Potential Therapeutic Applications of Gold Nanoparticle- Polythiophene (AuNP-PTh) Composite
4.3. Results and Discussion
Figure 4.2. Effect of combined treatment of AuNP-PTh nanocomposite and erythromycin on the growth of (a) E. coli MTCC 433 and (b) Y. enterocolitica MTCC 859.
The enhanced growth inhibition of the tested pathogenic bacteria in case of combination treatment with AuNP-PTh and erythromycin was also observed for various antibiotic concentrations (refer to Appendix, Figure A4.1). It is significant to mention here that the concentration of AuNP-PTh chosen for these experiments (15, 30 and 45 µM) was significantly lower than its MBC (112 µM). Interestingly, the MIC of erythromycin was reduced two-fold in combination with 15 µM AuNP-PTh and at the highest concentration of AuNP-PTh nanocomposite (45 µM) in combination, the reduction in MIC of erythromycin was a substantial eightfold (Table 4.1). From a therapeutic point of view, this observation is encouraging as it indicates the possibility of minimizing the dose of the antibiotic to eliminate the pathogen and thus addresses the growing concern related to excessive use of antibiotic in combating pathogenic bacteria. The ability of AuNP-PTh nanocomposite to potentiate the action of erythromycin at low concentrations highlights the therapeutic potential of the nanocomposite as an adjuvant in combination therapy.
Table 4.1. Fold reduction in MIC of erythromycin in combination with AuNP-PTh and determination of fractional inhibitory concentration (FIC) index.
Target bacteria AuNP-PTh (µM)
Folds reduction in MIC of erythromycin
FIC indexa Effectb
E. coli MTCC 433
15 30 45
2 4 8
0.6 0.5 0.5
A S S Y. enterocolitica
MTCC 859
15 30 45
2 2 8
0.3 0.5 0.5
S S S
a Fractional inhibitory concentration (FIC) index was assessed as described in section 4.2.5. b A:
Addition; S: Synergy.
The nature of interaction between AuNP-PTh nanocomposite and erythromycin was ascertained by determining the fractional inhibitory concentration (FIC) index against the target bacteria E. coli MTCC 433 and Y. enterocolitica MTCC 859. In case of E. coli MTCC 433 it was observed that in combination with increasing concentrations of AuNP- PTh nanocomposite there was a reduction in the MIC of erythromycin and the nature of the interaction was additive at lower concentration of the nanocomposite (15 µM) and synergistic at higher concentrations of the nanocomposite (30 µM and 45 µM) as indicated in Table 4.1. The reduction in MIC of erythromycin was also noted for Y. enterocolitica MTCC 859 and the interaction was synergistic at all the tested nanocomposite concentration (Table 4.1). The manifestation of a synergy effect suggested that AuNP-PTh nanocomposite perhaps initiates membrane damage in target pathogens, which in turn may augment the accessibility of the internal target for erythromycin, resulting in enhanced killing of the pathogens.
The therapeutic antibiotic polymyxin B is largely known for its potent activity against Gram-negative pathogenic bacteria while Gram-positive bacteria are mostly resistant (Zavascki et al., 2007). Based on the strong membrane-targeting activity of AuNP-PTh nanocomposite, it was construed that the nanocomposite could perhaps be explored to boost the activity of polymyxin B against Gram-positive pathogens. To test
Figure 4.3. Effect of combined treatment of AuNP-PTh nanocomposite and polymyxin B on the growth of (a) S. aureus MTCC 96 and (b) E. faecalis MTCC 439.
this premise, the bactericidal activity of polymyxin B was ascertained in combination with varying concentrations of AuNP-PTh nanocomposite. As evident from Figure 4.3, treatment of S. aureus MTCC 96 with 20 µg/mL polymyxin B could bring about only 17%
growth inhibition of the target pathogen, whereas in case of E. faecalis MTCC 439 around 34% growth inhibition was observed when treated with the same concentration of polymyxin B. However, a significant increase in the growth inhibition of the pathogens was observed when the cells were subjected to a combined treatment of AuNP-PTh nanocomposite and polymyxin B, as compared to either AuNP-PTh nanocomposite or antibiotic alone. As evident from Figure 4.3, this effect became more prominent by increasing the dose of AuNP-PTh nanocomposite in combination with 20 µg/mL of polymyxin B. Growth inhibition of the pathogen was also observed for various other concentrations of polymyxin B in combination with the nanocomposite (refer to Appendix, Figure A4.2). The nature of interaction between AuNP-PTh and polymyxin B was ascertained through fractional inhibitory concentration (FIC) index, which was
Table 4.2. Fold reduction in MIC of polymyxin B in combination with AuNP-PTh and determination of fractional inhibitory concentration (FIC) index.
Target bacteria AuNP-PTh (µM)
Folds reduction in MIC of Polymyxin B
FIC indexa Effectb
S. aureus MTCC 96
15 30 45
4 8 8
0.3 0.5 0.5
S S S E. faecalis
MTCC 439
15 30 45
2 4 4
0.6 0.5 0.5
A S S
a Fractional inhibitory concentration (FIC) index was assessed as described in section 4.2.5. b A:
Addition; S: Synergy.
estimated for both S. aureus and E. faecalis (Table 4.2). In case of S. aureus MTCC 96, a substantial reduction in the MIC of polymyxin B was evident with increasing concentrations of AuNP-PTh nanocomposite and the nature of interaction was synergy. In case of E. faecalis MTCC 439, the interaction of AuNP-PTh nanocomposite and the antibiotic was addition at the lowest nanocomposite concentration whereas at higher nanocomposite concentration the interaction was synergy (Table 4.2).
4.3.2. Antibacterial activity of AuNP-PTh nanocomposite in simulated gastric fluid (SGF) Pathogenic bacteria are highly enduring and have the ability to survive in various harsh environment. In this regard the ability of gastrointestinal pathogens to survive in the harsh acidic environment prevalent in the stomach. Although human gastric fluid has been acknowledged as a defense barrier against enteric pathogens owing to its highly acidic pH, (Tamplin, 2005) many bacterial enteric pathogens are endowed with evolved mechanisms to survive in the acidic milieu of the stomach prior to colonization of the small intestine (Foster, 1999; Foster, 2004; Cotter et al., 2000; Bavaro, 2009; Amieva and El-Omar, 2008;
Richard and Foster, 2004). An additional challenge in the elimination of acid-resistant pathogenic bacteria is that therapeutic agents such as antibiotics may suffer a loss of activity in acidic environment (Lamp et al., 1992; Mercier et al., 2002). It may be
Figure 4.4. (a) Viability of target pathogenic bacteria treated with AuNP-PTh in simulated gastric fluid. Statistically significant values derived by ANOVA are indicated by asterisk marks. * indicates p value <0.05 and ** indicates p value <0.001. (b) Time-dependent effect of AuNP-PTh treatment on E. coli MTCC 433 and L. monocytogenes Scott A cells in simulated gastric fluid (SGF).
mentioned here that with increasing concentration of AuNP-PTh (15-150 µM), the pH of PBS solution in which the target cells were suspended varied from 4.8-3.7. In the previous chapter (refer to Figure 3.7) it was observed that viable cell populations of bacterial pathogens were reduced on exposure to AuNP-PTh solution (15-150 µM), whose pH was in the acidic range (4.8-3.7) and it was also evident that the acidic pH per se did not account for the loss in viability of target pathogens on exposure to the AuNP-PTh nanocomposite. Hence it was conceived that AuNP-PTh could be a potential antibacterial agent to target acid-resistant bacteria. To evaluate the bactericidal efficacy of AuNP-PTh in an acidic niche, cells of E. coli MTCC 433 and L. monocytogenes Scott A were suspended in simulated gastric fluid (SGF) having a pH of 2.5 and treated with varying concentrations of AuNP-PTh nanocomposite for 2h. As evident from Figure 4.4a, a decrease in % viability of cells treated with AuNP-PTh was observed for both the pathogens and this reduction in viability was also observed to be dose-dependent and statistically significant based on ANOVA. A time-dependent decrease in cell viability
upon treatment with AuNP-PTh (112µM) in SGF was also observed (Figure 4.4b). There was only a one log reduction in the cell viability when the target bacterial cells were suspended in SGF alone for 2 h (Figure 4.4b). This suggested that the decrease in viability of AuNP-PTh-treated target pathogens in SGF was not influenced by pH. In the present set of experiments the target pathogens suspended in SGF were subjected to the bactericidal action of AuNP-PTh for 2 h. This time period is relevant considering that bacteria in food is likely to be subjected to a pH of 2.5 for approximately 2 h in the gastric environment (Gordon et al., 1993).
4.3.3. Anti-biofilm activity of AuNP-PTh nanocomposite
Bacterial biofilms have been associated with several chronic infections such as bacterial endocarditis and cystic fibrosis (Parsek and Singh, 2003; Tré-Hardy et al., 2009;
Fernández-Olmos et al., 2012). Opportunistic pathogens such as Pseudomonas aeruginosa, Staphylococcus epidermidis and Staphylococcus aureus are acknowledged as clinically most significant organisms that form biofilms. Owing to the significance of biofilms in human health and the high resistance of biofilms towards the action of antimicrobial agents, (Mah and O’Toole, 2001; Davies, 2003; Donlan and Costerton, 2002; Amini et al., 2011) there is considerable motivation in the development of bactericidal agents with anti- biofilm activity (Mahmoudi and Serpooshan, 2012). In the present study it was observed that AuNP-PTh nanocomposite displayed profound membrane-directed activity and could readily sensitize pathogenic bacteria to the action of antibiotic. It was thus envisioned that the nanocomposite could perhaps be explored as an anti-biofilm agent. To this end experiments were conducted to evaluate the effects of varying concentration of AuNP-PTh on P. aeruginosa biofilm. Treatment of P. aeruginosa biofilm with increasing concentration of AuNP-PTh nanocomposite resulted in a corresponding decrease in biofilm biomass as estimated by crystal violet staining (Figure 4.5a) and a reduction in elaboration of extracellular polysaccharide as ascertained by Congo red staining (Figure 4.5b). A one way ANOVA revealed that the reduction in biofilm biomass upon treatment with AuNP-PTh nanocomposite was significant (p value < 0.001) compared to untreated biofilm. Likewise, the reduction in the elaboration of extracellular polysaccharide on treatment of P. aeruginosa biofilm with AuNP-PTh nanocomposite was also statistically
Figure 4.5. Anti-biofilm activity of AuNP-PTh nanocomposite ascertained by (a) estimation of P.
aeruginosa MTCC 2488 biofilm biomass by crystal violet absorbance method and (b) Congo red binding assay. Statistically significant values derived by ANOVA are indicated by asterisk marks.
* indicates p value <0.001.
significant. Given the pivotal role of extracellular polysaccharides in constituting the structural framework of biofilms, protection against physical stress, such as shear and resistance against the action of antimicrobial agents such as antibiotics (Mah and O’Toole, 2001; Stewart, 2001) theability of AuNP-PTh nanocomposite to influence polysaccharide production in biofilms has significant therapeutic implications.
Fluorescence microscope analysis revealed a structured network of cFDA-SE stained P. aeruginosa cells suggesting the presence of a metabolically active biofilm, since cFDA fluorescence is an index of intracellular esterase activity of viable cells (Figure 4.6, panel a). Treatment of biofilm with 112 µM AuNP-PTh resulted in a drastic reduction of bacterial biofilm which manifested as sparse distribution of cFDA stained cells (Figure 4.6, panel b). Further, AuNP-PTh treated biofilm exhibited prominent PI staining, which clearly suggested the presence of membrane damage in nanocomposite-treated biofilm (Figure 4.6, panel d). These results indicated that AuNP-PTh could pervade across the extracellular matrix of P. aeruginosa biofilm to reach the embedded microcolonies and bring about copious membrane damage to the cells. Biofilms of pathogenic bacteria are known to be resistant to the action of antibiotics (Keren et al., 2004; Fux et al., 2005) and
Figure 4.6. Fluorescence microscope analysis of P. aeruginosa MTCC 2488 biofilm treated with 112 µM AuNP-PTh nanocomposite. Biofilm was visualized by (a-b) cFDA-SE staining and (c-d) PI staining. Scale bar for the images is 100 µm.
in this context, the ability of AuNP-PTh to infiltrate and eradicate bacterial biofilm holds special therapeutic interest.
Evidence for anti-biofilm activity of AuNP-PTh nanocomposite was further strengthened by FESEM analysis. As evident from Figure 4.7a, in case of untreated biofilm of P. aeruginosa MTCC 2488, the shape of the cells were quite distinct and the cells appeared to be enveloped by a slimy layer, which presumably is the polysaccharide elaborated by the biofilm. On the other hand, in the nanocomposite- treated samples, the biofilm architecture was apparently disintegrated and damaged cells with irregular shape were observed (Figure 4.7b).
To further explore the anti-biofilm activity of AuNP-PTh nanocomposite, experiments were conducted to ascertain the effect of sub-MIC levels of the nanocomposite on the action of tobramycin, an antibiotic which is known to exhibit anti- biofilm activity against P. aeruginosa (Musken et al., 2010, Tré-Hardy et al., 2009; Kim et al., 2008). The representative results of the experiments are shown in Figure 4.8. It can be
Figure 4.7. Field emission scanning electron microscope (FESEM) analysis of (a) untreated and (b) AuNP-PTh nanocomposite treated biofilm of P. aeruginosa MTCC 2488. Scale bar for the images is 1.0 µm.
Figure 4.8. Effect of combined treatment of AuNP-PTh nanocomposite and tobramycin on the growth of P. aeruginosa MTCC 2488 biofilm.
observed from the figure that treatment of P. aeruginosa MTCC 2488 biofilm with 0.078 and 0.156 µg/mL tobramycin resulted in about 17% and 30% growth inhibition, respectively. Interestingly, in a combinatorial assay, a progressive increase in the growth inhibition of biofilm was observed with increase in the concentration of AuNP-PTh nanocomposite. For instance, there was more than 90% growth inhibition of P. aeruginosa biofilm at a tobramycin concentration of 0.156 µg/mL in combination with 60 µM composite (Figure 4.8b). It is significant to mention here that the concentrations of AuNP- PTh composite and tobramycin used for the combinatorial assays were below their respective MIC values for the target pathogen. The ability of low levels of AuNP-PTh nanocomposite to potentiate the action of tobramycin reflects the therapeutic merit of the nanocomposite as an adjuvant in combination therapy for eradication of P. aeruginosa biofilm. Further, it also offered the opportunity to reduce the levels of tobramycin to be used for effective elimination of the biofilm. This is significant given that therapeutic applications of tobramycin at high concentrations may lead to unwarranted side effects (Thomsen and Friis, 1979).