1. INTRODUCTION:

Tuberculosis (TB) is a contagious airborne disease caused by Mycobacterium tuberculosis. When it affects mainly the lungs it is known as pulmonary TB and if other sites are involved, it is extra-pulmonary TB. Globally, around 10 million people are estimated to have fallen ill with TB of which around 558 000 people had rifampicin resistant tuberculosis (RR-TB) in 2017. Among RR-TB, 82% of patients had multidrug-resistant tuberculosis (MDR-TB) and only 55% of them were treated successfully^[1]. World Health Organization (WHO) reported 30 high TB burden countries accounted for 87% of the world cases of which India alone accounted for 27%. TB remains one of the top ten causes of death and it is estimated that around 1.6 million people had died from this disease among HIV-negative people in the year 2017. There is an increase in the rate of drug resistant TB that poses serious problem around the world. The treatment for tuberculosis requires very long duration with combination of antibiotics which are very expensive and toxic. The recommended treatment for drug susceptible TB is 6 months and 18-24 months for RR-TB/MDR TB.

The side effects caused by some anti-TB drugs lead to patients’ non-adherence to anti-TB therapy. Hence, development of new anti-mycobacterial formulations through novel strategies is needed to target M. tuberculosis more efficiently. In this connection, nanotechnology offers a novel and promising approach to develop nanoparticles for TB treatment including drug resistant TB by specifically targeting the infected sites and reducing the toxicity issues^[2].

Nanomaterials offer versatile applications that can be used as drug delivery systems and antibacterial agents. Nanomaterials are measuring less than 1000 nm in size as tiny objects that work as a whole unit. They can be made of very different materials including inorganic materials such as Ag, Au, Ga, Cu etc. These nanomaterials show anti-bacterial and anti-mycobacterial activity. They possess unique physical, chemical, electrical, optical, and mechanical properties. Regarding TB treatment, nanomaterials may be a useful strategy for their intrinsic anti-mycobacterial activity^[2]. Among the metallic nanomaterials, gold nanoparticles have numerous advantages and are well studied for their potential application in the field of medicine for centuries. Due to size, shape and unique physical and chemical properties gold of nanoparticles have triggered a wide interest in scientists. In addition, gold nanoparticles have been found to be taken up by cells without cytotoxic effects^[3,4]. Nanoparticles synthesis through physical, chemical and mechanical methodologies raised the concerns for various environmental hazards. In this concern, green chemistry approach using microbial resources for efficient and eco-friendly synthesis of nanomaterials has emerged.

Lactic acid bacteria (LAB) are group of gram positive bacteria suitable for various industrial applications viz. dairy industry, pharmaceutical industry and also considered as “Generally Recognized as Safe” (GRAS) microorganisms. They play a major role in maintaining healthy microbiota and have many benefits including managing diarrhoea, food allergies, inflammatory bowel diseases and gastrointestinal disorders. Lactobacilli are known to be highly suited vehicles for the delivery of compounds that are of pharmaceutical interest to the mucosa homeostasis^[5]. LAB cell wall consists mainly of peptidoglycans, (lipo)teichoic acids, proteins, and polysaccharides. Various compounds including organic acids, diacetyl hydrogen peroxide and bacteriocins have been produced by them that have been well documented ^[6]. LAB has been successfully demonstrated for their ability to accumulate and reduce gold ions to form nanoparticles. In addition, nanoparticles synthesized using LAB that are probiotic strains should be of better interest than others^[7,8]. Hence, in this study, lactic acid bacteria were used to synthesis gold nanoparticles and their characterization was done using different techniques. In addition, the gold nanoparticles were screened for their anti-mycobacterial activity using luciferase reporter phage (LRP) assay.

2. MATERIAL AND METHODS:

2.1 Biogenic synthesis of gold nanoparticles:

About five lactic acid bacteria (LAB) such as Enterococcus spp. (2), Aerococcus spp. 2), Lactobacillus spp.(1) were obtained from our in-house LAB culture collection repository based on their ability to produce bacteriocins active against M. tuberculosis in vitro. All the LAB isolates were used to produce gold nanoparticles with some modification. Briefly, 10% inocula of isolates were inoculated in MRS broth and incubated at 30^oC for 18 h. After incubation, cell biomass were collected by centrifugation at 5000 rpm for 10 min and washed twice with sterile distilled water and dissolved in 5mL of PBS (5mM, pH 6.5). The harvested bacterial biomass (1g wet wt.) was then resuspended in 10mL of 1mM aqueous HAuCl₄ solution in 100mL Erlenmeyer flasks. The whole mixtures were kept in incubated shaker at 30^◦C (150rpm). The biotransformation indicated by color change from pale yellow to purple was regularly monitored by visual observation. After this reaction period, the biomass was washed using sterile distilled water for further recovery of gold nanoparticles using ultra-sonication process^[9].

2.2 Characterization of Nanoparticles:

Further, The morphology of recovered gold nanoparticles was analyzed using HR-SEM attached with EDAX^[10]. The concentration of gold nanoparticles was measured by ICP-MS (Agilent 7700X). Briefly, 500μL of synthesized gold nanoparticles solution was digested with suprapur nitric acid (Merck Millipore) using microwave assisted auto digestion system (MARS 6) and concentration was measured.

2.3 Antimycobacterial activity:

The synthesized gold nanoparticles (L-AuNPs) were preliminarily analyzed for their anti-mycobacterial activity against M. smegmatis mc²155. Briefly, molten soft agar (1% agar) containing 200µL of M. smegmatis mc²155 suspension (McFarland No. 2) was prepared at 45˚C, poured immediately onto middlebrook 7H9 agar base plate and allowed for solidification. L-AuNPs was sonicated and two fold dilutions were made with sterile distilled water. About 10µL of undiluted (neat) and diluted samples (D1-D5) were spotted on the lawn. Then the plate was sealed and incubated at 37˚C for 48 hours. The titer was defined as the reciprocal of the highest dilution (2ⁿ) that resulted in inhibition of the M. smegmatis mc2155 lawn. The arbitrary unit (AU) of antimycobacterial activity per milliliter was calculated as follows: 2ⁿ X 1,000µl/10µl^[11,12]. Following preliminary anti-M.smegmatis activity, luciferase reporter phage (LRP) assay was applied to screen activity of L-AuNPs against M. tuberculosis H37Rv. To screen the anti-TB properties, about 350µL of sterile middlebrook 7H9 broth was aliquoted into to 4 sterile cryovials and marked 1-4. Each vial was added with 50 µL of L-AuNPs in order to get final concentration of 12.5µg/ml, 6.25µg/ml, 3.12µg/mL, 1.56µg/ml respectively in the assay. The control vial was aliquoted with 400µL of sterile middlebrook 7H9 broth and drug control vial consisted of 400µL of middle brook 7H9 broth containing standard drug rifampicin at 2μg/mL concentration. Hundred microliter of M. tuberculosis H37Rv cell suspension (#2 McFarland) was transferred to all cryovials and incubated at 37^oC. After 72 h of incubation, 50µL of high titre mycobacteriophage (titre - 6.5 × 10⁹pfu/mL) along with 40 µL of 0.1 M CaCl₂ solution was added into control, test and drug cryovials (cell-phage mixture) further incubating at 37°C for 4 h. Then 100µL of cell-phage mixture was pipetted out into luminometer cuvette and added with 100µL of D-Luciferin substrate. The relative light unit (RLU) was measured immediately at 10 seconds integration in luminometer (Model: LB9508 Lumat3; make: Berthold, Germany). Test RLU reduction by 50% or more when compared to control was considered as having anti-mycobacterial activity based on following formula: Percentage RLU reduction = Control RLU– Test RLU / Control RLU × 100^[13].

3. RESULTS AND DISCUSSION:

The production of gold nanoparticles was evaluated after exposure of the LAB cultures to gold based on the size, shape and the occurrence of the particles in the cells. Promising results were obtained with Lactobacillus spp. whereas other LAB cultures viz. Enterococcus spp. and Aerococcus spp. have not produced nanoparticles. The potent Lactobacillus spp. isolate was identified as Lactobacillus plantarum by 16s rRNA gene sequencing analysis and named as Lactobacillus spp.BFO21 (GenBank: MN367969.1). The biomass of Lactobacillus spp. was turned dark purple after 20 h exposure to HAuCl₄ while the solutions remained colorless which indicating the intracellular production of nanoparticles and named as L-AuNPs. Similar findings shown by Gericke and Pinches (2006), they reported that the biomass of fungal cultures turned dark purple while no colour changes observed in the solution^[9].

Figure 1. shows a) Biomass of Lactobacillus spp. in gold solution, b) Intra cellular synthesized Gold nanoparticles; c) recovered gold nanoparticles from cell; dande) HR-SEM images of gold nanoparticles and f) EDAX of gold nanoparticles.

Figure 2a. Inhibitory activities of ‘L-AuNPs’ against M. smegmatis Mc2155 by agar will diffusion

The pictorial representations of gold nanoparticles synthesis using lactic acid bacterial biomass are shown in figure 1a and b. Further, the biomass was ultrasonicated using bath ultrasonicator for 30 min facilitating the recovery of gold nanoparticles (Fig.1c). Structural morphology of intracellularly produced L-AuNPs was characterized using HR-SEM attached with EDAX. (Fig.1d and f). HR-SEM images of L-AuNPs show rod shaped morphology in the size range of 22 nm. The EDAX analysis was done to get an indication of the amount of gold nanoparticles which showed strong signals for gold atoms along with weak signals from oxygen and carbon. The concentration of synthesized gold nanoparticles was found to be 125µg/mL.

In this study, we have screened the synthesized gold nanoparticles (L-AuNPs) against M. smegmatis Mc²155 and M. tuberculosis H37Rv. Previously, authors have reported that gold nanoparticles synthesized using plants and biogenic synthesis of silver nanoparticles have shown antimycobacterial activity^[14-16]. However, this is first study describing antimycobacterial potential of gold nanoparticles synthesized using biomass of L. plnatarum. The preliminary activity of L-AuNPs was tested against M. smegmatis mc²155 and anti-mycobacterial titer was quantified to be 800AU/mL (Fig.2a).

The in vitro anti-mycobacterial activity against M. tuberculosis H37Rv was demonstrated by LRP assay. The percentage of RLU reduction in terms of inhibition was examined by relative light unit values (RLU) from the luminometer. We have observed that the growth of M. tuberculosis H37Rv was inhibited by L-AuNPs at concentration between 6.25µg/mL to 12.5µg/mL and percentage of RLU reduction was found to be 69.26 % to 98.01% respectively. Results of the L-AuNPs were in close concordance with 2µg/mL concentration of rifampicin which showed 90% RLU reduction (Fig.2b). In comparison, earlier study reported that AuNPs synthesized using plant extracts have MIC of >2.56 μg/mL against M. tuberculosis^[16].

4. CONCLUSION:

In conclusion, we have investigated the intracellular synthesis of gold nanoparticles in Lactobacillus plantarum. Remarkably, the synthesized L-AuNPs showed inhibitory activity against M. tuberculosis H37Rv. To the best of our knowledge, this is first study of LAB mediated gold nanoparticles that have been screened for anti-mycobacterial activity using LRP assay. Finding the mechanism of action and further research on conjugation of gold nanoparticles with anti-mycobacterial peptides produced by lactic acid bacteria will offer many solutions for potential TB therapeutic agents. Treating TB including the drug resistant one with either gold nanoparticles or peptide functionalized nanoparticles may pave the way for promising development of nanotechnology-mediated drug delivery systems in future.

5. ACKNOWLEDGMENT:

We thank the management of Sathyabama Institute of Science and Technology, Chennai for supporting the research work.

6. CONFLICT OF INTEREST:

All the authors declare that they have no conflict of interest.

7. REFERENCES:

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Received on 20.11.2019 Modified on 13.01.2020

Research J. Pharm. and Tech 2020; 13(9):4391-4394.

DOI: 10.5958/0974-360X.2020.00776.3