Metallo- β -lactamases are bacterial zinc-dependent enzymes involved in the hydrolysis of β -lactamic antibiotics representing the main cause of bacterial resistance to carbapenems, drugs of last resort for treating infections caused by multiresistant bacteria. We elaborated the hypothesis that it is possible to inhibit the enzymatic activity of metallo- β -lactamases by lowering the availability of zinc in the extracellular medium using metal chelating agents such as EDTA carried on nanoparticles. Chitosan, as linear cationic polysaccharide is frequently used in biomedical and pharmaceutical applications, has been studied as a biocompatible encapsulating agent in drug delivery systems and is an ideal transport agent for bioactive molecular complexes in antibiotic applica tions due to its ability to associate with negatively charged substances. We de veloped novel nanoparticles using chitosan as a transport matrix for β -lactamic antibiotics. Nanoparticles were synthesized according to the ion gelation method using tripolyphosphate as crosslinking agent. Nanoparticles were functionalized by the adsorption of EDTA, which acts as complexifying agent for Zn 2+ ions causing inhibition of metallo- β -lactamases activity. We evaluate the antimicrobial effects of EDTA-functionalized nanoparticles with an imipenem cargo on the clinical isolate P. aeruginosa AG1, a carbapenem-resistant high-risk clone ST-111 carrying both bla IMP-18 and bla VIM-2 metallo- β -lactamases genes.
Pseudomonas aeruginosa, a Gram-negative bacterial pathogen bacterium with extraordinary physiological and metabolic versatility found mostly in water reservoirs, causes severe nosocomial and community acquired infections at a variety of body sites including the urinary tract, surgical or burn wounds and the lower respiratory tract [
P. aeruginosa infections are a serious therapeutic challenge due to the difficulty in their treatment and control: acquired mechanisms of resistance found commonly in P. aeruginosa isolates often render ineffective known disinfectants and antibiotics [
β-lactamases confer significant antibiotic resistance to their bacterial hosts by hydrolyzing the amide bond of the four-membered β-lactam ring. These enzymes are especially important in P. aeruginosa as they constitute the major defense mechanism against β-lactam antibiotics such as carbapenems [
Class B enzymes, metallo-β-lactamases (MBLs), require a zinc divalent ion in order to perform their activity. With the aim of blocking the activity of MBLs, the search for activity inhibitors and deactivators along with their coupling with a β-lactam antibiotic has been proposed. The latter in promising in the sense of finding adequate molecules that can be added to carbapenems, resulting in combinations with the ability to overcome resistance mediated by metallo-β-lactamases [
A total of 198 non-duplicated P. aeruginosa AG1 isolates from a study in Costa Rica were evaluated for their susceptibility to beta-lactams, aminoglycosides and fluoroquinolones. From the sample, 63.1% were categorized as carbapenem-resistant and 88.8% of the carbapemen-resistant isolates were also resistant to ceftazidime, cefepime, aztreonam, ticarcillin/clavulanic acid, amikacin, gentamicin, tobramycin, ciprofloxacin and gatifloxacin. 81.6% of the carbapenem-resistant isolates showed MBL activity. The blaIMP and blaVIM genes were present in 94.1% of the MBL-producing isolates [
Chitosan is a linear polysaccharide that is composed of randomly distributed D-glycosamine and N-acetyl-glycosamine units linked in a β (1 → 4) manner [
These nanoparticles can achieve a size of up to 50 nm and a positive surface charge (reported as ζ potential) between +20 mV and +60 mV [
Ethylenediaminediacetic acid-disodium (EDTA) is a polyamino carboxylic acid and popularly known as chelating agent. The NH3+ moieties present in the quitosan were reported to react with COO− moieties of EDTA to form ionic EDTA-CHT complex. The EDTA-CHT was reported to show antimicrobial activity against Gram-negative and Gram-positive bacteria [
In the present study, EDTA-CHT nanopartices (EDTA-CHT NPs) were synthesized by an ionic gelation cross-linking method. Imipenem, a carbapenem, were loaded into the nanoparticles. The physicochemical properties of EDTA-CHT NPs and antimicrobial capability of imipenem-loaded EDTA-CHT NPs was also investigated against carbapenem-resistant P. aeruginosa.
Chitosan with molecular weight between 100,000 - 300,000 (ACROS OrganicsTM), sodium tripolyphosphate (Fischer scientific) and glacial acetic acid (J.T.Baker®) were used. Ultrapure water was obtained using the Milli-Q A10 system (Millipore). Imipenem monohydrate ≥ 98% (Sigma-Aldrich) was used as the antimicrobial agent. Disodium EDTA (J.T.Baker®) were used.
P. aeruginosa AG1, carrier of the blaIMP-18 and blaVIM-2 genes with expression of metallo-β-lactamase activity, was used as model for this study. P. aeruginosa PAO1 were used as control strains due to their susceptibility to carbapenems [
Chitosan-tripolyphosphate nanoparticles were produced using a modified ionic gelation method. Briefly, chitosan was dissolved at 5 mg/ml in 2% v/v acid acetic solution at pH = 5. Tripolyphosphate was dissolved in ultrapure water to obtain a 1.2 mg/ml concentration. 1 ml of tripolyphosphate solution was added dropwise to 1.5 ml of chitosan solution and was magnetically stirred at 500 stock/min for 1 h [
1 ml of imipenem (dissolved in a solution of MOPS, ethylene glycol and water 2:1:1) at different concentrations was added at constant stirring before polymer cross-linking. Later addition of sodium tripolyphosphate was required for encapsulation during ionic gelation [
A concentration gradient of EDTA was tested from 0.39 to 125 mM. We chose a concentration of EDTA for CHT NPs synthesis of 3 mM for surface functionalization. Surface functionalization of CHT NPs with chelating agent EDTA was carried out by adding 1 ml of EDTA at a concentration of 5 mg/ml after ionic gelation, the solution was magnetically stirred at 500 stock/min for 30 min more. The resulting reaction is acid-base leading to the formation of an amide bond between the free amino groups (−NH2) in chitosan and the carboxyl groups (−COOH) in EDTA [
The structural features of nanoparticles were estimated by Fourier transform infrared (FTIR) Thermo Scientific, Nicolet 6700. The samples for FTIR analysis are prepared by grinding the dry blended CHT and EDTA-CHT NPs using an attenuated total refraction.
Determination of particle size (apparent hydrodynamic diameter) was performed by dynamic light scattering (DLS), polydispersity index value (size distribution) and surface electric charge, reported as ζ potential or electrophoretic mobility [
In order to study the morphology of filtered (0.2 um) and diluted samples (1/10) of nanoparticles, topographic images of CHT-NPs were taken on a multimode atomic force microscope (AFM) Asylum Research MFP-3D. The AFM probes used for this study were rectangular silicon probes with a nominal spring constant of 40 nN/nm. Similarly, image visualization was carried out in a scanning electron microscope (SEM) Hitachi S-3700 with a 15 nm gold coating on the diluted samples (1/10) using a aluminum base at an acceleration voltage of 15 kV [
The encapsulation efficiency (EE) of the nanoparticles was determined according to the method described in the previous studies [
E E = F / T × 100 %
where, F is the free amount of imipenem in the supernatant and T is total amount of imipenem.
The in vitro release studies were carried out in PBS (pH 7.4) as followed: imipenem loaded EDTA-CHT NPs (1.5 ml) and 1.5 ml PBS were incubated at 37˚C and shaken at 200 stocks/min. Triplicate samples were analyzed at each time step, between 0 and 24 hours. The concentrations of the released imipenem into PBS were determined by HPLC-DAD S-200 Perkin-Elmer.
The spectrophotometrically adjusted inoculum (100 μl) of 104 bacterial cells was added to each well in the sterile flatbottomed microtiter plate containing the test CHT-NPs. The design of experiments includes duplicated wells of imipenem- loaded EDTA-CHT NPs with different concentrations of imipenem, two wells with imipenem as growth inhibition control, two wells containing bacterial suspension with CHT-NPs (growth control) and two wells containing only media (background control) were included in this plate. Dilutions were halved at each consecutive level in the gradient. Optical densities were measured for 12 hours at 37˚C using a multi-detection microplate reader Biotek Synergy HT at 600 nm and automatically recorded for each well every 30 min. Turbidimetric growth curves were obtained depending on the changes in the optical density of bacterial growth for each CHT NP sample and the drug-free growth control.
The morphological properties of CHT NPs and EDTA-CHT NPs (mean size, ζ potential and PDI) are indicated in
In present study the results obtained by DLS revealed that the EDTA-CHT NPs are larger than the CHT-NPs ones, possibly due to the EDTA surface adsorption during reaction time. ζ potential of CHT NPs can greatly influence their stability in suspension by means of electrostatic repulsion between the particles [
A batch of samples of CHT-NPs was synthesized for morphological analysis using the AFM.
EDTA functionalization has an aggregation effect on CHT NPs, as expected from literature [
The ability of the ionic gelation process to form EDTA-CHT NPs was assessed by employing Fourier transform infrared (FTIR) spectroscopy in order to determine EDTA-CHT interactions. The FTIR spectra of CHT NPs and EDTA- CHT NPs are shown in
In the present study, SEM images were taken in order to study the morphological properties and surface appearance of nanoparticles (
Size (nm) | ζ (mV) | PDI | ||||
---|---|---|---|---|---|---|
EDTA (%w/v) | avg | sd | avg | sd | avg | sd |
0.00 | 127.77 | 4.80 | 56.07 | 0.42 | 0.55 | 0.02 |
0.50 | 637.10 | 39.14 | 46.63 | 2.54 | 0.76 | 0.03 |
diameter of 100 nm. Size and texture is affected when EDTA is added to the synthesis process: larger and smoother particles are obtained, possibly due to the aggregation effects previously discussed (
The in vitro cumulative release profile of imipenem from the CHT NPs is shown in
In order to determine the range of EDTA concentrations at which growth of P. aeruginosa occur, a dilution assay with dilution ratio 0.5 and two replicate wells per sample was devised. EDTA concentrations with a maximum of 125 mM were utilized in this assay, which is pivotal to avoid reporting significant growth inhibition by EDTA alone.
An important consideration needs to be made in relation to the effective EDTA concentration in CHT NPs. A fraction of the EDTA molecules in the solution during functionalization process will attach to the surface of the nanoparticle, but not all of them. The concentration of EDTA carried by CHT NPs is a function of initial EDTA concentration, particle size and distribution of free amino groups on the surface. In general, if r1 and r2 represent the radii of two types of NPs where (without loss of generality) r2 > r1, the relation between the surface areas is (r2/r1)2. As SEM imaging reveals (
In order to determine the concentration of EDTA where synergistic effects were observed instead of EDTA leading to bacterial inhibition by itself, an assay with increasing EDTA concentrations was performed (
The comparison between CHP NPs, EDTA-CHT NPs and imipenem-loaded EDTA-CHT NPs with a concentration of 20 mg/ml (complete system) was performed (
complete system, growth curves were obtained respectively for each nanoparticle system (
The imipenem loaded CHT-EDTA NPs were successfully prepared based on ionic gelation by cross-linking with TPP to investigate the physicochemical properties of nanoparticles. The nanoparticles were stable and spherical in shape with a narrow size distribution. The release amounts of imipenem from the EDTA-CHT NPs suggested that nanoparticles have promising potential effect on antibiotic therapy.
The authors wish to acknowledge the Inter-University Fund for Higher Education (FEES) at the National Council of Rectors (CONARE) for financial support to this project through grant agreement No ACUERDO-VI-171-2014, the Research Center on Microscopic Structures (CIEMIC) at University of Costa Rica as well as, Maribel Chavarría (CIET), Rodrigo Muñoz (CENIBiot), Daniel Esquivel (CENIBiot) and Reinaldo Pereira (LANOTEC-CeNAT) for their collaboration and support with various aspects of this paper.
Porras-Gómez, M., Vega-Baudrit, J., García, F., Núñez-Corrales, S. and Madrigal-Carballo, S. (2018) Evaluation of the Synergistic Effect of EDTA-Functionalized Chitosan Nanoparticles on Imipenem Delivery in Pseudomonas aeruginosa Carbapenem-Resistant Strain AG1. Journal of Biomaterials and Nanobiotechnology, 9, 64-78. https://doi.org/10.4236/jbnb.2018.91006