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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: firstname.lastname@example.org) Received 8 March 2013; accepted for publication 17 May 2013. bs_bs_banner Animal Science Journal (2014) 85, 174–179 doi: 10.1111/asj.12098 © 2013 Japanese Society of Animal Science
(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
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
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
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. 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