In Vitro Activities of Antibiotics and Antimicrobial Cationic Peptides Alone and in Combination against Methicillin-Resistant Staphylococcus aureus Biofilms

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) strains are most often found as hospital- and community-acquired infections. The danger of MRSA infections results from not only the emergence of multidrug resistance but also the occurrence of bacteria that form strong biofilms. We investigated the in vitro activities of antibiotics (daptomycin, linezolid, teichoplanine, azithromycin, and ciprofloxacin) and antimicrobial cationic peptides {AMPs; indolicidin, CAMA [cecropin (1-7)–melittin A (2-9) amide], and nisin} alone or in combination against MRSA ATCC 43300 biofilms. The MICs and minimum biofilm eradication concentrations (MBECs) were determined by the broth microdilution technique. Antibiotic and AMP combinations were assessed using the checkerboard technique. For MRSA planktonic cells, MICs of antibiotics and AMPs ranged between 0.125 and 512 and 8 and 16 mg/liter, respectively, and the MBEC values were between 512 and 5,120 and 640 mg/liter, respectively. With a fractional inhibitory concentration of ≤0.5 as the borderline, synergistic interactions against MRSA biofilms were frequent with almost all antibiotic-antibiotic and antibiotic-AMP combinations. Against planktonic cells, they generally had an additive effect. No antagonism was observed. All of the antibiotics, AMPs, and their combinations were able to inhibit the attachment of bacteria at 1/10 MIC and biofilm formation at 1× MIC. Biofilm-associated MRSA was not affected by therapeutically achievable concentrations of antimicrobial agents. Use of a combination of antimicrobial agents can provide a synergistic effect, which rapidly enhances antibiofilm activity and may help prevent or delay the emergence of resistance. AMPs seem to be good candidates for further investigations in the treatment of MRSA biofilms, alone or in combination with antibiotics.

INTRODUCTION

Staphylococcus aureus is a human pathogen that can cause a range of illnesses, from minor skin infections to life-threatening diseases, such as pneumonia, meningitis, endocarditis, toxic shock syndrome (TSS), bacteremia, and sepsis. It is one of the most common causes of nosocomial infections and is often the cause of postsurgical wound infections. Methicillin-resistant S. aureus (MRSA) strains, which have become resistant to most antibiotics, are most often found associated with institutions such as hospitals, but they are also becoming increasingly prevalent in community-acquired infections (16, 20, 25). The danger of MRSA infections results from not only the emergence of multidrug resistance but also the occurrence of strong biofilm-forming bacteria.

A biofilm is a cluster of microorganisms that attaches to surfaces and produces extracellular polysaccharides. Biofilms may form on living or nonliving surfaces and can be prevalent in natural, industrial, and hospital settings. Biofilms pose a serious problem for public health because of the increased resistance of biofilm-associated organisms to antimicrobial agents and the potential for these organisms to cause severe infections in patients with indwelling inert surfaces, such as medical devices for internal or external use. An appreciation of the role of biofilms in infection should enhance the clinical decision-making process. Microbial cells growing in a biofilm are physiologically distinct from planktonic cells of the same organism. When a cell switches to the biofilm mode of growth, it undergoes a phenotypic shift in behavior in which large suites of genes are differentially regulated (10, 13).

Antimicrobial cationic peptides (AMPs) are major components of the innate immune system and play an important role in host defense against environmental microorganisms; they are widely distributed in nature, existing in organisms from insects to plants and microorganisms to mammals. AMPs have a rapid action and a broad spectrum of activity against infectious agents, including Gram-negative and Gram-positive bacteria, fungi, viruses, and parasites. Furthermore, cationic peptides are not affected by many antibiotic resistance mechanisms that now limit the use of other antibiotics (15, 33). Many kinds of AMPs have been found to be active against bacterial biofilms (7, 8, 17, 24). Therefore, we tried to determine whether the indolicidin cecropin A (1-7)–melittin A (2-9) amide (CAMA) and nisin possessed in vitro antibiofilm activities alone or in combination with the antibiotics that are routinely used against MRSA biofilms in clinical settings.

MATERIALS AND METHODS

Bacterial strains.

Methicillin-resistant S. aureus ATCC 43300 was used in this study, and S. aureus ATCC 29213 was used as a control to verify the accuracy of the microdilution test procedure for antibiotics.

Antimicrobial substances.

Two AMPs, CAMA and indolicidin, were obtained from Bachem AG, nisin was obtained from Sigma-Aldrich, and all other antibiotics, such as daptomycin, linezolid, teichoplanin, ciprofloxacin, and azithromycin, were kindly provided by their manufacturers. Stock solutions from dry powders were prepared according to the manufacturers' recommendations and stored frozen at −80°C for up to 6 months.

Media.

Media used in this study were tryptic soy broth supplemented with 1% glucose (TSB-glucose; Difco Laboratories) for biofilm production, cation-adjusted Mueller-Hinton broth (CAMHB; Difco Laboratories) for MIC and minimum biofilm eradication concentration (MBEC) determinations and the broth microdilution checkerboard technique, and tryptic soy agar (TSA; Difco Laboratories) for minimum bactericidal concentration (MBC) and MBEC determinations and colony counts.

MIC and MBC determinations.

MICs of antibiotics and AMPs were determined with the broth microdilution technique as described by the Clinical and Laboratory Standards Institute (CLSI) (5). The MIC was defined as the lowest concentration of antibiotic that produced complete inhibition of visible growth.

MBCs were determined at the end of the incubation period by removing two 10-μl samples from each well in which there was no visible growth and plating the samples onto TSA. Resultant colonies were counted after overnight incubation at 37°C. The MBC was defined as the lowest concentration of antimicrobial that produced at least 99.9% killing of the initial inoculum (19).

High-inoculum MIC determinations.

High-inoculum MICs of antibiotics and AMPs were determined by the broth microdilution technique as described by the CLSI (5) with the following modification. MRSA ATCC 43300 was diluted to give a final concentration of approximately 5 × 10⁷ CFU/ml, instead of 5 × 10⁵ CFU/ml, before addition to the wells. The MIC was defined as the lowest concentration of antibiotic that produced complete inhibition of visible growth.

Determinations of the FICI.

The effects of antibiotics and AMPs in combination were assessed by using the broth microdilution checkerboard technique (23). Each microtiter well containing the mixture of antibiotics was inoculated with a 4- to 6-h broth culture diluted to give a final concentration of approximately 5 × 10⁵ CFU/ml. After incubation at 37°C for 18 to 20 h, the fractional inhibitory concentration index (FICI) was determined as the inhibitory concentration of the combination divided by that of the single antibiotic. The combination index was derived from the highest dilution of antibiotic combination permitting no visible growth. With this method, synergy was defined as a FICI of ≤0.5, no interaction was defiend as a FICI of >0.5 to 4, and antagonism was defined as a FICI of >4.0.

Biofilm formation.

MRSA ATCC 43300 was cultured in 5 ml of TSB-glucose with rotation (50 rpm) at 360°, for 24 h at 37°C, and diluted 1/50 in fresh TSB-glucose to give a final concentration of approximately 1 × 10⁷ CFU/200 μl. This suspension was added to each well of a 96-well tissue culture microtiter plate (Greiner) and incubated at 37°C for 24 h. The negative control was TSB-glucose. After the incubation, the waste medium was aspirated gently, and the wells of the plates were washed three times with 250 μl physiological buffered saline (PBS) solution to remove unattached bacteria and air dried. A 200-μl volume of 99% methanol was added per well for 15 min for fixation, and then the well contents were aspirated and plates were allowed to dry. Wells were stained with 200 μl of 0.1% crystal violet (in water) for 5 min. Excess stain was gently rinsed off with tap water, and plates were air dried. Stain was resolubilized in 200 μl of 95% ethanol with shaking in an orbital shaker for 30 min, and then the optical density at 595 nm (OD₅₉₅) was measured (11).

Biofilm attachment assays.

Biofilm attachment assays were performed as previously described (22), with some modifications. An overnight culture of MRSA ATCC 43300 (diluted 1/50 to give 1 × 10⁷ CFU/200 μl in TSB-glucose) was added to each well of a 96-well tissue culture microtiter plate with 1/10 the MICs of AMPs, antibiotics, and their combinations. The plates were incubated for 1, 2, and 4 h at 37°C. Six wells were used for each AMP, antibiotic, or combination. The positive control was MRSA ATCC 43300 in TSB-glucose without peptide or antibiotic. After incubation, wells were washed with PBS solution and the OD₅₉₅ was measured.

Inhibition of biofilm formation.

MRSA ATCC 43300 (1 × 10⁵ CFU/200 μl) in TSB-glucose was incubated at 37°C, 24 h, with AMPs, antibiotics, or their combinations at 1× MIC, 1/10 the MIC, and 1/100 the MIC in 96-well tissue culture microtiter plates. Six wells were used for each AMP, antibiotic, or combination. The positive control was MRSA ATCC 43300 in TSB-glucose without peptide or antibiotic. After incubation, wells were washed with PBS solution and the OD₅₉₅ was measured.

MBEC determinations.

Measurements of the antimicrobial susceptibilities of MRSA ATCC 43300 biofilms were performed as previously described for MBEC assays (3), with the following modifications. The 24-h biofilms in a 96-well tissue culture microtiter plate were washed three times with 250 μl PBS solution and air dried. Serial 2-fold dilutions ranging from 640 to 0.06 mg/liter for AMPs and 5,120 to 5 mg/liter for antibiotics were prepared in CAMHB. A 200-μl sample of each concentration was added to a corresponding well, and plates were incubated for 24 h at 37°C. After the incubation, antibiotics were aspirated gently, plates were washed two times with sterile PBS solution, and wells were scraped thoroughly, with particular attention for the well edges. Well contents were removed to 1 ml of PBS solution and placed in a sonicating water bath (Bandelin sonopuls HD 2200) for 5 min to disrupt the biofilm, and 100-μl samples were plated on TSA. Colonies were counted after 24 h at 37°C. The MBEC was defined as the lowest concentration of AMP or antibiotic that prevented bacterial regrowth.

Antibacterial activities of combinations against biofilms.

The effects of antibiotics and AMPs in combinations against biofilms were determined by using the modified broth microdilution checkerboard technique (23). The 24-h biofilms in 96-well tissue culture microtiter plates were washed three times with 250 μl of PBS solution and air dried. Combinations of antibiotics and AMPs were tested over five concentrations, starting from 2× the MIC. After incubation of biofilms (including 1 × 10⁷ CFU/ml cells) with antibacterial combinations at 37°C for 24 h, antibiotics were aspirated gently, plates were washed two times with sterile PBS solution, and wells were scraped thoroughly, with particular attention to well edges. Well contents were removed into 1 ml of PBS solution and placed in a sonicating water bath for 5 min to disrupt the biofilm, and 100-μl samples were plated on TSA. Colonies were counted after 24 h at 37°C. The FICIs and definitions of the extent of inhibition were as described above in “Determinations of the FICI.”

Statistical analysis.

All experiments were analyzed in two independent assays. To determine MIC, MBC, MBEC, and FICI values, when the results were different in the two experiments we conducted another test for the final result. For the biofilm attachment and inhibition of biofilm formation assays, the results are shown as means ± standard deviations of the two independent experiments. A one-way analysis of variance with the Bonferroni multiple comparison test was used to compare differences between the control and antimicrobial-treated biofilms. A P value of <0.001 was considered statistically significant.

RESULTS

Susceptibility.

The in vitro activities of the studied antibiotics and AMPs against the MRSA ATCC 43300 planktonic cells, high-inoculum planktonic cells, and biofilms are summarized in Table 1. The MIC values of the antibiotics against the quality control strain S. aureus ATCC 29213 were within the accuracy range described by CLSI throughout the study (6).

Table 1.

In vitro antibacterial activities of antibiotics and AMPs against MRSA ATCC 43300

Antimicrobial agents	MIC (mg/liter)		MBEC (mg/liter)
	Standard inoculuma	High inoculumb
Antibiotics
Daptomycin	0.125	80	1,024
Linezolid	1	80	512
Teichoplanin	0.25	80	2,048
Ciprofloxacin	0.5	80	1,280
Azithromycin	512	1,024	5,120
Cationic peptides
Indolicidin	16	40	640
CAMA	8	20	640
Nisin	16	20	640

^aThe standard inoculum was 5 × 10⁵ CFU/ml.

^bThe high inoculum was 5 × 10⁷ CFU/ml.

There was no major difference between bactericidal and inhibitory concentrations of the antibiotics and AMPs. The MBC values were generally equal to or two times greater than the MIC values (data not shown).

Checkerboard assay results.

The results of combination studies, in which antibiotics were used in combinations with AMPs and antibiotics against MRSA ATCC 43300 planktonic cells and biofilms are shown in Table 2 and Table 3. With a FICI of ≤0.5 as the borderline, synergistic interactions against the MRSA ATCC 43300 biofilms were frequent with almost all of the combinations. No antagonism was observed with any combination.

Table 2.

In vitro activities of antibiotics in combination with AMPs against MRSA ATCC 43300 planktonic cells and biofilms

Antibiotic	FICI ratio
	Planktonic cells			Biofilm
	Indolicidin	CAMA	Nisin	Indolicidin	CAMA	Nisin
Daptomycin	2.0	2.0	2.0	0.50	0.375	0.375
Linezolid	1.0	1.0	1.0	1.5	0.375	0.375
Teichoplanin	2.0	2.0	0.75	0.375	0.50	0.25
Ciprofloxacin	1.0	0.75	1.0	0.50	0.125	0.25
Azithromycin	0.75	0.50	1.25	0.625	1.125	1.125

Table 3.

In vitro activities of antibiotics in combination with antibiotics against MRSA ATCC 43300 planktonic cells and biofilms

Antibiotic combinations	FICI ratio
	Planktonic cells	Biofilm
Daptomycin-teichoplanin	0.75	0.50
Daptomycin-linezolid	2.0	0.50
Daptomycin-ciprofloxacin	0.75	0.375
Daptomycin-azithromycin	1.25	0.375
Teichoplanin-linezolid	0.50	0.75
Teichoplanin-ciprofloxacin	1.0	0.375
Teichoplanin-azithromycin	1.25	0.25
Linezolid-ciprofloxacin	1.0	1.25
Linezolid-azithromycin	0.75	0.50
Ciprofloxacin-Azithromycin	1.0	0.25

Biofilm attachment assay.

When we incubated the 1/10 MICs of AMPs, antibiotics, and their combinations with MRSA ATCC 43300 for 1, 2, or 4 h at 37°C for adherence to the wells of tissue culture microtiter plates, all of the antimicrobial agents, alone or in combination, inhibited biofilm attachment in relation to incubation time (Fig. 1).

Fig 1.

Inhibition of MRSA ATCC 43300 attachment to the surface in wells containing the studied antimicrobials. (A) Antibiotics and AMPs alone; (B) antibiotic-antibiotic combinations; (C) AMP-antibiotic combinations. Control bars indicate MRSA without any antimicrobial agent, accepted as 100%. Each well of the 96-well plates contained 1/10 the MIC of AMPs, antibiotics, or their combinations and an inoculum of 1 × 10⁷ CFU/200 μl MRSA in TSB-glucose. The plates were incubated for 1, 2, or 4 h at 37°C. Six wells were used for each AMP, antibiotic, or their combination. Each experiment is representative of at least 2 independent tests, and the error bars indicate the standard deviations. All differences between the control and antimicrobial-treated biofilms were statistically significant (P < 0.001).

Inhibition of biofilm formation.

All of the studied AMPs, antibiotics, and their combinations showed significant inhibitory activity against MRSA ATCC 43300 biofilm formation at 24 h, in relation to their concentrations (Fig. 2).

Fig 2.

Inhibition of MRSA ATCC 43300 biofilm formation by the studied antimicrobials. (A) Antibiotics and AMPs alone; (B) antibiotic-antibiotic combinations; (C) AMP-antibiotic combinations. Control bars indicate MRSA cultures without any antimicrobial agent, accepted as 100%. Each well of the 96-well plates contained 1× MIC, 1/10× MIC, or 1/100× MIC of AMP, antibiotic, or their combination and an inoculum of 5 × 10⁵ CFU/200 μl MRSA in TSB-glucose. The plates were incubated for 24 h at 37°C. Six wells were used for each AMP, antibiotic, and the combination. Each experiment is representative at least 2 independent tests, and the error bars indicate the standard deviations. All differences between the control and antimicrobial-treated biofilms were statistically significant (P < 0.001).

DISCUSSION

The antibiofilm activities of antibiotics are becoming an important part of treating biofilm-related infections, such as chronic wounds or catheter-associated infections caused by MRSA. In this study, we investigated the in vitro activities of clinically available antibiotics and AMPs alone and in combination against MRSA planktonic cells (5 × 10⁵ [standard inoculum] or 5 × 10⁷ [high inoculum] CFU/ml per inoculum) and biofilms. We found that the planktonic MRSA ATCC 43300 cells were susceptible to all of the examined antibiotics, except for azithromycin, demonstrating MIC values between 0.125 and 1 mg/liter. On the other hand, when we considered the antibiofilm activities of these antibiotics, MBEC values ranged between 512 and 5,120 mg/liter, and the MBEC/MIC ratio was between 10- and 8,000-fold higher. Similar results have been noted by other researchers (18, 28). These results suggest that the high values might have been due to not only the biofilm structure but also the differences between the planktonic and biofilm bacteria counts (5 × 10⁵ to 5 × 10⁷ CFU/ml). We tried to determine the antibiotic activities against high-inoculum planktonic cells, and the MIC values for the antibiotics were found to be 80 mg/liter, except for azithromycin. These findings indicated that the size of the inoculum can change the MIC values by 80- to 640-fold in planktonic cells. Accordingly, the MBEC/MIC ratio was found to change between 5- and 26-fold.

When we determined three AMP's antimicrobial and antibiofilm activities against MRSA ATCC 43300, we found that the MIC, high-inoculum MIC, and MBEC values were within the ranges of 8 to 16, 20 to 40, and 640 mg/liter, respectively. According to these results, the MBEC/MIC ratio changes for standard and high-inoculum MICs were within the range of 40- to 80-fold and 16- to 32-fold, respectively. Although the MICs of the AMPs were not as low as with the other antibiotics that were used in this study, it was notable that the AMPs demonstrated activities with similar MIC, high-inoculum MIC, and MBEC values against MRSA and that there were not very high MBEC/MIC ratios, as seen with the antibiotics. The differences between antibiotic and AMP activities might have been due to their different structures and antibacterial mechanisms.

Because AMPs have desirable properties that make them excellent prospects for use as antimicrobial agents, they are considered one of the most preferred classes of antimicrobial substances for future use as treatments for serious infections, either alone or in combination with antibiotics, and have not demonstrated significant resistance problems (9). Among these, indolicidin, which is encoded by a member of the cathelicidin gene family and is classified as a cationic antimicrobial tridecapeptide amide, was isolated from cytoplasmic granules of bovine neutrophils (27). It is one of the shortest, a 13-residue AMP with extremely high tryptophan content that exhibits broad-spectrum antimicrobial and hemolytic activities (14). The antimicrobial actions of indolicidin are different from well-defined channel formation, as it creates pores through the cell membranes or causes the total disintegration of the membrane structures (12). CAMA is a cecropin-melittin hybrid peptide that contains portions of the amino acid sequences of cecropin-A and melittin that have different properties. A series of hybrid peptides were created that consisted of the amphipathic α-helical N-terminal region of cecropin-A and the hydrophobic N-terminal α-helix of the bee venom peptide melittin. These hybrids formed ion-permeable channels in model lipid membranes. Hybrid peptides have improved antimicrobial activities against Gram-positive bacteria, with a significant reduction of the toxicity that comes from melittin (2, 29). Nisin, which is a 34-residue peptide that can be isolated from the nonpathogenic bacterium Lactococcus lactis, belongs to a special group of AMPs called bacteriocins that, because of containing unusual amino acids (lanthionine and methyl lanthionine residues), emerged as one of the most extensively studied lantibiotics. Nisin has rapid bactericidal activities against Gram-positive bacteria, including multidrug-resistant pathogens. It is characterized by a dual mode of action against cell membranes. The first mode of action is characterized by the nonspecific recognition and binding of anionic lipids. The second mode of action has been described as specific recognition and binding of lipid II. The complexes then aggregate, incorporate additional peptides, and form a pore within the bacterial membrane (26, 30, 34).

As shown by our findings, which are similar to those noted by others (18, 28), biofilm-associated bacteria do not affect therapeutically achievable concentrations of antimicrobial agents, because of antimicrobial tolerance, persister cells, slow-growing cells, and the exopolysaccharide matrix. This biofilm-associated resistance, which is an excellent type of bacterial resistance, is provided by the sedentary lifestyle in biofilms (1). The development of antibiofilm therapeutics has generally focused on interfering with quorum sensing, inhibition of adhesion, enhancement of dispersion, or bacteriophage-based treatments, and now there is a new option for the species-specific control of biofilms, selective targeting of antimicrobial peptides (4). Another feasible way to overcome biofilm-associated resistance is through synergistic effects, created by the use of antimicrobial agents in combination, which can result in a rapid increase in antibiofilm activities and help to prevent or delay the emergence of resistance.

Because animal and human data are difficult to obtain, decisions about the selection of optimal combinations are based on in vitro information. The broth microdilution checkerboard assay is the most simple and widely used technique to assess antimicrobial combinations, even if it does not always reliably show additive effects when agents are combined (23, 31). Using the broth microdilution checkerboard technique with adequate controls and replicated experiments, synergistic interactions are frequently seen with either antibiotic-antibiotic or AMP-antibiotic combinations to treat MRSA ATCC 43300 biofilms. When we evaluated the same combinations against planktonic cells, the antimicrobial agents generally demonstrated additive effects, and synergism was rarely observed.

In addition to antimicrobial activities, AMPs serve as antiresistance compounds for classic antibiotics (32). They are able to interact with bacterial membranes, creating ion-permeable channels that can lead to increased cytoplasmic membrane permeability and bacterial cell death. They may also allow the entry of other substances, like antimicrobial agents, into the cell (15). This mechanism explains how AMPs act in synergy with conventional antibiotics against planktonic cells. On the other hand, antimicrobial agents exhibit different mechanisms in order to overcome biofilm resistance, such as the specific physiological properties of biofilms that limit the efficacies of antibiotics and prevent the antimicrobial substance from reaching its target, i.e., by limited diffusion or repulsion (21). The different checkerboard results that have been achieved against planktonic cells versus cells in biofilms can be explained by the differences between antibacterial and antibiofilm mechanisms that are employed by different antimicrobial agents.

The attachment of bacteria to a solid surface is critical for biofilm formation. Thus, many continuing studies target the inhibition of this essential step. In this study, all of the antibiotics, AMPs, and their combinations were able to inhibit the attachment of bacteria at 1/10 the MIC. Especially for azithromycin, CAMA, and their combinations with other antibiotics or AMPs, significantly decreased attachment of MRSA to the surface was observed within 1 to 2 h (P < 0.001). These inhibition levels suggest potential interactions between these AMPs and antibiotics that affect bacterial adhesion (7).

We were also able to determine that antibiotics, AMPs, and their combinations significantly inhibit biofilm formation, especially at 1× MIC and sub-MIC levels (P < 0.001). Although the inhibition of mature biofilms is very difficult and requires achievement of very high concentrations of antimicrobial agents, the inhibition of biofilm formation during the early stages seems more applicable. Interestingly, the inhibition levels of biofilm formation using a single antimicrobial agent or antibiotic-antibiotic or AMP-antibiotic combinations were not significantly different. These findings are comparable with the additive interactions against planktonic MRSA cells that were observed in broth microdilution checkerboard assays, while synergism was observed against cells in biofilms. These results suggest that the mechanisms involved in the eradication and inhibition of biofilms are very different.