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ARTICLE
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Antibacterial and synergic effects of gallic acid-grafted-chitosan with -lactams against methicillin-resistant Staphylococcus aureus (MRSA) Dae-Sung Lee, Sung-Hwan Eom, Young-Mog Kim, Hye Seon Kim, Mi-Jin Yim, Sang-Hoon Lee, Do-Hyung Kim, and Jae-Young Je
Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) is spreading worldwide, emphasizing the need to search for new antibiotics. The anti-MRSA activities of gallic acid-grafted-chitosans (GA-g-chitosans) were investigated against 2 MRSA standards and 10 MRSA clinical isolates by determining the minimum inhibitory concentrations (MICs). GA-g-chitosan (I), which has the highest gallic acid content, exhibited the strongest anti-MRSA activities, with MICs of 32–64 g/mL. A time-kill investigation revealed that GA-g-chitosan (I) exhibited a bactericidal effect at twice the MIC, also demonstrating good thermal and pH stability. Investigation of cell envelope integrity showed the release of intracellular components with an increasing absorbance value at 260 nm, indicating cell envelope damage caused by the GA-g-chitosan (I), which was further confirmed by transmission electron microscopy. When GA-g-chitosans were combined with -lactams, including ampicillin and penicillin, synergistic effects were observed on the 2 standard MRSA strains and on the 10 clinical isolates, with fractional inhibitory indices ranging from 0.125 to 0.625. In the time-kill dynamic confirmation test, synergistic bactericidal effects were observed for the combinations of GA-gchitosans with -lactams, and over 4.0 log CFU/mL reductions were observed after 24 h when combination treatment was used. These results may prove GA-g-chitosans to be a potent agent when combined with ampicillin and penicillin for the elimination of MRSA. Key words: chitosan, MRSA, antibacterial activity, cell envelope, synergy effect. Résumé : La propagation mondiale du Staphylococcus aureus résistant a` la méthicilline (SARM) rend la recherche de nouveaux antibiotiques plus urgente que jamais. On a étudié les activités de composés conjugués d’acide gallique greffé a` du chitosane (AG-g-chitosane) pour contrer 2 SARM typiques et 10 isolats cliniques de SARM, par la détermination de leur concentration inhibitrice minimale (CIM). L’AG-g-chitosane (I), qui contenait la plus grande quantité d’acide gallique, a présenté la plus forte activité anti-SARM, avec des CIM de 32–64 g/mL. Un essai de cinétique de létalité a révélé que l’AG-g-chitosane (I) présentait une activité bactéricide au double de sa CIM et démontrait une bonne stabilité thermique et selon le pH. Une étude de l’intégrité de l’enveloppe cellulaire a mis au jour une libération de composantes intracellulaires indiquée par une hausse de l’absorbance a` 260 nm, témoignant de dommages a` l’enveloppe cellulaire causés par l’AG-g-chitosane (I), phénomène qui a été confirmé par microscopie électronique a` transmission. Lorsque les composés d’AG-g-chitosane ont été combinés a` des -lactames, dont l’ampicilline et la pénicilline, on a observé des effets synergiques a` l’encontre des deux souches types de SARM et des dix isolats cliniques, les indices d’inhibition fractionnaire s’étalant entre 0,125 et 0,625. Pour ce qui est du test de confirmation de la dynamique de la cinétique de la létalité, on a observé des effets bactéricides synergiques chez les agencements de conjugués d’AG-g-chitosane et de -lactames; on a observé des baisses de plus de 4,0 log UFC/mL après 24 h suite au traitement combiné. Ces résultats apportent des éléments démontrant que les conjugués d’AG-g-chitosane sont de puissants antibactériens lorsque combinés a` l’ampicilline et la pénicilline et employés pour éliminer le SARM. [Traduit par la Rédaction] Mots-clés : chitosane, SARM, activité antibactérienne, enveloppe cellulaire, effet synergique.
Introduction Staphylococcus aureus is one of the most important human pathogens, responsible not only for severe infections of the skin but also for life-threatening diseases, such as erysipelas, osteomyelitis, pneumonia, septicemia, and endocarditis (Lowy 1998). Staphylococcus aureus causes both hospital-acquired and community-acquired infections, and invasive infections by this bacterium are associ-
ated with high morbidity and mortality (Van Bambeke et al. 2008). Since S. aureus was traditionally highly susceptible to penicillin, the subsequent abuse of penicillin resulted in the spread of staphylococcal resistance, conferred by the production of penicillinase. Penicillinase-stable antibiotics, such as methicillin and oxacillin, were discovered in the early 1960s; however, methicillin-resistant S. aureus (MRSA) emerged soon after the introduction of these
Received 28 April 2014. Revision received 7 August 2014. Accepted 12 August 2014. D.-S. Lee, H.S. Kim, and M.-J. Yim. Marine Biodiversity Institute of Korea, Seocheon 325-902, Republic of Korea. S.-H. Eom. Division of Platform Technology Research, Korea Food Research Institute, Sungnam 463-749, Republic of Korea. Y.-M. Kim. Department of Food Science and Technology, Pukyong National University, Busan 608-737, Republic of Korea. S.-H. Lee. Food Resource Research Center, Korea Food Research Institute, Sungnam 463-749, Republic of Korea; University of Science and Technology, Daejeon 305-350, Republic of Korea. D.-H. Kim. Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, Republic of Korea. J.-Y. Je. Department of Marine-Bio Convergence Science, Pukyong National University, Busan 608-737, Republic of Korea. Corresponding authors: Do-Hyung Kim (e-mail: [email protected]) and Jae-Young Je (e-mail: [email protected]). Can. J. Microbiol. 60: 629–638 (2014) dx.doi.org/10.1139/cjm-2014-0286
Published at www.nrcresearchpress.com/cjm on 18 August 2014.
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antibiotics. Most clinical MRSA isolates contain the mecA gene, which encodes penicillin-binding protein 2a (PBP2a), which has a lower affinity for -lactams than typical PBPs, making the bacterium resistant to this entire class of antibiotics (Chambers and Sachdeva 1990). MRSA has become a worldwide concern because it is highly prevalent and has become resistant to almost all available antibiotics, except glycopeptides such as vancomycin and teicoplanin, which are used as last-resort treatment against MRSA. However, the susceptibility of MRSA to vancomycin has also been decreasing with heavy use, leading to the emergence of vancomycinintermediate and -resistant S. aureus. Thus, alternative antibiotics are urgently needed. Chitosan is a natural cationic polymer derived by deacetylation of chitin and consists of 2-amido-2-deoxy-(1-4)--D-glucopyranose residues (D-glucosamine units). Since the antifungal activity of chitosan was first proposed by Allan and Hardwiger (1979), the antimicrobial activity of chitosan and its derivatives have been investigated extensively. The mode of chitosans’s antibacterial activity is still unclear, but the activity seems to be influenced by various factors, such as its polycationic structure, the number of amino groups at the C-2 position, environmental pH, and the different physical states and molecular masses of chitosan, as well as the microorganisms tested (Kong et al. 2010). Moreover, most antimicrobial activities of chitosan and its derivatives have been investigated against food spoilage bacteria, food-borne pathogens, and fungi. Thus, scanty information regarding antibacterial activity of chitosan and its derivatives against drug-resistant bacteria has been available, until now. Gallic acid (GA) is a natural phenolic antioxidant extractable from plants, especially green tea, and is widely used in foods, drugs, and cosmetics (Lu et al. 2006). Our group previously developed gallic acid-grafted-chitosans (GA-g-chitosans), which have a high antioxidant activity compared with unmodified chitosan (Cho et al. 2011b). GA-g-chitosans have also demonstrated hepatoprotective and acetylcholinesterase inhibitory activity (Cho et al. 2011a; Senevirathne et al. 2012). Thus, as part of our ongoing investigation on the bioactivity of GA-g-chitosans, the present investigation is the first report of the anti-MRSA activity of GA-gchitosans and their mode of action. In addition, the synergistic effects of GA-g-chitosans in combination with -lactams were also investigated.
Materials and methods Production of the different forms of GA-g-chitosans Four kinds of GA-g-chitosan were prepared for the anti-MRSA assay by the previously reported method, using different molar ratios of chitosan residues and GA (Cho et al. 2011b). To confirm successful synthesis, 1H NMR and thin layer chromatography were conducted, and the results were compared with those of Cho et al. (2011b). Unmodified chitosan: 1H NMR (400 MHz, D2O) ␦: 5.30 (1H, H-1), 3.63–4.35 (1H, H-2/6), 2.51 (H-Ac), 4.8 (D2O). GA-g-chitosan: 1H NMR (400 MHz, D2O) ␦: 7.63 (phenyl protons of GA), 5.33 (1H, H-1), 3.65– 4.36 (1H, H-2/6), 2.51–2.54 (H-Ac), 4.8 (D2O). The GA content in the GA-g-chitosans was determined by the Folin–Ciocalteu method to be 118.92 mg GA/g GA-g-chitosan for GA-g-chitosan (I), 82.91 for GA-g-chitosan (II), 67.62 for GA-g-chitosan (III), and 53.87 for GA-gchitosan (IV). Powder form of GA-g-chitosan was dissolved in 0.01 mol/L HCl with 10 mg/mL as stock solution and then kept at –20 °C until use. Bacterial strains and medium Two standard MRSA strains (KCCM 40510 and KCCM 40511) were purchased from the Korea Culture Center of Microorganisms (KCCM; Seoul, Korea), and 10 MRSA clinical isolates were kindly provided by the Donga-A University Hospital (Busan, Korea). The methicillin-susceptible Staphylococcus aureus (MSSA) tested in this
Can. J. Microbiol. Vol. 60, 2014
Fig. 1. (A) Structure of gallic acid-grafted-chitosan (GA-g-chitosan). (B) Photograph of the antibacterial activity of GA-g-chitosans against methicillin-resistant Staphylococcus aureus (MRSA). A, GA-g-chitosan (I); B, GA-g-chitosan (II); C, GA-g-chitosan (III); D, GA-g-chitosan (IV); PC, unmodified chitosan.
study was purchased from the Korean Collection for Type Cultures (Daejeon, Korea). All strains were grown aerobically at 37 °C in Mueller–Hinton broth (MHB; Difco, Sparks, Maryland, USA) and were subsequently used in experiments to measure antibacterial activity. Antibacterial activity screening and determination of MICs The paper disc diffusion method was employed to determine the antibacterial activity of the GA-g-chitosans. A 10 L volume of each GA-g-chitosan (10 mg/mL) was used against MRSA. The 2-fold serial dilution method was employed to determine the minimum inhibitory concentrations (MICs) of the GA-g-chitosans against MRSA (NCCLS 2005). The MIC is defined as the lowest concentration that demonstrates no visible growth after incubation for 24 h at 37 °C. Thermal and pH stability Thermal stability of the GA-g-chitosans was investigated with 2× MIC at various temperatures by incubating at 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 °C for 1 h, or at 121 °C for 15 min using an autoclave. To assess pH stability, the GA-g-chitosans (2× MIC) were suspended in 0.1 mol/L citrate phosphate buffer with a pH range of 4–7 and in 0.1 mol/L Tris–HCl buffer at pH 8–10. They were kept in each buffer for 1 h at room temperature, and the anti-MRSA activity was estimated turbidimetrically at 640 nm after treatment. Bactericidal assay The bactericidal activity of the GA-g-chitosans was evaluated with a time-kill experiment. Briefly, each GA-g-chitosan was added to MHB inoculated with an MRSA strain (KCCM 40510), which was Published by NRC Research Press
Lee et al.
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Table 1. Minimum inhibitory concentrations (MICs) of the gallic acid-grafted-chitosans (I), (II), (III), (IV) and -lactams.
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MIC (g/mL) Strain
mecAb
GAgC (I)
MSSA (KCTC 1927)
–
32
32
32
64
128
GA-g-chitosan (IV) > unmodified chitosan. The 2-fold serial dilution method was then implemented to determine the MIC values of the 4 GA-g-chitosans against 1 strain of MSSA, 2 MRSA standards, and 10 clinically isolated MRSA strains, as summarized in Table 1. Among the 13 strains tested, all MRSA strains were mecA positive, while the MSSA standard, used as a negative control, was mecA negative. This gene is responsible for production of the specific PBP2a that reduces the affinity of -lactams. The MIC values of the 4 GA-g-chitosans were determined as 32– 64 g/mL for GA-g-chitosan (I), 32–128 g/mL for GA-g-chitosan (II), 32–128 g/mL for GA-g-chitosan (III), and 64–256 g/mL for GA-gchitosan (IV), whereas the unmodified chitosan had MIC values ranging from 128 to 256 g/mL against the MSSA and MRSA strains. Also we used chloramphenicol, and this exhibited the MIC values of 2–32 g/mL. The anti-MRSA activity of the GA-g-chitosans was superior to that of the unmodified chitosan, indicating the anti-MRSA activity was increased by the conjugation of GA on the chitosan backbone. This result indicates that the GA content in GA-g-chitosan was a major factor affecting its anti-MRSA activity. The MIC values of 3 -lactam antibiotics, including ampicillin, penicillin, and oxacillin, were also determined. Being mecA negative, MSSA was highly sensitive to the -lactams (MIC value of 90 °C. After autoclaving at 121 °C, the anti-MRSA activity of GA-g-chitosan (I) decreased dramatically (0.5 to ≤1, additive; >1 to ≤2, independent; >2, antagonistic.
Table 3. Fractional inhibitory concentration (FIC) indices of 0, 8, 16 g/mL gallic acid-grafted-chitosan (II) (GAgC) in combination with -lactams against methicillin-resistant Staphylococcus aureus (MRSA). Ampicillin
Penicillin a
MIC (g/mL)
FIC index
MIC (g/mL)
Oxacillin a
FIC index
MIC (g/mL)
Chloramphenicol a
FIC index
MIC (g/mL)
FIC indexa
Strain
0 GAgC
8 GAgC
16 GAgC
8 GAgC
16 GAgC
0 GAgC
8 GAgC
16 GAgC
8 GAgC
16 GAgC
0 GAgC
8 GAgC
16 GAgC
8 GAgC
16 GAgC
0 GAgC
8 GAgC
16 GAgC
8 GAgC
16 GAgC
MRSA (KCCM 40510) MRSA (KCCM 40511) MRSA D-1 MRSA D-2 MRSA D-3 MRSA D-4 MRSA D-6 MRSA D-8 MRSA D-11 MRSA D-12 MRSA D-17 MRSA D-19
512 512 256 512 128 128 256 256 256 128 128 128
16 16 16 32 16 32 32 16 32 16 16 16
8 8 8 16 8 16 16 8 8 8 8 4
0.156 0.156 0.188 0.188 0.250 0.313 0.250 0.188 0.188 0.250 0.250 0.250
0.266 0.266 0.281 0.281 0.313 0.250 0.313 0.281 0.156 0.313 0.313 0.281
512 256 256 256 128 256 128 128 128 128 128 128
16 16 8 32 32 16 32 16 8 8 16 16
8 8 4 16 16 8 8 8 4 4 8 8
0.156 0.188 0.156 0.250 0.375 0.125 0.375 0.250 0.125 0.188 0.250 0.250
0.266 0.281 0.266 0.313 0.375 0.156 0.313 0.313 0.156 0.281 0.313 0.313
128 128 128 256 512 128 256 512 128 64 128 256
128 128 32 128 16 128 256 128 64 8 128 128
64 128 16 128 8 64 256 64 32 4 128 32
1.125 1.125 0.375 0.625 0.156 1.063 1.125 0.375 0.563 0.250 1.125 0.625
0.750 1.250 0.375 0.750 0.266 0.625 1.250 0.375 0.375 0.313 1.250 0.375
32 16 16 8 32 32 16 16 32 8 16 16
32 16 16 8 16 32 8 16 32 8 16 8
32 16 16 4 32 32 16 8 16 4 16 16
1.125 1.125 1.125 1.125 0.625 1.063 0.625 1.125 1.063 1.125 1.125 0.625
1.250 1.250 1.250 0.750 1.250 1.125 1.250 0.750 0.625 0.750 1.250 1.250
aThe
FIC index indicated synergy as follows: ≤0.5, synergic; >0.5 to ≤1, additive; >1 to ≤2, independent; >2, antagonistic.
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Table 2. Fractional inhibitory concentration (FIC) indices of 0, 8, 16 g/mL gallic acid-grafted-chitosan (I) (GAgC) in combination with -lactams against methicillin-resistant Staphylococcus aureus (MRSA).
Penicillin a
MIC (g/mL)
FIC index
MIC (g/mL)
Oxacillin a
FIC index
MIC (g/mL)
Chloramphenicol a
FIC index
MIC (g/mL)
FIC indexa
Strain
0 GAgC
16 GAgC
32 GAgC
16 GAgC
32 GAgC
0 GAgC
16 GAgC
32 GAgC
16 GAgC
32 GAgC
0 GAgC
16 GAgC
32 GAgC
16 GAgC
32 GAgC
0 GAgC
16 GAgC
32 GAgC
16 GAgC
32 GAgC
MRSA (KCCM 40510) MRSA (KCCM 40511) MRSA D-1 MRSA D-2 MRSA D-3 MRSA D-4 MRSA D-6 MRSA D-8 MRSA D-11 MRSA D-12 MRSA D-17 MRSA D-19
512 512 256 512 128 128 256 256 256 128 128 128
16 8 8 32 8 8 32 32 32 8 16 8
8 4 2 16 4 8 16 16 16 2 8 4
0.156 0.141 0.281 0.188 0.313 0.188 0.250 0.250 0.375 0.313 0.375 0.313
0.266 0.258 0.508 0.281 0.531 0.313 0.313 0.313 0.563 0.516 0.563 0.531
512 256 256 256 128 256 128 128 128 128 128 128
16 16 16 32 16 16 16 32 16 8 32 16
8 4 8 16 8 8 8 16 8 4 16 8
0.156 0.188 0.313 0.250 0.375 0.188 0.250 0.375 0.375 0.313 0.500 0.375
0.266 0.266 0.531 0.313 0.563 0.281 0.313 0.375 0.563 0.531 0.625 0.563
128 128 128 256 512 128 256 512 128 64 128 256
128 128 128 128 64 128 128 256 64 16 128 128
128 128 32 64 64 128 32 128 32 8 64 64
1.125 1.125 1.250 0.625 0.375 1.125 0.625 0.625 0.750 0.500 1.250 0.750
1.250 1.250 0.750 0.500 0.625 1.250 0.375 0.500 0.750 0.625 1.000 0.750
32 16 16 8 32 32 16 16 32 8 16 16
32 16 16 8 32 32 16 16 16 8 8 16
32 16 8 8 32 16 16 16 16 8 8 16
1.125 1.125 1.250 1.125 1.250 1.125 1.125 1.125 0.750 1.250 0.750 1.250
1.250 1.250 1.000 1.250 1.500 0.750 1.250 1.250 1.000 1.500 1.000 1.500
aThe
FIC index indicated synergy as follows: ≤0.5, synergic; >0.5 to ≤1, additive; >1 to ≤2, independent; >2, antagonistic.
Table 5. Fractional inhibitory concentration (FIC) indices of 0, 16, 32 g/mL gallic acid-grafted-chitosan (IV) (GAgC) in combination with -lactams against methicillin-resistant Staphylococcus aureus (MRSA). Ampicillin
Penicillin a
MIC (g/mL)
FIC index
MIC (g/mL)
Oxacillin a
FIC index
MIC (g/mL)
Chloramphenicol a
FIC index
MIC (g/mL)
FIC indexa
Strain
0 GAgC
16 GAgC
32 GAgC
16 GAgC
32 GAgC
0 GAgC
16 GAgC
32 GAgC
16 GAgC
32 GAgC
0 GAgC
16 GAgC
32 GAgC
16 GAgC
32 GAgC
0 GAgC
16 GAgC
32 GAgC
16 GAgC
32 GAgC
MRSA (KCCM 40510) MRSA (KCCM 40511) MRSA D-1 MRSA D-2 MRSA D-3 MRSA D-4 MRSA D-6 MRSA D-8 MRSA D-11 MRSA D-12 MRSA D-17 MRSA D-19
512 512 256 512 128 128 256 256 256 128 128 128
64 64 64 128 32 32 64 64 64 32 32 32
32 64 32 64 16 32 16 32 32 16 32 16
0.250 0.250 0.500 0.375 0.313 0.375 0.500 0.375 0.375 0.375 0.375 0.375
0.313 0.375 0.625 0.375 0.250 0.500 0.563 0.375 0.375 0.375 0.500 0.375
512 256 256 256 128 256 128 128 128 128 128 128
64 64 64 64 32 64 32 32 32 32 32 32
32 64 32 64 16 16 16 32 16 32 16 16
0.250 0.375 0.500 0.375 0.313 0.375 0.500 0.375 0.375 0.375 0.375 0.375
0.313 0.500 0.625 0.500 0.250 0.313 0.625 0.500 0.375 0.500 0.375 0.375
128 128 128 256 512 128 256 512 128 64 128 256
64 64 64 128 256 64 128 256 64 64 64 128
64 32 32 64 128 64 128 128 64 32 32 64
0.625 0.625 0.750 0.625 0.563 0.625 0.750 0.625 0.625 1.125 0.625 0.625
0.750 0.500 0.750 0.500 0.375 0.750 1.000 0.500 0.750 0.750 0.500 0.500
32 16 16 8 32 32 16 16 32 8 16 16
32 16 16 8 32 32 16 8 16 8 16 16
32 8 16 8 32 32 8 8 16 8 8 16
1.125 1.125 1.250 1.125 1.063 1.125 1.250 0.625 0.625 1.125 1.125 1.125
1.250 0.750 1.500 1.250 1.125 1.250 1.000 0.750 0.750 1.250 0.750 1.250
aThe
FIC index indicated synergy as follows: ≤0.5, synergic; >0.5 to ≤1, additive; >1 to ≤2, independent; >2, antagonistic. 635
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Ampicillin
Lee et al.
Table 4. Fractional inhibitory concentration (FIC) indices of 0, 16, 32 g/mL gallic acid-grafted-chitosan (III) (GAgC) in combination with -lactams against methicillin-resistant Staphylococcus aureus (MRSA).
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Fig. 6. Time-kill curves of the synergic activity of the combination of gallic acid-grafted-chitosans (GAgC) and the selected -lactams (ampicillin and penicillin). -Lactams with (A) GAgC (I), (B) GAgC (II), (C) GAgC (III), and (D) GAgC (IV).
after 24 h. In the case of combining 0.25× MIC of the GA-gchitosan (I) with 0.03125× MIC of ampicillin or penicillin, the timekilling curve showed that the combination resulted in a 5.4 log reduction after 9 h (Fig. 6A), and a 6.4 log reduction (ampicillin) and 6.6 log reduction (penicillin) were observed after 24 h, indicating synergic bactericidal effects. In combination with GA-gchitosan (II), a 6.3 log reduction (ampicillin) and 6.1 log reduction (penicillin) were observed after 12 h, whereas the reduction was slightly decreased to 5.7 log for ampicillin and 5.6 log for penicillin after 24 h (Fig. 6B). A 6.4 log reduction (ampicillin) and 6.1 log reduction (penicillin) were observed with GA-g-chitosan (III) after 15 h, whereas the reduction was also slightly decreased after 24 h (Fig. 6C). GA-g-chitosan (IV) also revealed synergic bactericidal effects in combination with ampicillin and penicillin, but the effects were lower than those of the other GA-g-chitosans tested (Fig. 6D).
Discussion Staphylococcus aureus has a single membrane composed of phosphatidylglycerol and cardiolipin surrounded by a thick cell wall of peptidoglycan, which is essential for cell survival, exerts selective permeability, and protects the bacteria from foreign factors (Epand et al. 2007). Thus, disruption of the cell envelope is a major
goal of antibiotics, and many antibiotics act on the enzymes involved in peptidoglycan synthesis (Silver 2006). However, MRSA produces -lactamases that inactivate -lactam antibiotics and also produces the cell-wall-synthetizing enzyme PBP2a whose transpeptidase domain has a reduced affinity for -lactam antibiotics. PBP2a can still link the long glycan strands of peptidoglycan with peptide crosslinks even in the presence of -lactam antibiotics, thus providing resistance (Van Bambeke et al. 2008). Our previous study showed that MRSA strains with the mecA gene are highly resistant to -lactam antibiotics, such as ampicillin, penicillin, and oxacillin, and that the MIC values of the -lactam antibiotics were 64–512 g/mL, while the MSSA strain is highly sensitive to the -lactam with MIC values
ARTICLE
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Antibacterial and synergic effects of gallic acid-grafted-chitosan with -lactams against methicillin-resistant Staphylococcus aureus (MRSA) Dae-Sung Lee, Sung-Hwan Eom, Young-Mog Kim, Hye Seon Kim, Mi-Jin Yim, Sang-Hoon Lee, Do-Hyung Kim, and Jae-Young Je
Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) is spreading worldwide, emphasizing the need to search for new antibiotics. The anti-MRSA activities of gallic acid-grafted-chitosans (GA-g-chitosans) were investigated against 2 MRSA standards and 10 MRSA clinical isolates by determining the minimum inhibitory concentrations (MICs). GA-g-chitosan (I), which has the highest gallic acid content, exhibited the strongest anti-MRSA activities, with MICs of 32–64 g/mL. A time-kill investigation revealed that GA-g-chitosan (I) exhibited a bactericidal effect at twice the MIC, also demonstrating good thermal and pH stability. Investigation of cell envelope integrity showed the release of intracellular components with an increasing absorbance value at 260 nm, indicating cell envelope damage caused by the GA-g-chitosan (I), which was further confirmed by transmission electron microscopy. When GA-g-chitosans were combined with -lactams, including ampicillin and penicillin, synergistic effects were observed on the 2 standard MRSA strains and on the 10 clinical isolates, with fractional inhibitory indices ranging from 0.125 to 0.625. In the time-kill dynamic confirmation test, synergistic bactericidal effects were observed for the combinations of GA-gchitosans with -lactams, and over 4.0 log CFU/mL reductions were observed after 24 h when combination treatment was used. These results may prove GA-g-chitosans to be a potent agent when combined with ampicillin and penicillin for the elimination of MRSA. Key words: chitosan, MRSA, antibacterial activity, cell envelope, synergy effect. Résumé : La propagation mondiale du Staphylococcus aureus résistant a` la méthicilline (SARM) rend la recherche de nouveaux antibiotiques plus urgente que jamais. On a étudié les activités de composés conjugués d’acide gallique greffé a` du chitosane (AG-g-chitosane) pour contrer 2 SARM typiques et 10 isolats cliniques de SARM, par la détermination de leur concentration inhibitrice minimale (CIM). L’AG-g-chitosane (I), qui contenait la plus grande quantité d’acide gallique, a présenté la plus forte activité anti-SARM, avec des CIM de 32–64 g/mL. Un essai de cinétique de létalité a révélé que l’AG-g-chitosane (I) présentait une activité bactéricide au double de sa CIM et démontrait une bonne stabilité thermique et selon le pH. Une étude de l’intégrité de l’enveloppe cellulaire a mis au jour une libération de composantes intracellulaires indiquée par une hausse de l’absorbance a` 260 nm, témoignant de dommages a` l’enveloppe cellulaire causés par l’AG-g-chitosane (I), phénomène qui a été confirmé par microscopie électronique a` transmission. Lorsque les composés d’AG-g-chitosane ont été combinés a` des -lactames, dont l’ampicilline et la pénicilline, on a observé des effets synergiques a` l’encontre des deux souches types de SARM et des dix isolats cliniques, les indices d’inhibition fractionnaire s’étalant entre 0,125 et 0,625. Pour ce qui est du test de confirmation de la dynamique de la cinétique de la létalité, on a observé des effets bactéricides synergiques chez les agencements de conjugués d’AG-g-chitosane et de -lactames; on a observé des baisses de plus de 4,0 log UFC/mL après 24 h suite au traitement combiné. Ces résultats apportent des éléments démontrant que les conjugués d’AG-g-chitosane sont de puissants antibactériens lorsque combinés a` l’ampicilline et la pénicilline et employés pour éliminer le SARM. [Traduit par la Rédaction] Mots-clés : chitosane, SARM, activité antibactérienne, enveloppe cellulaire, effet synergique.
Introduction Staphylococcus aureus is one of the most important human pathogens, responsible not only for severe infections of the skin but also for life-threatening diseases, such as erysipelas, osteomyelitis, pneumonia, septicemia, and endocarditis (Lowy 1998). Staphylococcus aureus causes both hospital-acquired and community-acquired infections, and invasive infections by this bacterium are associ-
ated with high morbidity and mortality (Van Bambeke et al. 2008). Since S. aureus was traditionally highly susceptible to penicillin, the subsequent abuse of penicillin resulted in the spread of staphylococcal resistance, conferred by the production of penicillinase. Penicillinase-stable antibiotics, such as methicillin and oxacillin, were discovered in the early 1960s; however, methicillin-resistant S. aureus (MRSA) emerged soon after the introduction of these
Received 28 April 2014. Revision received 7 August 2014. Accepted 12 August 2014. D.-S. Lee, H.S. Kim, and M.-J. Yim. Marine Biodiversity Institute of Korea, Seocheon 325-902, Republic of Korea. S.-H. Eom. Division of Platform Technology Research, Korea Food Research Institute, Sungnam 463-749, Republic of Korea. Y.-M. Kim. Department of Food Science and Technology, Pukyong National University, Busan 608-737, Republic of Korea. S.-H. Lee. Food Resource Research Center, Korea Food Research Institute, Sungnam 463-749, Republic of Korea; University of Science and Technology, Daejeon 305-350, Republic of Korea. D.-H. Kim. Department of Aquatic Life Medicine, Pukyong National University, Busan 608-737, Republic of Korea. J.-Y. Je. Department of Marine-Bio Convergence Science, Pukyong National University, Busan 608-737, Republic of Korea. Corresponding authors: Do-Hyung Kim (e-mail: [email protected]) and Jae-Young Je (e-mail: [email protected]). Can. J. Microbiol. 60: 629–638 (2014) dx.doi.org/10.1139/cjm-2014-0286
Published at www.nrcresearchpress.com/cjm on 18 August 2014.
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antibiotics. Most clinical MRSA isolates contain the mecA gene, which encodes penicillin-binding protein 2a (PBP2a), which has a lower affinity for -lactams than typical PBPs, making the bacterium resistant to this entire class of antibiotics (Chambers and Sachdeva 1990). MRSA has become a worldwide concern because it is highly prevalent and has become resistant to almost all available antibiotics, except glycopeptides such as vancomycin and teicoplanin, which are used as last-resort treatment against MRSA. However, the susceptibility of MRSA to vancomycin has also been decreasing with heavy use, leading to the emergence of vancomycinintermediate and -resistant S. aureus. Thus, alternative antibiotics are urgently needed. Chitosan is a natural cationic polymer derived by deacetylation of chitin and consists of 2-amido-2-deoxy-(1-4)--D-glucopyranose residues (D-glucosamine units). Since the antifungal activity of chitosan was first proposed by Allan and Hardwiger (1979), the antimicrobial activity of chitosan and its derivatives have been investigated extensively. The mode of chitosans’s antibacterial activity is still unclear, but the activity seems to be influenced by various factors, such as its polycationic structure, the number of amino groups at the C-2 position, environmental pH, and the different physical states and molecular masses of chitosan, as well as the microorganisms tested (Kong et al. 2010). Moreover, most antimicrobial activities of chitosan and its derivatives have been investigated against food spoilage bacteria, food-borne pathogens, and fungi. Thus, scanty information regarding antibacterial activity of chitosan and its derivatives against drug-resistant bacteria has been available, until now. Gallic acid (GA) is a natural phenolic antioxidant extractable from plants, especially green tea, and is widely used in foods, drugs, and cosmetics (Lu et al. 2006). Our group previously developed gallic acid-grafted-chitosans (GA-g-chitosans), which have a high antioxidant activity compared with unmodified chitosan (Cho et al. 2011b). GA-g-chitosans have also demonstrated hepatoprotective and acetylcholinesterase inhibitory activity (Cho et al. 2011a; Senevirathne et al. 2012). Thus, as part of our ongoing investigation on the bioactivity of GA-g-chitosans, the present investigation is the first report of the anti-MRSA activity of GA-gchitosans and their mode of action. In addition, the synergistic effects of GA-g-chitosans in combination with -lactams were also investigated.
Materials and methods Production of the different forms of GA-g-chitosans Four kinds of GA-g-chitosan were prepared for the anti-MRSA assay by the previously reported method, using different molar ratios of chitosan residues and GA (Cho et al. 2011b). To confirm successful synthesis, 1H NMR and thin layer chromatography were conducted, and the results were compared with those of Cho et al. (2011b). Unmodified chitosan: 1H NMR (400 MHz, D2O) ␦: 5.30 (1H, H-1), 3.63–4.35 (1H, H-2/6), 2.51 (H-Ac), 4.8 (D2O). GA-g-chitosan: 1H NMR (400 MHz, D2O) ␦: 7.63 (phenyl protons of GA), 5.33 (1H, H-1), 3.65– 4.36 (1H, H-2/6), 2.51–2.54 (H-Ac), 4.8 (D2O). The GA content in the GA-g-chitosans was determined by the Folin–Ciocalteu method to be 118.92 mg GA/g GA-g-chitosan for GA-g-chitosan (I), 82.91 for GA-g-chitosan (II), 67.62 for GA-g-chitosan (III), and 53.87 for GA-gchitosan (IV). Powder form of GA-g-chitosan was dissolved in 0.01 mol/L HCl with 10 mg/mL as stock solution and then kept at –20 °C until use. Bacterial strains and medium Two standard MRSA strains (KCCM 40510 and KCCM 40511) were purchased from the Korea Culture Center of Microorganisms (KCCM; Seoul, Korea), and 10 MRSA clinical isolates were kindly provided by the Donga-A University Hospital (Busan, Korea). The methicillin-susceptible Staphylococcus aureus (MSSA) tested in this
Can. J. Microbiol. Vol. 60, 2014
Fig. 1. (A) Structure of gallic acid-grafted-chitosan (GA-g-chitosan). (B) Photograph of the antibacterial activity of GA-g-chitosans against methicillin-resistant Staphylococcus aureus (MRSA). A, GA-g-chitosan (I); B, GA-g-chitosan (II); C, GA-g-chitosan (III); D, GA-g-chitosan (IV); PC, unmodified chitosan.
study was purchased from the Korean Collection for Type Cultures (Daejeon, Korea). All strains were grown aerobically at 37 °C in Mueller–Hinton broth (MHB; Difco, Sparks, Maryland, USA) and were subsequently used in experiments to measure antibacterial activity. Antibacterial activity screening and determination of MICs The paper disc diffusion method was employed to determine the antibacterial activity of the GA-g-chitosans. A 10 L volume of each GA-g-chitosan (10 mg/mL) was used against MRSA. The 2-fold serial dilution method was employed to determine the minimum inhibitory concentrations (MICs) of the GA-g-chitosans against MRSA (NCCLS 2005). The MIC is defined as the lowest concentration that demonstrates no visible growth after incubation for 24 h at 37 °C. Thermal and pH stability Thermal stability of the GA-g-chitosans was investigated with 2× MIC at various temperatures by incubating at 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 °C for 1 h, or at 121 °C for 15 min using an autoclave. To assess pH stability, the GA-g-chitosans (2× MIC) were suspended in 0.1 mol/L citrate phosphate buffer with a pH range of 4–7 and in 0.1 mol/L Tris–HCl buffer at pH 8–10. They were kept in each buffer for 1 h at room temperature, and the anti-MRSA activity was estimated turbidimetrically at 640 nm after treatment. Bactericidal assay The bactericidal activity of the GA-g-chitosans was evaluated with a time-kill experiment. Briefly, each GA-g-chitosan was added to MHB inoculated with an MRSA strain (KCCM 40510), which was Published by NRC Research Press
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Table 1. Minimum inhibitory concentrations (MICs) of the gallic acid-grafted-chitosans (I), (II), (III), (IV) and -lactams.
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MIC (g/mL) Strain
mecAb
GAgC (I)
MSSA (KCTC 1927)
–
32
32
32
64
128
GA-g-chitosan (IV) > unmodified chitosan. The 2-fold serial dilution method was then implemented to determine the MIC values of the 4 GA-g-chitosans against 1 strain of MSSA, 2 MRSA standards, and 10 clinically isolated MRSA strains, as summarized in Table 1. Among the 13 strains tested, all MRSA strains were mecA positive, while the MSSA standard, used as a negative control, was mecA negative. This gene is responsible for production of the specific PBP2a that reduces the affinity of -lactams. The MIC values of the 4 GA-g-chitosans were determined as 32– 64 g/mL for GA-g-chitosan (I), 32–128 g/mL for GA-g-chitosan (II), 32–128 g/mL for GA-g-chitosan (III), and 64–256 g/mL for GA-gchitosan (IV), whereas the unmodified chitosan had MIC values ranging from 128 to 256 g/mL against the MSSA and MRSA strains. Also we used chloramphenicol, and this exhibited the MIC values of 2–32 g/mL. The anti-MRSA activity of the GA-g-chitosans was superior to that of the unmodified chitosan, indicating the anti-MRSA activity was increased by the conjugation of GA on the chitosan backbone. This result indicates that the GA content in GA-g-chitosan was a major factor affecting its anti-MRSA activity. The MIC values of 3 -lactam antibiotics, including ampicillin, penicillin, and oxacillin, were also determined. Being mecA negative, MSSA was highly sensitive to the -lactams (MIC value of 90 °C. After autoclaving at 121 °C, the anti-MRSA activity of GA-g-chitosan (I) decreased dramatically (0.5 to ≤1, additive; >1 to ≤2, independent; >2, antagonistic.
Table 3. Fractional inhibitory concentration (FIC) indices of 0, 8, 16 g/mL gallic acid-grafted-chitosan (II) (GAgC) in combination with -lactams against methicillin-resistant Staphylococcus aureus (MRSA). Ampicillin
Penicillin a
MIC (g/mL)
FIC index
MIC (g/mL)
Oxacillin a
FIC index
MIC (g/mL)
Chloramphenicol a
FIC index
MIC (g/mL)
FIC indexa
Strain
0 GAgC
8 GAgC
16 GAgC
8 GAgC
16 GAgC
0 GAgC
8 GAgC
16 GAgC
8 GAgC
16 GAgC
0 GAgC
8 GAgC
16 GAgC
8 GAgC
16 GAgC
0 GAgC
8 GAgC
16 GAgC
8 GAgC
16 GAgC
MRSA (KCCM 40510) MRSA (KCCM 40511) MRSA D-1 MRSA D-2 MRSA D-3 MRSA D-4 MRSA D-6 MRSA D-8 MRSA D-11 MRSA D-12 MRSA D-17 MRSA D-19
512 512 256 512 128 128 256 256 256 128 128 128
16 16 16 32 16 32 32 16 32 16 16 16
8 8 8 16 8 16 16 8 8 8 8 4
0.156 0.156 0.188 0.188 0.250 0.313 0.250 0.188 0.188 0.250 0.250 0.250
0.266 0.266 0.281 0.281 0.313 0.250 0.313 0.281 0.156 0.313 0.313 0.281
512 256 256 256 128 256 128 128 128 128 128 128
16 16 8 32 32 16 32 16 8 8 16 16
8 8 4 16 16 8 8 8 4 4 8 8
0.156 0.188 0.156 0.250 0.375 0.125 0.375 0.250 0.125 0.188 0.250 0.250
0.266 0.281 0.266 0.313 0.375 0.156 0.313 0.313 0.156 0.281 0.313 0.313
128 128 128 256 512 128 256 512 128 64 128 256
128 128 32 128 16 128 256 128 64 8 128 128
64 128 16 128 8 64 256 64 32 4 128 32
1.125 1.125 0.375 0.625 0.156 1.063 1.125 0.375 0.563 0.250 1.125 0.625
0.750 1.250 0.375 0.750 0.266 0.625 1.250 0.375 0.375 0.313 1.250 0.375
32 16 16 8 32 32 16 16 32 8 16 16
32 16 16 8 16 32 8 16 32 8 16 8
32 16 16 4 32 32 16 8 16 4 16 16
1.125 1.125 1.125 1.125 0.625 1.063 0.625 1.125 1.063 1.125 1.125 0.625
1.250 1.250 1.250 0.750 1.250 1.125 1.250 0.750 0.625 0.750 1.250 1.250
aThe
FIC index indicated synergy as follows: ≤0.5, synergic; >0.5 to ≤1, additive; >1 to ≤2, independent; >2, antagonistic.
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Table 2. Fractional inhibitory concentration (FIC) indices of 0, 8, 16 g/mL gallic acid-grafted-chitosan (I) (GAgC) in combination with -lactams against methicillin-resistant Staphylococcus aureus (MRSA).
Penicillin a
MIC (g/mL)
FIC index
MIC (g/mL)
Oxacillin a
FIC index
MIC (g/mL)
Chloramphenicol a
FIC index
MIC (g/mL)
FIC indexa
Strain
0 GAgC
16 GAgC
32 GAgC
16 GAgC
32 GAgC
0 GAgC
16 GAgC
32 GAgC
16 GAgC
32 GAgC
0 GAgC
16 GAgC
32 GAgC
16 GAgC
32 GAgC
0 GAgC
16 GAgC
32 GAgC
16 GAgC
32 GAgC
MRSA (KCCM 40510) MRSA (KCCM 40511) MRSA D-1 MRSA D-2 MRSA D-3 MRSA D-4 MRSA D-6 MRSA D-8 MRSA D-11 MRSA D-12 MRSA D-17 MRSA D-19
512 512 256 512 128 128 256 256 256 128 128 128
16 8 8 32 8 8 32 32 32 8 16 8
8 4 2 16 4 8 16 16 16 2 8 4
0.156 0.141 0.281 0.188 0.313 0.188 0.250 0.250 0.375 0.313 0.375 0.313
0.266 0.258 0.508 0.281 0.531 0.313 0.313 0.313 0.563 0.516 0.563 0.531
512 256 256 256 128 256 128 128 128 128 128 128
16 16 16 32 16 16 16 32 16 8 32 16
8 4 8 16 8 8 8 16 8 4 16 8
0.156 0.188 0.313 0.250 0.375 0.188 0.250 0.375 0.375 0.313 0.500 0.375
0.266 0.266 0.531 0.313 0.563 0.281 0.313 0.375 0.563 0.531 0.625 0.563
128 128 128 256 512 128 256 512 128 64 128 256
128 128 128 128 64 128 128 256 64 16 128 128
128 128 32 64 64 128 32 128 32 8 64 64
1.125 1.125 1.250 0.625 0.375 1.125 0.625 0.625 0.750 0.500 1.250 0.750
1.250 1.250 0.750 0.500 0.625 1.250 0.375 0.500 0.750 0.625 1.000 0.750
32 16 16 8 32 32 16 16 32 8 16 16
32 16 16 8 32 32 16 16 16 8 8 16
32 16 8 8 32 16 16 16 16 8 8 16
1.125 1.125 1.250 1.125 1.250 1.125 1.125 1.125 0.750 1.250 0.750 1.250
1.250 1.250 1.000 1.250 1.500 0.750 1.250 1.250 1.000 1.500 1.000 1.500
aThe
FIC index indicated synergy as follows: ≤0.5, synergic; >0.5 to ≤1, additive; >1 to ≤2, independent; >2, antagonistic.
Table 5. Fractional inhibitory concentration (FIC) indices of 0, 16, 32 g/mL gallic acid-grafted-chitosan (IV) (GAgC) in combination with -lactams against methicillin-resistant Staphylococcus aureus (MRSA). Ampicillin
Penicillin a
MIC (g/mL)
FIC index
MIC (g/mL)
Oxacillin a
FIC index
MIC (g/mL)
Chloramphenicol a
FIC index
MIC (g/mL)
FIC indexa
Strain
0 GAgC
16 GAgC
32 GAgC
16 GAgC
32 GAgC
0 GAgC
16 GAgC
32 GAgC
16 GAgC
32 GAgC
0 GAgC
16 GAgC
32 GAgC
16 GAgC
32 GAgC
0 GAgC
16 GAgC
32 GAgC
16 GAgC
32 GAgC
MRSA (KCCM 40510) MRSA (KCCM 40511) MRSA D-1 MRSA D-2 MRSA D-3 MRSA D-4 MRSA D-6 MRSA D-8 MRSA D-11 MRSA D-12 MRSA D-17 MRSA D-19
512 512 256 512 128 128 256 256 256 128 128 128
64 64 64 128 32 32 64 64 64 32 32 32
32 64 32 64 16 32 16 32 32 16 32 16
0.250 0.250 0.500 0.375 0.313 0.375 0.500 0.375 0.375 0.375 0.375 0.375
0.313 0.375 0.625 0.375 0.250 0.500 0.563 0.375 0.375 0.375 0.500 0.375
512 256 256 256 128 256 128 128 128 128 128 128
64 64 64 64 32 64 32 32 32 32 32 32
32 64 32 64 16 16 16 32 16 32 16 16
0.250 0.375 0.500 0.375 0.313 0.375 0.500 0.375 0.375 0.375 0.375 0.375
0.313 0.500 0.625 0.500 0.250 0.313 0.625 0.500 0.375 0.500 0.375 0.375
128 128 128 256 512 128 256 512 128 64 128 256
64 64 64 128 256 64 128 256 64 64 64 128
64 32 32 64 128 64 128 128 64 32 32 64
0.625 0.625 0.750 0.625 0.563 0.625 0.750 0.625 0.625 1.125 0.625 0.625
0.750 0.500 0.750 0.500 0.375 0.750 1.000 0.500 0.750 0.750 0.500 0.500
32 16 16 8 32 32 16 16 32 8 16 16
32 16 16 8 32 32 16 8 16 8 16 16
32 8 16 8 32 32 8 8 16 8 8 16
1.125 1.125 1.250 1.125 1.063 1.125 1.250 0.625 0.625 1.125 1.125 1.125
1.250 0.750 1.500 1.250 1.125 1.250 1.000 0.750 0.750 1.250 0.750 1.250
aThe
FIC index indicated synergy as follows: ≤0.5, synergic; >0.5 to ≤1, additive; >1 to ≤2, independent; >2, antagonistic. 635
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Ampicillin
Lee et al.
Table 4. Fractional inhibitory concentration (FIC) indices of 0, 16, 32 g/mL gallic acid-grafted-chitosan (III) (GAgC) in combination with -lactams against methicillin-resistant Staphylococcus aureus (MRSA).
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Fig. 6. Time-kill curves of the synergic activity of the combination of gallic acid-grafted-chitosans (GAgC) and the selected -lactams (ampicillin and penicillin). -Lactams with (A) GAgC (I), (B) GAgC (II), (C) GAgC (III), and (D) GAgC (IV).
after 24 h. In the case of combining 0.25× MIC of the GA-gchitosan (I) with 0.03125× MIC of ampicillin or penicillin, the timekilling curve showed that the combination resulted in a 5.4 log reduction after 9 h (Fig. 6A), and a 6.4 log reduction (ampicillin) and 6.6 log reduction (penicillin) were observed after 24 h, indicating synergic bactericidal effects. In combination with GA-gchitosan (II), a 6.3 log reduction (ampicillin) and 6.1 log reduction (penicillin) were observed after 12 h, whereas the reduction was slightly decreased to 5.7 log for ampicillin and 5.6 log for penicillin after 24 h (Fig. 6B). A 6.4 log reduction (ampicillin) and 6.1 log reduction (penicillin) were observed with GA-g-chitosan (III) after 15 h, whereas the reduction was also slightly decreased after 24 h (Fig. 6C). GA-g-chitosan (IV) also revealed synergic bactericidal effects in combination with ampicillin and penicillin, but the effects were lower than those of the other GA-g-chitosans tested (Fig. 6D).
Discussion Staphylococcus aureus has a single membrane composed of phosphatidylglycerol and cardiolipin surrounded by a thick cell wall of peptidoglycan, which is essential for cell survival, exerts selective permeability, and protects the bacteria from foreign factors (Epand et al. 2007). Thus, disruption of the cell envelope is a major
goal of antibiotics, and many antibiotics act on the enzymes involved in peptidoglycan synthesis (Silver 2006). However, MRSA produces -lactamases that inactivate -lactam antibiotics and also produces the cell-wall-synthetizing enzyme PBP2a whose transpeptidase domain has a reduced affinity for -lactam antibiotics. PBP2a can still link the long glycan strands of peptidoglycan with peptide crosslinks even in the presence of -lactam antibiotics, thus providing resistance (Van Bambeke et al. 2008). Our previous study showed that MRSA strains with the mecA gene are highly resistant to -lactam antibiotics, such as ampicillin, penicillin, and oxacillin, and that the MIC values of the -lactam antibiotics were 64–512 g/mL, while the MSSA strain is highly sensitive to the -lactam with MIC values
