Antimicrobial effects of lysophosphatidylcholine on methicillin-resistant Staphylococcus aureus

Abstract

Objectives:

Methicillin-resistant Staphylococcus aureus (MRSA) is an important health care-associated and community-associated pathogen and causes a large number of infections worldwide. For the purpose of application to topical treatment of MRSA infection, we examined the antimicrobial effects of lysophosphatidylcholine (LPC) on MRSA strains. We also investigated the combination effect of LPC and gentamicin on MRSA growth.

Methods:

The LPC minimum inhibitory concentrations (MIC) for Gram-positive (S. aureus, Staphylococcus epidermidis, and Streptococcus pneumoniae) and Gram-negative (Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, and Pseudomonas aeruginosa) bacteria were measured by the broth microdilution method. The mechanism of LPC-mediated MRSA killing was investigated by membrane permeability analysis with DiSC3(5) fluorescence and growth curve analysis. Lastly, the effects of LPC on gentamicin-induced bactericidal activity were determined in combination treatment studies with 15 gentamicin-resistant MRSA isolates from the skin, nose, or ears.

Results:

The LPC MIC for Gram-positive bacteria varied between 32 µg/ml and >2048 µg/ml, whereas that for all Gram-negative bacteria was >2048 µg/ml. Consistently, membrane permeability analysis showed that LPC was substantially more effective in inducing membrane permeability in Gram-positive bacteria than in Gram-negative counterparts. Growth curve analysis in cotreatment studies demonstrated that LPC has intrinsic bactericidal effects and can also potentiate gentamicin sensitivity in resistant MRSA strains.

Conclusions:

Our study demonstrates that LPC exhibits intrinsic antimicrobial effects and can enhance the antimicrobial effects of gentamicin for resistant MRSA strains, suggesting that LPC may be a beneficial additive in topical antibiotics for superficial skin infections.

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of both health care-associated and community-associated (CA) infections and has also developed resistance to various antimicrobial agents (β-lactams, quinolones, and aminoglycosides).¹ CA-MRSA has become increasingly prevalent in Japan,² and almost all healthy individuals are colonized with CA-MRSA. However, some strains can cause skin and soft-tissue infections and life-threatening necrotizing pneumonia even in the absence of predisposing conditions.³ The rising number of antimicrobial-resistant strains has limited the treatment options in infections and is associated with clinical treatment failure.⁴ ‘Anti-infectious drugs’ include not only compounds that inhibit the growth of pathogenic microorganisms statically or kill them but also compounds that control microbial survival or pathogenicity, potentiate the activities of known antimicrobials, or enhance the activity of the host immune system against microbial infection.

Lysophosphatidylcholine (LPC; also called lysolecithin) is a component of phospholipids in eukaryotic cells and is synthesized by the liver in mammals from phosphatidylcholine through phospholipase A2-mediated hydrolysis. LPC has been reported to reduce mortality in animal models of sepsis induced from both Gram-positive and Gram-negative bacteria, and modulation of inflammation accounted for the survival effect.⁵ A recent clinical study suggested that the serum level of LPC might be a mortality predictor in septic shock syndrome.⁶

Although LPC has been considered an immune modulator and shown to stimulate certain leukocyte activities critical for the initiation and maintenance of inflammation,⁷ information about the direct antimicrobial effects of LPC is limited. Here, we studied the antimicrobial efficacy of LPC on MRSA to evaluate LPC as a topical ointment.

Materials and methods

Strains

Thirty-two strains of clinically isolated MRSA from Tokyo Medical University Hospital and two reference strains (ATCC 33591 and ATCC 43300) were used in the present study. In addition, various other strains were included in a comparative analysis, including methicillin-sensitive S. aureus (MSSA; 20 clinical isolates, ATCC 25923, and ATCC 29213), Staphylococcus epidermidis (16 clinical isolates and ATCC 29887), Streptococcus pneumoniae (18 clinical isolates), Escherichia coli (19 clinical isolates and ATCC 25922), Enterobacter cloacae (20 clinical isolates), Klebsiella pneumoniae (20 clinical isolates), and Pseudomonas aeruginosa (19 clinical isolates and ATCC 27853).

Antimicrobial susceptibility testing

The minimum inhibitory concentration (MIC) of LPC (Egg Yolk Lysolecithin LPC-1, Kewpie) for each isolate was determined following a broth microdilution method according to the Clinical and Laboratory Standards Institute protocol with some modifications. The inoculum was adjusted to a cell density of 5 × 10⁵ CFU/ml in Mueller-Hinton broth (Eiken Chemical) supplemented with 25 mg/l Ca²⁺ and 12.5 mg/l Mg²⁺ (CA-MHB). Bacteria were plated in triplicate, incubated for 20 h at 35°C, and then judged visually.

Membrane permeability assay

The effects of LPC on bacterial membrane permeability were assessed with the membrane potential-sensitive fluorescent dye DiSC3(5) (3,3′-dipropylthiadicarbocyanine iodide; Invitrogen), as described previously.⁸ Bacteria were grown at 35°C in CA-MHB, with shaking, to mid-logarithmic phase (0.5–0.6 OD₆₀₀). The cells were harvested, washed once with buffer (5 mM HEPES pH 7.2, 5 mM glucose), and resuspended in the same buffer, and then supplemented with 100 mM KCl and 2 µM DiSC3(5) to 0.05 OD₆₀₀. The mixture was then incubated for 15 min at room temperature to enable dye uptake and fluorescence quenching. The change in fluorescence was measured immediately after the addition of serial concentrations of LPC (16–256 µg/ml), using a fluorescence microplate reader (excitation: 622 nm; emission: 670 nm; Mithras LB940, Berthold Technologies). Measurements were recorded every minute for 10 min to analyse treatment kinetics. Saline-treated bacteria served as a negative control.

Colony enumeration

The effect of LPC treatment on MRSA ATCC 33591 growth was examined by colony counting. Bacteria were cultured to log-phase growth in CA-MHB and then diluted to a final concentration of 10⁶ cells/ml. The cultures were subsequently treated with LPC at 128 or 256 µg/ml; incubated at 35°C for 0, 3, 6, and 24 h; serially diluted; and then plated on heart infusion agar (Eiken Chemical). The plates were incubated at 35°C for 24 h, and the surviving MRSA cells were counted.

LPC and gentamicin combination treatment

To determine the bactericidal efficacy of LPC in combination with gentamicin (Gentamicin Sulfate, Wako) in a topical ointment, we analysed the combined effects in vitro on CA-MRSA isolated from skin, nose, or ears. Among the 24 CA-MRSA isolates tested (Table 1), 15 were gentamicin-resistant (MIC ⩾ 16 µg/ml). MIC of gentamicin combined with 32 µg/ml LPC was measured for 15 gentamicin-resistant CA-MRSA isolates, following a broth microdilution method.

Table 1.

CA-MRSA isolates from skin, nose, or ear.

Strain no.	Age	Sex	Sample	SCC mec	MIC of LPC (A)	MIC of GM (B)	MIC of GM combined with 32 µg/ml of LPC (C)	C/B
1	2	F	Skin	Type II	128	0.25	–
2	7	M	Pus	Type II	>2048	8	–
3	0	M	Nose	Type II	>2048	>256	64	<1/4
8	27	M	Ear	Type V	64	256	64	1/4
12	7	F	Pus	Type IV	64	64	32	1/2
13	64	M	Ulcer	Type IV	32	0.25	–
18	6	M	Nose	Type II	64	128	32	1/4
19	63	F	Ear	Type II	64	>256	256	1/2
22	4	M	Nose	Type II	64	256	128	1/2
24	7	F	Ear	Type V	128	256	32	1/8
26	68	M	Nose	Type IV	32	4	–
27	68	F	Ear	Type IV	64	128	32	1/4
29	2	M	Skin	Type V	2048	0.25	–
31	0	M	Nose	Type IV	32	4	–
35	5	M	Wound	Type IV	256	8	–
36	45	M	Skin	Type IV	64	0.25	–
40	34	M	Nose	Type V	2048	0.25	–
41	0	F	Nasal discharge	Type V	128	128	4	1/32
43	0	F	Nose	Type II	2048	256	64	1/4
46	37	F	Pus	Type V	128	128	16	1/8
47	3	F	Pus	Type V	>2048	256	16	<1/16
48	1	F	Skin	Type V	>2048	256	16	<1/16
49	53	M	Pus	Type IV	2048	128	32	1/4
50	66	M	Skin	Type IV	>2048	256	32	<1/8

GM, gentamicin; LPC, lysophosphatidylcholine.

Results

LPC MIC

As shown in Figure 1, the LPC MIC for Gram-positive bacteria varied between 32 µg/ml and >2048 µg/ml. The MIC₅₀ of MRSA and MSSA was 64 µg/ml, which was lower than that of S. epidermidis (512 µg/ml) and S. pneumoniae (256 µg/ml). All strains of Gram-negative bacteria showed an LPC MIC >2048 µg/ml.

Figure 1.

Minimum inhibitory concentrations (MICs) for lysophosphatidylcholine (LPC) for various Gram-positive and Gram-negative bacteria. The LPC MIC for each isolate was determined according to a broth microdilution method.

LPC-induced bacterial membrane permeability

Figure 2 shows the results from membrane permeability assays for four species of bacteria. MRSA ATCC33591 (LPC MIC, 128 µg/ml) exhibited membrane depolarization with LPC in a concentration-dependent manner. Furthermore, Gram-positive methicillin-resistant S. epidermidis (MRSE) ATCC 29887 also showed membrane depolarization, which was not observed in the Gram-negative bacteria E. coli ATCC 25922 and P. aeruginosa ATCC 27853.

Figure 2.

LPC-induced bacterial membrane permeability. Membrane permeability was determined by DiSC3(5) fluorescence analysis after treatment with serial concentration of LPC or saline vehicle. Data represent the mean ± standard deviation from three experiments.

Effect of LPC on bacterial growth

Figure 3 shows the growth curve of MRSA ATCC 33591. Notably, cell growth markedly decreased 6 h after exposure to the MIC and 2× MIC of LPC, demonstrating the bactericidal effect of LPC on MRSA.

Figure 3.

Effect of LPC on MRSA ATCC 33591 growth. Cells (10⁶ cells/ml) were treated with LPC and enumerated by serial dilution colony counting. Gray square, 128 µg/ml LPC; gray triangle, 256 µg/ml LPC; black diamond, untreated control. Data represent the mean ± standard deviation from three experiments.

Efficacy of LPC and gentamicin combination treatment

As shown in Table 1, the addition of 32 µg/ml LPC was sufficient to decrease the gentamicin MIC for all 15 gentamicin-resistant CA-MRSA by ⩽ 1/2 compared to that observed with gentamicin alone, indicating that LPC showed a beneficial effect on gentamicin efficacy.

Discussion

LPC is an important lipid mediator involved in the pathogenesis of various diseases, including diabetes, atherosclerosis, rheumatoid arthritis, bronchial asthma, psoriatic skin, and biliary tract cancer.^9–13 The anti-infective effect of LPC is mainly attributed to modulation of inflammation such as upregulation of adhesion molecules, monocyte chemotactic protein-1, and growth factors.^14–16

It has been proposed that LPC interacts primarily with the membrane interface and promotes bilayer damage. As shown in our study, Gram-negative bacteria had uniformly high MICs for LPC in vitro, which may have resulted from the outer membrane preventing the interaction between LPC and bacterial cytoplasmic membrane. The beneficial effects of LPC alone and the antibiotic combination treatment against Gram-negative species such as Acinetobacter baumannii and E. coli have been previously reported in murine models of sepsis and pneumonia.^17–19 These reports suggested that LPC can effectively treat sepsis and microbial infections by stimulating immune cells that regulate cytokine homeostasis in some Gram-negative infections.

Our in vitro study showed that 2 mg/ml LPC suppressed the growth of most MRSA. Because MRSA is a Gram-positive bacterium, LPC can directly induce MRSA killing by interacting with the cytoplasmic membrane and inducing membrane depolarization and increased membrane permeability, as shown in our permeability assays. However, the MIC of LPC for MRSA strains ranged from 32 µg/ml to >2048 µg/ml and the bactericidal effect differed depending on strains. We did observe some discrepancy between membrane depolarization and growth inhibition. For instance, a few MRSA isolates exhibited high LPC resistance (MIC > 2048 µg/ml), but membrane depolarization was observed with LPC. These results suggested that MRSA strains may have LPC resistance mechanisms other than membrane interaction. Furthermore, we also showed that LPC acts in synergy with gentamicin for inhibition of CA-MRSA growth. All gentamicin-resistant strains showed decrease of the MIC by ⩽ 1/2 for the combination with LPC compared with that for gentamicin alone. It is believed that the membrane perturbation induced by LPC facilitates the entry of the antimicrobial agent into the cells and makes it more effective.²⁰

Because LPC is known to have low toxicity to the skin and is used as a component in cosmetics, it is also appropriate as a component of ointments. Although the mechanism underlying this bactericidal effect remains poorly understood, our results suggests that topical use of LPC may be potentially beneficial in the treatment of superficial skin MRSA infections. Further studies including in vivo evaluation are urgently required.