Development of Cefotaxime Impregnated Chitosan as Nano-antibiotics: De Novo Strategy to Combat Biofilm Forming Multi-drug Resistant Pathogens

Abstract

Frequent incidents of antibiotic-resistant biofilm forming pathogens in community-associated and hospital-acquired infections have become a global concern owing to failure of conventional therapies. Nano-antibiotics (NABs) are de novo tools to overcome the multi-drug resistant mechanisms employed by the superbugs. Inhibition of biofilm formation is one of those strategies to curb multi drug resistance phenomenon. In the current study, the anti-biofilm and antibacterial potential of newly synthesized cefotaxime loaded chitosan based NABs have been investigated. Both bare and cefotaxime loaded NABs were prepared by ionotropic gelation method. They were found carrying positive zeta potential of more than +50 mV, indicating highly stable nano-dispersion. Moreover, microscopic studies revealed their size as less than 100 nm. NABs were tested against clinical isolates of multi drug resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and methicillin resistant Staphylococcus aureus and wherein they demonstrated broad-spectrum anti-biofilm and anti-pathogenic activity. Thus, in vitro synergistic action of cephalosporin drugs and chitosan polymer at nano-scale in contrast to free antibiotics can be an improved broad-spectrum strategy to thwart resistance mechanisms in both Gram-positive and Gram-negative resistant pathogens.

Keywords: biofilm; cephalosporins; chitosan nano-carriers; drug resistance; growth kinetics; zeta potential.

FIGURE 1

Zones of Inhibitions formed on nutrient agar surface by different antibiotic disks against (A) Methicillin-resistant Staphylococcus aureus (MRSA), (B) Klebsiella pneumoniae (C) Escherichia coli displaying extended spectrum beta lactamases (ESBL) phenomenon exhibited by extension in zone of inhibition formed between augmentin and cephalosporins (D) Pseudomonas aeruginosa and (E) Listeria monocytogenes, after incubation at 37°C after 24 h.

FIGURE 2

Atomic force microscopic images of surface topography and 3-Dimensional (3D) structures of empty CSNPs (A,B) and cefotaxime loaded CSNPs (C,D) respectively. A small drop of sample was dried on glass slide and all images were taken at room temperature without any sample treatment. Results obtained depict height of 50 and 51 nm.

FIGURE 3

Scanning electron microscopy (SEM) of bare (A) and drug loaded CSNPs (B). SEM images were taken by placing a tiny droplet of sample on l × l cm glass slide and it was spreaded evenly. After gold sputtering, images were taken at ambient conditions. SEM images depicted spherical particles having diameter of less than 100 nm.

FIGURE 4

Fourier Transform Infrared Spectroscopy spectra (A) Chitosan raw material (B) Cefotaxime raw material (C) bare CSNPs (D) cefotaxime loaded CSNPs. FTIR spectrum of powders were taken after mixing raw material with KBr. Whereas liquid samples were analyzed directly by palcing a tiny drop over glass assembly. FTIR spectra indicated no new bond formation so the drug is not reacting chemically with nano-scaffolds.

FIGURE 5

Zeta potential. Measured by Malvern Zeta sizer at ambient conditions (A) empty CSNPs (B) β-lactam drug loaded CSNPs. Both empty and drug loaded CSNPs are displaying zeta potential values greater than 50 mV. It indicates highly stable colloidal dispersion.

FIGURE 6

Comparative Anti-pathogenic ability of Aqueous solution of Antibiotic and Antibiotic loaded CSNPs against pathogens (A) Methicillin-resistant Staphylococcus aureus (MRSA), (B) Escherichia coli (E. coli) (C), Klebsiella pneumoniae (KP), (D) Pseudomonas aeruginosa (Pseudo) and (E) Listeria monocytogenes (Listeria). Pathogens were exposed to Antibiotic solution (AB), blank chitosan nano-particles (CSNPs), and antibiotic loaded CSNPs (NAB). All samples were incubated at 37°C for 144 h and after every 24 h reading were taken on ELISA plate reader at 595 nm. Negative control contains only the media and no inoculum where as positive control contain inoculated media.

FIGURE 7

Colony forming unit (CFU) assay. CFU assay was performed to count the viable bacteria after reaction of CSNPs and drug loaded CSNPs with pathogens. CFU was done by plating 10μL of sample from each test tube after serial dilutions in normal saline (0.9% NaCl). All plates were then incubated for 48 h and colonies were counted manually. CSNP, Chitosan nano-particles, NAB, Nano-antibiotic; KP, Klebsiella pneumoniae; Pseudo, Pseudomonas aeruginosa and MRSA for Methicillin-resistant S. aureus.

FIGURE 8

Anti-biofilm activity of antibiotic suspension (AB) Chitosan nano-particles (CSNPs) and Cefotaxime loaded CSNPs (NAB) against pathogens. Methicillin-resistant S. aureus (MRSA), Klebsiella pneumoniae (KP), Escherichia coli (E. coli), Pseudomonas aeruginosa (Pseudo), and Listeria monocytogenes (Listeria) were incubated on ELISA plates at 37°C for 24 h. Biofilms were stained by crystal violet stain and removed by 30% acetic acid solution. This solution was then measured on ELISA plate reader at 595 nm.