Int. J. Life. Sci. Scienti. Res., 4(4): 1897-1904, July 2018

Studies on Bacterial Synthesis of Silver Nanoparticles and its Synergistic Antibacterial effect with antibiotics against Selected MDR Enteric Bacteria

Payal Agrawal^1*, Nikhilesh Kulkarni²

¹Microbiology Research Laboratory, Department of Microbiology, R. A. College, Washim-444505 (M.S), India

² Head, Department of Microbiology, Microbiology Research Laboratory, R. A. College, Washim-444505 (M.S), India

 

Address for correspondence- Dr. Payal N Agrawal, Research Student, Department of Microbiology, Microbiology Research Laboratory, R. A. College, Washim-444505 (M.S), India

 

ABSTRACT: In the present study, the extracellular synthesis of Silver nanoparticles was done using two different bacterial strains viz. Bacillus flexus and Bacillus pseudomycoides. The silver nanoparticles were confirmed by in color test and characterized by UV-Visible spectroscopy, the λ_max observed at 430 nm and 410 nm confirmed the synthesis of AgNPs. FTIR analysis confirms the presence of elemental silver and reveals the dual function of the biological molecule responsible for the reduction and stabilization of AgNPs in the aqueous medium. The XRD showed that silver nanoparticles produced are crystalline in nature with size ranges from 30 to 70 nm. The SEM shows that produced silver nanoparticles are spherical, Pseudo spherical in shape with traces of agglomeration. Further through investigation of Antibiotic Sensitivity/Resistant pattern expressed that out of eighteen virulent enteric bacterial isolates, three isolates showed MAR index equal to 1, which indicates the presence of multiple drug resistance (MDR). MIC values of AgNPs against MDR isolate E7 and K3 was established to be 80 μg/ml whereas, for isolate Sa1 the MIC value was 70 μg/ml. The synergistic effect of antibiotics in conjugation with biologically synthesized AgNPs encourage the susceptibility amongst the tested bacterial cultures; viz. Salmonella followed by Klebsiella and E. coli.

 KEYWORDS: Biosynthesis, Synergistic activity, Antibacterial activity, Silver nanoparticles, Multidrug-resistant (MDR)

 

INTRODUCTION- Silver nanoparticles are having great interest today due to its different properties such as good conductivity, chemical stability, catalytic and antibacterial activity. Nanotechnology provides a good platform to modify metal in the form of nanoparticles. An important area of research in nanotechnology is the biosynthesis and Characterization of nanoparticles such as nanosilver. It was reported that highly stable silver nanoparticles (40 nm) could be synthesized by bioreduction of aqueous silver ions with a culture supernatant of some nonpathogenic and pathogenic Bacteria ^[1]. Silver nanoparticles (AgNPs) have emerged as an arch product from the field of therapeutic nanotechnology.  Resistance in human pathogens is a big challenge in pharmaceutical and biomedicine. The present study is focused on antibiotic-resistant enteric bacteria as these represent the most immediate urgent global concern ^[2,3]. Enteric diseases are among the most common causes of morbidity and mortality in low-income nations, strangely affecting children under the age of five ^[4]. The Silver-Nanoparticles against Multidrug-resistant enteric human pathogens have received minor attention by means of published citations. Hence, the biosynthesis of silver nanoparticles from bacteria with special reference to Potentiation of antibiotic activity against Multidrug-resistant enteric human pathogen has investigated.

 

  MATERIALS AND METHODS

Synthesis of silver nitrate reductase enzyme- The silver nanoparticles were synthesized from two different silver-resistant bacterial isolates viz. Bacillus flexus, Bacillus pseudomycoides ^[5]. Intended for the biosynthesis of silver nanoparticle, the bacterial cell-free extract was prepared by separately inoculating the bacterial isolates in 100 ml LB broth followed by shaking incubation at 220 rpm for 24 hours. The cell free extract was separated by ultracentrifugation at 20,000 rpm for 10 minutes and use as a crude source of reductase enzyme for the extracellular synthesis of nanoparticles.

 

Biosynthesis of silver nanoparticles ^- In a typical biosynthesis production scheme of silver nanoparticles, 2 ml of reductase enzyme was mixed separately with 100ml of 1mM aqueous solutions of filtered sterilized AgNO₃, in 250ml conical flasks and the reaction mixture was further incubated on incubator shaker at 150 rpm (Remi make) at 37˚C up to 72 hours and allow for reduction. The set without AgNO₃ was maintained as Control. The work was done adopting the method suggested by Das et al., (2013) with slight modifications ^[5].

 

Purification of silver nanoparticles- The silver nanoparticles were purified by three successive ultra centrifugations at 20,000 rpm for 15 minutes at 40˚C the supernatant clear suspension was redispersed in sterile deionized water to remove the residual biological molecules. The process was repeated thrice for complete removal of redundant residual entities from the silver nanoparticles. The purified solution was then dried to form the powder using hot air oven at 60˚C for overnight ^[6].

 

Characterization of silver nanoparticles- The dried powder of Silver nanoparticles was then mixed with 10 ml of deionized water and kept on a sonicator to prevent aggregation of molecules and further Characterized by UV- Visible spectroscopic analysis; FT-IR analysis; XRD analysis, and SEM analysis.

 

Antibacterial activity of Silver Nanoparticles against MDR Enteric bacteria

Isolation and Identification of Enteric Human Pathogens- The isolation of pathogens was done for three consecutive years on selective as well as differential enteric media. Frequently reported enteric human pathogens viz. E. coli, Klebsiella, Salmonella, and Shigella species were isolated from urine, stool and sewage samples respectively ^[7]. All the isolates were further screened for the virulence by India ink degradation. The obtained virulence strains were identified by the conventional method. The multidrug resistance strains were screened adopting antibiotic susceptibility test ^[8]. The assays were implemented in triplicate and expressed in terms of central tendency. The S/R blueprints of the isolates were determined by comparing the values of inhibition zones with “Disc diffusion supplemental table” ^[9] MAR (Multiple antibiotic resistance) indexes were calculated by standard formula ^[10]. The isolate showing MAR indexes equal to 1 were selected for further analysis.

 

MAR Index = Number of antibiotics to which isolates showed resistance/ Total number of antibiotics tested

 

Independent and Synergistic Antibacterial activity of Silver Nanoparticles- Standard stock solutions of different concentration (100 μg/ml–10 μg/ml) of obtained silver nanoparticles were prepared. The control was used as autoclaved deionize water. The suspensions were sonicated for 20 minutes to avoid deposition of AgNPs and use for disc impregnate. The AgNPs impregnated discs were placed aseptically on MH agar plates speeded with test pathogens and incubated at 37˚C for 16 to 18 hours. Post incubation, the zone of inhibition was measured and MIC of AgNPs was determined. Assays were implemented in triplicate and expressed in terms of central tendency.

For determining synergistic effects, each standard antibiotic disc was impregnated with respective MIC of AgNPs against test MDR bacteria viz., E. coli (E7), Klebsiella (K3) and Salmonella (Sa1). The impregnated discs were placed aseptically on MH agar plates speeded with test pathogens and incubated at 37˚C for 16 to 18 hours. Further, the zone of inhibition was measured as mm diameter. The assays were implemented in triplicate and expressed in terms of central tendency. Both the readings obtained were then compared and expressed in terms of fold area increase in antibacterial activity, by using the formula ^[11]. Where a and b are the zone of inhibition (mm) obtained for antibiotic alone and antibiotic in combination with AgNPs, respectively.

Increase in fold area = (b² - a²) / a²

 

A and b= Zone of inhibition (mm)

 

RESULTS

Biosynthesis of Silver Nanoparticles- The isolates Bacillus flexus and Bacillus pseudomycoides showed the reduction of Ag+ ions, since, visualizing the change in color from colorless to dark brown. The results revealed the possible use of the bacterial strains for rapid synthesis of Silver nanoparticles hence conceivably to be used in biosynthesis process for large-scale production.

 

Characterization of synthesized Silver Nanoparticles- The purified dried AgNPs powder samples viz. AK1 and AK2 were characterized by means of UV-Visible spectrum graphically represented in Fig. 1. During, which two strong peaks were observed at 430 nm and 410 nm which confirmed the synthesis of AgNPs ^[8].

 

         

Fig. 1: UV-Visible absorbance spectra of synthesized silver nanoparticles

The results of FTIR for two AgNPs samples (viz. AK1 and AK2) were represented in Fig. 2, the bands obtained at 591.86 cm^-1and 577.46 cm^-1. Hence the FTIR analysis confirms the presence of elemental silver, ^[12].

Fig. 2: FTIR Spectrum of Silver nanoparticles synthesized from Bacillus flexus and Bacillus pseudomycoides

 

The XRD pattern obtained for two AgNPs samples (viz. AK1 and AK2) were represented in Fig. 3. Comparisons of XRD spectrum with the standard powder diffraction card of Joint Committee on Powder Diffraction Standards (JCPDS), silver file No. 04-0783, confirms that the silver nanoparticles found in the present study were in the form of nano-crystals as evident from the peak at 2θ values (111), (200), (220), (311) respectively for silver and are in accordance with calculated particle size calculated. Table (1), It was also observed that all the samples contain different sizes of Silver nanoparticle with size ranges from 30 to 70 nm.

 

Fig. 3: XRD Spectrum of Silver nanoparticles synthesized from Bacillus flexus and Bacillus pseudomycoides

Table 1: Peak indexing from d-spacing and particle size of synthesized silver nanopowder

2θ	θ	D	1000/d2	(1000/d2)/60.62	Hkl	FWHM(β)	β cos θ	Size of the particle (D) nm
Sample AK1
37.91	18.955	2.371	177.904	2.934	111	0.0041	0.00407	34
43.98	21.99	2.056	236.574	3.902	200	0.0048	0.00479	29
64.21	32.105	1.449	476.417	7.859	220	0.0038	0.00292	47
77.20	38.6	1.234	657.030	10.838	311	0.0034	0.00210	66
Sample AK2
38	19	2.366	178.667	2.947	111	0.0041	0.00405	34
44.15	22.075	2.049	238.208	3.929	200	0.0048	0.00478	29
64.36	32.18	1.446	478.468	7.892	220	0.0038	0.00274	50
77.28	38.64	1.233	657.894	10.852	311	0.0034	0.00200	69

 

In present study Fig. 4 shows representative SEM images recorded at high magnifications of biosynthesized silver nanoparticles, it was observed that the produced silver nanoparticles were scattered as well as in aggregates of varying sizes.

Antibacterial activity of Silver Nanoparticles against MDR Enteric bacteria

Isolation and Identification of Enteric Human Pathogens- In favor of the antibacterial study of AgNPs against enteric human pathogens viz, E. coli, Klebsiella, Salmonella and Shigella species. All the isolates were further confirmed by screening the virulence adopting India ink degradation. The obtained virulence strains were identified and labeled as E1 to E10 for E. coli as well as K1 to K4 for Klebsiella, Sa1, Sa2 for Salmonella, Sh1, Sh2 for Shigella species respectively. The findings on antimicrobial susceptibility testing are graphically presented in Fig. 5, from the figure, maximum isolates (88%) among tested pathogens showed resistance to Gentamycin, Co-Trimoxazole and Tetracyclin followed by 83% isolates showed resistance to Nitrofurantoin and Ceftriaxone. Whereas, in case of Azithromycin and Chloramphenicol, the (72%) isolates among the test pathogens showed at par resistance against both the antibiotics.

 

 

Fig 5: Antibiotic Sensitivity/Resistant Pattern of the isolated human enteric pathogens

 

 

 The resistance was exhibited by only 22 - 50% of isolates under study against Ampicillin, Amoxicillin-clavulanate, Ceftazidime, Imipenem, Amikacin, Ciprofloxacin, and Ofloxacin. The results on MAR index of test isolates are graphically presented in Fig. 6.  From the figure it was established that out of eighteen isolates studied, isolates E7, K3 and Sa1 showed the MAR index equal to one, which indicates the presence of multiple drug resistance (MDR) in these isolates and their origin from a high-risk source of contamination where antibiotics are often used ^[13]. Hence, only E7, K3, and Sa1 isolates were used for further investigations.

 

Fig 6: MAR Index of the isolated human enteric pathogens

 

Independent and Synergistic Antibacterial activity of Silver Nanoparticles- The individual antibacterial activity of AgNPs against test pathogens viz. E7, K3, and Sa1 are depicted in Fig. 7, MIC values for isolate E7 and K3 were recorded to be 80 μg/ml whereas, for isolate Sa1 MIC value was 70 μg/ml. The results obtained on combined antibacterial activity are depicted in Fig. 8, from the results; it was observed that in case of study on antibacterial activity of antibiotics alone all the selected MDR isolates exhibited resistance(R). In case of study on antibacterial activity of AgNPs alone, mild bactericidal activities were observed in terms of zone of inhibition ranging from 10-11 mm.

 

Fig 8:  Increase in Fold Area of Antibiotics in combination with AgNPS

Notes: In the absence of bacterial growth inhibition zones (NI), the disc’s diameter (6mm) was used to calculate the fold increases ^[11]. Increase in fold area= (b²-a²)/a². (R)- Resistance, (S)- Sensitive, (I)- Intermediate

 

However, in case of the combined activity of Antibiotics along with AgNPs; in case of isolate E7, the maximum increase i.e. (2.3) in fold area inhibition was recorded due to Cotrimoxazole-AgNPs combination followed by Tetracyclin-AgNPs combination (1.7). The remaining combinations showed the increase in fold area inhibition in the range of (0.1) to (0.9). However, in case of Amikacin- AgNPs and Azithromycin-AgNPs conjugates, no change in fold area inhibition was observed. Similarly, in case of Isolate K3 maximum increase in fold area inhibition (3) was observed in Tetracyclin-AgNPs combination followed by Cotrimoxazole-AgNPs (1.8), Amoxicillin-clavulanate-AgNPs (1.13) and Nitrofurantoin- AgNPs (1.08) combinations respectively. The remaining combinations showed the increase in the fold is inhibition in the range of (0.5) to (0.1) and Azithromycin-AgNPs showed no change in increase fold area inhibition. Isolate Sa1 showed the maximum increase in fold area inhibition (9.03) with Azithromycin-AgNPs combination followed by Gentamycin-AgNPs (9.02). Chloramphenicol-AgNPs and Ofloxacin-AgNPs combination showed at par increase in fold area inhibition of (8). Cotrimoxazole-AgNPs and Tetracyclin-AgNPs combination showed at par results (7.03), all of the remaining combinations showed the increase in inhibition fold area inhibition greater than (1) except Nitrofurantoin-AgNPs and Ciprofloxacin-AgNPs combination which showed the increase in fold area inhibition of (0.21). Hence, a Maximum synergistic antibacterial activity of Cotrimoxazole-AgNPs combination was observed against isolate E7, Tetracyclin-AgNPs combination against K3 and Azithromycin- AgNPs combination against Sa1.

 

DISCUSSION- The AgNPs were synthesized by using two bacterial strains viz. Bacillus flexus and Bacillus pseudomycoides, characterization by UV-Visible spectrometry and FTIR revealed the presence of AgNPs in Synthesized samples. The overall result of XRD explained that silver nanoparticles found in the present study were in the form of nano-crystals with varying sizes ^[14] ; the scanning images  showed the agglomeration which may be due to the fact that silver nanoparticles have the tendency to agglomerate due to their high surface energy and high surface tension of the ultrafine nanoparticles ^[15]. The research findings on Antibiotic susceptibility testing of enteric human pathogens reported the persistence of antibiotic resistance in enteric human pathogens ^[16-19]. The consistency and overuse of antibiotics as well resistant gene transfer from animals to man via Food chain might be the reason for resistance traits in pathogens. Our findings on Minimum Inhibitory Concentration of AgNPs reported the lethal effect of silver nanoparticles against different pathogens with MIC values in the range of the 50 to 75 μg/ml ^[20,21]. The results enlightened that the synergistic effect of antibiotics in conjugation with biologically synthesized AgNPs, increased the susceptibility among the tested MDR enteric bacteria in the following sequence; viz. Salmonella species followed by Klebsiella species and E. coli species respectively. These results are in line with the findings of Birla et al. ^[11] who mentioned increasing efficacies percentage of different antibiotics when used in combination with AgNPs against P. aeruginosa, S. aureus, and E. coli. In a similar study, the antimicrobial activities of biologically synthesized AgNPs were assessed with commercially available antibiotics against G- and G+ bacteria ^[22].

 

CONCLUSIONS- The present study even though is very preamble; the studies enlighten the Potentiating of antibiotics activity due to presence of silver nanoparticles and provide helpful insights to the development of novel antimicrobial agents in combination with silver nanoparticle. This synergistic antibacterial effect may be considered as beneficial for the management of multiple drug resistance enteric pathogens however; more elaborate experimental shreds of evidence will be needed.

The focus may also be given towards the Toxicity studies of silver nanoparticles on human pathogen in relation to human physiology which may open a door for new combinational range of antibacterial agents using nanoparticles.

 

ACKNOWLEDGEMENT- Authors thanks to the Rajasthan Education Society, for providing all the facility and requirement required during performing the research work.

 

CONTRIBUTION OF AUTHOR- The conception or design of the work, Data collection, Data analysis and interpretation for the work was done by P. N Agrawal.  Whereas, Drafting of the article, Critical revision of the article for important intellectual content was done by N. S Kulkarni.

 

 REFERENCES

1.      Kalishwaralal K, Deepak V, Ramkumarpandian S, Nellaiah H. and Sangiliyandi G. Extracellular biosynthesis of silver nanoparticles by the culture supernatant of Bacillus licheniformis. Mater Lett., 2008b; 62: 4411-4413.

2.      World Health Organization. WHO report. Antimicrobial Drug Resistance. Geneva, http://apps.who.int/gb/ebwha/pdf_files/EB134/B134_37-en.pdf, 2013 (December): 1–5.

3.      World Health Organization. WHO report. Antimicrobial Resistance: Global Report on Surveillance (Summary). 2014:3–6.

4.      Alliance for the Prudent Use of Antibiotics (APUA). Final Report. Situation Analysis and Needs Assessment of Antibiotic Resistance in Uganda and Zambia. 2011:1–22.

5.      Das Vidhya Lakshmi, Roshmi Thomas, Rintu T. Varghese, E. V. Soniya, Jyothis Mathew, E. K. Radhakrishnan. Extracellular synthesis of silver nanoparticles by the Bacillus strain CS 11 isolated from industrialized area. 3 Biotech., 2013; DOI 10. 1007/s13205-013-0130-8.

6.      Nagarajan S, Kuppusamy KA. Extracellular synthesis of zinc oxide nanoparticles using seaweeds of Gulf of Mannar, India.  Journal of Nanobiotechnology., 2013; 11(39): 1-11.

7.      Forbes BA, Sahm DF, Weissfeld AS. Bailey and Scott’s Diagnostic Microbiology.12th edition. Mosby Elsevier, China. 2007.

8.      Bauer AW, Kirby WMM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Amer. J. Clin. Pathol., 1966; 4: 493-496.

9.      Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing.15th Informational Supplement, 2013; 25, M100-S15.

10.  Wei LS, Musa N, Wee W. Bacterial flora from a healthy fresh water Asian sea bass (Lates calcarifer) Fingerling hatchery with emphasis on their antimicrobial and heavy metal resistance pattern. Vet. Archiv, 2010; 80(3): 411- 420.

11.  Birla SS, Tiwari VV, Gade AK, Inglex AP, Yadav AP, Rai MK. Fabrication of silver nanoparticles by Phoma glomerata and its combined effect against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Lett Appl Microbiol., 2009; 48: 173–179.

12.  Jeevan P, Ramya K, Edith Rena A. Extracellular biosynthesis of silver nanoparticles by culture supernatant of Pseudomonas aeruginosa. Indian Journal of Biotechnology, 2012; 11:72-76.

13.  Wei L S and Wee W.  Antibiogram and heavy metal resistance pattern of Salmonella spp. isolated from wild Asia sea bass (Lates calcarifer) from Tok Bali, Kelantan, Malaysia. Jordan J. Bio. Sci., 2011; 4(3): 125-128.

14.  Theivasanthi T, Alagar M. Electrolytic Synthesis and Characterizations of Silver Nanopowder, 2010.

15.  Malarkodi C, Annadurai G. A novel biological approach on extracellular synthesis, and characterization of semiconductor zinc sulfide nanoparticles, Appl Nanosci., 2012.

16.  Ballal M, Devadas SM, Chakraborty R, Shetty V. Emerging Trends in the Etiology and Antimicrobial Susceptibility Pattern of Enteric Pathogens in Rural Coastal India. International Journal of Clinical Medicine, 2014; 5: 425-432.

17.  Ikpeme E, Nfongeh J, Enyi-Idoh K, Eja M E and Etim L,. Antibiotic suspectibility profile of Enteric bacteria isolates from Dumpsite Utisols and Water sources in a Rular Community in cross River State, Southern Nigeria. Nature and Science., 2011; 9(5) 46-50.

18.  Sumathi, B.R. Antibiogram profile based dendrogram analysis of Escherichia coli serotypes isolated from bovine mastitis. Vet.Wld., 2009;1(2), 37-39.

19.  Farzana, K. Prevalence and antibiotic resistance of bacteria in two ethnic milk based products. Pak. J. Bot. 2009; 4(2):935-943.

20.  Humberto H. Lara, Nilda V. Ayala-Nunez, Liliana del Carmen Istepan Turrent, Cristina Rodrigues Padilla. Bactericidal effect of Silver nanoparticles against Multidrug- resistant bacteria. World J Microbiol Biotechnol., 2010; 26: 615-62.

21.  Kim JS, Kuk E, Yu KN, Kim JH, Park SJ, Lee HJ, Kim SH, Park YK., Antimicrobial effects of silver nanoparticles. Nanomedicine: Nanotechnology, Biology and Medicine, 2007; 3(1): 95–101.

22.  Fayaz AM, Balaji K, Girilal M, Yadav R, Kalaichelvan PT, Venketesan R. Biogenic synthesis of silver nanoparticles and their synergistic effect with antibiotics: a study against gram-positive and gram-negative bacteria. Nanomedicine., 2010; 6(1):103–109.