Antimicrobial Compound Cladding by Successive Ionic Layer Adsorption and Reaction (SILAR) Method for Antifomite Clinical Tools

Surgical site infections are a serious iatrogenesis caused by surgical fomites at large which should be culminated by finding proper solution [1]. Prevention of hospital acquired cross infection within the hospital is of a top priority as it is widely known that clinical pathogens are mostly multidrug resistant and also with high virulent factor [2,3]. The infection is caused by the catheters used for a longer period in dialysis procedures and in some cases permanent catheters known as permcath when contaminated spread dreadful bacteremia and peritonitis [4-6]. Post operative keratosis caused by the microkeratome blade infections were proven by many workers [7,8]. Affirming non infective surgical sites by specially focussing on the infections caused by the surgical blades which resulted in 80% contamination as reported in a prospective study carried out in a surgical centers [9]. Antifoul coating surgical blades with antibacterial substance would pave the way for the successful presurgical and post surgical management in mitigating the intricacies in surgical site recovery and precludes hospital acquired infections due to contaminated fomites [10].


Introduction
Surgical site infections are a serious iatrogenesis caused by surgical fomites at large which should be culminated by finding proper solution [1]. Prevention of hospital acquired cross infection within the hospital is of a top priority as it is widely known that clinical pathogens are mostly multidrug resistant and also with high virulent factor [2,3]. The infection is caused by the catheters used for a longer period in dialysis procedures and in some cases permanent catheters known as permcath when contaminated spread dreadful bacteremia and peritonitis [4][5][6]. Post operative keratosis caused by the microkeratome blade infections were proven by many workers [7,8]. Affirming non infective surgical sites by specially focussing on the infections caused by the surgical blades which resulted in 80% contamination as reported in a prospective study carried out in a surgical centers [9]. Antifoul coating surgical blades with antibacterial substance would pave the way for the successful presurgical and post surgical management in mitigating the intricacies in surgical site recovery and precludes hospital acquired infections due to contaminated fomites [10].
Infectious bacterial pathogens like Achromobacter xylosoxidans [11], Staphylococcus aureus [12], Pseudomonas aeruginosa [13], Salmonella typhimurium [14], Escherichia coli [13], Shigella flexneri and Klebsiella pneumonia [15] are gram negative bacterial pathogens which are potential threat posing secondary infection to the patients across the healthcare facilities. Recent approaches in bioprospecting new antibacterial small molecules and compounds from plants, bacteria, fungi etc. against the above mentioned pathogens have been proven to have antimicrobial activity [16,17]. Bacteria mediated infections on the surgical tools and implants were subjected for experiment with bactericidal substance coating such as antibiotic-loaded silk fibroin/ hyaluronic acid polyelectrolyte [18], titanium based on nanoporous silica and silver nanoparticles [19,20] gentamicin-loaded coating against S. aureus [21], layer by layer antibacterial polysaccharide-based coatings [22] immobilization and release of bactericidal substances such as metal derivatives and polyammonium salts were used as antibiotic coating [23] and application of furanones to screen pathogenic bacterial biofilm formation on the surfaces [24] was reported.
Thin film layering of the various molecules and quantum dots on the metals and successfully sensitising them using the successive ionic layer adsorption and reaction (SILAR) method [25][26][27] was studied recently in various metallurgic and chemical departments. But utilisation of this technique for the medical purpose is not substantial. Hence this present study focus on (1) isolation of anti-bacterial compound dipropyl-S-propyl ester (C4) from bacteria Exiguobacterium mexicanum (2) thin layer coating of the antibacterial compound on the catheters and surgical blades using the successive ionic layer adsorption and reaction method. (3) Bacterial colonisation evaluation of the coated materials. (4) Adverse drug reaction and affinity interactome.

Bacterial source and compound isolation
The clinical bacterial pathogens Escherichia coli strain ATCC 25922. The beneficial bacterium Exiguobacterium mexicanum (GenBank Ac No: KF471138) was isolated and from rhizosphere soil. 48 h culture filtrate of E. mexicanum was prepared and separated with the ethyl acetate in separating funnel individually in the ratio of 1:1 (v/v) followed by shaking for 30 min in a separating funnel. The extraction was repeated thrice and the solvents with the fractions were pooled and evaporated under vacuum using a rotary evaporator (Buchi type, Switzerland). The concentrated crude extract was mixed with chloroform-silica gel slurry and loaded onto a silica gel 60-120 mesh (E-Merck, Darmstadt, Germany) column, packed in ethyl acetate: chloroform: acetic acid (5:4:1) as the solvent system (the dimension of the column was 450 × 30 mm). One hundred tubes of 10 milliliters of the fractions were collected and analyzed by TLC. Fractions showing similar spots with the same R f values were pooled and concentrated by a speed-Vac under low pressure with an evaporating temperature of 40°C.

Characterization of the metabolite
The purified fractions were subjected to HPLC (on the bondapak column with a flow rate 1.5 ml/min and pressure up to 300 psi) using acetonitrile (9:1) as elutant to obtain the pure sample. The single fraction, which eluted was evaporated to dryness and subjected to Fourier Transform Infra-Red (FTIR). Infrared spectral data were measured on Perkin-Elmer 1600 series FTIR Spectrometer (Nujol, KBr disks). To determine the molecular weight, the samples were subjected to Electro Spray Ionization quadrupole Mass Spectrometry (ESIMS) Finnigan LCQ MS Detector. Source conditions were set as follows: Voltage 5 KV: nitrogen sheath gas pressure 60 psi: heated capillary temperature 200°C: full scan 50 to 2000 m/z. 13 C-NMR and 1 H-NMR spectra were recorded on a Bruker 300 MHz instrument using Tetra Methyl Silane (TMS) as the internal standard. Optical density was estimated using UV spectrometry (Shimadzu) Using the spectral data of FTIR, ESIMS, 1 H-NMR, 13 C-NMR and UV spectrum the structure of the active principles was determined.

Thin film compound layering by SILAR method
Successive ionic layer adsorption and reaction (SILAR) method was modified after [28,29]. The surgical blades (Surgeon -no: 23) and catheters (Dispovan) were immersed in 40% of compound C4 at 75°C for 15 minutes which acted as a precursor later dipped in deionised water for 15 minutes and followed by 15 minutes (S2) cation wash which was prepared by dissolving 0.16 M Na 2 S in deionised water. The cycle was repeated 20 times.

Antimicrobial analysis
Coated surgical blades and catheters using procedure mentioned above was analysed for coating properties on surgical blades only as the needles cannot be subjected for testing under atomic force microscopy-AFM (NTEGRA; NT-MDT, Moscow, Russia). Antibacterial activity of C4 coated and uncoated blades and catheters (negative control) were tested on an nutrient agar (NA) plate spread with 1µL of 0.2 OD 600 , surgical blades and catheters were incubated three replication batch for 1 hr, 2 hr and 3 hr per batch at 37°C. After incubation the coated and uncoated instruments were introduced into sterile nutrient broth and OD 600 bacterial growth was recorded and parallely plated on NA to estimate the CFU/ml to verify bacterial surface colonisation.

Adverse drug reaction (ADRs) analysis
The off-target adverse drug reaction on humans protein was screened using the insilico tool named DRAR-CPI [30].

Data analysis
All the parameters were replicated five times and activity data were subjected to Duncan multiple range test (DMRT) to draw relative statistical differences among the treatment using the SPSS 16.0 version (Statistical package social sciences, USA). Results at P<0.05 were considered to be statistically significant.

Bactericidal coating and antisurface colonising activity
Thin film coating of the compound was confirmed with AFM thickness record of 2.9 µm (Figure 1) with surface roughness average of 0.071 µm. The compound C4 had demonstrated significant antimicrobial and antisurface colonisation activity against the bacterial pathogens tested. Among the three incubation time there were no bacterial colonisation found in first two hours incubation of both coated catheters and surgical blades and the uncoated instruments have shown the bacterial colonisation with 3.5 ± 0.2 cfu/ml after 1 hr incubation, 3.5 ± 0.3 cfu/ml after 2 hr incubation and 4.5 ± 0.5 cfu/ ml after 3 hr incubation in case of uncoated surgical blades ( Figures  2 and 3). Whereas the uncoated catheters with 0.5 ± 0.3 cfu/ml after 1 hr incubation, 1.5 ± 0.3 cfu/ml after 2 hr incubation and 1.5 ± 0.5 cfu/ml after 3 hr incubation. Even though the coated materials did not show any surface colonisation for first two hours of incubation time both surgical blades (1.0 ± 0.1 cfu/ml in first wash and 0.5 ± 0.1 cfu/ ml in second wash) and catheter (0.3 ± 0.1 cfu/ml in both wash) have developed comparatively less bacterial colonisation.

Compound identification
Based on the polyphasic chemical characterization the compound C4 is identified as Dipropyl-S-propyl ester and its chemical structure was derived based on results of multi-technique analysis as follows, the RT value for compound was 3.0 in single peak in HPLC and the UV spectrometer reading for the compound 4 was 231 nm.

Discussion
Recent findings on the surface contamination of surgical equipments which potentially transmits the infectious pathogens within the clinical facilities. About 66 bacterial pathogens were isolated from 44 surgical equipment's with 0 to 296 colony-forming units predominantly grampositive cocci [31]. During surgical procedures, the instruments will penetrate the skin or the external layer before reaching the actual point of contact which facilitates these pathogenic transfer from the external surface to inside and spreads infection. In case of dental tools sterilization can be tedious due their micro structure and complex architecture like it is in dental burs and endodontic files [32].

Adverse drug reaction and affinity interactome
Putative target function of the important protein interactome inferred no adverse drug reaction and were presented according to the highest affinity (Figure 3) based on the docking score and -Z'-score for following human proteins 1. Aldose reductase which catalyzes the NADPH-dependent reduction of a wide variety of carbonyl-containing compounds to their corresponding alcohols with a broad range of catalytic efficiencies. -44.1354 and -2.36354 ( Figure 4).
2. Voltage-gated potassium channel sub unit beta-2 is an accessory potassium channel protein which modulates the activity of the poreforming alpha subunit. -43.5778 and -2.15928. Prevention of infection caused by the vascular catheters across the clinical procedures is felt very important by the physicians after witnessing the bacterial sepsis in patients [33][34][35]. Need for coating the antibacterial substance on the surgical tools to prevent the infections was illustrated by many researchers [36]. In the present study, we report the optimal surface coating methodology of the compound dipropyl-S-propyl ester and its antisurface colonising property against clinically important bacteria pathogens E. coli. Similar work was described earlier to prevent the surface bacterial surface colonisation by coating the clinical tools with plasma-sprayed silver-doped hydroxyapatite [37] which have proven resistance against the bacterial adhesion. Recently to curtail the implant associated infection, work related to biolfilm prevention on the medical oriented material surfaces is being carried out with the different types of nanoparticles [38] which was found to be active against Enterobacter sp. and Pseudomonas sp.

Ubiquitin carboxyl-terminal hydrolase 14 which is proteasome-
Instead of using the more toxic metallic substance for bactericidal coating, we have used bioactive compound isolated from the beneficial bacterium E. mexicanum in the present study. Many metabolites produced by the microbial agents which could be used as antibacterial materials such as siderophores, bacteriocines, lysosomes, proteases and organic acids are present in natural ecosystems. But there are no significant quantity of novel antibiotic discovery in recent past in correspondence with the evolving pathogenic strains. This is because of various challenges in the antibacterial drug discovery [39,40]. There were no major class of antimicrobials discovered between 1962 to 2000 [41,42] to combat the drug resistant pathogens. Only improved version of the available drugs were registered and released instead of screening for novel drugs which could prevent adverse drug reaction.
Adverse drug reaction is the major cause of deaths and economic burden in many developed countries which spends 15% to 20% of the hospital budget to treat ADRs [43,44]. About 408 ADR cases were recorded in South India in 12 month studies and most of the cases were rated to be preventable [45]. These can be prevented by using recent pharmacokinetics and insilico analytics [46,47]. The possible human protein affinity studies on the isolated compound dipropyl-S-propyl ester taken up in the present studies has shown its highest interaction potential with aldose reductase with highest docking score of -44.1354 and lowest score was found in macrophage migration inhibitory factor with -35.7365 docking score. All the intermediates were also evaluated and found no adverse drug reaction according to insilico analysis [48][49][50][51][52][53][54][55][56][57][58][59].

Conclusion
In recent past the technical advancement has increased the rate of the surgical procedures in saving lives. As many long duration surgical procedures were done in a shorter period, nowadays people's acceptance level to comply with surgeries instead of undergoing long term medication is higher. Along with this kind of clinical progress, there is also a demerit of cross infection because of the surgical tools. From the present study antifouling coating with dipropyl-S-propyl ester is a novel technique that had effective resistance against the clinical pathogens. This study suggests that the compounds isolated and coated may be potentially used by the clinical tool manufacturing industries to develop antifouling devices against bacterial pathogens.