NANOSPIDER TECHNOLOGY FOR CONTROLLING OF PSEUDOMONAS CICHORII AND DICKEYA DADANTII BY ELECTROSPUN NANOFIBERS OF NYLON-6/CHITOSAN BLENDS

This is the first report on the use of electrospun nanofibers which could be of considerable interest to the development of new antibacterial compounds against two species of bacteria: Pseudomonas cichorii causing bacterial leaf spot (bacterial midrib rot) and Dickeya dadantii (Erwinia chrysanthemi) causing bacterial bligh. Electrospun nylon-6/chitosan (nylon-6/Ch) nanofibers were obtained using formic acid as a single solvent. Surface modification of electrospun nylon-6/chitosan nanofibers was performed by soaking the mat in an aqueous solution of glycidyltrimethylammonium chloride (GTMAC) at room temperature overnight to give nylon-6/N-[(2-hydroxy-3-trimethyl ammonium)propyl] chitosan chloride (nylon-6/HTCC). The morphological, structural and thermal properties of the nylon-6/chitosan nanofibers were studied by field-emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), Fourier transform-infrared (FT-IR) spectroscopy, and thermogravimetric analysis (TGA). Biological screening demonstrated that Nylon-6/HTCC mat exhibited high potential antibacterial activity on protein synthesis of bacteria Pseudomonas cichorii and Dickeya dadantii. Bacteria examined using SEM were totally deformed and exhibited symptoms of severe destruction.


Introduction
Bacterial pathogens employ diverse strategies to infect their host plants.
Losses due to postharvest disease may occur at any time during postharvest handling, from harvest to consumption.When estimating postharvest disease losses, it is important to consider reductions in fruit quantity and quality, as some diseases may not render the produce unsalable, yet still reduce product value (De Britto et al., 2011).For example, blemished fruit may not be sold as fresh fruit but may still be suitable for processing, in which case, it lowers the price.It is also important to take into account costs of harvesting, packaging and transport when determining the value of produce loss as a result of postharvest wastage (Bradbury, 1986;Mandavia et al., 1999).Aside from direct economic considerations, diseased produce poses a potential health risk.The bacteria that cause postharvest diseases of vegetables and fruits belong to five of the six genera of bacteria that cause plant diseases.Among them, members of the genera Pseudomonas and Erwinia are responsible for the necrotic lesions on leaves, stems, and fruits, and internal discolorations and decay.The Gram-negative bacterium Dickeya dadantii (Erwinia chrysanthemi) is a phytopathogenic bacterium causing soft rot diseases on many crops.E. chrysanthemi has a world-wide distribution.The disease causes the destruction of many flower and ornamental crops, particularly carnation and chrysanthemum in rooting beds.Losses are also recorded in different glasshouse ornamentals (Saintpaulia ionantha, Kalanchoe spp.), as well as in tuber production of potato and Dahlia spp.On potatoes, it causes soft rot and blackleg like E. carotovora (Pérombelon and Kelman, 1987), but the symptoms tend to be expressed at higher temperatures.Seed potato certification schemes producing material for warmer countries should take account of E. chrysanthemi (Pérombelon et al., 1987;Smith et al., 1988;Mirik et al., 2011).
Chitosan can be used in agriculture as a seed treatment and biopesticide, helping plants to fight off bacterial infections (El-Shafei et al., 2008).In agriculture, chitosan is used primarily as a natural seed treatment and plant growth enhancer, and as an ecologically friendly biopesticide substance that boosts the innate ability of plants to defend themselves against bacterial infections (Fouda et al., 2009).The chitin and chitosan are found in the shells of crustaceans, such as lobsters, crabs, and shrimp, and many other organisms, including insects and bacteria.It is one of the most abundant biodegradable materials in the world (Hebeish et al., 2011a;El-Newehy et al., 2011).As a natural polymer, chitosan possesses biodegradable, biocompatible, antimicrobial activity, non-toxicity and versatile chemical and physical properties (Fahmy and Fouda, 2008;Fouda et al., 2009;Abdel-Halim et al., 2010;Abdel-Halim and Al-Deyab, 2011;Hebeish et al., 2011b;Nirmala et al., 2011c).On the other hand, nylon-6 is a biodegradable, biocompatible and synthetic polymeric material which has good mechanical and physical properties (Ma et al., 2008).
The application of electrospinning to biologically significant polymers has increased since the electrospun membranes were identified as a candidate for tissue engineering constructs (Kim et al., 2005;Nirmala et al., 2011b).Electrospun nanofibers have become promising materials for many biomedical applications such as wound dressing (Hebeish et al., 2011b) drug delivery, scaffold for tissue engineering (Dizman et al., 2004;Xu et al., 2004;Binulal et al., 2010;Nirmala et al., 2011c).
In this work, we report on the preparation of nylon-6/chitosan nanofibers as a candidate for antibacterial agents.We were able to synthesize nylon-6/chitosan composite nanofibers by blending chitosan (8%) with our Nanospider setup.The morphology of the resulting nanofibers before and after surface modification was analyzed by field-emission scanning electron microscopy (FE-SEM).Then, the structural and thermal properties of electrospun nylon-6/chitosan nanofibers were demonstrated using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and thermogravimetric analysis (TGA).The antibacterial activity of this new finding was demonstrated against Pseudomonas cichorii and Dickeya dadantiithe main causes of several vegetable and cash crop diseases.

Material and Methods
Nylon-6 (Ultramid ® B24 N 03) was purchased from BASF.Low molecular weight chitosan was purchased from Aldrich.Glycidyltrimethylammonium chloride (GTMAC) (technical, ≥ 90%) and formic acid were purchased from Fluka.All materials and solvents were used as received without further purification.
Nylon-6/chitosan nanofibrous mat was fabricated using electrospinning process in which, nylon-6 was dissolved in formic acid at 60-70°C with gentle stirring in order to prepare 12% (w/v) homogeneous solution.Chitosan with concentration of 8% (w/w) was mixed with the nylon-6 solution with gentle stirring until complete dissolution.The prepared solution was then used for electrospinning at room temperature.
Nylon-6/chitosan nanofibers were electrospun using nanospider technology.The main advantage of utilizing the Nanospider technology is to synthesise composite nanofibers by blending higher weight percentage chitosan (8%) which is difficult to spin with the conventional electrospinning process.The electrospinning was accomplished with the following conditions; active electrode for collecting electrode distance is 14 cm long at a driving voltage of 75 kV, active electrode speed is 2.2 rpm and humidity of 37%.The electrospun nanofiber was collected on aluminum sheet and was dried in hood at room temperature until constant weight was reached.
Electrospun nylon-6/chitosan nanofibers mat was treated with excess GTMAC in water with stirring for 24 h and then the mat was washed with excess water and finally dried in hood at room temperature.
Bacterial cultures of the Gram-negative bacteria Pseudomonas cichorii and Dickeya dadantii were provided from the culture collection of College of Science, Botany and Microbiology Department, King Saudi University, Riyadh, KSA.The enrichment of the culture was carried out according to the method described by Abdel-Megeed (2011).Hence, 300 µL of each stock-culture were added to 3 mL of Luria-Bertani (LB) broth.The enrichment of the bacteria was carried out in LB broth for 16 h at 27ºC in an orbital shaker at 200 rpm.Overnight, cultures were kept for 24 h at 37°C ± 1°C and the purity of cultures was checked after 8 h of incubation.After 24 h of incubation, bacterial suspension was diluted with sterile physiological solution and was ready for furher experiment.The bacterial stock was maintained at 4°C on nutrient agar slants.
The assay of antibacterial activity of nylon-6/HTCC against Pseudomonas cichorii and Dickeya dadantii was performed by agar disc diffusion method (Hayat et al., 1981).Muller Hinton agar medium was seeded with 100 µl of inoculum (1×10 8 CFU/ml).A weight of 38 g of Muller Hinton agar medium was suspended in 1,000 ml distilled water.After heating to dissolve the medium completely, it was sterilized by autoclaving at 15 lbs pressure (121°C) for 15 minutes.Then it was mixed well before pouring.The impregnated discs (5 mm) of nylon-6/HTCC mat were placed on the agar medium seeded with tested microorganisms.Standard plates with nylon-6/Ch were assessed in parallel as negative control.The plates were then incubated at 37°C for 24 hr to allow maximum growth of the microorganisms.The antibacterial activity of the test samples was determined by measuring the diameter of zone of inhibition expressed in millimeter.
Total protein concentration of Pseudomonas cichorii and Dickeya dadantii of the crude homogenate was determined by the method of Bradford (1976).
Samples of 50 µg of Pseudomonas cichorii and Dickeya dadantii of crude protein preparation were separated by electrophoresis in 12% sodium dodecyl sulphate polyacrylamide gels (SDS-PAGE) according to Laemmli (1970) at 150 volts for 90 minutes.Separated proteins are visualized by Coomassie Brilliant Blue R-250 staining.
The bacterial cells were prepared according to the initial fixation and dehydration steps previously described (Tung et al., 2005).The cells were fixed at 24°C for 60 minutes with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (20.15 g sodium cacodylate trihydrate, 0.1 mL HCl in 250 ml distilled water, pH = 7.4) and then dried by a critical point dryer (HCP2; Hitachi Company, Japan).The dried cell samples were coated with gold, and examined using a scanning electron microscope (S-4100, Hitachi, Japan).

Results and Discussions
In this study, we successfully obtain electrospun nylon-6/chitosan nanofibers with 8% (w/w) chitosan concentration of nylon-6 using Nanospider technology as a needless electrospinning technique in which we used up to 75 kV of electrostatic force.The main idea of increasing the chitosan concentration is to use its functional amino group as a carrier for antimicrobial agents.The antimicrobial agent was introduced via surface modification of the electrospun nylon-6/Ch nanofibers by treatment in aqueous solution of GTMAC at room temperature.The surface modification was confirmed by different methods including FT-IR spectroscopy, XRD, TGA as well as FE-SEM.

Scanning electron microscopy
Figure 1 shows the FE-SEM images of electrospun nylon-6/chitosan nanofibers before and after surface modification (nylon-6/Ch and nylon-6/HTCC), respectively.The images showed nanofibers with a smooth surface and uniform diameters along their lengths.The crystalline structures such as electrospun nylon-6/Ch and nylon-6/HTCC nanofibers were characterized by XRD, and the result was compared with that acquired from the pristine.The XRD patterns of the electrospun nylon-6/Ch nanofibers before and after surface modification are shown in Figure 2. The diffraction pattern of nylon-6/Ch nanofibers exhibited a narrow peak at 2θ = 21°.On the other hand, a peak at 2θ = 42° corresponding to the characteristic of the γ phase was also too feeble (Abel et al., 2002).This result clearly confirms that the surface treatment efficiently occurred via immobilization of quaternary ammonium salt group onto the electrospun nylon-6/HTCC nanofibers.

FT-IR spectra
The FTIR of the electrospun nanofibers as shown in Figure 3 showed a resonance band at 1,168 cm -1 characteristic of its saccharide structure (asymmetric stretching of C-O-C bridges).The bands at 1,645 cm -1 are assigned to C = O stretching of the secondary amide band (amide I) and the -NH 2 bending of the primary amino groups as well as the stretching vibrations C = O of polyamide.Peaks at 1,120 and 1,074 cm -1 were due to the skeletal vibrations involving the C = O stretching.The absorption band in the 2,856 cm -1 region is characteristic of the stretching vibrations -CH 2 -in polyamide backbone.The FTIR spectrum also shows an evidence for introduction of the quaternary ammonium salt group onto chitosan backbone; a new peak occurred at 1,462 cm -1 confirms the C-H bending of trimethylammonium group.The peak at 3,298 cm -1 is assigned to the hydroxyl group stretching.It should be also noted that the N-H bending at 1,541 cm -1 is assigned to the stretching of the secondary amine due to the change of the primary amine to the secondary amine.According to the FTIR spectrum, the epoxide group of GTMAC has reacted with the NH 2 groups rather than with the OH groups of chitosan.This confirmed the occurrence of the N-alkylation reaction in chitosan.

Thermogravimetric analysis (TGA)
The TGA thermogram of electrospun nanofibers before and after modification (nylon-6/Ch and nylon-6/HTCC) is shown in Figure 4.The thermogram of nylon-6/Ch shows a weight loss of 1.1 % in the range starting from 25 to 100°C due to the evaporation of the residual absorbed solvents, followed by slow weight loss of 7.6% in the range starting from 100 to 335°C due to the decomposition of polymer with low molecular weight chitosan, dehydration of the saccharide rings, depolymerization and decomposition of the acetylated and deacetylated units of the chitosan.In addition, a maximum weight loss was observed at the temperature range of 335-500°C which may be due to the degradation of nylon-6.On the other hand, the thermogram of nylon-6/HTCC shows similar results to those of nylon-6/Ch in addition to slow weight loss of 4.6% in the range starting from 100 to 223ºC due to the degradation of the immobilized active group.Moreover, the mat leaves a residue of 7.0-14.2%at 500ºC, whereas the significant weight loss at 422 and 366°C indicates the onset (T on ) of electrospun nylon-6/Ch and nylon-6/HTCC, respectively.Table 1 summarizes the TGA results of nylon-6/Ch and nylon-6/HTCC electrospun nanofibers.
The results confirmed the surface modification of amino group of chitosan with biologically active group which leads to the loss of thermal stability of nylon-6/HTCC compared to the original nylon-6/Ch.

Antibacterial assessment
The biological assessments explored by the disc diffusion method revealed that nylon-6/HTCC mat exhibited potential antibacterial activity against P. cichorii and D. dadantii.The reduction of the colony formation was observed 24 hours after treatment.The zone diameter was 20 and 22 mm, respectively (Figure 5).The control sample (nylon-6/Ch) exhibits no inhibition activity after checking bacterial activity inside inhibition zones near the disc.
Figure 5. Inhibition zones of nylon-6/HTCC mat against Dickeya dadantii (right) and Pseudomonas cichorii (left) by disc diffusion method on nutrient agar at 37°C.This leads to the general observation that Pseudomonas cichorii and Dickeya dadantii examined using SEM were totally deformed and exhibited severe destruction (Figure 6).In case of Pseudomonas cichorii, the surfaces of the bacterial cells were totally damaged.It was also found that the intact cells had a smooth surface, while most of the exposed bacterial cells exhibited severe destruction.Furthermore, the damage to the surface structure of Pseudomonas cichorii cells may, therefore, be the main reason for exposure by nylon-6/HTCC.
Nylon-6/HTCC exposed cells remained unlysed in suspension, in some cases of the cells.It was found that intact cells had a smooth surface with overall intact morphology.As regards Pseudomonas cichorii, it was observed that many cells were enlarged, elongated and highly irregular.However, a pronounced deformation and visible shrinkage were observed and these were mainly due to the binding of antimicrobial agents to the certain receptors of the bacterial membrane that leads to the disruption of the cytoplasmic membrane and thus inhibits the growth.The lethal action of polycationic biocides can be interpreted and identified as modes of action: (1) adsorption onto the bacterial cell surface; (2) diffusion through the cell wall; (3) binding to the cytoplasmic membrane; (4) disruption of the cytoplasmic membrane; (5) release of the cytoplasmic constituents such as K + ions, DNA, RNA; and (6) death of the cell (Katchalsky, 1964;Dizman et al., 2004).

Control
Nylon-6/HTCC Dickeya dadantii cells become rough and swollen, the structure of the cell wall surface layer was wrinkled, and round pores were partially deformed, indicating that the cytoplasmic structures were flushed out of the cells but they were unlysed.In fact, it is well known that bacterial cell surfaces are negatively charged.Therefore, adsorption onto the negatively charged cell surface (process 1) is expected to be enhanced with the increasing charge density of the cationic biocides.Therefore, it is reasonable to assume that process 1 is much more enhanced for polymers than for model compounds (Anguiz, 1989).A similar situation can also be expected in process 3 because there are many negatively charged species present in the cytoplasmic membrane, such as acidic phospholipids and some membrane proteins (Kenawy et al., 2002;Ghazzali et al., 2012).The disruption of the membrane (process 4) is a result of the interaction of the bound polymers with the membrane disruption and, therefore, is expected to be facilitated by increasing amounts of the bound polymers.other micro-organisms.Nylon-6/HTCC has been engineered to mimic antimicrobial peptides which are used by the immune systems of living things to kill bacteria (Emily et al., 2010).They are active against micro-organisms by interaction with the cellular membrane and aimed to kill micro-organisms (Haziza-Laskar et al., 1993).
In fact, polymers containing antibiotics are the ones of our most important weapons in fighting bacterial infections and have greatly improved the healthrelated quality of human life since their introduction.However, over the past few decades these health benefits are under threat as many commonly used antibiotics have become less and less effective against certain illnesses not only because many of them cause toxic reactions but also due to the emergence of drug resistant bacteria.It is essential to investigate newer polymers containing drug with lesser resistance.Systematic studies among various pharmacological compounds have revealed that any drug may have the possibility of possessing diverse functions and thus may have useful activity in medical and agricultural fields.Therefore, the results of the present investigation are successful in identifying new antibacterial activity of polymer and in ascertaining its value in the development of new anti-microbial materials.Finally, we present an appropriated chemical reaction for the possible surface modification which is illustrated in Figure 8.As shown in the figure, the quaternary ammonium salt group (-NH-CH 2 -CH(OH)-N + (CH 3 ) 3 Cl -) was introduced onto chitosan backbone during surface modification process.This proposed mechanism is in good agreement with the XRD and FTIR data as shown in Figures 2 and 3.

Conclusion
Thus, the study ascertains the value of the use of electrospun nanofibers which could be of considerable interest to the development of new antimicrobial materials for biomedical applications.SEM image of the affected microbes was totally deformed and exhibited severe destruction.Abnormal cell division was observed at high frequencies among cells that tried to divide in the presence of the polymer.Many cells were enlarged, elongated, empty ghosts, or fragmented, consistent with low viability of the modified technique of electrospun nanofibers.Therefore, this finding sheds light on the nanotechnology applications in the fields of medical and life sciences.Moreover, these adverse effects of nylon-6/Ch could be also used in general toxicology of nanoscale materials.

Figure 6 .
Figure 6.Scanning Electron Micrograph (SEM) demonstrating the effect of nylon-6/HTCC mat on Dickeya dadantii (a) and Pseudomonas cichorii (b).The arrows refer to deformation and visible shrinkage of the bacterial cell (a), and to total damage of the bacterial cell (b).The bacterial cells were taken from the affected cells on the treated bacteria cultivated in the Petri dishes.
* Represents degradation of chitosan in the temperature range of 100-335°C.