Anti-pathogenic efficacy of biogenic silver nanoparticles through adherence and biofilm inhibition in multidrug resistant ESKAPE pathogens

The current study aimed to produce AgNPs through a biogenic approach and assessed for their significant anti-pathogenic activities against multi-drug resistant ESKAPE pathogens. The biogenic AgNPs were synthesized through non-toxic manner and characterized by using UV-vis, XRD, DLS, TGA-DTA, FTIR, SEM along with EDX and UHRTEM were used to determine the absorption spectra, shape, size, thermal behaviour, functional groups, morphology, elemental constituents and defined particle size distribution profile, respectively. The AgNPs were evaluated for their anti-pathogenic effects against eleven strains of multi drug resistant ESKAPE pathogens by growth inhibition, biofilm adhesion, growth kinetics and Live/dead assays.Results The inhibitory range of AgNPs concentration was investigated as higher zones at escalating concentration (50 to 200 µg/ml). The growth kinetics of inhibition of all tested pathogens occurred after 4 hrs of treatment with AgNPs. Adherence assay exhibited highest inhibition in E. faecium (MCC 2763), P. aeruginosa (MTCC 1688) and E. species (MCC 2296) at 100µg/ml of AgNPs. The exposure of AgNPs increased the dead cell and consequently reduced cells density with AgNPs comparable with the effect of commercial antibiotics. The selected pathogens were found more sensitive to AgNPs than Cefotaxime/AgNO3 with the statistically significant (P < 0.05).Conclusion The emergence of drug resistance in ESKAPE pathogens are the extending reason for nosocomial infections, limiting the choice of antibiotics. Nanomaterials have been considered potential agents to prevent infections. Therefore, present study showed the broad spectrum potential and anti-pathogenic potency of biogenic AgNPs as an alternative to conventional antimicrobial agents. two significant


Background
Most of the chronic and opportunistic infections arise through health care units included pneumonia, bacteremia, urinary tract/gastrointestinal infections, osteomyelitis, meningitis and endocarditis [1]. The ESKAPE pathogens are the main agents in a nosocomial infection to spread in hospital settings, compelling the patient for a longer stay [2]. These pathogens are highly susceptible to genetic modifications leading to resistance and able to survive in harsh conditions. Most of them are having a tendency to form biofilms on various surfaces including catheters, eye lenses, thermometers and artificial heart valves [3,4]. The formation of biofilm on the medical devices leads to the development of chronic infections and more suitable with immune-compromised patients. Therefore, actions to reduce opportunistic infection or prevent the formation of biofilm should be directed by the alternative of the anti-adherent agents. The development of a biocompatible and cost-effective method are being considered to treat multi drug resistant (MDR) human pathogens, and are indispensable to save various lives [5]. The technology of extremely small things in the form of nano has been an unavoidable boon to humanity in therapeutics, pharmaceuticals and biocatalysis [6]. The availability of natural resources for the green synthesis of nanoparticles is very common like plants and their products, bacteria, algae, yeast fungi and viruses [7]. The most efficient method is bacterial mediated synthesis in which the genetic manipulation is also possible for the production of nonmaterial. In addition to this the high yield, low toxicity, less time consuming, cost-effective and its biocompatibility synergizes to its values [8,9].
Recent decade, AgNPs have led focus of the nano investigators and regarded as an emerging field of nanomedicine in targeting drug resistant pathogens to treat chronic infections/inflammations [10]. In the field of medicine, experimental reports showed that silver representing a broad-spectrum potency and effective against more than 650 pathogens [11]. The use of silver in the form nano enhances their multiple properties in a ubiquitous range of effectiveness. Generally silver nitrate is also effective against microbes but at higher concentrations while silver nanoparticles are enough to show its efficacy, owing to their low volume and a large surface area that are available to expose and bind with microbial populations [12]. The appropriate rating of AgNPs as antimicrobial agents can form one of the potential alternative strategies towards combating drug resistant microorganisms especially ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) describes a group of pathogens which are most difficult to treat with available antibiotics and responsible for hospital raises infections [13]. Based on the present scenario, AgNPs considered one of the most viable alternatives to available antibiotics because it seems to have a high potential with the possible mode of actions to solve the problem of multidrug resistance in several strains of the pathogens [14].
In this context, we reported a conventional method for the synthesis of AgNPs and evaluated their antimicrobial impression on ESKAPE pathogens. The efficacy of AgNPs was quantified and tested by well diffusion assay, anti-adherence assay, time kinetics growth inhibition and Live/dead assay against multi drug resistant ESKAPE pathogens. The biologically synthesized nanoparticles were characterized by UV-Vis spectrophotometer, FTIR, XRD, DLS, TGA, HRSEM with EDX and HRTEM analysis. Therefore, the present study is a naive attempt to develop a non-toxic based approach to cure the diseases caused by drug resistant pathogens. for the evaluation of AgNPs.

Screening of bacteria and biogenic production of AgNPs:
Most silver nitrate salt tolerant bacteria (AgTB), isolated from pond water, contaminated and polluted with the sewage and garbage. The screened bacteria were grown on assorted concentrations of AgNO 3 . A pure colony of AgTB (Bacillus cereus) was selected for the non-toxic biogenic synthesis of AgNPs [15]. The healthy colony of Bacillus cereus inoculated at 37 0 C, 100 rpm for 36 hrs in incubator Shaker (New Brunswick ™ Innova® 40, Eppendorf New York USA).
Then, the culture was centrifuged at 10000 rpm for 10 mins (Eppendorf 5804R Refrigerated Centrifuge). Whatman No.1 filter paper was used to obtain the final filtrate collection.
Bacterial supernatant and AgNO 3 was utilized to standardize the reaction parameters. The supernatants were taken in the different volumes (1,5,10,20,30,40, and 50%) and mixed with freshly prepared 1.5 mM AgNO 3 solution maintaining the total volume 100 ml in sterile flasks. In this non-toxic synthesis method, the supernatant-AgNO 3 reaction mixture was subjected to incubator shaker at 37 0 C. Different set of experiments were carried out and the aqueous extracellular material along with AgNO 3 solution was used as control up to the desired reaction period (12 hrs). The experiments were performed under 6 visually observation and photographed for changes in colour from light yellow to dark brown [16,17]. The colored solution containing AgNPs was centrifuged at 10000 rpm, 6 0 C for 10 mins followed by washing (thrice) with sterile UPH 2 O to remove biological interactive molecules. Finally, dry powder form of AgNPs was stored for characterization techniques and the antimicrobial applications. The UV-vis analysis was determined by sampling from the reaction mixture periodically, the measurement of spectra was recorded at the wavelength ranges between 350-800 nm. Double beam UV-vis spectrophotometer (Jasco V-730 from Jasco Corporation Tokyo, Japan) was used to record the spectra at different time points (0, 0.5, 1, 3, 6, 9 and 12 hrs) with varying conditions and parameters. The sterile UPH 2 O was used as a blank. The UV-vis spectra of the supernatant and AgNO 3 solution were also recorded [18].
For XRD measurements the crystalline nature of the AgNPs were subjected to XRD analysis by coating the dried powder on XRD grid. The spectra were recorded by XRD system (Rigaku Corporation, Tokyo, Japan) by using Scherrer's formula: D= 0.9λ/βcosθ operating at 40 kV and a current of 30 mA with Cu Ka radiation. The diffracted pattern was scanned in 2θ ranges from 20° to 80° [19]. Dynamic light scattering measurements were carried out by using DLS (Zetasizer Nano ZSP, Malvern-Worce, UK). The reference dispersive medium, ultrapure water used as a dispersive medium with a refractive index of 1.330, a viscosity of 0.8872 cP, and PDI were analyzed in acquisition time 60 s, at 25 °C. Three measurements were performed and the average particle size of AgNPs calculated [20].
Thermal gravimetric analysis (TGA) was used to determine the thermal stability of surface capped AgNPs and its fraction of volatile components by monitoring the weight loss that occurs as the sample is heated. The sample was subjected to TGA using (TG/DTA 6200 SII Nanotechnology, Japan) instrument over a temperature range of 30-800 0 C at a heating rate of 20°C/min under nitrogen gaseous atmosphere [21]. FTIR analysis carried out find the possible functional groups responsible for surface capping of AgNPs in biomolecules present in bacterial supernatant. The measurement of spectra at a scanning speed 2 mm/sec in transmittance mode from 400 to 4000 cm -1 at 4 cm -1 resolution with 32 scans by using (JASCO-FTIR 6300 Type A, Tokyo, Japan) instrument.
The morphological evaluation of biosynthesized AgNPs was carried out by using

Adherence inhibition assay-
The assay was performed to determine the adherence pattern inhibition of ESKAPE pathogens by AgNPs on the glass surface. Young cultures, grown over night containing 10 7 CFU/ml were added to glass test tubes at different concentrations of AgNPs (5, 25, 50 and 100 μl/ml) and maintained a total volume 5 ml, in each test tube including positive (Hydrogen peroxide) and negative (sterile Viability Kit (Thermo Fisher Scientific), for quantitative assays [26]. The wells were filled 200μl of PBS (1X) by adding 0.1 μl stain in each well and kept for 1 hr in an aseptic condition to quantify the live/dead cells. Multi-detector microplate reader -VICTOR™ X3 (PerkinElmer) was used to measure the fluorescence intensity. The wells containing untreated cells were considered as a control.

Statistical analysis:
In the present study, the results of all experiments were carried out in triplicate and the means along with ± SD of the data sets were calculated in duplicates and employed. The appropriate statistical proficiencies were adopted by using  UV-vis spectroscopy analysis represents the pictures and graphs which are showing that the supernatant and AgNO 3 mixed in a set (1,5,10,20,30,40, and 50%) of experiment displayed a gradual change in their appearance. A UV-vis spectra shows a strong peak at 428 nm is imputed to the surface plasmon resonance of AgNPs. The intensity of the functionalized AgNPs change in colour was found in an increased manner with the contact time, the sharper peak was observed. The reduction gradually started after 0.5 and 1 hr, and small peaks started to appears at 424 nm. A shift in peaks was also reported from 426 nm to 448 nm but after the reaction period of 3 and 6 hrs. The maximum colour intensity was attained after 12 hrs and the maximum absorbance reported at 428 nm and there was no shift observed in peaks upto ending of incubation period. Bacterial supernatant and AgNO 3 solution was also scanned individually but there no any peak observed Figure 1 SEM analysis of AgNP showed in micrographs of the dried silver nanoparticles which expresses morphology and size distribution of nanoparticles Figure 4 (A). In SEM analysis, the images elucidating the particles are predominantly irregular, cuboids and spherical in shape and aggregates into larger particles with no well-defined morphology was observed.
The estimation of elemental composition and the purity was determined by EDX analysis.
The strong signals of capped AgNPs were observed from the silver atoms at 3 KeV, which indicates the crystalline property of silver nanocrystals Figure 4 (B). The determination of the mass of total product was estimated as the strong signal for Silver (88.62%) and weak signals of Carbon (6.14%) Oxygen (2.57%) and Sulphur (2.67%) Figure 4 (C).
In UHRTEM analysis, the microgram of UHRTEM exhibit pleomorphic morphology including rectangular and oval shape but the majority of particles are in the spherical shape with a smooth surface Figure 5

Discussion
Bacterial mediated AgNPs applications is a relatively growing field and have a great attention to rid off exiting toxicological consequence and also to increase attention towards the nanotherapy. The procedure for biogenic production of AgNPs was standardized with the UV-vis analysis, in which the spectra were recorded time to time to represent the approaches. The change in colour was due to the excitation of surface plasmon resonance by newly formed nuclei within 30 mins in the mixed solution reveals the reduction of silver ions into silver nanoparticles [27]. The formation of AgNPs in an aqueous medium was confirmed by the UV-visible spectra and the maximum colour intensity was attained after 12 hrs. The change in colour was due to reduction of silver ion by the biomolecules and enzymes present in the cell-free extracts [28]. The presences of free electrons in silver nanoparticles are responsible to raise absorption band with the combined vibration of electrons of metal nanoparticles [29]. The reaction time completed in 12 hrs and spectra were becomes sharper which indicate the formation of mono dispersed nanoparticles [30]. Based on enzyme kinetics action it is clear that the highest optical density found upto at 1.5 mM by attending high intensity of reduction while beyond that of the concentrations of silver nitrate shows gradually decreased bio-reduction of particles [31].
The synthesized AgNPs were characterized by XRD to determine the translational symmetry-size and shape incurred from the peak positions of diffractograms pattern. The XRD confirmed the synthesized particles as metallic silver and Braggs's reflections of silver nanocrystals which are already reported [32]. The XRD results showed typical pattern of planes found to report the face centred cubic (fcc) silver, respectively [33].
Thus the XRD analysis strongly suggested the crystallinity of AgNPs [34]. In DLS analysis, the high intensity distribution at a lower range of particle size and a single peak indicates the lower range of size and the quality of the particles respectively [35]. The TGA results shows loss of 8.4% up to 300°C and 10.6% up to 800°C. DTA plot gives information about the simultaneous occurrence of complete thermal decomposition and crystallization of the sample. An approximate of no weight loss was observed below 200°C which can be largely linked to the evaporation of water and organic components [36]. The presence of an exothermic peak between 200°C and 400°C in the DTA plot can be associated with the crystallization of silver nanoparticles [37].The FTIR analysis of the powder sample used to investigate the action of biomolecules and responsible factor for capping and stabilization of nanoparticles. In current study, FTIR peaks correspond to alkyl halides (C-Cl and C-F) responsible to make compounds bioactive in a medium, Amine (C-N) responsible for increase polarity, reactivity and labeling of peptides/proteins [38]. Aromatics (C=C) substances contain alternating single and double bonds in its chemical structure so these bonds may break with the extracellular materials and produces odor in medium and forms a stability with the particles [39]. Ketonic group (C=O) the group refers to amino and amino-methyl stretching groups of protein. The presence of carbonyl groups of the amino acid residues and the peptides have a strong ability to bind to the silver [40], alkynes (C≡C-) contributing in tagging of biomolecules including proteins and lipids [41], nitriles (C≡N) helping in making up a proper structural format of the synthesized nanoparticles with vibrational probes of proteins [42], alkanes (C-H) responsible for saturation and instauration of molecules and also reported that the proteins can bind to AgNPs either through free amine or cysteine groups in proteins present in medium [43], basically these are enzyme-mediated and produced by bacteria [44], amides (N-H) responsible for increase polarity and labelling of peptides/proteins [38] alcoholic group (O-H) and carboxylic acid (O-H) an active role played by the groups in reduction of metal ions and oxidation of biomolecules, followed by formation of nanoparticles [45] respectively.
Finding of these groups in medium confers the stability of the synthesized AgNPs for a period time and keeps functional in both wet and dried conditions [46]. Intermolecular forces are another factor may involve in prevention of nanoparticles to aggregate in the medium. The possible interactions between silver and bioactive molecules and capping agents could be responsible for the synthesis and stabilization of AgNPs [47].
Biomolecules present on the surface of nanoparticles leads to agglomeration structure. In SEM measurements, on the surface of slide the larger view of silver particles may be due to the aggregation of the smaller ones. Similar image illustrations were also reported by Vibrio alginolyticus mediated synthesis of AgNPs [48].The weak signals were also observed during the EDX analysis, strongly suggested the capping of the particles with indication for the presence of biomolecules released in bacterial supernatant contributed in stabilization of the particles [49]. SEM images shows pleomorphic morphology of particles and expressing some difference in size with DLS results, the variation in size and shape may be due to the dispersion and various biological reductants used in the synthesis process [50]. Through TEM images, biological component as a capping were spotted which can be observed as ring masks on the surface of particles. The size measured through DLS is slightly larger than the size evaluated by TEM analysis because of bio-capping on their surface [51]. Similar studies have been done with MDR strains by using biosynthesized nanoparticles [52]. Therefore, the small amount of AgNPs can be rid off MDR pathogens and may prove an effective besides combating the problematic pathogenic microorganisms [10]. The anti adherence effect of antimicrobial agent on any surface directly affects the accumulation of microbes and biofilms formation which can be reservoirs for pathogenicity. The anti-adherence potential of nano materials may be attributed to their ability to inhibit the attachment of bacteria on a glass surface [54]. The anti-adherence behavior of AgNPs suggested that this could be useful for the development of biologically functionalized silver nanoantibiotics [55].
In live/ dead staining assay, the biofilm was grown upto 72 hrs and stained with LIVE/DEAD BacLight TM Bacterial Viability Kit and treated with increasing concentrations of AgNPs, the SYTO-9 dye specific for live cells to provide substantial resolution between live and dead cells population [24]. The impacts of biogenic AgNPs on MDR pathogen biofilms formed by ESKAPE pathogens showed statistically significant depletion in their population.

Conclusions
The biogenic synthesis of AgNPs presented structural uniquity with specific binding sites on their surface to interact with the complimentary object. In this context, AgNPs were