A novel antibacterial approach of Cecropin-B peptide loaded on chitosan nanoparticles against MDR Klebsiella pneumoniae isolates

Abstract

Egypt has witnessed the emergence of multidrug-resistant (MDR) Klebsiella pneumoniae, which has posed a serious healthcare challenge. The proper treatment choice for MDR-KP infections is not well determined which renders the problem more complicated, thus making the control of such infections a serious challenge for healthcare professionals. This study aims to encapsulate the cationic antimicrobial peptide; Cecropin-B (Cec-B), to increase its lifetime, drug targeting, and efficacy and study the antimicrobial effect of free and encapsulated recombinant rCec-B peptide on multidrug-resistant K. pneumoniae (MDR-KP) isolates. Fifty isolates were collected from different clinical departments at Theodore Bilharz Research Institute. Minimal inhibitory concentrations (MICs) of rCec-B against MDR-KP isolates were determined by the broth microdilution test. In addition, encapsulation of rCec-B peptide into chitosan nanoparticles and studying its bactericidal effect against MDR-KP isolates were also performed. The relative expression of efflux pump and porin coding genes (ArcrB, TolC, mtdK, and Ompk35) was detected by quantitative PCR in treated MDR-KP bacterial isolates compared to untreated isolates. Out of 60 clinical MDR isolates, 50 were MDR-KP. 60% of the isolates were XDR while 40% were MDR. rCec-B were bactericidal on 21 isolates, then these isolates were subjected to treatment using free nanocapsule in addition to the encapsulated peptide. Free capsules showed a mild cytotoxic effect on MDR-KP at the highest concentration. MIC of encapsulated rCec-B was higher than the free peptide. The expression level of genes encoding efflux and porin (ArcrB, TolC, mtdK, and Ompk35) was downregulated after treatment with encapsulated rCec-B. These findings indicate that encapsulated rCec-B is a promising candidate with potent antibacterial activities against drug-resistant K. pneumoniae.

Keywords: Antibacterial; Bactericidal; Cationic antimicrobial peptide; Cecropin; Chitosan; Egypt; Klebsiella pneumoniae; Multidrug-resistant; Nanocapsule; PCR.

Fig. 1

This chromatogram represents the purification of rCec-B using the SP-FF cation exchange column. The X-axis represents time in minutes (min), the primary Y-axis represents the absorbance UV at 190 nm, the red curve represents the absorbance UV at 210 nm, the green curve represents the gradient increasing in salt concentration using elution buffer (0–100% of 1M NaCl), and the secondary Y-axis represents buffer conductivity in Sm/cm

Fig. 2

Nano-chitosan (CF)—size: 177.6 nm, PDI: 0.412, and zeta potential: + 29.2 mV

Fig. 3

Chitosan/Cec-B nanoparticles (CB)—size = 205.4 nm, PDI: 0.469, and zeta potential =  + 31.3 mV

Fig. 4

The FTIR spectra of chitosan (NCLCs), chitosan unloaded (CLCs) NPs, and chitosan/Cec-B (CLCs–CB) NPs

Fig. 5

The swelling kinetics of chitosan NPs A: effect of temperature, B effect of pH, and (C) effect of ionic strength

Fig. 6

The in vitro release profile of chitosan–rCec-B nanoparticles in PBS at pHs 2.5, 5.5, 7.4, and 8.5

Fig. 7

In vitro RBCs toxicity of free chitosan, rCec-B, and encapsulated rCec-B. (p value = 0.0002). (*) comparison between rCec-B and chitosan–rCec-B: p value < 0.05. (b) and (c) comparison between chitosan and chitosan–rCec-B: p value < 0.01 and p value < 0.05, respectively

Fig. 8

Bactericidal of rCec-B peptide MDR K. pneumoniae isolates. The fraction of the total calculation was 0.58 for nontoxic rCec-B and 0.42 for toxic rCec-B

Fig. 9

Effect of the chitosan nanocapsule at the highest concentration used (100 µg/ml) on the 21 MDR K. pneumoniae isolates. Each dot represents a different isolate. (p value < 0.0001)

Fig. 10

MIC of free and encapsulated rCec-B on the 21 MDR K. pneumoniae isolates

Fig. 11

Effect of encapsulated rCec-B on MDR K. pneumoniae isolate. (p value < 0.0001)

Fig. 12

The relative expression levels of MDR-related genes in untreated and treated K. pneumoniae isolates. (p vlaue < 0.0002), a: p vlaue < 0.0001

Fig. 13

Downregulation of ArcrB, TolC, mtdK, and Ompk35 genes’ expression as a result of treatment with encapsulated rCec-B