Antimicrobial activities of nanostructured polyanilines doped with aromatic nitro compounds

2.5.1 FT-IR spectroscopy

FT-IR spectra of all PANIs were recorded as KBr pellets on a Perkin Elmer RXI FT-IR spectrophotometer.

2.5.2 Powder X-ray diffraction (XRD)

Powder X-ray diffraction measurements were made with a Rigaku Ultima III X-ray diffractometer using Cu Kα target (λ = 1.5418 Å) at a scanning rate of 10° per min.

2.5.3 Scanning electron microscopy (SEM)

SEM micrographs of PANI salts at different magnifications were recorded using JEOL JSM-5600LV Scanning Electron Microscope. Fe-SEM micrographs of PANIDN were obtained using JEOLJSM-5610LV field emission scanning electron microscope. The PANI samples were gold coated for 1 min using a magnetron sputter coater before SEM analysis.

2.5.4 UV–Visible spectroscopy

UV–Visible spectra of the PANI solutions in DMSO (1 mg/25 ml) were recorded in the region 300–1100 nm using Perkin–Elmer Lambda 25 spectrophotometer.

3.5.1 Antibacterial properties of PANIs

The susceptibility of bacteria against PANIs is species dependant. The antibacterial activities observed in the current study for PANIPI, PANIDN, PANICl and PANIEB are evaluated from the zone of inhibition diameter and MIC values (Table 2). The PANIs at 100 μg ml⁻¹ concentration exhibited maximum zone of inhibition diameter of 14–24 mm (Table 2) tested for the gram negative and gram positive bacteria and hence the as-prepared PANIs synthesized in the present work can be considered to possess strong antibacterial activities (Chohan and Nasser, 2007).

3.5.2 Antibacterial activities of PANIs on gram negative bacteria

S. dysenteriae is inhibited strongly (18–20 mm) by PANI salts while PANIEB showed only a moderate activity (14 mm) at the maximum concentration tested (100 μg ml⁻¹, Fig. 9a). The growth of S. enterica another rod shaped gram negative bacteria closely related to S. dysenteriae is inhibited by all the PANIs with inhibitory zone diameter lying in the range from 17 to 19 mm (Table 2). K. pneumoniae, a causative agent for many respiratory infections is also inhibited by the PANIs strongly (16–17 mm). It is surprising to find that the growth of P. aeruginosa, a multidrug resistant bacteria is inhibited strongly by PANIEB (19 mm) even though it is hydrophobic in nature. The MIC values for S. dysenteriae, S. enterica and K. pneumoniae of all the PANIs are less than 25 μg ml⁻¹ (Fig. 6A–C). MIC needed for inhibiting P. aeruginosa bacteria is 50 and 75 μg ml⁻¹ of PANIPI (Fig. 6D₂) and PANIDN (Fig. 6D₃) respectively. The MIC for E. coli needed is 50 μg ml⁻¹ of PANICl, PANIPI and PANIDN (Fig. 6E₁) while the MIC of PANIEB (Fig. 6E₂) is less than 25 μg ml⁻¹.

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Figure 9 A comparative study on the antimicrobial activities of polyaniline emeraldine salts and base at 100 μg ml⁻¹. A – Shigella dysenteriae, B – Salmonella enterica, C – Klebsiella pneumoniae, D – Pseudomonas aeruginosa, E – Escherichia coli, F – Staphylococcus aureus, G – Streptococcus pyogenes, H – Bacillus subtilis, I – Enterococcus faecalis, J – Candida albicans. Error bars show the standard deviation (S.D = 0.1–0.25) of 3 replicated data.

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3.5.3 Antibacterial activities of PANIs on gram positive bacteria

The gram positive bacteria, S. aureus is inhibited by PANICl and PANIPI with inhibitory zone of 17 and 19 mm respectively (Table 2, Fig. 9b) at 100 μg ml⁻¹. At this concentration, PANIDN exhibited lower activity with the zone of inhibition diameter of 15 mm. The bacterial strains such as S. pyogenes and B. subtilis are inhibited by all the PANI salts and base (14–17 mm) to the same extent. An important finding of this work is the maximum inhibitory zone diameter of 24 mm at 100 μg ml⁻¹ (Table 2) observed for PANIDN against E. faecalis among all PANI salts implying that reactions of 3,5-dinitrobenzoic acid with the contents of the cell sap occur effectively. At this concentration, a zone of inhibition of 16 mm is found against all the bacterial strains when PANIEB is used. All the PANIs exhibit a MIC of less than 25 μg ml⁻¹ (Fig. 7F) for S. aureus. The growth of S. pyogenes is prevented by PANIPI and PANIDN at a MIC of 50 μg ml⁻¹ (Fig. 7G₂). The growth of B. subtilis and E. faecalis is prevented by PANICl and PANIDN at a MIC less than 25 μg ml⁻¹ (Fig. 7H and I) while PANIPI is effective only at a concentration of 50 μg ml⁻¹ (Fig. 7).

In the present work, the zone of inhibition (11–13 mm) obtained for PANIs against the S. aureus at 25 μg ml⁻¹ is higher than that reported for PANI and PANI-mupirocin (7.3 and 7.6 mm) at 30 μg ml⁻¹ by earlier workers (Jotiram et al., 2012). A PANI composite with Ag and polyvinyl alcohol (Ghaffari-Moghaddam and Eslahi, 2014) has been reported to have a inhibition zone of 8–15 mm and 7–12 mm for S. aureus and E. coli respectively at 40 μg ml⁻¹, whereas in the present work, PANIs exhibit a higher inhibition diameter of 12–14 and 11–14 mm for the same species at 50 μg ml⁻¹. Moreover, the results of PANIs obtained in the present work by the well diffusion method agree closely with the results of the colony forming reduction method reported by Seshadri and Baht (2005). Hence, from the merits of the results obtained, the as-prepared PANIs and PANIEB possess good antibacterial efficacies.

The cell walls of different types of microbes may not be the same. Primarily gram-positive and gram-negative bacteria are categorized on the basis of cell wall structures. Gram negative bacteria contain a lipopolysaccharide layer at the exterior, with a thin layer (7–8 nm) of peptidoglycan present below the lipopolysaccharide layers (Madigan and Martinko, 2005). Thus gram negative bacteria that are rich in negatively charged lipopolysaccharides (Salton et al., 1996) can attract the PANI chains. PANI chains with positive imino sites bind to the negatively charged outer walls of the cells (Scheme 2). The PANI chains anchor to the cell walls at several sites and cause damage to the membrane, by changing the potential gradients across the ion channels present on the cell membranes. On the other hand, gram-positive bacteria are composed of a thick layer (20–80 nm) of peptidoglycan, consisting of linear polysaccharide chains cross-linked by short peptides to form a three dimensional rigid structure (Baron, 1996). The rigid and extended crosslinking not only endows the cell wall with fewer anchoring sites for the PANIs but also makes it difficult for the dopant ions (X⁻) to penetrate. Thus, the growth of gram negative bacteria is inhibited to a greater extent compared to gram positive bacteria.

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Scheme 2 Bactericidal activity of PANI.

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3.5.4 Antifungal properties of PANIs

PANI salts are also effective against the fungus C. albicans. For PANICl, the average zone inhibition diameter is 16 mm (Table 2) at 100 μg ml⁻¹. For PANIPI and PANIDN, it is 17 mm. Nitro compounds doped PANIs are more effective than PANICl at 100 μg ml⁻¹ (Fig. 9c). For PANIEB, inhibitory zone diameter is 13 mm only. Thus it can be inferred that the antifungal properties of PANI salts are higher than PANIEB. The MIC values required for PANIDN are found to be 50 μg ml⁻¹ (Fig. 8J₂) compared to other PANIs (<25 μg ml⁻¹, Fig. 8J₁).

In the well diffusion technique, the zone of inhibition is considered for comparing the efficacy of PANI salts. The efficiency of PANI salts against gram negative bacteria S. dysenteriae determined from average zone of inhibition values follows an order of PANICl > PANIPI > PANIDN > PANIEB implying that PANI emeraldine salts are strongly active compared to PANIEB. Among the hydrophilic PANI salts, PANICl is strongly active due to the presence of small dopant chloride ions compared to large nitro compounds as it can penetrate the cell membrane easily. As PANIEB is hydrophobic it is less active. The bacterial cells tend to show greater adhesion and more proliferation on more hydrophilic surfaces and the cell proliferation increases with increasing surface hydrophilicity (Nam et al., 2014). Thus, in consonance with earlier workers, PANI salts being more hydrophilic are found to exhibit greater zone of inhibition than PANIEB which is hydrophobic in nature. On comparing the activities of PANIs prepared in this work with picric acid derivatives and the standard antibacterial drug, streptomycin (Sheth et al., 2010), a higher zone of inhibition is observed for PANIs. PANIs also exhibit comparable activity similar to piperazine derivatives and the standard antifungal drug miconazole (Thriveni et al., 2014) indicating that PANIs possess good antifungal properties.

3.5.5 Comparison of antimicrobial activities of PANIDN and 3,5-dinitrobenzoic acid

Acids are known to be effective bactericidal compounds (Cunha et al., 2007). In this work, 3,5-dinitrobenzoic acid is also used as an antimicrobial agent and the zone of inhibition produced by the free acid is compared with PANIDN. The zone of inhibition diameter (Table 3) for free acid is almost twice than that of the polymer at 100 μg ml⁻¹ (Fig. 10). The lower activity of PANIDN may be due to the slow release of 3,5-dinitrobenzoic acid from the polymer matrix even though PANIDN has two reactive 3,5-dinitrobenzoate molecules as dopant ions on the imino sites.

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Figure 10 A comparative study on the antimicrobial activities of PANI-3,5-dinitrobenzoate and 3,5-dinitrobenzoic acid at 100 μg ml⁻¹. A – Shigella dysenteriae, B – Salmonella enterica, C – Klebsiella pneumoniae, D – Pseudomonas aeruginosa, E – Escherichia coli, F – Staphylococcus aureus, G – Streptococcus pyogenes, H – Bacillus subtilis, I – Enterococcus faecalis, J – Candida albicans. Error bars show the standard deviation (S.D = 0.1–0.25) of 3 replicated data.

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3.5.6 Mechanism of bactericidal activity

The mechanism of antibacterial effects of PANI (Scheme 2) arises due to two independent phenomena. PANI chains are positive in the salt form and neutral in the base form. Both the positively charged chains and the PANIEB forms can affect the cell membranes. In the first step, PANI matrix bind to the cell walls through electrostatic interactions (Shi et al., 2006; Gizdavic-Nikolaidis et al., 2011) and get anchored to the cell walls at several sites on the membranes (Scheme 2). The ion channels present on the cell wall experience a change in the potential gradient as a result of which the normal influx and efflux of electrolytes are disturbed (step 2). It is well established that PANI materials are highly electroactive at low pH values (Syed and Dinesan, 1991). Thus PANIs bring about a change in the pH levels of the cell due to the release of the dopants. The permeability of the cell membranes on the bacterial cells is affected. The released dopant ions (picrate, 3,5-dinitrobenzoate, chloride) from the PANI matrix (Deepa et al., 2014; Dhivya et al., 2014) penetrate the semi-permeable membranes and disturb the functions of the ion-gates. The dopant ions react with enzymes, amino acids, nucleic acids, DNA and electrolytes present in the cell sap and may alter the cell composition leading to cell lysis.

The properties of PANIs can be tuned via molecular architecture and are thus suitable candidates for preparing diverse antimicrobial drugs. Electrochemical switching property of PANI helps in the movement of dopant ions in and out of the cell membranes. Further, the unique redox properties of PANI, result in controlled ionic transport through the polymer membrane to the cells leading to enhanced antimicrobial effects.