Microbial glycolipoprotein-capped silver nanoparticles as emerging antibacterial agents against cholera

Abstract

Background: With the increased number of cholera outbreaks and emergence of multidrug resistance in Vibrio cholerae strains it has become necessary for the scientific community to devise and develop novel therapeutic approaches against cholera. Recent studies have indicated plausibility of therapeutic application of metal nano-materials. Among these, silver nanoparticles (AgNPs) have emerged as a potential antimicrobial agent to combat infectious diseases. At present nanoparticles are mostly produced using physical or chemical techniques which are toxic and hazardous. Thus exploitation of microbial systems could be a green eco-friendly approach for the synthesis of nanoparticles having similar or even better antimicrobial activity and biocompatibility. Hence, it would be worth to explore the possibility of utilization of microbial silver nanoparticles and their conjugates as potential novel therapeutic agent against infectious diseases like cholera.

Results: The present study attempted utilization of Ochrobactrum rhizosphaerae for the production of AgNPs and focused on investigating their role as antimicrobial agents against cholera. Later the exopolymer, purified from the culture supernatant, was used for the synthesis of spherical shaped AgNPs of around 10 nm size. Further the exopolymer was characterized as glycolipoprotein (GLP). Antibacterial activity of the novel GLP-AgNPs conjugate was evaluated by minimum inhibitory concentration, XTT reduction assay, scanning electron microscopy (SEM) and growth curve analysis. SEM studies revealed that AgNPs treatment resulted in intracellular contents leakage and cell lysis.

Conclusion: The potential of microbially synthesized nanoparticles, as novel therapeutic agents, is still relatively less explored. In fact, the present study first time demonstrated that a glycolipoprotein secreted by the O. rhizosphaerae strain can be exploited for production of AgNPs which can further be employed to treat infectious diseases. Although this type of polymer has been obtained earlier from marine fungi and bacteria, none of these reports have studied the role of this polymer in AgNPs synthesis and its application in cholera therapy. Interestingly, the microbial GLP-capped AgNPs exhibited antibacterial activity against V. cholerae comparable to ciprofloxacin. Thus the present study may open up new avenues for development of novel therapeutic agents for treatment of infectious diseases. Graphical abstract Development of novel therapeutic agents for treatment of cholera.

Graphical abstract

Development of novel therapeutic agents for treatment of cholera

Fig. 1

Neighbour-joining tree based on 16S rRNA gene sequences showing relationships of isolate ARC-61 with closely related species of the genus Ochrobactrum. Bar 0.01 indicates substitutions per nucleotide position. Bradyrhizobium japonicum (ATCC 10324^T) was used as an out-group. Bootstrap values (>70 %) based on 100 re-sampled datasets are shown at branch nodes

Fig. 2

a Synthesis of AgNPs by culture broth at 37 °C. Change in color of AgNO₃ solution from colorless to dark brown after 24 h. b UV–visible spectra of AgNPs synthesized via supernatant (SN). c TEM micrograph of SN–AgNPs. Scale bars correspond to 50 nm. d DLS histogram of SN–AgNPs indicates size distribution by number

Fig. 3

DPPH radical scavenging activity of GLP polymer. Each value represents the average of three experiments. Error bars represent standard deviation

Fig. 4

a Characteristic surface plasmon resonance (SPR) of AgNPs synthesized via GLP. b TEM micrograph of GLP-AgNPs. Scale bars correspond to 50 nm. c DLS histogram of GLP–AgNPs indicates size distribution by number

Fig. 5

EDX spectrum of AgNPs stabilized by GLP molecule

Fig. 6

FT-IR spectra of a GLP only and b GL–AgNPs

Fig. 7

a Zone of inhibition of AgNPs against Vibrio cholerae 1. (SN–AgNPs, 2. Supernatant, Silver nitrate (AgNO₃), GLP–AgNPs). b Microtitre plate demonstrates MIC value (no color change in first 7 wells) using XTT assay. Lane B–D—GLPAgNPS; Lane E–G—SNAgNPs; Lane A and H—LB broth (Blank) c Antibacterial activity of GLP–AgNPs and SN–AgNPs against V. cholerae and MIC values for growth as determined by XTT-reduction assay. d Growth kinetics of V. cholerae in LB (negative control), with GLP and SN alone, with ciprofloxacin (positive control), with GLP–AgNPs and SN–AgNPs at two-fold and four-fold lower concentration than MICs

Fig. 8

SEM images after 5 h incubation a Untreated V. cholerae cells b GLP–AgNPs treated cells (at two-fold lower concentration than MIC values) c GLP–AgNPs treated cells (at MIC value). Arrows indicate cell lysis