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. 2025 Aug 27;10(35):40271–40281. doi: 10.1021/acsomega.5c05384

Biological Merits and Antibiofilm Mechanisms of Action of a Specially Designed Double-Shelled Microparticle

Chengju Sheng †,‡,§, Chao Zhou ∥, Mingming Guo §, David C McGiffin ⊥, David M Kaye #, Vinh X Truong , Changrui Qian ∇,○, Xenia Kostoulias ∇,⧫,††, Anton Y Peleg ∇,⧫,††, John S Forsythe †,‡,††,*, Timothy F Scott †,‡,§,††,*, Yue Qu ∇,○,⧫,††,*
PMCID: PMC12423863  PMID: 40949289

Abstract

We recently fabricated a double-shelled microparticle (DSMP) incorporating a quaternary ammonium compound (QAC) and quinine, which demonstrated promising anti-infective properties. In this study, we aimed to elucidate biological merits and antibiofilm mechanisms of action of the specifically designed DSMP. Antimicrobial activities of the DSMP against 32 Gram-positive clinical isolates and laboratory strains were assessed by determining the minimum inhibitory concentrations (MICs). Antibiofilm efficacies of the DSMP were evaluated by using a tetrazolium salt reduction assay and confocal laser scanning microscopy. The fractional inhibitory concentration index was determined to clarify drug–drug interactions between QAC and quinine. Evolution of DSMP resistance was experimentally assessed by using an adaptive laboratory evolutionary assay. Molecular mechanisms underlying DSMP resistance was investigated by whole genome sequencing. The DSMP demonstrated strong activity against antibiotic-susceptible clinical isolates of Staphylococcus species, Enterococcus faecalis and Enterococcus faecium, with MICs of 4–8 μg/mL, and notable antibiofilm capacities. Weakly positively charged DSMP readily penetrated through the negatively charged biofilm matrix in 3 h and accumulated in biofilm water pores by 24 h, thereby killing biofilm-embedded cells. The copresence of the QAC and quinine demonstrated synergistic effects against Gram-positive bacteria while simultaneously mitigating the development of resistance. Whole-genome sequences revealed genetic mutations in efflux pumps that are associated with cross-resistance to antibiotics and the DSMP components. Co-presence of the QAC and quinine in the DSMP lays the foundation for its antimicrobial and antibiofilm properties against Gram-positive bacteria, allowing the DSMP to maintain efficacy while limiting the development of resistance.

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1. Introduction

Antimicrobial resistance (AMR) has emerged as a major threat to global health. In addition to the microbial genetic evolution that allows pathogenic microorganisms to resist conventional antimicrobials, biofilm formation is another important microbial strategy that is associated with treatment failure of many medical device-related infections. Novel interventions have been appealed to combat biofilm-associated AMR and infections; among them are drugs with biofilm-specific resilience mechanisms and drug delivery systems. ,

Whereas intensive research is currently underway to develop novel antimicrobials that can be systemically administrated for acute infections, fewer efforts have been made for biofilm-related chronic infections. Conventional antibiotic formulations have limitations such as low achievable concentrations in the serum or at infection sites, short half-life, limited efficacy against biofilms, inevitable host toxicities, poor patient compliance, and high risks of inducing AMR. , All above-mentioned limitations of conventional antibiotics may be addressed by developing innovative drug delivery systems. A biofilm is a unique microbial growth mode characterized by highly dense microbial growth embedded in the matrix of extracellular polymeric substances (EPS), altered genetic expression and metabolic status of embedded cells, and high resistance to antimicrobials and human immune responses. , Indeed, biofilm infection sources are often characterized by poor antibiotic penetration and low bacterial metabolic activities. Although numerous drug delivery systems have been developed and experimentally assessed for biofilm-associated infections, many of these systems failed to provide satisfying therapeutic effects due to their low bioavailability, high host toxicity, irrational selection of incorporated drugs, and suboptimal release-dynamic of incorporated drugs. − Novel anti-infective drug delivery systems that are highly effective and biocompatible are still urgently needed for biofilm infections. To achieve an optimal treatment outcome, a smart and effective drug delivery system should have high drug-loading capacity, biofilm-specific drug release dynamics, reduced dosage intervals, improved drug bioavailability, and low potential to induce AMR. −

Nanostructured polymer-based particles with unique architectures and loading capacities are of great importance as drug delivery materials. − These nanostructured polymer particles often have great stability and controllable physicochemical properties and have been used to fabricate drug delivery platforms with subcellular sizes and morphologies, ideal surface area to volume ratio, biocompatibility, and biodegradability. ,, As compared to bulk counterparts, nanostructured polymer particles have controlled release dynamics for embedded drugs, improved mechanical properties, swelling properties, and a better drug-loading efficiency and can effectively deliver antibiotics to infection sites and maintain high drug concentrations in situ with reduced side effects.

We recently developed a novel anti-infective nanostructured polymer particle system with adequate drug stability, reduced premature degradation, and sustained release dynamics. This double-shelled microparticle (DSMP) consisting of a quaternary ammonium compound (QAC) outer shell and a quinine inner shell demonstrated antimicrobial characteristics even without preloaded antibiotics. It was hypothesized that the outer-shelled hydrophobic QAC interacted with bacterial cell membranes, compromising the integrity of the cell envelope, ultimately leading to the death of targeted microorganisms. , While the structural characteristics of the DSMP have been unveiled, , its antimicrobial activity against clinical isolates, efficacy against microbial biofilms, and resilience toward AMR development remains to be elucidated. This study aims to determine antimicrobial and antibiofilm activities of the DSMP against a collection of Gram-positive clinical isolates causing hospital-acquired infections and to elucidate their biological merits and mechanisms of action against biofilms. Our ultimate goal is to showcase design rules and biological principles that can be used to develop anti-infective microparticles.

2. Materials and Methods

2.1. Preparing Anti-infective DSMP and Zeta Potential Measurement

The DSMP was synthesized as previously described. Surface charges of the DSMP were assessed by measuring the zeta potential of the microparticle prepared as a suspension in phosphate-buffered saline (PBS, pH = 7.4 and pH = 5.5, respectively) at 64 μg/mL, using the Malvern Zetasizer ZEN3600 Particle Analyzer (Malvern Panalytical, UK). The measurement was carried out for three independent samples.

2.2. Microorganisms and Growth Media

Thirty-two bacterial strains, across two genera and seven species, were selected for this study, including twenty-nine hospital isolates from a range of clinical presentations and three laboratory reference strains from the American Type Culture Collection (ATCC). The clinical isolates were obtained from the Royal Children’s Hospital and The Alfred Hospital, Melbourne, Australia (see Table ). Muller–Hinton broth (MHB, Oxoid, UK) was used for antimicrobial and antibiofilm susceptibility testing, and a tryptic soya broth (TSB, Oxoid, UK) was used for biofilm growths.

1. Antimicrobial Activities of the DSMP against Gram-Positive Clinical Isolates and Reference Strains .

strain ID
 species
 clinical/laboratory
 source
 MIC (μg/mL)
        Van Oxa Cip DSMP
RCH 1 S. warneri clinical RCH 2 >128 0.25 >128
RCH 2 S.hemolyticus clinical RCH 1 16 0.25 8
RCH 3 S. epidermidis clinical RCH 2 1 0.25 8
RCH 5 S. epidermidis clinical RCH 2 >128 0.25 >128
RCH 6 S. capitis clinical RCH 2 8 4 >128
RCH 8 S. capitis clinical RCH 0.25 >128 0.25 >128
RP62A S. epidermidis laboratory ATCC 2 128 0.5 32
AH17 × 1071 MRSA clinical Alfred 1 4 8 16
AH17 D014 MRSA clinical Alfred 1 >128 >32 64
AH17 D028 MSSA clinical Alfred 2 2 16 16
AH17 1003 MRSA clinical Alfred 2 >128 0.5 16
AH17 5008 MRSA clinical Alfred 1 64 16 32
AH17 5054 MRSA clinical Alfred 2 32 16 128
AH17 5058 MRSA clinical Alfred 4 16 16 64
AH18 1009 MRSA clinical Alfred 1 16 32 32
AH18 1061 MRSA clinical Alfred 2 >128 16 >128
AH18 H052 MRSA clinical Alfred 2 64 >32 >128
AH17 Goss MSSA clinical Alfred 0.5 0.5 1 2
AH17 5026 MSSA clinical Alfred 2 2 0.5 2
AH17 H009 MSSA clinical Alfred 1 0.25 0.5 1
AH17 K025 MSSA clinical Alfred 1 0.5 0.5 0.5
AH17 D009 MSSA clinical Alfred 1 0.25 1 0.25
AH17 B026 MSSA clinical Alfred 1 1 0.5 0.5
AH17 × 10117 MSSA clinical Alfred 0.25 0.25 0.5 0.25
AH17 × 1061 MSSA clinical Alfred 1 1 1 0.25
AH17 G022 MSSA clinical Alfred 2 0.25 8 0.5
AH17 F053 MSSA clinical Alfred 2 2 2 1
ATCC 25923 MSSA laboratory ATCC 1 0.5 0.5 0.5
ATCC 19433 E. faecalis laboratory ATCC 1 32 2 16
AP067 E. faecium clinical Alfred >128 >128 >128 >128
AP068 E. faecium clinical Alfred >128 >128 >128 >128
AP069 E. faecalis clinical Alfred 2 16 1 16
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MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus; Van, vancomycin; Oxa, oxacillin; Cip, ciprofloxacin; DSMP, double-shelled microparticle. RCH, Royal Children’s Hospital, Melbourne; Alfred, the Alfred Hospital, Melbourne; and ATCC, the ATCC. Minimum inhibitory concentrations (MICs) in bold indicate resistance to this antibiotic.

2.3. Antimicrobial Susceptibility Tests

The minimum inhibitory concentrations (MICs) of the DSMP were determined using the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guideline M07-A9. Bacterial growth was examined with the aid of a microplate reader (Tecan, Infinite M Plex). The MIC was defined as the lowest concentration of the DSMP, resulting in no obvious growth of bacterial cells in the microwell. Three conventional antibiotics, vancomycin, oxacillin, and ciprofloxacin, were included as comparators. MHB supplemented with 2% NaCl was used for the oxacillin MIC testing.

2.4. DSMP Susceptibility Testing against Staphylococcal Biofilms

The antibiofilm activity of the DSMP was evaluated against ten biofilm-producing representative bacterial strains. Biofilms were grown in tissue-culture-treated 96-well polystyrene microplates with flat bottom as previously described. The suspensions were aspirated, and the microwells were washed with PBS to remove nonadherent cells. Two hundred microliter aliquots of 2-fold serial dilutions of the DSMP or antibiotics were added into each well, and the treatment lasted for 18–24 h. After washing with PBS, antibiofilm efficacies of the DSMP or antibiotics were assessed using the tetrazolium salt XTT (sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis­(4-methoxy6-nitro)­benzenesulfonic acid hydrate) reduction assay. The biofilm MIC80 (BMIC80) was defined as the lowest concentration of antimicrobial agents that inhibited ≥ 80% of biofilm growth.

2.5. Extraction of the Biofilm EPS Matrix

Staphylococcal biofilms were grown in 6-well microplates with TSB for 24 h and transferred into sterile tubes containing 2 mL of PBS (pH = 5.5 and pH = 7.2, respectively) using sterile cotton swabs. The biofilm EPS matrix was dissociated from bacterial cells by vortex (30s × 4) and sonication (42 kHz, 10 min) and collected as previously described.

2.6. Fluorescent Labeling of the DSMP and CLSM

Fifty-five microliters of the DSMP suspension (solid content = 18%) was dispersed in 1 mL of dimethylformamide and mixed with ethylene diamine (1.2 mg, 0.02 mmol). The fluorescent dye 7-methoxycoumarin-3-carboxylic acid N-succinimidyl ester (3.5 mg, 0.01 mmol) was added, and the dispersion was heated to 90 °C for 24 h. The microparticles were sequentially washed with dimethylformamide (DMF) and ethanol to remove excess dye. The fluorescently labeled DSMP was dispersed in water and stored in the dark.

For confocal laser scanning microscopy (CLSM), bacterial biofilms were grown on medical-grade silicone discs in a 24-well microplate using TSB as the growth medium. The discs were washed once with 0.9% saline to remove nonadherent microorganisms and treated with either the DSMP at 64 μg/mL or vancomycin at 1024 μg/mL for 3 or 24 h. The treated biofilms were stained with the LIVE/DEAD BacLight Bacterial Viability Kit (L7007, Invitrogen) at 37 °C for 30 min in the dark, washed twice with saline, and examined with a Leica SP5 inverted microscope, using 20× and 60× objectives, respectively. All samples were sequentially scanned at 488, 561, and 380 nm. Three-dimensional biofilm images were reconstructed with the LAS X Life Science Microscopy software (Leica Microsystems) and Imaris 10.1 (Oxford Instruments, Abingdon, UK).

2.7. QAC and Quinine Drug Interaction Testing

The checkerboard method was adopted to assess drug interactions between the two key components of the DSMP, the QAC and quinine. The fractional inhibitory concentration (FIC) index was calculated by comparing the MIC for each component with the combination-derived MIC. The combination of QAC and quinine was considered to be synergistic when an FIC ≤ 0.5 was reported. FICs between 0.5 and 1.0 suggested no synergistic or only additive effects between the two components. FICs ranging from 1 to 4 were defined as indifferent, while those >4 were antagonistic.

2.8. Adaptive Laboratory Evolution of AMR

Two methicillin-susceptible Staphylococcus aureus isolates, ATCC 25923 and AH17 D009, were selected for the adaptive laboratory evolution assay. Bacterial strains were exposed to oxacillin, the QAC, quinine, and the DSMP at a sub-MIC concentration (1/2 MIC) for 15 passages to induce AMR in vitro, following a published method. In brief, 96-well microplates containing serially diluted antimicrobial solutions were inoculated with 100 μL of freshly prepared bacterial suspensions at 1 × 106 CFU/mL and incubated at 37 °C overnight. Contents in the microwell with distinct bacterial growth at the highest drug concentration were collected. Half of the content was used to inoculate a new microplate with fresh antimicrobial solutions, and the remaining half was stored at −80 °C in 20% glycerol for downstream MIC testing. This was used to confirm the induction of AMR.

2.9. Extraction of Bacterial Genomic DNA, Sequencing, and Bacterial Genome Comparison

Whole genome sequencing (WGS) was carried out for all clinical isolates and laboratory reference strains used in this study. The DNeasy Blood & Tissue Kit (Qiagen) was used for bacterial DNA extraction, as per manufacturer’s instructions. WGS was performed by using the Illumina HiSeq 150 bp paired-end platform (Genewiz). Bacterial genomes were independently assembled from the reads and examined for genes encoding AMR against commonly used antibiotics, including QAC genes qacA, qacC, and qacR.

2.10. Data Analysis

All experiments were carried out in three independent biological repeats. MICs were expressed as the geometric means of three repeats.

3. Results

3.1. Structural Composition and Zeta Potential of the DSMP

Figure shows the synthetic process and structural composition of DSMP. Styrene (St) was used as the base substrate to synthesize the hollow microparticle. Two chemical agents, a polymerizable QAC [2-(methacryloyloxy)­ethyl] trimethylammonium chloride and quinine, were grafted on the outer and inner shells of the DSMP for structural stability and antimicrobial properties, utilizing the polystyrene-bound functionalities on the outer shell and SO4 2– on the inner shell, respectively.

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Schematic illustration of the synthetic process and structural characteristics of the DSMP. Styrene; NIPAM, N-isopropylacrylamide; DMF, dimethylformamide; EGDMA, ethylene glycol dimethyl acrylate; and MTCl, [2-(methacryloyloxy)­ethyl] trimethylammonium chloride.

3.2. DSMP Showed Good Activities against Antibiotic-Susceptible Gram-Positive Clinical Isolates

In our preliminary tests, the DSMP showed strong antimicrobial activity against S. aureus ATCC 25923 (MIC = 0.5 μg/mL), a representative Gram-positive bacterium, and no effect on Pseudomonas aeruginosa PAO1 (MIC ≥ 128 μg/mL), a model Gram-negative bacterium. We thus decided to focus on the DSMP study on Gram-positive bacteria. MICs of the DSMP, vancomycin, oxacillin, and ciprofloxacin were determined, respectively, against 32 Gram-positive bacterial isolates, including three laboratory reference strains and 29 clinical isolates from two Australian tertiary referral hospitals (Table ). The DSMP demonstrated strong antimicrobial activity against 11 methicillin-susceptible S. aureus (MSSA) strains that were susceptible to all three antibiotics, with MICs ranging from 0.25 to 2 μg/mL. Another MSSA strain AH17 D028 was resistant to ciprofloxacin and had a higher DSMP MIC of 16 μg/mL. The DSMP had lower activity against the other 20 strains that were resistant to at least one antibiotic, with MICs between 8 and >128 μg/mL.

To understand the molecular mechanisms underpinning bacterial resistance to the DSMP, all clinical and laboratory isolates underwent WGS at the Alfred Hospital. qacA and qacC encoding efflux pumps previously implicated in reduced staphylococcal susceptibilities to QACs, were identified in only 4/20 DSMP-resistant strains. Three of these four isolates also carried the regulatory gene qacR; repression of qacR is known to lead to qacA overexpression and increased efflux pump activity. It is evident that the presence of qac genes could not solely explain the bacterial resistance to the DSMP. Other important AMR genes known to encode efflux pumps, including cadD, lmrS, tetK/38, and mepA, were also identified in almost all DSMP-resistant isolates, suggesting the importance of multiple efflux pumps in bacterial resistance to the DSMP. WGS results of AMR genes for all 32 studied strains were summarized and can be found in the Supporting Information.

3.3. DSMP Demonstrated Promising Activities against Biofilms Formed by Gram-Positive Bacteria

Antibiofilm efficacies of the DSMP were assessed for seven representative clinical isolates and three ATCC strains using the XTT reduction assay (Table ). Our preliminary crystal-violet-based biofilm assay suggested that all selected strains were able to form biofilms in 96-well microplates. The DSMP showed strong antimicrobial activity against biofilms produced by 3 MSSA strains and Enterococcus faecalis ATCC 19433, with BMIC80 ranging from 16 to 32 μg/mL. At a higher concentration of 1024 μg/mL, the DSMP effectively killed biofilms formed by methicillin-resistant S. epidermidis RP62A and RCH5, and E. faecalis AP069. In contrast, conventional first-line antibiotics vancomycin and oxacillin required at least 1024 μg/mL or higher to inhibit the biofilm growth of these strains by 80% (Table ).

2. Antibiofilm Activities of the DSMP against Gram-Positive Bacteria .

isolate ID
 species
 clinical/laboratory
 BMIC80 (μg/mL)
      Van Oxa Cip DSMP
ATCC 25923 MSSA laboratory >1024 >1024 >1024 32
RP62A S. epidermidis laboratory >1024 >1024 >1024 1024
ATCC 19433 E. faecalis laboratory 1024 1024 64 32
AH17 1003 MRSA clinical >1024 >1024 >1024 >1024
AH18 1009 MRSA clinical >1024 >1024 >1024 >1024
AH17 D009 MSSA clinical 1024 1024 512 16
AH17 × 10117 MSSA clinical 1024 >1024 1024 16
RCH3 S. epidermidis clinical >1024 >1024 >1024 >1024
RCH5 S. epidermidis clinical 1024 >1024 32 1024
AP069 E. faecalis clinical >1024 >1024 64 1024
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MRSA, methicillin-resistant S. aureus; MSSA, methicillin-susceptible S. aureus; Van, vancomycin; Oxa, oxacillin; Cip, ciprofloxacin; and DSMP, double-shelled microparticle.

Qualitative CLSM in combination with BacLight Live/Dead staining further confirmed the antimicrobial activity of the DSMP at 64 μg/mL against mature biofilms grown by methicillin-susceptible S. aureus ATCC 25923 and clinical isolate AH17 D009, S. epidermidis RP62A, and E. faecalis AP069 (Figure ). In comparison, conventional antibiotic vancomycin at 1024 μg/mL showed very limited activity in killing preformed biofilms of these isolates (Figure ).

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Killing of Gram-positive bacterial biofilms by the DMSP and vancomycin. Biofilms were formed on medical grade silicone discs and treated with the DSMP at 64 μg/mL or vancomycin at 1024 μg/mL for 24 h. Biofilms were stained with the LIVE/DEAD BacLight Bacterial Viability Kit and imaged by CLSM. The DSMP at 64 μg/mL efficiently killed biofilms formed by S. aureus ATCC 25923, S. epidermidis RP62A, S. aureus AH17 D009, and E. faecalis AP069 after overnight treatment. Vancomycin had little effect on these biofilms. Three-dimensional image reconstructions show staining pattern for the DSMP (coumarin, blue), live cells (stained with SYTO-9, green), and dead cells (stained with propidium iodide, red).

3.4. Mechanisms of Action of the DSMP Support Its Antibiofilm Potentials

An ideal antibiofilm agent must possess important characteristics including good penetration through the biofilm EPS matrix and the capability to kill metabolically inactive biofilm cells. To further elucidate antibiofilm mechanisms of the DSMP, we assessed the DSMP diffusion through the biofilm EPS matrix using fluorescently tagged DSMP and CLSM at two different post-treatment time periods. At 3 h, the DSMP was found within and across the biofilm structure, with stronger signals found on the surface of the biofilm.

This suggested that the DSMP was able to extensively bind to the biofilm surface, penetrate through the biofilm EPS matrix, and reach the proximity of the deeply embedded biofilm cells (Figure ). At 24 h, the DSMP self-accumulated into large blocks in the water channels or pores, crossing the full thickness of the biofilm structures (Figure ).

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Interactions between the DSMP and microbial biofilms at 3 h post-treatment. The DSMP extensively bound to biofilms formed by S. aureus ATCC 25923, S. epidermidis RP62A, S. aureus AH17 D009, and E. faecalis AP069 and penetrated through the biofilm EPS matrix by 3 h. The biofilms were stained with the LIVE/DEAD BacLight Bacterial Viability Kit (SYTO-9 for live cells, green signals; propidium iodide stains dead cells, red signals). The DSMP were tagged with fluorescence dye coumarin, blue signals). Imaris 10 was used to reconstruct the 3D images. Each panel contains the top-bottom view (images on the top) showing the overall binding of the DSMP with biofilms and cross-sectional view (images in the bottom) highlighting the distribution of the DSMP throughout the biofilms.

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Accumulation of the DSMP in biofilm water channels or pores (white arrows) at 24 h. The biofilms were stained with the LIVE/DEAD BacLight Bacterial Viability Kit (SYTO-9 for live cells, green signals; propidium iodide stains dead cells, red signals). The DSMPs were tagged with fluorescence dye coumarin (blue signals). Imaris 10 was used to reconstruct the 3D images. The orthogonal views of the control biofilms clearly showed the presence of water channels or pores in the biofilm structure. The images of orthogonal view and 3D reconstruction of biofilms exposed to the DSMP demonstrated the accumulation of the DSMP in the water channels or pores of the biofilms. E. faecalis AP069 did not form a large number of biofilms on medical-grade silicone discs and was not included in this experiment.

To further gain an understanding of how the DSMP diffuses through staphylococcal biofilms, we assessed the possible electrostatic interactions between the DSMP and the EPS matrix of staphylococcal biofilms. Biofilms are known to have highly heterogeneous pH landscapes, often showing neutral pHs on their surface and acidic pHs within the structure. A DSMP suspension of 64 μg/mL prepared in PBS appeared to be weakly positively charged, showing zeta potentials of 3.2 ± 0.3 mV at pH = 7.2 and 2.6 ± 0.56 mV at pH = 5.5, respectively (Figure ).

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Zeta potentials of the staphylococcal biofilm EPS matrix and the DSMP at neutral and acidic environments.

The EPS matrix isolated from mature biofilms formed by S. aureus ATCC 25923 and S. epidermidis RP62A was found to be both negatively charged, with zeta potentials of −10.8 ± 1.2 and −8.4 ± 0.9 mV, respectively, at pH = 7.2 and −11.0 ± 1.2 mV and −10.00 ± 0.03 mV at pH = 5.5. The weakly positively charged nature and high water solubility of the DSMP may allow the microparticle to adhere to the negatively charged biofilm surface to an extent that it can still freely diffuse through the biofilm matrix via its water channels, eventually accumulated in the water pores.

The low metabolic state of biofilm cells is another important mechanism mediating biofilm resistance to antimicrobials. Testing the minimum bactericidal concentrations under the nutrient-depleted and low-bacterial-metabolism growth conditions (1% MHB + 99% PBS) and the nutrient-rich (100% MHB) conditions, respectively, found no difference for the DSMP against 5/9 bacterial strains, suggesting a weakly metabolism-dependent characteristic of bacterial lethality of the DSMP (Table ). Vancomycin, in contrast, showed at least 16-fold differences in its bacterial lethality under these two conditions for all tested strains, reflecting a strongly metabolism-dependent trait for its bactericidal property (Table ).

3. Bacterial Metabolism Dependence of Drug Lethality .

isolate ID
 species
 MBC (μg/mL)
    vancomycin
 DSMP
    0.1% MHB 100% MHB fold changes in log2 0.1% MHB 100% MHB fold changes in log2
ATCC 25923 MSSA 8 >128 >5 16 16 1
RP62A S. epidermidis 8 128 5 32 >128 >2
ATCC 19433 E. faecalis 16 >128 >4 32 32 1
AH17 1003 MRSA 16 128 4 32 >128 >5
AH18 1009 MRSA 8 >128 >5 64 >128 >2
AH17 D009 MSSA 8 128 5 4 4 1
AH17 × 10117 MSSA 4 64 4 8 8 1
RCH3 S. epidermidis 8 >128 >5 8 >128 >5
RCH5 S. epidermidis 16 >128 >4 >128 >128 NA
AP069 E. faecalis 8 >128 >5 128 128 1
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0.1% MHB was used to represent a nutrient-depleted infection environment and a low metabolic status of bacterial cells. 100% MHB was used to represent a nutrient-rich environment and a high metabolic status of bacterial cells. NA, not applicable.

3.5. Synergistic Interactions of the QAC and Quinine Contribute to the Antimicrobial and Antibiofilm Activities of the DSMP

It was noticed that the DSMPs have superior activity against Gram-positive bacteria relative to that of the QAC and quinine alone (Table ). We thus assessed the interactions between the QAC and quinine that were copresented in the DSMP. A synergistic interaction between the QAC and quinine was found for 6/10 tested bacterial strains (see FICs and their interpretation in Table ). An additive effect or an indifferent effect was observed for the remaining four strains. The overall synergistic interactions between QAC and quinine may explain the great antimicrobial and antibiofilm activities of the DSMP.

4. Drug Interactions between the QAC and Quinine.

isolate ID
 species
 sources
 MIC (μg/mL)
 FIC
interpretation
      QAC quinine DSMP    
ATCC 25923 MSSA laboratory 8 >128 0.5 ≤0.5 synergetic
RP62A S. epidermidis laboratory 64 >128 32 >0.5–1 additive
ATCC 19433 E. faecalis laboratory 64 >128 16 ≤0.5 synergetic
AH17 1003 MRSA clinical 16 >128 16 1–4 indifferent
AH18 1009 MRSA clinical 32 128 32 1–4 indifferent
AH17 D009 MSSA clinical 4 >128 0.25 ≤0.5 synergetic
AH17 × 10117 MSSA clinical 8 128 0.25 ≤0.5 synergetic
RCH3 S. epidermidis clinical 16 >128 2 ≤0.5 synergetic
RCH5 S. epidermidis clinical >128 >128 >128 1–4 indifferent
AP069 E. faecalis clinical 64 >128 16 ≤0.5 synergetic
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Fractional inhibitory concentration (FIC) calculation FIC=MICAcombinationA&BMICagentA+MICBcombinationA&BMICagentB

3.6. Co-presence of the QAC and Quinine in the DSMP Lowers the Risk of Inducing Bacterial AMR

A major concern in fabricating novel antimicrobial agents or vehicles is the adaptation of microorganisms to the drug after long-term exposure and the development of AMR. We assessed AMR evolution of two MSSA strains, S. aureus AH17 D009 and S. aureus ATCC 25923, under the stress of oxacillin, the QAC, quinine, or the DSMP at a subinhibitory concentration for a period of 15 days. Continuous exposure of the two staphylococcal strains to the QAC, quinine, or oxacillin alone induced AMR toward respective agents after 8–11 passages. Both bacteria developed AMR after being exposed to the DSMP for 13 passages, 2–3 passages later than that for the QAC alone, or 5 passages slower than that for quinine or oxacillin (Figure A,B).

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In vitro evolution of antimicrobial resistance. Bacterial strains were exposed to different antimicrobial agents at a subinhibitory concentration for 15 passages and MICs were determined for survivors from each passage. (A) S. aureus ATCC 25923 and (B) S. aureus AH17-D009.

4. Discussion

The burden of AMR has triggered global attention and actions. One of the proposed mitigation strategies is to enrich the antimicrobial arsenal with novel modes of delivery, either to decelerate the evolution of AMR or to increase the treatment efficacy. We have previously developed a novel double-shelled antimicrobial microparticle. This microparticle incorporates two antimicrobial chemicals, a QAC on the outer shell and a quinine on the inner shell as the structural backbone. The developed DSMP presented strong electrostatic interactions with bacterial cells and disrupted their membrane integrity, ultimately causing cell death. ,

In this study, we intended to comprehensively understand the “real” anti-infective performance and to unveil the biological merits of the DSMP, with particular interest in microbial biofilms that are often considered as the root cause of many difficult-to-treat infections. Our pilot experiment suggested that the DSMP had great activity against S. aureus ATCC 25923 but not P. aeruginosa PAO1. This is not surprising as the QAC and quinine, two major agents incorporated into the DSMP for both structural stability and antimicrobial activities, have been found to be more effective against Gram-positive bacteria relative to Gram-negative bacteria. Among Gram-positive pathogens, the DSMP showed strong activity against antibiotic-susceptible bacteria and limited activity against antibiotic-resistant strains. The co-occurrence of bacterial resistance to the DSMP and conventional antibiotics was observed in many clinical isolates in our study. WGS analyses suggested that the cross-resistance to the DSMP and conventional antibiotics in Gram-positive bacteria was possibly mediated by the presence of numerous efflux pumps.

The DSMP has a default antibiofilm characteristic and outperformed vancomycin in killing staphylococcal or enterococcal biofilms. It was evident that the DSMPs were able to bind to the surface of biofilms formed by Gram-positive bacteria, possibly due to the electrostatic interaction between the positively charged DSMP and negatively charged biofilm EPS. The DSMP also demonstrated great capacities in penetrating through the biofilms, as found in our CLSM study. The penetration of microparticles through biofilms is known to be related to the combinational chemical (e.g., osmotic pressure) and electrical gradients (electrophoresis). , The weak positive charge that the DSMP carried allowed the microparticle to overcome the electrostatic attractions between the particle and negatively charged biofilm EPS materials and freely diffuse into the biofilm. Although it is challenging for a particle of ∼500 nm to pass through the bacterial clusters, the presence of water channels of variable sizes in mature biofilms empowered the penetration of the highly soluble DSMP to the deeper sections of the biofilm structure. Inner-biofilm water channels and pore often act as the specific pathways or storage that facilitate antimicrobial access to deeply embedded biofilm cells. We propose that the buildup of antimicrobial microparticles within the biofilm water channels, potentially influenced by their size and charge, offers an effective mechanism for biofilm disruption.

QACs have been widely used in hospitals or aged care settings for disinfection purposes. High dose of QACs has been recommended due to their low activities and may inevitably lead to high toxicity and risk of promoting AMR. Simultaneous incorporation of the QAC and quinine in the DSMP not only allows structural stability of the microparticle but possibly lowered the effective and safe dose of the QAC required for the antimicrobial activities of the DSMP. Although the exact quantity of the QAC in a single copy of the DSMP cannot be determined due to technical limitations, the amount of the DSMP required to inhibit bacterial growth was far below that of the QAC or quinine alone. The synergistic interaction between the QAC and quinine at least partially explains the higher antimicrobial activities of the DSMP in comparison with those of the QAC alone. Combining QACs and quinine also appeared to decelerate AMR evolution when compared with oxacillin, quinine, and the QAC alone. Resistance to quinine and oxacillin in staphylococci appeared after 8 passages and that to the QAC emerged after 10 or 11 passages in our study, similar to what was reported by others. Placing the QAC and quinine in proximity in the DSMP showed delayed induction of AMR that emerged after 13 passages. It is believed that grafting antibiotics on the DSMP will further postpone the development of AMR. The unique distribution dynamics of the DSMP through biofilms formed by Gram-positive bacteria, the presence of antibiotic-binding docks on the shells, the default antibiofilm characteristics, and the tolerance of the microparticle to AMR induction render the DSMP an ideal platform for the treatment of biofilm-related infections, preferentially as an antibiotic-delivering vehicle.

The strength of our study is the multidisciplinary efforts that were spent; microbiological assays were cautiously designed or adopted to study the biological characteristics of the DSMP. An evident limitation of this study is the limited microbial spectrum that we covered. Our study only focuses on Gram-positive bacteria, and the applicability of the DSMP against Gram-negative pathogens should be further investigated.

In this study, we rationalized the use of QAC and quinine for the DSMP and explored clinical potentials of the system. We also demonstrated how to successfully design anti-infective microparticles as antibiotic vehicles from a biological perspective.

Supplementary Material

ao5c05384_si_001.pdf (709.1KB, pdf)

Acknowledgments

The authors thank MMI for the facility to perform CLSM. We would also like to acknowledge Dr. Matthew Parker and Dr. Ruzeen Patwa from the Department of Infectious Diseases for their assistance in performing whole genome sequencing.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c05384.

  • WGS of all clinical isolates and laboratory reference strains used in this study (PDF)

CRediT Conceptualization: CS, YQ, JSF, and TFS; data curation: CS, CZ, MG, DCM, DMK, VXT, CQ, XK, and AYP; formal analysis: CS, YQ, XK, and CQ; funding acquisition: YQ, DCM, DMK, AYP, JSF, and TFS; investigation: CS, YQ, JSF, and TFS; methodology: YQ, CS, JSF, and TFS; project administration: YQ, JSF, and TFS; resources: YQ, JSF, TFS, and AYP; supervision: YQ, JSF, and TFS; validation: JSF, TFS, and YQ; writingoriginal draft; and YQ and CS: writingreview and editing, all coauthors.

This work was supported by fundings from the Artificial Heart Frontier Program, the Monash Institute of Medical Engineering and the Department of Infectious Diseases of Monash University.

No authors have no conflicts of interest to disclose. None of the authors have a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose. The authors acknowledge the financial assistance provided by the Department of Infectious Diseases, and Department of Microbiology, Monash University, Australia. The funding organizations listed above had no role in the collection of data, its analysis, and interpretation and in the right to approve or disapprove publication of the finished manuscript.

The authors declare no competing financial interest.

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Supplementary Materials

ao5c05384_si_001.pdf (709.1KB, pdf)

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