Enzyme-mediated formulation of stable elliptical silver nanoparticles tested against clinical pathogens and MDR bacteria and development of antimicrobial surgical thread

The silver and gold nanoparticles have been largely explored in biomedical sector due to its great potential as a broad spectrum antimicrobial agent [1–4]. Despite of its wide use, antibacterial activity of silver nanoparticles is poorly understood with few implicit mechanisms including (1) generations of reactive oxygen species (ROS) intracellularly and/or extracellularly, (2) cellular uptake of silver ions (3) cascade of intracellular reactions and (4) interactions between nanoscale silver and cell membranes [5, 6]. Recently, Hsueh et al. [7] reported the release of Ag+ ions intracellular or extracellular from the AgNPs, highlights the precise antimicrobial mechanism. Based on these mechanisms, AgNPs have been employed and established as an antibacterial agent in wound treatment, medical devices, cosmetics, water purification, air treatment and clothing [8, 9]. Presently, >30% of AgNPs are utilized by the healthcare sector due to impressive demand of antimicrobial consumable stuff. Thus, the growing demand of antimicrobial materials in healthcare applications is projected to drive silver nanoparticles market to reach USD 2.45 billion by 2022 (http://www.grandviewresearch.com/press-release/global-silver-nanoparticles-market). This ongoing demand of the AgNPs will promote the exploration of various new synthesis routes.

Nanobiotechnological production of nanoparticle using biomolecules offers the characteristic of specificity, benign nature, viability and conceivability for functionalization [2]. Biocatalysis dependent nanoparticles synthesis was found to be more pharmacologically active than chemical route formulation and holds promise for amended biocompatible nanoparticles production [10]. Few green approaches have been tested for metal nanoparticles synthesis, which includes the use of plant extract, enzyme, biomolecule, biosurfactant and microorganisms [11–15]. In vitro synthesis process is new developed concept which eliminates the culturing of the microorganisms and extraction of their metabolites/macromolecules. Several enzymes have been explored for lab scale as well as bulk production of AgNPs including keratinase, nitrate reductase, alpha-amylase and sulfite reductase [16–19]. Enzyme-mediated nanoparticle formation is widely utilized methods as it is simple, eco-friendly, inexpensive and easily scaled-up process for bulk production of functionalized and stable nanoparticles [16–18]. The size and shape controlled synthesis of the AgNPs is the key issues to determined their efficacy and applications. Such controlled synthesis of AgNPs can be formulated through the optimization of process variables including pH, temperature, reaction time, enzyme concentration and substrate concentration.

Biosynthesis of the size and shape controlled nanoparticles are yet the challenging task for the nanotechnologiest. The size of the AgNPs determines their effectiveness against pathogenic bacteria. Smaller than 50 nm of AgNPs are reported as a best antimicrobial weapon against bacterial pathogens [20]. The shape of AgNPs also play the significant role to kill the microorganisms, sphere-shaped nanoparticles are considered to be the best-suited for antimicrobial applications in either colloidal form or immobilized state [21]. Elliptical or oval AgNPs may also more antimicrobial than other reported shape due to more surface area to interact with cell membrane and ease to inter into cell. Morphological variation of the AgNPs is not only the determinant of microbial susceptibility, but the microbial cell structure and type (i.e. Gram positive and negative), are also play a significant role [22]. Earlier studies of AgNPs as bactericides have been largely explored through the model microbial species—Escherichia coli [23]. The outputs of such experiments are not adequate to confirm the AgNPs as antimicrobials. Additionally, the susceptibility of MDR bacteria against antimicrobials is very negligible than the non-MDR bacteria. So the multiple non-MDR and MDR bacterial species need to test against the AgNPs to establish it as antimicrobials.

Currently, the outbreak of various MDR strains globally at the alarming rate resulted in treatment difficulties which have imposed a burden on health care systems and simultaneously intensifying the need for new antimicrobial agents [24]. Recently, WHO list out the 12 MDR bacterial species (http://www.who.int/mediacentre/news/releases/2017/bacteria-antibiotics-needed/en/) that pose a greatest threat to human health suggest the prompt need of new therapy to manage this antimicrobial crisis. The discovery of the new antimicrobials is quite slower than the emerging rate of the antimicrobial resistance among the bacteria. Provisionally, the use of the metal nanoparticles to achieve the success in antimicrobial crisis can be a viable option. Due to the interaction of nano-silver with multiple microbial target sites, it is expected to be feasible and promising alternative against most common MDR strains such E. coli, Staphylococcus aureus and Streptococcus pneumoniae. Moreover, the synergistic activity of AgNPs coupled with antibiotics is also an advisable approach to combat against various MDR strains. Therefore, stable AgNPs are effective bactericidal materials due to its combined effects with antibacterial agents and are the robust ammunition against the MDR strains [25].

In present research, attempt was made to in vitro synthesis of AgNPs using macerating enzymes. AgNPs characterization was investigated to confirm the size, shape, and nature. The antimicrobial activity of AgNPs was studied against pathogenic bacteria, yeast and molds. The key investigation was carried out by testing the antimicrobial activity against three MDR bacterial species E. coli, S. aureus and S. pneumoniae.

AgNO₃ reduction and AgNPs formation was found to temperature and time dependent. The increase in the reaction temperature and the time, increase the accessibility of the silver ions for the rapid reduction. The synthesis of AgNPs at elevated temperature is due to the accessibility of thiol groups of macerating enzymes. High temperature unfolds the structure of enzymes and makes an easy availability of thiol groups for the bioreduction of metal (i.e. silver) [27, 28]. However, there are many possibilities for reduction using enzymes. The optimum temperature was 80 °C and time was 8 h. The further increase up to 90 °C caused the broadening of the peak suggesting the bigger and heterogeneous particles formation that led the decrease in absorption [29]. The sharpness of the absorbance peak is dependent to size of the AgNPs because the particle size may be smaller at the higher temperature consequently narrow and sharp SPR band of AgNPs was observed [30]. Beside temperature, reaction time is another factor to determine the controlled synthesis. The steady increasing of absorbance spectrum with increasing the reaction time resulted in the colorless reaction mixture shifts to pinkish-orange with the duration of incubation indicates the increased concentrations of the AgNPs. Thus, the rate of synthesis can be accelerated by increasing the temperature and contact time [31].

Size distribution profile of the colloidal nanoparticles showed narrow monodispersity with an average size of 63.65 ± 12.71 nm by DLS. Nucleation of the silver ions and growth of nanoparticles is the key processes to determine the size. In biosynthesis route the amino and carboxyl group are usually involved into nucleation process. However, the mechanism of biogenic AgNPs is poorly understood. Earlier reports of enzymes assisted AgNPs synthesis suggested the involvement of various amino acids and their functional groups for the AgNPs formation [32, 33]. The result of EDX proved the presence of nearly 40% silver and 60% other trace metals (O, K, Cl) by weight; this is primarily due to the biogenic root of synthesis. Biological derived impurities like carbon, oxygen, potassium are usually involves in stabilizing the particles by capping on the surface of AgNPs [34]. Absence of oxides indicated the sample contain pure silver. TEM image confirmed elliptical shape and average size of particles is 38.26 ± 0.4 nm. TEM also indicate the good stability of particles with negligible aggregation after 1 year may be due to the presence of Cl ions. The XRD pattern reveals the AgNPs correspond to face-centered cubic (fcc) crystalline phase. Few minor unassigned peaks (*) were due to the formation of the crystalline bioorganic compounds by the enzyme [35].

Antimicrobials resistance (AMR) in pathogenic microorganisms is ever challenging global issue. A wide range of antimicrobial compounds and their combination are used to control AMR in the MDR bacteria and the searching of new antimicrobials for MDR species are yet ongoing practices. One of the best substitutions to combating the resistant problem in different microbes is use of metal nanoparticles due to the multifaceted antimicrobial mechanisms of metals nanoparticles against wide range of microbes [36, 37]. Silver is known to be used for centuries as a potent antimicrobial weapon because of its growth hindrance abilities against a diverse microorganism by exerting its effect at multiple site. AgNPs synthesized by various routes and of different size and shape was tested against diverse clinical pathogenic MDR strains [4, 8, 12, 13, 16, 25, 31]. Due to failure of first and second line antibiotics, the exploration of AgNPs against various MDR strains is the new emerging trend. These suggest the further exploration of silver or other nanoparticles for treatment of various MDR, extensively drug-resistant (XDR) and pan drug resistance (PDR) pathogens. The remarkable antimicrobial activity of 2.1 µg of AgNPs was reported against S. aureus, B. subtilis, P. aeruginosa, P. vulgaris, M. racemosus, P. chrysogenum and R. stolonifer. The minimum inhibitory concentration of AgNPs is found to be varies in literature and it depends on coating medium, route of synthesis, types of strains, different size and shape of nanoparticles [4, 8, 16, 30, 38–40]. Result of antimicrobial activity against E. coli ESBL positive, S. aureus (MRSA) and MDR S. pneumoniae were also very excellent. The antimicrobial activities against all the Gram-negative and Gram-positive bacteria are more than 12.50 ± 0.5 which is far better than the earlier report [20]. The toxicity of AgNPs is due to the release of intracellular Ag ions in bacterial cell but its orders of magnitude is different into different bacterial species because the intracellular bioavailability of Ag ions liberated from AgNPs is bacterial strain-specific [41]. Moreover, extracellular secretion i.e. organic acids, peptides, biosurfactants and cellular uptake of Ag ions via cell-nanoparticles interaction my affects the toxicity. Additionally, the AgNPs-cell contact is the key phenomenon to determine the toxicity of nanoparticle [41]. The formation of biocorona by hydrogen bonding and hydrophobic interactions between cellular peptides/proteins and AgNPs may varies on cell to cell, consequently the toxicity will be varies among the different microbial cell [42]. Thus, the binding behaviors and kinetics of AgNPs with proteins are important aspects in realizing the toxicity of AgNPs in biosystem [43]. The size and shape determine the surface-to-volume ratio of AgNPs and impacting on its effectiveness. AgNPs are readily interact with organic materials and mounted in fabric for slow release to exert their long-lasting antimicrobial effect. Thus, the safe use and enduring antimicrobial properties can be achieved by tethering the AgNPs on organic nanosheets [44]. Antimicrobial property of nano-coated surgical threads is a key outcome of the present study which is promising tool to control the spreading of MDR infections over a long time. The nano-silver coated nylon thread is useful in the healing process as AgNPs not allows the tissue adherence and thereby helps in healing process [45, 46]. The antimicrobial dressing materials developed through the tethering of the AgNPs can be easily use for biomedical application.

Although, the AgNPs are broad spectrum antimicrobials, it is hard to conclude using clinical and laboratory control strains that it can be substitutes the antibiotics as their toxicity is still a big challenge for their clinical and biomedical applications. Furthermore, there are few controversies related to safe use of AgNPs in human diseases treatment and health care. In the present study, use of only three MDR strains may be inadequate and in future we will conduct the study with few more MDR strains. Nevertheless, the study provides the baseline information to develop the prototype for antimicrobial medical materials.

Macerating enzymes are good reductant of AgNO₃ for in vitro synthesis of silver nanoparticles. The adopted process was environment-friendly, simple, rapid, and easy to perform. Bioformulated AgNPs were well dispersed, small in size, elliptical and stable over a year suggest promising application in the biomedical and healthcare sector. Synthesized AgNPs showed antimicrobial activity against various non-MDR and MDR bacteria, yeast and molds. This study confirms the biologically synthesized AgNPs are safe and viable option against pathogens. So it is proved that the AgNPs has a great potential to be used as an antimicrobial agent. The nano-silver treated nylon thread can be useful especially for the stitching the surgery cuts to avoids the secondary infection.