Insights Into The Broad Antibacterial Spectrum Of Bacteriocin Isolated From Probiotic Lactobacillus Fermentum LMEM22 Strain

This communication aims to validate the probiotic attributes of the Lactobacillus fermentum LMEM22 strain, safety proling, 16S rRNA gene sequence analysis, and bacteriocin characterization. The bacteriocin of 13.1 kDa (based on the glycine-SDS-PAGE analysis) was isolated from L. fermentum LMEM22 curd strain (NCBI GenBank with accession No. MH380182), the identity for which was conrmed by 16S rRNA gene sequence analysis. The probiotic properties and safety proles were authenticated in vitro systems. The zone diameter of inhibition from the L. fermentum LMEM22 bacteriocin action, following agar-well diffusion, ranged 19 – 23 mm and 17 – 24 mm, respectively, for gram-negative and gram-positive bacteria. On enzymatic treatment with proteinase K and trypsin the bacteriocin lost the bacterial growth inhibition capacity, and it was found insensitive against α-amylase action, authenticating its proteinaceous nature. The γ-haemolytic L. fermentum LMEM22 strain, for which gelatinase and DNase activities were negative, had tolerance to high sodium chloride concentration range (2 – 6.5%), low pH (2 – 4%), and bile salts (0.125 – 0.5%). This study, thus, authenticated the probiotic attributes of L. fermentum LMEM22 strain for safe consumption by the people, and the usefulness of bacteriocin isolated, as a valued protein antibiotic for the prevention and treatment of multidrug resistant bacterial infection.


Introduction
The antagonism of probiotic LAB (lactic acid bacteria) strains against human pathogenic bacteria has been reported to be due to their capacity to produce hydrogen peroxide, organic acids, such as lactic acid, and bacteriocins. The bacteriocins are antimicrobial peptides of low molecular weights and produced by gram-negative as well as gram-positive bacteria (Usui et al., 2012;Bosak et al., 2018); however, those produced by LAB remain the most valuable compounds in food and medicine (Dobson et al., 2012), because the majority of such producers are 'generally regarded as safe' and designated as probiotics.
Therefore, the one plausible opportunity to curb the ARPB (antibiotic resistant pathogenic bacteria) infections to humans is to deploy LAB strains, or to utilize bacteriocins produced by them, as protein antibiotics (Das and Goyal, 2014;Gupta et al.,, 2017). The probiotic potentiality assessment of different LAB strains takes an account of testing for physiological stressors tolerance, safety aspects and functionality (Halder et al.,, 2017), and before in vivo application, in order to get a bene t for a given health disorder or pathogenic infection to humans in vitro testing is mandatory in substantiating the preferred outcome (Gareau et al.,, 2010).
The gram-negative ARBP that pose severe global health threats include Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Salmonella enterica serovar Typhi and Proteus vulgaris, while among the gram-positive bacteria, the MDR strains of Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus and Listeria monocytogenes cause threats to the patients' safety. Antibiotics remain the basis of all kinds of therapies in modern healthcare systems, such as enabling the treatment life-threatening bacterial infection to humans (Van Boeckel et al.,, 2014). However, the global emergence of multiple antibiotic resistant pathogenic bacteria necessitates for novel antimicrobials safe for human usage. The bacteriocin producing probiotic lactobacilli have been proved to be safe and suitable for biotherapy because of their capacity to display antibacterial activity against clinically relevant bacteria, or spoilage as well as food-borne pathogenic bacteria (Das and Goyal, 2014).
Many authors isolated and identi ed, following conventional phenotypic characterization as well as 16S rRNA gene sequencing, LAB strains from varied sources and characterized bacteriocins produced by them (Ge et al., 2016;Oliveira et al., 2017).Earlier, we have identi ed (by phenotypic characterization) a LAB strain, Lactobacillus fermentum LMEM22 procured from commercially available curd (West Bengal, India), checked the antibiogram and validated its antagonistic capacity against human pathogenic bacteria (Halder and Mandal, 2018). The current study was, thus, prompted to be undertaken in order to authenticate the probiotic attributes of the Lactobacillus fermentum LMEM22 strain through stressors tolerance, safety pro ling, 16S rRNA gene sequence analysis, and bacteriocin characterization.

Bacterial strains and media
The LAB strain, which was isolated from curd, identi ed as L. fermentum LMEM22 by conventional phenotypic characterization (Halder and Mandal, 2018), and maintained in MRS broth as well as in MRS agar stab in freezing, was utilized in the instant study. The indicator bacteria used include the randomly selected gram-negative (Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Salmonella enterica serovar Typhi and Proteus vulgaris) and gram-positive (Bacillus cereus, Enterococcus faecalis, Staphylococcus aureus) clinical bacterial isolates from the laboratory stock cultures, and Listeria monocytogenes MTCC657 standard strain, which were maintained in cystine tryptone agar stabs, in refrigeration.

Molecular identity of lactic acid bacterium
The identity authentication of the bacteriocinogenic LAB strain was done by 16S rRNA gene sequence and phylogenetic analyses. The 16S rDNA (≈1.5 kb fragment) was PCR ampli ed (from the genomic DNA extracted from the LAB) and thereafter sequenced using 16S rRNA speci c primers (forward primer: 5′-AGHGTBTGHTCMTGNCTCAS-3′ and reverse primer: 5′-TRCGGYTMCCTTGTWHCGACTH-3′) from Chromous Biotech Pvt Ltd, India.
The sequenced data (partial 16S rRNA gene sequence: 672 bp) was aligned using ClastalW (Thompson et al.,, 1994), and in the NCBI (National Center for Biotechnology Information) GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) the closest known relatives of the sequence obtained (from the test LAB strain) were determined through nucleotides homology search, with BLAST (Basic Local Alignment Search Tool), for nucleotide sequences (BLASTN).
The phylogenetic tree, based upon 16S rRNA gene sequences was constructed within the SeaView version-4 software (Gouy et al., 2010),following maximum likelihood method with PhyML GTR model (bootstrap with 1000 replicates). The partial 16S rRNA gene sequence of the test LAB strain has been deposited with the NCBI Genbank accession No. MH380182.

Probiotic characterization
The probiotic features of the test L. fermentum LMEM22 strain was substantiated through physiological stressors tolerance tests and safety pro ling. The L. fermentum LMEM22 was subjected to probiotic characterization by performing bile salt and low-pH (acid) tolerance tests according to Liong and Shah (2005), and sodium chloride tolerance test following Chowdhury et al. (2012), with some alteration as cited before (Halder and Mandal, 2015;Halder et al.,, 2017).
In order to check the viability of the LAB at different incubation hours, the L. fermentum LMEM22 strain, with ≈5 × 10 5 CFU/ml (5.698 log 10 CFU/ml) inocula, was allowed to grow in presence of varied

Isolation and quanti cation of bacteriocin
Bacteriocin from the L. fermentum LMEM22 strain was extracted following Ismail et al. (2013), with modi cations as explained below. Following subcultures of L. fermentum LMEM22 strain twice in MRS broth, 100 µl was inoculated into 25 ml of MRS broth, and after incubation at 37°C for 48 h, cell free supernatant (CFS) was prepared by centrifugation (at 10,000 rpm, for 10 minutes at 4°C) and syringe ltration. The CFS was treated with 60% ammonium sulfate at 4°C for 24 h to get precipitated the bacteriocin, which by centrifugation (at 12,000 rpm for 15 minutes at 4°C), was extracted. The isolated bacteriocin was processed by washing with sterilized double distilled water and centrifuging (at 10,000 rpm for 10 minutes at 4°C) for thrice, in order to remove the impurities in bacteriocin. At the nal stage, pure bacteriocin pellet was mixed with phosphate buffer solution (1 ml, pH 7.2), with 0.6 % SDS, and stored at 4°C for further usage.
The bacteriocin yield was quanti ed spectrophotometrically, following Lowry et al. (1951), using bovine serum albumin as the standard.

SDS-PAGE analysis of bacteriocin
The molecular weight of bacteriocin isolated from L. fermentum LMEM22 strain was approximated by glycine-SDS-PAGE (glycine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis) analysis (Laemmli, 1970), using a vertical slab gel apparatus (Tarsons, India) with 7.5% stacking and 12.5 % separating gels, and high range protein molecular markers (Hi-Media, India). Following electrophoresis for 4 h at 130 V, the gel was subjected to Coomassie brilliant blue (Hi-Media, India) staining (for 30 min) and thereafter de-staining in 20% (v/v) methanol / 7.5% (v/v) glacial acetic acid until the bands were clearly visible. The molecular weight of L. fermentum LMEM22 bacteriocin was calculated from the relative mobility of the molecular weight markers in the gel.

Antibacterial activity of bacteriocin
The antibacterial activity of L. fermentum LMEM22 bacteriocin was determined by agar-well diffusion following Tagg and McGiven (Tagg and McGiven, 1971), using 66.67-µg bacteriocin per well (6 mm diameter), prepared on nutrient agar plate, which was swabbed with the overnight grown culture of indicator bacterial strains in nutrient broth, and the ZDIs were measured (in nearest whole in millimeter), in order to interpret the effectiveness of bacteriocin, following the criteria mentioned earlier 6 , as less active, moderately active, or highly active through ZDIs ≤ 10 mm, 11-14 mm, and ≥ 15 mm, respectively.

Results
Based upon the 16S rRNA gene sequence and phylogeny analysis, the LAB strain of curd origin, LMEM22, has been found to be closest to Lactobacillus fermentum hani.A ton 2 KM214424, and was identi ed as Lactobacillus fermentum LMEM22 (Fig. 1), the NCBI GenBank accession No. for which is MH380182. The antibacterial bacteriocin producing γ-haemolytic L. fermentum strain LMEM22 (Fig.1), for which gelatinase and DNase activities were negative, had tolerance to high sodium chloride concentration (2 % -6.5 %) and temperature (15 o C-42 o C) range, acidic milieu (pH: 2.0-4.0) and bile salts (0.125-0.5 %), as represented in Table 1.
The effect of enzymes on the L. fermentum LMEM22 bacteriocin, in terms of the antibacterial activity, has been represented in Table 2. When treated with enzymes, proteinase K and trypsin, the bacterial growth inhibition capacity (in terms of ZDI) of L. fermentum LMEM22 bacteriocin, against gram-positive: Staphylococcus aureus and Listeria monocytogenes as well as gram-negative: Acinetobacter baumannii and Escherichia coli, test bacteria was reduced by 3-5 mm (both for gram-positive and gram-negative bacteria) compared to the untreated bacteriocin activity, while mostly the bacteriocin activity was unaffected by α-amylase.

Discussion
This study authenticates the probiotic competency of a native L. fermentum LMEM22 strain from curd by means of safety pro ling and functionality (stability against selective physiological stress and antibacterial e cacy) testing, and the identity con rmation, as well through 16S rRNA gene sequencing. The sequence determination of 16S rRNA genes of the genus Lactobacillus provides precise information on their phylogeny and identi cation (Vandamme et al.,, 1996). The 16S rRNA gene analysis, using the universal primers (forward: 5′-AGAGTTTGATCCTGGCTCAG-3′ and reverse: 5′-TACGGCTACCTTGTTACGACTT-3′), of two heterofermentative lactobacilli isolates showed 99 % similarity to L. fermentum, while the two homofermentative isolates had similarity with L. plantarum, as has been demonstrated by Patil et al. (2010). A set of forward primer: 5′-AGHGTBTGHTCMTGNCTCAS-3′ and reverse primer: 5′-TRCGGYTMCCTTGTWHCGACTH-3′ has been utilized in the PCR ampli cation of 16S rRNA in order to recognize the identity of LAB strains isolated from a variety of sources (Roy and Rai, 2017). A non-spore forming gram-positive, but catalase and oxidase negative rod shaped LAB strain, LMEM22, which was conventionally identi ed as L. fermentum LMEM22 (Das and Goyal, 2014), has been subjected to molecular based identi cation, in this study, by 16S rRNA gene sequencing and phylogeny analysis. The identity of the test strain, being closest to Lactobacillus fermentum hani.A ton 2 KM214424, was con rmed as L. fermentum LMEM22 (the NCBI GenBank accession number for which is MH380182).
Based upon the physiological stressor tolerance (bile salts, low-pH and sodium chloride) and safety pro les (γ-haemolysis and inept to gelatinase and DNase production), the L. fermentum LMEM22 has been substantiated as a potential probiotic strain, following the criteria mentioned earlier (Gareau et al.,, 2010;Halder et al.,, 2017). In addition, a vital probiotic feature that signi es the safe consumption of indigenous LAB strain is the antibiotic sensitivity (to avoid the risk of resistance transferability) and the intrinsic resistance (chromosomally conferred due to point mutation) as well (Georgieva et al., 2015;Jose et al.,, 2015), for co-administration of probiotic with antibiotic (Mombelli and Gismondo, 2000). The isolated L. fermentum LMEM22 strain had been found to be safe, on the basis of non-transferable nature of antibiotic resistance (Ammor et al., 2008;Imperial and Ibana, 2016), and thus, the current study validated the single-strain based (L. fermentum LMEM22) probiotic bene t for people in our part of the globe (Malda, West Bengal state, India).
In order to muddle through the escalating incidence of antimicrobial resistance (AMR) among human pathogenic bacteria a class of protein antibiotics, the bacteriocins, produced especially by probiotic LAB strains, has been paid an immense attention, because of their stupendous capacity to antagonize the human pathogenic bacteria, including the WHO priority pathogens (Mycobacterium tuberculosis, Clostridium di cile and Staphylococcus aureus) too (Cotter et (Shelburne et al.,, 2007). As per the report of Sharma et al. (2018), a bacteriocin of ≈6.5 kDa from Bacillus subtilis GAS101, has been found to be the potential inhibitor of Staphylococcus epidermidis and Escherichia coli. The bacteriocin of 2.7 kDa isolated from Weissella confusa A3 strain had antibacterial activity against gram-positive (Bacillus cereus ATCC14579 and Micrococcus luteus ATCC10240) and gram-negative (Escherichia coli UT181 and Pseudomonas aeruginosa PA7) bacteria, displaying MICs of 9.25 μg/ml and 18.5 μg/ml, respectively, while the ZDIs ranged 7.98 -11.83 mm (Goh and Philip, 2015). Elayaraja et al.(2014) demonstrated the antibacterial activity of 21 kDa Lactobacillus murinus AU06 bacteriocin against gram-positive (S. aureus, Micrococcus sp., Bacillus licheniformis, Enterococcus faecalis, Listeria monocytogenes) and gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria, with ZDIs 10-28 and 18-22 mm, respectively. The bacteriocin, paracin 1.7 of ≈10 kDa, produced by L. paracasei HD1-7 strain, had a wide spectrum of growth-inhibitory effect on Staphylococcus, Micrococcus and Bacillus (gram-positive bacteria) as well as Proteus, Escherichia, Enterobacter, Pseudomonas and Salmonella (gram-negative bacteria), as has been reported by Ge et al. (2009). The ≈13 kDa L. fermentum LMEM22 bacteriocin, in the current study, had broad antibacterial capacity (against bacteria of clinical relevance) displaying ZDIs of 17-23 mm and MICs of 41-199 µg/ml.
The protein/peptide nature of LAB bacteriocin has been speci ed previously through enzyme (proteolytic) sensitivity testing, wherein the disbanding of antibacterial capacity of such protein antimicrobials by enzymes (proteinase K, chymotrypsin and trypsin) validated their (bacteriocins) proteinaceous nature (Xiraphi et al.,, 2008;Ge et al., 2016). The bacteriocin of L. fermentum sl36 strain isolated from goat milk, which inhibited the growth of food-borne bacteria (Enterococcus faecalis, Listeria monocytogenes, Listeria innocua, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli),, lost the antibacterial activity, against gram-positive strains, after treatment with trypsin (Mitjans et al.,, 2018). The antimicrobial bacteriocin Lac-B23, of ≈6.73 kDa from L. plantarum J23 strain, demonstrated complete loss of anti-Listeria monocytogenes activity on being treated with proteinase K, trypsin, and proteinase E, indicating the complete disruption of bacteriocin Lac-B23, by proteolytic enzymes, due to its proteinaceous nature (Zhang et al.,, 2018).As has been reported by Todorov et al. (2013), the Lactobacillus sakei strains ST22Ch, ST153Ch and ST154Ch (from fermented meat products), containing bacteriocin of 3 kDa, 10 kDa and 3 kDa, respectively (in tricine/SDS-PAGE), which inhibited the growth of gram-positive (Enterococcus spp., Staphylococcus spp., Listeria spp. and Streptococcus spp.) and gramnegative (Escherichia coli, Pseudomonas spp. and Klebsiella spp.) bacteria, demonstrating a decrease of antibacterial action with proteinase K and pronase treatment of the bacteriocin, but not due to α-amylase treatment. The bacteriocins of L. plantarum, L. pentosus and L. paracasei displaying growth inhibitory effect against Salmonella and S. aureus with ZDIs 13.08±0.15-15.22±0.13 mm, lost their antimicrobial action after treatment with proteolytic enzymes (proteinase K, pepsin, and papain), as has been reported by Ren et al. (2018). In the current study, on enzymatic treatment with proteinase K and trypsin, the L. fermentum LMEM22 bacteriocin activity has been found to be decreased when tested against grampositive (Staphylococcus aureus and Listeria monocytogenes) as well as gram-negative (Acinetobacter baumannii and Escherichia coli) indicator bacteria, and the bacteriocin was found insensitive against αamylase activity, authenticating its proteinaceous nature, which was in accordance to the report of the earlier authors (

Conclusion
The isolated L. fermentum LMEM22 bacteriocin (13.1 kDa) of proteinaceous nature had broad spectrum antibacterial activity against an array of gram-positive (ZDIs: 17-24 mm; MICs: 41-132 µg/mL) and gram-negative (ZDIs: 19-23 mm; MICs: 50-199 µg/ml) bacteria of clinical importance, and the bacteriocinogenic LAB L. fermentum LMEM22 strain accomplished the probiotic attributes along with the indispensable safety pro les so as to be safe for human consumption, at least in our developing part of the globe. However, in order to resolve whether the bene cial effect is shaped enough by the live form of L. fermentum LMEM22 strain of curd origin and/or the bacteriocin produced by this LAB further studies (animal model/clinical trials) are warranted for global usage.

Declarations Physiological stressors
Level of stressors exposed log 10 CFU/mL at different hours of incubation   Figure 1 The 16S rRNA gene sequence based phylogenetic tree for Lactobacillus fermentum LMEM22 strain compared with the sequences of closely related reference bacterial strains retrieved from NCBI GenBank database.