1. ORIGINAL ART ICLE
Susceptibility difference between methicillin-susceptible
and methicillin-resistant Staphylococcus aureus to a
bovine myeloid antimicrobial peptide (BMAP-28)
Shiaki TAKAGI,1
Junko NISHIMURA,2
Lanlan BAI,1
Hikaru MIYAGI,1
Kengo KURODA,1
Shunji HAYASHI,3
Hiroshi YONEYAMA,1
Tasuke ANDO,1
Hiroshi ISOGAI4
and Emiko ISOGAI1
1
Laboratory of Animal Microbiology and 2
Technical Division, Graduate School of Agricultural Science, Tohoku
University, Sendai, 3
Division of Bacteriology, Department of Infection & Immunity, Jichi Medical University,
Tochigi, and 4
Division of Animal Experimentation, Sapporo Medical University, Sapporo, Japan
ABSTRACT
A bovine myeloid antimicrobial peptide antimicrobial peptide (BMAP-28) is a member of the cathelicidin family and acts
as a component of innate immunity. There are few reports of susceptibility difference of methicillin-resistant Staphylococ-
cus aureus (MRSA) and susceptible strains (MSSA) against BMAP-28. This study aims to clarify how a few amino acid
substitutions of BMAP-28 are related to its antimicrobial activity using four analog peptides of BMAP-28. We also
compared cellular fatty acid components of MSSA and MRSA using gas chromatography. We found that a few amino acid
substitutions of BMAP-28 do not change antimicrobial activity. It was also revealed that the percentage of cis-11-eicosenoic
acid in total detected fatty acids of MRSA was significantly higher than that of MSSA. In addition, the percentage of
palmitic acid in total detected fatty acids of MRSA tended to be lower than that of MSSA. Our results will provide new
information to deal with the question of differences in bacterial susceptibility against BMAP-28.
Key words: amino acid substitution, antimicrobial peptide, drug-resistant bacteria, fatty acid component, susceptibility.
INTRODUCTION
Antimicrobial peptides (AMPs) are components of
innate immunity. A bovine myeloid antimicrobial
peptide (BMAP-28) is a member of the cathelicidins
which is one of the families of mammalian AMPs
(Zanetti 2004). Recently, we and many other
reseachers reported that BMAP-28 exerts a potent
antimicrobial activity against Gram-negative and
Gram-positive bacteria, including drug-resistant bacte-
ria (Skerlavaj et al. 1996; Takagi et al. 2012). It is also
known as one of the representatives of the α-helical
type of cathelicidins (Zanetti 2004). The activities of
AMPs are generally dependent upon their interaction
with bacterial cell membranes (Kang et al. 2012).
There have been some models of interaction between
bacterial membrane and this type of AMPs. However,
the precise mechanism for their interaction remains to
be clarified.
Staphylococcus aureus is a potentially pathogenic
bacterium that causes a broad spectrum of diseases
(Deurenberg et al. 2006). In the case of cows, it is one
of the major bacteria which cause mastitis. The disease
brings huge economic losses to farms. S. aureus can
rapidly adapt to antibiotics, and this has resulted
in emergence and spread of methicillin-resistant
S. aureus (MRSA) (Deurenberg et al. 2006). In addi-
tion, hospital-associated MRSA (HA-MRSA) and
community-associated MRSA (CA-MRSA) have also
emerged. In general, CA-MRSA is more virulent com-
pared to HA-MRSA due the presence of various viru-
lence factors (Deurenberg & Stobberingh 2008). To
make matters worse, MRSA has been detected from
livestock animals such as pigs, dairy cows, veal calves
and fowl (Kluytmans 2010). This is so-called livestock-
associated MRSA (LA-MRSA). In association with this
fact, people working with live pigs or veal calves were
found to be colonized from animal reservoirs with
MRSA ST398, a relatively new type of LA-MRSA
Correspondence: Emiko Isogai, Laboratory of Animal Micro-
biology, Department of Microbial Biotechnology, Graduate
School of Agricultural Science, Tohoku University, 1-1
Tsutsumidori Amamiya-machi, Aoba, Sendai, Miyagi 981-
8555, Japan. (Email: emiko@bios.tohoku.ac.jp)
Received 8 March 2013; accepted for publication 17 May
2013.
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Animal Science Journal (2014) 85, 174–179 doi: 10.1111/asj.12098
© 2013 Japanese Society of Animal Science

2. (Kluytmans 2010). Furthermore, it has been reported
that LA-MRSA strains were isolated from retail meats
in many countries (Vanderhaeghen et al. 2010). Thus,
it is an emergency that effective strategies are needed
to protect not only humans but also livestock animals
from MRSA.
In spite of so many studies about the anti-MRSA
effect of AMPs, there are few reports dealing with
the differences in susceptibility between MRSA and
methicillin-susceptible S. aureus (MSSA). Therefore, in
this study, we compared MRSA and MSSA with regard
to their AMP susceptibility and composition of cellular
fatty acids. We used four analogs of BMAP-28 in order
to find the effects of amino acid substitution on their
potency to kill MRSA and MSSA. We also investigated
the composition of cellular polar lipids of both MRSA
and MSSA by gas chromatography.
MATERIALS AND METHODS
Bacterial strains
We used MSSA and MRSA which were isolated from
humans. The bacteria were grown in brain heart infusion
(BHI) broth for 18 h at 37°C.
Peptides
The four analog peptides of BMAP-28 were synthesized by
the method previously described by Isogai et al. (2009). The
sequences are shown in Table 1. The peptides were more than
96.5% purified by reverse-phase high-performance liquid
chromatography (Model LC-8A; Shimadzu Corporation,
Kyoto, Japan) on a YMC-A 302 column. The final products
were confirmed by electrospray ionization mass spectrometry
and were supplied as trifluoroacetate. They were conserved by
suspension in Hanks’ Balanced Salt Solution (pH 7.4; Gibco,
Grand Island, NY, USA) and stored at −20°C.
Growth inhibition test
The experiments were performed as previously described
(Takagi et al. 2012). The optical density (OD) 660 nm of pre-
cultured bacteria was measured by the Ubest-35 (JASCO
Corporation, Tokyo, Japan). By adding BHI broth, the OD
660 nm was adjusted to 0.5. The bacteria were diluted to a
final concentration of 1–5 × 103
colony forming units
(CFU)/mL with BHI broth, after which 1 mL of bacterial
suspension and 1 mL of each kind of peptide solution were
mixed together. The peptide solutions were prepared by two-
fold dilution in BHI broth, while each of four peptide solu-
tions were prepared to final peptide concentrations of 20, 10,
5, 2.5 and 1.25 μg/mL. After mixing bacterial suspension and
peptide solutions, the mixtures were incubated at 37°C. The
OD of the cell suspension was measured at 660 nm every 3 h
after 15 h incubation. A control was prepared by mixing
1 mL of bacterial suspension, 0.9 mL of BHI broth and
0.1 mL of Hanks’ Balanced Salt Solution. The minimal
inhibitory concentration (MIC) of the peptides was defined
as the lowest concentration of peptides which showed OD
660 nm of less than 0.2, where the control cells showed the
absorbance of more than 0.7 (0.7–1.0). MIC50 was calculated
by Probit analysis when a linear regression curve was
obtained (significant at P < 0.05).
Fatty acid composition analysis
Both MSSA and MRSA were incubated in BHI broth for 18 h.
After incubation, the bacterial cells were washed three times
with PBS and then lyophilized. Serial experiments were done
according to the method of Bligh and Dyer (1959) and
Nishimura et al. (2012). Briefly, the lipids of the cells were
extracted in 1:2:0.8 (v:v:v) chloroform : methanol : water.
Separation of neutral and polar lipids was carried out using a
Sep-Pak Plus NH2 Cartridge (Waters Corporation, Milford,
MA, USA). Then, samples were converted to fatty acid
methyl esters and analyzed using Hitachi G-6000 gas chro-
matograph equipped flame ionization detectors (Hitachi
High-Technologies Corporation, Tokyo, Japan). The column
used for the experiment was a TC-70 column (0.25 mm ×
60 m; GL Sciences Inc., Tokyo, Japan) with He as a carry gas.
The temperature of injection and detection port was 260°C,
and the column temperature was maintained at 140°C for
5 min, increasing at a rate of 4°C/min to 180°C. Then, the
column temperature was kept at 180°C for 10 min, increas-
ing at 20°C/min to 250°C and kept at 250°C for 10 min.
Peaks were identified by comparing the retention times
to a FAME standard mixture containing 37 components
(Supelco®
37 Component FAME Mix; Sigma-Aldrich Japan
Corporation, Tokyo, Japan).
RESULTS
Antimicrobial activity of
BMAP-28 analogs
In the growth inhibition test, MICs of A837 peptide for
10 strains of MSSA were ranged from 1.25 μg/mL to
more than 5 μg/mL, while MICs of the other three
peptides were all ranged from 2.5 μg/mL to more than
5 μg/mL. For MRSA, MICs of A840 peptide were
ranged from 5 μg/mL to more than 5 μg/mL. The MICs
of the other three peptides were all more than 5 μg/
mL. The value of MIC50 of the analogs were the same
Table 1 Sequences and physical information of the peptides
Peptide Sequence Net charge† Average
hydrophobicity‡
BMAP-28 GGLRS LGRKI LRAWK KYGPI IVPII RI +7 0.256
A837 GGLRK LGRKI LRAWK KYGPI IVPII RI +8 0.141
A838 KGLRK LGRKI LRAWK KYGPI IVPII RI +9 0.011
A839 GGLRS LGRKI LRAWK KGGPI IVPII RI +7 0.289
A840 KGLRK LGRKI LRAWK KGGPI IVPII RI +9 0.044
†(Lysine + arginine) – (aspartic acid + glutamine). ‡Average hydrophobicity was estimated by SOSUI (Nagoya University, Aichi, Japan).
SUSCEPTIBILITY DIFFERENCE OF MSSA AND MRSA 175
© 2013 Japanese Society of Animal ScienceAnimal Science Journal (2014) 85, 174–179

3. among the four peptides. They were 3.1 (A837), 2.8
(A838), 2.4 (A839) and 3.2 (A840) μg/mL for MSSA
and more than 5 μg/mL for MRSA, respectively
(Table 2).
Cumulative inhibition rates are shown in Figure 1.
Statistical analyses were carried out by chi-square test
between MSSA and MRSA and between BMAP-28
and each of the four analog peptides. There were no
significant differences of the cumulative inhibition rate
between BMAP-28 and the four analog peptides.
However, in the cases of A837, A838 and A840
peptides, there were significant differences between
MSSA and MRSA at the concentrations of 2.5 and
5 μg/mL (P < 0.05). In addition, A839 peptide showed
significant differences between MSSA and MRSA at
the concentration of 2.5 μg/mL (P < 0.05).
Table 2 Growth inhibition of MSSA and MRSA
Peptides MIC range
(μg/mL)
MIC50 and 95%
confidence (μg/mL)
MSSA MRSA MSSA MRSA
A837 2.5–> 5 5–> 5 3.1 (2.16–5.46) >5
A838 1.25–> 5 5–> 5 2.8 (1.94–4.63) >5
A839 1.25–> 5 5–> 5 2.4 (1.59–4.54) >5
A840 1.25–> 5 >5 3.2 (2.06–7.89) >5
For each of MSSA and MRSA, 10 strains of bacteria were used. The experiment was performed one time for each strain. MSSA,
methicillin-susceptible Staphylococcus aureus; MRSA, methicillin-resistant S. aureus; MIC, minimal inhibitory concentration. MIC50 of MSSA
was calculated by Probit analysis. In MRSA, a linear regression curve could not be obtained.
0
20
40
60
80
100
0 0.6 1.25 2.5 5 >5
Cumulativeinhibitionrate(%)
Concentration of peptide (µg/mL)
A837
MSSA MRSA MSSA MRSA
MSSA MRSA MSSA MRSA
*
*
A
0
20
40
60
80
100
0 0.6 1.25 2.5 5 >5
Cumulativeinhibitionrate(%)
Concentration of peptide (µg/mL)
A838B
*
*
0
20
40
60
80
100
0 0.6 1.25 2.5 5 >5
Cumulativeinhibitionrate(%)
Concentration of peptide (µg/mL)
A839
*
C
0
20
40
60
80
100
0 0.6 1.25 2.5 5 >5
Cumulativeinhibitionrate(%)
Concentration of peptide (µg/mL)
A840
*
D
*
Figure 1 Antimicrobial activity of analog peptides of BMAP-28 against methicillin-susceptible Staphylococcus aureus (MSSA)
and methicillin-resistant S. aureus (MRSA). The cumulative inhibition rate was calculated from the results of growth inhibition
tests of the peptides against MSSA (n = 10) and MRSA (n = 10). Only the results of the peptides which showed significant
difference between MSSA and MRSA are shown. A is the result of peptide A837, B is that of peptide A838, C is that of
peptide A839 and D is that of peptide A840. *P < 0.05 chi-square test between MSSA and MRSA.
176 S. TAKAGI et al.
© 2013 Japanese Society of Animal Science Animal Science Journal (2014) 85, 174–179

4. Fatty acid composition of MRSA
and MSSA
It has been reported that ceragenins, antimicrobial
agents which are cationic bile salt derivatives showed
different levels of leakage activity against liposomes
which have different lipid compositions (Epand et al.
2007). Therefore, we studied the relationship between
bacterial susceptibility of BMAP-28 and the composi-
tion of bacterial cellular fatty acids which are related to
bacterial membrane composition. The fatty acid com-
positions of three strains of MRSA and MSSA were
compared. The representative data of both MSSA and
MRSA are shown in Figure 2. The data is shown as
relative ratio of each fatty acid in the whole of fatty
acids detected. In the case of cis-11-eicosenoic acid
(C20:1), the relative ratio of it to the total detected
fatty acids in MRSA was 8.1% and that in MSSA was
3.5% (Fig. 3A). The ratio of MRSA was significantly
higher than that of MSSA. On the other hand, the
relative ratio of palmitic acid (C16:0) in MRSA was
21.0% and that in MSSA was 33.0% (Fig. 3B). The
ratio of MRSA tended to be lower than that of MSSA
although there was no statistical significance. In
respect of other fatty acids, the diversity between
strains was extensive. Therefore, no significant differ-
ence and tendency between MSSA and MRSA were
observed.
DISCUSSION
We examined antimicrobial activity of four analog
peptides of BMAP-28 against MRSA and MSSA in this
study. Each of the four analogs has the sequence
which substituted one to three amino acid residues for
the original sequence of BMAP-28. It has also been
suggested that there is a critical threshold for the net
positive charge or positive charge density on a given
α-helical AMP that governs antimicrobial and
hemolytic activities (Jiang et al. 2008; Huang et al.
2010). The net charge of four analogs of BMAP-28
ranged from +7, the same value as that of BMAP-28,
to +9. Within the range of the net charge, there was no
significant difference of MIC among these analogs. On
the other hand, A837, A838 and A840 peptides
showed significantly higher growth inhibition against
MSSA than against MRSA at 2.5 and 5 μg/mL. A839
peptide also showed significantly higher growth inhi-
bition against MSSA than against MRSA at 2.5 μg/mL
and although there was no significance, it tended to
inhibit the growth of MSSA more than that of MRSA
at 5 μg/mL. We have previously reported that
BMAP-28 showed significantly higher antimicrobial
effect against MSSA than that against MRSA at
2.5 μg/mL and 5 μg/mL (Takagi et al. 2012). Based on
these results, the increase of the net charge of
BMAP-28 in this range did not raise its antimicrobial
activity.
From the viewpoint of average hydrophobicity, only
A839 had a higher value than BMAP-28 (Table 1). It
has been found that there was an optimal hydropho-
bicity window in which antimicrobial activity could
be obtained (Chen et al. 2007; Huang et al. 2010).
Decreased or increased hydrophobicity beyond this
Figure 2 Gas chromatograph (GC) chromatograms of fatty
acid analysis of the cells of methicillin-susceptible
Staphylococcus aureus (MSSA) (A) and methicillin-resistant
S. aureus (MRSA) (B).
0
5
10
15
20
25
30
35
40
45
MSSA MRSA
Percentageoffattyacid(%)
Bacteria
C16:0B
0
2
4
6
8
10
12
MSSA MRSA
Percentageoffattyacid(%)
Bacteria
C20:1A
Figure 3 Relative area ratio of fatty acids in bacterial cells
of methicillin-susceptible Staphylococcus aureus (MSSA) and
methicillin-resistant S. aureus (MRSA). The area ratio was
analyzed by gas chromatography. Total area of all the
detected peaks was calculated to be 100%. Each graph
shows relative area ratio of C20:1 of MSSA and MRSA (A)
and that of C16:0 of MSSA and MRSA (B).
SUSCEPTIBILITY DIFFERENCE OF MSSA AND MRSA 177
© 2013 Japanese Society of Animal ScienceAnimal Science Journal (2014) 85, 174–179

5. window dramatically compromised the antimicrobial
activity (Huang et al. 2010). In our research, although
the four analog peptides had various levels of hydro-
phobicity, there were no significant differences in the
antibacterial activity among the peptides compared to
BMAP-28. This means, in the case of BMAP-28, the
substitution of a few amino acids does not affect the
antimicrobial activity of the AMP.
The analogs tended to inhibit the growth of MSSA at
a lower concentration in comparison to their effects on
MRSA growth (Table 2). This accorded with the anti-
microbial activity of BMAP-28. It was indicated that
the differences of cumulative inhibition rate among
the analog peptides was caused by the substitution of
amino acids. Because there was no statistical signifi-
cance in the differences, amino acid substitutions of
the four analog peptides did not affect the tendency of
BMAP-28 to inhibit the growth of MSSA in lower
concentrations above that for inhibiting the growth of
MRSA.
There has been some research about the cellular
fatty acid compositions of S. aureus (Theodore & Panos
1973; Asai et al. 1993). Theodore and Panos reported
that pentadecanoic acid (C15:0), heptadecanoic acid
(C17:0) and nonadecanoic acid (C19:0) fatty acids
were dominant in the plasma membrane of S. aureus
(Theodore & Panos 1973). The study about the cellular
fatty acid composition of S. aureus showed that the
major fatty acids were myristic acid (C14:0), C15:0,
stearic acid (C18:0) and arachidic acid (C20:0) (Asai
et al. 1993). It has been reported that the percentage of
the C15:0 fatty acid component of MRSA strains was
higher than that of MSSA strains (Asai et al. 1993).
Asai et al. also reported the percentage of the C20:0
fatty acid component of MRSA strains was lower than
that of MSSA strains.
In contrast to these facts, we found that the major
fatty acids of strains we used were C16:0 and C18:0.
Their percentage was more than 10% in each strain.
This difference seemed to be caused by the strains used
and detailed culture conditions. Our results also did
not show significant differences of C15:0 and C18:0
percentages between MRSA and MSSA. Instead, our
results indicated the new candidate fatty acids, C16:0
and C20:1, to be applied to examine susceptibility of
AMP. This means there is a possibility that AMP sus-
ceptible strains of S. aureus tend to contain relatively
high percentages of C16:0 and relatively low percent-
ages of C20:1. However, according to our study and
other previous studies, the cellular fatty acid compo-
sitions of bacteria have various patterns. Therefore,
further studies are needed which contain greater
numbers of strains and more sensitive and multiple
detection systems.
In this study, we found the following facts. First, a
few amino acid substitutions of BMAP-28 do not
convert antimicrobial activity. MSSA strains were
more susceptible to the four analog peptides than
MRSA strains, similar to the case of BMAP-28. This
indicates the changes in amino acids, of net charge and
of hydrophobicity in this range, do not affect the ten-
dency to susceptibility of S. aureus. Second, the cellular
fatty acid compositions of MSSA and MRSA, especially
the percentage of C16:0 and C20:1 fatty acids may be
the marker to predict this bacteria’s susceptibility to
AMPs.
Recently, AMPs have been getting more and more
attention from researchers as new antibiotics. In order
to deal with the problems of drug-resistant bacteria, it
is very important to study bacterial susceptibility
against AMPs. When the mechanisms of difference in
drug susceptibility have been revealed, we will be a
step closer to achieving new medicines.
ACKNOWLEDGMENTS
This work was supported by the grant of Kieikai
Research Foundation and a part of grant-aid
(25292178) from Ministry of Education, Culture,
Sports, Science and Technology of Japan.
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