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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

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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