*3.5. Quantitative Comparisions*

There is no general consensus evolved for the comparison of efficiency of antibacterial activity of polymers surfaces between the research groups. However, most studies on antibacterial activity are interpreted by the number of surviving colony forming unit CFU/mL<sup>í</sup><sup>1</sup> or per unit area. Kubacka *et al.* [42], studied the antibacterial effect of isotactic polypropylene (iPP) polymeric matrix incorporated with anatase-TiO2 against *Pseudomonas aeruginosa* (Gram negative) and *Enterococcus faecalis* (Gram positive). They reported a maximum reduction by *ca.* 8–9 log in 30 min in case of *P. aeruginosa.* Francolini *et al.* [19] evaluated the effect of (+)-usnic acid incorporated into modified polyurethane surfaces on the biofilm forming ability of *S. aureus*. After three days postinoculation, they found culturable biofilm cell concentration of *S. aureus* on the untreated polymer was 7.3 log10 CFU/cm2 compared to 0.9 log10 CFU/cm2 on the (+)-usnic acid-containing polymer. Cen *et al.* [20] introduced pyridinium groups at 15 nmol/cm2 on the surface of poly(ethylene terephthalate) (PET) film and demonstrated its bactericidal effect against *Escherichia coli*. Jansen *et al.* introduced silver ions by plasma-induced grafting onto polyurethane films which was found to reduce adherent viable bacteria from initial 10<sup>4</sup> cells/cm2 to zero within 48 h [43]*.* Jiang *et al.* [44] coated silver on silicon rubber substrates and showed decline in number of *L. monocytogenes* cells post 6 h. After 12 h, there was a reduction of over 2-log10 CFU/chip, and no viable bacteria were detected on both types of silver-coated SR after 18 and 24 h. Sambhy *et al.* [21] demonstrated antibacterial activity of composites consisting of poly(4-vinyl-N-hexylpyridinium bromide) (NPVP) embedded with silver bromide nanoparticles. They observed no biofilm formation on 1:1 AgBr/21% NPVP-coated surfaces after 72 h when incubated for 24–72 h with *P. aeruginosa* suspension (107 CFU/mL) in LB broth. Pant *et al.* [45] have demonstrated the ability to eliminate up to 99.9% of pathogenic bacteria on the surface of siloxane epoxy system containing quaternary ammonium moieties. In another work involving epoxy system, Perk *et al.* [46] observed fungicide, carbendazim supported on poly (ethylene-*co*-vinyl alcohol) and epoxy resin coating showed the antifungal activity contingent upon release from their polymer supports.

Coatings and thin films based on titania photoctalysts (Ag+-doped TiO2/Ag-TiO2/TiO2) that kills microbes under UV and visible light illumination, also have been actively investigated in recent years. Studies by Necula *et al.* [47], with TiO2-Ag composite coating prepared by plasma electrolytic oxidation on implantable titanium substrate, showed the ability to completely kill methicillin-resistant *S. aureus* (MRSA) within 24 h. In yet another investigation by Necula *et al.* [25], they examined the ion release and antibacterial activity of porous TiO2-Ag coating on biomedical alloy disk. Each evaluated samples could release 20.82 and 127.75 μg of Ag+ per disk and showed markedly enhanced killing of the MRSA inoculums with 98% and >99.75% respectively within 24 h of incubation, while their silver free counterpart sample allowed the bacteria to grow up to 1000-fold. The non-cumulative release of silver ions of 0.4 ppm, 0.26 ppm and 0.005 ppm for 1 h, 24 h and 7 days respectively after immersion in water, from nanometer scale Ag-TiO2 composite film was demonstrated by Yu *et al.* [34] and they also reported that 0.4 ppm released silver from Ag-TiO2 composite film is sufficient to cause almost 100% killing of *E. coli* when exposed to UV for 1 h. Studies by Jamuna-Thevi *et al.* [48], reported nanostuctured Ag+ doped TiO2 coatings deposited by RF magnetron on stainless steel, with overall Ag+ ion release measured between 0.45 and 122 ppb. They also noted that at least 95 ppb Ag+ ion released in buffered saline was sufficient for 99.9% of reduction against *S. aureus* after 24 h of incubation. Biological activity of silver-incorporated bioactive glass studies conducted by Balamurugan *et al.* [49] assessed *in vitro* antibacterial bioactive glass system elicited a rapid bactericidal action. Antimicrobial efficacy of these silver-incorporated bioglass suspension at 1 mg/mL for *E. coli* was estimated to be >99% killing, and the amount of Ag+ released from silver-incorporated glass was up to 0.04 mM after 24 h. In yet another study involving silver ions release by Liu *et al.* [35], the amount of silver released form the mesoporous TiO2 and Ag/TiO2 composites was measured to be 1.6 × 10<sup>í</sup><sup>8</sup> mol after 20 days. The photo-bactericidal activity on composite films was extremely high and displayed bactericidal activity even in the dark; they further reported that the survival rate was only 9.2% in the dark, and the *E. coli* cells were totally killed in UV light. Sun *et al.* [50] reported killing of bacteria on Ag-TiO2 thin film, even in the absence of UV irradiation against *S. aureus* and *E. coli* with significant antibacterial rate about 91% and 99% after 24 h respectively due to release of silver, and the concentration of silver ions released from the Ag-TiO2 film was 0.118 ȝg/mL during 192 h. Akhavan [51], reported that a concentration of 2.8 to 2.5 nM/mL completely killed 107 CFU/mL *E. coli* with visible light response photocatalytic Ag-TiO2/Ag/a-TiO2 material in 110 min. However, in most of the cases reports are based on planktonic studies and the release of silver is dependent upon the method employed for coating, thickness, conditions for gradient formation and silver source used. Nevertheless, release of silver ions frombare Ag*/*TiO2 composite layers reported above, obtained by methods *viz*., impregnation, deposition and nano-coatings gradually diminish over the time.

Bacterial biofilms are often more difficult to eradicate unlike planktonic cells. Until now, there have been very few reports that shown to resist biofilm formation on titania based polymer-nanocomposites. In one such study, Kubacka *et al*. [52] have demonstrated photocatalysis using ethylene-vinyl alcohol copolymer (EVOH) embedded with Ag-TiO2 nanoparticles (*ca.* 10<sup>í</sup><sup>2</sup> wt%) that showed outstanding resistance to biofilm formation by bacteria and yeast, upon ultraviolet (UV) light activation. In the present study, although the release kinetics of silver was not established but comparing to above studies which established the antimicribial threshold concentration of silver and efficacy of killing with different bare Ag-TiO2 (Ag/Ag-TiO2 nanofilms), the polymer composite system reported here which released 6.4 to 16.8 ȝg/mL of silver seems adequate [53], when the overall biocidal ability (to prevent bacterial attachment) of the composite during 48 h period in combination with radical-mediated photocatalytic action. Practically, the added strengths of the polymer-based Ag-TiO2 nanocomposite coatings as compared to bare TiO2/Ag-TiO2 coatings are its wear stability, flexibility, permeability and optical properties.

But the main objective of the disinfection technology in ensuring microbiological safety is to; set a standard for achieving a required logarithm of reduction of the microbial consortia. The microbial cells, which are not inactivated by the antimicrobial coatings adhering onto the testing surface over the different irradiation time, were able to grow on the agar plates. Quantifying their reduction in number (for quantitative assessment) of surviving CFU on a bactericidal surface compared with a non-bactericidal (neat epoxy) surface revealed reduction of microbial cells. In the present study, epoxy/Ag-TiO2 with 1.0 wt% loading was found to cause a reduction of CFU on agar plates by approximately 6-log in case of *E. coli* and the same effected *ca.* 4-log reduction in case of *S. aureus*  after 48 h of incubation, while epoxy/TiO2 with 1.0 wt% loading exhibited lesser inhibition of biofilm formation, see Table 2*.* 

There was an initial slower decrease in bacterial load by all the composites, *i.e.*, below 1-log reduction observed up to 18 h exposure followed by a rapid microbial decrease up to 6-log in 48 h for both 1.0 wt% of TiO2 and Ag-TiO2 loaded epoxy composites. Incomplete inhibition of biofilm formation was observed with lesser Ag-TiO2 loading, but complete inhibition of both *E. coli* and *S. aureus* was possible for composites with above 1.5 wt% of Ag-TiO2 after 24 h with UV irradiation. Strikingly, for the composite coating with 2.0 wt% epoxy/Ag-TiO2 showed highest antibiofilm effectiveness with 1-log reduction in 18 h, *i.e*., the shortest period with maximum inhibition. In addition, after 48 h of irradiation against both *S. aureus* and *E. coli* with very few surviving CFUs and complete inhibition (biofilm formation) and 7-log reduction was observed, relative to that in control plates as shown in Table 2. However, the present study results take into consideration only biofilm phase inhibition, and the obtained concentrations of range 6.4–16.8 μg/mL (ppm) Ag+ is very high (many times above minimum biocidal concentration levels) to radically prevent microbial cell viability. The polymer-based nanocomposite reported here obtained by dispersion of the Ag-TiO2 nanoparticles into epoxy manifest a real potential as photobiocidal coatings in a wide variety of settings that prevents biofilm formation by a wide range of Gram-positive and Gram-negative bacteria.


**Table 2.** Different nanocomposite materials and their antibiofilm efficacy for 18 and 48 h irradiation time.

a Percent reduction in biofilm formation as determined by Crystal Violet assay; b Mean value ± SD for the group Log10 reduction in CFU/plate.
