essential oil의 항균력를 확인하기 위해서
Manuka essential oil의 minimum inhibitory concentrations (MICs)를 Agar
dilution법을 이용하여 측정하였으며 그 결과를 Cephalexin의 MIC와
비교하였다. 50개의 S.pseudintermedius에 대한 Manuka essential oil과
Cephalexin의 MIC는 각각 2-6
~ 2- 9
(v/v)와 0.125 ~ 128 mg/ml 였다. 분
리된 S.pseudintermedius중 41개의 분리균만 Cephalexin에 감수성을
보였으나(82%) Manuka essential oil은 임상적으로 분리된 MRSP
를 포함한 50개의 S.pseudintermedius 모두에 대해 항균력을 나타냈다.
Biofilm형성억제능력은 Crystal violet염색을 이용한 Colarimetric
법을 이용하여 평가 하였다. 각각 0.01%, 0.1%, 1% 농도의
Manuka essential oil과 반응한 S.pseudintermedius는 통계적으로 유
의적으로 Manuka essential oil ( p < 0.05 ) 농도와 비례하여 Biofilm 형
성이 억제 되었으며 이러한 결과들을 통해 Manuka essential oil이
S.pseudintermedius에 대해 항균력과 Biofilm 형성억제효과가 있음을
확인하였고 개에서 S.pseudintermedius 감염을 치료할 수 있는 잠재적
인 치료물질이 될 수 있을 것이라고 생각된다.
주요어: Manuka essential oil, S.pseudintermedius, Antimicrobial
activity, Biofilm, dogs 학 번 : 2011-21672