Effective release of Ciprofloxacin and Rifampicin antibiotics from alginate-chitosan complex and its application against clinical strains of Staphylococcus aureus

doi:10.21203/rs.3.rs-3116546/v1

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Research Article

Effective release of Ciprofloxacin and Rifampicin antibiotics from alginate-chitosan complex and its application against clinical strains of Staphylococcus aureus

https://doi.org/10.21203/rs.3.rs-3116546/v1

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published 05 Dec, 2023

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This study aimed to characterize the antimicrobial and anti-biofilm activity of ciprofloxacin (CIP) and rifampicin (RMP) loaded on the calcium alginate (CaAlg) backbone through the electrostatic interaction of CaAlg/antibiotic (s) and chitosan-TPP nanoparticles (Cs-TPP NPs). The shape and functional groups were identified by scanning electron microscope (SEM), Fourier transform infrared (FT-IR) spectroscopy. SEM images show that the nanoparticles are spherical and granular with an average size of about 100–500 nm. FT-IR results showed bonds corresponding to the Cs-TPP NPs and CaAlg-Antibiotics (CaAlg-CIP, CaAlg-RMP) after the loading process. The tube adherence method (TM) was used to screen potent biofilm producers. The anti-biofilm capacity of Cs/CaAlg-CIP and Cs/CaAlg-RMP, along with free antibiotics was used against clinical strains in the tube method and results showed that the use of sub-MICs of nano-drugs (¹/₂ MIC) inhibited biofilm formation in the tested strains compared to free CIP and RMP (P < 0.05). The potential antimicrobial effect of Cs/CaAlg-CIP and Cs/CaAlg-RMP NPs against clinical strains was also evaluated using minimum inhibitory concentration (MICs) and a time-depending killing method. A robust inhibitory effect on bacterial growth even at low concentrations of Cs/CaAlg-CIP and Cs/CaAlg-RMP NPs (CONC 4 and 2 µg.mL^─1) was observed compared with free drugs (CIP at 32 and RMP at 8.0 µg.mL^─1), (P < 0.05). The time-kill kinetics assay showed that Cs/CaAlg-CIP and Cs/CaAlg-RMP NPs had faster and more lasting antibacterial effects than the free drug (s) on the tested strains. This study shows that loading CIP and RMP into CaAlg scaffolds and complexes with chitosan-TPP nanoparticles using the emulsification/internal gelation method increases the antimicrobial activity of antibiotics against clinical strains of S. aureus and thus might serve as a new type of antimicrobial compounds for the treatment of bacterial infections.

Ciprofloxacin

Rifampicin

Chitosan-TPP

Staphylococcus aureus

Cs/CaAlg-CIP

Cs/CaAlg-RMP

Emulsification

An essential feature of medical science in the twentieth century was the use of antibiotics to treat bacterial infections. However, shortly after their introduction into clinical practice, the first bacterial strains became resistant to antibiotics, the most important of which was methicillin-resistant Staphylococcus aureus (Davies et al., 2010; Costa et al., 2013). According to research conducted around the world, methicillin-resistant S. aureus (MRSA) is one of the primary pathogens in both hospital and community-associated infections it was officially ranked as one of the 12 deadliest drug-resistant bacteria by WHO, which imposes high costs on countries around the world (Abdulgader., et al 2015; Lee et al., 2013; Köck et al., 2010 and Hua et al., 2018). The significant infections caused by S. aureus include wound infections and toxins-mediated syndromes, as well as systemic and life-threatening diseases (Wu et al., 2018). There are several strategies used for drug resistance by S. aureus, the most important of which are outer membrane permeability, excessive production of β-Lactamases, resistance by mutations, acquisition of resistance genes, and biofilm-mediated resistance (Guo et al., 2020).

In general, biofilm formation allows single-celled bacteria to adopt a temporary multicellular lifestyle, in which cell aggregation in one place will enable them to survive in improper conditions and facilitates growth (Pratt and Kolter 1998). The formation of biofilms in organisms was defined as forming a community of microorganisms attached to a surface, as a complex growth process that is multifaceted and dynamic. Transfer from planktonic (freely moving) to group growth occurs in response to changes in the environment and involves several intricate regulatory networks that lead from the physiological level to the genomic level and subsequently to the proteomics, thus allowing bacteria to adapt to the environment optimally (Kostakioti et al., 2013). Hence, the biofilm formation in S. aureus is one of the most critical factors in giving bacteria antibiotic resistance and protection against the host immune system (Torres et al., 2019). The infections are very diverse and can include acute conditions such as bacteremia and skin abscesses, which are usually secreted by planktonic cells through the production of toxins and exo-enzymes (Lister and Horswill., 2014). On the other hand, chronic infections are correlated with a biofilm formation, in which S. aureus can attach and insist on host tissues, such as bone and heart valves, to cause osteomyelitis and endocarditis, respectively, or on implanted materials, such as catheters, prosthetic joints, and pacemakers (Lister and Horswill., 2014, Parsek and Singh, 2003; Kiedrowski and Horswill, 2011).

Ciprofloxacin (CIP) is an effective antibiotic against S. aureus. Still, today, ciprofloxacin-resistant (CIP-R) strains are reported from all over the world, and this resistance is considered a rapid increase in resistance to fluoroquinolones and is one of the best examples of biological evolution (Campion et al., 2004). Two main mechanisms have been reported for fluoroquinolone resistance, the first involves point mutations in the grlA/grlB and gyrA/gyrB genes, which encode the subunits of topoisomerase IV and DNA gyrase, respectively, and the second mechanism involves in the extrusion of the fluoroquinolone-based antimicrobials by the bacterial efflux pump, especially the NorA protein, which is chromosomally encoded by the norA gene. The NorA efflux pump can extrude fluoroquinolones and other structurally unrelated compounds out of the bacterial cell, thus causing drug resistance (Campion et al., 2004). Likewise, rifampicin (RMP) is another transcription inhibitor antibiotic used to treat a variety of bacterial infections and is commonly used in combination with other antimicrobial agents for severe S. aureus infections that inhibits bacterial transcription by binding to the β-subunit of the bacterial DNA-dependent RNA polymerase (RNAP), which is encoded by the rpoB gene, and rifampicin-resistance in S. aureus is closely associated with mutations which occur in the rpoB gene (Wang et al., 2019; Zhou et al., 2012). Therefore, given the widespread drug resistance and therapeutic failure that is being reported daily around the world to commonly used antibiotics, the need to find an alternative way to maximize the effectiveness of antibiotics is more needed than ever to review (https://www.who.int/publications/i/item/9789240021303).

Chitosan (Cs) contain β-1,4-linked 2-amino-2-deoxy-β-D-glucose (deacetylated D-glucosamine) and N-acetyl-D-glucosamine units are biocompatible, biodegradable, and non-toxic compounds which are derived from chitin, so that it can be used in numerous medical applications as antitumor, immunoenhancing, antifungal, antimicrobial, antioxidant and wound healing biomaterials (Shariatinia, 2019; Pourebrahim et al., 2021). Structurally, chitosan nanoparticles are obtained using chitosan and sodium tripolyphosphate (STPP) by the ionic gelation method. The formulations displayed particle size in the nanometer range with high loading and encapsulation efficiency with natural and/or artificial components (Bangun et al., 2018). Regarding the interaction of chitosan with the bacterial cell, it has been suggested that positive charges on the surface of chitosan make it possible to bind to the negative part of the bacterial cell membrane (Pourebrahim et al., 2021).

This study aimed to develop controlled drug delivery based on chitosan-alginate nanoparticles. First, antibiotics (CIP, RMP) were loaded with calcium alginate (CaAlg) and the resulting structure (CaAlg-CIP; CaAlg-RMP) were covered by chitosan nanoparticles to enhance antibiotics’ antimicrobial and anti-biofilm activity compared to free drugs. The interaction between the CaAlg-Antibiotic (s) and the chitosan NPs forms a polyelectrolyte complex through the ionic interaction between the carboxyl alginate residue and chitosan’s amino residue. The reason for using CIP and RMP antibiotics was that both of them are transcriptional inhibitors and have a target site inside the cell, such as type II topoisomerase (DNA gyrase) and bacterial DNA-dependent RNA polymerase (RNAP), respectively so that we can have an accurate evaluation of drug delivery to target sites compared to free drugs.

Reagents

Ciprofloxacin hydrochloride (CIP HCl), rifampicin (RMP), low viscosity sodium alginate (SALG) polymer molecular weight (MW, 70 kDa), Calcium chloride (CaCl₂), sodium tripolyphosphate (TPP), phosphate buffer saline (PBS), and acetic acid were purchased from Sigma-Aldrich. Chitosan from the chitin of black soldier fly (75–85% deacetylated) was purchased from Zist Tabdil Gilan Company, Iran.

Preparation of chitosan nanoparticles

Briefly, a 1% (v/v) solution of acetic acid in 50 mL of hot water was used to dissolve 0.5 g of chitosan (pH 4.5) under stirring at 45°C for 24 h to obtain a homogeneous solution. On the other side of the reaction, 0.1% w/v tripolyphosphate (TPP) was dissolved in deionized water for 4 h using a low-speed magnetic stirrer and then, sonicated for 20 min to improve solubility. Then, TPP was added drop-wise to the aqueous solution of chitosan under severe magnetic stirring (700–1200 rpm) at room temperature (25°C). The resulting suspension was sonicated for 20 min and then, was centrifuged at 15,000 rpm at 4°C for 10 min. The supernatant was discarded and the resulting particles were dried at 45°C for 24 h (Bangun et al., 2018).

Preparation of antibiotics-loaded alginate and formation of an alginate-antibiotics complex with chitosan-TPP nanoparticles

Preparation of CaAlg-CIP and CaAlg-RMP NPs was performed using the method described by Scolari et al., (2020) with minor modifications. Initially, sodium alginate (0.5 g), antibiotics (200 mg), and, 0.05% w/v CaCl₂ aqueous solution were prepared separately in distilled water (20 mL) and the mixture of each reaction (60 mL) was homogenized in an ultrasonic bath (Branson CPX-952-238R) for 15 min to obtain the CIP and RMP loaded Alg NPs, respectively. CaCl₂ were used for alginate gelation to increase the stability of the alginate-antibiotic (s) complex. Then, the suspension was centrifuged at 15,000 rpm at 4°C for 15 min and the resulting particles were harvested, washed thrice with distilled water, and dried at 45°C for 10 h. The obtained particles (CaAlg-CIP, CaAlg-RMP) were coated with chitosan-TPP NPs at the 2:1 molar ratio (pH 4.5) with stirring at 400 rpm for 60 min at 37°C. The obtained suspension was left under ultra-sonication for 20 min and then was centrifuged at 15,000 rpm for 10 min at 4°C. The supernatant was discarded and nanoparticles with a unique structure (Cs/CaAlg-CIP; Cs/CaAlg-RMP) were dried at 45°C for 24 h and kept at 4°C for further study. A simple overview of the synthesis of chitosan-TPP nanoparticles, the loading process of antibiotic (s) in CaAlg scaffolds, and the chitosan-TPP complex with CaAlg-antibiotic (s) are shown in Fig. 1.

Characterization of the nanoparticles

FT-IR Spectroscopy was used to describe and provide information on the vibrational state of functional groups in the molecular chains of chitosan-TPP NPs, sodium alginate, and final nano-drugs formulation (Cs/CaAlg-CIP, Cs/CaAlg-RMP). FTIR measurements were performed over the range of 500─4,000 cm^─1 with a resolution of 1 cm^─1 on a Thermo Scientific Nicolet Nexus, Smart Orbit spectrometer 380, using the KBr disk method. The surface morphologies (size and shape) of pure chitosan, chitosan-TPP NPs, as well as Cs/CaAlg-CIP and Cs/CaAlg-RMP NPs were characterized by scanning electron microscopy (SEM), (Philips, XL30).

Antimicrobial susceptibility testing (AST)

S. aureus strains (n = 12) were isolated from different clinical specimens of patients referred to the Razi Central Hospital, Rasht, Iran. The isolated strains were named BI, BII, BIII, BIV, BV and, BVI from blood samples; UI from urine samples; SI, SII and, SIII from skin samples; OCI and SCI from ovarian and scalp cysts were stored at -80°C, respectively. The antibiotic resistance profile of S. aureus strains was determined using Kirby Bauer’s disk diffusion method (DDM) according to Clinical & Laboratory Standards Institute guidelines (CLSI, 2020). In this study, different classes of antibiotic (s) with various mechanisms of action were used, which are: Transcription-inhibitory antibiotics such as Ciprofloxacin (CIP 5µg), Nalidixic acid (NAL 30µg), Novobiocin (NB 5µg), Rifampicin (RMP 5µg); translational-inhibitory antibiotics such as Tetracycline (TET 30µg) and inhibitors of the biosynthesis of the bacterial cell wall, Oxacillin (OXA 1µg) and Vancomycin (VAN 30µg). S. aureus (ATCC® 25923) was used as an endogenous control strain in antimicrobial susceptibility testing. In this study, only strains with high resistance to antimicrobial compounds were exposed to nanoparticles for a more appropriate evaluation of the antibacterial activity of Nano-drug formulations. The resistance phenotypes determined for S. aureus isolates are shown in Table 1.

Table 1

Shows the patterns of antibiotic resistance as well as the minimum inhibitory concentration and the minimum bacterial concentration of antimicrobial agents against clinical strains of *S. aureus*. **Abbreviations**: CRI = Catheter-related infection; TKA = Total knee arthroplasty; pt. = patient; S = Susceptibility; I = Intermediate and R = resistance. CIP, ciprofloxacin; NB, novobiocin; NAL, nalidixic acid; RMP, rifampicin, OXA, oxacillin, TET, tetracycline, VAN, vancomycin.
		Inhibition Zones (mm²)							MICs and MBCs (µg.mL^− 1)
Strains	Type & source	Antibiotics (µg/discs)							Cs/CaAlg-CIP		Cs/CaAlg-RMP		Cs-TPP		Free CIP		Free RMP
Strains	Type & source	CIP	NB	NAL	RMP	OXA	TET	VAN	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
ATCC 25923	─	S	S	S	S	S	S	S	2	4	1	2	32	64	16	32	8	16
BI	Blood; pt. with CRI	R	R	R	R	R	R	I	8	16	4	8	256	512	256	512	128	256
BII	Blood; pt. with CRI	R	S	I	R	R	R	S	32	64	16	32	512	1,024	256	512	64	128
BIII	Blood; pt. with CRI	R	S	R	S	I	R	I	16	32	4	8	128	256	64	128	16	32
BIV	Blood; pt. with CRI	R	S	R	R	R	R	I	64	128	16	32	> 512	1,024	256	512	128	256
BV	Blood; pt. with CRI	S	R	I	R	R	I	R	16	32	8	16	256	512	128	256	64	128
BVI	Blood; pt. with CRI	S	R	R	R	R	I	R	4	8	32	64	128	256	32	64	128	256
UI	Urinary tract infection (UTI)	S	I	R	S	R	S	S	4	8	2	4	32	64	32	64	8	16
SI	Septic TKA	R	S	R	R	I	R	S	32	64	8	16	512	1,024	128	256	64	128
SII	Septic TKA	R	S	R	S	R	R	I	16	32	4	8	256	512	128	256	64	128
SIII	Septic TKA	I	I	R	S	R	S	I	8	16	2	4	64	128	64	128	16	32
OCI	Ovarian Cyst; with surgery	R	S	R	S	I	R	I	16	32	4	8	128	256	128	256	32	64
SCI	Scalp cyst; with surgery	R	I	R	S	I	R	I	32	64	8	16	128	256	256	512	64	128

Minimum inhibitory concentration (MICs) & minimum bactericidal concentration (MBCs) determination

The MICs and MBCs of Cs/CaAlg-CIP and Cs/CaAlg-RMP nanoparticles in comparison with Cs-TPP NPs, free ciprofloxacin and rifampicin were determined using the broth micro-dilution method according to CLSI instructions (CLSI, 2020). 96-well plates were used vertically to dilute the agents so that the first column had the highest concentration and the other column had the lowest concentration of antimicrobial agents, respectively, (1 to 10). Columns 11 and 12 served as positive and negative controls and were incubated with medium and bacterial inoculums without antimicrobial agents and medium only, respectively. In summary, 100 MHB medium was poured into all wells and 10-fold of each agent (CONC 10.240 µg.mL^–1) was prepared separately and diluted 2:3 (v/v) in distilled water (CONC of 4.096 µg.mL^–1). Then, 100 µL of antimicrobial compounds were poured into columns 1_A to 1_H and diluted sequentially in bottom wells. Then, 100 µL of adjusted bacterial inoculums (10⁶ CFU/mL) was poured into 96-well plates and the plate was incubated at 37°C for 24 h. The MICs value was considered to be the lowest concentration of antibacterial compounds that prevented the visible growth of bacteria. Finally, to quantify the growth rate of nanoparticle-treated bacteria, wells without and/or low bacterial growth were inoculated on Tryptic Soy Agar (TSA) and incubated at 37°C for 24 h and monitored for bacterial growth. The MBCs were specified as the concentration of the antimicrobial compounds that killed 99.9% of the start inoculum.

Time-dependent killing

Based on our previous work (Pourebrahim et al., 2021), ½ MICs of Cs/CaAlg-CIP, Cs/CaAlg-RMP nanoparticles compared with Cs-TPP NPs, and free antibiotics (CIP, RMP) were used to plot the dynamic curve against clinical and susceptible strain to antimicrobial compounds (SI and ATCC-25923). After obtaining a fresh bacterial culture in Mueller Hinton Broth (MHB), the bacterial cells were harvested by centrifugation (15,000 rpm/10 min), and collected cells were washed twice with distilled water (dH2O) and the bacterial inoculums were adjusted to 10⁶ CFU/mL. 100 µL of MHB medium was poured into each well and 100 µL of antimicrobial compounds was poured into the wells (½ MICs). Next, 100 µL of cell suspension was poured into each well and the plates were incubated at 37°C, and the OD was measured at 600 nm using an ELISA plate reader (ELx808™, BioTek®) in an 18─h period with an interval of 60 min. Positive and negative controls were incubated with medium and bacteria inoculation without antimicrobial agents and only with culture medium, respectively.

Screening of biofilm-producing strains

The described tube method (TM) by Thilakavathy et al., (2015) was used to detection of biofilm formation with a few modifications. Briefly, fresh bacterial culture was performed and a 0.5 McFarland standard was developed for all strains. Then, 100 µL of bacterial suspension was added to each tube containing 5 mL of trypticase soy broth (TSB), and the tubes were incubated at 37°C for 24 h. The bacterial suspension was then discarded and tubes were washed twice using distilled water and were stained with crystal violet (1%) for 10 min at room temperature (25°C) and washed with distilled water several times (4─5) and then were inverted to dry. Biofilm formation in the strains was considered positive when the wall and bottom of the tube were covered by a visible film. The tube containing medium without bacterial induction was considered negative control. Biofilm formation in the wall and bottom of the tube was considered positive by an observable film and the amount of biofilm formed was rated as none/weak (o, I), medium (II), and strong (III). Subsequently, antimicrobial compounds were used at ½ MICs in the test tube to evaluate the anti-biofilm activity of Cs/CaAlg-CIP, and Cs/CaAlg-RMP nanoparticles compared to free antibiotics (CIP and RMP) according to the above instructions. Tubes containing medium & bacterial inoculation without antimicrobial compounds were considered positive control. Biofilm formation and potential anti-biofilm effects in bacterial strains are shown in Fig. 6.

Biofilm formation assay in 96-well microtiter plate

Anti-biofilm activity of nanoparticles was performed based on our previous work by the crystal violet method in a polystyrene 96-well microtiter plate (Pourebrahim et al., 2021). The clinical strain (SI) that showed the highest resistance to antibiotics and biofilm production in the tube test method (TM) was exposed to antimicrobial compounds. Briefly, fresh bacterial culture was performed in Trypticase soy broth (TSB) and the 0.5 MacFarland standard (OD₆₀₀) was established and the bacterial inoculums were adjusted to 10⁶ CFU/mL. 100 µL of the bacterial inoculums was then poured into wells previously diluted with sub-MICs of antimicrobial compounds (CON, ¹/₈, ¼, and ½ MICs), and the plates were incubated at 37°C for 24 h. The free-flowing bacteria in each well were removed and the wells were washed three times with distilled water and allowed to dry at room temperature for 10 min. The wells were then stained with 125 µL crystal violet (0.1%) for 15 min at room temperature (25°C). The dye was removed, and the wells were washed immediately with distilled water. To fixation, 95% ethanol was added to each well and incubated for 10 min. Finally, the contents of each well (crystal violet/ethanol) were transferred to a separate well on a 96-well plate and the reaction mixture was read at OD₅₉₅ nm using an ELISA reader (ELx808™ Absorbance Microplate Reader, BioTek®). The bacterial adhesion index to the wells was considered as follows: Mean OD values →Biofilm production.

< 0.120 Non/Weak

0.120─0.240 Moderate

& >0.240 High

Disk-diffusion method

Disk-diffusion method was used to calculate the potent antimicrobial activity of Cs/CaAlg-CIP, and Cs/CaAlg-RMP nanoparticles in comparison with free antibiotics against resistant strain (SI) and ATCC-25923 as a control sample. Initially, a fresh culture of bacteria was performed and bacterial cells were suspended in PBS, (1.5×10⁶ CFU/mL). The bacterial suspension was uniformly inoculated into a prepared MHA plate (4 mm depth) using a sterile swap. Antimicrobial compounds with ¹/₈, ¹/_4, and ¹/₂ MICs were then added to each 6 mm diameter filter-paper disc. Finally, plates were incubated at 37°C for 24 h, and a clear inhibition zone (diameter, mm) was evaluated around the discs. Antibiotics (CIP 5µg/disc; RMP 5µg/disc) and PBS (10%) were used as positive and negative controls, respectively.

Statistical Analysis

GraphPad Prism 8.0 was used to analyze the data and differences between Cs-TPP NPs, Cs/CaAlg-CIP, Cs/CaAlg-RMP nanoparticles as well as free antibiotics (treated), and the control sample was analyzed using a t-test. P-values of < 0.05 were considered statistically significant. Bacterial growth curves against antimicrobial agents were determined by plotting the log CFU/mL versus time. Triplicate experiments were performed for each reaction.

Characterization of nanoparticles

The FT-IR spectrum shows a broad bond at 3433 cm^─1 which is attributed to the asymmetric and symmetric stretching bonds of NH₂ groups which are overlapped with O-H groups (Fig. 2, a). The first vibration of NH₂ groups can be observed at 1642 cm^─1 and the bond at 1567 cm^─1 may be attributed to the asymmetric fixation of NH₃⁺ groups (Fig. 2, a). The vibration of the PO₄^3─ groups is commonly visible between 1100 − 940 cm^─1 and there is a bond at 1074 cm^─1 that could be attributed to the PO₄^3─ groups.

Sodium alginate contains hydroxyl and carboxylate groups (Wang et al., 2018). The bond at 3443 cm^─1 belongs to the sheet change vibration of O-H groups (Fig. 2, b). A bond at 1623 cm^─1 is assigned to asymmetric stretching at CO₂^─ and symmetric stretching can be seen at 1418 cm^─1. A cyclic-etheric band (medium band) is substituted at 1032 cm^─1 (Fig. 2, b). Ca²⁺ ions replacing Na⁺ ions can only split the carboxylate bond to a weaker number. There is also a weak bond at 1725 cm^─1 indicating that some carboxylate groups are protonated and converted to carboxylic acid groups.

The FT-IR spectrum of chitosan-TPP nanoparticles in combination with CaAlg-rifampicin (Cs/CaAlg-RMP) shows a suitable formula between these two components (Fig. 2, d). The CH₃ state change bond of rifampicin closely can be observed at 2927 cm^─1 and ester groups (R-CO-OR) are observed at 1727 cm^─1 (Fig. 2, d). The carbonyl and amide groups are seen at 1628 cm^─1 (Fig. 2, d).

In the case of Cs/CaAlg-CIP, there is a number of free carboxylic acid (R − CO₂H) groups. The sharp bond at 1728 cm^─1 can be assigned to those groups (Fig. 2, c). However, there is a synergistic effect between the carboxylic and carbonyl groups of Cs/CaAlg-CIP, which leads to a strong and large bond at 1728 cm^─1 (Fig. 2, c). All these correlations indicate that CaAlg-CIP is covered with chitosan-TPP.

The scanning electron microscope (SEM) technique was used to evaluate the surface morphology of the pure chitosan, chitosan-TPP, and Cs/CaAlg-CIP as well as Cs/CaAlg-RMP nanoparticles. SEM of the Cs/CaAlg-CIP and Cs/CaAlg-RMP nanoparticles showed that they are approximately spherical and granular in shape with an average size of about 100–500 nm (Fig. 3: e, f and g, h). SEM results also showed that CaAlg-CIP and CaAlg-RMP nanoparticles were covered by chitosan-TPP.

Antimicrobial resistance patterns

The results of the primary screening of bacteria for different antibiotics including ciprofloxacin and rifampin are shown in Table 1. According to the results, out of 12 strains exposed to ciprofloxacin 8.0 clinical strains showed strong resistance to the antibiotic, and the frequency of susceptible and intermediated strains was 3.0 and 1.0, respectively. The frequency of strains resistant to rifampicin was observed as 6.0 resistant, 6.0 susceptible, and zero intermediate strains.

Disk-diffusion method for the determination of the antibacterial activity of nanoparticles

Disk-diffusion also confirmed the inhibitory effect of nano-drug formulation, even at low concentrations (¹/₈ MIC), more antibacterial activity than free antibiotics was observed in the tested strain (SI) Fig. 4. After 24 h of incubation of strain SI in the presence of Cs/CaAlg-RMP nanoparticles, zone of inhibition (ZOI) with diameters of 19, 23, and 29 mm were observed compared to free RMP with ZOI of 15 mm, and the results show an inhibition zone of ~ 14% larger than of free RMP. However, there was no inhibition of bacterial growth by PBS. The antibiotic CIP at a concentration of 5µg/disc displayed an inhibition zone of 11 mm, compared to Cs/CaAlg-CIP reaching 10, 13, and 19 mm (¹/₈, ¹/₄, and ¹/₂ MIC), showing an inhibition zone ~ 8% larger than free CIP. The results showed that the inhibitory effect of nanoparticles depends on the concentration and with increasing concentration, more inhibition is applied. The results of the disc-diffusion showed that antibiotics loaded in Cs-CaAlg structure have more antibacterial activity than free antibiotics and, this increase in inhibition was observed by about ~ 14% and ~ 8%, compared free RMP and CIP, respectively. In fact, chitosan/alginate nanoparticles act as a scaffold and release the loaded drugs around the disc, and thus nano-drugs create an inhibitory zone by electrostatically binding to the bacterial cell wall and passing through the plasma membrane, and acting on the target site. Meanwhile, the highest antimicrobial activity was observed near the discs containing nano-drugs, and, naturally, the lowest amount of antimicrobial activity was observed at the end of the inhibitory areas, which could be due to the dilution of the drugs relative to the center.

The minimum inhibitory and minimum bactericidal concentration of nanoparticles

The potent antibacterial activity of antibiotic (s) loaded into the chitosan-alginate backbone compared to free antibiotic (s) and Cs NPs is shown in Table 1. According to the results, compared to free RMP, treated strains with Cs/CaAlg-RMP nanoparticles reduced bacterial growth by about 7-folds in isolated strains from blood samples, 4-folds in urine, 10-folds in the skin, and 8-folds in cyst isolates. In this regard, samples treated with Cs/CaAlg-CIP nanoparticles inhibited bacterial growth approximately by 7-folds in blood samples, 8-folds in the urine samples, 11-folds in the skin, and 8-folds in cyst isolated strains compared to free ciprofloxacin. Overall, the results showed a 6.9-folds reduction in the growth of clinical strains. MICs values lower than 1–2 µg.mL^─1 were also observed in the ATCC strain in the presence of Cs/CaAlg-RMP and Cs/CaAlg-CIP compared to free RMP (8.0 µg.mL^─1) and CIP (16 µg.mL^─1), respectively. Indeed, the loaded nanoparticles reduced bacterial growth by several times compared to free antibiotics and chitosan nanoparticles, even at low concentrations (2.0 µg.mL^─1), loaded nanoparticles inhibited the growth of drug-resistant strains (blood, skin samples) Table 1.

A time-kill kinetics assay in the presence of antimicrobial compounds

The growth curve of bacteria against antimicrobial compounds was drawn and antibiotic (s) loaded with chitosan-alginate structure showed a more lasting antimicrobial effect than free antibiotics. As shown in Fig. 5, the effect of antimicrobial compounds after incubation failed to inhibit the growth of bacteria at the indicated time, so after 3─4 h we observed gradual growth in both clinical (SI) and standard strains. However, nano-drugs with unique formulations were able to inhibit bacterial growth at a constant rate of up to 12 h after incubation. The growth rate of the clinical strain (SI) after 12 h of incubation was observed as OD 1.41 for the control sample, CIP, OD 1.075; RMP, OD 0.961; Cs/CaAlg-CIP, OD 0.298 and, Cs/CaAlg-RMP, OD 0.225. The Growth rate for the standard sample (ATCC) was observed as OD 1.42 for the untreated sample, CIP, OD 0.744; RMP, OD 0.382; Cs/CaAlg-CIP, OD 0.201 and Cs/CaAlg-RMP, OD 0.135. In fact, free antibiotics can take effect shortly after incubation and lose their antibacterial activity over time, so the bacteria can return to their natural cycle. Meanwhile, CIP and RMP loaded in the alginate-chitosan scaffold with a unique formulation can inhibit bacterial growth in a period of 18─24 h.

Anti-biofilm activity of Nano-drug formulations

The biofilm formation capability of the isolated strains is shown in Fig. 6A. All clinical strains produced biofilm, so the highest level of biofilm production was observed in SI, SII and, SIII as well as SCI and, OCI strain with the optical density of 1.013, 0.960, 0.724, 0.743, and 0.712, respectively. The optical density of the strains isolated from the blood sample was observed as, BI OD _0.670, BII OD _0.624, BIII OD _0.573, BIV OD _0.582, BV OD _0.654, and, BVI OD _0.712, respectively. To evaluate the biological activity of Cs/CaAlg-CIP and Cs/CaAlg-RMP during biofilm formation, the capacity of biofilm formation in the presence of sub-MICs of nano-drugs was compared to free antibiotics. As shown in Fig. 6. The ½ MICs of Cs/CaAlg-CIP and Cs/CaAlg-RMP significantly induced biofilm formation compared to free antibiotics. The potent biological activity of nanoparticles compared to free drugs was also investigated in a 96-well polystyrene plate against the clinical strain (SI strain) and the result showed that antimicrobial activity was concentration-dependent and with increasing concentration, more biological activity was observed (Fig. 7). For example, rifampin loaded into the chitosan-alginate structure inhibited ~ 76.4, ~ 154.6, and ~ 287.6% of bacterial growth and thus biofilm formation at sub-MICs (¹/₈, ¹/₄, and ¹/₂ MICs) compared to free drug. Regarding the antimicrobial effect of loaded ciprofloxacin, it inhibited bacterial growth by ~ 35.7, ~ 85.3, and ~ 136.3% compared to the free drug, respectively, (Fig. 7). Overall, compared to ciprofloxacin, rifampin exerted a greater antibacterial effect on biofilm formation even at ¼ MICs.

This study was conducted to determine the antibacterial and anti-biofilm properties of alginate-antibiotic (s) coated with chitosan-TPP nanoparticles. For this purpose, three goals have been pursued: I) synthesis of chitosan nanoparticles by ionotropic gelation of chitosan with tripolyphosphate (TPP), II) loading of ciprofloxacin and rifampicin antibiotics separately in the alginate structure in the presence of CaCl₂ ions and III) coating of alginate-antibiotic complex (Cs/CaAlg-CIP; Cs/CaAlg-RMP) with chitosan-TPP nanoparticles. The hypothesis was that by loading antibiotics into CaAlg scaffolds and covering them with chitosan nanoparticles, the antibacterial properties of antibiotics would increase and antibiotics could be available and more effective at the target sites.

The formulation of polymeric nanoparticles such as chitosan brings advantages such as low toxicity, biocompatibility, incorporation of both lipophilic and hydrophilic drugs, and the ability to release the loaded drug with a controlled rate and sustained release, thereby reducing its side effects (Herdiana et al., 2021). The desire for controlled drug release has made chitosan nanoparticles a promising carrier in therapeutic applications (Herdiana et al., 2021). In this regard, one of the most important features that give chitosan such a special place compared to other carriers is the reduction of toxicity, stability, and improvement of drug solubility. Mucoadhesion properties of chitosan and alginate increase the residence time in the target areas, increase the absorption of antibiotics and reduce the frequency of dosing (Costa et al., 2017; Sarkar et al., 2017).

The reduction of bacterial growth can be considered as a result of the synergistic formulation of nanoparticles (Cs/CaAlg-drug). In this regard, studies have reported that microspheres obtained from the combination of these two natural polymers (chitosan & alginate) have more physical stability than nanoparticles prepared with synthetic polymer (Scolari et al., 2020). The anionic property of alginate with carboxylic groups classified it as a good adhesive agent in pharmaceutical applications. Therefore, the inherent adhesion of alginate particles to the mucous tissue can be considered one of its important features in drug loading to the target cell (George and Abraham., 2006 and Adrian Martău et al., 2019).

The exact mechanisms of chitosan nanoparticles against bacteria are still unknown. However, small Cs NPs are able to cross physical barriers such as cell walls and plasma membrane and act on their target sites (Ali and Ahmad. 2018). According to the results obtained in this study, the fabricated nanoparticles are in the Nano-size range, which enables them to easily pass through the physical barriers of the cell membranes. The electrostatic interaction between anions such as N-acetylmuramic acid, sialic acid, and neuraminic acid, present in the bacterial cell wall and/or the cell membrane and cationic amino group of chitosan nanoparticles is considered an important hypothesis regarding the antibacterial effect (Strand et al., 2001). Hence, the electrostatic interaction (EIs) between the amino groups of Cs NPs (δ⁺) with the cell membranes (δ^─) of bacteria was demonstrated as a model of antimicrobial effect (Fig. 8). This interaction causes joint changes at the cell surface and leads to changes in membrane permeability, which in turn stimulates osmotic imbalance and excretion of intracellular substances, leading to cell death (Chandrasekaran et al., 2020). With an electrostatic bond between chitosan nanoparticles and bacterial cell wall, this interaction causes active accumulation in the interaction area and causes the permeability of the plasma membrane. Therefore, nanoparticles with low molecular weight are able to affect intracellular organelles without a physical barrier, the most important of which is the effect of nanoparticles on DNA, proteins, and key enzymes in cell replication machinery (Birsoy et al., 2015). The chelating capacity of chitosan towards metal ions such as Ca²⁺,Zn²⁺,Ni²⁺,Mg²⁺ and, Fe²⁺ is another important feature of chitosan nanoparticles, enables it to enclose metal ions and disrupt the flow of vital nutrients for bacterial growth by forming a metal complex (Ma et al., 2017).

Considering the anti-biofilm effects of chitosan nanoparticles, it is assumed that the attachment of Cs NPs to the surface of the biofilm matrix allows penetration into the polysaccharide matrix. In fact, polymer nanoparticles such as chitosan are capable of delivering drugs to the biofilm matrix (Tan et al., 2018). Positively charged chitosan nanoparticles can bind to negatively charged compounds in the biofilm matrix, the most important of which are polysaccharide intercellular adhesin (PIA), extracellular-DNA (eDNA), proteins, and amyloid fibrils (Tan et al., 2018; Arciola et al., 2015). In addition, the structure of chitosan allows it to facilitate drug delivery to the cell and the target site after binding to the surface of the biofilm matrix (Tan et al., 2018).

Chitosan has been used as a carrier molecule for the controlled release of several drug formulations, which provides higher stability, lower toxicity, and prolonged antibacterial activity of the drugs (Khan et al., 2020).

In a similar work, it was observed that chitosan nanoparticles could facilitate the diffusion of oxacillin into the S. aureus biofilm matrix and efficiently inhibit bacterial biofilm (Tan et al., 2018). The combination of two drugs in which one disrupts the biofilm matrix through anti-biofilm and anti-quorum sensing activities to facilitate the penetration of other drugs is a recent antibacterial chemotherapy strategy. It was found that immobilization of antibiotics to the chitosan nanoparticles is a very efficient approach to inhibiting bacterial biofilm, followed by the complete eradication of bacterial cells in a synergic way (Khan et al., 2020). In addition to biofilm formation, many virulence determinants are controlled by bacterial quorum sensing systems. Some previous studies revealed that, due to the anti-quorum sensing activity of chitosan nanoparticles, several virulent features of pathogenic bacteria, including biofilm formation, drug resistance, and extracellular toxins are prohibited. Moreover, biofilm formation is a critical factor that is strongly associated with drug resistance. Therefore, synergism between chitosan nanoparticles and antibiotics could provide efficient antibacterial chemotherapy (Jamil et al., 2016; Zhang et al., 2013). The drug complex formed between the CaAlg-antibiotic (s) and the Cs-TPP nanoparticles enables antibiotics to penetrate into the bacterial cell when the physical barriers such as cell walls and plasma membranes are destroyed in contact with Cs nanoparticles. In such a case, the antibiotics loaded in the CaAlg-chitosan scaffold can be released into the cytoplasm and negatively affect the target site (Pourebrahim et al., 2021).

The antibiotics used in this study were broad-spectrum antibiotics, and each inhibits bacteria with an independent strategy. Ciprofloxacin acts by inhibiting type II topoisomerase (DNA gyrase) and topoisomerase IV, and rifampicin inhibits bacterial DNA-dependent RNA synthesis by inhibiting bacterial DNA-dependent RNA polymerase (RNAP), respectively, (Campion et al., 2004; Wang et al 2019; Zhou et al., 2012). Considering the resistance of isolated strains to antimicrobial compounds such as ciprofloxacin and rifampicin in the initial screening of microbial activity in our study, it can be considered that since the target sites of the ciprofloxacin and rifampicin are DNA gyrase and DNA-dependent RNA polymerase, respectively, (Drlica and Zhao 1997; Calvori et al., 1965), they must penetrate into the cell, while the bacterium develops processes that inactivate the antibiotics, the most important of which are changes in the target site by mutation and enzymatic degradation of antibacterial compounds (Egorov et al., 2018). Meanwhile, the antibiotics encapsulated in the alginate structure are protected from the activities of various bacterial enzymes and can exert their antimicrobial activity more effectively.

In this study, an ionic gelation method was developed to generate stable chitosan-based nanoparticles. Covering alginate-antibiotic (s) complex with chitosan NPs in our study exerted more antibacterial and anti-biofilm activity than free antibiotics (CIP, RMP) against clinical strains of S. aureus. Even at a low concentration (¹/₄, ½ MICs) of Cs/CaAlg-CIP and Cs/CaAlg-RMP, bacterial growth was inhibited and thus no biofilm was formed. MICs showed a several-fold reduction in bacterial growth compared to the free antibiotic. This antibacterial effect may have originated from the alginate-antibiotic (s) complex and chitosan NPs, which kept the drug away from the destructive activities of the certain enzyme, and naturally, the antibiotic could have acted more effectively on the site of its effect. Hence, the antibiotic-alginate complex coated with the chitosan NPs in this study can be a good candidate against S. aureus bacteria, which are widely resistant to ciprofloxacin and rifampicin. Further studies can confirm the antimicrobial and anti-biofilm properties against the phenotype at the genotypic level.

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Competing interests:

Authors express no conflict of interest.

Data availability:

The data produced in this research are ready by the corresponding author on request.

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Not applicable.

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Author Contribution:

A.S. and A.N. :Conceptualization. A.S. and A.N. :Methodology. A.S. and F.R.M.:Formal analysis and investigation. A.S. and S.D.M.: Writing Original Draft Preparation. S.D.M and F.R.M. .:Resources. A.S. and A.N.::Supervision

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Effective release of Ciprofloxacin and Rifampicin antibiotics from alginate-chitosan complex and its application against clinical strains of Staphylococcus aureus

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Abstract

Figures

Introduction

Materials and methods

Reagents

Preparation of chitosan nanoparticles

Preparation of antibiotics-loaded alginate and formation of an alginate-antibiotics complex with chitosan-TPP nanoparticles

Characterization of the nanoparticles

Antimicrobial susceptibility testing (AST)

Minimum inhibitory concentration (MICs) & minimum bactericidal concentration (MBCs) determination

Time-dependent killing

Screening of biofilm-producing strains

Biofilm formation assay in 96-well microtiter plate

Disk-diffusion method

Statistical Analysis

Results

Characterization of nanoparticles

Antimicrobial resistance patterns

Disk-diffusion method for the determination of the antibacterial activity of nanoparticles

The minimum inhibitory and minimum bactericidal concentration of nanoparticles

A time-kill kinetics assay in the presence of antimicrobial compounds

Anti-biofilm activity of Nano-drug formulations

Discussion

Conclusion

Declarations

References

Additional Declarations

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