Identification of the selected isolate using polyphasic approach

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2 SUMMARY The rapid emergence of drug resistance among pathogenic bacteria, especially multi drug resistant bacteria, has emerged as one of the pre-eminent public health concerns of the 21st century. The highly concerned resistant bacteria causing community and hospital acquired infections include Methicillin Resistant Staphylococcus aureus (MRSA), Vancomycin Resistant Enterococcus (VRE), Vancomycin Resistant Staphylococcus aureus (VRSA), Extended Spectrum β- Lactamase (ESBL) producing Escherichia coli and Klebsiella spp., Haemophilus influenzae, Multi-Drug Resistant Mycobacterium tuberculosis (MDRTB, Pseudomonas aeruginosa, Acinetobacter baumannii and Enterobacter spp. Hence, it is imperative to search new, efficient and safe antimicrobials to combat the problem of microbial resistance and to develop new antibiotics to substitute the ineffective ones. Microbial natural products appear as the most promising sources for developing future antibiotics and special attention has been directed towards actinobacteria as they are the major source of novel bioactive metabolites. Among actinobacteria, genus Streptomyces has long been recognized as inexhaustible producer of useful secondary metabolites and produces more than half of the known microbial bioactive substances. Nevertheless, they are still a rich source of novel bioactive compounds and many more antibiotics remain to be identified from them. Keeping all these factors in mind, the present study was planned to isolate an actinobacterium producing novel and potent antimicrobial compound/s, and research work was carried out on the following lines: Isolation of the potent actinobacterial isolate, exhibiting broad spectrum antimicrobial activity against drug resistant bacteria, especially MRSA. Identification of the selected isolate, using polyphasic approach. Optimization of antimicrobial production using classical and statistical approaches, and recovery of compound(s). Purification of antimicrobial compound/s by using various chromatographic techniques. Characterization of active compound(s) using SDS-PAGE, MALDI-TOF-MS, MS/MS sequencing and GC-MS. 173

3 Safety evaluation of purified compound/s by Ames mutagenicity test, DNA nicking assay and in vitro cytotoxicity assay. Studies of protoplast regenerated strains. Isolation, screening and selection of potential isolate In the present study, 134 different actinobacterial isolates were isolated from the soil samples collected from different localities of Punjab and Himachal Pradesh. In order to identify potential antibiotic producers, all isolates were screened for antimicrobial activity against various test organisms viz. Bacillus megaterium (MTCC 428), Bacillus subtilis (MTCC 619), Enterobacter aerogenes (MTCC 111), Escherichia coli (MTCC 1885), Klebsiella pneumoniae sub sp. pneumoniae (MTCC 109), Proteus mirabilis (MTCC 1429), Salmonella typhi (MTCC 733), Candida albicans (MTCC 3017), Rhodotorula rubra (MTCC 248) and clinical isolates procured from local hospitals: Enterococcus sp., multi drug resistant E. coli and MRSA. Among 134 isolates, forty four (32.8%) exhibited antimicrobial activity in culture supernatant and six (2A, R3YS, N23, A26, A27 and A13) showed promising broad spectrum activity against different test organisms. When grown in Starch Casein Nitrate (SCN) broth all showed maximal antibiotic production on fifth day of incubation. Statistical analysis showed significant interaction between actinobacterial isolates and test organisms. Out of them, isolate 2A, procured from a soil sample collected from Guru Nanak Dev University, Amritsar, Punjab (India) and showing strong antimicrobial activity against all the tested bacteria and fungi was chosen for further studies. Identification of the selected isolate using polyphasic approach The isolate 2A was characterized using polyphasic taxonomic approach which included the complete 16S rrna gene sequencing and its comparative analysis by constructing phylogenetic trees, DNA-DNA hybridization studies with related strains, along with chemotaxonomical, biochemical, physiological and morphological studies. It showed a range of chemotaxonomic and morphological properties consistent with its classification in the genus Streptomyces. It formed rectiflexibilis-type spore chains with smooth spore surface ornamentation and grew well on most of the tested media, except ISP 5 (International Streptomyces Project). 174

4 Isolate 2A had cell wall chemotype I which is characteristic of the Streptomyces genus. The cell wall contained LL-diaminopimelic acid and glycine, and no characteristic sugar was detected in whole cell hydrolysates. The menaquinones present were MK-9(H 6 ) (58.2%), MK-9(H 8 ) (28.3%) and MK-9(H 10 ) (13.5%). The major phospholipids detected were phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol (phosholipid type II). In addition, one unknown glyco lipid and five unknown phospholipids were also present. The major fatty acids found were anteiso-c 15: 0 (29.2%), C 16: 0 (16.0%) and iso-c 16: 0 (12.1%). The DNA G+C content of strain was estimated to be 70.5 ± 1.0 mol%. Comparison of 16S rrna gene sequence (1463 bp, GenBank accession no GQ906975) of the isolate with those of other closely related taxa retrieved from the EzTaxon database showed the highest similarity i.e. 99.9%, 99.5% and 99.5% with the 16S rrna gene sequences of the type strains Streptomyces flavotricini DSM T, Streptomyces toxytricini DSM T and Streptomyces globosus DSM T, respectively. No significant differences were observed in topologies of phylogenetic trees generated by using neighbour-joining and maximum-parsimony methods. The isolate formed a distinct cluster in both phylogenetic trees. The low bootstrap values (<50%) in maximum-parsimony tree do not reliably indicate its specific relatedness with closely related species. The taxonomic integrity of the isolate was also supported by the DNA-DNA relatedness data as it shared DNA-DNA homology of only 37.6± (0.6)%, 34.4± (0.5)% and 33.1± (0.4)% with the closely related type strains, S. flavotricini DSM T, S. globosus DSM T and S. toxytricini DSM T, respectively. These values are clearly well below the 70 % cut-off point recommended for the assignment of strains to the same genomic species. The isolate was further distinguished from the phylogenetic close relatives on the bases of a variety of morphological, physiological and biochemical properties. Based on the genotypic and phenotypic characteristics, isolate 2A was identified as a novel species of the genus Streptomyces, for which the name Streptomyces amritsarensis sp. nov. is proposed, with the type strain 2A T (=MTCC T = JCM T ). Besides above differences, isolate 2A and related Streptomyces species type strains exhibited difference in their antimicrobial activity spectrum. Isolate 2A 175

5 demonstrated potent antimicrobial activity against all the test microorganisms whereas related type strains showed weak activity. Optimization of antimicrobial production The traditional one-variable-at-a-time optimization strategy and statistically based experimental designs, Placket-Burman Design (PBD) and Response Surface Methodology (RSM), were applied for optimization of process parameters for antimicrobial production from S. amritsarensis. By using traditional strategy maximum production was achieved by growing S. amritsarensis at 28ºC in a medium with initial ph between under shaking conditions (180 rpm) using 2.0% inoculum. Among the various carbon and nitrogen sources studied, starch and KNO 3 were found to be the best carbon and nitrogen sources, respectively. Optimum growth and production were achieved using % (w/v) starch and 0.2% (w/v) KNO 3 in production medium. PBD analysis values (t-values) demonstrated significant positive influence of KNO 3, K 2 HPO 4, NaCl and starch on the production with main effects ranging from 1.0 to The data obtained showed a range of positive main effect values indicating the presence of high levels of KNO 3, K 2 HPO 4, NaCl and starch in the medium positively effecting the production; while casein, MgSO 4.7H2O, FeSO 4.7H 2 O and CaCO 3 had negative effect on the production. In PBD optimized medium antimicrobial activity increased by 1.06 folds. For determination of optimal combination of KNO 3, K 2 HPO 4, and NaCl for antimicrobial production response surface methodology was applied. The t-test and p-values were used to identify the effect of each factor on production. The R 2 values were found to be 95.2, 92.9 and 92.8% for B. subtilis, K. pneumoniae and MRSA, respectively which reflected a very good fit between the observed and predicted responses, and implied that the model was suitable for the optimization of antimicrobial production by S. amritsarensis. Comparison of antibacterial activity in basal, classically, PBD and RSM optimized media revealed significant i.e. 1.5-fold enhancement in antimicrobial production (P 0.05). In addition, optimization enhanced the antimicrobial activity spectrum. When S. amritsarensis was grown in unoptimized production medium, the culture supernatant did not show any 176

6 activity against P. aeruginosa (MTCC 1688) but after optimization culture supernatant showed activity against P. aeruginosa. Production and purification of antimicrobial compounds For large scale production of antimicrobial compounds by S. amritsarensis, fermentation was carried out in SCN broth at 28ºC for 4 days at ph 7.0 under shaking conditions (180 rpm). The active compounds from culture supernatant were recovered by adsorption using adsorbent resin XAD-4 (5%). From methanol extract two antimicrobial compounds were purified using silica gel chromatography, size exclusion chromatography, and reverse phase-hplc using C18 columns (Pursuit10 C18 and ZORBAX 300SB-C18) and identified to be i) peptide and ii) lipopeptide. Characterization of purified peptides The peptide was characterized using Tricine-SDS-PAGE analysis and MALDI- TOF. The precise molecular mass of the peptide was found to be 5.6 kda as determined by MALDI-TOF. It showed broad spectrum antimicrobial activity i.e. against bacteria viz. MRSA, VRE, multidrug resistant E. coli, P. aeruginosa (resistant to cefepime and imipenum), Klebsiella sp. (resistant to cefepime) and Mycobacterium smegmatis (MTCC 6), fungi viz. Colletotrichum acutatum (MTCC 1037), Cercospora beticola (GenBank accession no KJ461435), Fusarium oxysporum f.sp. dianthi (MTCC 6659), and Alternaria brassicicola (MTCC 2102), yeasts viz. C. albicans and R. rubra, and Streptomyces spp. viz. Streptomyces olivaceus (MTCC 1392), Streptomyces lavendulae sub sp. lavendulae (MTCC 706) and Streptomyces hydrogenans (DH16 JX123130). The MIC values of this bacteriocin-like peptide against various test organisms varied from 2-32 µg ml -1 depending upon the sensitivity of the tested microorganism. It showed the lowest MIC value of 2µg ml -1 against B. subtilis and the highest against C. acutatum i.e. 32 µg ml -1. It was completely stable at 80 C for 1h with half life of 15 min at 121 C, and was found to be sensitive to proteolytic enzymes (trypsin and proteinase K). The structure of lipoppeptide was elucidated by MS/MS analysis which revealed that it had amino acid sequence as Ala-Thr-Gly-Ser-His-Gln and a long chain fatty acid 177

7 tail with six times repeated the molecular mass of 161 Da which was corresponding to -C 12 H 19. Based on the molecular mass (878.5 Da) and amino acid composition, the lipopeptide was identified as a novel lipopeptide. It showed antimicrobial activity only against Gram-positive bacteria viz. B. subtilis, Staphylococcus epidermidis, Mycobacterium smegmatis and MRSA, and the MIC values against them were found to be 10, 15, 25 and 45 µg ml -1, respectively. It was completely stable at 70 C for 1 h and retained 81.8% activity after autoclaving (121 C for 15 min). It did not show any change in its activity profile between ph It was found to be resistant to trypsin and lipase, and negligible loss in activity was observed after treatment with proteinase K. Safety evaluation of peptides Safety evaluation by Ames test revealed that the antimicrobial peptides from S. amritsarensis were non mutagenic at two concentrations (50 and 100 µg 0.1 ml -1 ) used in the experiment. However, both the peptides showed antimutagenicity and were found to be more effective against indirect-acting mutagen that is 2-aminofluorene (2-AF), in the presence of S9, as compared to direct acting mutagen, 4-Nitro-o-phenylenediamine (NPD). The highest antimutagenic response of and 97.93% against 2-AF was obtained with peptide and lipopeptide, respectively at concentration of 50 µg 0.1 ml -1. The results of the DNA nicking assay showed that both the peptides provided protection to the super coiled pbr 322 DNA from devastating effects of hydroxyl radicals generated by Fenton s reagent. It was found that when the plasmid DNA was dissolved in the Fenton s reaction mixture, there was an increase of single stranded and double stranded nicked and linear forms of DNA (Forms II and III, respectively) due to hydroxyl radicals generated in reaction mixture. However, addition of peptides to the reaction mixture reduced the hydroxyl radical mediated strand breaking and conversion of supercoiled DNA to Forms II and III DNA. In vitro sulforhodamine B assay revealed that the peptide was cytotoxic and showed significant cytotoxicity i.e 81.3 and 82.1% against Prostrate (PC3) cell line and Chinese Hamster Ovary (CHO) cell line, respectively. However, lipopeptide showed insignificant cytotoxicity i.e. 42.3% inhibition against PC3 cell line and 30.1% inhibition against CHO cell line. 178

8 Emulgel formation and its characterization The emulgel formulation of the peptides produced by S. amritsarensis was developed and characterized for physical appearance, ph, extrudability, viscosity and antimicrobial activity. It was opaque whiter in its appearance and had ph 6.8 ± 0.2, reflecting that the gel would be non irritant to the skin. The emulgel showed good extrudability and its viscosity was found to be 1500 ± 120 cp. It showed antimicrobial activity against microorganisms used for testing its activity viz. MRSA, B. subtilis, E. coli, K. pneumoniae, S. typhi, C. albicans and R. rubra. The skin irritancy as determined by Draize test in male albino rats revealed that emulgel formulation as well as 0.5% solution of peptides did not cause any type of skin irritation. Thus emulgel formulation of these peptides could be safely applied for treatment of skin infections. Studies on protoplast regenerated strains To obtain the strain with enhanced antimicrobial production, protoplasts were prepared from S. amritsarensis and regenerated, and the effect of regeneration on antimicrobial activity was studied. The maximum number of protoplasts (63.1 x 10 4 ml -1 ) was achieved when 30 h old mycelium (taken from early logarithmic phase of growth), grown in S-medium containing 0.4% glycine, was incubated for 45 min in the presence of 10 mg ml -1 lysozyme. Regenerants showed considerable cultural and morphological variations when grown on various ISP media and SCNA. The wild strain formed rectiflexibilis-type spore chains, the regenerants with enhanced activity (R1 b, R1 a, R2 and R3) formed rectiflexibilis-type spore chains with a loop at the tip and non active regenerants (R4, R2) formed straight to flexuous spore chains. There was also variation in spore size: hyperactives had large spores i.e. 1.2 x 0.6 µm as compared to wild strain (0.9 x 0.5 µm) whereas nonactive had spores of 0.8 x 0.4 µm size. S. amritsarensis formed light pinkish colonies with regular margins. However, hyper producers formed thin light grayish color colonies with regular edges and non active formed thick white color colonies with irregular margins. The protoplast regenerated strains demonstrated significant enhancement in antimicrobial production (P 0.05) and maximum i.e 2.0 fold increase in activity in terms of the inhibition zone (mm) was observed in R1 b. 179

9 FUTURE PROSPECTS S. amritsarensis produces two novel compounds, one peptide exhibiting broad spectrum activity and another lipopeptide exhibiting activity against Gram-positive bacteria. They are thermostable and active over a broad ph range. Lipopeptide is nontoxic and demonstrates good antimutagenic and DNA protective properties. Thus it might serve as a new pharmacological agent to fight infections caused by Gram-positive bacteria, especially MRSA. It might also be used in the cosmetics industry for developing skin-care products and shampoos, and as an emulsifier and bio-preservative in the food industry. The chemical modifications in the lipid tail length of the lipopeptide could enhance its antimicrobial spectrum. The peptide showed antimutagenic and DNA protective properties and good anti-cancerous activity against PC3 cell line. Therefore, it provides a ray of hope for development of novel anti-cancerous agent and might serve as new antimicrobial agent for fighting infections caused by wide range of drug resistant bacteria. Besides antibacterial activity it also possesses antifungal activity against phytopathogens hence, it could also be used as a potent biocontrol agent to control fungal plant pathogens. 180