Antibacterial activity of chitosan-based nanohybrid membranes against drug-resistant bacterial isolates from burn wound infections

Biocompatible and non-toxic properties of chitosan make it a candidate with excellent application prospects in developing wound dressing conjugate compounds. Six different chitosan-based nanohybrid membranes were evaluated against multidrug-resistant bacterial isolates. Different combinations of chitosan, ciprofloxacin (CIP), biofunctionalized montmorillonite (MMT), and montmorillonite with sulfate chains (SMMT) were provided, and their antibacterial activity was assessed using the colony count method. Totally, 27 drug-resistant isolates, including 6x methicillin-resistant Staphylococcus aureus, 7 vancomycin-resistant Enterococcus faecalis, 4 Acinetobacterbaumannii, and 10 Pseudomonas aeruginosa isolates were identified from burn wound infections. Chitosan and montmorillonite did not show significant antibacterial effect (p > 0.05), but chitosan/SMMT/CIP was the most effective nanocomposite (p < 0.01). Chitosan-based nanocomposites with ciprofloxacin could effectively reduce the susceptibility of drug-resistant bacterial isolates. Bacterial targeting using nanosystems provides an opportunity for effective antibiotic treatment by improving antibacterial efficacy.


Introduction
Burn patients comprise a large population that faces lifethreatening conditions around the world (Mock et al. 2008) Loss of the natural cutaneous barrier, immune system dysregulation, and prolonged hospitalization predispose the patients to bacterial colonization and infection. Spread of the infection throughout the body and subsequent complications as bacteriemia, sepsis, and organ dysfunction result in an increased risk of patient death (Lachiewicz et al. 2017). Antimicrobial resistance (AMR) has emerged as one of the most serious global public health threats of the twenty-first century (Prestinaci et al. 2015) and the challenging issue in burn wound infection control is the increasing resistance to antibiotics (Lari et al. 2005;van Langeveld et al. 2017). Multidrug-resistant organisms (MDROs) have been frequently reported from different countries, including Pseudomonas aeruginosa, Acinetobacter baumannii, and Staphylococcus aureus, which are resistant to several classes of antimicrobial agents and increase the risk of antibiotic treatment failure (Estahbanati et al. 2002;Lachiewicz et al. 2017;Lari et al. 2005;Heidari et al. 2016).
To overcome the drug resistance, antimicrobial biomaterials have been introduced recently (Hall et al. 2020).
Pouya Amiri and Jalil Kardan-Yamchi contributed equally to this work.
In vitro and in vivo antimicrobial activity of chitosan, a polycationic biopolymer, has been demonstrated previously (Rashki et al. 2020). The biocompatible, biodegradable, nonantigenic, and non-toxic properties placed chitosan as an essential target at the Siebel Center for antibacterial research (Pangestuti and Kim 2010). The antimicrobial activity of this natural polysaccharide was investigated in different formulations with different combinations, and wound dressing application of chitosan nanohybrids was proposed (Kravanja et al. 2019;Qin and Li 2020).
Therefore, the antibacterial activity of montmorillonite (MMT) and ciprofloxacin-conjugated chitosan membranes against multidrug-resistant (MDR) isolates of S. aureus, Enterococcus faecalis, P. aeruginosa, and A. baumannii from burn wound infections was evaluated in this study.

Susceptibility testing of nanohybrid membranes
Susceptibility testing of all nanohybrids was assessed using the colony counting method. Briefly, a 0.5 McFarland suspension of all bacterial strains was prepared from new colonies. One mL of a 1:20 dilution of 0.5 McFarland suspension was added to 9 mL of MHB having 5 mg of each membrane and put in a shaker incubator at 35 °C/24 h/100 rpm. Then, 100 µL of serially diluted culture suspensions was spread over the surface of MHA, and bacterial colonies were counted after incubation at 35 °C for 24 h. At least one log reduction of bacteria load was considered an antibacterial activity of the nanohybrid membranes (Pinto et al. 2012).

Statistical analysis
The mean differences in the growth of S. aureus, E. faecalis, A. baumannii, and P. aeruginosa isolates against the initial inoculation in the presence of chitosan, chitosan/MMT, chitosan/SMMT, chitosan/CIP, chitosan/MMT/CIP, and chitosan/SMMT/CIP nanohybrid membranes were analyzed by T test using SPSS statistical software version 20.

Antibiotic resistance pattern
The distribution of the isolates was as follows: six methicillin-resistant S. aureus (MRSA), seven vancomycin-resistant E. faecalis (VRE), four A. baumannii, and ten P. aeruginosa multidrug-resistant (MDR), and were tested. Ciprofloxacin resistance of S. aureus, E. faecalis, A. baumannii, and P. aeruginosa was found in two, two, two, and four isolates, respectively. The resistance pattern of the isolates is shown in Table 1.

Antibacterial activity of nanohybrid membranes
Pure chitosan showed a bacteriostatic effect on the growth of all the testing Gram-positive and Gram-negative isolates, but this was not significant (< 1 log reduction, p > 0.05). Four nanohybrid membranes, including chitosan/SMMT, chitosan/CIP, chitosan/MMT/CIP, and chitosan/SMMT/ CIP, had a significant effect against the testing isolates. E. faecalis, S. aureus, and P. aeruginosa isolates were the most affected bacteria with significant growth reduction (p < 0.01) in the presence of nanohybrid membranes except for pure chitosan. However, despite > 1 log growth reduction of A. baumannii isolates in the presence of different nanohybrid films, mean differences in these isolate's growth were not statistically significant when chitosan/MMT chitosan/CIP    Figure 1 shows the log reduction of six different nanohybrid membranes against Gram-positive and Gramnegative bacterial isolates.

Discussion
Nano-based drug delivery to targeting bacteria has apparent advantages over overexposing bacteria to higher drug concentration and enhancing existing antibiotics' ability to treat infections (Yeh et al. 2020). Chitosan with polycationic properties attacks the negatively charged bacterial cell wall to demonstrate its antimicrobial activity, leading to reduced susceptibility and an effective combination with available antibiotics (Rogers et al. 2012). Herein, the antibacterial efficacy of different chitosan nanohybrid membranes was evaluated and a nanocomposite of the chitosan in combination with SMMT and ciprofloxacin was found as the most antibacterial agent against multidrug bacterial isolates. Pure chitosan showed bacteriostatic effects by inhibiting the growth of both Gram-negative and Gram-positive bacterial isolates tested, which agrees with previous experiments (Sobhani et al. 2017). Also, MMT did not significantly increase the antibacterial activity in the chitosan biocomponent structure. Sandri G et al. investigated the antibacterial activity of montmorillonite-chitosan-silver sulfadiazine nanocomposites against standard strains of S. aureus, Streptococcus pyogenes, Escherichia coli, and P. aeruginosa (Sandri et al. 2014). Our experiment indicated that neither montmorillonite nor chitosan itself possesses significant antibacterial activity against targeting strains. However, SMMT showed better results in improving the antibacterial effect of the chitosan-based nanohybrids. Thus, better results were obtained when chitosan/SMMT was used with ciprofloxacin antibiotics.
Among the bacterial species considered, some ciprofloxacin-resistant/-intermediate susceptible isolates became susceptible in the presence of chitosan/SMMT/ CIP. This observation is a clear benefit of nano-based composites in increasing the efficacy of antibiotic therapy. Sobhani Z et al. showed that ciprofloxacin-loaded chitosan nanoparticles had 50% lower MICs than the ciprofloxacin hydrochloride alone in standard strains of E. coli and S. aureus (Sobhani et al. 2017). Furthermore, four and two times decreased MICs of P. aeruginosa and Klebsiella. Pneumoniae species were reported by Farhangi M et al. by chitosan-ciprofloxacin conjugates against the free drug (Farhangi et al. 2018).
Burn wounds can be quickly colonized by different bacterial species and are at risk for developing secondary sepsis to pneumonia; then, an urgent management is needed (Bowler et al. 2012). Moreover, co-infection and co-existing within a biofilm community are often common in burn wounds, resulting in complicated conditions and limited therapeutic options (Church et al. 2006). Promising results of chitosan-based nanocomposites with a broadspectrum antibiotic, ciprofloxacin, were obtained against different common bacterial pathogens of burn wounds in the present study. In addition, the anti-biofilm activity of chitosan conjugates has been successfully investigated previously (Regiel et al. 2012).
In conclusion, chitosan nanohybrids showed satisfying improvement in drug susceptibility of Gram-negative/positive multidrug-resistant bacterial isolates. Despite the low antibacterial activity of chitosan and MMT, nanoparticle combinations of chitosan/SMM/CIP significantly inhibited the growth of four bacterial species tested. The biocompatible and non-toxic nature of chitosan and the obtained antibacterial activity of nanohybrid membranes converge to propose especially that chitosan/SMM/CIP has a wound-healing potential.
Funding This project was supported by Lorestan University of Medical Sciences (Project No. 391).