Centre for Drug Discovery and Development Sathyabama Institute of Science and Technology, Chennai- 600119, India

Article Publishing History

INTRODUCTION

Tuberculosis (TB) poses serious epidemics around the world caused by the pathogen Mycobacterium tuberculosis (Mtb). The increasing rate of HIV-related TB, multi-drug resistant TB (MDR- TB) and Extensively Drug Resistant TB (XDR-TB) also concerned globally. Treatment for tuberculosis requires 6-8 months for new cases and 18-24 months for MDR TB whereas the treatment for XDR-TB is ineffective making the treatment options seriously limited. The big challenge here has been to find new drugs effective against tuberculosis. In this context, antimicrobial peptides such as bacteriocins produced by lactic acid bacteria has been emphasized for their prominent antimycobacterial properties due to cationic characteristics which likely binds to the anionic lipids in membrane (Sivaraj et al., 2018). There are only few studies carried out on bacteriocins focused on their antimicrobial or antitubercular activity but many studies focused on their potential applications as food preservatives (Perez et al., 2018).  Bacteriocins exhibit significant potency against multidrug-resistant bacteria and also offer promising lead compound as substitutes or conjugates to current therapeutic compounds (Meade et al., 2020).

The concept of research on bacteriocins applications are expanding from food preservative to human health including novel drug delivery systems, anti-tubercular treatment and anticancer treatment applications (Meade et al., 2020). Lactic Acid Bacteria (LAB) is a group of Gram positive, catalase negative, often non-motile organisms that are grouped in to two distinct phyla, such as Firmicutes and Actinobacteria. Around 15 different genera of LAB have been reported and some of them are dominant genera viz. Lactobacillus, Streptococcus, Pediococcus, Enterococcus, Aerococcus, etc. LAB have been isolated from various sources like raw milk, cultured milk products, meat products, fish, grains, green plants, fermenting vegetables, mucosal surface of animals (Lindgren and Dobogosz, 1990).

Fish gut is considered as complex ecosystem that contains numerous microorganisms (Wong et al., 2013). The microbiota of fishes helps in antagonistic activity against pathogens and is in various immune responses (Huber et al. 2004).  LAB isolated from the gastrointestinal tract (GIT) of fishes had been reported to have the potential as probiotic agents by protecting the aquatic species from various aquatic infections and also by killing the pathogens involved in the spoilage of fish products (Meade et al., 2020).

Broad range of various lactic acid bacteria species found in the GIT of various fish species which include both fresh water and marine water species (Merrifield et al., 2014). It was believed that LAB obtained from the GIT of fishes has the potential to develop as ideal probiotic agents (Gomez‑Sala et al., 2015). LAB is considered as “Generally Recognized as Safe (GRAS)” microorganisms, which produce various compounds during lactic acid fermentation including organic acids, diacetyl hydrogen peroxide, bacteriocins, etc. Bacteriocins produced by LAB are considered as small peptides (<10 kDa) that have greater antibacterial activity by means of their unique characteristics such as cationic in nature, heat-stable, amphiphilic and adsorption to the gram-positive cell surfaces. LAB and their metabolic products usages are generally considered as safe (Zacharof and Lovitt, 2012). LAB-bacteriocins are emerging as a novel alternative to antibiotics and known to exert either bacteriostatic or bactericidal activity toward sensitive organisms. Bacteriocins of LAB target specific species and do not affect other population within the same ecosystem (Vieco-Saiz et al., 2019).

Bacteriocins are secreted in the logarithmic growth phase of bacteria with increasing bacterial numbers and optimal culture conditions promoting increasing peptide secretion (Ge et al., 2019; Anbarasu et al., 2020). Bacteriocins are extracellularly released peptides and they are mainly categorized into class I, class II, class III and class IV based on the host producer, molecular weight and amino acid sequence (Meade et al., 2020). The electrostatic interaction between positive charge of bacteriocins and the negative charge of bacterial cell membranes plays a significant role in the initial interaction thereby facilitating the binding of the molecules to the membranes of target cells (Perez et al., 2015).

LAB isolated from GI of estuarine fish and freshwater fishes have showed significant antimicrobial activity against various aquaculture pathogens (Sahoo et al., 2015; Hagi et al.,2004; Ghosh et al., 2014). The antimycobacterial properties of bacteriocins isolated from lactic acid bacteria have been studied by few researchers globally. Lantibiotics are class I bacteriocins that certainly possess sufficient potential for treating tuberculosis in future. Bacteriocins from lactic acid bacteria have showed greater antimycobacterial activity than equal concentrations of rifampicin in vitro model (Carroll and Jim O’Mahony, 2011; Sosunov et al., 2007).

Pérez et al., (2018) demonstrated synergistic actions of enterocin AS-48 (bacteriocins produced by LAB) with ethambutol against Mycobacterium tuberculosis and revealed its potential role in tuberculosis treatment. In this context, the present study was aimed to isolate lactic acid bacteria from fish gut and to evaluate their bacteriocins for anti-tubercular activity against M. tuberculosis using luciferase reporter phage (LRP) assay.

MATERIAL AND METHODS

The fishes such as Rastrelliger kanagurta, Tilapia, Centropristis striata and Teuthida were aseptically dissected to collect intestinal contents, weighed and homogenized. They were each transferred aseptically to 10 ml of saline, shaken well and about 1 ml aliquots were transferred to 10ml of MRS broth each and incubated at 30˚C for 18 hours. After incubation, ten-fold serial dilution of samples was carried out and plated onto MRS agar containing bromocresol (0.04mg/ml) and incubated at 30˚C for 24 hours. Colonies showing yellow zones were selected and sub-cultured on to MRS agar plate. Isolates that were catalase negative and gram positive further got identified at genus level according to method described by Nikita and Hemangi (2012) and Kalschne et al., (2015).

To test the antimicrobial and antimycobacterial activities, one ml of overnight grown LAB culture was transferred to 100 ml of MRS broth (Himedia, India) and incubated at 30˚C in shaker (100rpm). After 18 hours of incubation, cell free supernatant (CFS) was collected by centrifugation at 5000rpm for 10mins (4˚C). Then pH of CFS was adjusted to 6.5 and treated with catalase (1mg/ml) for testing their inhibitory activity against non-mycobacterial strains such as S. aureus, B. cereus, E. coli and K. pnemuoniae and mycobacterial strains, M. smegamtis mc²155 by agar well diffusion assay (Anbarasu et al., 2019). Briefly, about 5 mm diameter wells were cut on nutrient agar medium containing non-mycobacterial cultures and middlebrook 7H9 agar plate containing M. smegamtis mc²155. About 100µl of treated CFS from all isolates were added into each well. Nutrient agar plates were incubated at 30˚C for 18 hours and middlebrook 7H9 agar plates were incubated for 48 hours at 37˚C. Zones of inhibition were measured in mm.

LAB isolate (BFO21) showing activity against M. smegmatis mc²155 was identified by 16s rRNA gene sequencing. Briefly, genomic DNA was isolated using a modified phenol-chloroform protocol.  PCR was performed using the following primer pair –  forward primer 27F (5’-AGA GTT TGA TCC TGG CTC AG-3’) and  reverse primer 1492R (5’-TAC GGT TAC CTT GTT ACG ACT T-3’) at the  following conditions: initial denaturation (97°C for 5 min followed by 35 cycles at 94°C for 2 min), annealing (51°C for 1 min) extension (72°C for 2 min) and  final extension (72 °C for 5 min).  PCR products were purified and subjected to sanger sequencing for 16s rRNA gene sequence analysis. The homology of the obtained sequence was analyzed using NCBI BLAST.

Lactobacillus spp. (BFO21) was subjected to partial purification of bacteriocin using solvent extraction method. Briefly, overnight grown LAB isolate was inoculated to 500ml of MRS broth and incubated at 30°C for 18 hours in shaker, 100 rpm. CFS were collected by centrifugation at 5000 rpm for 15minutes at 4^oC, added with equal amount of chloroform and kept in magnetic stirrer for 20minutes at 4°C. Resulting white precipitate was collected by centrifugation at 5000rpm for 30 minutes, lyophilized and stored in PBS buffer (pH 6.0) at -20^oC. The precipitated bacteriocin was filtered through 0.45micron syringe driven filter and subjected to antimycobacterial screening.

To screen for anti-tubercular activity of partially purified bacteriocin against M. tuberculosis H37Rv, LRP assay was used as mentioned (Anbarasu et al., 2019). Briefly, 400µl of middelbrook 7H9 broth was added to two cryo vials (Control), 400µl of middlebrook 7H9 broth containing rifampicin at 2µg/ml concentration (drug control). The test cryo vial was added with 350µl of middlebrook 7H9 broth and 50µl of partially purified bacteriocin (10mg/ml). About 100µl of M. tuberculosis H37Rv suspension (Mcfarland Unit 2) was added to all the vials and incubated at 37˚C. After 72 hours of incubation, 40µl of 0.1M CaCl₂ and 50µl of mycobacteriophage (phAE202) were added to all the vials and incubated for 4 hours at 37˚C. Then 100µl of the cell-phage mixture from each vial was added with 100µl of D-Luciferin and Relative Light Units (RLU) was measured using Luminometer (model: Lumat³ LB 9508, make: Berthold).

The inhibitory activity against M. tuberculosis was calculated based on the formula: Percentage RLU reduction = Control RLU– Test RLU / Control RLU × 100. The test RLU reduction by 50% or more when compared to control RLU was considered as positive for anti-tubercular activity.

RESULTS AND DISCUSSION

In the present study, Pediococcus sp. (2) was isolated from Centropristis striata and Teuthida fish intestinal tract. Lactobacillus sp. (2) were isolated from Tilapia. Aerococcus sp. and Streptococcus spp. were isolated from the gut of Rastrelliger kanagurta (Table 1a; Table 1b). The antibacterial and antimycobacterial activity of treated CFS samples are summarized in Table 2.  CFS of five LAB isolates viz. BFO21, BFO22, BFO27, BFO29, BFO30 showed activity against B. cereus alone whereas BFO28 isolate have not shown inhibitory activity against any of the tested organisms. Plantaricin LPL-1 produced by L. plantarum have showed significant antibacterial activity against S. aureus, L. monocytogenes, B. pumilus, B. amyloliquefaciens, E. faecalis (Wang et al., 2018). In our study, BFO21 (Lactobacillus sp.) alone showed inhibition against M. smegmatis mc²155 by agar well diffusion assay indicating its antimycobacterial potential. Comparative 16s rRNA gene sequence analysis confirmed that Lactobacillus spp. BFO21 displayed 99.86% homology with Lactobacillus plantarum (GenBank accession number: MN367969). Silva et al., (2014) revealed that L. plantarum is one of the most important and versatile species and has many applications in various industries including pharmaceutical industries (Bravo et al., 2019).

Table 1a. Isolation and characterization of lactic acid bacteria from fish gut

S. No	Reference ID	Source (Fish)	Morphology	Gas Production	Growth @10˚C	Growth @45˚C	Growth @pH 4.4	Growth @pH 9.6	Growth @6.5% NaCl	Growth @18% NaCl
1	BFO21	Tilapia	Gram positive bacilli	–	–	+	+	+	+	+
2	BFO22	Tilapia	Gram positive bacilli	–	+	+	+	+	+	+
3	BFO27	Rastrelliger kanagurta	Gram positive cocci (tetrads)	–	–	–	–	+	+	–
4	BFO28	Rastrelliger kanagurta	Gram positive cocci (tetrads)	–	+	+	–	+	–	–
5	BFO29	Centropristis striata	Gram positive cocci (tetrads)	–	+	+	+	+	+	–
6	BFO30	Teuthida	Gram positive cocci (tetrads)	–	+	+	+	+	–	–

Table 1b. LAB identified at genus level by biochemical methods

S. No	Reference ID	Genus Identified
1	BFO21	Lactobacillus sp.
2	BFO22	Lactobacillus sp.
3	BFO27	Streptococcus sp.
4	BFO28	Aerococcus sp.
5	BFO29	Pediococcus sp.
6	BFO30	Pediococcus sp.

Table 2. Inhibitory activity of cell free supernatant by agar well diffusion assay

S. No	Reference ID	Zone of Inhibition (mm)
		S. aureus	B. cereus	E. coli	K. pneumoniae	M. smegmatis
1	BFO21	–	11	–	–	12
2	BFO22	–	10	–	–	–
3	BFO27	–	11	–	–	–
4	BFO28	–	–	–	–	–
5	BFO29	–	8	–	–	–
6	BFO30	–	10	–	–	–

Table 3. Anti-tubercular activity of partially purified bacteriocin by LRP assay

S. No	Reference ID	Percentage of Reduction in RLU	Result
1	Rifampicin	91.93	Inhibition
2	BFO21*	98.00	Inhibition

*Bacteriocins produced by L. plantarum.

The isolated Lactobacillus plantarum BFO21 was chosen for further purification of bacteriocin. Chloroform solvent extraction method was applied to purify the bacteriocin and the resultant lyophilized bacteriocin was screened against M. tuberculosis H37Rv using LRP assay. Bacteriocin of Lactobacillus plantarum BFO21 has shown reduction in RLU values by 98.00% at concentration of 35.53µg/ml. The result was compared with known anti-TB drug rifampicin at 2µg/ml that showed 91.93% of RLU reduction (Table 3). Previous studies have found that lactobacilli isolated from badger feces, wild boar feces or fermented milk products exhibited antimycobacterial activity against BCG and M. bovis (Mariam, 2009; Macuamule et al., 2016; Stedman et al., 2018; Bravo et al., 2019). To the best of our knowledge, this is the first study that showed the partially purified bacteriocin from Lactobacillus of Tilapia fish intestinal tract showing anti-tubercular properties (Bravo et al., 2019).

CONCLUSION

The burden of tuberculosis was reported high in India and treatment requires 6-8 months for new cases and 18-24 months for MDR TB. Lactic acid bacteria (LAB) and their bacteriocins received significant attention in the past decade due to its Generally Recognized as Safe (GRAS) status. Hits with improved cell penetration and with activity against M. tuberculosis should be prioritized. Bacteriocins of LAB certainly possess sufficient potential to merit hope for future therapies for treating intracellular infections. In this study, the Lactobacillus plantarum BFO21 obtained from fish gut has shown to produce antibacterial and antimycobacterial bacteriocins. The in vitro LRP assay of partially purified bacteriocins have shown significant anti-tubercular activity against M. tuberculosis H₃₇Rv. Further purification and characterization of bacteriocins should be done to explore the possibility to develop the same as lead anti-tubercular compounds.

ACKNOWLEDGMENTS

The authors thank the management of Sathyabama Institute of Science and Technology, Chennai for providing the research facilities.

Compliance with Ethical Standards: There are no laboratory animals and human subjects involved. The lactic acid bacteria were isolated from the intestinal tract of fishes obtained from fish slaughter-house.

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

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