A Novel Approach for Combating Klebsiella pneumoniae Biofilm Using Histidine Functionalized Silver Nanoparticles

Abstract

Treating pathogens is becoming challenging because of multidrug resistance and availability of limited alternative therapies which has further confounded this problem. The situation becomes more alarming when multidrug resistant pathogens form a 3D structure known as biofilm. Biofilms are formed in most of the infections especially in chronic infections where it is difficult to eradicate them by conventional antibiotic therapy. Chemically synthesized nanoparticles are known to have antibiofilm activity but in the present study, an attempt was made to use amino acid functionalized silver nanoparticles alone and in combination with gentamicin to eradicate Klebsiella pneumoniae biofilm. Amino acid functionalized silver nanoparticles were not only able to disrupt biofilm in vitro but also led to the lowering of gentamicin dose when used in combination. To the best of our knowledge, this is the first study demonstrating the application of amino acid functionalized silver nanoparticles in the eradication of young and old K. pneumoniae biofilm.

Keywords: antibacterial agents; antibacterial nanoparticles; antibiofilm agents; bacterial; biofilm; drug resistance.

FIGURE 1

(A,B) Are representative pictures of Congo red agar plates in which blackening was observed confirming the EPS production by K. pneumoniae. (C,D) Show less black colonies on agar plates containing 0.9 mg per ml of H-AgNPs, decreased EPS production by K. pneumoniae.

FIGURE 2

Biofilm formation by K. pneumoniae in the wells of 96-well micro titer plate under static conditions as determined by viable count and crystal violet (OD₅₉₅). The experiment was performed in duplicate and repeated at least three times on different days. Error bars represent the mean ± of standard deviation.

FIGURE 3

Bacterial count [Log 10 (CFU/ml)] in K. pneumoniae biofilm grown for different days and treated with H-AgNPs. The experiment was performed in duplicate and repeated at least three times on different days. Bars represents standard deviation and ^∗∗ denotes P-value (P < 0.01).

FIGURE 4

FE-SEM images of bioflim of K. pneumoniae on fifth day Figure 9 (A), (B), and (C) (at × 4-25k magnification) show that bioflim forms 3D network and water channels (red arrow) along with EPS matrix.

FIGURE 5

FESEM images (A,B) H-AgNPs were seen interacting with cells of K. pneumoniae (×22k-30k magnification at 15 kV). (C–E) Different fields showing loss of biofilm integrity (× 4k magnification at 15 kV).

FIGURE 6

(A) EDS graph represents the count per second on x-axis and the kilo electron volt which it is generated is on y-axis. (B) Showing percentage of elements present in control biofilm.

FIGURE 7

(A) EDS graph represents the count second on x-axis and the kilo electron volt on which it is generated is on y-axis. (B) Showing percentage of elements present in treated biofilm.

FIGURE 8

The effect of H-AgNPs alone [fractional inhibitory concentration (FIC)], antibiotic alone (FIC), and H-AgNPs +antibiotic treatment (FIC value) on biofilm. The experiment was performed in duplicate and repeated at least three times on different days. Bars represent standard deviation, ^∗ denotes (P < 0.05) and ^∗∗ denotes (P < 0.01).

FIGURE 9

K. pneumoniae bioflim stained with LIVE/DEAD Baclight kit and treated with H-AgNPs and gentamicin, alone or in combination. The bioflim observed under CLSM (40x) shows distribution of live/dead bioflim cells on different days of incubation.

FIGURE 10

DNA fragmentation following treatment with H-AgNPs. Lane 1 shows DNA control sample of untreated bacterial cells, lane 2 DNA of bacterial pellet treated with the lowest concentration (0.45 mg per ml) of H-AgNPs, lane 3 DNA of bacterial pellet treated with H-AgNPs (0.9 mg per ml), and lane 4 contains the DNA of bacterial pellet treated with H-AgNPs (1.8 mg per ml).