Nanocomposites: A Novel Strategy to Arrest Bacterial Pathogens Infestation

This work is an extension to our previous work. Montmorillonite/poly(o-phenylenediamine) (MMT/POPD) Nanocomposites (NC) were synthesized by Riaz et al. 2016 and evaluated for the antimicrobial potential. Antibacterial potential of NC were investigated by the broth microdilution and agar well diffusion methods. Further, effective concentration (EC 50 ) and minimum inhibitory concentration (MIC) of the NC were determined. Intercalation and in-situ polymerization of POPD within MMT was successfully obtained using sonochemical technique. The TEM micrograph of MMT/POPD and POPD/MMT conrmed the size in the range of 20-90 nm and the shape observed was spherical (Riaz et al. 2016). The NC was then checked for antimicrobial activity against four bacterial strains and they showed potent antibacterial activity in both broth and on agar plate. Quantitative test in liquid media clearly showed that NC had viable cells reduction ability for testing strains. Results obtained here concluded that the NC can prevent the infestation of various bacterial pathogens effectively. Therefore, we propose that they are useful as effective and non-cytotoxic biologically active agents and can be used as nanocarriers with dual role i.e. in addition to carrying drugs, carrier will itself act as antibacterial agent. This dual nature of NC could be an alternative to target multi-drug resistant bacterial strains and can further be explored for their mode of action against bacterial pathogens.


Introduction
The excessive use of antibiotics over the years is now under scrutiny of a subsequent propagation of antimicrobial resistance. Antimicrobial substances are capable of growth inhibition or even elimination of certain groups of microorganisms. The exploration of novel products with potent antimicrobial activity has acquired a particular interest in numerous operations, viz. raw materials for pharmaceuticals and cosmetics, clinical and veterinary products, etc. Indigenous population globally has consumed clay for remedial purposes throughout the history. Bandages of French green clay was in use for healing the Buruli ulcer (Williams et al. 2004) which is a necrotizing fasciitis caused by Mycobacterium ulcerans. French clays were proved to be of antibacterial nature ) whereas other French green clays augmented the bacterial growth compared to controls ). Continued examination of clays globally has disclosed only a few numbers of deposits that possess antibacterial property. All deposits are mineralogically diverse but they belong to hydrothermally altered volcanic atmosphere; either transformed pyroclastic matter or bentonite (volcanic ash).To point out the antibacterial properties of an inert substance like clay, it is important to design nanocomposites which have exhibited bacteriostatic activity (Corrales et al. 2012). Clay minerals have been employed to produce organic-inorganic hybrid nanocomposites because of their ultra-ne particle size, intercalation property and large surface area. . For a better understanding for the clay's role, conducting polymer as a ller and as well as a matrix, in this research paper, the nanocomposites of poly(ophenylenediamine) (POPD) and MMT were prepared by means of sonochemical intercalation with the help of two methods, rst by taking POPD to act in one case as a ller and matrix in another one. The investigation of spectral, uorescence and spectral features of the synthesized nanocomposites led to explore the clay's optimum loading and conducting polymer for designing nanocomposites possessing controlled self-assembled morphology which can be utilized in optoelectronic devices (Riaz et al. 2016).
In recent years, clay mineral-based antibacterial complexes have been prepared by a series of processes between the clay minerals and antibacterial substances. Clay minerals are being scrutinized because of the nontoxic and eco-friendly properties and an effortless production through intercalation with the use of antimicrobial organic modi ers (Bertagnolli et al. 2011). Recent investigations have revealed that the reason behind the antimicrobial property is because of the contact between organoclay and the cells,

Synthesis and characterization of NC by sonochemical dispersion method
The NC were prepared as per our previously published protocol (Riaz et al. 2016). POPD/MMT NC was synthesized by dissolving MMT in distilled water followed by the addition of POPD. Whereas, MMT/POPD based NC was synthesized by dissolving MMT in distilled water followed by the addition of o-phenylenediamine and ferric chloride (oxidant). The NC were then characterized for their size and morphology using TEM and further evaluated for their antimicrobial potential.The powdered NC were soluble in double distilled water, dimethyl sulfoxide (DMSO), N-Methyl-2-pyrrolidone (NMP) upto 5 mg/ml. Overnight culture was diluted with fresh LB media to approximately 10 4 colony-forming units (CFU) and incubated at 37ºC for 12-14 hrs in the presence of NC. The growth of bacterial culture obtained (test) was compared to the growth obtained in the presence of ampicillin (negative control). The experiment was performed in triplicates. For each replicates, the average values were recorded. The growth of positive control culture was also determined where only media and primary culture was taken. Bacterial growth was determined spectrophotometrically by taking absorbance at 600nm. The % mean growth inhibition (%MGI) was calculated by using the formula: MGI (%) = (Ac -As/ Ac) x 100, where Ac and As represent absorbance of control and treated sample strains respectively.
The EC 50 value (concentration causing 50% reduction in the bacterial growth) was calculated using Microsoft o ce Excel 2007, based on the readings.

Determination of Minimum Inhibitory Concentration (MIC)
The determination of MIC was carried out by technique of Broth dilution as explained in method M07-A9 from the Clinical and Laboratory Standards Institute (CLSI) (CLSI 2012). Brie y, 300µl of media was placed in each well of 96 wells plate. Further, 150 µl of the NC were added to the rst well and then sample was serially diluted. The extra sample from last well was discarded to make nal volume as 300µl in all well). ~10µl of bacterial cell culture (normally to an OD600 0.1) was added to each well and OD 600 was measured after 12 hours of incubation at 37°C at incubator. The MIC 80 was de ned as the concentration at which ~80% of the bacterial growth was inhibited.

Agar well-diffusion method
Agar well-diffusion method was used to determine the antibacterial activity by the method as described in method M07-A9 from the Clinical and Laboratory Standards Institute (CLSI) (CLSI 2012). Ampicillin (stock, 2mg ml −1 ) was used as standard antibacterial drug. The log phase bacterial cultures (secondary culture) were spread on LB agar medium plates using a sterile spreader in order to get a uniform bacterial growth on test plates. Wells were punched over the agar plates using sterile cork borer. Stock solution of each nanocomposite was prepared at a concentration of 2 mg ml −1 in distilled water. Approximately 40-50 µl NC (~80 µg) and Ampicillin solution was added with the aid of a sterile syringe into the wells and diffusion was allowed to carry out at room temperature for 2 hours. Control experiments comprising LB media was also set up. The plates were incubated at 37°C for 18-24 h. The diameter of the inhibition zone (mm) was measured. Experiment was done in triplicates. The results (zone of inhibition) were compared with the activity of the standards, ampicillin (2 mg ml −1 ).

Statistical Analysis
The experiments were performed in triplicates (n=3). Graph Pad Prism Software was used to prepare Graphs and to calculate the statistical parameters such as mean, standard error of mean (SEM) etc. The data obtained were expressed as 'mean±SEM'.

Results
In this study, the nanocomposites of POPD and MMT were successfully synthesized via intercalation and in-situ polymerization of o-phenylenediamine within MMT using sonochemical technique by taking the former to be as ller in one case and as matrix in the other. The results con rmed the formation of NC which is published in our previously paper (Riaz et al., 2016).

TEM analysis
TEM micrograph of the different ratios of POPD:MMT and MMT:POPD (i.e. 1:0.25, 1:0.50, 1:1) exhibited particle size in the range of 20-90 nm and showed the spherical chain like structure (Fig. 1). POPD has shown the formation of core and MMT was found to encapsulate the dense POPD particles like a shell. The POPD:MMT-1:1 and MMT:POPD-1:1 NC showed clustering and aggregation of the spherical nanoparticles of 50-90 nm range (Fig. 1d and 1g). The results obtained were in compliance with as described in our previously published paper (Riaz et al., 2016).

Assessment of antibacterial activity
To check the e cacy of NC against bacterial strains, antibacterial tests were performed by two independent methods viz. broth micro-dilution and well diffusion assay.

Broth microdilution Assay
The effects of different ratios of MMT/POPD and POPD/MMT and standard antibiotic drug (ampicillin) on bacterial strains were determined by measuring postincubation absorbance readings at 600 nm and %MGI was calculated. The results were compared with standard ampicillin as shown in Fig. 2 (Table 1a and 1b).
Both the assays con rmed that NC is capable to inhibit the growth of bacterial strains in a range of 15.625-2000 μg ml -1 . The results showed that B. subtilis is the highest sensitive strains among all NC and exhibited the highest % inhibition i.e. sensitivity towards all the ratios of NC that's why MIC was performed for this strain only in presence of all NC. However, the results also proved that NC was bacteriostatic to B. subtilis at its MIC 80 concentration where its 80% growth was inhibited and the clear solution was obtained. Inhibitory concentration of B. subtilis was evaluated by determining MIC 80 as depicted in Fig. 4 and 5.
The antibacterial activity of NC showed that, B. subtilis displayed susceptibility to all NC ranging from 15-2000 μg ml -1 . This method is based on a broth micro dilution method in 96-well microtitre plates. The MIC (MIC 80 ) of MMT/POPD and POPD/MMT against the B. subtilis was shown below (Table 1c). No matter whichever the NC were tested, all of them exhibited good antibacterial activity and a broad spectrum of activity.
Determination of zone of inhibition by agar well-diffusion method NC which was synthesized from MMT and POPD has shown good zone of inhibitions which were provided in Table 2 and Fig. 6, 7, 8. The antibacterial activity was conducted against the pathogenic bacterial strains such as E. coli, S. aureus, P. aeruginosa and B. subtilis. The well-diffusion method also showed the highest antimicrobial activity against B. subtilis followed by P. aeruginosa, E. coli, and S. aureus. The stock concentration used for different ratios of MMT:POPD and POPD:MMT were 2 mg ml -1 and approximately 40 μl of the NC were diffused into each well.
NC had showed various degree of zone of inhibition against tested bacterial strains and among them, B. subtilis showed the highest inhibition zone whereas S. aureus possess the minimum zone towards the NC. When MMT: POPD -1:0.25, MMT: POPD -1:0.5 and MMT: POPD -1:1 were diffused into the wells, they showed the maximum zone of inhibition in B. subtilis i.e. 30, 35 and 32 mm respectively whereas they showed minimum inhibition zone in S. aureus which were 21, 24 and 23 mm respectively (Fig. 6).
When ~40 μl of POPD: MMT -1:0.25, POPD: MMT -1:0.5, and POPD: MMT -1:1 was diffused into each well of agar plate of separate bacterial strains, they showed the highest susceptibility towards B. subtilis and hence maximum zone of inhibition was observed i.e. 20, 25 and 24 mm respectively whereas S. aureus showed minimum zone of inhibition on agar plate towards these POPD/MMT NC. The observed inhibition zone in S. aureus was approximately 16, 20 and 17 mm respectively. The results were quite comparable with standard ampicillin which showed maximum zone of inhibition in B. subtilis (34 mm) and minimum zone of inhibition in S. aureus (30 mm) ( Fig. 7 and 8). The positive control where the LB broth was used, there were no zone of inhibition produced suggesting that the NC are antibacterial agent as they have cleared the bacterial growth zone on agar plate.
The synthesized NC was energetically involved in the antibacterial activity against B. subtilis, P. aeruginosa, E. coli and S. aureus. B. subtilis had the maximum zone of inhibition among all NC tested and S. aureus had the minimum zone of inhibition because of the maximum resistant capacity of the bacterial isolates and having lowest growth inhibition with all NC (Table 2a and 2b).
The organic-inorganic hybrid nanocomposites generation using clay minerals was aided by their properties like large surface area, ultra-ne particle size, and intercalation property. Although, in construction of these nanocomposites comprising controlled properties the interaction between matrix and ller serves as a substantial feature. Hence, for a better understanding of the role played by play as well as conducting polymer as a matrix and ller, in this research paper nanocomposites of poly(ophenylenediamine) (POPD) and MMT have been synthesized using sonochemical intercalation methods via two methods -by keeping the former as a ller in one case and as matrix in other. To navigate the optimum loading of clay and conducting polymer for the construction of nanocomposites with property of self-assembled morphology so that they could be employed in optoelectronic devices, the spectral, uorescence, and morphological properties were investigated ( Some of the chemical antimicrobial agents are irritant and toxic, while plants are easily available, safe, and nontoxic in most cases, but do not have antimicrobial potential as effective as other chemical agents. Therefore, there is vital need and much interest in nding ways to formulate new types of safe and cost-effective biocidal materials. This study helped us to use these NC as safe and strong antimicrobial material. It may be due to the strong adsorption and immobilization capacity of modi ed layered silicates (Guo et al. 2005;Hu et al. 2005). It is a well-established fact that at pH 7.4, a net negative charge is exhibited by the parent layered silicate (Nzengung et al. 1996). Bacteria possessing negative charge will not be adsorbed notably onto the clay under these conditions. Nevertheless, after modi cation of POPD, nanocomposites were synthesized with several degrees of hydrophobicity. The diameters exhibited by inhibition zone fell between 10 and 14 mm. Furthermore, Gram-positive bacteria displayed a greater effect than the Gram-negative bacteria. The basic difference is the presence of an outer membrane in Gram-negative whereas absent in Gram-positive bacteria. Dissimilar barricades serves as the explanation of variation in their sensitivity as their capability to avoid the entrance of microbicides varies. Antimicrobial agents kill bacteria through various means depending on the type of bacteria. Most antiseptics and disinfectants kill bacteria immediately on contact by causing the bacterial cell to burst, or by depleting the nutrients preventing bacterial reproduction such as bacterial conjugation. Antimicrobial polymers might kill bacteria by adsorbing onto the bacterial cell wall. Most bacterial surfaces are negatively charged; therefore, the adsorption of polymeric cations has proved to be more effective than adsorption of polymeric anions. The antimicrobial agent must then diffuse through the bacterial cell wall and adsorb onto the cytoplasmic membrane. This might lead to the disruption of the cytoplasmic membrane and subsequent leakage of cytoplasmic constituents leads to the death of the cell (Nonaka et al. 2003;Uemura et al. 1999).

Conclusion
From the executed study, it can be inferred that NC showed potent antimicrobial activity and hence it can be concluded that the NC may be used as novel antimicrobial agents against wide range of microbes over conventional antibiotics that combined the bene t of organic materials with the excellence of inorganic materials prepared via simple solution intercalation technique as they have shown good antimicrobial e cacy. MMT/POPD and POPD/MMT NC have shown the %MGI in the order of B. subtilis> P. aeruginosa> E. coli> S aureus. In the future, nontoxic and biocompatible NC may replace some antibiotics or medicines to destroy pathogenic bacterial strains in the gastrointestinal tract of animals.
Results concluded that with the development of multidrug-resistant strains of bacteria, NC could be good alternatives in the prevention of MDR strains infections because of their dual role i.e. the NC are good nanocarriers and the carrier itself will act as strong antibacterial agent. Hence, this study open the new gateways for future research such as mechanism of action of these NC, drug carrying capability and inhibition of MDR strains.  Tables   Tables 1 and 2 are not available with this version.    MMT -1:0.5, and POPD: MMT -1:1) against B. subtilis (MTCC 736) was determined by the broth microdilution method. Turbidity was measured by recording absorbance at 600 nm. Graph was plotted between OD600 vs different concentration of NC (μg ml -1 ).

Figure 6
Agar well diffusion assay showing zone of inhibition in separate agar plate for all four strains where positive control (LB broth) and negative control (ampicillin) were diffused into well.